HK1149767A - Monoclonal antibody capable of binding to anexelekto, and use thereof - Google Patents

Description

Monoclonal antibody that binds to Anexelekto and use thereof

Technical Field

The present invention relates to a monoclonal antibody that binds to Anexelekto, a pharmaceutical agent containing the antibody as an active ingredient, and a method for using the antibody.

Background

Anexelekto (also called AXL, UFO, ARK or TYRO7, hereinafter referred to as AXL) was cloned as an oncogene from a patient with chronic myelogenous leukemia, which can transform mouse NIH3T3 cells, due to its high expression (non-patent documents 1 and 2). The AXL protein is a 140kDa receptor tyrosine kinase (non-patent document 3), and has a function of transmitting a signal to a downstream molecule through autophosphorylation of the protein by binding to Gas6 (growth arrest-specific gene 6) as a ligand (non-patent document 4). Among receptor tyrosine kinases having Gas6 as a ligand, there are known receptor tyrosine kinases called Sky and Mer in addition to AXL (non-patent document 5).

As a molecular function of AXL, it is suggested that it is involved in promoting cell proliferation, inhibiting apoptosis, cell migration, and cell adhesion. Experimental facts as a basis for the presumption of these functions are: a phenomenon which occurs in cells treated with Gas6 protein. To date, the following experimental results have been reported: inhibition of cell death and hyperproliferation of vascular smooth muscle in rats (non-patent documents 6 and 7); enhancing cell proliferation and suppressing cell death of mouse NIH3T3 cells after serum starvation (non-patent documents 8 and 9); promoting proliferation of mouse cardiac fibroblasts (non-patent document 10); promoting the proliferation of cells derived from human prostate cancer (non-patent document 11); promoting proliferation of cells derived from human gastric cancer, promoting cell infiltration, and inhibiting cell death (non-patent document 12); enhancing cell migration ability of vascular smooth muscle cells of human and rat (non-patent document 13); enhancing the cell migration ability of mouse nerve cells (non-patent document 14); cells of mouse AXL-highly expressing cells were aggregated (non-patent document 15).

Similarly, the PI3K-Akt pathway and the MAPK pathway function as downstream pathways of AXL-mediated signaling by intracellular signaling molecule analysis after Gas6 treatment (non-patent document 5). An experiment confirming the existence of direct intermolecular interactions in these downstream pathways was conducted by analyzing yeast double-hybridization using the intracellular region of AXL as a bait region, and a molecular group called GrbB2/PI3K/p55 γ/SOCS-1/NcK2/RanBP2/C1-TEN was identified (non-patent document 16). The interaction of these proteins suggests that the intracellular signaling pathway exists downstream of AXL signaling, while supporting putative molecular functions on AXL-involved in promoting cell proliferation, inhibiting apoptosis, cell migration, cell adhesion. AXL was also identified as a gene highly expressed when IL-15 inhibits TNF α -induced cell death of mouse fibroblasts, and inhibition of expression of AXL could release inhibition of TNF α -induced cell death, and experimental facts that IL-15 treatment increased phosphorylation of IL-15 receptor and AXL suggested that there was signaling mediated by conjugation of AXL and IL-15 receptor (non-patent document 17).

It has been reported that: in a dominant deviant human glioma strain in which AXL is overexpressed, phosphorylation of AXL by Gas6 is inhibited, and tumorigenicity in nude mice disappears (non-patent document 18). However, there has been no report showing whether a component inhibiting phosphorylation is present in an anti-AXL antibody.

AXL is a primary transmembrane receptor tyrosine kinase, and the extracellular region is composed of two immunoglobulin-like domains (IgD 1 and IgD2, respectively) and two fibronectin type III domains (FND 1 and FND2, respectively) from the N-terminal side (non-patent document 19). FND is known to be expressed in molecules such as nerve cell adhesion molecules associated with intercellular adhesion and nephrotic protein (nephrin), but the details of the function of FND in AXL are not known (non-patent document 20).

