Antibodies binding to axl

Abstract

The present invention relates to antibodies that bind AXL. The present invention relates to anti-AXL antibodies, immunoconjugates, compositions, and methods of using such anti-AXL antibodies, immunoconjugates, or compositions to treat cancer.

Description

[0001] This application is a divisional application of the international application PCT / EP2015 / 065900, filed on July 10, 2015, which entered into China with application number 201580045131.4 and entitled "Antibody that binds to AXL". Technical Field

[0002] This invention relates to antibodies that bind to AXL, immunoconjugates, compositions comprising such antibodies or immunoconjugates, and the use of said antibodies and immunoconjugates. Background Technology

[0003] The TAM subfamily of mammalian receptor tyrosine kinases (RTKs) consists of AXL, Tyro3, ​​and Mer. AXL is a 104–140 kDa transmembrane protein with transforming capabilities[1]. AXL can be activated by binding to its ligand, vitamin K-dependent growth arrest-specific factor 6 (Gas6). Gas6 binding to AXL leads to AXL dimerization, autophosphorylation, and subsequent activation of intracellular signaling pathways such as PI3K / AKT, mitogen-activated protein kinase (MAPK), STAT, and NF-κB cascades[2]. In cancer cells, AXL enhances tumor cell motility, invasion, migration, and is involved in epithelial-mesenchymal transition (EMT)[3]. Furthermore, AXL expression has been considered to be involved in resistance to chemotherapy and targeted therapies, such as epidermal growth factor receptor (EGFR) targeted therapy (Wilson 2014, Brand 2013, Zhang 2012) or inhibitors of the B-raf (BRAF) pathway (Muller, 2014).

[0004] The extracellular domains of TAM receptor family members consist of a combination of two N-terminal immunoglobulin (Ig)-like domains and two fibronectin type III (FNIII) repeat sequences [1]. The ligand Gas6 binds to Ig-like domains I and II of AXL

[14] .

[0005] Upregulation of AXL has been reported in a variety of cancers, including gastric cancer, prostate cancer, ovarian cancer and lung cancer[1]. In addition, AXL is overexpressed in breast cancer and pancreatic cancer and is significantly associated with higher metastasis frequency and poorer overall survival[2].

[0006] Targeted inhibition of RTK may be as effective as antitumor and / or metastasis therapy. Such targeted inhibition of AXL and / or ligand Gas6 includes small molecules and anti-AXL antibodies [3]. Anti-AXL antibodies have been described to attenuate in vivo growth of non-small cell lung cancer xenografts by downregulating receptor expression, thereby reducing tumor cell proliferation and inducing apoptosis [4]. In addition, a number of monoclonal antibodies that block the binding of ligand Gas6 to AXL have been described [2], [5] and [7].

[0007] Anti-AXL antibodies have been described previously [8]-

[13] . However, there is still a need for anti-AXL antibodies with improved antitumor activity. Summary of the Invention

[0008] The objective of this invention is to provide anti-AXL antibodies. Therefore, in one aspect, the invention relates to antibodies that bind to AXL, wherein said antibodies do not compete with ligand growth arrest-specific 6 (Gas6) for binding to AXL.

[0009] In another aspect, the present invention relates to bispecific antibodies comprising a first binding region of the antibody according to the invention, and a second binding region that binds a target or epitope different from the first antigen binding region.

[0010] On the other hand, the present invention relates to immunoconjugates comprising an antibody or bispecific antibody according to the invention, and a therapeutic component such as a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressant, antibiotic or radioisotope.

[0011] In another respect, the present invention relates to compositions comprising antibodies, bispecific antibodies or immunoconjugates according to the invention.

[0012] In another respect, the present invention relates to pharmaceutical compositions comprising antibodies, bispecific antibodies or immunoconjugates according to the invention, and pharmaceutically acceptable carriers.

[0013] On the other hand, the present invention relates to nucleic acid constructs that encode antibodies according to the present invention.

[0014] In another respect, the present invention relates to expression vectors comprising one or more nucleic acid constructs according to the present invention.

[0015] In another respect, the present invention relates to a host cell comprising a carrier according to the invention.

[0016] In another respect, the present invention relates to hybridomas that produce antibodies according to the present invention.

[0017] In another aspect, the present invention relates to the use of antibodies, bispecific antibodies or immunoconjugates according to the present invention as pharmaceuticals.

[0018] In another aspect, the present invention relates to antibodies, bispecific antibodies or immunoconjugates according to the present invention for the treatment of cancer.

[0019] In another aspect, the present invention relates to a method for treating cancer, comprising administering to an individual in need an antibody, bispecific antibody, immunoconjugate, composition, or pharmaceutical composition according to the invention.

[0020] In another aspect, the present invention relates to a method for diagnosing a disease characterized by the involvement or accumulation of cells expressing AXL, the method comprising administering to an individual an antibody, bispecific antibody, immunoconjugate, composition, or pharmaceutical composition according to the invention, optionally wherein the antibody is labeled with a detectable reagent, and wherein the amount of cells expressing AXL is associated with or indicates the disease.

[0021] In another aspect, the present invention relates to a method for inhibiting the growth and / or proliferation of tumor cells expressing AXL, comprising administering to an individual in need an antibody, bispecific antibody, immunoconjugate, composition, or pharmaceutical composition according to the invention.

[0022] In another aspect, the present invention relates to a method for producing antibodies according to the invention, the method comprising the steps of: a) culturing host cells or hybridomas according to the invention, and b) purifying the antibody from the culture medium.

[0023] In another respect, the present invention relates to diagnostic compositions comprising antibodies or bispecific antibodies according to the present invention.

[0024] In another aspect, the present invention relates to a method for detecting the presence of AXL antibodies or AXL-expressing cells in a sample, comprising the steps of: a) contacting the sample with an antibody, bispecific antibody, or immunoconjugate according to the invention (under conditions that allow the formation of a complex between the antibody, bispecific antibody, or immunoconjugate and AXL); and b) analyzing whether a complex has been formed.

[0025] In another aspect, the present invention relates to a kit for detecting the presence of AXL antigen or AXL-expressing cells in a sample, comprising: i) an antibody, bispecific antibody or immunoconjugate according to the present invention; and ii) instructions for use of the kit.

[0026] In another aspect, the present invention relates to anti-idiotype antibodies that combine with anti-AXL antibodies according to the present invention. Attached Figure Description

[0027] Figure 1Binding curves of anti-AXL antibody with HEK293 cells transfected with the following: (A) human AXL-ECD, (B) cynomolgus monkey AXL-ECD, or (C) mouse AXL-ECD. The data shown are mean fluorescence intensities (MFI) of a representative experiment, as described in Example 2.

[0028] Figure 2 The binding of anti-AXL antibodies to mouse-human AXL chimeras was performed as described in Example 3. The following human AXL (hsAXL) and mouse AXL (mmAXL) chimeric proteins were tested: (A) hsAXL and mimics, (B) hsAXL-mmECD, (C) hsAXL-mmIg1, (D) hsAXL-mmIg2, (E) hsAXL-mmFN1, and (F) hsAXL-mmFN2.

[0029] Figure 3 Antibody-dependent cell-mediated cytotoxicity induced by anti-AXL antibody in A431 cells. Antibody-dependent cell-mediated cytotoxicity induced by anti-AXL antibody in A431 cells was determined as described in Example 4.

[0030] Figure 4 Binding properties of AXL antibody-drug conjugate (AXL-ADC). Binding of AXL-ADC to HEK293T cells transiently transfected with human AXL was determined as described in Example 5. The data shown are mean fluorescence intensity (MFI) of a representative experiment.

[0031] Figure 5 In vitro cytotoxicity induced by the AXL antibody-drug conjugate. The cytotoxicity induced by the AXL antibody-drug conjugate was determined as described in Example 6.

[0032] Figure 6 : Allows binding of AXL antibody VH and VL variants. Antibodies with the same VL or VH region were compared, and differences in the VH (AD) or VL (E) sequences were identified and marked with boxes in the figure. CDR regions are underlined.

[0033] Figure 7 As described in Example 8, ADC-induced cytotoxicity was measured in LCLC-103H cells.

[0034] Figure 8 As described in Example 9, the antitumor activity of the MMAE-conjugated AXL antibody in the therapeutic LCLC-103H xenograft model.

[0035] Figure 9Immunohistochemical staining of frozen PAXF1657 tumor sections (pancreatic cancer PDX model) was performed using a set of AXL monoclonal antibodies as described in Example 10.

[0036] Figure 10 : Mean tumor size after therapeutic treatment with AXL-ADC (PAXF1657 model). Uncoupled

[0037] AXL Humab and the untargeted ADC did not show antitumor activity, indicating that the therapeutic efficacy of AXL-ADC depends on the cytotoxic activity of MMAE and on targeted binding. The error bar represents SEM.

[0038] Figure 11 The binding of anti-AXL antibodies to mouse-human AXL chimeras was performed as described in Example 11. The following human AXL (hsAXL) and mouse AXL (mmAXL) chimeric proteins were tested: (A) hsAXL and mimics, (B) hsAXL-mmECD, (C) hsAXL-mmIg1, (D) hsAXL-mmIg2, (E) hsAXL-mmFN1, and (F) hsAXL-mmFN2.

[0039] Figure 12 The binding of human Gas6 (hGas6) to A431 cells, which had been pre-incubated with an antibody binding to the Ig1 domain of AXL. The data shown are the mean fluorescence intensity (MFI) of a representative experiment.

[0040] Figure 13 As described in Example 13, the antitumor activity of the MMAE-conjugated AXL antibody was demonstrated in a therapeutic A431 xenograft model (which produces high levels of endogenous Gas6). Figures A and B show results from two independent experiments.

[0041] Figure 14 As described in Example 13, the antitumor activity of the MMAE-conjugated AXL antibody in a therapeutic LCLC-103H xenograft model (which expresses low levels of endogenous Gas6) was demonstrated. Panels A and B show results from two independent experiments.

[0042] Figure 15 As described in Example 8, the cytotoxicity induced by AXL-ADC in A431 cells (A) and MDA-MB231 cells (B) was measured.

[0043] Figure 16AXL staining in thyroid cancer, esophageal cancer, ovarian cancer, breast cancer, lung cancer, pancreatic cancer, cervical cancer, and endometrial cancer. The mean AXL staining intensity (OD) of AXL-positive cells is plotted on the X-axis, while the percentage of AXL-positive tumor cells is plotted on the Y-axis. Each point represents a tumor core from an individual patient.

[0044] Figure 17 Representative examples of tumor cores identified by AXL immunostaining, indicating different types of tumors.

[0045] Figure 18 The AXL antibody specifically binds to AXL, but not to other members of the TAM receptor family. The HuMab-AXL antibody binds to HEK293 cells transfected with: human AXL (A), human MER (B), human TYRO3 (C), or untransfected HEK293 cells (D). To confirm proper expression in transfected cells, untransfected HEK293F cells and cells transfected with AXL (E), MER (F), or TYRO3 (G) were stained with MER-specific and TYRO3-specific antibodies. The data shown are mean fluorescence intensities (MFI) of a representative experiment, as described in Example 15.

[0046] Figure 19 AXL antibodies were detected on the plasma membrane of tumor cell lines that had been incubated with AXL antibodies at 4°C for 1 hour, followed by overnight incubation at 4°C or 37°C. In both MDA-MB-231 cells (A and B) and Calu-1 cells (C and D), more antibodies were detected on the plasma membrane of cells incubated at 4°C compared to cells incubated at 37°C, indicating internalization of membrane-bound antibodies at 37°C.

[0047] Figure 20 Geometric mean fluorescence intensity of LCLC-103H cells after incubation with AXL antibody (which was conjugated with Fab-TAMRA / QSY7). Individual IgG1-b12 and Fab-TAMRA / QSY7 were included as negative controls.

[0048] Figure 21 (A) Mean tumor size following therapeutic treatment with IgG1-AXL-107-vcMMAE (in the esophageal cancer PDX model ES0195). IgG1-b12 and IgG1-b12-MMAE were included as isotype control antibody and isotype control ADC, respectively. (B) Tumor size in individual mice on day 32 following injection of MDA-MB-231-luc D3H2LN tumor cells into the mammary fat pads of female SCID mice. * p < 0.05; ** p < 0.0001.

[0049] Figure 22 The therapeutic effect of AXL-ADC in a patient-derived cervical cancer xenograft model.

[0050] (A) Mean tumor size after therapeutic treatment with IgG1-AXL-183-vcMMAE or IgG1-AXL-726-vcMMAE (in the cervical cancer PDX model CEXF 773). IgG1-b12 and IgG1-b12-MMAE were included as isotype control antibody and isotype control ADC, respectively. (B) Tumor size in individual mice on day 28 after the start of treatment (in the cervical cancer PDX model CEXF 773). * p < 0.001.

[0051] Figure 23 Therapeutic activity of AXL-ADC in an orthotopic breast cancer xenograft model. (A) Mean tumor size after therapeutic treatment with IgG1-AXL-183-vcMMAE or IgG1-AXL-726-vcMMAE (in an orthotopic MDA-MB-231-lucD3H2LN xenograft model). IgG1-b12 and IgG1-b12-MMAE were included as allotype control antibody and allotype control ADC, respectively. (B) Tumor size in individual mice on day 32 following injection of MDA-MB-231-lucD3H2LN tumor cells into the mammary fat pads of female SCID mice. * p < 0.001.

[0052] Figure 24 Cytotoxicity of IgG1-AXL-107-vcMMAE in human tumor cell lines with different AXL expression levels on the plasma membrane. AXL expression in the plasma membrane of human tumor cell lines was assessed using Qifikit analysis, and the cytotoxicity of IgG1-AXL-107-vcMMAE was expressed as the percentage of viable tumor cells remaining in the cell culture after exposure to 1 µg / mL IgG1-AXL-107-vcMMAE. Detailed Implementation

[0053] Antibody

[0054] In one aspect, the present invention relates to antibodies that bind to AXL, wherein the antibodies do not compete with ligand growth arrest-specific 6 (Gas6) for binding to AXL.

[0055] The term "antibody," as used herein, is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, which has the ability to specifically bind an antigen under typical physiological and / or tumor-specific conditions and has a significant half-life, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about eight hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally defined time period (such as the time sufficient to induce, promote, enhance, and / or regulate the physiological response associated with antibody binding to the antigen and / or the time sufficient to allow the antibody to be internalized). The binding region that interacts with the antigen (or the binding domain that may be used herein, both having the same meaning) includes the variable regions of both the heavy and light chains of the immunoglobulin molecule. The constant region of an antibody (Ab) mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (which is the first component in the classical pathway of complement activation). As stated above, the term antibody, as used herein, unless otherwise stated or clearly contradicted by the context, includes an antibody fragment that maintains the ability to specifically interact with, such as bind, an antigen. It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antibody” include: (i) Fab’ or Fab fragments, which are monovalent fragments consisting of VL, VH, CL and CH1 domains, or monovalent antibodies as described in

[15] ; (ii) F(ab’)2 fragments, which are bivalent fragments consisting of two Fab fragments connected by disulfide bridges in the hinge region; (iii) Fd fragments, which consist primarily of VH and CH1 domains; (iv) Fv fragments, which consist primarily of VL and VH domains of a single arm of an antibody; (v) dAb fragments

[16] , which consist primarily of the VH domain and are also referred to as domain antibodies

[17] ; (vi) camel antibodies or nanobodies

[18] ; and (vii) separated complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined together using recombination methods via synthetic linkers that allow them to be made into single-protein chains, wherein the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain antibody or single-chain Fv (scFv), see, for example,

[19] and

[20] ). Unless otherwise stated or clearly contradicted by the context, the term antibody encompasses such single-chain antibodies. While such fragments are generally included within the meaning of antibody, they are unique features of the present invention in general and individually, possessing different biological properties and uses. These and other antibody fragments that may be used in the context of the present invention are discussed further herein.It should also be understood that the term antibody, unless otherwise specified, includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like peptides (such as chimeric antibodies and humanized antibodies), and 'antibody fragments' or 'fragments thereof' provided by any known technology (such as enzyme cleavage, peptide synthesis, and recombinant technology) that retain the ability to specifically bind to antigens (antigen-binding fragments) and retain the ability to conjugate to toxins. The resulting antibodies can be of any isotype.

[0056] The term “immunoglobulin heavy chain” or “heavy chain of immunoglobulins,” as used herein, is intended to refer to one of the heavy chains of immunoglobulins. A heavy chain typically consists of a heavy chain variable region (abbreviated VH herein) and a heavy chain constant region (abbreviated CH herein), which defines an isotype of immunoglobulin. The heavy chain constant region typically consists of three domains: CH1, CH2, and CH3. The term “immunoglobulin,” as used herein, is intended to refer to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains: a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four chains potentially interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized (see, for example,

[21] ). Within the structure of an immunoglobulin, the two heavy chains are interconnected by disulfide bonds in so-called “hinge regions.” Like the heavy chain, each light chain typically consists of several regions: a light chain variable region (abbreviated VL herein) and a light chain constant region. The light chain constant region typically consists of a single domain, CL. Furthermore, the VH and VL regions can be further subdivided into highly variable regions (or hypervariable regions, which can be highly variable in the form of sequence- and / or structurally defined rings), also known as complementarity-determining regions (CDRs), scattered within more conserved regions called framework regions (FRs). Each VH and VL typically 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 CDR sequence is defined according to IMGT (see

[22] and

[23] ).

[0057] The terms "antigen-binding region" or "binding region," as used herein, refer to the region of an antibody capable of binding to an antigen. An antigen can be any molecule, such as a polypeptide present in, for example, cells, bacteria, or viral particles. Unless otherwise specified in the context, the terms "antigen" and "target" are used interchangeably in the context of this invention.

[0058] The term "binding," as used herein, refers to the binding of an antibody to a predetermined antigen or target, typically corresponding to the following K... D Binding affinity: approximately 10- 6 M or lower, such as 10- 7 M or lower, such as about 10- 8 M or lower, such as about 10- 9M or lower, approximately 10- 10 M or lower, or about 10- 11 M or even lower (when the antigen is used as a ligand and the protein as an analyte in a BIAcore 3000 instrument using, for example, surface plasmon resonance (SPR) technology), and K corresponding to its affinity for the predetermined antigen compared to its affinity for non-specific antigens (e.g., BSA, casein) that are different from or closely related to the predetermined antigen. D At least ten times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower. The amount by which the affinity is lower depends on the protein's K. D This makes the protein's K D At extremely low levels (i.e., when the protein is highly specific), the affinity for the antigen can be at least 10,000 times lower than the affinity for non-specific antigens. The term "K" D "(M), as used in this paper, refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, and is determined by k d Divide by k a get.

[0059] The term "k" d "(Second -1 (), as used herein, refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also referred to as k. off Value or dissociation rate.

[0060] The term "k" a (M) -1 x seconds -1 (), as used herein, refers to the binding rate constant of a specific antibody-antigen interaction. This value is also referred to as k. on Value or binding rate.

[0061] The term "K" A (M) -1 (), as used herein, refers to the binding equilibrium constant of a specific antibody-antigen interaction, and is expressed by k a Divide by k d get.

[0062] The term “AXL”, as used herein, refers to the protein named AXL, also known as UFO or JTK11, which is a protein containing 894 amino acids and a molecular weight of 104–140 kDa, and is part of the mammalian TAM receptor tyrosine kinase (RTK) subfamily. The molecular weight may vary due to potential differences in the glycosylation of the protein. The AXL protein consists of two extracellular immunoglobulin-like (Ig-like) domains at the N-terminus of the protein, two juxtamembranous extracellular fibronectin type III (FNIII) domains, a transmembrane domain, and an intracellular kinase domain. When bound to its ligand Gas6, AXL is activated by: ligand-independent homophilic interactions between the extracellular domains of AXL, by autophosphorylation in the presence of reactive oxygen species

[24] , or by transactivation via EGFR (Meyer, 2013), and is aberrantly expressed in several tumor types. In humans, the AXL protein is encoded by a nucleic acid sequence that encodes the amino acid sequence shown in SEQ ID NO:130 (human AXL protein: Swissprot P30530; cynomolgus monkey AXL protein: Genbank accession number HB387229.1).

[0063] The term “ligand-independent homophilic interaction,” as used herein, refers to the association between two AXL molecules (expressed on adjacent cells) that occurs in the absence of a ligand.

[0064] The term "antibody that binds to AXL," as used herein, refers to any antibody that binds to an epitope on the extracellular portion of AXL.

[0065] The term "epitope" refers to a protein determinant that specifically binds to an antibody. Epitopes typically consist of surface components of a molecule (such as amino acids, glycosidic side chains, or combinations thereof) and usually possess specific three-dimensional structural characteristics and specific charge properties. The difference between conformational and non-conformational epitopes is that binding to the former is lost in the presence of denaturing solvents, while binding to the latter is lost. Epitopes may contain amino acid residues directly involved in binding and other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked or covered by a specific antigen-binding peptide (in other words, the amino acid residue is located within the covered region of the specific antigen-binding peptide).

[0066] The term "ligand," as used herein, refers to a substance, such as a hormone, peptide, ion, drug, or protein, that specifically and reversibly binds to another protein, such as a receptor, to form a larger complex. A ligand bound to a receptor can alter its chemical conformation and determine its functional state. For example, ligands can function as agonists or antagonists.

[0067] The term "Growth Arrest Specific 6" or "Gas6," as used herein, refers to a protein containing 721 amino acids and a molecular weight of 75-80 kDa that acts as a ligand for the TAM receptor family, including AXL. Gas6 consists of an N-terminal region containing multiple γ-carboxyglutamate (Gla) residues responsible for specific interactions with negatively charged phospholipid membranes. While this Gla domain is not essential for Gas6 binding to AXL, it is necessary for AXL activation. Gas6 may also be referred to as an "AXL ligand."

[0068] The terms “monoclonal antibody,” “monoclonal Ab,” “monoclonal antibody composition,” “mAb,” etc., as used herein, refer to antibody molecular preparations having a single molecular composition. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope. Therefore, the term “human monoclonal antibody” refers to an antibody exhibiting single binding specificity, having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced from hybridomas comprising B cells derived from transgenic or transchromosomally transgenic non-human animals (such as transgenic mice) whose genome contains human heavy chain and light chain transgenes, fused into immortalized cells.

[0069] In one embodiment, the maximum antibody binding in the presence of Gas6 is at least 90%, such as at least 95%, such as at least 97%, such as at least 99%, such as at least 100%, as determined by the method disclosed in Example 2, in the absence of Gas6.

[0070] The competition for AXL between anti-AXL and the ligand Gas6 can be determined as described under the heading "Interference of Gas6 Binding with Anti-AXL Binding" in Example 2. Therefore, in one embodiment, the antibody does not compete with the ligand Gas6 for AXL binding, wherein said competitive binding is determined in an assay comprising the following steps:

[0071] i) Incubate AXL-expressing cells with Gas6.

[0072] ii) Add the anti-AXL antibody to be tested.

[0073] iii) Add the fluorescently labeled secondary reagent for detecting anti-AXL antibodies, and

[0074] iv) Analyze the cells using FACS.

[0075] In another embodiment, the antibody does not competitively bind to the ligand Gas6, wherein the competitive binding is determined in an assay comprising the following steps:

[0076] i) Incubate AXL-expressing cells together with anti-AXL antibody.

[0077] ii) Add Gas6,

[0078] iii) Add the fluorescently labeled secondary reagent for detecting Gas6, and

[0079] iv) Analyze the cells using FACS.

[0080] In one embodiment, the antibody has a binding affinity (K) for AXL. D The range is 0.3x10 -9 Up to 63x10 - 9 M, and wherein the binding affinity is measured using the soluble AXL extracellular domain using biomembrane interferometry.

[0081] The binding affinity can be determined as described in Example 2. Therefore, in one embodiment, the antibody's binding affinity to the antigen is 0.3 x 10⁻⁶. -9 Up to 63x10 -9 M, wherein the binding affinity is determined by a method comprising the following steps:

[0082] i) Load the anti-AXL antibody onto the anti-human Fc capture biosensor, and

[0083] ii) The binding and dissociation of the extracellular domains of soluble recombinant AXL at different concentrations were determined using biomembrane interferometry.

[0084] The term “soluble recombinant AXL extracellular domain”, as used herein, refers to an AXL extracellular domain that has been recombinantly expressed. Since there are no transmembrane and intracellular domains, the recombinant AXL extracellular domain does not attach to, for example, the cell surface but remains in solution. How proteins are recombinantly expressed is well known, see, for example

[25] , and therefore, providing such recombinant AXL extracellular domains is within the knowledge of a person skilled in the art.

[0085] In one embodiment, the antibody dissociates at a rate of 6.9 x 10⁻⁶ for AXL. -5 s -1 Up to 9.7x10 -3 s -1 The dissociation rate was measured using soluble recombinant AXL extracellular domains via biomembrane interferometry.

[0086] The binding affinity can be determined as described above (and in Example 2). Therefore, in one embodiment, the dissociation rate of the antibody against AXL is 6.9 x 10⁻⁶.-5 s -1 Up to 9.7x10 -3 s -1 And said dissociation rate is measured by a method comprising the following steps:

[0087] i) Load the anti-AXL antibody onto the anti-human Fc capture biosensor, and

[0088] ii) The binding and dissociation of the extracellular domains of recombinant AXL at different concentrations were determined using biomembrane interferometry.

[0089] The term "dissociation rate," as used herein, refers to the rate at which an antigen-specific antibody bound to its antigen dissociates from that antigen, and is expressed as s. -1 Therefore, in the context of antibodies binding to AXL, the term "dissociation rate" refers to the dissociation of the AXL-binding antibody from the recombinant AXL extracellular domain and is denoted as s. -1 .

[0090] In one implementation, AXL is human AXL. The amino acid sequence of AXL is based on Swissprot P30530.

[0091] In one implementation, AXL is the cynomolgus monkey AXL (Genbank accession number HB387229.1).

[0092] In one embodiment, the antibody comprises at least one binding region comprising variable heavy chain (VH) CDR1, CDR2, and CDR3 sequences having at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, sequence identity with sequences selected from the following:

[0093] a) SEQ ID No: 36, 37 and 38

[107] ;

[0094] b) SEQ ID No: 93, 94 and 95

[613] ;

[0095] c) SEQ ID No: 93, 126 and 127 [613 / 608-01 / 610-01 / 620-06];

[0096] d) SEQ ID No: 46, 47 and 48

[148] ;

[0097] e) SEQ ID No: 57, 58 and 59

[171] ;

[0098] f) SEQ ID No: 78, 79 and 80

[187] ;

[0099] g) SEQ ID No: 46, 119, and 120 [148 / 140];

[0100] h) SEQ ID No: 51, 52 and 53

[154] ;

[0101] i) SEQ ID No: 72, 73 and 75

[183] ;

[0102] j) SEQ ID No: 72, 74 and 75 [183-N52Q];

[0103] k) SEQ ID No: 114, 115 and 116

[733] ;

[0104] l) SEQ ID No: 123, 124 and 125 [171 / 172 / 181];

[0105] m) SEQ ID No: 108, 109 and 110

[726] ;

[0106] n) SEQ ID No: 108, 121 and 122 [726 / 187];

[0107] o) SEQ ID No: 41, 42 and 43

[140] ;

[0108] p) SEQ ID No: 62, 63 and 64

[172] ;

[0109] q) SEQ ID No: 67, 68 and 69

[181] ;

[0110] r) SEQ ID No: 51, 52 and 54 [154-M103L];

[0111] s) SEQ ID No: 78, 79 and 80

[187] ;

[0112] t) SEQ ID No: 83, 84 and 85 [608-01];

[0113] u) SEQ ID No: 88, 89 and 90[610-01];

[0114] v) SEQ ID No: 98, 99, and 100 [613-08];

[0115] w) SEQ ID No: 103, 104 and 105 [620-06]; and

[0116] x) SEQ ID No: 108, 109 and 111 [726-M101L].

[0117] In one embodiment, the antibody comprises at least one binding region comprising variable heavy chain (VH) CDR1, CDR2, and CDR3 sequences, each of which has a total of up to five mutations or substitutions, such as up to four mutations or substitutions, up to three mutations or substitutions, up to two mutations or substitutions, or up to one mutation or substitution, throughout the entire CDR sequence selected from the variable heavy chain.

[0118] a) SEQ ID No: 36, 37 and 38

[107] ;

[0119] b) SEQ ID No: 93, 94 and 95

[613] ;

[0120] c) SEQ ID No: 93, 126 and 127 [613 / 608-01 / 610-01 / 620-06];

[0121] d) SEQ ID No: 46, 47 and 48

[148] ;

[0122] e) SEQ ID No: 57, 58 and 59

[171] ;

[0123] f) SEQ ID No: 78, 79 and 80

[187] ;

[0124] g) SEQ ID No: 46, 119, and 120 [148 / 140];

[0125] h) SEQ ID No: 51, 52 and 53

[154] ;

[0126] i) SEQ ID No: 72, 73 and 75

[183] ;

[0127] j) SEQ ID No: 72, 74 and 75 [183-N52Q];

[0128] k) SEQ ID No: 114, 115 and 116

[733] ;

[0129] l) SEQ ID No: 123, 124 and 125 [171 / 172 / 181];

[0130] m) SEQ ID No: 108, 109 and 110

[726] ;

[0131] n) SEQ ID No: 108, 121 and 122 [726 / 187];

[0132] o) SEQ ID No: 41, 42 and 43

[140] ;

[0133] p) SEQ ID No: 62, 63 and 64

[172] ;

[0134] q) SEQ ID No: 67, 68 and 69

[181] ;

[0135] r) SEQ ID No: 51, 52 and 54 [154-M103L];

[0136] s) SEQ ID No: 78, 79 and 80

[187] ;

[0137] t) SEQ ID No: 83, 84 and 85 [608-01];

[0138] u) SEQ ID No: 88, 89 and 90[610-01];

[0139] v) SEQ ID No: 98, 99, and 100 [613-08];

[0140] w) SEQ ID No: 103, 104 and 105 [620-06]; and

[0141] x) SEQ ID No: 108, 109 and 111 [726-M101L].