AXL, which is identified as an oncogene that retains the original cell transforming ability, is an analyte of canceration-related molecules, and many expression analyses are reported in terms of the amount of protein and mRNA. Examples of the expression in human cancer tissues or human cancer cells are confirmed: high expression of AXL protein was confirmed in cancer types such as lung cancer (non-patent document 21), breast cancer (non-patent document 22), ovarian cancer (non-patent document 23), thyroid cancer (non-patent document 24), melanoma (non-patent document 24), kidney cancer (non-patent document 25), stomach cancer (non-patent document 12), and glioma (non-patent document 26), and high expression of AXL protein was suggested by the presence of high levels of AXLmRNA in esophageal cancer (non-patent document 27), colorectal cancer (non-patent document 28), and acute myelogenous leukemia (non-patent document 29). In addition to the above facts, there are reports that: inhibition of AXL expression via RNAi in HUVEC cells, resulting in obstruction of luminal formation (non-patent document 30); inhibition of constant AXL expression leads to a decrease in the ability of human breast cancer-derived cells to form tumors in mice (non-patent document 30); high expression of constant dominant negative mutants resulted in a decrease in the ability of human glioma cells to form tumors in mice (non-patent document 26), strongly indicating that AXL molecular function is involved in tumor proliferation.

It has been reported that: polyclonal antibodies against the extracellular domain of AXL inhibit migration of the highly invasive breast cancer cell system (patent document 1). However, it is known that a drug showing an effect of inhibiting cancer cell migration does not necessarily show an antitumor activity in a non-clinical test. For example, matrix metalloproteases (hereinafter referred to as MMPs) are known to promote tumor infiltration and migration, and therefore, various matrix metalloprotease inhibitors that inhibit the enzymatic activity of MMPs have been attracting attention as candidates for anticancer drugs, and various drugs such as Batimastat (Batimastat), Marimastat (Marimastat), and Prinomastat (Prinomastat) have been clinically tested. However, no antitumor effect was found in clinical practice (non-patent document 31).

Therefore, it has not been reported and confirmed whether an anti-AXL antibody has an anti-tumor effect, an anti-AXL antibody has an angiogenesis inhibitory effect, or an anti-AXL antibody has a cancer inhibitory effect, particularly in vivo.

Patent document 1: WO 2004/008147

Non-patent document 1: liu et al Proc Natl Acad Sci USA 1988; 85: 1952 to 6.

Non-patent document 2: janssen et al Oncogene 1991; 6: 2113 to 20.

Non-patent document 3: o' Bryan et al Mol Cell Biol 1991; 11: 5016 to 31.

Non-patent document 4: varum et al Nature 1995; 373: 623 to 626.

Non-patent document 5: hafizi et al FEBS J2006; 273: 5231 to 5244.

Non-patent document 6: nakano et al FEBS Lett 1996; 387: 78-80.

Non-patent document 7: nakano et al J Biol Chem 1995; 270: 5702-5.

Non-patent document 8: goruppi et al Mol Cell Biol 1997; 17: 4442 to 53.

Non-patent document 9: bellosta et al Oncogene 1997; 15: 2387 to 97.

Non-patent document 10: stenhoff et al Biochem Biophys Res Commun 2004; 319: 871-8.

Non-patent document 11: sainaghi et al J Cell Physiol 2005; 204: 36-44.

Non-patent document 12: sawabu et al Mol Carcinog 2007; 46: 155 to 164.

Non-patent document 13: fridell et al J Biol Chem 1998; 273: 7123-6.

Non-patent document 14: allen et al Mol Cell Biol 2002; 22: 599 to 613.

Non-patent document 15: McCloskey et al J Biol Chem 1997; 272: 23285-91.

Non-patent document 16: hafizi et al Biochem Biophys Res Commun 2002; 299: 793 to 800.

Non-patent document 17: budagian et al Embo J2005; 24: 4260 to 70.

Non-patent document 18: vajkoczy P et al Proc Natl Acad Sci USA 2006; 103: 5799-804.

Non-patent document 19: o' Bryan et al Mol Cell Biol 1991; 11: 5016 to 31.