[0142] An implementation scheme is provided in which up to five mutations or substitutions are permitted in the three CDR sequences of the entire variable heavy chain. The mutations or substitutions may be mutations or substitutions of conserved, physical, or functional amino acids, such that the mutations or substitutions do not alter the epitope or preferably alter the binding affinity of the epitope by no more than 30%, such as no more than 20% or no more than 10%. The conserved, physical, or functional amino acids are selected from 20 natural amino acids, namely Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr, and Val.

[0143] In one embodiment, the antibody comprises at least one binding region comprising sequences selected from the following variable heavy chain (VH) CDR1, CDR2, and CDR3 sequences:

[0144] a) SEQ ID No: 36, 37 and 38

[107] ;

[0145] b) SEQ ID No: 93, 94 and 95

[613] ;

[0146] c) SEQ ID No: 93, 126 and 127 [613 / 608-01 / 610-01 / 620-06];

[0147] d) SEQ ID No: 46, 47 and 48

[148] ;

[0148] e) SEQ ID No: 57, 58 and 59

[171] ;

[0149] f) SEQ ID No: 78, 79 and 80

[187] ;

[0150] g) SEQ ID No: 46, 119, and 120 [148 / 140];

[0151] h) SEQ ID No: 51, 52 and 53

[154] ;

[0152] i) SEQ ID No: 72, 73 and 75

[183] ;

[0153] j) SEQ ID No: 72, 74 and 75 [183-N52Q];

[0154] k) SEQ ID No: 114, 115 and 116

[733] ;

[0155] l) SEQ ID No: 123, 124 and 125 [171 / 172 / 181];

[0156] m) SEQ ID No: 108, 109 and 110

[726] ;

[0157] n) SEQ ID No: 108, 121 and 122 [726 / 187];

[0158] o) SEQ ID No: 41, 42 and 43

[140] ;

[0159] p) SEQ ID No: 62, 63 and 64

[172] ;

[0160] q) SEQ ID No: 67, 68 and 69

[181] ;

[0161] r) SEQ ID No: 51, 52 and 54 [154-M103L];

[0162] s) SEQ ID No: 78, 79 and 80

[187] ;

[0163] t) SEQ ID No: 83, 84 and 85 [608-01];

[0164] u) SEQ ID No: 88, 89 and 90[610-01];

[0165] v) SEQ ID No: 98, 99, and 100 [613-08];

[0166] w) SEQ ID No: 103, 104 and 105 [620-06]; and

[0167] x) SEQ ID No: 108, 109 and 111 [726-M101L].

[0168] In one specific implementation, the VH CDR1, CDR2, and CDR3 are selected from a), d), g), or k).

[0169] In one embodiment, the at least one binding region comprises a VH region and a variable light chain (VL) region, which has at least 95%, such as at least 97%, such as at least 99%, such as at least 100% sequence identity with a sequence independently selected from:

[0170] a) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 36, 37 and 38 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 39, GAS and 40 respectively

[107] ;

[0171] b) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively

[148] ;

[0172] c) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 114, 115 and 116 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 117, DAS and 118 respectively

[733] ;

[0173] d) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 51, 52 and 53 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 55, GAS and 56 respectively

[154] ;

[0174] e) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 51, 52 and 54 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 55, GAS and 56 respectively [154-M103L];

[0175] f) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 57, 58 and 59 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 60, GAS and 61 respectively

[171] ;

[0176] g) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 62, 63 and 64 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 65, GAS and 66 respectively

[172] ;

[0177] h) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 67, 68 and 69 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 70, GAS and 71 respectively

[181] ;

[0178] i) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 72, 73 and 75 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 76, ATS and 77 respectively

[183] ;

[0179] j) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 72, 74 and 75 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 76, ATS and 77 respectively [183-N52Q];

[0180] k) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 78, 79 and 80 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 81, AAS and 82 respectively

[187] ;

[0181] l) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 83, 84 and 85 respectively; and VL region, which contains sequences having SEQ ID No: 86, GAS and 87 [608-01] respectively;

[0182] m) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 88, 89 and 90 respectively; and VL region, which contains sequences having SEQ ID No: 91, GAS and 92 [610-01] respectively;

[0183] n) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 93, 94 and 95 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 96, GAS and 97 respectively

[613] ;

[0184] o) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 98, 99 and 100 respectively; and VL region, which contains sequences having SEQ ID No: 10, DAS and 102 [613-08] respectively;

[0185] p) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 103, 104 and 105 respectively; and VL region, which contains sequences having SEQ ID No: 106, GAS and 107 [620-06] respectively;

[0186] q) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 108, 109 and 110 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 112, AAS and 113 respectively

[726] ;

[0187] r) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 108, 109 and 111 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 112, AAS and 113 respectively [726-M101L];

[0188] s) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 41, 42 and 43 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 44, AAS and 45 respectively

[140] ;

[0189] t) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 93, 94 and 95 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 128, XAS (where X is D or G) and 129 respectively [613 / 613-08];

[0190] u) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 119 and 120 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively [148 / 140];

[0191] v) The VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 123, 124, and 125, respectively; and the VL region contains CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 60, GAS, and 61, respectively [171 / 172 / 181]; and

[0192] w) VH region, comprising CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 121, 109, and 122, respectively; and VL region, comprising CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 112, AAS, and 113, respectively [726 / 187]; and

[0193] x) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 93, 126 and 127 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 96, GAS and 97 respectively [613 / 608-01 / 610-01 / 620-06];

[0194] In one embodiment, the at least one binding region comprises a VH region and a variable light chain (VL) region having a total of up to five mutations or substitutions selected from conserved, physical, or functional amino acids, such as up to four mutations or substitutions selected from conserved, physical, or functional amino acids, such as up to three mutations or substitutions selected from conserved, physical, or functional amino acids, such as up to two mutations or substitutions selected from conserved, physical, or functional amino acids, such as up to one mutation or substitution selected from conserved, physical, or functional amino acids.

[0195] a) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 36, 37 and 38 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 39, GAS and 40 respectively

[107] ;

[0196] b) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively

[148] ;

[0197] c) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 114, 115 and 116 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 117, DAS and 118 respectively

[733] ;

[0198] d) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 51, 52 and 53 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 55, GAS and 56 respectively

[154] ;

[0199] e) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 51, 52 and 54 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 55, GAS and 56 respectively [154-M103L];

[0200] f) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 57, 58 and 59 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 60, GAS and 61 respectively

[171] ;

[0201] g) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 62, 63 and 64 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 65, GAS and 66 respectively

[172] ;

[0202] h) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 67, 68 and 69 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 70, GAS and 71 respectively

[181] ;

[0203] i) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 72, 73 and 75 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 76, ATS and 77 respectively

[183] ;

[0204] j) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 72, 74 and 75 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 76, ATS and 77 respectively [183-N52Q];

[0205] k) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 78, 79 and 80 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 81, AAS and 82 respectively

[187] ;

[0206] l) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 83, 84 and 85 respectively; and VL region, which contains sequences having SEQ ID No: 86, GAS and 87 [608-01] respectively;

[0207] m) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 88, 89 and 90 respectively; and VL region, which contains sequences having SEQ ID No: 91, GAS and 92 [610-01] respectively;

[0208] n) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 93, 94 and 95 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 96, GAS and 97 respectively

[613] ;

[0209] o) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 98, 99 and 100 respectively; and VL region, which contains sequences having SEQ ID No: 10, DAS and 102 [613-08] respectively;

[0210] p) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 103, 104 and 105 respectively; and VL region, which contains sequences having SEQ ID No: 106, GAS and 107 [620-06] respectively;

[0211] q) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 108, 109 and 110 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 112, AAS and 113 respectively

[726] ;

[0212] r) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 108, 109 and 111 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 112, AAS and 113 respectively [726-M101L];

[0213] s) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 41, 42 and 43 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 44, AAS and 45 respectively

[140] ;

[0214] t) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 93, 94 and 95 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 128, XAS (where X is D or G) and 129 respectively [613 / 613-08];

[0215] u) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 119 and 120 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively [148 / 140];

[0216] v) The VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 123, 124, and 125, respectively; and the VL region contains CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 60, GAS, and 61, respectively [171 / 172 / 181]; and

[0217] w) VH region, comprising CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 121, 109, and 122, respectively; and VL region, comprising CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 112, AAS, and 113, respectively [726 / 187]; and

[0218] x) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 93, 126 and 127 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 96, GAS and 97 respectively [613 / 608-01 / 610-01 / 620-06];

[0219] This document provides an implementation scheme in which up to five mutations or substitutions are permitted in the three CDR sequences of the entire variable heavy chain and the variable light chain. These up to five mutations or substitutions may be distributed across the three CDR sequences of the entire variable heavy chain and the three CDR sequences of the variable light chain. These up to five mutations or substitutions may also be distributed across the six CDR sequences of the entire binding region. The mutations or substitutions may be mutations or substitutions of conserved, physical, or functional amino acids, such that the mutation or substitution does not alter the epitope or preferably alters the binding affinity of the epitope by no more than 30%, such as no more than 20% or no more than 10%. The conserved, physical, or functional amino acids are selected from 20 known naturally occurring amino acids, namely Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr, and Val.

[0220] In a specific implementation, the at least one binding region comprises a VH region and a variable light chain (VL) region selected from the following:

[0221] a) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 36, 37 and 38 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 39, GAS and 40 respectively

[107] ;

[0222] b) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively

[148] ;

[0223] c) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 114, 115 and 116 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 117, DAS and 118 respectively

[733] ;

[0224] d) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 51, 52 and 53 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 55, GAS and 56 respectively

[154] ;

[0225] e) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 51, 52 and 54 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 55, GAS and 56 respectively [154-M103L];

[0226] f) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 57, 58 and 59 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 60, GAS and 61 respectively

[171] ;

[0227] g) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 62, 63 and 64 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 65, GAS and 66 respectively

[172] ;

[0228] h) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 67, 68 and 69 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 70, GAS and 71 respectively

[181] ;

[0229] i) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 72, 73 and 75 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 76, ATS and 77 respectively

[183] ;

[0230] j) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 72, 74 and 75 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 76, ATS and 77 respectively [183-N52Q];

[0231] k) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 78, 79 and 80 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 81, AAS and 82 respectively

[187] ;

[0232] l) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 83, 84 and 85 respectively; and VL region, which contains sequences having SEQ ID No: 86, GAS and 87 [608-01] respectively;

[0233] m) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 88, 89 and 90 respectively; and VL region, which contains sequences having SEQ ID No: 91, GAS and 92 [610-01] respectively;

[0234] n) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 93, 94 and 95 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 96, GAS and 97 respectively

[613] ;

[0235] o) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 98, 99 and 100 respectively; and VL region, which contains sequences having SEQ ID No: 10, DAS and 102 [613-08] respectively;

[0236] p) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 103, 104 and 105 respectively; and VL region, which contains sequences having SEQ ID No: 106, GAS and 107 [620-06] respectively;

[0237] q) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 108, 109 and 110 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 112, AAS and 113 respectively

[726] ;

[0238] r) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 108, 109 and 111 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 112, AAS and 113 respectively [726-M101L];

[0239] s) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 41, 42 and 43 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 44, AAS and 45 respectively

[140] ;

[0240] t) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 93, 94 and 95 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 128, XAS (where X is D or G) and 129 respectively [613 / 613-08];

[0241] u) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 119 and 120 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively [148 / 140];

[0242] v) The VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 123, 124, and 125, respectively; and the VL region contains CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 60, GAS, and 61, respectively [171 / 172 / 181]; and

[0243] w) VH region, comprising CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 121, 109, and 122, respectively; and VL region, comprising CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 112, AAS, and 113, respectively [726 / 187]; and

[0244] x) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 93, 126 and 127 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 96, GAS and 97 respectively [613 / 608-01 / 610-01 / 620-06].

[0245] In one embodiment, the at least one binding region comprises a VH region and a VL region selected from the following:

[0246] a) The VH region containing SEQ ID No: 1 and the VL region containing SEQ ID No: 2

[107] ;

[0247] b) The VH region containing SEQ ID No: 5 and the VL region containing SEQ ID No: 6

[148] ;

[0248] c) The VH region containing SEQ ID No: 34 and the VL region containing SEQ ID No: 35

[733] ;

[0249] d) The VH region containing SEQ ID No: 7 and the VL region containing SEQ ID No: 9

[154] ;

[0250] e) The VH region containing SEQ ID No: 10 and the VL region containing SEQ ID No: 11

[171] ;

[0251] f) The VH region containing SEQ ID No: 16 and the VL region containing SEQ ID No: 18

[183] ;

[0252] g) The VH region containing SEQ ID No: 25 and the VL region containing SEQ ID No: 26

[613] ;

[0253] h) The VH region containing SEQ ID No: 31 and the VL region containing SEQ ID No: 33

[726] ;

[0254] i) The VH region containing SEQ ID No: 3 and the VL region containing SEQ ID No: 4

[140] ;

[0255] j) The VH region containing SEQ ID No:8 and the VL region containing SEQ ID No:9 [154-M103L];

[0256] k) The VH region containing SEQ ID No:12 and the VL region containing SEQ ID No:13

[172] ;

[0257] l) The VH region containing SEQ ID No:14 and the VL region containing SEQ ID No:15

[181] ;

[0258] m) Contains the VH region of SEQ ID No:17 and the VL region of SEQ ID No:18 [183-N52Q];

[0259] n) The VH region containing SEQ ID No:19 and the VL region containing SEQ ID No:20

[187] ;

[0260] o) The VH region containing SEQ ID No:21 and the VL region containing SEQ ID No:22 [608-01];

[0261] p) The VH region containing SEQ ID No:23 and the VL region containing SEQ ID No:24 [610-01];

[0262] q) The VH region containing SEQ ID No:27 and the VL region containing SEQ ID No:28 [613-08];

[0263] r) The VH region containing SEQ ID No:29 and the VL region containing SEQ ID No:30 [620-06]; and

[0264] s) The VH region containing SEQ ID No:32 and the VL region [726-M101L] containing SEQ ID No:33.

[0265] In one embodiment, the at least one binding region comprises a variable heavy chain (VH) region and a variable light chain (VL) region having at most 10 mutations or substitutions, at most 5 mutations or substitutions, at most 4 mutations or substitutions, at most 3 mutations or substitutions, at most 2 mutations or substitutions, or at most 1 mutation or substitution throughout the entire variable heavy chain and variable light chain sequence selected from the following:

[0266] In one embodiment, the at least one binding region comprises a VH region and a VL region selected from the following:

[0267] a) The VH region containing SEQ ID No: 1 and the VL region containing SEQ ID No: 2

[107] ;

[0268] b) The VH region containing SEQ ID No: 5 and the VL region containing SEQ ID No: 6

[148] ;

[0269] c) The VH region containing SEQ ID No: 34 and the VL region containing SEQ ID No: 35

[733] ;

[0270] d) The VH region containing SEQ ID No: 7 and the VL region containing SEQ ID No: 9

[154] ;

[0271] e) The VH region containing SEQ ID No: 10 and the VL region containing SEQ ID No: 11

[171] ;

[0272] f) The VH region containing SEQ ID No: 16 and the VL region containing SEQ ID No: 18

[183] ;

[0273] g) The VH region containing SEQ ID No: 25 and the VL region containing SEQ ID No: 26

[613] ;

[0274] h) The VH region containing SEQ ID No: 31 and the VL region containing SEQ ID No: 33

[726] ;

[0275] i) The VH region containing SEQ ID No: 3 and the VL region containing SEQ ID No: 4

[140] ;

[0276] j) The VH region containing SEQ ID No:8 and the VL region containing SEQ ID No:9 [154-M103L];

[0277] k) The VH region containing SEQ ID No:12 and the VL region containing SEQ ID No:13

[172] ;

[0278] l) The VH region containing SEQ ID No:14 and the VL region containing SEQ ID No:15

[181] ;

[0279] m) Contains the VH region of SEQ ID No:17 and the VL region of SEQ ID No:18 [183-N52Q];

[0280] n) The VH region containing SEQ ID No:19 and the VL region containing SEQ ID No:20

[187] ;

[0281] o) The VH region containing SEQ ID No:21 and the VL region containing SEQ ID No:22 [608-01];

[0282] p) The VH region containing SEQ ID No:23 and the VL region containing SEQ ID No:24 [610-01];

[0283] q) The VH region containing SEQ ID No:27 and the VL region containing SEQ ID No:28 [613-08];

[0284] r) The VH region containing SEQ ID No:29 and the VL region containing SEQ ID No:30 [620-06]; and

[0285] s) The VH region containing SEQ ID No:32 and the VL region [726-M101L] containing SEQ ID No:33.

[0286] An implementation scheme is provided in which up to 10 mutations or substitutions are permitted throughout the variable heavy and variable light chains. These up to 10 mutations or substitutions may be distributed throughout the entire length of the variable heavy and variable light chains in each binding region. The mutations or substitutions may be mutations or substitutions of conserved, physical, or functional amino acids, such that the mutations or substitutions do not alter the epitopes and preferably alter the binding affinity of the epitopes by no more than 30%, such as no more than 20% or no more than 10%. The conserved, physical, or functional amino acids are selected from 20 naturally occurring amino acids, namely Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr, and Val.

[0287] In one embodiment, the at least one binding region comprises a variable heavy chain (VH) region and a variable light chain (VL) region having at most 10 mutations or substitutions selected from conserved, physical, or functional amino acids, at most 5 mutations or substitutions selected from conserved, physical, or functional amino acids, at most 4 mutations or substitutions selected from conserved, physical, or functional amino acids, at most 3 mutations or substitutions selected from conserved, physical, or functional amino acids, at most 2 mutations or substitutions selected from conserved, physical, or functional amino acids, or at most 1 mutation or substitution selected from conserved, physical, or functional amino acids.

[0288] In one embodiment, the at least one binding region comprises a VH region and a VL region selected from the following:

[0289] a) The VH region containing SEQ ID No: 1 and the VL region containing SEQ ID No: 2

[107] ;

[0290] b) The VH region containing SEQ ID No: 5 and the VL region containing SEQ ID No: 6

[148] ;

[0291] c) The VH region containing SEQ ID No: 34 and the VL region containing SEQ ID No: 35

[733] ;

[0292] d) The VH region containing SEQ ID No: 7 and the VL region containing SEQ ID No: 9

[154] ;

[0293] e) The VH region containing SEQ ID No: 10 and the VL region containing SEQ ID No: 11

[171] ;

[0294] f) The VH region containing SEQ ID No: 16 and the VL region containing SEQ ID No: 18

[183] ;

[0295] g) The VH region containing SEQ ID No: 25 and the VL region containing SEQ ID No: 26

[613] ;

[0296] h) The VH region containing SEQ ID No: 31 and the VL region containing SEQ ID No: 33

[726] ;

[0297] i) The VH region containing SEQ ID No: 3 and the VL region containing SEQ ID No: 4

[140] ;

[0298] j) The VH region containing SEQ ID No:8 and the VL region containing SEQ ID No:9 [154-M103L];

[0299] k) The VH region containing SEQ ID No:12 and the VL region containing SEQ ID No:13

[172] ;

[0300] l) The VH region containing SEQ ID No:14 and the VL region containing SEQ ID No:15

[181] ;

[0301] m) Contains the VH region of SEQ ID No:17 and the VL region of SEQ ID No:18 [183-N52Q];

[0302] n) The VH region containing SEQ ID No:19 and the VL region containing SEQ ID No:20

[187] ;

[0303] o) The VH region containing SEQ ID No:21 and the VL region containing SEQ ID No:22 [608-01];

[0304] p) The VH region containing SEQ ID No:23 and the VL region containing SEQ ID No:24 [610-01];

[0305] q) The VH region containing SEQ ID No:27 and the VL region containing SEQ ID No:28 [613-08];

[0306] r) The VH region containing SEQ ID No:29 and the VL region containing SEQ ID No:30 [620-06]; and

[0307] s) The VH region containing SEQ ID No:32 and the VL region [726-M101L] containing SEQ ID No:33.

[0308] An embodiment is hereby provided in which up to 10 mutations or substitutions are permitted throughout the variable heavy chain and variable light chain. These up to 10 mutations or substitutions may be distributed throughout the variable heavy chain and variable light chain. These up to 10 mutations or substitutions may be distributed throughout the binding region. The mutations or substitutions may be mutations or substitutions of conserved, physical, or functional amino acids, such that the mutations or substitutions do not alter the epitope or the binding to the epitope.

[0309] In one aspect, the present invention relates to an antibody that binds to the extracellular domain of AXL without competing with Gas6 for AXL binding or interfering with Gas6 binding to AXL. In a specific embodiment, the antibody binds to an Ig1-like extracellular domain without competing with Gas6 for AXL binding or interfering with Gas6 binding to AXL. In one embodiment, the antibody binds to the Ig1-like extracellular domain and exhibits a reduction in the maximum binding of Gas6 to AXL of no more than 20%. In one embodiment, the antibody exhibits a reduction in the maximum binding of Gas6 to AXL of no more than 15%. In one embodiment, the antibody exhibits a reduction in the maximum binding of Gas6 to AXL of no more than 10%. In one embodiment, the antibody exhibits a reduction in the maximum binding of Gas6 to AXL of no more than 5%. In one embodiment, the antibody exhibits a reduction in the maximum binding of Gas6 to AXL of no more than 4%. In one embodiment, the antibody exhibits a reduction in the maximum binding of Gas6 to AXL of no more than 2%. In one embodiment, the antibody exhibits a reduction in the maximum binding of Gas6 of no more than 1%. In one embodiment, the antibody binds to the extracellular Ig2-like domain without competing with Gas6 for AXL binding or interfering with Gas6 binding to AXL. In one embodiment, the antibody binds to the Ig2-like extracellular domain and exhibits a reduction in the maximum binding of Gas6 to AXL of no more than 20%, such as no more than 15%, such as no more than 10%, such as no more than 5%, such as no more than 4%, such as no more than 2%, such as no more than 1%. The ability of this embodiment to compete with or reduce Gas6 binding can be determined as disclosed in Example 2 or Example 12. In one embodiment, the antibody binds to the extracellular Ig2-like domain without competing with Gas6 for AXL binding or interfering with the maximum binding of Gas6 to AXL.

[0310] In one embodiment, the antibody binds to an epitope on AXL, the epitope being recognized by the antibody described herein.

[0311] Methods for determining the epitopes to which antibodies bind are well known in the art, and thus, those skilled in the art will know how to determine such epitopes. However, an example of determining whether an antibody binds to any epitope as defined herein would be through point mutation of the AXL extracellular domain. Introducing a point mutation into the AXL extracellular domain and testing antibody binding to the point-mutated AXL extracellular domain is within the knowledge of those skilled in the art. When referring to amino acid positions within the AXL protein in the context of epitopes, numbering has been determined as described in Example 7. Therefore, the numbering of amino acid positions defining epitopes is based on… Figure 6 The sequence shown is complete, that is, the first amino acid in the sequence is numbered as position '1', the second as position '2', and so on.

[0312] In one embodiment, the antibody binds to an epitope within the Ig1-like domain of AXL, the epitope comprising or requiring amino acids corresponding to positions L121 to Q129 or T112 to Q124 of human AXL. In another embodiment, the antibody binds to an epitope within the Ig1-like domain of AXL, the epitope comprising or requiring one or more amino acids corresponding to positions L121 to Q129 or T112 to Q124 of human AXL. In one embodiment, the epitope comprises one or more amino acids located at L121, G122, H123, Q124, T125, F126, V127, S128, Q129, or multiple amino acids located at T112, G113, Q114, Y115, Q116, C117, L118, V119, F120, L121, G122, H123, Q124.

[0313] In another embodiment, the antibody binds to an epitope within the Ig2-like domain of AXL, the epitope containing or requiring amino acids corresponding to a combination of positions D170 or D179 with T182 to R190 in human AXL.

[0314] In one embodiment, the antibody binds to an epitope within the Ig2-like domain of AXL, the epitope comprising or requiring an amino acid corresponding to position D170 or D179 of human AXL and a combination of one or more amino acids corresponding to positions T182 to R190. In one embodiment, the epitope comprises one or more amino acids located at T182, A183, P183, G184, H185, G186, P187, Q189, and R190.

[0315] In another embodiment, the antibody binds to an epitope within the FN1-like domain of human AXL, the epitope containing or requiring amino acids corresponding to positions Q272 to A287 and G297 to P301 of human AXL.

[0316] In another embodiment, the antibody binds to an epitope within the FN2-like domain of human AXL, the epitope containing or requiring amino acids corresponding to positions A359, R386, and Q436 to K439 of human AXL.

[0317] In one embodiment, the antibody binds to an epitope within the FN1-like domain of human AXL.

[0318] In one embodiment, the antibody binds to an epitope on AXL, the epitope being recognized by any of the antibodies defined below:

[0319] a) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 36, 37 and 38 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 39, GAS and 40 respectively

[107] ;

[0320] b) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively

[148] ;

[0321] c) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 114, 115 and 116 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 117, DAS and 118 respectively

[733] ;

[0322] d) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 51, 52 and 53 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 55, GAS and 56 respectively

[154] ;

[0323] e) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 51, 52 and 54 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 55, GAS and 56 respectively [154-M103L];

[0324] f) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 57, 58 and 59 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 60, GAS and 61 respectively

[171] ;

[0325] g) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 62, 63 and 64 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 65, GAS and 66 respectively

[172] ;

[0326] h) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 67, 68 and 69 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 70, GAS and 71 respectively

[181] ;

[0327] i) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 72, 73 and 75 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 76, ATS and 77 respectively

[183] ;

[0328] j) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 72, 74 and 75 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 76, ATS and 77 respectively [183-N52Q];

[0329] k) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 78, 79 and 80 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 81, AAS and 82 respectively

[187] ;

[0330] l) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 83, 84 and 85 respectively; and VL region, which contains sequences having SEQ ID No: 86, GAS and 87 [608-01] respectively;

[0331] m) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 88, 89 and 90 respectively; and VL region, which contains sequences having SEQ ID No: 91, GAS and 92 [610-01] respectively;

[0332] n) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 93, 94 and 95 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 96, GAS and 97 respectively

[613] ;

[0333] o) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 98, 99 and 100 respectively; and VL region, which contains sequences having SEQ ID No: 10, DAS and 102 [613-08] respectively;

[0334] p) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 103, 104 and 105 respectively; and VL region, which contains sequences having SEQ ID No: 106, GAS and 107 [620-06] respectively;

[0335] q) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 108, 109 and 110 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 112, AAS and 113 respectively

[726] ;

[0336] r) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 108, 109 and 111 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 112, AAS and 113 respectively [726-M101L];

[0337] s) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 41, 42 and 43 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 44, AAS and 45 respectively

[140] ;

[0338] t) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 93, 94 and 95 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 128, XAS (where X is D or G) and 129 respectively [613 / 613-08];

[0339] u) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 119 and 120 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively [148 / 140];

[0340] v) The VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 123, 124, and 125, respectively; and the VL region contains CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 60, GAS, and 61, respectively [171 / 172 / 181]; and

[0341] w) VH region, comprising CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 121, 109, and 122, respectively; and VL region, comprising CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 112, AAS, and 113, respectively [726 / 187]; and

[0342] x) VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 93, 126 and 127 respectively; and VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 96, GAS and 97 respectively [613 / 608-01 / 610-01 / 620-06].

[0343] In one embodiment, the antibody comprises an isotype heavy chain selected from IgG1, IgG2, IgG3, and IgG4.

[0344] The term "isotype," as used herein, refers to an immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by a heavy chain constant region gene, or any of its allotypes such as IgG1m(za) and IgG1m(f). Furthermore, each heavy chain isotype can be combined with either a κ(κ) or λ(λ) light chain.

[0345] In one embodiment, the isotype is IgG1, optionally allotype IgG1m(f).

[0346] In one embodiment, the antibody is a full-length monoclonal antibody, optionally a full-length monoclonal IgG1,κ antibody.

[0347] The term "full-length antibody," as used herein, refers to an antibody (e.g., a parental or variant antibody) containing all the constant and variable heavy and light chain domains corresponding to those typically present in wild-type antibodies of that isotype. The full-length antibody according to the invention can be produced by a method comprising the steps of: (i) cloning a CDR sequence into a suitable vector containing the complete heavy and light chain sequences, and (ii) expressing the complete heavy and light chain sequences in a suitable expression system. Producing a full-length antibody from a CDR sequence or a complete variable region sequence is within the knowledge of those skilled in the art. Therefore, those skilled in the art will know how to generate the full-length antibody according to the invention.

[0348] In one embodiment, the antibody is a human antibody.

[0349] The term "human antibody," as used herein, is intended to include antibodies having variable and frame regions derived from human germline immunoglobulin sequences, as well as constant domains of human immunoglobulins. The human antibodies of the present invention may contain amino acids not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which a CDR sequence derived from another non-human species (e.g., mouse) has been grafted onto a human frame sequence.

[0350] As used herein, a human antibody is "derived" from a specific germline sequence if the antibody is derived from a system using a human immunoglobulin sequence, such as by immunizing transgenic mice carrying a human immunoglobulin gene or by screening a human immunoglobulin gene library, and wherein the selected human antibody is at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99%, identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, human antibodies derived from a specific human germline sequence, excluding the heavy chain CDR3, will show a difference of no more than 20 amino acids from the amino acid sequence encoded by the germline immunoglobulin gene, such as no more than 10 amino acids, such as no more than 9, 8, 7, 6, or 5, such as no more than 4, 3, 2, or 1 amino acid.

[0351] The antibodies according to the present invention may contain amino acid modifications in the heavy and / or light chains of immunoglobulins. In a specific embodiment, the amino acids in the Fc region of the antibody may be modified.

[0352] The term "Fc region," as used herein, refers to a region that includes at least a hinge region, a CH2 region, and a CH3 region along the N-to-C-terminus of an antibody. The Fc region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system.

[0353] The term “hinge region”, as used herein, refers to the hinge region of the immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 (according to Eu numbering as shown in Kabat)

[26] . However, the hinge region may also be any other subtype as described herein.

[0354] The term “CH1 region” or “CH1 domain”, as used herein, refers to the CH1 region of the immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 (according to Eu numbering as shown in Kabat)

[26] . However, the CH1 region may also be any other subtype as described herein.

[0355] The term “CH2 region” or “CH2 domain”, as used herein, refers to the CH2 region of the immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 (according to Eu numbering as shown in Kabat)

[26] . However, the CH2 region may also be any other subtype as described herein.

[0356] The term “CH3 region” or “CH3 domain”, as used herein, refers to the CH3 region of the immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 (according to Eu numbering as shown in Kabat)

[26] . However, the CH3 region may also be any other subtype as described herein.

[0357] In another embodiment, the antibody is an effector-deficient antibody, a stable IgG4 antibody, or a monovalent antibody.

[0358] In one specific implementation, the heavy chain has been modified to the point that the entire hinge area is missing.

[0359] In one embodiment, the antibody sequence has been modified to not contain any N-linked glycosylated receptor sites.

[0360] In one embodiment, the antibody is a single-chain antibody.

[0361] In another aspect, the present invention relates to multispecific antibodies comprising a first binding region of an antibody according to at least any aspect or embodiment described herein, and a second binding region binding to a target or epitope different from the first binding region. The term "multispecific antibody," as used herein, refers to an antibody in which the binding region binds to at least two (e.g., at least three) different antigens or at least two (e.g., at least three) different epitopes on the same antigen.

[0362] In one embodiment, the present invention relates to a bispecific antibody comprising a first binding region of an antibody according to any aspect or embodiment described herein, and a second binding region that binds a target or epitope different from the first binding region.

[0363] The term “bispecific,” as used herein, refers to a binding molecule, such as an antibody, in which the binding region of the binding molecule binds to two different antigens or two different epitopes on the same antigen.

[0364] The term "bispecific antibody" refers to an antibody that is specific to at least two different (usually non-overlapping) epitopes. These epitopes may be on the same or different targets. If the epitopes are on different targets, these targets may be on the same or different cells, cell types, or structures (such as extracellular tissues).

[0365] The term "different target," as used in this article, refers to another protein, molecule, etc., that is different from AXL or AXL fragment.

[0366] Examples of bispecific antibody molecules that can be used in this invention include: (i) monoclonal antibodies having two arms comprising different antigen-binding regions; (ii) single-chain antibodies that are specific for two different epitopes, for example, via two scFvs tandemly linked by an additional peptide linker; and (iii) dual variable domain antibodies (DVD-Ig). TM(iv) chemically linked bispecific (Fab')2 fragments; (v) Tandab®, a tetravalent bispecific antibody obtained by fusing two single-chain biantibodies, each having two binding sites against each target antigen; (vi) flexibody, a multivalent molecule obtained by combining scFv with biantibodies; (vii) a so-called "dock-and-lock" molecule (Dock-and-Lock®), based on the "dimerization and docking domain" in protein kinase A, which, when applied to Fab, can produce a trivalent bispecific binding protein composed of two identical Fab fragments linked to different Fab fragments; (viii) a so-called Scorpion molecule, containing, for example, two scFvs fused to the ends of human Fab arms; and (ix) biantibodies.

[0367] In one embodiment, the bispecific antibody of the present invention is a bispecific antibody, a cross-body antibody such as CrossMab, or a bispecific antibody obtained via controlled Fab arm exchange (as described in

[30] ).

[0368] Examples of different classes of bispecific antibodies include, but are not limited to: (i) IgG-like molecules having complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual-targeting molecules, wherein each side of the molecule contains Fab fragments or portions of Fab fragments of at least two different antibodies; (iii) IgG fusion molecules, wherein a full-length IgG antibody is fused to an additional Fab fragment or portion of a Fab fragment; (iv) Fc fusion molecules, wherein a single-chain Fv molecule or a stable biantibody is fused to a heavy chain constant domain, Fc region, or a portion thereof; (v) Fab fusion molecules, wherein different Fab fragments are fused together to a heavy chain constant domain, Fc region, or a portion thereof; and (vi) ScFv-based antibodies and biantibody-based antibodies and heavy chain antibodies (e.g., domain antibodies, Nanobodies®), wherein different single-chain Fv molecules or different biantibodies or different heavy chain antibodies (e.g., domain antibodies, Nanobodies®) are fused together or fused to another protein or carrier molecule to a heavy chain constant domain, Fc region, or a portion thereof.

[0369] Examples of IgG-like molecules with complementary CH3 domains include, but are not limited to: Triomab® (TrionPharma / Fresenius Biotech,

[31] ), Knobs-into-Holes (Genentech,

[32] ), CrossMAb (Roche,

[33] ), and electrostatically matched (Amgen,

[34] and

[35] ; Chugai,

[36] ; Oncomed,

[37] ), LUZ-Y (Genentech), DIG-body and PIG-body (Pharmabcine), Strand Exchange Engineered Domain Body (SEEDbody) (EMD Serono,

[38] ), Biclonics (Merus), FcΔAdp (Regeneron,

[39] ), Bispecific IgG1 and IgG2 (Pfizer / Rinat,

[40] ), Azymetric scaffold (Zymeworks / Merck,

[41] ), mAb-Fv (Xencor,

[42] ), bivalent bispecific antibody (Roche

[43] ), and DuoBody® molecule (Genmab A / S,

[30] ).

[0370] Examples of recombinant IgG-like dual-targeting molecules include, but are not limited to: dual-targeting (DT)-Ig (GSK / Domantis), dual-antibody (Genentech), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star,

[44] ), and Zybodies. TM (Zyngenia), using the same light chain method (Crucell / Merus,

[45] ), κλBodies (NovImmune) and CovX-body (CovX / Pfizer).

[0371] Examples of IgG fusion molecules include, but are not limited to: dual variable domain (DVD)-Ig TM(Abbott,

[46] ), bidomain bispecific antibody (Unilever; Sanofi Aventis,

[47] ), IgG-like bispecific antibody (ImClone / EliLilly), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec,

[48] ), scFv fusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc,

[49] ) and TvAb (Roche,

[50] ,

[51] ).

[0372] Examples of Fc fusion molecules include, but are not limited to: ScFv / Fc fusion (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), and dual-affinity retargeting technology (Fc-DART). TM ) (MacroGenics,

[52] ,

[53] ) and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine – China).

[0373] Examples of Fab fusion bispecific antibodies include, but are not limited to: F(ab)2 (Medarex / AMGEN), bifunctional or dual-Fab (Genentech), Dock-and-Lock® (DNL) (ImmunoMedics), bivalent bispecific (Biotecnol), and Fab-Fv (UCB-Celltech).

[0374] Examples of ScFv-based antibodies, bispecific antibodies, and domain-specific antibodies include, but are not limited to: Bispecific T-cell conjugate (BiTE®) (Micromet, Tandem Diabody (Tandab)). TM (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-Chain Biantibodies (Academic), TCR-like Antibodies (AIT, Receptor Logics), Human Serum Albumin ScFv Fusion (Merrimack), and COMBODY (EpigenBiotech), Dual-Targeting Nanobodies® (Ablynx), and Dual-Targeting Heavy Chain-Only Domain-Containing Antibodies.

[0375] The bispecific antibody according to the present invention can be generated by introducing modifications into the constant region of the antibody.

[0376] Unless otherwise stated or conflicted with the context, the amino acids in the constant region sequence are numbered herein according to the Eu number index (described in

[26] ). The terms “Eu number index” and “Eu number as shown in Kabat” are used interchangeably and have the same meaning and purpose. Thus, when an amino acid or segment in one sequence “corresponds” to an amino acid or segment in another sequence, it means that, using standard sequence alignment programs (such as ALIGN, ClustalW, etc.), it is typically aligned with the other amino acid or segment by default and has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. How to align sequences or segments in sequences and thereby determine the corresponding positions of the amino acid positions according to the invention in the sequence is well known in the art.

[0377] The term “amino acid at the corresponding position”, as used herein, refers to the amino acid position number in the human IgG1 heavy chain.

[0378] The present invention also provides antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of the antibodies in these examples. The functional variants of the VL, VH, or CDR used in the context of AXL antibodies still allow the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) of the affinity / affinity and / or specificity / selectivity of the parent antibody, and in some cases, such AXL antibodies may have greater affinity, selectivity, and / or specificity than the parent antibody.

[0379] Such functional variants typically maintain significant sequence identity with the parent antibody. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account both the number of vacancies and the length of each vacancy, which needs to be introduced for optimal alignment of the two sequences. Comparing sequences and determining the percentage identity between two sequences can be accomplished using mathematical algorithms well known in the art.

[0380] The VH, VL, and / or CDR sequences of the variant may differ from those of the parent antibody sequence by mostly conserved substitutions; for example, at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more (e.g., about 65-95%, such as about 92%, 93%, or 94%) of the substitutions in the variant are conserved amino acid residue substitutions.

[0381] The VH, VL, and / or CDR sequences of the variants may differ from those of the parent antibody sequences by mostly conserved substitutions; for example, 10 or fewer of the variant substitutions, such as 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 is a conserved amino acid residue substitution.

[0382] The terms “amino acid” and “amino acid residue” are used interchangeably in this document and should not be construed as restrictive.

[0383] In the context of this invention, amino acids can be defined as conserved or non-conserved amino acids, and can be classified accordingly. Amino acid residues can also be classified into categories defined by optional physical and functional properties. Therefore, the categories of amino acids can be reflected in one or both of the following tables:

[0384] Conserved amino acid residues

[0385] acid residues D and E basic residues K, R and H Hydrophilic uncharged residues S, T, N and Q Aliphatic uncharged residues G, A, V, L and I Nonpolar uncharged residues C, M and P Aroma residues F, Y and W

[0386] Optional physical and functional amino acid residues

[0387] alcohol residues S and T aliphatic residues I, L, V and M Cycloalkenyl-related residues F, H, W and Y hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively charged residues D and E polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K and R small residues A, C, D, G, N, P, S, T and V ultrasmall residues A, G and S Residues involved in corner formation A, C, D, E, G, H, K, N, Q, R, S, P and T flexible residues Q, T, K, S, G, P, D, E, and R

[0388] In the context of this invention, substitution in the antibody is referred to as:

[0389] Original amino acid – position – replaced amino acid;

[0390] Referring to accepted amino acid nomenclature, any amino acid residue is represented using a three-letter code or a single-letter code (including the codes "Xaa" or "X"). Therefore, Xaa or X can generally represent any of the 20 naturally occurring amino acids. The term "naturally occurring," as used herein, refers to any of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, proline, tryptophan, phenylalanine, tyrosine, methionine, and cysteine. Therefore, the symbol "K409R" or "Lys409Arg" indicates that the antibody contains an arginine substitution for lysine at amino acid position 409.

[0391] Replacing an amino acid at a given position with any other amino acid is called:

[0392] Original amino acid – position; or for example, “K409”.

[0393] The original amino acid and / or the replaced amino acid may contain more than one, but not all, amino acid modifications, separated by commas or slashes. For example, the substitution of lysine at position 409 with arginine, alanine, or phenylalanine is:

[0394] “Lys409Arg, Ala, Phe” or “Lys409Arg / Ala / Phe” or “K409R, A, F” or “K409R / A / F” or “K409 to R, A or F”.

[0395] Such reference numerals are interchangeable in the context of this invention but have the same meaning and purpose.

[0396] Furthermore, the term "substitution" includes substitution with any of the other nineteen natural amino acids, or substitution with other amino acids such as non-natural amino acids. For example, substitution of amino acid K at position 409 includes each of the following substitutions: 409A, 409C, 409D, 409E, 409F, 409G, 409H, 409I, 409L, 409M, 409N, 409Q, 409R, 409S, 409T, 409V, 409W, 409P, and 409Y. Incidentally, this is equivalent to designation 409X, where X represents any amino acid other than the original amino acid. These substitutions may also be labeled K409A, K409C, etc., or K409A, C, etc., or K409A / C / etc. This also applies to each and all positions mentioned herein, to specifically include any of such substitutions herein.

[0397] The antibody according to the invention may also contain deletions of amino acid residues. Such deletions may be denoted as "del" and include, for example, K409del. Thus, in such embodiments, the lysine at position 409 has been deleted from the amino acid sequence.

[0398] In one specific embodiment, the bispecific antibody comprises first and second heavy chains, each of which includes at least a hinge region, a CH2 region, and a CH3 region, wherein in the first heavy chain, at least one of the amino acids selected from the following positions corresponding to the human IgG1 heavy chain has been substituted: K409, T366, L368, K370, D399, F405, and Y407, and in the second heavy chain, at least one of the amino acids selected from the following positions corresponding to the human IgG1 heavy chain has been substituted: F405, T366, L368, K370, D399, Y407, and K409, and wherein the substitutions in the first and second heavy chains do not occur at the same positions.

[0399] In one embodiment, in the first heavy chain, the amino acid at the position corresponding to K409 of the human IgG1 heavy chain is not K, L, or M, but optionally the amino acid at the position corresponding to F405 of the human IgG1 heavy chain is F, and in the second heavy chain, the amino acid at the position corresponding to F405 of the human IgG1 heavy chain is not F, but the amino acid at the position corresponding to K409 of the human IgG1 heavy chain is K.

[0400] In one embodiment, in the first heavy chain, the amino acid at position F405 corresponding to the human IgG1 heavy chain is not F, R, or G, and in the second heavy chain, the amino acids at positions corresponding to the human IgG1 heavy chain selected from the following have been replaced: T366, L368, K370, D399, Y407, and K409.

[0401] In one embodiment, in the first heavy chain, the amino acid at the position corresponding to K409 of the human IgG1 heavy chain is not K, L, or M, and in the second heavy chain, the amino acid at the position corresponding to F405 of the human IgG1 heavy chain is not F, while optionally the amino acid at the position corresponding to K409 of the human IgG1 heavy chain is K.

[0402] In one embodiment, in the first heavy chain, the amino acid at position F405 corresponding to the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at position K409 corresponding to the human IgG1 heavy chain is R, or vice versa.

[0403] Therefore, in one embodiment, in the first heavy chain, the amino acid at position K409 corresponding to the human IgG1 heavy chain is R, and in the second heavy chain, the amino acid at position F405 corresponding to the human IgG1 heavy chain is L.

[0404] In another embodiment, both the first and second binding regions of the bispecific antibody bind to AXL. However, the first binding region contains a different set of CDR sequences than the second binding region. Therefore, in a specific embodiment, the bispecific antibody comprises first and second binding regions and first and second heavy chains, wherein the first and second binding regions each comprise VH and VL regions selected from:

[0405] a) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 46, 47, and 48, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 49, AAS, and 50, respectively

[148] ;

[0406] b) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 114, 115, and 116, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 117, DAS, and 118, respectively

[733] ;

[0407] c) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 41, 42, and 43, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 44, AAS, and 45, respectively

[140] ;

[0408] d) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 51, 52, and 55, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 55, GAS, and 56, respectively

[154] ;

[0409] e) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 51, 52, and 54, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 55, GAS, and 56, respectively [154-M103L];

[0410] f) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 57, 58, and 59, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 60, GAS, and 61, respectively

[171] ;

[0411] g) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 62, 63, and 64, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 65, GAS, and 66, respectively

[172] ;

[0412] h) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 67, 68, and 69, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 70, GAS, and 71, respectively

[181] ;

[0413] i) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 72, 73, and 75, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 76, ATS, and 77, respectively

[183] ;

[0414] j) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 72, 74, and 75, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 76, ATS, and 77, respectively [183-N52Q];

[0415] k) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 78, 79, and 80, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 81, AAS, and 82, respectively

[187] ;

[0416] l) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38 respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40 respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 83, 84, and 85 respectively; and a second VL region comprising sequences having SEQ ID No: 86, GAS, and 87 respectively [608-01];

[0417] m) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 88, 89, and 90, respectively; and a second VL region comprising sequences having SEQ ID No: 91, GAS, and 92, respectively [610-01];

[0418] n) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 94, 95, and 95, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 96, GAS, and 97, respectively

[613] ;

[0419] o) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38 respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40 respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 98, 99, and 100 respectively; and a second VL region comprising sequences having SEQ ID No: 101, DAS, and 102 respectively [613-08];

[0420] p) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 103, 104, and 105, respectively; and a second VL region comprising sequences having SEQ ID No: 106, GAS, and 107, respectively [620-06];

[0421] q) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 108, 109, and 110, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 112, AAS, and 113, respectively

[726] ;

[0422] r) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 36, 37, and 38, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 39, GAS, and 40, respectively

[107] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 108, 109, and 111, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 112, AAS, and 113, respectively [726-M101L];

[0423] s) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 46, 47, and 48, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 49, AAS, and 50, respectively

[148] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 114, 115, and 116, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 117, DAS, and 118, respectively

[733] ;

[0424] t) A first VH region comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; a first VL region comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively

[148] ; a second VH region comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 41, 42 and 43 respectively; and a second VL region comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 44, AAS and 45 respectively

[107] ;

[0425] u) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 46, 47, and 48, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 49, AAS, and 50, respectively

[148] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 51, 52, and 55, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 55, GAS, and 56, respectively

[154] ;

[0426] v) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 46, 47, and 48, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 49, AAS, and 50, respectively

[148] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 51, 52, and 54, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 55, GAS, and 56, respectively [154-M103L];

[0427] w) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 46, 47, and 48, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 49, AAS, and 50, respectively

[148] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 57, 58, and 59, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 60, GAS, and 61, respectively

[171] ;

[0428] x) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 46, 47, and 48, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 49, AAS, and 50, respectively

[148] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 62, 63, and 64, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 65, GAS, and 66, respectively

[172] ;

[0429] y) A first VH region comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; a first VL region comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively

[148] ; a second VH region comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 67, 68 and 69 respectively; and a second VL region comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 70, GAS and 71 respectively

[181] ;

[0430] z) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 46, 47, and 48, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 49, AAS, and 50, respectively

[148] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 72, 73, and 75, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 76, ATS, and 77, respectively

[183] ;

[0431] aa) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 46, 47, and 48, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 49, AAS, and 50, respectively

[148] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 72, 74, and 75, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 76, ATS, and 77, respectively [183-N52Q];

[0432] bb) A first VH region comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; a first VL region comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively

[148] ; a second VH region comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 78, 79 and 80 respectively; and a second VL region comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 81, AAS and 82 respectively

[187] ;

[0433] cc) First VH region, comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and first VL region, comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively

[148] ; and second VH region, comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 83, 84 and 85 respectively; and second VL region, comprising sequences having SEQ ID No: 86, GAS and 87 respectively [608-01];

[0434] dd) First VH region, comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and first VL region, comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively

[148] ; and second VH region, comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 88, 89 and 90 respectively; and second VL region, comprising sequences having SEQ ID No: 91, GAS and 92 respectively [610-01];

[0435] ee) First VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and first VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively

[148] ; and second VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 94, 95 and 95 respectively; and second VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 96, GAS and 97 respectively

[613] ;

[0436] ff) First VH region, comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and first VL region, comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively

[148] ; and second VH region, comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 98, 99 and 100 respectively; and second VL region, comprising sequences having SEQ ID No: 101, DAS and 102 respectively [613-08];

[0437] gg) First VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and first VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively

[148] ; and second VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 103, 104 and 105 respectively; and second VL region, which contains sequences having SEQ ID No: 106, GAS and 107 respectively [620-06];

[0438] hh) First VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and first VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 49, AAS and 50 respectively

[148] ; and second VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 108, 109 and 110 respectively; and second VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 112, AAS and 113 respectively

[726] ;

[0439] ii) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 46, 47, and 48, respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 49, AAS, and 50, respectively

[148] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 108, 109, and 111, respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 112, AAS, and 113, respectively [726-M101L];

[0440] jj) A first VH region containing CDR1, CDR2 and CDR3 sequences having SEQ ID No: 114, 115 and 116 respectively; a first VL region containing CDR1, CDR2 and CDR3 sequences having SEQ ID No: 117, DAS and 118 respectively

[733] ; a second VH region containing CDR1, CDR2 and CDR3 sequences having SEQ ID No: 41, 42 and 43 respectively; and a second VL region containing CDR1, CDR2 and CDR3 sequences having SEQ ID No: 44, AAS and 45 respectively

[140] ;

[0441] kk) A first VH region containing CDR1, CDR2 and CDR3 sequences having SEQ ID No: 114, 115 and 116 respectively; a first VL region containing CDR1, CDR2 and CDR3 sequences having SEQ ID No: 117, DAS and 118 respectively

[733] ; a second VH region containing CDR1, CDR2 and CDR3 sequences having SEQ ID No: 51, 52 and 55 respectively; and a second VL region containing CDR1, CDR2 and CDR3 sequences having SEQ ID No: 55, GAS and 56 respectively

[154] ;

[0442] ll) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 114, 115, and 116 respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 117, DAS, and 118 respectively

[733] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 51, 52, and 54 respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 55, GAS, and 56 respectively [154-M103L];

[0443] The first VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 114, 115, and 116 respectively; and the first VL region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 117, DAS, and 118 respectively

[733] ; and the second VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 57, 58, and 59 respectively; and the second VL region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 60, GAS, and 61 respectively

[171] ;

[0444] nn) A first VH region containing CDR1, CDR2 and CDR3 sequences having SEQ ID No: 114, 115 and 116 respectively; a first VL region containing CDR1, CDR2 and CDR3 sequences having SEQ ID No: 117, DAS and 118 respectively

[733] ; a second VH region containing CDR1, CDR2 and CDR3 sequences having SEQ ID No: 62, 63 and 64 respectively; and a second VL region containing CDR1, CDR2 and CDR3 sequences having SEQ ID No: 65, GAS and 66 respectively

[172] ;

[0445] oo) First VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 114, 115 and 116 respectively; and first VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 117, DAS and 118 respectively

[733] ; and second VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 67, 68 and 69 respectively; and second VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 70, GAS and 71 respectively

[181] ;

[0446] pp) First VH region, comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 114, 115 and 116 respectively; and first VL region, comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 117, DAS and 118 respectively

[733] ; and second VH region, comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 72, 73 and 75 respectively; and second VL region, comprising CDR1, CDR2 and CDR3 sequences having SEQ ID No: 76, ATS and 77 respectively

[183] ;

[0447] qq) First VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 114, 115 and 116 respectively; and first VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 117, DAS and 118 respectively

[733] ; and second VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 72, 74 and 75 respectively; and second VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 76, ATS and 77 respectively [183-N52Q];

[0448] The first VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 114, 115, and 116, respectively; and the first VL region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 117, DAS, and 118, respectively

[733] ; and the second VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 78, 79, and 80, respectively; and the second VL region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 81, AAS, and 82, respectively

[187] ;

[0449] ss) First VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 114, 115 and 116 respectively; and first VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 117, DAS and 118 respectively

[733] ; and second VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 83, 84 and 85 respectively; and second VL region, which contains sequences having SEQ ID No: 86, GAS and 87 respectively [608-01];

[0450] The first VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 114, 115, and 116, respectively; and the first VL region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 117, DAS, and 118, respectively

[733] ; and the second VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 88, 89, and 90, respectively; and the second VL region contains sequences having SEQ ID No: 91, GAS, and 92, respectively [610-01];

[0451] The first VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 114, 115, and 116 respectively; and the first VL region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 117, DAS, and 118 respectively

[733] ; and the second VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 94, 95, and 95 respectively; and the second VL region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 96, GAS, and 97 respectively

[613] ;

[0452] vv) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 114, 115, and 116 respectively; a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 117, DAS, and 118 respectively

[733] ; a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 98, 99, and 100 respectively; and a second VL region comprising sequences having SEQ ID No: 101, DAS, and 102 respectively [613-08];

[0453] The first VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 114, 115, and 116 respectively; and the first VL region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 117, DAS, and 118 respectively

[733] ; and the second VH region contains CDR1, CDR2, and CDR3 sequences having SEQ ID No: 103, 104, and 105 respectively; and the second VL region contains sequences having SEQ ID No: 106, GAS, and 107 respectively [620-06];

[0454] xx) A first VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 114, 115, and 116 respectively; and a first VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 117, DAS, and 118 respectively

[733] ; and a second VH region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 108, 109, and 110 respectively; and a second VL region comprising CDR1, CDR2, and CDR3 sequences having SEQ ID No: 112, AAS, and 113 respectively

[726] ; and

[0455] yy) First VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 114, 115 and 116 respectively; and first VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 117, DAS and 118 respectively

[733] ; and second VH region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 108, 109 and 111 respectively; and second VL region, which contains CDR1, CDR2 and CDR3 sequences having SEQ ID No: 112, AAS and 113 respectively [726-M101L];

[0456] Anti-AXL antibody drug conjugate - immunoconjugate

[0457] Antibodies according to any aspect or embodiment of the invention may be conjugated to therapeutic or diagnostic components, such as cytotoxic agents, chemotherapeutic drugs, cytokines, immunosuppressants, antibiotics, or radioisotopes. Such conjugates are referred to herein as “immunoconjugates.” Immunoconjugates containing one or more cytokines are referred to as “immunotoxins.” Antibodies conjugated to cytotoxic agents, drugs, etc., are also referred to as antibody-drug conjugates (ADCs). Immunoconjugates can have such long half-lives that they allow the antibody-drug conjugate to be internalized, degraded, and induce cell killing through the released toxins.

[0458] Therefore, in another aspect, the present invention relates to immunoconjugates comprising an antibody or a bispecific antibody according to any aspect or embodiment described herein, and a therapeutic component such as a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressant, antibiotic, or radioisotope. The cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressant, antibiotic, or radioisotope may be conjugated to the antibody or the bispecific antibody via a linker.

[0459] ADCs are often designed so that the cytotoxic payload is inactivated when conjugated to an antibody. The cytotoxic payload can be released intracellularly either upon binding to the cell membrane and subsequent internalization or in response to proteolytic activity in the tumor microenvironment. The term “internalization” or “internalization”, as used herein, refers to the biological process in which molecules (such as antibodies according to the invention) are encapsulated by the cell membrane and drawn into the cell interior. It is also referred to as “endocytosis.”

[0460] Therefore, antibodies according to any aspect or embodiment of the present invention can be internalized into cells when they bind to the target AXL.

[0461] In some cases, it may be necessary to use antibodies that undergo internalization. Such antibodies with good internalization properties are suitable for conjugation to cytotoxic agents, drugs, etc., optionally via a linker designed for intracellular lysis.

[0462] Once internalized, ADCs can, in most cases, be delivered to lysosomes, where the release of the effective drug utilizes the catabolic environment within these organelles. Antibodies are typically linked to cytotoxic agents via linkers. Therefore, specialized linkers have been designed to be cleaved only within or on target tumor cells in specific microenvironments or within the tumor microenvironment. Examples include linkers cleaved by acidic conditions, reducing conditions, or specific proteases.

[0463] The stability of antibody-linker-drug in circulation is important because it allows antibody-mediated drug delivery to specific target cells. In addition, the long circulating half-life of ADCs provides exposure for several days to several weeks after injection. Drugs coupled via non-cleavable linkers and protease-cleavable linkers are generally more stable in circulation than disulfide linkers and hydrazone linkers, but the stability of the latter two linkers can be modulated by altering adjacent chemical structures [6].

[0464] In one embodiment, the treatment component is a cytotoxic agent.