Non-patent document 20: yamagata et al curr. opin. cell Biol 2003; 15: 621 to 632.

Non-patent document 21: shieh et al Neopalasia 2005; 7: 1058 to 1064.

Non-patent document 22: meric et al Clin Cancer Res 2002; 8: 361 to 367.

Non-patent document 23: sun et al Oncology 2004; 66: 450 to 457.

Non-patent document 24: ito et al, Thyroid 2002; 12: 971 to 975.

Non-patent document 25: chung et al DNA Cell Biol 2003; 22: 533 to 540.

Non-patent document 26: vajkoczy et al Proc Natl Acad Sci USA 2006; 103: 5799-804.

Non-patent document 27: nemoto et al Pathobiology 1997; 65: 195 to 203.

Non-patent document 28: craven et al Int J Cancer 1995; 60: 791 to 797.

Non-patent document 29: neubauer et al Blood 1994; 84: 1931 to 1941.

Non-patent document 30: holland et al Cancer Res 2005; 65: 9294-9303.

Non-patent document 31: pavlaki et al Cancer Metastasis Rev.2003; 22: 177 to 203.

Disclosure of Invention

The invention is to solveSubject matter

The present invention addresses the problem of providing an anti-AXL antibody and use thereof, and more specifically, aims to provide a method for inhibiting angiogenesis, a method for inhibiting cell proliferation, a method for inhibiting the function of AXL, a method for enhancing the function of AXL, and a method for reducing the expression level of AXL using an anti-AXL antibody. It is also intended to provide an anti-AXL antibody having a novel effect.

Means for solving the problems

The present inventors have conducted extensive studies and, as a result, have succeeded in obtaining an anti-AXL antibody having a specific function, and have further found that the antibody has an angiogenesis inhibitory effect and an anticancer effect, thereby completing the present invention. More specifically, the present invention includes the following inventions (1) to (56).

(1) A monoclonal antibody that binds to AXL.

(2) The antibody according to (1), wherein the antibody has a cell proliferation inhibitory activity.

(3) The antibody according to (1), wherein the antibody inhibits the proliferation of cancer cells.

(4) The antibody according to any one of (1) to (3), which is bound to FND 1.

(5) An antibody prepared by using a peptide having all or at least 5 or more consecutive amino acid sequences of FND1 as an immunogen.

(6) The antibody according to any one of (1) to (5), which has an agonistic activity against AXL.

(7) The antibody according to any one of (1) to (5), which has an antagonistic activity against AXL.

(8) The antibody according to (7), which is obtained by: the cells expressing AXL were contacted with the AXL ligand, and antibodies in which no phosphotyrosine was detected in AXL were selected.

(9) The antibody according to any one of (1) to (8), which has an activity of reducing the expression level of AXL.

(10) The antibody according to any one of (1) to (9), which has an angiogenesis inhibitory activity.

(11) An antibody according to any one of the following (a) to (j):

(a) an antibody produced by the hybridoma deposited under accession number FERM BP-10858(Ax 285);

(b) an antibody produced by the hybridoma deposited under accession number FERM BP-10859(Ax 292);

(c) an antibody (Ax223) produced by the hybridoma deposited under accession number FERM BP-10853;

(d) an antibody produced by the hybridoma deposited under accession number FERM BP-10852(Ax 96);

(e) an antibody produced by the hybridoma deposited under accession number FERM BP-10856(Ax 258);

(f) an antibody produced by the hybridoma deposited under accession number FERM BP-10857(Ax 284);

(g) an antibody produced by the hybridoma deposited under accession number FERM BP-10850(Ax 7);

(h) an antibody produced by the hybridoma deposited under accession number FERM BP-10851(Ax 51);

(i) an antibody produced by the hybridoma deposited under accession number FERM BP-10854(Ax 225);

(j) an antibody (Ax232) produced by the hybridoma deposited under accession number FERM BP-10855.

(12) An antibody that binds to the same epitope as any of the antibodies of (11).

(13) An antibody having the same CDR sequence as any of the antibodies of (11).