[0465] Cytotoxins or cytotoxic agents include any agent that is harmful to cells (e.g., kills). Cytotoxic agents suitable for forming the immunoconjugates of this invention include: paclitaxel, tubulysins, duostatins, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxydiketone anthrax, maytansine or its analogues or derivatives, mitoxantrone, sclerosomycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin; cazithromycin or its analogues or derivatives; antimetabolites (such as methotrexate, 6-mercaptopurine) 1,0-dimethylguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine C, mitomycin C, cisplatin and other platinum derivatives such as carboplatin; and docalamycin A, docalamycin SA, CC-1065 (also known as rachelmycin) or analogs or derivatives of CC-1065), dolastatin, auritastatin (aur Istatin, pyrrolo[2,1-c][1,4]benzodiazepines (PDB), indobenzobenzodiazepines (IGN) or their analogues; antibiotics (such as actinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, procainox, amoxicillin (AMC)); antimitotic agents (e.g., tubulin targets), such as diphtheria toxin and related molecules (e.g., diphtheria A chain and its active fragments and hybrid molecules); ricin (e.g., ricin A or deglycosylated ricin A chain toxin), cholera toxin, shiga-like toxin (SLT) -I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alarin, saponin, Capsula root toxin, gelanin, Abrus precatorius toxin A chain, Capsula root toxin A chain, α-Azotocin, Tung oil protein, Caryophyllin protein, Phytolacca acinosa protein (PAPI, PAPII and PAP-S), bitter melon inhibitor, Jatropha curcas toxin, Croton toxin, Fructus philodendron inhibitor, White tree toxin, Mitogellin, Localized trachomatis, Phenyromycin and Enoxacin toxin.Other suitable conjugate molecules include: antimicrobial / cleavage peptides such as CLIP, Magaiin 2, melitin, silkworm antimicrobial peptide, and P18; ribonucleases (RNases), DNase I, staphylococcal enterotoxin-A, pokeweed antiviral protein, diphtheria toxin, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell. 47 , 641 (1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 44 , 43 (1994). Therapeutic agents that can be administered in combination with the anti-AXL antibodies or antibody-drug conjugates of the present invention as described elsewhere herein, such as, for example, anticancer cell factors or chemokines, are also candidates for use in therapeutic portions conjugated with the antibodies disclosed in the present invention.

[0466] The term "cytotoxic agent," as used herein, refers to any agent that is harmful to cells (e.g., kills them). For a description of these classes of drugs well known in the art and their mechanisms of action, see

[54] . Additional techniques related to the preparation of antibody immunotoxins are provided, for example, in

[55] and

[56] .

[0467] In one embodiment, the cytotoxic agent is linked to the antibody or a fragment thereof via a cleavable linker, such as 4-(2-pyridinedithio)-pentanoic acid. N -Succinimide ester (SSP), Maleimide hexanoyl-valine-citrulline- p -Aminobenzyloxycarbonyl (mc-vc-PAB) or AV-1 K-lock valine-citrulline.

[0468] The term "cleavable linker," as used herein, refers to a subset of linkers that are catalyzed by specific proteases in target cells or the tumor microenvironment, resulting in the release of a cytotoxic agent. Examples of cleavable linkers are linkers based on chemical motifs, including disulfides, hydrazones, or peptides. Another subset of cleavable linkers adds an additional linker motif between the cytotoxic agent and the primary linker, i.e., attaching the linker-drug combination to the antibody site. In some embodiments, the additional linker motif may be cleaved by a lysing agent present in the intracellular environment (e.g., lysosomes, endosomes, or endothelial spaces). The linker may be, for example, a peptide linker cleaved by intracellular peptidases or proteases (including, but not limited to, lysosomal proteases or endosomal proteases). In some embodiments, the peptide linker is at least two or at least three amino acids in length. Lysing agents may include cathepsins B and D, as well as plasmin, all known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug within target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). In specific embodiments, the peptide linker cleavable by intracellular proteases is a Val-Cit (valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker (see, for example, US6214345, which describes the synthesis of doxorubicin containing a Val-Cit linker). An advantage of using intracellular proteolytically hydrolyzed therapeutic agents is that the agents are typically attenuated upon conjugation, and the conjugate generally exhibits high serum stability.

[0469] In another embodiment, the cytotoxic agent is linked to the antibody or a fragment thereof via a non-cleavable linker, such as 4( N -maleimide-methyl)cyclohexane-1-carboxylic acid succinate imide (MCC) or maleimide-hexanoyl (MC).

[0470] The term “non-cleavable linker,” as used herein, refers to a subset of linkers that, in contrast to cleavable linkers, do not contain motifs that are specifically and predictively recognized by intracellular or extracellular proteases. Therefore, an ADC based on a non-cleavable linker will not be released from or cleaved from the antibody until the entire antibody-linker-drug complex is degraded in the lysosomal cavity. An example of a non-cleavable linker is a thioether. In yet another embodiment, the linker unit is non-cleavable, and the drug is released by antibody degradation (see

[57] ). Typically, such linkers are substantially insensitive to the extracellular environment. As used herein, “substantially insensitive to the extracellular environment” in the context of a linker means that, when the antibody-drug conjugate is present in an extracellular environment (e.g., plasma), no more than 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linker is cleaved in a sample of the antibody-drug conjugate. Whether the linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating the antibody-drug conjugate with plasma for a predetermined time (e.g., 2, 4, 8, 16, or 24 hours) and subsequently quantifying the amount of free drug present in the plasma.

[0471] In one embodiment, the cytotoxic agent is selected from: DNA-targeting agents, such as DNA alkylating agents and cross-linking agents, such as chazim, docamycin, rachelmycin (CC-1065), pyrrolo[2,1-c][1,4]benzodiazepine (PBD) and indobenzobenzodiazepine (IGN); microtubule-targeting agents, such as duostatin (e.g., duostatin-3), aurestatin (e.g., monomethylaurestatin E (MMAE) and monomethylaurestatin F (MMAF)), dolalastatin, maytansine, etc. N (2')-Deacetylated- N (2')-(3-mercapto-1-oxopropyl)-matansin (DM1) and tubulysin; and nucleoside analogs; or analogs, derivatives or prodrugs thereof.

[0472] In one embodiment, the immunoconjugate comprises the following combination:

[0473] i) Cytotoxic agents with said cleavable connectors that have bystander-killing capabilities;

[0474] ii) Cytotoxic agents with said cleavable connectors that do not have bystander-killing capabilities;

[0475] iii) Cytotoxic agents with said non-lytically cleavable connectors that have bystander-kill capability; or

[0476] iv) Cytotoxic agents with the aforementioned non-lytically cleavable connectors that do not have bystander-kill capability.

[0477] The terms “bystander killing effect,” “bystander killing,” “bystander killing capability,” or “bystander cytotoxicity,” as used herein, refer to an effect in which a cytotoxic agent coupled to an antibody via a cleavable or non-cleavable linker is capable of diffusing across the cell membrane after release from the antibody, thereby causing the killing of adjacent cells. When a cytotoxic agent is coupled via a cleavable or non-cleavable linker, it can be a cytotoxic agent-only with bystander killing capability or a cytotoxic agent containing a portion of said linker. The ability to diffuse across the cell membrane is related to the hydrophobicity of the cytotoxic agent or the combination of the cytotoxic agent and the linker. Such cytotoxic agents can advantageously be membrane-permeable toxins, such as MMAEs that have been released from the antibody via a protease. Bystander killing effects may be desirable, particularly in tumors with heterogeneous target expression and in solid tumors where antibody penetration may be limited.

[0478] The terms “bystander-killing capability,” “bystander-killing effect,” “bystander-killing,” or “bystander-cytotoxicity,” as used herein, refer to an effect in which a cytotoxic agent coupled to an antibody via a cleavable or non-cleavable linker is unable to diffuse across the cell membrane after release from the antibody. Therefore, such cytotoxic agents, or combinations of cytotoxic agents with linkers, will fail to kill adjacent cells upon release from the antibody. It is believed, without being bound by theory, that such combinations of cytotoxic agents with cleavable or non-cleavable linkers will kill only cells expressing the target to which the antibody is bound.

[0479] A stable linker between the antibody and the cytotoxic agent is a crucial factor for antibody-drug conjugates (ADCs). Both cleavable and non-cleavable linkers have been proven safe in preclinical and clinical trials.

[0480] In one embodiment, the cytotoxic agent is selected from microtubule-targeting agents, such as aurestatin and maytanol.

[0481] The term "microtubule-targeting agent," as used herein, refers to an agent or drug that inhibits mitosis (cell division). Microtubules are essential structures for the proper separation of DNA during cell division, and their function is critically dependent on 'dynamic instability,' the process by which microtubule structures are continuously elongated and shortened. Microtubule-targeting agents disrupt or stabilize microtubules, preventing the formation of the mitotic spindle, leading to mitotic arrest and apoptosis. These agents can be derived from, for example, natural substances such as alkaloids, and inhibit cell mitosis by disrupting or stabilizing microtubule polymerization, thereby preventing the formation of the mitotic spindle and subsequent cell division, resulting in inhibition of cancer growth. Examples of microtubule-targeting agents include: paclitaxel, docetaxel, vincristine, vinorelbine, duostatins, auristatin, maytanol, tubulysins, and dolalastatin.

[0482] In one embodiment, the cytotoxic agent is aurestatin or an aurestatin peptide analog and derivative (

[131] ;

[132] ). Aurestatin has been shown to interfere with microtubule dynamics, GTP hydrolysis, and cell nucleus and cell division

[133] , and has anticancer

[134] and antifungal activities

[135] . The aurestatin drug moiety can be attached to an antibody via a linker through the N (amino) terminus or C (carboxyl) terminus of the peptide drug moiety.

[0483] Exemplary auristatin implementations include N-terminally linked monomethyl auristatin pharmaceutical portions DE and DF, disclosed in

[136] and described in

[137] .

[0484] In a specific implementation scheme, the cytotoxic agent is monomethylaurestatin E (MMAE).

[0485]

[0486] The antibody is attached to the nitrogen (N) on the left-hand side of the above chemical structure of MMAE via a suitable linker.

[0487] In one embodiment, the cytotoxic agent monomethylauratestatin E (MMAE) is linked to the antibody via a valine-citrulline (VC) linker.

[0488] In another embodiment, the cytotoxic agent monomethylaurestatin E (MMAE) is linked to the antibody via a valine-citrulline (VC) linker and a maleimide hexanoyl (MC) linker, wherein the combination of the cytotoxic agent and the linkers has a chemical structure;

[0489]

[0490] MAb is an antibody.

[0491] In one embodiment, the cytotoxic agent is monomethylaurestatin F (MMAF).

[0492]

[0493] The antibody is attached to the nitrogen (N) on the left-hand side of the above chemical structure of MMAF via a suitable linker.

[0494] In one embodiment, the cytotoxic agent monomethylaurestatin F (MMAF) is linked to the antibody via a maleimide hexanoyl (mc) linker, wherein the combination of the cytotoxic agent and the linker has a chemical structure;

[0495]

[0496] MAb is an antibody.

[0497] In one embodiment, the cytotoxic agent is duostatin3.

[0498] In another specific implementation, the cytotoxic agent is a DNA-targeting agent.

[0499] The term "DNA-targeting agent," as used herein, refers to a specific class of cytotoxic agents capable of alkylating and / or cross-linking DNA. Examples of such DNA-targeting agents are IGN agents comprising indobenzodiazepine dimers and pyrrolo[2,1-c][1,4]benzodiazepines (PBD), which are highly effective due to their ability to alkylate and cross-link DNA. Another example is IGN agents comprising indobenzodiazepine dimers, which are highly effective due to their ability to alkylate DNA only. Docamycin is another class of DNA-targeting agents. Docamycin is a small, synthetic DNA minor groove binding alkylating agent. These compounds are suitable for targeting solid tumors as well as hematologic malignancies.

[0500] In one embodiment, the immunoconjugate comprises two to four cytotoxic molecules / antibodies. Depending on the chemical properties of the toxin and adapter-toxin combination, two to four cytotoxic molecules / antibodies may be superior to conjugates with a larger loading (which are cleared from circulation more rapidly than conjugates with a smaller loading). The cytotoxic agent loading is represented by p and is the average number of cytotoxic agent moieties / antibodies in the molecule (also known as the drug-antibody ratio, DAR). The cytotoxic agent loading ranges from 1 to 20 drug moieties / antibodies and may be present on amino acids containing useful functional groups (such as, but not limited to, amino or thiol groups), such as lysine or cysteine.

[0501] In one embodiment, the number of cytotoxic agents / antibodies is 1 to 8, such as 2 to 7, such as 2 to 6, such as 2 to 5, such as 2 to 4, and such as 2 to 3.

[0502] In another embodiment, the immunoconjugate comprises four to eight cytotoxic molecules / antibodies. In yet another embodiment, the immunoconjugate comprises six to ten cytotoxic molecules / antibodies. In still another embodiment, the immunoconjugate comprises 10 to 30, such as 15 to 25, such as 20 cytotoxic molecules / antibodies.

[0503] Depending on the conjugation method, p can be limited by the number of attachment sites on the antibody, for example, when the attachment is a cysteine ​​thiol or lysine. Typically, antibodies do not contain many free and reactive cysteine ​​thiol groups, which can be linked to the drug moiety as disulfide bridges, just like most cysteine ​​thiol residues in the antibody. Therefore, in these embodiments, when the cytotoxic agent is conjugated via cysteine ​​thiols, the antibody can be reduced under partial or complete reduction conditions by a reducing agent (such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP)) to generate reactive cysteine ​​thiol groups. In some embodiments, the drug loading of the ADC of the present invention ranges from 1 to approximately 8, because up to 8 free cysteine ​​thiol groups become available after (partial) reduction of the antibody (8 cysteine ​​residues are involved in interchain disulfide bonding).

[0504] In one embodiment, the drug linker portion is vcMMAE. The vcMMAE drug linker portion and conjugation method are disclosed in

[27] ,

[28] ,

[145] ,

[146] , and

[147] , which are incorporated herein by reference. The vcMMAE is formed by conjugating the linker mc-vc-PAB to the cytotoxic portion MMAE, and the vcMMAE drug linker portion is bound to the cysteine ​​residue of the anti-AXL antibody using a method similar to those disclosed in the literature.

[0505] In one embodiment, the drug linker portion is mcMMAF. The mcMMAF drug linker portion and conjugation method are disclosed in

[138] ,

[139] and

[140] (which are incorporated herein by reference), and the mcMMAF drug linker portion is bound to the cysteine ​​residue of the anti-AXL antibody using methods similar to those disclosed in the literature.

[0506] In one embodiment, the cytotoxic agent is delivered via K-Lock as described in

[58] ,

[148] and

[149] . TMOne or two lysine residues are coupled and linked to the amino acid sequence of the antibody, and duostatin3 (also known as Duo3) is bound to the lysine residues of the anti-AXL antibody using methods similar to those disclosed in the literature.

[0507] Other linker technologies can be used in the anti-AXL antibody drug conjugates of the present invention, such as linkers containing hydroxyl groups.

[0508] In one embodiment, the adapter is attached to a free cysteine ​​residue of an anti-AXL antibody obtained by (partial) reduction of the anti-AXL antibody.

[0509] In a specific implementation, the connector is mc-vc-PAB and the cytotoxic agent is MMAE; or the connector is SSP and the cytotoxic agent is DM1.

[0510] In a specific implementation, the connector is MMC and the cytotoxic agent is DM1; or the connector is MC and the cytotoxic agent is MMAF.

[0511] In a specific implementation, the connector is a cleavable connector AV1-K lock, and the cytotoxic agent is duostatin3.

[0512] In one embodiment, the immunoconjugate comprises a linker mc-vc-PAB, a cytotoxic agent MMAE, and an antibody, wherein the at least one binding region comprises a VH region and a VL region selected from:

[0513] a) The VH region containing SEQ ID No: 1 and the VL region containing SEQ ID No: 2

[107] ;

[0514] b) The VH region containing SEQ ID No: 5 and the region containing SEQ ID No: 6

[148] ;

[0515] c) The VH region containing SEQ ID No: 34 and the VL region containing SEQ ID No: 35

[733] ;

[0516] d) The VH region containing SEQ ID No: 7 and the VL region containing SEQ ID No: 9

[154] ;

[0517] e) The VH region containing SEQ ID No: 10 and the VL region containing SEQ ID No: 11

[171] ;

[0518] f) The VH region containing SEQ ID No: 16 and the VL region containing SEQ ID No: 18

[183] ;

[0519] g) The VH region containing SEQ ID No: 25 and the VL region containing SEQ ID No: 26

[613] ;

[0520] h) The VH region containing SEQ ID No: 31 and the VL region containing SEQ ID No: 33

[726] ;

[0521] i) The VH region containing SEQ ID No: 3 and the VL region containing SEQ ID No: 4

[140] ;

[0522] j) The VH region containing SEQ ID No:8 and the VL region containing SEQ ID No:9 [154-M103L];

[0523] k) The VH region containing SEQ ID No:12 and the VL region containing SEQ ID No:13

[172] ;

[0524] l) The VH region containing SEQ ID No:14 and the VL region containing SEQ ID No:15

[181] ;

[0525] m) Contains the VH region of SEQ ID No:17 and the VL region of SEQ ID No:18 [183-N52Q];

[0526] n) The VH region containing SEQ ID No:19 and the VL region containing SEQ ID No:20

[187] ;

[0527] o) The VH region containing SEQ ID No:21 and the VL region containing SEQ ID No:22 [608-01];

[0528] p) The VH region containing SEQ ID No:23 and the VL region containing SEQ ID No:24 [610-01];

[0529] q) The VH region containing SEQ ID No:27 and the VL region containing SEQ ID No:28 [613-08];

[0530] r) The VH region containing SEQ ID No:29 and the VL region containing SEQ ID No:30 [620-06]; and

[0531] s) The VH region containing SEQ ID No:32 and the VL region [726-M101L] containing SEQ ID No:33.

[0532] In another alternative embodiment, the anti-AXL antibody drug conjugate disclosed in this invention comprises a conjugated nucleic acid or nucleic acid-related molecule. In one such embodiment, the conjugated nucleic acid is: a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (e.g., siRNA molecule), or an immunostimulatory nucleic acid (e.g., a DNA molecule containing an immunostimulatory CpG motif).

[0533] In another alternative embodiment, the anti-AXL antibody of the present invention is coupled to an aptamer or ribozyme or a functional peptide analog or derivative thereof.

[0534] In another alternative embodiment, an anti-AXL antibody-drug conjugate comprising one or more radiolabeled amino acids is provided. The radiolabeled anti-AXL antibody can be used for both diagnostic and therapeutic purposes (conjugation to a radiolabeled molecule is another possible feature). Non-limiting examples of labeling for peptides include: 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc and 125 I, 131 I and 186 Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, for example,

[59] and

[60] ,

[61] ,

[62] ,

[63] ,

[64] and

[65] , US 5,697,902). For example, radioisotopes can be coupled via the chloramine-T method.

[0535] In one embodiment, the antibody is conjugated to a radioisotope or a chelate containing a radioisotope. For example, the antibody may be conjugated to a chelating agent linker, such as DOTA, DTPA, or tiuxetan, which allows the antibody to complex with the radioisotope. The antibody may also, or alternatively, contain or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. Radiolabeled anti-AXL antibodies can be used for both diagnostic and therapeutic purposes. Non-limiting examples of radioisotopes include: 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 125 I, 111 In、 131 I, 186 Re、 213 Bs、 225 Ac and 227 Th.

[0536] Anti-AXL antibodies can also be chemically modified, for example, by covalently coupling to a polymer, to extend their cycling half-life. Exemplary polymers and methods of attaching them to peptides are illustrated, for example, in

[66] ,

[67] ,

[68] , and

[69] . Other polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., PEG with a molecular weight between about 1,000 and about 40,000, such as PEG between about 2,000 and about 20,000). This can be used, for example, if the anti-AXL antibody is a fragment.

[0537] Any method known in the art for conjugating the anti-AXL antibody according to the invention to a conjugating molecule (as described above) may be employed, including the methods described in

[70] ,

[71] , and

[72] . Such antibodies may be produced by chemically conjugating other portions to the N-terminal or C-terminal side of the anti-AXL antibody (e.g., the H or L chain of the anti-AXL antibody) (see, for example,

[73] ). Where appropriate, such conjugated antibody derivatives may also be generated by conjugating internal residues or sugars, or non-naturally present amino acids or other amino acids that have been introduced into the constant structural domain of the antibody.

[0538] The reagent can be directly or indirectly linked to the anti-AXL antibody disclosed in this invention. One example of indirect linking of the second reagent is via a spacer portion to a cysteine ​​or lysine residue in the antibody. In one embodiment, the anti-AXL antibody is coupled to a prodrug molecule (which can be activated in vivo to become a therapeutic agent) via a spacer or adapter. Upon administration, the spacer or adapter is cleaved by tumor cell-associated enzymes or other tumor-specific conditions, thereby forming the active drug. Examples of such prodrug techniques and adapters are described in

[74] ,

[75] ,

[76] ,

[77] ,

[78] , and

[79] (all incorporated herein by reference). Suitable antibody-prodrug techniques and docalamycin analogs are also described in

[80] (incorporated herein by reference).

[0539] In one embodiment, the anti-AXL antibody of the present invention is attached to a chelating agent linker, such as tiuxetan, which allows the antibody to be coupled to a radioisotope.

[0540] Composition

[0541] In another aspect, the present invention relates to compositions comprising the antibodies, bispecific antibodies, or immunoconjugates of the present invention.

[0542] In another aspect, the present invention relates to pharmaceutical compositions comprising antibodies, bispecific antibodies or immunoconjugates according to the present invention, and pharmaceutical carriers.

[0543] The pharmaceutical composition may be formulated with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients according to conventional techniques, such as those disclosed in

[81] .

[0544] The pharmaceutically acceptable carrier or diluent, as well as any other known adjuvants and excipients, shall be suitable for the antibodies or antibody-drug conjugates of the present invention and the selected administration method. The suitability of the carrier and other components of the pharmaceutical composition is determined based on the absence of a significant negative impact on the desired biological properties of the selected compound or pharmaceutical composition of the present invention (e.g., no substantial effect on antigen binding (relative inhibition of 10% or less, relative inhibition of 5% or less, etc.)).

[0545] The pharmaceutical compositions of the present invention may further comprise diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or non-protein amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in the pharmaceutical composition.

[0546] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition, and administration method without toxicity to that patient. The selected dose level will depend on various pharmacokinetic factors, including: the activity of the specific composition of the present invention or its amide used, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, and factors well known in the medical field such as the age, sex, weight, condition, general health, and medical history of the patient receiving treatment.

[0547] The pharmaceutical composition may be administered via any suitable route and mode. Suitable routes for in vivo and in vitro administration of the compounds of the present invention are well known in the art and may be selected by those skilled in the art.

[0548] In one embodiment, the pharmaceutical composition of the present invention is administered parenterally.

[0549] The terms “parenteral administration” and “post-gastrointestinal administration”, as used herein, refer to administration methods other than enteric and local administration, typically by injection, and include: epidermal, intravenous, intramuscular, intra-arterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, tracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, spinal, intracranial, intrathoracic, epidural, and intramembranous injections and infusions.

[0550] In one embodiment, the pharmaceutical composition of the present invention is administered by intravenous or subcutaneous injection or infusion.

[0551] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, antioxidants, and absorption delay agents that are physiologically compatible with the compounds of the present invention.

[0552] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include: water, saline, phosphate-buffered saline, ethanol, glucose, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof; vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil and sesame oil; carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters such as ethyl oleate, and / or various buffer solutions. Other carriers are well known in the pharmaceutical field.

[0553] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersants and sterile powders used for the ad hoc preparation of sterile injectable solutions or dispersants. The use of such media and reagents for pharmaceutically active substances is known in the art. The use of such conventional media or reagents in the pharmaceutical compositions of the present invention is contemplated unless they are incompatible with the active compound.

[0554] This can be achieved, for example, by using coating materials (such as lecithin), by maintaining the desired particle size in the dispersed state, and by using surfactants to maintain appropriate flowability.

[0555] The pharmaceutical compositions of the present invention may further comprise pharmaceutically acceptable antioxidants such as: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbate palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0556] The pharmaceutical compositions of the present invention may also contain isotonic agents, such as sugars, polyols (such as mannitol, sorbitol, glycerol) or sodium chloride.

[0557] The pharmaceutical compositions of the present invention may also contain one or more adjuvants suitable for the chosen route of administration, such as preservatives, wetting agents, emulsifiers, dispersants, or buffers, which may improve the shelf life or efficacy of the pharmaceutical composition. The compounds of the present invention may be prepared together with a carrier into controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems, wherein the carrier protects the compound from rapid release. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers (such as vinyl acetate, polyanhydride, polyglycolic acid, collagen, poly-orthoesters, and polylactic acid) either alone or with waxes, or other materials well known in the art. Methods for preparing such formulations are well known to those skilled in the art. See, for example,

[82] .

[0558] In one embodiment, the compounds of the present invention may be formulated to ensure proper in vivo distribution. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or ad hoc preparations of dispersants and sterile powders for sterile injectable solutions or dispersants. The use of such media and reagents for pharmaceutically active substances is known in the art. The use of such conventional media or reagents in the pharmaceutical compositions of the present invention is contemplated unless they are incompatible with the active compound. Other active or therapeutic compounds may also be incorporated into the compositions.

[0559] Pharmaceutical compositions for injection must generally be sterile and stable under manufacturing and storage conditions. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier may be an aqueous or non-aqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof; vegetable oils, such as olive oil; and injectable organic esters, such as ethyl oleate. Appropriate flowability may be maintained, for example, by using coatings (such as lecithin), by maintaining the desired particle size in the dispersed state, and by using surfactants. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols (such as glycerol, mannitol, sorbitol), or sodium chloride. Extended absorption of the injectable composition may be achieved by including agents that delay absorption (e.g., monostearate and gelatin) in the composition. Sterile injectable solutions may be prepared by combining the desired amount of the active compound in a suitable solvent with one or a combination of the ingredients listed above, and, if necessary, by sterile microfiltration. Dispersants are typically prepared by incorporating an active compound into a sterile medium containing a base dispersion medium and other desired components, such as those listed above. Examples of preparation methods for sterile powders used to prepare sterile injectable solutions include vacuum-drying and freeze-drying (lyophilization), methods that yield a powder of the active ingredient from a previously sterile filtered solution, plus any other desired components.

[0560] Sterile injectable solutions can be prepared by combining the desired amount of an active compound in a suitable solvent with one or a combination of the components listed above, followed by sterile microfiltration as needed. Typically, dispersants are prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and any other desired components from those listed above. Examples of methods for preparing sterile powders for sterile injectable solutions include vacuum drying and freeze-drying (lyophilization), methods that yield a powder of the active ingredient from a previously sterile filtered solution plus any other desired components.

[0561] The pharmaceutical compositions of the present invention may contain: the antibody, bispecific antibody or ADC of the present invention; a combination of the antibody, bispecific antibody or ADC of the present invention with another therapeutic compound; or a combination of compounds of the present invention.

[0562] Nucleic acid constructs, expression vectors, and host cells

[0563] In one aspect, the present invention relates to nucleic acid constructs that encode one or more sequences shown in Table 1. Therefore, the present invention relates to nucleic acid constructs that encode any one of the sequences shown in SEQ ID No: 1 to 135. In one embodiment, the nucleic acid construct encodes at least one sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, The amino acid sequences of 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, and 135. Therefore, in one embodiment, the nucleic acid construct encodes an antibody according to any aspect or embodiment described herein.

[0564] In a specific implementation, the nucleic acid construct encodes at least one amino acid sequence selected from SEQ ID NO: 46, 47, 48, 49, 50, 36, 37, 38, 39, 40, 114, 115, 116, 117 and 118.

[0565] In another aspect, the present invention relates to expression vectors encoding the antibodies of the present invention. Thus, the expression vector comprises one or more nucleic acid constructs according to any aspect or embodiment described herein. In one embodiment, such an expression vector may be used to express the anti-AXL antibody of the present invention. The expressed anti-AXL antibody may then be conjugated to a portion as described herein. In another embodiment, the anti-AXL antibody may then be used to generate a bispecific antibody as described herein.

[0566] In one embodiment, the expression vector of the present invention comprises a nucleic acid sequence encoding one or more of the following heavy chain (VH)CDR3 amino acid sequences: SEQ ID No: 38, 43, 48, 53, 54, 59, 64, 69, 75, 80, 85, 90, 95, 100, 105, 110, 111, 116, 120, 122, 125, and 127.

[0567] In a specific implementation, the expression vector of the present invention comprises a nucleic acid sequence encoding one or more of the following VH CDR1, CDR2 and CDR3 amino acid sequences: SEQ ID No: 36-38, 41-43, 46-48, 51-54, 57-59, 62-64, 67-69, 72-75, 78-80, 83-85, 88-90, 93-95, 98-100, 103-105, 108-110 and 114-116.

[0568] In one embodiment, the expression vector of the present invention comprises a nucleic acid sequence encoding one or more of the following light chain (VL)CDR3 amino acid sequences: SEQ ID No: 40, 45, 50, 56, 61, 66, 71, 77, 82, 87, 92, 97, 102, 107, 113 and 118.

[0569] In another specific embodiment, the expression vector of the present invention comprises a nucleic acid sequence encoding one or more of the following VH amino acid sequences: SEQ ID No: 1, 3, 5, 7, 8, 10, 12, 14, 16, 17, 19, 21, 23, 25, 27, 29, 31, 32 and 34.

[0570] In another specific embodiment, the expression vector of the present invention comprises a nucleic acid sequence encoding one or more of the following VL amino acid sequences: SEQ ID No: 2, 4, 6, 9, 11, 13, 15, 18, 20, 22, 24, 26, 28, 30, 33 and 35.

[0571] In one embodiment, the expression vector of the present invention comprises a nucleic acid sequence encoding one or more of the following amino acid sequences: SEQ ID No: 1 to 35.