(14) An antibody, wherein the sequence of the heavy chain CDR1, 2,3 is SEQ ID NO: 4. 5 and 6.

(15) An antibody having a heavy chain CDR comprising: an amino acid sequence obtained by substituting, deleting, inserting and/or adding 1 or more amino acid sequences in the heavy chain CDR amino acid sequence of the antibody of (14).

(16) An antibody, wherein the light chain CDR1, 2,3 has the sequence of SEQ ID NO: 8. 9 and 10.

(17) An antibody having a light chain CDR comprising: the light chain CDR amino acid sequence of the antibody of (16) has an amino acid sequence in which 1 or more amino acid sequences are substituted, deleted, inserted and/or added.

(18) The antibody according to any one of (13) to (17), which is a chimeric antibody.

(19) The antibody according to any one of (13) to (17), which is a humanized antibody.

(20) A hybridoma described in any one of the following (a) to (j):

(a) the hybridoma deposited under accession number FERM BP-10858(Ax 285);

(b) the hybridoma deposited under accession number FERM BP-10859(Ax 292);

(c) the hybridoma deposited under accession number FERM BP-10853(Ax 223);

(d) the hybridoma deposited under accession number FERM BP-10852(Ax 96);

(e) the hybridoma deposited under accession number FERM BP-10856(Ax 258);

(f) the hybridoma deposited under accession number FERM BP-10857(Ax 284);

(g) the hybridoma deposited under accession number FERM BP-10850(Ax 7);

(h) the hybridoma deposited under accession number FERM BP-10851(Ax 51);

(i) the hybridoma deposited under accession number FERM BP-10854(Ax 225);

(j) the hybridoma (Ax232) deposited under accession number FERM BP-10855.

(21) An angiogenesis inhibitor comprising an anti-AXL antibody as an active ingredient.

(22) The angiogenesis inhibitor according to (21), wherein the antibody is the antibody according to any one of (1) to (19).

(23) A cell growth inhibitor comprising an anti-AXL antibody as an active ingredient.

(24) The inhibitor according to (23), wherein the cell is a cancer cell.

(25) The inhibitor according to (23), wherein the antibody is the antibody according to any one of (1) to (19).

(26) The inhibitor of (23), wherein the anti-AXL antibody is an antibody that binds to FND 1.

(27) The inhibitor according to (23), which comprises an antibody that binds to IgD2 and has an inhibitory activity on phosphorylation as an active ingredient.

(28) An agent for inducing the phosphorylation activity of AXL, which comprises an anti-AXL antibody as an active ingredient.

(29) The inducer of (28), wherein the anti-AXL antibody is an antibody that binds to IgD.

(30) The inducer of (28), wherein the antibody is the antibody of (6).

(31) An inhibitor of the phosphorylation activity of AXL, which comprises an anti-AXL antibody as an active ingredient.

(32) The inhibitor of (31), wherein the anti-AXL antibody is an antibody that binds to IgD 2.

(33) The inhibitor of (31), wherein the antibody is the antibody according to any one of (7) or (8).

(34) An agent for reducing the expression level of AXL, which comprises an anti-AXL antibody as an active ingredient.

(35) The expression level reducing agent of (34), wherein the anti-AXL antibody is an antibody that binds to FND 1.

(36) The agent for reducing an expression level of (34), wherein the antibody is the antibody of (9).

(37) A method of inducing phosphorylation of AXL using an anti-AXL antibody.

(38) A method for reducing the expression level of AXL by using an anti-AXL antibody.

(39) Methods of inhibiting the phosphorylation of AXL using an anti-AXL antibody.

(40) An anticancer agent comprising an anti-AXL antibody as an active ingredient.

(41) The anticancer agent according to (40), wherein the antibody is the antibody according to any one of (1) to (19).

(42) The anticancer agent according to item (40), which comprises an antibody having a phosphorylation activity inhibitory activity, which binds to IgD2, as an active ingredient.

(43) The anticancer agent according to item (40), wherein the cancer is pancreatic cancer, gastric cancer, lung cancer, osteosarcoma, colorectal cancer, prostate cancer, melanoma, endometrial cancer, ovarian cancer, uterine leiomyoma, thyroid cancer, stem cell cancer, breast cancer, bladder cancer, renal cancer, glioma, neuroblastoma, or esophageal cancer.

(44) The anticancer agent of (42), wherein the cancer is glioma, gastric cancer, endometrial cancer, non-small cell lung cancer, pancreatic cancer or breast cancer.

(45) The anticancer agent according to (43), wherein the cancer is pancreatic cancer or breast cancer.

(46) The antibody according to (1), wherein the antibody has an effect of inhibiting AXL phosphorylation.

(47) Methods of inhibiting angiogenesis using anti-AXL antibodies.

(48) An anti-AXL antibody and a method for using the same in the preparation of an angiogenesis inhibitor.

(49) Methods of inhibiting cell proliferation using anti-AXL antibodies.

(50) Methods of treatment and/or prevention of cancer using anti-AXL antibodies.

(51) An anti-AXL antibody, and a method for producing a cell proliferation inhibitor using the same.

(52) An anti-AXL antibody for use in the preparation of an anti-cancer agent.

(53) An application method of an anti-AXL antibody in preparing a phosphorylation inducer.

(54) An anti-AXL antibody and its application in preparing phosphorylation inhibitor are provided.

(55) An anti-AXL antibody, and a method for producing an AXL expression level-reducing agent using the same.

(56) A method for the preparation of an anti-AXL specific antibody, the method comprising the steps of:

(a) a step of immunizing a non-human animal with a peptide having all or at least 5 or more consecutive amino acid sequences of FND 1; and

(b) recovering the antibody from the non-human animal of (a), or recovering antibody-producing cells and recovering the antibody produced by the antibody-producing cells.

Drawings

Fig. 1a is a photograph showing the results of an evaluation experiment of the activity of the anti-AXL monoclonal antibody (Ax292) of the present invention in inducing AXL phosphorylation in cancer cells. This antibody was shown to induce phosphorylation of the kinase domain of AXL.

FIG. 1b is a photograph showing the results of an evaluation experiment of the activity of the anti-AXL monoclonal antibody (Ax258) of the present invention in inducing AXL phosphorylation in cancer cells. This antibody was shown to induce phosphorylation of the kinase domain of AXL.

FIG. 1c is a photograph showing the results of an evaluation experiment of the activity of the anti-AXL monoclonal antibody (Ax285) of the present invention in inducing AXL phosphorylation in cancer cells. This antibody was shown to induce phosphorylation of the kinase domain of AXL.

FIG. 1d is a photograph showing the results of an evaluation experiment of the activity of the anti-AXL monoclonal antibody (Ax223) of the present invention in inducing AXL phosphorylation in cancer cells. This antibody was shown to induce phosphorylation of the kinase domain of AXL.

Fig. 1e is a photograph showing the results of an evaluation experiment of the activity of the anti-AXL monoclonal antibody (Ax96) of the present invention in inducing AXL phosphorylation in cancer cells. This antibody was shown to induce phosphorylation of the kinase domain of AXL.

Fig. 2a is a photograph showing the results of an evaluation experiment of the activity of the anti-AXL monoclonal antibody (Ax51) of the present invention in inhibiting the ligand-dependent phosphorylation in cells. The antibody was shown to inhibit ligand-dependent phosphorylation of the kinase domain of AXL.

Fig. 2b is a photograph showing the results of an evaluation experiment of the activity of the anti-AXL monoclonal antibody (Ax7) of the present invention in inhibiting the ligand-dependent phosphorylation in cells. The antibody was shown to inhibit ligand-dependent phosphorylation of the kinase domain of AXL.

Fig. 3a is a photograph showing the results of an evaluation test of the activity of the anti-AXL monoclonal antibody (Ax292) of the present invention to induce AXL degradation in cancer cells. This antibody was shown to induce the breakdown of AXL protein.

FIG. 3b is a photograph showing the results of an evaluation test of the activity of the anti-AXL monoclonal antibody (Ax284) of the present invention for inducing the decomposition of AXL in cancer cells. This antibody was shown to induce the breakdown of AXL protein.