[0572] In specific embodiments, the expression vector of the present invention comprises a nucleic acid sequence encoding one or more variants of the above-mentioned amino acid sequences, the variant having up to 25 amino acid modifications, such as 20, or up to 15, 14, 13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid modification, such as deletion or insertion, preferably substitutions such as conserved or non-conserved substitutions, or having at least 80% identity with any of the above sequences, or having at least 85%, 90%, or 95% identity with any of the above-mentioned amino acid sequences, such as 96%, 97%, 98%, or 99% identity. The present invention also relates to nucleic acid sequences different from the above-mentioned nucleic acid sequences, but encoding the same amino acid sequence as the antibodies of the present invention due to differences in the genetic code. For example, the nucleic acid sequence may be varied but yields the same amino acid sequence as any of the amino acid sequences described herein. It is well known to those skilled in the art how to identify such additional nucleic acid sequences based on the genetic code.

[0573] In another embodiment, the expression vector further comprises a nucleic acid sequence that encodes a constant region of the light chain, heavy chain, or both of the light and heavy chains of an antibody (e.g., a human IgG1,κ monoclonal antibody).

[0574] Such expression vectors as described above can be used for recombinant production of the antibodies of the present invention.

[0575] In the context of this invention, the expression vector can be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (containing a nucleic acid sequence with a suitable set of expression control elements). Examples of such vectors include: derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding an anti-AXL antibody is contained in a naked DNA or RNA vector, including, for example: linear expression elements (as described in, for example,

[83] ), dense nucleic acid vectors (as described in, for example,

[84] and / or

[85] ), plasmid vectors such as pBR322, pUC19 / 18, or pUC 118 / 119, “midge” size minimized nucleic acid vectors (as described in, for example,

[86] ), or as a precipitated nucleic acid vector construct, such as a CaPO4 precipitated construct (as described in, for example,

[87] ,

[88] ,

[89] , and

[90] ). Such nucleic acid vectors and their uses are well known in the art (see, for example,

[91] and

[92] ).

[0576] In one embodiment, the vector is suitable for expressing anti-AXL antibodies in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (

[93] , pET vectors (Novagen, MadisonWI, etc.).

[0577] The expression vector may also be, or optionally, a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters (such as α-factors, alcohol oxidases, and PGH) (reviewed in

[94] and

[95] ).

[0578] Nucleic acid constructs and / or vectors may also contain nucleic acid sequences encoding secretion / localization sequences that can target polypeptides (such as nascent polypeptide chains) into the periplasmic space or into the cell culture medium. Such sequences are known in the art and include: secretion leader or signal peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER residency signals, mitochondrial transport sequences, chloroplast transport sequences), membrane localization / anchoring sequences (e.g., stop-transfer sequences, GPI anchoring sequences), etc.

[0579] In the expression vector of the present invention, the nucleic acid encoding the anti-AXL antibody may contain any suitable promoter, enhancer, and other elements that facilitate expression, or are associated with it. Examples of such elements include: strong expression promoters (e.g., human CMV IE promoter / enhancer and RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, origin of replication of plasmid products in E. coli, antibiotic resistance genes as selection markers, and / or convenient cloning sites (e.g., polymer adapters). The nucleic acid may also contain inducible promoters, such as CMV IE, as opposed to constitutive promoters (those skilled in the art will recognize that such terms are practical descriptions of the degree of gene expression under specific conditions).

[0580] In one embodiment, the expression vector encoding the anti-AXL antibody may be located to and / or delivered to a host cell or host animal via a viral vector.

[0581] In even further aspects, the present invention relates to recombinant eukaryotic or prokaryotic host cells, such as transfected tumors, that produce anti-AXL antibodies of the present invention as defined herein or bispecific molecules of the present invention as defined herein. Examples of host cells include yeast, bacteria, and mammalian cells, such as CHO or HEK cells or derivatives thereof. For example, in one embodiment, the present invention provides cells comprising nucleic acids stably integrated into the cell genome, said nucleic acids containing sequences encoding the expression of the anti-AXL antibody of the present invention. In another embodiment, the present invention provides cells comprising non-integrated nucleic acids (such as plasmids, viscera, phages, or linear expression elements) containing sequences encoding the expression of the anti-AXL antibody of the present invention.

[0582] In another aspect, the present invention relates to host cells comprising vectors according to any aspect and embodiment described herein. In one embodiment, the anti-AXL antibody described herein is provided by producing the antibody using recombinant eukaryotic, prokaryotic, or microbial host cells. Therefore, the present invention provides recombinant host cells, such as recombinant prokaryotic, recombinant eukaryotic, or recombinant microbial host cells. Examples of host cells include yeast, bacteria, and mammalian cells, such as CHO or HEK-293 cells. For example, in one embodiment, the host cell comprises nucleic acids stably integrated into the cell genome, the nucleic acids comprising a sequence encoding the expression of the anti-AXL antibody described herein. In one embodiment, the host cell comprises nucleic acids stably integrated into the cell genome, the nucleic acids comprising a sequence encoding the expression of the first or second polypeptide described herein. In another embodiment, the host cell comprises non-integrated nucleic acids (such as plasmids, granules, phages, or linear expression elements), the nucleic acids comprising a sequence encoding the expression of the anti-AXL antibody, the first or second polypeptide described herein.

[0583] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to cells in which an expression vector has been introduced. It should be understood that this term is intended to refer not only to the specific cell discussed but also to the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may indeed differ from the parent cells but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example: transfected tumors such as CHO cells, HEK-293 cells, PER.C6, and NSO cells; and lymphocytes; as well as prokaryotic cells such as Escherichia coli and other eukaryotic hosts such as plant cells and fungi.

[0584] The term “transfected tumor”, as used herein, includes recombinant eukaryotic host cells that express antibodies or target antigens, such as CHO cells, PER.C6, NS0 cells, HEK-293 cells, plant cells, or fungi, including yeast cells.

[0585] In another aspect, the present invention relates to hybridomas that produce antibodies according to any aspect or embodiment described herein. Thus, antibodies can be obtained from hybridomas prepared from mouse spleen B cells derived from mice immunized with a target antigen, for example, in the form of cells expressing the antigen on their surface or nucleic acids encoding the target antigen. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals such as rabbits, rats, dogs, primates, etc.

[0586] Human antibodies can be generated using transgenic or transchromosomal mice, such as HuMAb mice, which carry portions of the human immune system rather than the mouse system. The HuMAb mice contain mini-sites of human immunoglobulin genes encoding unrearranged human (μ and γ) heavy chain and κ light chain immunoglobulin sequences, and targeted mutations that inactivate endogenous μ and κ chain sites

[96] . Thus, the mice have reduced mouse IgM or κ expression, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutations to generate high-affinity human IgG,κ monoclonal antibodies

[96] ,

[97] ,

[98] , and

[99] . The preparation of HuMAb mice is described in detail in

[100] ,

[101] ,

[102] ,

[103] , and

[104] . See also

[105] through

[121] . Spleen cells from these transgenic mice can be used to generate hybridomas that secrete human monoclonal antibodies according to well-known techniques.

[0587] In addition, the human antibodies of the present invention or the antibodies of the present invention derived from other species can be identified by display techniques, including but not limited to: phage display, retrovirus display, ribosome display, mammalian display, yeast display, and other techniques known in the art, and the resulting molecules can undergo additional maturation, such as affinity maturation, which is well known in the art.

[0588] Therefore, in another aspect, the present invention relates to a method for producing antibodies according to any aspect or embodiment described herein, comprising the following steps:

[0589] a) Culturing host cells or hybridomas according to any aspect or implementation scheme described herein; and

[0590] b) Purify the antibody from the culture medium.

[0591] Therapeutic applications

[0592] In another aspect, the present invention relates to antibodies, bispecific antibodies or immunoconjugates or ADCs of the present invention as defined in any aspect or embodiment herein, used as pharmaceuticals.

[0593] The anti-AXL antibody of the present invention can be used to treat or prevent diseases involving cells expressing AXL. For example, the antibody can be administered to cells in culture (e.g., in vitro or ex vivo) or to a human individual (e.g., in vivo) to treat or prevent diseases involving cells expressing AXL. As used herein, the term "individual" generally refers to a human to whom the anti-AXL antibody or ADC is administered. An individual may, for example, include a human patient suffering from a disease that can be directly or indirectly corrected or improved by modulating AXL function or by killing cells expressing AXL.

[0594] In one aspect, the present invention relates to antibodies, bispecific antibodies, or immunoconjugates of the present invention as defined in any aspect or embodiment herein for the treatment of cancer.

[0595] In one aspect, the present invention provides a method for modulating AXL-related signaling in cells expressing AXL, said method being achieved by contacting said cells with an anti-AXL antibody or ADC of any aspect or embodiment described herein.

[0596] In one embodiment, the present invention provides a method for killing cells expressing AXL, said method being achieved by contacting said cells with an anti-AXL antibody or ADC of the present invention. Unbound from theoretical constraints, antibody-mediated or ADC-mediated cross-linking or aggregation of AXL molecules on the cell surface (e.g., due to the Fc region of an AXL-bound antibody binding to cells expressing FcR) can lead to apoptosis of said cells.

[0597] In one embodiment, the present invention provides a method for killing cells expressing AXL, the method being achieved by contacting the cells with an AXL-specific antibody or ADC of the present invention in the presence of effector cells (such as ADCC or ADCP) capable of inducing an Fc-dependent cellular response. In this embodiment, the antibody is typically full-length and has an isoform such as, for example, IgG1,κ isoform that induces an ADCC or ADCP response.

[0598] In one embodiment, the present invention provides a method for killing cells expressing AXL, the method being achieved by contacting the cells with an AXL-specific antibody or ADC of the present invention in the presence of a complement protein (such as a complement protein present in normal human serum), which can be activated and thereby induce CDC upon binding of the AXL-specific antibody or ADC to the plasma membrane of the AXL-expressing cells. In this embodiment, the antibody is typically full-length and has an isoform capable of inducing complement system activation, such as, for example, the IgG1,κ isoform.

[0599] The anti-AXL antibody of the present invention is characterized in that it is internalized after binding to AXL, making it suitable for ADC methods using ADCs as described in any aspect or embodiment herein.

[0600] Therefore, in one embodiment, the present invention provides a method for killing cells expressing AXL, the method being achieved by contacting the cells with an ADC of the present invention, the ADC being internalized and delivered to liposomes for specific (i.e., cleavable linker) or non-specific (non-cleavable linker) protein hydrolysis of the anti-AXL antibody-linker-drug complex.

[0601] In one embodiment, the present invention relates to a method for interfering with the regulation of AXL-mediated intrinsic or adaptive immune responses, said method being achieved, for example, by binding an AXL-specific antibody or ADC according to the invention to macrophages, dendritic cells or NK cells expressing AXL.

[0602] In another embodiment, the present invention provides a method for killing cells expressing AXL, said method being achieved by contacting said cells with an ADC of the present invention, wherein said anti-AXL antibody is linked to a treatment site via a linker, thereby allowing drug release once said ADC is internalized, for example by a pH change or reducing conditions. Suitable linker technologies are known in the art, as described above.

[0603] In another aspect, the present invention provides a method for treating or preventing diseases involving cells expressing AXL in an individual, the method comprising administering to the individual in need a therapeutically effective amount of the anti-AXL antibody, bispecific antibody, or ADC of the present invention. The method generally involves administering to the individual an amount of the anti-AXL antibody, bispecific antibody, or ADC according to the present invention in a dose effective in treating or preventing the disease.

[0604] In specific terms, prophylactic administration of anti-AXL antibodies or ADCs can reduce the risk of developing cancer, delay the onset of cancer progression events, or reduce the risk of recurrence when cancer is in remission and / or the primary tumor has been surgically removed. In the latter case, the anti-AXL antibody may be administered, for example, in conjunction with surgery (i.e., before, during, or after surgery). Prophylactic administration can also be used in patients in whom tumors are difficult to locate, and which are believed to be present due to other biological factors.

[0605] Cells with high AXL expression (such as overexpression or aberrant expression of AXL, as found in some cancer cells) are particularly good targets for the anti-AXL antibody, bispecific antibody, or ADC of the present invention because more antibody or ADC / tumor cells can bind. Heterogeneously AXL-expressing tissues, such as tumor tissues, may also be suitable targets for the anti-AXL antibody, bispecific antibody, ADC, or anti-AXL-ADC of the present invention. Thus, in one aspect, the disease involving AXL-expressing cells is cancer, i.e., a tumorigenic disease, such as a disease characterized by the presence of tumor cells expressing AXL, including, for example, diseases in which said cells are derived from solid tumors or hematologic malignancies. AXL expression has been described, for example, in non-small cell lung cancer (NSCLC;

[122] ), pancreatic cancer

[123] , esophageal cancer

[124] , and endometrial cancer

[125] .

[0606] Therefore, exemplary cells expressing AXL include cancer cells, such as those from non-small cell lung cancer, pancreatic cancer, and esophageal cancer.

[0607] In one aspect, the present invention provides a method for treating or preventing cancer, comprising administering to an individual in need an antibody, bispecific antibody, immunoconjugate, composition, or pharmaceutical composition according to the invention.

[0608] In one embodiment, the cancer is a solid tumor expressing AXL or a hematologic malignancy expressing AXL. In one embodiment, the hematologic malignancy is acute myeloid leukemia (AML). In one embodiment, the solid tumor expressing AXL is lung cancer or epidermoid carcinoma.

[0609] Therefore, the present invention relates to a method comprising administering a therapeutically effective amount of the anti-AXL antibody or ADC of the present invention to an individual in need.

[0610] In one aspect, the present invention relates to a method for inhibiting the growth and / or proliferation of tumor cells expressing AXL, comprising administering to an individual in need an antibody, bispecific antibody, immunoconjugate, composition, or pharmaceutical composition according to any aspect or embodiment described herein.

[0611] In one aspect, the present invention relates to a method for inhibiting the migration and / or invasion of tumor cells expressing AXL, comprising administering to an individual in need an antibody, bispecific antibody, immunoconjugate, composition, or pharmaceutical composition according to any aspect or embodiment described herein.

[0612] In one aspect, the present invention relates to a method for inhibiting resistance to targeted therapies (such as EGFR- or BRAF-targeted therapies) or to chemotherapeutic agents, comprising administering to an individual in need an antibody, bispecific antibody, immunoconjugate, composition, or pharmaceutical composition according to any aspect or embodiment described herein.

[0613] In one aspect, the present invention relates to a method for targeting or inhibiting tumor-associated macrophages, comprising administering to an individual in need an antibody, bispecific antibody, immunoconjugate, composition, or pharmaceutical composition according to any aspect or embodiment described herein.

[0614] In one aspect, the present invention provides a method for treating or preventing solid tumors, the method comprising administering a therapeutically effective amount of the present invention's anti-AXL antibody or ADC to an individual in need, wherein said solid tumor is melanoma, malignant tumor, sarcoma, adenoma, and / or glioma. In one embodiment, the cancer is selected from: endometrial / cervical cancer, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), thyroid cancer, colon cancer, kidney cancer, ovarian cancer, breast cancer, esophageal cancer, skin cancer, malignant melanoma, and pancreatic cancer.

[0615] In one embodiment, the cancer is pancreatic cancer, such as unresectable advanced or metastatic pancreatic cancer. In other separate and specific embodiments, the cancer is endometrial / cervical cancer or lung cancer. In one embodiment, the cancer is thyroid cancer. In one embodiment, the cancer is colon cancer. In one embodiment, the cancer is kidney cancer. In one embodiment, the cancer is ovarian cancer. In one embodiment, the cancer is breast cancer, such as estrogen receptor alpha-negative or estrogen receptor alpha-positive cancer. In one embodiment, the cancer is triple-negative breast cancer (i.e., breast cancer that is negative for all three of the following: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)). In one embodiment, the cancer is esophageal cancer. In one embodiment, the cancer is skin cancer. In one embodiment, the cancer is melanoma, such as malignant melanoma. In one embodiment, the cancer is acute myeloid leukemia (AML). In one embodiment, the cancer is resistant to chemotherapy, tyrosine kinase inhibitors, and / or BRAF inhibitors. In one embodiment, the cancer is resistant to EGFR-targeted therapy.

[0616] In one aspect, the present invention relates to antibodies that bind to extracellular domains of AXL (such as the Ig1-like domain, the Ig2-like domain, the FN1 domain, or the FN2 domain of AXL), wherein said antibody is used as a drug.

[0617] In a specific embodiment, the antibody comprises at least one binding region, the at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2 and CDR3 sequences having SEQ ID No: 36, 37 and 38 respectively; and the VL region comprises sequences having SEQ ID No: 39, GAS and 40, wherein the antibody is used as a drug.

[0618] In a specific implementation, the antibody comprises at least one binding region, the at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2 and CDR3 sequences having SEQ ID No: 36, 37 and 38 respectively; and the VL region comprises sequences having SEQ ID No: 39, GAS and 40, wherein the antibody is used to treat or prevent cancer.

[0619] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is used to treat or prevent thyroid cancer.

[0620] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is used to treat or prevent colon cancer.

[0621] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is used to treat or prevent renal cell carcinoma.

[0622] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is used to treat or prevent ovarian cancer.

[0623] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is used to treat or prevent breast cancer.

[0624] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is used to treat or prevent triple-negative breast cancer.

[0625] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is used to treat or prevent esophageal cancer.

[0626] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is used to treat or prevent skin cancer.

[0627] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is used to treat or prevent melanoma such as malignant melanoma.

[0628] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is used to treat or prevent lung cancer (e.g., small cell lung cancer or non-small cell lung cancer (NSCLC)).

[0629] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is used to treat or prevent acute myeloid leukemia (AML).

[0630] This document provides implementation schemes for treating or preventing cancer. These schemes may be coupled to or linked to cytotoxic agents to enhance the efficacy or effect of the treatment.

[0631] In a specific aspect of the invention, the antibodies disclosed herein may be linked to cytotoxic agents such as auristatin (e.g., monomethylaurestatin E (MMAE)) to form immunoconjugates for use as medicines, such as for treating or preventing cancer.

[0632] In one aspect, the present invention relates to immunoconjugates comprising antibodies that bind to AXL, wherein the antibodies do not compete with the ligand Gas6 for binding to AXL.

[0633] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2 and CDR3 sequences having SEQ ID No: 36, 37 and 38 respectively; and the VL region comprises sequences having SEQ ID No: 39, GAS and 40, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used as a drug.

[0634] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0635] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of thyroid cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0636] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of colon cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0637] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of renal cell carcinoma. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0638] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of ovarian cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0639] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of breast cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0640] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of triple-negative breast cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0641] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of lung cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0642] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of esophageal cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0643] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of melanoma such as malignant melanoma. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0644] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of lung cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0645] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 36, 37, and 38, respectively; and the VL region comprises sequences having SEQ ID Nos: 39, GAS, and 40, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used to treat or prevent acute myeloid leukemia. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0646] An embodiment is provided herein, wherein the antibody is linked to a cytotoxic agent (such as auratestatin) to form an immunoconjugate. The antibody and the cytotoxic agent can be coupled via maleimide hexanoyl-valine-citrulline- p -Aminobenzyloxycarbonyl (mc-vc-PAB) connector connection.

[0647] In another aspect of the invention, the invention relates to antibodies that bind to extracellular domains of AXL (such as the Ig2-like domain of AXL), wherein said antibodies are used as drugs.

[0648] In a specific embodiment, the antibody comprises at least one binding region, the at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and the VL region comprises sequences having SEQ ID No: 49, AAS and 50, wherein the antibody is used as a drug.

[0649] In a specific implementation, the antibody comprises at least one binding region, the at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and the VL region comprises sequences having SEQ ID No: 49, AAS and 50, wherein the antibody is used to treat or prevent cancer.

[0650] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is used to treat or prevent thyroid cancer.

[0651] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is used to treat or prevent colon cancer.

[0652] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is used to treat or prevent renal cell carcinoma.

[0653] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is used to treat or prevent ovarian cancer.

[0654] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is used to treat or prevent breast cancer.

[0655] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is used to treat or prevent triple-negative breast cancer.

[0656] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is used to treat or prevent esophageal cancer.

[0657] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is used to treat or prevent skin cancer.

[0658] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is used to treat or prevent melanoma such as malignant melanoma.

[0659] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is used to treat or prevent lung cancer (e.g., small cell lung cancer or non-small cell lung cancer).

[0660] In one embodiment, the antibody comprises at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is used to treat or prevent acute myeloid leukemia (AML).

[0661] This document provides implementation schemes for treating or preventing cancer. These schemes may be coupled or linked to cytotoxic agents to enhance the efficacy or effect of the treatment.

[0662] In a specific aspect of the invention, the antibodies disclosed herein can be linked to cytotoxic agents such as auristatin (e.g., monomethylaurestatin E (MMAE)) to form immunoconjugates for use as medicines, such as for treating or preventing cancer.

[0663] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and the VL region comprises sequences having SEQ ID No: 49, AAS and 50, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used as a drug.

[0664] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2 and CDR3 sequences having SEQ ID No: 46, 47 and 48 respectively; and the VL region comprises sequences having SEQ ID No: 49, AAS and 50, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of cancer.

[0665] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of thyroid cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0666] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of colon cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0667] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of renal cell carcinoma. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0668] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of ovarian cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0669] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of breast cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0670] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of triple-negative breast cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0671] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of lung cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0672] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of esophageal cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0673] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of melanoma such as malignant melanoma. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0674] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of lung cancer. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0675] In one embodiment, the present invention relates to an immunoconjugate comprising an antibody comprising at least one binding region comprising a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 sequences having SEQ ID Nos: 46, 47, and 48, respectively; and the VL region comprises sequences having SEQ ID Nos: 49, AAS, and 50, wherein the antibody is linked to monomethylaurestatin E to form an immunoconjugate, and the immunoconjugate is used for the treatment or prevention of acute myeloid leukemia. In one embodiment, the antibody is linked to monomethylaurestatin E via the linker mc-vc-PAB.

[0676] An embodiment is provided herein in which the antibody is linked to a cytotoxic agent (such as aurestatin) to form an immunoconjugate. The antibody and the cytotoxic agent are linked via a maleimide hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PAB) linker.

[0677] In implementations, patients selected for anti-AXL antibody treatment are based on the level of AXL expression in a sample (such as a sample containing tumor cells), or by detecting AXL-expressing tumors using labeled anti-AXL antibodies or antibody fragments (such as those of the present invention). Exemplary diagnostic assays for determining AXL expression using the AXL antibodies of the present invention are described herein. Effective doses and dosing regimens of anti-AXL antibodies or ADCs depend on the disease or condition to be treated and can be determined by those skilled in the art.

[0678] Physicians with ordinary skill in the art can easily determine and prescribe the effective amount of the desired pharmaceutical composition. Therefore, when referring to a pharmaceutical composition, it should be understood that such compositions are also included, or vice versa. For example, regarding the dosage of anti-AXL antibody used in a pharmaceutical composition, a physician may start below the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally, the appropriate dosage of the pharmaceutical composition of the present invention will be the amount of the lowest dose of compound that effectively produces a therapeutic effect according to a specific dosage regimen. Such an effective dosage will generally depend on the factors described above.

[0679] For example, a therapeutically effective amount can be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer can be evaluated, for example, in animal model systems to predict its efficacy in human tumors. Alternatively, the properties of a compound can be evaluated by examining its ability to inhibit cell growth or induce cytotoxicity using in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise improve an individual's symptoms. Those skilled in the art will be able to determine such amounts based on factors such as individual size, the severity of individual symptoms, and the specific composition or route of administration chosen.

[0680] Exemplary, non-limiting ranges for the therapeutically effective amount of the anti-AXL antibody of the present invention are about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, such as about 0.1-20 mg / kg, such as about 0.1-10 mg / kg, such as about 0.5, about 0.3, about 1, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg.

[0681] Exemplary, non-limiting ranges for the therapeutically effective amount of the anti-AXL ADC of the present invention are about 0.02-100 mg / kg, such as about 0.02-30 mg / kg, for example about 0.05-10 mg / kg or 0.1-3 mg / kg, for example about 0.5-2 mg / kg.

[0682] The application can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, and may be applied near the target site.

[0683] Dosing regimens for the above treatment methods and uses can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus injection can be administered, several fractional doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation.

[0684] In one implementation, the efficacy-safety window is optimized by reducing specific toxicities, such as by reducing the drug-antibody ratio (DAR) and / or by mixing the anti-AXL ADC with an unlabeled anti-AXL antibody.

[0685] In one embodiment, the efficacy of the treatment is monitored during treatment (e.g., at predetermined time points). In one embodiment, efficacy can be monitored by measuring the level of AXL in a sample containing tumor cells; by visualizing the disease area; or by other diagnostic methods further described herein, such as by performing one or more PET-CT scans, for example, using a labeled anti-AXL antibody, a fragment, or a mini-antibody derived from the AXL-specific antibody of the present invention.

[0686] If desired, the effective daily dose of the pharmaceutical composition may be administered throughout the day in two, three, four, five, six or more fractional doses (optionally, in unit dosage form) at appropriate intervals. In another embodiment, the anti-AXL antibody may be administered by a slow, continuous infusion over a prolonged period (e.g., more than 24 hours) to minimize any undesirable side effects.

[0687] Although the compounds of the present invention may be administered alone, they are preferably administered in the form of a pharmaceutical composition as described above.

[0688] An effective dose of the anti-AXL antibody, bispecific antibody, or ADC of the present invention can also be administered using weekly, bi-weekly, or bi-weekly dosing periods. The dosing period may be limited, for example, to 8 weeks, 12 weeks, or until clinical progress has been established.

[0689] For example, in one embodiment, the anti-AXL antibody, bispecific antibody, or ADC is administered at a concentration of 10 to 500 mg / m². 2 such as 200 to 400 mg / m 2 The weekly dose is administered by infusion. Such administration may be repeated, for example, 1 to 8 times, or 3 to 5 times. The administration may be carried out by continuous infusion over a period of 1 to 24 hours, or 1 to 12 hours.

[0690] In another embodiment, the anti-AXL antibody, bispecific antibody, or ADC is administered at a dose of 10 to 500 mg / m² every three weeks. 2 such as 50-200 mg / m 2 The dose is administered by infusion. Such administration may be repeated, for example, 1 to 8 times, or 3 to 5 times. The administration may be carried out by continuous infusion over a period of 1 to 24 hours, or 1 to 12 hours.

[0691] In one embodiment, the anti-AXL ADC is administered weekly or every three weeks at a single dose of about 0.1-10 mg / kg, such as about 1-3 mg / kg, up to twelve times, up to eight times, or until clinical progression. The administration can be carried out over a period of 1 to 24 hours, such as 1 to 12 hours, via continuous infusion. If necessary, such a regimen can be repeated once or more after, for example, 6 months or 12 months. The dosage can be determined or adjusted, for example, by collecting biological samples and using an anti-idiotype antibody targeting the antigen-binding region of the anti-AXL antibody of the present invention, by measuring the amount of the compound of the present invention in the blood at the time of administration.

[0692] In one implementation, the anti-AXL antibody is administered as maintenance therapy, such as, for example, once weekly for six months or longer.

[0693] As a non-limiting example, the treatment according to the invention can provide a daily dose of the compound of the invention of about 0.1-100 mg / kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg. mg / kg / day, at least one of the following dates after the start of treatment: days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40; or as an alternative, at least one of the following dates: weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; or any combination thereof, every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof, using a single dose or fractional dose.

[0694] For ease of administration and uniformity of dosage, parenteral compositions can be formulated in unit dosage form. As used herein, unit dosage form refers to physically discrete units suitable for use as an integrated dosage form for the individual to be treated; each unit contains a calculated predetermined amount of active compound to bind with the desired drug carrier to produce the desired therapeutic effect. The specifications of the unit dosage form of the present invention are limited by and directly depend on: (a) the unique properties of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art in combining such active compounds for individual sensitive treatments.

[0695] Diagnostic applications

[0696] The anti-AXL antibody of the present invention can also be used for diagnostic purposes, using a composition comprising the anti-AXL antibody as described herein. Therefore, the present invention provides diagnostic methods and compositions using the anti-AXL antibody described herein. Such methods and compositions can be used for purely diagnostic purposes, such as detecting or identifying diseases involving cells expressing AXL, and for monitoring the progress of therapeutic treatment, monitoring disease progression, assessing post-treatment status, monitoring disease relapse, evaluating the risk of disease development, and so on.

[0697] In one aspect, the anti-AXL antibody of the present invention is used in vitro for diagnosing diseases in which AXL-expressing cells indicate disease or are involved in the pathogenesis, such as by detecting AXL levels in samples taken from patients or the levels of cells expressing AXL on their cell surface. This can be achieved, for example, by contacting the sample to be tested (optionally along with a control sample) with the anti-AXL antibody under conditions that allow the antibody to bind to AXL. Complex formation can then be detected (e.g., using an ELISA). When the control sample is used along with the test sample, the levels of anti-AXL antibody or anti-AXL antibody-AXL complex in both samples are analyzed, and a statistically significantly higher level of anti-AXL antibody or anti-AXL antibody-AXL complex in the test sample indicates a higher AXL level in the test sample compared to the control sample.

[0698] Examples of conventional immunoassays that can use the anti-AXL antibody of the present invention include, but are not limited to: ELISA, RIA, FACS assays, plasma resonance assays, chromatographic assays, tissue immunohistochemistry, Western blotting and / or immunoprecipitation.