FIG. 3c is a photograph showing the results of an evaluation test of the activity of the anti-AXL monoclonal antibody (Ax285) of the present invention for inducing the decomposition of AXL in cancer cells. This antibody was shown to induce the breakdown of AXL protein.

FIG. 3d is a photograph showing the results of an evaluation test of the activity of the anti-AXL monoclonal antibody (Ax223) of the present invention for inducing degradation of AXL in cancer cells. This antibody was shown to induce the breakdown of AXL protein.

Fig. 3e is a photograph showing the results of an evaluation test of the activity of the anti-AXL monoclonal antibody (Ax7) of the present invention in inducing AXL degradation in cancer cells. This antibody was shown to induce the breakdown of AXL protein.

FIG. 3f is a photograph showing the results of an evaluation test of the activity of the anti-AXL monoclonal antibody (Ax225) of the present invention for inducing degradation of AXL in cancer cells. This antibody was shown to induce the breakdown of AXL protein.

FIG. 3g is a photograph showing the results of an evaluation test of the activity of the anti-AXL monoclonal antibody (Ax96) of the present invention for inducing degradation of AXL in cancer cells. This antibody was shown to induce the breakdown of AXL protein.

FIG. 3h is a photograph showing the results of an evaluation test of the activity of the anti-AXL monoclonal antibody (Ax258) of the present invention for inducing the decomposition of AXL in cancer cells. This antibody was shown to induce the breakdown of AXL protein.

Fig. 4 is a graph and a photograph showing the results of an evaluation experiment of the in vitro angiogenesis inhibitory activity of the anti-AXL monoclonal antibodies (Ax232, Ax292, Ax285, Ax284) of the present invention. The antibody was shown to have in vitro inhibitory activity against angiogenesis.

Fig. 5 is a graph showing the measurement results of the antitumor effect of the anti-AXL monoclonal antibodies of the present invention (Ax223, Ax285, Ax96, Ax292, Ax258, Ax7, Ax51, Ax284, Ax225) on a human pancreatic cancer transplant mouse model.

Fig. 6 is a graph showing the measurement results (2) of the antitumor effect of the anti-AXL monoclonal antibodies of the present invention (Ax223, Ax285, Ax96, Ax292, Ax258, Ax7, Ax51, Ax284, Ax225) on a human pancreatic cancer transplant mouse model, the binding domains of the antibodies, and the phosphorylation inhibitory activity of the antibodies.

FIG. 7 is a graph showing the results of measurement of the antitumor effect of the anti-AXL monoclonal antibody (Ax225) of the present invention on a human breast cancer transplant mouse model.

Detailed Description

The present invention provides novel anti-AXL antibodies. And the present invention provides a novel use of the anti-AXL antibody.

The anti-AXL antibody of the present invention is not particularly limited as long as it binds to AXL, and is not particularly limited, regardless of its origin (human, mouse, rat, rabbit, chicken, etc.), type (polyclonal antibody, monoclonal antibody), shape (altered antibody, modified antibody, antibody fragment, small molecule antibody, etc.), and the like. The anti-AXL antibody of the present invention is not particularly limited, and specifically binds to anexeekto, or is preferably a monoclonal antibody.

It is also preferable that the anti-AXL antibody of the present invention has a cell proliferation inhibitory activity.

One of the preferred modes of the anti-AXL antibody of the present invention may be: an anti-AXL antibody that binds to FND 1.

As will be clear from the examples described below: in particular, an antibody binding to FND1, which has a remarkably high antitumor activity in vivo as compared with other antibodies.

The binding activity of an anti-AXL antibody to FND1 can be evaluated according to a method known to those skilled in the art, and for example, the following method can be used. FND1 was electrophoresed and subjected to western blotting with an anti-AXL antibody, whereby the binding activity of the anti-AXL antibody to FND1 was confirmed.