[0699] Therefore, in one embodiment, the present invention relates to a method for diagnosing a disease characterized by the involvement or accumulation of cells expressing AXL, the method comprising administering to an individual an antibody, bispecific antibody, immunoconjugate, composition, or pharmaceutical composition according to any aspect or embodiment described herein, optionally wherein the antibody is labeled with a detectable marker, and wherein the amount of cells expressing AXL is associated with or indicates the disease.

[0700] In one embodiment, the present invention relates to a method for detecting the presence of AXL antigen or AXL-expressing cells in a sample, comprising:

[0701] - The sample is contacted with the anti-AXL antibody of the present invention under conditions that allow the anti-AXL antibody to bind to AXL in the sample; and

[0702] - Analyze whether a complex has formed. Typically, the sample is a biological sample. The term "AXL antigen," as used in this context, refers to both soluble and cell-bound AXL antigen.

[0703] In one embodiment, the sample is a tissue sample known or suspected of containing AXL antigen and / or cells expressing AXL. For example, in situ detection of AXL expression can be achieved by removing a histological specimen from a patient and providing such a specimen with the antibody of the present invention. The antibody can be provided by applying it to the specimen or by covering it with the specimen, followed by detection using a suitable method. Subsequently, not only the presence of AXL or AXL-expressing cells can be determined, but also the distribution of AXL or AXL-expressing cells in the examined tissue can be determined (e.g., in the context of assessing cancer cell dispersal). Using the present invention, those skilled in the art will readily understand that any of a wide variety of histological methods (such as staining procedures) can be modified to achieve such in situ detection.

[0704] In the above assay, the anti-AXL antibody can be labeled with a detectable substance to allow the detection of antibodies bound to AXL. Alternatively, the bound (first) anti-AXL antibody can be detected by a second antibody labeled with a detectable substance and binding to the first antibody. Furthermore, in the above assay, diagnostic compositions comprising antibodies or bispecific antibodies according to any aspect or embodiment described herein can be used. Therefore, in one aspect, the present invention relates to diagnostic compositions comprising antibodies or bispecific antibodies according to any aspect or embodiment described herein.

[0705] The level of AXL in a sample can also be estimated using a competitive immunoassay employing AXL standards labeled with a detectable substance and unlabeled anti-AXL antibodies. In this type of assay, the biological sample, labeled AXL standards, and anti-AXL antibodies are combined, and the amount of labeled AXL standards bound to the unlabeled anti-AXL antibodies is measured. The amount of AXL in the biological sample is inversely proportional to the amount of labeled AXL standards bound to the anti-AXL antibodies.

[0706] Labeling suitable for anti-AXL antibodies, secondary antibodies, and / or AXL standards used in in vitro diagnostic techniques includes, but is not limited to, various enzymes, cofactors, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include: horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable cofactor complexes include: streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include: umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine fluorescein, dansyl chloride, and phycoerythrin; examples of luminescent materials include luminescent ammonia; and examples of suitable radioactive materials include: 125 I, 131 I, 35 S and 3 H.

[0707] In one aspect, the anti-AXL antibody of the present invention is used for in vivo imaging of tissues (such as tumors) expressing AXL. For in vivo methods, antibody fragments such as (Fab')2, Fab, and Fab' fragments are particularly advantageous due to their rapid distribution kinetics.

[0708] In vivo imaging can be performed using any suitable technique. For example, using... 99 Tc, 131 I, 111 Anti-AXL antibodies labeled with In or other gamma-ray emitting isotopes (e.g., fragments) can be used to image the accumulation or distribution of anti-AXL antibodies in tissues expressing AXL (e.g., tumors), employing a gamma scintillation camera (e.g., the Elscint Apex 409ECT device), typically using a low-energy, high-resolution collimator or a low-energy universal collimator. Alternatively, using... 89 Zr、 76 Br、 18 Labeling with F or other positron-emitting radionuclides can be used to image the distribution of anti-AXL antibodies or antibody fragments in tumors using positron emission tomography (PET). Images obtained using such techniques can be used to assess the biodistribution of AXL in patients, mammals, or tissues, for example, in the context of using AXL as a biomarker for the presence of cancer cells. Variations of this technique may include the use of magnetic resonance imaging (MRI) to obtain images superior to gamma camera techniques. Conventional immunoscintillation methods and principles are described, for example, in

[126] ,

[127] , and

[128] . Furthermore, such images can, or optionally, serve as the basis for surgical techniques for tumor removal. In addition, such in vivo imaging techniques can allow for the identification and localization of tumors in cases where a patient is identified as having a tumor (due to the presence of other biomarkers, metastases, etc.) but the tumor cannot be identified by conventional analytical techniques. All of these methods are features of the present invention.

[0709] The in vivo imaging and other diagnostic methods provided by this invention are particularly useful for detecting micrometastases in human patients (e.g., patients who have not been previously diagnosed with cancer or patients in the recovery / remission phase of cancer).

[0710] In one embodiment, the present invention provides an in vivo imaging method in which the anti-AXL antibody of the present invention is conjugated to a radiopaque reagent that facilitates detection, the conjugated antibody is administered to a host (e.g., by injection into the bloodstream), and the presence and location of the labeled antibody in the host are determined. Through this technique, and any other diagnostic methods provided herein, the present invention provides methods for screening the presence of disease-related cells in human patients or biological samples taken from human patients and / or for assessing the distribution of anti-AXL antibodies prior to anti-AXL ADC therapy.

[0711] For diagnostic imaging, radioisotopes can be directly or indirectly bound to anti-AXL antibodies using intermediate functional groups. Available intermediate functional groups include chelating agents such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (see, for example,

[129] ).

[0712] In addition to radioisotopes and radiopaque reagents, diagnostic methods may also utilize anti-AXL antibodies conjugated to dyes (such as biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancement agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions) (see, for example,

[130] , which describes MRI techniques and the preparation of antibodies conjugated to MRI enhancement agents). Such diagnostic / detection reagents may be selected from reagents and fluorescent compounds used for MRI. To attach a radioactive metal or paramagnetic ion to an anti-AXL antibody, it may be necessary to react it with a reagent having a long tail with various chelating groups attached for binding the ion. Such a tail may be a polymer such as polylysine, a polysaccharide, or another derived or derivatizable chain with side groups that may bind chelating groups such as, for example, porphyrins, polyamines, crown ethers, thioureas, polyoximes, etc., known to be suitable for this purpose. Standard chemistry can be used to link the chelate to the anti-AXL antibody.

[0713] Therefore, the present invention provides a diagnostic anti-AXL antibody, wherein the anti-AXL antibody is conjugated to a contrast agent (such as a contrast enhancer for magnetic resonance imaging, computed tomography or ultrasound) or a radionuclide (which may be, for example, γ-, β-, α-, Auger electron-, or positron-emitting isotopes).

[0714] In another aspect, the present invention relates to a kit for detecting the presence of AXL antigen or AXL-expressing cells in a sample, comprising:

[0715] - The anti-AXL antibody, bispecific antibody, or immunoconjugate or ADC of the present invention; and

[0716] - Instructions for use of the kit.

[0717] In one embodiment, the present invention provides a kit for diagnosing cancer, comprising: a container containing an anti-AXL antibody and one or more reagents for detecting the binding of the anti-AXL antibody to AXL. The reagents may include, for example, fluorescent tags, enzyme tags, or other detectable tags. The reagents may also include a second or third antibody or reagent for an enzyme reaction, wherein the enzyme reaction produces a visualized product. In one embodiment, the present invention provides a diagnostic kit comprising, in a suitable container, one or more anti-AXL antibodies of the present invention in labeled or unlabeled form, an incubation reagent for indirect assay, and a substrate or derivatizing reagent (depending on the nature of the label) for detection in such assays. A control reagent and instructions for use may also be included.

[0718] Diagnostic kits can also be provided for use with anti-AXL antibodies (such as conjugated / labeled anti-AXL antibodies) to detect the presence of AXL in tissue samples or the host. Therefore, the anti-AXL antibody according to the invention can also be used, for example, as part of a companion diagnostic, such as as a first antibody in immunohistochemical assays (designed to detect AXL expression in biopsies of solid tumors, lymph nodes, or other tissues). Alternatively, the anti-AXL antibody according to the invention can be used as a first antibody in flow cytometry or immunocytochemical assays to identify AXL-expressing cells in blood, bone marrow, fine needle aspirates (e.g., lymph node aspirates), or peritoneal fluid to identify AXL-expressing tumor cells. The anti-AXL antibody according to the invention can be used, for example, to identify soluble AXL in ELISA-based assays. The anti-AXL antibody according to the invention can be used as a companion diagnostic, such as as a radioconjugate, which can be used for imaging studies of patients. In such diagnostic kits and in therapeutic kits described elsewhere herein, anti-AXL antibodies are typically provided in a container, either alone or in combination with additional antibodies specific to target cells or peptides, in lyophilized form. Typically, pharmaceutically acceptable carriers (e.g., inert diluents) and / or components thereof, such as Tris, phosphate or carbonate buffers, stabilizers, preservatives, biocides, inert proteins such as serum albumin (typically in a separate container for mixing), and additional reagents (also typically in a separate container) are also included. In some kits, a second antibody capable of binding to an AXL-specific Ab (typically provided in a separate container) is also included. This second antibody is typically conjugated to a label and formulated in a manner similar to the anti-AXL antibody of the present invention. Using the methods described above and elsewhere herein, anti-AXL antibodies can be used to define subsets of cancer / tumor cells and characterize such cells and associated tumor tissues.

[0719] Anti-idiotype antibodies

[0720] In another aspect, the present invention relates to anti-idiotype antibodies that combine with the anti-AXL antibody of the present invention as described herein.

[0721] Anti-idiotype (Id) antibodies are antibodies that recognize unique determinants typically associated with the antigen-binding site of the antibody. Anti-Id antibodies can be prepared by immunizing animals of the same species and genotype as the source of anti-AXL monoclonal antibodies, wherein the monoclonal antibody is targeted by the prepared anti-Id. The immunized animals can recognize and respond to the idiotype determinants of the immunizing antibody by producing antibodies against these idiotype determinants (anti-Id antibodies). Such antibodies are described, for example, in US 4,699,880. Such antibodies are a further feature of the present invention.

[0722] Anti-Id antibodies can also be used as “immunogens” to induce an immune response in yet another animal, producing so-called anti-anti-Id antibodies. Anti-anti-Id antibodies may be epitopes identical to the original monoclonal antibody that induces said anti-Id antibody. Therefore, by using antibodies targeting the idiotype determinants of monoclonal antibodies, it is possible to identify other clones expressing antibodies with the same specificity. Anti-Id antibodies can be varied (thus producing anti-Id antibody variants) and / or derived by any suitable technique (such as those described elsewhere herein relative to the AXL-specific antibodies of the present invention). For example, monoclonal anti-Id antibodies can be ligated to a vector such as keyhole hemocyanin (KLH) and used to immunize BALB / c mice. Serum from these mice will typically contain anti-anti-Id antibodies with binding characteristics similar to (if not identical to) the original / parental anti-AXL antibody.

[0723] sequence

[0724]

[0725]

[0726]

[0727]

[0728]

[0729]

[0730]

[0731]

[0732]

[0733]

[0734]

[0735]

[0736]

[0737]

[0738] The present invention is further illustrated by the following embodiments, which should not be construed as further limiting. Example

[0739] Example 1 - Immunization and Generation of AXL Antibodies

[0740] AXL's expression constructs

[0741] The following codon-optimized constructs were generated to express various full-length AXL variants: human (Homo sapiens) AXL (Genbank accession number NP_068713.2), human-cynomolgus monkey chimeric AXL, wherein the human extracellular domain (ECD) is replaced by a cynomolgus monkey (Homo sapiens) chimeric AXL. Macaca fascicularisECD replacement of AXL (translation of Genbank login HB387229.1; aa 1-447), human-mouse chimeric AXL, in which the human ECD is replaced by the mouse (house mouse) AXL ECD (Genbank login NP_033491.2; aa1-441), human-mouse chimeric AXL, in which the human Ig-like domain I (aa 1-134, also referred to as "Ig1 domain" in this paper) is replaced by the mouse AXL Ig-like domain I, human-mouse chimeric AXL, in which the human Ig-like domain II (aa 148-194, also referred to as "Ig2 domain" in this paper) is replaced by the mouse AXL Ig-like domain II, human-mouse chimeric AXL, in which the human FNIII-like domain I (aa 227-329, The FN1 domain (also referred to herein as the "FN1 domain") was replaced by the FNIII-like domain I of mouse AXL, and human-mouse chimeric AXL, in which the human FNIII-like domain II (aa 340-444, also referred to herein as the "FN2 domain") was replaced by the FNIII-like domain II of mouse AXL. Additionally, codon-optimized constructs of various AXL ECD variants were generated: the extracellular domain (ECD) of human AXL (aa 1-447) with a C-terminal His tag (AXLECDHis), the FNIII-like domain II of human AXL (aa 327-447) with an N-terminal signal peptide and a C-terminal His tag (AXL-FN2ECDHis), and the Ig1 and Ig2-like domains of human AXL (aa 1-227) with a C-terminal His tag (AXL-Ig12ECDHis).

[0742] The construct contained restriction sites suitable for cloning and an optimal Kozak (GCCGCCACC) sequence

[141] . The construct was cloned into the mammalian expression vector pcDNA3.3 (Invitrogen).

[0743] AXL expression in EL4 cells

[0744] EL4 cells were stably transfected with the pcDNA3.3 vector containing the complete human AXL coding sequence, and stable clones were selected after selection with the antibiotic reagent G418 (Geneticin).

[0745] Purification of His-tagged AXL

[0746] AXLECDHis, AXL-FN2ECDHis, and AXL-Ig12ECDHis were expressed in HEK-293F cells. The His tagging enabled purification using immobilized metal affinity chromatography. In this method, Co... 2+The cations charge the chelating agent immobilized on the chromatography resin. A supernatant containing a His-tagged protein is incubated with the resin in batch mode (i.e., solution). The His-tagged protein binds strongly to the resin beads, while other proteins present in the culture supernatant do not bind or bind weakly compared to the His-tagged protein. After incubation, the beads are recovered from the supernatant and packed into a column. The column is washed to remove weakly bound proteins. Then, a column containing imidazole (which competes with His for binding to Co...) is used. 2+ The elution buffer is used to elute strongly bound His-tagged proteins. The eluent is removed from the proteins by buffer exchange on the desalting column.

[0747] immunity

[0748] Antibodies IgG1-AXL-061, IgG1-AXL-107, IgG1-AXL-183, IgG1-AXL-613, and IgG1-AXL-726 were derived from the following immunizations: HCo12-BalbC (IgG1-AXL-107), HCo17-BalbC (IgG1-AXL-183, IgG1-AXL-726), and HCo20 (IgG1-AXL-061, IgG1-AXL-613) transgenic mice (Medarex, San José, CA, USA) were alternately immunized at 14-day intervals with 20 µg of AXLECDHis protein (IgG1-AXL-511, IgG1-AXL-613, IgG1-AXL-183), 20 µg of AXL-FN2ECDHIS plus 20 µg of AXL-Ig12ECDHis (IgG1-AXL-726), or 20 µg of AXLECDHis protein (IgG1-AXL-511, IgG1-AXL-613, IgG1-AXL-183). Intraperitoneal (IP) immunization was performed using µg AXL-Ig12ECDHis (IgG1-AXL-107), followed by subcutaneous (SC; at the tail root) immunization using the same protein. A total of eight immunizations were administered: four IP immunizations and four SC immunizations. For most immunizations, the first immunization was performed with complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA), while all subsequent immunizations were performed with incomplete Freund's adjuvant (IFA; Difco Laboratories, Detroit, MI, USA). Antibody IgG1-AXL-183 was derived from immunizations performed using the Sigma adjuvant system (Sigma-Aldrich, St. Louis, MO, USA).

[0749] Antibodies IgG1-AXL-137, IgG1-AXL-148, IgG1-AXL-154, IgG1-AXL-171, and IgG1-AXL-733 were derived from the following immunizations: HCo12-BalbC (IgG1-AXL-137, IgG1-AXL-148), HCo17-BalbC (IgG1-AXL-154, IgG1-AXL-733), and HCo20-BalbC (IgG1-AXL-171). Transgenic mice (Medarex, San José, CA, USA) were immunized with 20 µg of AXLECDHis protein in CFA. Subsequently, the mice were immunized intraperitoneally (IP) with full-length human AXL-transfected EL4 cells in PBS at 14-day intervals, and subcutaneously (SC; at the base of the tail) with AXLECDHis protein in IFA.

[0750] Mice with at least two consecutive AXL-specific antibody titers of 200 (serum dilution 1 / 200) or higher, which were detected in the antigen-specific screening FMAT assay described below, were boosted 3–4 days prior to fusion (by intravenous injection of 10 µg of AXL-derived protein in PBS).

[0751] Allogeneic specific screening assay

[0752] The presence of anti-AXL antibodies in the serum of immunized mice or in the culture supernatant of HuMab (human monoclonal antibody) hybridomas or transfected tumors was determined using fluorescence microassay (FMAT; Applied Biosystems, Foster City, CA, USA) as an alloantigen-specific screening assay. Two different assay designs with combinations of 4- or 8-cell-based assays were used for this purpose.

[0753] The 4-cell assay design was used to test serum from immunized mice and as a primary screening test for hybridoma or transfected tumor culture supernatants. In the 4-cell assay design, the binding of human antibodies in the samples to A431 (DSMZ) and MDA-MB-231 cells (both expressing AXL on their cell surface) and to TH1021-AXL (HEK-293F cells transiently expressing full-length human AXL; generated as described above) and HEK293 wild-type cells (negative control, which does not express AXL) was analyzed.

[0754] Hybridoma or transfected tumor culture supernatant samples were subjected to an additional 8-cell assay design. In this 8-cell assay design, human antibodies were analyzed in the samples in relation to TH1021-hAXL (HEK-293F cells transiently expressing human AXL), TH1021-cAXL (HEK-293F cells transiently expressing a human-cynomolgus AXL chimera in which the human ECD has been replaced by a cynomolgus AXL ECD), TH1021-mAXL (HEK-293F cells transiently expressing a human-mouse AXL chimera in which the human ECD has been replaced by a mouse AXL ECD), TH1021-mIg1 (HEK-293F cells transiently expressing human AXL in which the Ig-like domain I has been replaced by the mouse AXL Ig-like domain I), and TH1021-mIg2. The combination of HEK-293F cells transiently expressing human AXL, in which Ig-like domain II was replaced by Ig-like domain II of mouse AXL, TH1021-mFN1 (HEK-293F cells transiently expressing human AXL, in which FNIII-like domain I was replaced by FNIII-like domain I of mouse AXL), TH1021-mFN2 (HEK-293F cells transiently expressing human AXL, in which FNIII-like domain II was replaced by FNIII-like domain II of mouse AXL) and HEK293 wild-type cells (negative control, which do not express AXL).

[0755] Samples were added to cells to allow binding to AXL. HuMab binding was then detected using a fluorescent conjugate (goat anti-human IgG Fcγ-DyLight649; Jackson ImmunoResearch). AXL-specific humanized mouse antibody A0704P (produced in HEK-293F cells) was used as a positive control, while pooled serum from HuMab mice and ChromPure human IgG, whole molecule (Jackson ImmunoResearch), were used as negative controls. Samples were scanned using an Applied Biosystems 8200 Cell Detection System (8200 CDS), and mean fluorescence was used as the data readout. A sample was considered positive when the count was above 50 and the count x fluorescence was at least three times higher than the negative control.

[0756] HuMab hybridoma generation

[0757] HuMab mice with sufficient antigen-specific titers developed (as described above) were sacrificed, and spleens and lymph nodes adjacent to the abdominal aorta and vena cava were collected. Spleen cells and lymph node cells were fused into a mouse myeloma cell line (SP2.0 cells) via electrofusion using a CytoPulse CEEF 50 electrofusion system (Cellectis, Paris, France), essentially according to the manufacturer's instructions. Subcloning of primary wells was then performed using the ClonePix system (Genetix, Hampshire, UK). For this purpose, specific primary well hybridomas were seeded in a semi-solid medium prepared from 40% CloneMedia (Genetix, Hampshire, UK) and 60% HyQ 2x complete medium (Hyclone, Waltham, USA). Subclones were retested according to the antigen-specific binding assay described above and scanned using an IsoCyte system (Molecular Devices, LLC, Sunnyvale, CA). IgG levels were measured using Octet (Fortebio, Menlo Park, USA) to select the optimal production clone / native well for further expansion. The resulting HuMab hybridomas were further expanded and cultured according to standard protocols (e.g., as described in *Current Protocols in Immunology*, edited by Coligan J.E., Bierer, BE, Margulies, DH, Shevach, EM, and Strober, W., John Wiley & Sons, Inc., 2006). The clone obtained by this method was named PC1021.

[0758] Mass spectrometry of purified antibodies

[0759] Hybridoma supernatant containing small 0.8 mL aliquots of sample antibody from 6-well or Hyperflask stages was purified using a PhyTip column packed with G protein resin (PhyNexus Inc., San Jose, USA) on a Sciclone ALH 3000 workstation (Caliper Lifesciences, Hopkinton, USA). The PhyTip column was used according to the manufacturer's instructions, but the buffers were replaced with the following: binding buffer PBS (B. Braun, Medical BV, Oss, Netherlands) and elution buffer 0.1 M glycine-HCl pH 2.7 (Fluka Riedel-de Haën, Buchs, Germany). After purification, the sample was neutralized with 2 M Tris-HCl pH 9.0 (Sigma-Aldrich, Zwijndrecht, Netherlands). Alternatively, in some cases, larger volumes of culture supernatant were purified using protein A affinity column chromatography.

[0760] After purification, the sample was placed in a 384-well plate (Waters, 100 µl square plate, part number 186002631). The sample was deglycosylated overnight at 37°C using N-glycosidase F. DTT (15 mg / ml) (1 µl / well) was added and incubated at 37°C for 1 h. The sample (5 or 6 µl) was desalted on an Acquity UPLC™ column (Waters, Milford, USA) using a BEH300C18, 1.7 µm, 2.1 x 50 mm column at 60°C. MQ water containing 0.1% formic acid (Fluka, catalog number 56302, Buchs, Germany) and LC-MS grade acetonitrile (Biosolve, catalog number 01204101, Valkenswaard, The Netherlands) were used as eluents A and B, respectively. Time-of-flight electrospray ionization mass spectra were recorded online on a microOTOF™ mass spectrometer (Bruker, Bremen, Germany) operating in positive ion mode. Prior to analysis, the 900–3000 m / z scale was calibrated using an ES calibration mixture (Agilent Technologies, Santa Clara, USA). The mass spectra were deconvolved using DataAnalysis™ software v. 3.4 (Bruker), and the maximum entropy algorithm was used to find molecular weights in the range of 5–80 kDa.

[0761] After deconvolution, the heavy and light chain masses of all resulting samples were compared (under reducing conditions) to identify repeat antibodies. In the comparison of heavy chains, possible C-terminal lysine variants were taken into account. This yielded a list of unique antibodies, where unique was defined as a unique combination of heavy and light chains. In cases where repeat antibodies were found, results from other tests were used to determine which antibody was the optimal material for further experiments.

[0762] Sequence analysis of the variable domain of the AXL antibody and its cloning into the expression vector.

[0763] From 0.2 to 5x10 6 Total RNA was prepared from hybridoma cells, and 5'-RACE-complementary DNA (cDNA) was prepared from 100 ng of total RNA using the SMART RACE cDNA amplification kit (Clontech) according to the manufacturer's instructions. VH and VL cloning regions were amplified by PCR and cloned directly into pG1f and pKappa expression vectors within the frame by ligase-free cloning (Aslanidis, C. and PJ de Jong, Nucleic Acids Res 1990;18(20): 6069-74). For each antibody, 12 VL clones and 12 VH clones were sequenced. The resulting sequences are shown in Table 1. CDR sequences were defined according to IMGT

[22] and

[23] . Clones with the correct open reading frame (ORF) were selected for further study and expression. Vectors with all heavy and light chain combinations identified were transiently co-expressed in Freestyle™ 293-F cells using 293fectin.

[0764] For antibodies IgG1-AXL-154, IgG1-AXL-183, and IgG1-AXL-726, the following variants with point mutations in the variable domains were generated: IgG1-AXL-154-M103L, IgG1-AXL-183-N52Q, and IgG1-AXL-726-M101L. Mutants were generated through site-directed mutagenesis using the Quickchange II mutagenesis kit (Stratagene).

[0765] AXL control antibody

[0766] In some embodiments, a comparative antibody against AXL (IgG1-YW327.6S2), which has been previously described

[142] and

[143] , was used. The VH and VL sequences of these AXL-specific antibodies were cloned into pG1f and pKappa expression vectors.

[0767] b12 antibody

[0768] In some embodiments, antibody b12 (a gp120-specific antibody

[144] ) is used as a negative control.

[0769] Express

[0770] The antibody is designated IgG1,κ. Essentially as the manufacturer describes, a mixture of plasmid DNA encoding both the heavy and light chains of the antibody is transiently transfected into FreestyleHEK293F cells (Invitrogen, US) using 293fectin (Invitrogen, US).

[0771] Antibody purification

[0772] The culture supernatant was filtered through a 0.2 µm terminal filter, loaded onto a 5 mL MabSelect SuRe column (GE Health Care), and eluted with 0.1 M sodium citrate-NaOH, pH 3. The eluent was immediately neutralized with 2 M Tris-HCl, pH 9, and dialyzed overnight to 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B. Braun). Alternatively, after purification, the eluent was loaded onto a HiPrep desalting column and the antibody was exchanged to 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B. Braun) buffer. After dialyzing or buffer exchange, the sample was sterilely filtered through a 0.2 µm terminal filter. Purity was determined by SDS-PAGE, and IgG concentration was measured using Octet (Fortebio, Menlo Park, USA). The purified antibody was stored at 4 °C.

[0773] Antibody IgG1-AXL-511 is generated by the following method:

[0774] AXL's expression constructs

[0775] The following codon-optimized constructs were generated to express various full-length AXL variants: human (Homo sapiens) AXL (Genbank accession number NP_068713.2), human-cynomolgus monkey chimeric AXL, wherein the human extracellular domain (ECD) is replaced by a cynomolgus monkey (Homo sapiens) chimeric AXL. Macaca fascicularisECD replacement of AXL (translation of Genbank login HB387229.1; aa 1-447), human-mouse chimeric AXL, in which the human ECD is replaced by the mouse (house mouse) AXL ECD (Genbank login NP_033491.2; aa 1-441), human-mouse chimeric AXL, in which the human Ig-like domain I (aa 1-147, also referred to as "Ig1 domain" in this paper) is replaced by the mouse AXL Ig-like domain I, human-mouse chimeric AXL, in which the human Ig-like domain II (aa 148-227, also referred to as "Ig2 domain" in this paper) is replaced by the mouse AXL Ig-like domain II, human-mouse chimeric AXL, in which the human FNIII-like domain I (aa 228-326, ... The FN1 domain (also referred to herein as the "FN1 domain") was replaced by the FNIII-like domain I of mouse AXL, and human-mouse chimeric AXL, in which the human FNIII-like domain II (aa 327-447, also referred to herein as the "FN2 domain") was replaced by the FNIII-like domain II of mouse AXL. Additionally, codon-optimized constructs of various AXL ECD variants were generated: the extracellular domain (ECD) of human AXL (aa 1-447) with a C-terminal His tag (AXLECDHis), the FNIII-like domain II of human AXL (aa 327-447) with an N-terminal signal peptide and a C-terminal His tag (AXL-FN2ECDHis), and the Ig1 and Ig2-like domains of human AXL (aa 1-227) with a C-terminal His tag (AXL-Ig12ECDHis).

[0776] The construct contained restriction sites suitable for cloning and the optimal Kozak (GCCGCCACC) sequence (Kozak et al. (1999) Gene 234: 187-208). The construct was cloned into the mammalian expression vector pcDNA3.3 (Invitrogen).

[0777] AXL expression in EL4 cells

[0778] EL4 cells were stably transfected with the pcDNA3.3 vector containing the full-length human AXL coding sequence, and stable clones were selected after selection with the antibiotic reagent G418 (Geneticin).

[0779] Purification of His-tagged AXL

[0780] AXLECDHis, AXL-FN2ECDHis, and AXL-Ig12ECDHis were expressed in HEK293F cells and purified by immobilized metal affinity chromatography.

[0781] immunity

[0782] Material from four transgenic mice expressing human antibody gene sequences was used to select antibodies. Mice were selected that were immunized with various immunization regimens and exhibited various antibody responses and yielded various antibody numbers from the conventional hybridoma method. Mouse A (3.5% hit in the hybridoma method) was an HCo17-BALB / c transgenic mouse (Bristol-Myers Squibb, Redwood City, CA, USA) that was immunized intraperitoneally (IP) with 20 µg AXL-FN2ECDHIS plus 20 µg AXL-Ig12ECDHis at 14-day intervals and subcutaneously (SC) with the same protein at the base of the tail. A total of eight immunizations were performed: four IP immunizations and four SC immunizations. For most immunizations, the first immunization was performed with complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA), while all subsequent immunizations were performed with incomplete Freund's adjuvant (IFA; Difco Laboratories, Detroit, MI, USA). Mouse B (0% hit in the hybridoma method) was an HCo12 transgenic mouse (Medarex) immunized with 20 µg of AXLECDHis protein using a similar immunization regimen to mouse A. Mouse C (38% hit in the hybridoma method) was an HCo12-BALB / c mouse immunized alternately at 14-day intervals via intraperitoneal (IP) immunization with full-length human AXL-transfected EL4 cells in PBS and subcutaneous (SC; at the tail base) immunization with AXLECDHis protein in IFA. Mouse D (0% hit in the hybridoma method) was an HCo12 transgenic mouse (Medarex) immunized with 20 µg of AXL-Ig12ECDHis protein using a similar immunization regimen to mouse A.

[0783] At least two mice with consecutive AXL-specific antibody titers of 200 (serum dilution 1 / 200) or higher were boosted 3-4 days before fusion (10 µg of AXL-derived protein was injected intravenously in PBS).

[0784] RNA was isolated from spleen cells.

[0785] Total RNA was isolated from spleen cells using the Mini RNA Simple Kit (Qiagen). First-strand cDNA of the 5'-RACE region was synthesized using the SMART RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) with 150 ng of RNA, PrimeScript reverse transcriptase (Clontech), and SMARTIIA oligo and oligodT as primers. The VL coding region was amplified by PCR using Advantage 2 polymerase (Clontech), primers RACEkLIC4shortFW2 (320 nM), RACEkLIC4LongFW2 (80 nM), and RACEkLICRV_PmlA3 (400 nM), for 35 cycles at 95°C for 30 seconds and 68°C for 1 minute. The VH coding region was amplified by PCR using Pfu Ultra II fusion HS DNA polymerase (Stratagene), primers RACEG1LIC3shortFW (320 nM), RACEG1LIC3longFW (80 nM), and RACEG1LIC3RV2 (400 nM), for 40 cycles of the following: 95°C for 20 seconds, 66°C for 20 seconds, and 72°C for 30 seconds, ending with a final extension step of 72°C for 3 minutes. The PCR products encoding VH or VL were separated by agarose gel electrophoresis, and DNA products of the desired size were excised from the gel and purified using the Qiagen MiniElute kit. The VH and VL coding regions amplified by PCR were cloned within the framework into mammalian expression vectors pG1f (containing a DNA sequence encoding the human IgG1 constant region) (for the VH region) and pKappa (containing a DNA sequence encoding the κ light chain constant region) (for the VL region) via ligase-free cloning (Aslanidis, C. and PJde Jong, Nucleic Acids Res 1990;18(20): 6069-74), and single colonies containing a single HC or LC expression vector were obtained in Escherichia coli strain DH5αT1R (Life technologies).

[0786] Primer sequence

[0787]

[0788] LEE PCR

[0789] Linear expression elements (LEEs) are produced by amplifying fragments containing a CMV promoter, an HC or LC coding region, and a polyadenosine monophosphate (PAM) signal from expression plasmids. For this purpose, the amplification of these regions was performed using AccuprimeTaq DNA polymerase (Life Technologies) and primers CMVPf(BsaI)2 and TkpA(BsaI)r, for 35 cycles of the following: 94°C, 45 sec; 55°C, 30 sec; and 68°C, 2 min (LC) or 3 min (HC), using plasmids contained in *E. coli* (strain DH5α) colony material as DNA templates.

[0790] Transient expression in HEK-293 cells

[0791] The antibody was denoted as IgG1,κ. A mixture of plasmid DNA encoding both the heavy and light chains of the antibody was transiently transfected into Freestyle293-F (HEK293F) cells (Lifetechnologies, USA) using 293fectin (Lifetechnologies), essentially as described by Vink, T., et al. (2014) ('A simple, robust and highly efficient transient expression system for producing antibodies', Methods, 65 (1), 5-10).

[0792] For LEE expression of Ab, 1 µl of HC LEE PCR reaction mixture, 1 µl of LC PCR reaction mixture, and 1 µl of 30 ng / µl of the enhancement complex as described in Vink, T., et al. (2014) (which contains a mixture of three expression enhancement plasmids) were mixed and transfected into HEK293F cells in a total volume of 100 µl using 293 fectin as the transfection reagent, according to the manufacturer's (Life Technologies) instructions, using 96-well plates as containers, essentially as described above.

[0793] AXLECDHis ELISA

[0794] Spread 0.5 µg / ml AXLECDHis in phosphate-buffered saline (PBS) at 100 µl / well onto ELISA plates (Greiner, Netherlands) and incubate at room temperature (RT) for 16 hours. Remove the coating solution and block the wells by adding 150 µl PBSTC (PBS containing 0.1% Tween-20 and 2% chicken serum) / well and incubate at RT for 1 hour. Wash the plates three times with 300 µl PBST (PBS containing 0.1% Tween-20) / well and add 100 µl of test solution, then incubate at RT for 1 hour. After washing three times with 300 µl PBST / well, add 100 µl of horseradish peroxidase-conjugated goat anti-human IgG antibody (1 / 3000 dilution) and incubate at RT for 1 hour. After washing three times with 300 µl of PBST per well, 100 µl of ABTS (1 mg / mL) solution was added and the plate was incubated at RT until a sufficient signal was observed. The reaction was stopped by adding 100 µl of 2% oxalic acid solution. The 96-well plate was measured at 405 nm on an ELISA reader.

[0795] Diversity screening

[0796] The antibodies in the samples were analyzed in relation to TH1021-hAXL (HEK293F cells transiently expressing human AXL), TH1021-cAXL (HEK293F cells transiently expressing a human-cynomolgus monkey AXL chimera, in which the human ECD has been replaced by a cynomolgus monkey AXL ECD), TH1021-mAXL (HEK293F cells transiently expressing a human-mouse AXL chimera, in which the human ECD has been replaced by a mouse AXL ECD), TH1021-mIg1 (HEK293F cells transiently expressing human AXL, in which Ig-like domain I has been replaced by mouse AXL Ig-like domain I), TH1021-mIg2 (HEK293F cells transiently expressing human AXL, in which Ig-like domain II has been replaced by mouse AXL Ig-like domain II), and TH1021-mFN1. (HEK293F cells transiently expressing human AXL, in which FNIII-like domain I is replaced by FNIII-like domain I of mouse AXL), TH1021-mFN2 (HEK293F cells transiently expressing human AXL, in which FNIII-like domain II is replaced by FNIII-like domain II of mouse AXL) and the binding of HEK293F cells (negative control, which does not express AXL).

[0797] Samples from LEE expression were added to these cells to allow binding to various AXL constructs. Antibody binding was then detected using a fluorescent conjugate (goat anti-human IgG Fcγ-DyLight649; Jackson ImmunoResearch). Samples were scanned using an Applied Biosystems 8200 Cell Detection System (8200 CDS), and mean fluorescence was used as the data readout. A sample was considered positive when the count was above 50 and the count x fluorescence was at least three times higher than the negative control.

[0798] Preparing HC and LC sets:

[0799] For each mouse, colonies containing the 352 HC expression vector and those containing the 384 LC expression vector were selected and amplified by LEE PCR. A portion of the LEE reaction was sequenced (AGOWA). The percentage of constructs containing the correct VH insert varied considerably among the four mice: mouse A (50%), mouse B (23%), mouse C (90%), and mouse D (14%), and was similar to the differences in hits obtained in the hybridoma approach, see above. HC diversity in mice with only a limited number of correct inserts was dominated by the same large class: 65 / 83 (mouse B) and 46 / 49 (mouse D). For mice B and D, unique HCs were selected (9 for mouse B and 4 for mouse D). For mice A and C, no selection was made.

[0800] Co-transfection of HC and LC sets

[0801] The LEE transfection scheme is used to co-transfect the HC of a single LEE with the LC of 96 LEEs.

[0802] HC selection of antibodies against AXL

[0803] For mice B and D, AXL binding of antibody mixtures generated from the supernatant co-transfected with LEE from a single HC and a pooled LC was analyzed by AXL ELISA. Seven of the nine HCs from mouse B resulted in AXL binding, while all four of the four HCs from mouse D resulted in AXL binding.

[0804] For mice A and C, AXL binding of antibody mixtures generated from LEE co-transfection supernatants of individual HCs and aggregated LCs was analyzed by diversity screening. This screening allowed for the identification of AXL-binding HCs and coarse epitope localization by recognizing the loss of binding to AXL variants. Approximately 40% of HCs from mouse A bound to human AXL, most of which lost binding to the Ig1 or FNIII-2 domains when these domains were replaced by mouse equivalents. Approximately 70% of HCs from mouse C bound to human AXL, most of which lost binding to the Ig1 or Ig2 domains when these domains were replaced by mouse equivalents. Based on binding as determined by AXL ELISA or diversity screening, HC sequence information, and the loss of binding to specific AXL domains in the diversity screening, a total of 12 unique HCs were selected to determine the optimal LC.

[0805] Co-transfection of HC and a single LC

[0806] Each individual HC LEE from the 12 unique selected HCs was co-transfected with 96 individual LC LEEs from the corresponding mouse LC set.

[0807] LC selection of antibodies against AXL

[0808] AXL binding of antibodies generated in the supernatant of LEE expression from a single HC / LC combination was analyzed by AXL ELISA. For each HC, at least six LCs were identified based on ELISA results and LC sequence information, and a single LC was selected as the optimal one. Antibodies binding to AXL were identified from all four mice, even those that were not successful in the hybridoma method.

[0809] Binding affinity of antibody 511

[0810] The affinity of an anti-AXL antibody (clone 511) was determined.

[0811] Affinity was determined using biomembrane interferometry on a ForteBio OctetRED384. hIgG (1 µg / mL) was loaded onto an anti-human Fc capture (AHC) biosensor (ForteBio, Portsmouth, UK; catalog 18-5064) for 150 s, targeting a loading response of 1 nm. Binding (1000 s) and dissociation (2000 s) of AXLECDHis were measured after baseline (150 s) (as described in Example 1) using a 2-fold dilution step, employing concentration ranges from 10 µg / mL to 0.16 µg / mL (218 nM – 3 nM). For calculations, the theoretical molecular mass of AXLECDHis, 46 kDa, based on its amino acid sequence, was used. Experiments were performed on an OctetRED384 with simultaneous shaking at 1000 rpm at 30°C. Each antibody was tested in three independent experiments.

[0812] Data was analyzed using ForteBio data analysis software v7.0.3.1. Unless otherwise specified, a 1:1 mode and global fit were used, with a binding time of 1000 s and a dissociation time of 1000 s. The 1000 s dissociation time (instead of the acquired 2000 s dissociation time) was used because it yielded a better fit. The data trajectory was corrected by subtracting the reference curve (antibody excluding AXLECDHis) to align the Y-axis with the baseline for the last 5 s, and inter-level correction and Savitzky-Golay filtering were applied.

[0813] Clone 511's affinity for AXL (K D ) is 23*10 -9 M (k on 1.7*10 5 1 / Ms and k dis 3.9*10 -3 1 / s).

[0814] Duostatin-3 Synthesis

[0815] Preparation of compound 3:

[0816]

[0817] To 30 mL of a dichloromethane (DCM) solution of Boc-L-phenylalanine 1 (5.36 g, 20.2 mmol), carbonyl diimidazole (CDI, 4.26 g, 26.3 mmol) was added and the mixture was stirred for 1 hour. Then, 15 mL of a DCM solution of Boc-L-phenylalanine 1 (3.67 g, 30.3 mmol) and 2,4-diaminobutyric acid (DBU, 4.5 mL, 30 mmol) was added. The mixture was heated at 40 °C for 16 hours. The mixture was diluted with 60 mL of DCM and 40 mL of water, and then neutralized to pH 7 with concentrated HCl. The DCM extract was collected, washed with 0.2 M HCl (60 mL) followed by brine (60 mL), dried over Na₂SO₄, and evaporated to give 7.47 g of Boc-protected sulfonamide. This material was suspended in 40 mL of methanol, and then 200 mL of 6N HCl / isopropanol was added, and the mixture was stirred for 2 hours. The solvent was evaporated under vacuum, and then 100 mL of ether was added. The precipitate was collected by filtration and dried to give the HCl salt of compound 3 (5.93 g, 96%); MS m / z 269.1 (M+H).

[0818] Preparation of compound 5:

[0819]

[0820] To a 10 mL solution of compound 4 (1.09 g, 1.6 mmol) in N,N-dimethylformamide (DMF), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluranium hexafluorophosphate (HATU, 0.61 g, 1.6 mmol), diisopropylethylamine (DIEA, 0.56 mL), and compound 3 (0.49 g, 1.6 mmol) were added sequentially. The mixture was stirred for 1 hour and diluted with 100 mL of water and 4 mL of acetic acid. The precipitate was collected by filtration, dried under vacuum, and added to 10 mL of 4M HCl / dioxane. After 30 min, 200 mL of ether was added, and the insoluble precipitate was collected and purified by HPLC to give the tetrahydrofuran salt of compound 5 (TFA, 1.3 g, 88%); MS m / z 835.5 (M+H). Throughout the manuscript, compound 5 is referred to as duostatin-3.

[0821] Preparation of compound 7:

[0822]

[0823] Compound 6 (483 mg, 0.631 mmol), N-hydroxybenzotriazole (HOBt, 40 mg, 0.296 mmol), and DIEA (0.27 mL) were added to 5 mL of a DMF solution of compound 5 (500 mg, 0.527 mmol). The mixture was stirred for 16 hours, followed by the addition of 0.4 mL of piperidine. After 1 hour, the mixture was diluted with 100 mL of ether, and the precipitate was collected and dried to give the HCl salt of compound 7 (640 mg, 95%); MS m / z 1240.7 (M+H).

[0824] Preparation of compound 9:

[0825]

[0826] HATU (236 mg, 0.62 mmol), DIEA (0.15 mL), and compound 7 (316 mg, 1.6 mmol) were added to 5 mL of a DMF solution of compound 8 (219 mg, 0.62 mmol). After 1 hour, 0.2 mL of piperidine was added and the mixture was stirred for 30 min. The mixture was then purified by HPLC to obtain the TFA salt of compound 9 (235 mg, 64%); MS m / z 1353.8 (M+H).

[0827] Preparation of compound 11:

[0828]

[0829] To a solution of compound 9 (235 mg, 0.16 mmol) in 2 mL of methanol and 1 mL of water, dialdehyde 10 (0.3 M, in 1.6 mL of iPrOH) and NaCNBH3 (180 mg, 2.85 mmol) were added. The mixture was stirred at RT for 2 hours and subsequently purified by HPLC to obtain the TFA salt of compound 11 (126 mg, 50%); MS m / z 1465.8 (M+H).

[0830] Generate AXL-specific antibody-drug conjugates (ADCs).

[0831] Purified AXL antibodies IgG1-AXL-148, IgG1-AXL-183 and IgG1-AXL-726, as well as negative control antibody IgG1-b12, were covalently coupled to Duostatin-3 by Concortis Biosystems, Inc. (San Diego, CA) using a K-lockAV1-valine-citrulline (vc) linker

[58] ,

[148] and

[149] .

[0832] The concentration of the anti-AXL antibody-drug conjugate (by absorbance at 280 nm), drug-antibody ratio ('DAR') (by reversed-phase HPLC and hydrophobic interaction chromatography (HIC)), amount of unconjugated drug (by reversed-phase chromatography), aggregation percentage (by size exclusion chromatography, SEC-HPLC), and endotoxin level (by LAL) were then analyzed. The results are shown in Table 2:

[0833] Table 2

[0834] IgG1-AXL-148-vcDuostatin3 IgG1-AXL-183-vcDuostatin3 IgG1-AXL-726-vcDuostatin3 IgG1-b12-vcDuostatin3 Concentration (mg / mL) 6.57 3.40 5.93 3.36 DAR (via HIC-HPLC) 1.71 1.79 1.77 2.05 % Unconjugated drugs 6.67 4.16 5.38 4.19 % Aggregates (by SEC-HPLC) 3.71% 3.35 3.42 1.75

[0835] Example 2 - Binding characteristics of AXL antibody

[0836] Binding affinity of AXL antibodies

[0837] Affinities were determined for a group of nine anti-AXL antibodies and three variants of these antibodies (with a single amino acid mutation in the variable domain) (IgG1-AXL-154-M103L, IgG1-AXL-183-N52Q, IgG1-AXL-726-M101L).

[0838] Affinity was determined using biomembrane interferometry on a ForteBio OctetRED384. hIgG (1 µg / mL) was loaded onto an anti-human Fc capture (AHC) biosensor (ForteBio, Portsmouth, UK; catalog 18-5064) for 150 s, targeting a loading response of 1 nm. Binding (1000 s) and dissociation (2000 s) of AXLECDHis were measured after baseline (150 s) (as described in Example 1) using a 2-fold dilution step, employing concentration ranges from 10 µg / mL to 0.16 µg / mL (218 nM – 3 nM). For calculations, the theoretical molecular mass of AXLECDHis, 46 kDa, based on its amino acid sequence, was used. Experiments were performed on an OctetRED384 with simultaneous shaking at 1000 rpm at 30°C. Each antibody was tested in three independent experiments.

[0839] Data was analyzed using ForteBio data analysis software v7.0.3.1. Unless otherwise specified, a 1:1 mode and global fit were used, with binding and dissociation times of 1000 s each. The 1000 s dissociation time (instead of the acquired 2000 s dissociation time) was used to achieve a better fit. For antibodies IgG1-AXL-154 and IgG1-AXL-154-M103L, a dissociation time of 500 s was used. For IgG1-AXL-012 and IgG1-AXL-094, a dissociation time of 200 s was used. The data trajectory was corrected by subtracting the reference curve (antibody excluding AXLECDHis) to align the Y-axis to the baseline for the last 5 s, and inter-level correction and Savitzky-Golay filtering were applied.

[0840] Affinity (K) of anti-AXL antibody D The range is 0.3*10 -9 M to 63*10 -9 M (Table 3). For the mutant IgG1-AXL-183-N52Q, its K D The dissociation rate was lower than that of wild-type IgG1-AXL-183, due to a dissociation rate approximately 2.5 times higher. The kinetics of the other two mutants observed were similar to those of wild-type IgG.

[0841] Table 3

[0842]

[0843] Binding of AXL antibody to human, mouse, and cynomolgus monkey AXL

[0844] HEK293T cells were transiently transfected with expression constructs of full-length human AXL, human AXL with the cynomolgus monkey extracellular domain (ECD), or human AXL with the mouse ECD (see Example 1). The binding of the HuMab-AXL antibody to these cells was evaluated by flow cytometry. Transfected HEK293 cells were incubated at 4°C for 30 min with serially diluted AXL-antibody solutions (final concentrations ranging from 0.0024 to 10 µg / mL). After washing three times in PBS / 0.1% BSA / 0.02% azide, the cells were incubated with goat anti-human IgG F(ab')2 conjugated to R-phycoerythrin (PE) in PBS / 0.1% BSA / 0.02% azide (final volume 100 µL). Next, the cells were washed twice in PBS / 0.1% BSA / 0.02% azide, resuspended in 120 μL PBS / 0.1% BSA / 0.02% azide, and analyzed on FACS Cantoll (BDBiosciences).

[0845] The binding curves were analyzed using nonlinear regression (with variable slope Sigmoidal dose-response) with GraphPad Prism V5.04 software (GraphPad Software, San Diego, CA, USA).

[0846] Figure 1 A showed that the HuMab-AXL antibody bound to HEK293 cells expressing human AXL-ECD in a dose-dependent manner. Furthermore, the HuMab-AXL antibody recognized AXL cells with cynomolgus monkey ECDs, whose ECDs... 50 The value is within the same range as that of a fully developed person (AXL). Figure 1 B). In contrast, HuMab binds poorly to AXL with mouse ECD (IgG1-AXL-107, IgG1-AXL-154, IgG1-AXL-154-M103L, IgG1-AXL-733, IgG1-AXL-183, IgG1-AXL-183-N52Q) or is undetectable (IgG1-AXL-171, IgG1-AXL-613, IgG1-AXL-726, IgG1-AXL-726-M101L, IgG1-AXL-148); Figure 1 C). As expected, the negative control antibody IgG1-b12 did not bind to cells expressing any AXL variant. Figure 1Table 4 shows the EC50 values ​​and standard deviations (determined in at least 3 experiments) for the binding of anti-AXL antibodies to human AXL or human AXL with cynomolgus monkey ECD. The EC50 values ​​for binding to human AXL with mouse AXL ECD could not be determined due to extremely low or no binding.

[0847] Table 4

[0848]

[0849] AXL antibody competitively binds to Gas6.

[0850] The presence or absence of the AXL ligand Gas6 was investigated to determine whether it interfered with the binding of the AXL antibody to AXL. AXL-positive A431 cells were incubated with 10 µg / mL recombinant human Gas6 (R&D Systems, Abingdon, UK; catalog number 885-GS) at 4°C for 15 minutes. Subsequently, serial dilutions of the AXL antibody (final concentrations ranging from 0.014 to 10 µg / mL) were added to the cells and incubated at 4°C for 30 minutes. After washing three times with PBS / 0.1% BSA / 0.02% azide, the cells were incubated with a secondary antibody in 100 μL at 4°C in the dark for 30 minutes. Goat anti-human IgG F(ab')2 (Jackson ImmunoResearch Laboratories, Inc., WestGrove, PA; Catalogue No. 109-116-098), conjugated with R-phycoerythrin (PE) at a 1 / 100 dilution in PBS / 0.1% BSA / 0.02% azide, was used as a secondary antibody to bind to the Fc region. Next, the cells were washed twice in PBS / 0.1% BSA / 0.02% azide, resuspended in 120 μL of PBS / 0.1% BSA / 0.02% azide, and analyzed on FACS Cantoll (BD Biosciences).

[0851] As an alternative, A431 cells were pre-incubated with 10 µg / mL AXL antibody (15 min, 4°C) to assess whether the AXL ligand Gas6 could still bind in the presence of AXL antibody. After antibody pre-incubation, serial dilutions of recombinant human Gas6 (R&D Systems, Abingdon, UK; catalog number 885-GS) were added to the cells at final concentrations ranging from 0.001 to 20 µg / mL and incubated at 4°C for 30 min. After washing three times in PBS / 0.1% BSA / 0.02% azide, the cells were incubated with mouse anti-Gas6 IgG2a (R&D Systems; cat no. MAB885) at 4°C for 30 min. After washing three times in PBS / 0.1% BSA / 0.02% azide, the cells were incubated with FITC-labeled goat anti-mouse IgG (Dako, Heverlee, Belgium; catalog number F049702) at 4°C in the dark for 30 min. Next, the cells were washed twice in PBS / 0.1% BSA / 0.02% azide, resuspended in 120 μL PBS / 0.1% BSA / 0.02% azide, and analyzed on FACS Cantoll (BD Biosciences).

[0852] The binding curves were analyzed using nonlinear regression (with variable slope Sigmoidal dose-response) with GraphPad Prism V5.04 software (GraphPad Software, San Diego, CA, USA).

[0853] In the experiment where A431 cells were pre-incubated with Gas6 (n=3), the maximum binding value of anti-AXL antibody was comparable to that without Gas6 (the maximum binding after Gas6 pre-incubation was 90-108% of the binding without Gas6 pre-incubation) (Table 4). EC2 cells bound by AXL antibody with or without Gas6 pre-incubation. 50 The values ​​were within the same range, or slightly increased after preheating with Gas6 (Table 5).

[0854] In the presence of Gas6, the binding of the control AXL antibody YW327.6S2 to A431 cells was significantly reduced compared to the absence of Gas6. The maximum binding of YW327.6S2 in the presence of Gas6 was only 19% of that without Gas6, while the EC50 value of binding to A431 cells was 21-fold higher after pre-incubation with Gas6.

[0855] Gas6 binding was evaluated in experiments involving pre-incubation of A431 cells with anti-AXL antibody (n=3). Gas6 binding to A431 cells was similar with and without pre-incubation with HuMab-AXL antibody. When cells were pre-incubated with HuMab, the mean EC50 concentration of Gas6 binding (0.34–0.83 µg / mL) and the maximum Gas6 binding were similar to those in the presence of the negative control antibody b12 (EC50 concentration: 0.40 µg / mL; 95–115% of Gas6 binding in the presence of b12 control antibody). Compared to pre-incubation with b12, Gas6 binding to A431 cells was significantly reduced in the presence of the control AXL antibody YW327.6S2 (EC50 concentration 14-fold higher). The maximum Gas6 binding in the presence of the control antibody YW327.6S2 was 17% of the binding in the presence of the negative control antibody b12.

[0856] Table 5

[0857]

[0858] a na, not applicable

[0859] * EC50 values ​​are not very accurate due to low binding.

[0860] Example 3 – Epitope Localization Study of Anti-AXL Antibody Group

[0861] AXL domain specificity was determined using human-mouse AXL chimeric molecules.

[0862] The AXL domain specificity of the AXL antibody was determined using a group of human-mouse chimeric AXL mutants. Five different chimeric AXL molecules were generated, in which human Ig-like domain I (Ig1), Ig-like domain II (Ig2), human FNIII-like domain I (FN1), or human FNIII-like domain II (FN2) was replaced by its mouse homologue.

[0863] The following codon-optimized constructs for expressing the AXL human-mouse chimera were generated and expressed in HEK293F cells as described in Example 1:

[0864] Homo sapiens AXL (p33-HAHs-AXL): (SEQ ID NO:130)

[0865]

[0866] House mouse AXL (p33-HAMm-AXL): (SEQ ID NO:131)

[0867]

[0868] Homo sapiens AXL-mIg1 domain (p33-AXL-mIg1): (SEQ ID NO:132)

[0869]

[0870]

[0871] Homo sapiens AXL-mIg2 domain (p33-AXL-mIg2): (SEQ ID NO:133)

[0872]

[0873] Homo sapiens AXL-mFN1 domain (p33-AXL-mFN1): (SEQ ID NO:134)

[0874]

[0875] Homo sapiens AXL-mFN2 domain (p33-AXL-mFN2): (SEQ ID NO:135)

[0876]

[0877]

[0878] The binding of 1 µg / mL anti-AXL antibody to human-mouse AXL chimeras was determined by flow cytometry, as described in Example 2. IgG1-b12 was included as an isotype control IgG1.

[0879] All anti-AXL antibodies bind to human AXL ( Figure 2 A), while when human AXL ECD was replaced by its mouse homologue, the binding was eliminated or greatly reduced ( Figure 2 B). This included the human-mouse cross-reactive monoclonal AXL antibody YW327.6S2 to confirm the expression of hsAXL-mmECD.

[0880] The binding of anti-AXL antibodies 107 and 613 to hsAXL-mmIg1 was significantly reduced. Figure 2 C) indicates recognition of epitopes within the AXL Ig1 domain. The binding of IgG1-AXL-148 and IgG1-AXL-171 to hsAXL-mmIg2 is significantly reduced ( Figure 2D) indicates recognition of epitopes within the AXL Ig2 domain. The binding of IgG1-AXL-154, IgG1-AXL-183, and IgG1-AXL-733 to hsAXL-mmFN1 is significantly reduced ( Figure 2 E), indicating recognition of epitopes within the AXL FN1 domain. Finally, the binding of IgG1-AXL-726 to hsAXL-mmFN2 is lost ( Figure 2 F) indicates the identification of epitopes within the FN2 structural domain.

[0881] The AXL domain specificity of all anti-AXL antibodies is summarized in Table 6.

[0882] Table 6

[0883] Antibody AXL domain specificity AXL aa involving combination IgG1-AXL-107 Ig1 L121-Q129 IgG1-AXL-148 Ig2 D170-R190 IgG1-AXL-154 Fn1 Q272-A287, G297-P301 IgG1-AXL-154-M103L <![CDATA[n.d. a ]]> nd IgG1-AXL-171 Ig2 P170, T182-R190 IgG1-AXL-183 Fn1 It is still unclear IgG1-AXL-183-N52Q nd nd IgG1-AXL-613 Ig1 T112-Q124 IgG1-AXL-726 Fn2 A359, R386, Q436-K439 IgG1-AXL-726-M101L nd nd IgG1-AXL-733 Fn1 It is still unclear IgG1-AXL-061 Ig1 I97-Q124 IgG1-AXL-137 Ig1 Q57, E92-T105 YW327.6S2 Ig1 G39-D59

[0884] a nd, cannot be determined.

[0885] High-resolution epitope localization to identify amino acids in the extracellular domain of AXL involved in AXL antibody binding.

[0886] To identify amino acids in the AXL extracellular domain involved in anti-AXL antibody binding, an AXL sequence variant library was generated by recombining AXL sequences from species with variable homology levels with human AXL sequences in the extracellular domain. Briefly, an expression plasmid encoding human AXL (Hs) was mixed with cloning plasmids encoding AXL homologs of mice (Mm), opossums (Md; opossum), anoles (Ac; lizard), and pufferfish (Tn; pufferfish), or vice versa. PCR amplification of the AXL extracellular domain (ECD) was performed using a combination of two primers specific to either the cloning or expression vector, employing a shortened extension time in the PCR cycle to force the nascent DNA replication strands to unwind and re-anneal. The full-length ECD was amplified using nested PCR (again, specific to recombinant products containing ends from both vectors).

[0887] The resulting AXL ECD PCR product was cloned into an expression vector that generated full-length AXL, and the resulting plasmids were sequenced and sorted according to the greatest difference from the template vector. The smallest set with the highest resolution was selected. Following the manufacturer's (Life Technologies) instructions, plasmids encoding AXL homologues from Hs, Mm, Md, Ac, and Tn, four human / mouse chimeric plasmids encoding Hs AXL with mouse Ig1, Ig2, Fn1, or Fn2 domains, and sixteen most discriminative plasmids from the recombinant library were transfected into HEK293-F cells. FACS binding data using 1 µg / mL anti-AXL antibody were unconvolved using a score / amino acid (if the mutation was associated with (+1) or not with (-1) binding loss) followed by baseline correction and normalization to a scale of -5 to +5 to obtain the influence score / amino acid across the entire ECD.

[0888] The deconvolution binding data are summarized in Table 6 in the form of the amino acids involved in the binding. Antibodies whose binding sites could not be located at high resolution due to the lack of recombination events near the binding site were marked as unclear.

[0889] Example 4 – Fc-mediated effector function

[0890] Antibody-dependent cell-mediated cytotoxicity (ADCC)

[0891] The ability of anti-AXL antibody to induce ADCC in A431 epidermal-like cancer cells was determined as described below. Peripheral blood mononuclear cells from healthy volunteers (UMC Utrecht, The Netherlands) were used as effector cells.

[0892] Label target cells

[0893] A431 cells (5x10) 6 Cells were collected in culture medium (RPMI 1640 medium supplemented with 10% fetal bovine serum (FSC)) containing 100 µCi. 51 Cr (Chromium-51; Amersham BiosciencesEurope GmbH, Roosendaal, The Netherlands) and incubate the mixture in a 37°C water bath with shaking for 1 hour (hr). After washing the cells (twice, in PBS, 1200 rpm, 5 min), resuspend the cells in RPMI 1640 / 10% FSC and count them using the trypan blue exclusion method. Dilute the cells to 1x10⁻⁶. 5 Density of cells / mL.

[0894] Preparation of effector cells

[0895] Peripheral blood mononuclear cells (PBMCs) were isolated from 45 mL of freshly drawn heparinized blood using Ficoll (Bio Whittaker; Lymphocyte Separation Culture Medium, Catalogue No. 17-829E) from a healthy volunteer (UMC Utrecht, Utrecht, The Netherlands) via Ficoll (Bio Whittaker; Lymphocyte Separation Culture Medium, Catalogue No. 17-829E). After resuspending the cells in RPMI 1640 / 10% FSC, the cells were counted using the trypan blue exclusion assay and diluted to 1 x 10⁻⁶ cells / mL. 7 Density of cells / mL.

[0896] ADCC settings

[0897] 50 µl of 51 Cr-labeled target cells were transferred to 96-well plates, and 50 µl of antibody was added, diluted in RPMI 1640 / 10% FSC (3-fold dilution in the final concentration range of 0.01–10 µg / mL). Cells were incubated at room temperature (RT) for 15 min, and 50 µl of effector cells were added to obtain an effector-to-target ratio of 100:1 (to determine maximum lysis, 100 µl of 5% Triton-X100 was added instead of effector cells; to determine spontaneous lysis, 50 µL of target cells and 100 µL of RPMI 1640 / 10% FSC were used). Cells were incubated overnight at 37°C and 5% CO2. After centrifugation (1200 rpm, 10 min), 70 µL of supernatant was harvested into a microtube and counted using a gamma counter. The percentage of specific lysis was calculated as follows:

[0898] % Specific lysis = (cpm sample - cpm target cells only) / (cpm maximum lysis - cpm target cells only)

[0899] Where cpm stands for count per minute.

[0900] IgG1-AXL-183-N52Q and IgG1-AXL-733 induced 15 to 21% ADCC in A431 cells at a concentration of 10 µg / mL. Figure 3 IgG1-AXL-148, IgG1-AXL-726-M101L, IgG1-AXL-171, IgG1-AXL-613, IgG1-AXL-107, and IgG1-AXL-154-M103L, at concentrations up to 10 µg / mL, failed to induce significant ADCC in A431 cells. Figure 3 ).

[0901] Example 5 – Binding characteristics of AXL antibody-drug conjugate (AXL-ADC)

[0902] HEK293T cells were transiently transfected with a full-length human AXL expression construct (see Example 1). The binding of anti-AXL antibody and AXL-ADC to these cells was evaluated by flow cytometry. Transiently transfected HEK293 cells were incubated at 4°C for 30 min with serial dilutions of anti-AXL antibody or AXL-ADC (4-fold dilution; final concentration range 0.003–10 µg / mL). After washing three times in PBS / 0.1% BSA / 0.02% azide, cells were incubated in 100 μL of a secondary antibody at 4°C in the dark for 30 min. R-phycoerythrin (PE) conjugated to goat anti-human IgG F(ab')2 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; catalog number 109-116-098) diluted 1 / 100 in PBS / 0.1% BSA / 0.02% azide was used as the secondary antibody. Next, the cells were washed twice in PBS / 0.1% BSA / 0.02% azide, resuspended in 120 μL PBS / 0.1% BSA / 0.02% azide, and analyzed on FACS Cantoll (BDBiosciences).

[0903] The binding curves were analyzed using nonlinear regression (with variable slope Sigmoidal dose-response) with GraphPad Prism V5.04 software (GraphPad Software, San Diego, CA, USA).

[0904] Figure 4 The results showed that the binding of anti-AXL antibody to HEK293 cells expressing human AXL-ECD was similar to the binding of AXL-ADC.

[0905] Example 6 – In vitro cytotoxicity induced by AXL-specific antibody-drug conjugate

[0906] LCLC-103H cells (human large cell lung cancer) were cultured in RPMI1640 (Cambrex; catalog number BE12-115F) containing L-glutamine supplemented with 10% (vol / vol) heat-inactivated enhanced fetal bovine serum (Perbio; catalog number SH30087.03), 2 mM L-glutamine (Cambrex; catalog number US17-905C), 50 IU / mL penicillin and 50 μg / mL streptomycin (Cambrex; catalog number DE17-603E). MDA-MB-231 cells (human breast cancer) were cultured in DMEM (Cambrex ... LCLC-103H and MDA-MB-231 cells were cultured to near confluence, then treated with trypsin, resuspended in culture medium, and passed through a cell filter (BD Falcon, catalog number 352340) to obtain a single-cell suspension. 1 x 102 cells were seeded into each well of a 96-well plate. 3 Cells were collected and incubated at room temperature for 30 min, followed by incubation at 37°C and 5% CO2 for 5 hours to allow them to adhere to the plate.

[0907] A series of dilutions (4-fold; final concentration range 0.00015 to 10 µg / mL) of the AXL antibody-drug conjugate (AXL-ADC; see Example 1) were prepared in culture medium and added to plates. Cells incubated with 1 µM astrococcus (#S6942-200, Sigma) were used as a reference for 100% tumor killing. Untreated cells were used as a reference for 0% tumor cell killing. The plates were incubated at 37°C and 5% CO2 for 5 days. Next, CellTiter-Glo reagent (Promega; catalog number G7571) (20 µL / well) was added to the wells, and the plates were incubated at 37°C and 5% CO2 for 1.5 hours. Subsequently, 180 µL / well was transferred to white 96-well Optiplate™ plates (PerkinElmer, Waltham, MA; catalog number 6005299) and incubated at room temperature for 30 min. Finally, the emission was measured on an EnVision multi-panel reader (Envision, Perkin Elmer).

[0908] AXL-ADC IgG1-AXL-148-vcDuo3, IgG1-AXL-183-vcDuo3, and IgG1-AXL-726-vcDuo3 induced cytotoxicity in LCLC-103H cells, with IC50 values ​​ranging from 0.01 to 0.06 µg / mL. Figure 5 As shown in A. Similarly, Figure 5 B showed that these AXL-ADCs induced cytotoxicity in MDA-MB-231 cells, with IC50 values ​​ranging from 0.005 to 0.015 µg / mL.

[0909] Example 7 – VH and VL antibody variants that allow binding to AXL

[0910] The protein sequences of the VH and VL regions of the anti-AXL antibody group (described in Example 1) were compared, and AXL binding was compared to identify key or permissible amino acid residue changes in the VH or VL regions. Therefore, antibodies with the same VH or VL region were grouped, and their binding to human AXL and differences in the VL or VH sequences were compared, respectively. Binding to transiently expressed human AXL from HEK-293F cells was evaluated in an alloantigen-specific screening assay as described in Example 1. The amino acid positions numbered in this example are based on... Figure 6 The sequence shown is complete, that is, the first amino acid in the sequence is numbered as position '1', the second as position '2', and so on.

[0911] First, antibodies with the same VL sequence are classified.

[0912] It was found that IgG1-AXL-148 and IgG1-AXL-140 have the same VL sequence and differ by one amino acid in the HC CDR3 region (at amino acid position 109, F replaces I). Figure 6 A). Both antibodies bound to human AXL (Table 7), indicating that the amino acid at position 109 is not essential for antibody binding, whereby the mutation identified in the CDR2 region (at amino acid position 56, G replaces A) does not compensate for the loss of binding. Figure 6 A).

[0913] It was found that IgG1-AXL-726 and IgG1-AXL-187 have the same VL sequence, and both antibodies bind to human AXL (Table 7). Two amino acid residues are allowed to change in the HC CDR3 region (R replacing S at position 97 and A replacing T at position 105); Figure 6 B) without loss of binding, wherein it is assumed that mutations identified in the CDR1 region (where Y replaces H at position 32) and / or the framework regions (P3Q, V24I, Y25D, T86A, and T117A) do not compensate for the loss of binding. Figure 6 B).

[0914] It was found that IgG1-AXL-171, IgG1-AXL-172, and IgG1-AXL-181 have the same VL sequence, and all antibodies bind to human AXL (Table 7). The CDR3 regions of these three antibodies are identical, but a one-amino acid residue change is allowed in the HC CDR1 region (where S replaces N at position 31) or the framework region (where H replaces Q at position 82) without loss of binding. Figure 6 C).

[0915] It was found that IgG1-AXL-613, IgG1-AXL-608-01, IgG1-AXL-610-01 and IgG1-AXL-620-06 have the same VL sequence and a difference of one amino acid in the HC CDR3 region (at amino acid position 101, N replaces D); Figure 6 D). All antibodies bound to human AXL (Table 7), indicating that the amino acid at position 101 is not essential, where the mutations identified in the HC CDR2 region (where V replaces A at amino acid position 58) and / or the framework regions (N35S, M37V, A61V, L70I, S88A) do not compensate for the loss of binding. Figure 6 D).

[0916] Next, antibodies with the same VH sequence will be classified.

[0917] It was found that IgG1-AXL-613 and IgG1-AXL-613-08 have the same VH sequence and a five-amino acid difference in the CDR3 region of LC (at positions 92 to 96, RSNWL replaces YGSSY). Figure 6 E). Both antibodies bound to human AXL (Table 7), indicating that amino acid changes at positions 92 to 96 are permissible and do not affect antibody binding, where it is assumed that mutations identified in the CDR1 region (with S deleted at position 30), the CDR2 region (G51D), and / or the frame regions (G9A, S54N, R78S, Q100G, L104V) do not compensate for the loss of binding. Figure 6 E).

[0918] Table 7

[0919] Antibody EC50 (µg / mL) Maximum assimilation (any unit) IgG1-AXL-140 0.0026 2889 IgG1-AXL-148 0.0036 3499 IgG1-AXL-171 0.003 2575 IgG1-AXL-172 0.0055 5378 IgG1-AXL-181 0.008 3598 IgG1-AXL-187 0.0065 2563 IgG1-AXL-608-01 0.0035 3318 IgG1-AXL-610-01 0.0023 2947 IgG1-AXL-613 0.0072 5211 IgG1-AXL-613-08 0.0242 2209 IgG1-AXL-620-06 0.0034 4352 IgG1-AXL-726 0.0471 3154

[0920] Example 8 – In vitro cytotoxicity induced by MMAE-conjugated AXL antibody

[0921] Conjugation of MMAE with anti-AXL antibody

[0922] The anti-AXL antibody was purified by protein A chromatography according to a standard procedure and conjugated to vcMMAE. The drug-linker vcMMAE was alkylated to the cysteine ​​residue of the reduced antibody according to a procedure described in the literature (see

[150] ,

[151] , and

[152] ). The reaction was quenched by adding excess N-acetylcysteine. Any residual unconjugated drug was removed by purification, and the final anti-AXL antibody-drug conjugate was prepared in PBS. The concentration of the anti-AXL antibody-drug conjugate (by absorbance at 280 nm), drug-antibody ratio (DAR) (by reversed-phase HPLC and hydrophobic interaction chromatography (HIC)), amount of unconjugated drug (by reversed-phase chromatography), percentage of aggregation (by size exclusion chromatography, SEC-HPLC), and endotoxin level (by LAL) were then analyzed. The results are shown in Table 8 below.

[0923] Table 8 - Overview of the different properties of antibody-drug conjugates.

[0924]

[0925] Cell culture

[0926] LCLC

[0927] -103H cells (human large cell lung cancer) and A431 cells (DMSZ, Braunschweig, Germany) were cultured in L-glutamine-containing RPMI 1640 (Cambrex; catalog number BE12-115F) supplemented with 10% (vol / vol) heat-inactivated enhanced fetal bovine serum (Perbio; catalog number SH30087.03), 2 mM L-glutamine (Cambrex; catalog number US17-905C), 50 IU / mL penicillin and 50 μg / mL streptomycin (Cambrex; catalog number DE17-603E). MDA-MB231 cells were cultured in DMEM containing high glucose and HEPES (Lonza #BE12-709F), iron-containing donor bovine serum (LifeTechnologies #10371-029), 2 mM L-glutamine (Lonza #BE17-605E), 1 mM sodium pyruvate (Lonza #BE13-115E), and MEM non-essential amino acid solution (LifeTechnologies #11140). These cell lines were maintained in a humidified incubator at 37°C and 5% (vol / vol) CO2. LCLC-103H, A431, and MDA-MB231 cells were cultured to near confluence, followed by trypsin treatment, resuspending in culture medium, and passing through a cell filter (BD Falcon, catalog number 352340) to obtain a single-cell suspension. 1 x 102 cells were seeded in each well of a 96-well plate. 3 Cells were collected and incubated at room temperature for 30 min, followed by incubation at 37°C and 5% CO2 for 5 hours to allow them to adhere to the plate.

[0928] Cytotoxicity assay

[0929] Serial dilutions of MMAE-conjugated AXL antibody (final concentrations ranging from 0.00015 to 10 µg / mL) were prepared in culture medium and added to plates. Cells incubated with 1 µM astrococcus (#S6942-200, Sigma) were used as a reference for 100% tumor killing. Untreated cells were used as a reference for 100% cell growth. Plates were incubated at 37°C and 5% CO2 for 5 days. Next, CellTiter-Glo reagent (Promega; catalog number G7571) (20 µL / well) was added to the wells, and the plates were incubated at 37°C and 5% CO2 for 1.5 hours. Subsequently, 180 µL / well was transferred to white 96-well Optiplate™ plates (PerkinElmer, Waltham, MA; catalog number 6005299) and incubated at room temperature for 30 min. Finally, luminescence was measured on an EnVision multiplate reader (Envision, Perkin Elmer).

[0930] MMAE-conjugated AXL antibody induced 50% cell killing in LCLC-103H cells at concentrations ranging from 0.004 to 0.219 µg / mL, as shown in Table 9. Figure 7 As shown in the image.

[0931] Similarly, AXL-ADC in A431 cells (Table 10 and Figure 15 A) and MDA-MB231 cells (Table 10 and Figure 15 B) effectively induced cytotoxicity.

[0932] Table 9 – Cytotoxicity of MMAE-conjugated AXL antibody in LCLC-103H cells (EC50 values)

[0933] ADC EC50 (µg / mL) IgG1-AXL-613-vcMMAE 0.004 IgG1-AXL-148-vcMMAE 0.012 IgG1-AXL-171-vcMMAE 0.018 IgG1-AXL-726-M101L-vcMMAE 0.018 IgG1-AXL-107-vcMMAE 0.022 IgG1-AXL-511-vcMMAE 0.032 IgG1-AXL-154-M103L-vcMMAE 0.044 IgG1-AXL-183-N52Q-vcMMAE 0.113 IgG1-AXL-733-vcMMAE 0.219

[0934] Table 10: Cytotoxicity (EC50 value) of MMAE-conjugated AXL antibody in A431 and MDA-MB-231 cells.

[0935]

[0936] Example 9 – Therapeutic treatment of LCLC-103H tumor xenografts in SCID mice with MMAE-conjugated anti-AXL antibody

[0937] The in vivo efficacy of MMAE-conjugated anti-AXL antibody was determined in subcutaneous (SC) LCLC-103H xenograft tumors established in SCID mice. The antibody was prepared in 200 μL PBS at a concentration of 5 x 10⁻⁶ ppm. 6LCLC-103H (large cell lung cancer) tumor cells (obtained from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH (DSMZ)) were subcutaneously injected into the right side of female SCID mice. Initiation was initiated 14–21 days post-inoculation, when the average tumor size was >100–200 mm. 3 Upon observation of significant tumo...

Claims

1. An antibody that binds AXL, wherein the antibody does not compete for binding to AXL with ligand growth arrest-specific factor 6 (Gas6), and wherein the antibody comprises at least one binding region comprising a variable heavy (VH) region and a variable light (VL) region selected from the group consisting of: a) a VH region comprising CDR1, CDR2, and CDR3 sequences of SEQ ID Nos: 46, 47, and 48, respectively; and a VL region comprising CDR1, CDR2, and CDR3 sequences of SEQ ID Nos: 49, AAS, and 50, respectively; and b) a VH region comprising CDR1, CDR2, and CDR3 sequences of SEQ ID Nos: 57, 58, and 59, respectively; and a VL region comprising CDR1, CDR2, and CDR3 sequences of SEQ ID Nos: 60, GAS, and 61, respectively.

2. The antibody according to claim 1, wherein the maximum antibody binding to AXL in the presence of Gas6 is at least 90% of the binding in the absence of Gas6, wherein the competition between the antibody that binds AXL and the Gas6 is determined on A431 cells pre-incubated with Gas6 or not.

5. The antibody according to any one of claims 1 to 2, wherein AXL is human AXL as specified in SEQ ID NO:

130.

6. The antibody according to any one of claims 1 to 2, wherein AXL is cynomolgus AXL as specified in SEQ ID NO:

147.

3. The antibody according to any one of claims 1 to 2, wherein the binding affinity (K) of the antibody to human AXL is... D The range is 0.3x10 -9 Up to 63x10 -9 M, and wherein the binding affinity is measured using the soluble AXL extracellular domain using biomembrane interferometry.

4. The antibody according to any one of claims 1 to 2, wherein the dissociation rate of the antibody against AXL is 9.7 x 10⁻⁶. -5 Up to 4.4x10 -3 s -1 The dissociation rate was measured using soluble recombinant AXL extracellular domains via biomembrane interferometry.

7. The antibody according to any one of claims 1 to 2, wherein AXL is human AXL as specified in SEQ ID NO: 130 and cynomolgus AXL as specified in SEQ ID NO:

147.

8. The antibody according to any one of claims 1 to 2, wherein the at least one binding region comprises a VH region and a VL region selected from the group consisting of: a) a VH region comprising SEQ ID No: 5 and a VL region comprising SEQ ID No: 6; and b) a VH region comprising SEQ ID No: 10 and a VL region comprising SEQ ID No:

11.

9. The antibody according to any one of claims 1 to 2, wherein the antibody comprises a heavy chain of an isotype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

10. The antibody according to claim 9, wherein the isotype is IgGl.

11. The antibody according to claim 10, which is isotype IgGl m(f).

12. The antibody according to any one of claims 1 to 2, which is a full-length monoclonal antibody.

13. The antibody according to claim 12, wherein the full-length monoclonal antibody is a full-length monoclonal IgGl, kappa antibody.

14. The antibody according to any one of claims 1 to 2, wherein the antibody is an effector function deficient antibody, a stable IgG4 antibody, or a monovalent antibody.

15. The antibody according to claim 14, wherein the heavy chain has been modified such that the entire hinge region has been deleted. ​ ​ ​ ​ 16. The antibody according to claim 14, wherein the antibody sequence has been modified such that it does not comprise any N-linked glycosylation acceptor sites.

17. The antibody according to any one of claims 1 to 2, wherein the antibody is a single chain antibody.

18. A bispecific antibody comprising a first binding region of an antibody according to any one of claims 1-17, and a second binding region that binds a different target or epitope than the first binding region.

19. The bispecific antibody according to claim 18, wherein the bispecific antibody comprises a first and a second heavy chain, each of which comprises at least a hinge region, a CH2 and a CH3 region, wherein in the first heavy chain at least one of the amino acids in a position corresponding to a position selected from the group consisting of K409, T366, L368, K370, D399, F405 and Y407 in a human IgGl heavy chain has been substituted, and in the second heavy chain at least one of the amino acids in a position corresponding to a position selected from the group consisting of F405, T366, L368, K370, D399, Y407 and K409 in a human IgGl heavy chain has been substituted, and wherein the substitutions of the first and the second heavy chain are not in the same position.

20. The bispecific antibody according to claim 19, wherein in the first heavy chain the amino acid in a position corresponding to K409 in a human IgGl heavy chain is R, and in the second heavy chain the amino acid in a position corresponding to F405 in a human IgGl heavy chain is L, or wherein in the first heavy chain the amino acid in a position corresponding to F405 in a human IgGl heavy chain is L, and in the second heavy chain the amino acid in a position corresponding to K409 in a human IgGl heavy chain is R.

21. An immunoconjugate comprising an antibody according to any one of claims 1 to 17 or a bispecific antibody according to any one of claims 18 to 20, and a therapeutic moiety.

22. The immunoconjugate according to claim 21, wherein the therapeutic moiety is a chemotherapeutic drug.

23. The immunoconjugate according to claim 21, wherein the therapeutic moiety is a cytotoxic agent, a cytokine, an immunosuppressant, an antibiotic or a radioisotope.

24. The immunoconjugate according to claim 21, wherein the therapeutic moiety is a cytotoxic agent.

25. The immunoconjugate according to any one of claims 23 to 24, wherein the cytotoxic agent is linked to the antibody by a cleavable linker.

26. The immunoconjugate according to claim 25, wherein the cleavable linker is 4-(2- pyridyldithio)-pentanoic acid N - succinimidyl or maleimidocaproyl-valine-citrulline p - aminobenzyloxycarbonyl (mc-vc-PAB).

27. The immunoconjugate according to any one of claims 23 to 24, wherein the cytotoxic agent is linked to the antibody or fragment thereof by a non-cleavable linker.

28. The immunoconjugate according to claim 27, wherein the non-cleavable linker is 4( N - maleimidomethyl)cyclohexane-1 -carboxylate succinimidyl ester or maleimidocaproyl (MC).

29. The immunoconjugate according to any one of claims 23 to 24, wherein the cytotoxic agent is selected from the group consisting of: a DNA-targeting agent; a microtubule-targeting agent; and a nucleoside analogue.

30. The immunoconjugate according to any one of claims 23 to 24, wherein the immunoconjugate comprises a combination of: i) the cytotoxic agent with bystander killing capacity and a cleavable linker; ii) said cytotoxic agent without bystander killing ability and a cleavable linker; iii) said cytotoxic agent with bystander killing ability and a non-cleavable linker; or iv) said cytotoxic agent without bystander killing ability and a non-cleavable linker.

31. The immunoconjugate according to any one of claims 23 to 24, wherein the linker is mc-vc-PAB and the cytotoxic agent is MMAE; or the linker is 4-(2-pyridyldithio)-pentanoic acid N - succinimidyl ester and the cytotoxic agent is DM1.

32. The immunoconjugate according to any one of claims 23 to 24, wherein said immunoconjugate comprises the linker mc-vc-PAB, the cytotoxic agent MMAE and said antibody, wherein said at least one binding region comprises a VH region and a VL region selected from the group consisting of: a) a VH region comprising SEQ ID No: 5 and a VL region comprising SEQ ID No: 6; and b) a VH region comprising SEQ ID No: 10 and a VL region comprising SEQ ID No:

11.

33. The immunoconjugate according to claim 23, wherein the linker is MC and said cytotoxic agent is MMAF.

34. The immunoconjugate according to claim 23, wherein the number of cytotoxic agent / antibodies is from 1 to 8.

35. The immunoconjugate according to claim 34, wherein the number of cytotoxic agent / antibodies is from 2 to 7.

36. The immunoconjugate according to claim 34, wherein the number of cytotoxic agent / antibodies is from 2 to 6.

37. The immunoconjugate according to claim 34, wherein the number of cytotoxic agent / antibodies is from 2 to 5.

38. The immunoconjugate according to claim 34, wherein the number of cytotoxic agent / antibodies is from 2 to 4.

39. The immunoconjugate according to claim 34, wherein the number of cytotoxic agent / antibodies is from 2 to 3.

40. A composition comprising an antibody according to any one of claims 1 to 17, a bispecific antibody according to any one of claims 18 to 20, or an immunoconjugate according to any one of claims 21 to 39.

41. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 17, a bispecific antibody according to any one of claims 18 to 20, or an immunoconjugate according to any one of claims 21 to 39, and a pharmaceutically acceptable carrier.

42. A nucleic acid construct encoding a bispecific antibody according to any one of claims 18 to 20.

43. A nucleic acid construct encoding an antibody according to any one of claims 1 to 17.

44. An expression vector comprising one or more nucleic acid constructs according to any one of claims 42 and 43.

45. A host cell comprising a vector according to claim 44.

46. The host cell according to claim 45, wherein said host cell is a recombinant host cell.

47. The host cell according to claim 46, wherein said recombinant host cell is a recombinant prokaryotic, recombinant eukaryotic or recombinant microbial host cell.

48. The host cell according to any one of claims 45 to 46, wherein said host cell produces an antibody according to any one of claims 1 to 17, or a bispecific antibody according to any one of claims 18 to 20.

49. A hybridoma producing an antibody according to any one of claims 1 to 17. ​ ​ 50. Use of an antibody according to any one of claims 1 to 17, a bispecific antibody according to any one of claims 18 to 20, or an immunoconjugate according to any one of claims 21 to 39 for the manufacture of a medicament for the treatment of a cancer, wherein the cancer is a solid tumor expressing AXL, and wherein the cancer is selected from the group consisting of a colorectal cancer, a breast cancer, a cervical cancer, a lung cancer, an ovarian cancer, a pancreatic cancer, an esophageal cancer, a skin cancer, and a soft tissue sarcoma.

51. Use according to claim 50, wherein the colorectal cancer is a colorectal adenoma, wherein the breast cancer is a triple negative breast cancer, wherein the lung cancer is NSCLC, wherein the pancreatic cancer is a pancreatic adenocarcinoma, and wherein the skin cancer is a malignant melanoma.

52. Use according to claim 50, wherein the lung cancer is a lung squamous cell carcinoma.

53. Use according to claim 50, wherein the pancreatic cancer is a pancreatic ductal carcinoma.

54. Use of an antibody according to any one of claims 1 to 17, a bispecific antibody according to any one of claims 18 to 20, an immunoconjugate according to any one of claims 21 to 39, a composition according to claim 40, or a pharmaceutical composition according to claim 41 for the manufacture of a medicament for the diagnosis of a disease characterized by the involvement or accumulation of cells expressing AXL, wherein the disease is a solid tumor expressing AXL, and wherein the disease is selected from the group consisting of a colorectal cancer, a breast cancer, a cervical cancer, a lung cancer, a skin cancer, an ovarian cancer, a pancreatic cancer, and an esophageal cancer, wherein the diagnosis of the disease comprises the administration of the antibody to an individual.

55. Use according to claim 54, wherein the antibody is labeled with a detectable agent, and wherein the amount of cells expressing AXL correlates with or is indicative of the disease.

56. Use of an antibody according to any one of claims 1 to 17, a bispecific antibody according to any one of claims 18 to 20, an immunoconjugate according to any one of claims 21 to 39, a composition according to claim 40, or a pharmaceutical composition according to claim 41 for the manufacture of a medicament for the inhibition of the growth and / or proliferation of tumor cells expressing AXL, wherein the tumor cells are from a solid tumor expressing AXL and selected from the group consisting of a colorectal cancer, a breast cancer, a cervical cancer, a lung cancer, a skin cancer, an ovarian cancer, a pancreatic cancer, and an esophageal cancer, which use comprises the administration to an individual in need thereof.

57. A method of producing an antibody according to any one of claims 1 to 17, the method comprising the steps of: a) culturing a host cell according to any one of claims 45 to 48 or a hybridoma according to claim 49, and b) purifying the antibody from the culture medium.

58. A diagnostic composition comprising an antibody according to any one of claims 1 to 17 or a bispecific antibody according to any one of claims 18 to 20.

59. Use of an antibody according to any one of claims 1 to 17, a bispecific antibody according to any one of claims 18 to 20, or an immunoconjugate according to any one of claims 21 to 39 for the manufacture of a medicament for detecting the presence of AXL antigen or AXL-expressing cells in a sample, wherein the use comprises: a) contacting the sample with the antibody, bispecific antibody or immunoconjugate under conditions allowing the formation of a complex between the antibody, bispecific antibody or immunoconjugate and AXL; and b) analyzing whether a complex has been formed.

60. A kit for detecting the presence of AXL antigen or AXL-expressing cells in a sample, comprising: i) an antibody according to any one of claims 1 to 17, a bispecific antibody according to any one of claims 18 to 20, or an immunoconjugate according to any one of claims 21 to 39; and ii) instructions for use of the kit.

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