One of the preferred modes of the anti-AXL antibody of the present invention is: an anti-AXL antibody having agonistic activity against AXL. An anti-AXL antibody having agonistic activity against AXL is an antibody that induces AXL-mediated phosphorylation, particularly tyrosine phosphorylation reaction, when the anti-AXL antibody binds to AXL. The subject to which an anti-AXL antibody having agonistic activity induces phosphorylation reaction is not particularly limited, and examples thereof include autophosphorylation of AXL.

Whether an anti-AXL antibody has agonistic activity can be determined according to a method known to those skilled in the art, and for example, the following method can be used. The anti-AXL antibody to be tested is contacted with cells expressing AXL (e.g., Calu-1, MDA-MB-231, DU-145, etc.), and AXL is then extracted from the cells. It was confirmed that tyrosine in AXL extracted with an anti-phosphotyrosine antibody was phosphorylated. More specifically, whether an anti-AXL antibody has agonistic activity can be confirmed according to the method described in the examples.

Examples of the anti-AXL antibody having agonistic activity include the following antibodies (a) to (g):

(a) an antibody produced by the hybridoma deposited under accession number FERM BP-10858(Ax 285);

(b) an antibody produced by the hybridoma deposited under accession number FERM BP-10852(Ax 96);

(c) an antibody produced by the hybridoma deposited under accession number FERM BP-10856(Ax 258);

(d) an antibody produced by the hybridoma deposited under accession number FERM BP-10859(Ax 292);

(e) an antibody (Ax223) produced by the hybridoma deposited under accession number FERM BP-10853;

(f) an antibody that recognizes the same epitope as any of the antibodies (a) to (e);

(g) an antibody having a CDR sequence identical to that of any of the antibodies (a) to (e).

An antibody recognizing the same epitope as the above antibody can be obtained, for example, by the following method.

The test antibody shares an epitope with a particular antibody, and this can be confirmed by competition between the two antibodies for the same epitope. Competition between antibodies can be detected by cross-blocking assays and the like. For example, a competitive ELISA assay is a preferred cross-blocking assay. Specifically, in the cross-blocking assay, AXL protein coated in wells of a microplate is pre-incubated in the presence or absence of candidate competing antibodies, and then the anti-AXL antibody described above is added. The amount of the above-mentioned anti-AXL antibody that binds to the AXL protein in the well is indirectly related to the binding capacity of the candidate competitor antibody (test antibody) that competes for binding to the same epitope. That is, the greater the affinity of the test antibody for the same epitope, the lower the amount of binding between the anti-AXL antibody and the AXL protein-coated well, and the greater the amount of binding between the test antibody and the AXL protein-coated well.

The amount of antibody bound to the well can be easily determined by labeling the antibody in advance. For example, biotin-labeled antibodies can be determined by using avidin peroxidase conjugates and appropriate substrates. Cross-blocking assays that utilize enzymatic labels such as peroxidase are particularly referred to as competitive ELISA assays. The antibody may be labeled with other labels that can be detected or measured. Specifically, radioactive labels, fluorescent labels, and the like are known.

And when the test antibody has a constant region derived from a different species from the anti-AXL antibody, the amount of antibody bound to the well can be measured by a labeled antibody that recognizes the constant region of the antibody. Or even when the antibody is of the same origin, the amount of the antibody bound to the well can be measured by recognizing each type of antibody when the classification (class) is different.

A candidate competing antibody is an antibody that binds essentially to the same epitope as the anti-AXL antibody described above, or competes for binding to the same epitope, if the candidate antibody blocks at least 20%, preferably at least 20-50%, more preferably at least 50% of the binding of the anti-AXL antibody compared to the binding activity obtained in a control assay performed in the absence of the candidate competing antibody.

Determination of CDR sequences to obtain an antibody having the same CDR sequences as an antibody can be carried out according to methods known to those skilled in the art. For example, CDR sequences can be determined by determining the amino acid Sequence of the full-length antibody or the amino acid Sequence of the variable region, and applying the determined amino acid Sequence to the database of amino acid sequences of antibodies prepared by Kabat et al ("Sequence of Proteins of Immunological Interest," US depth.health and Human Services, 1983), to investigate the homology. The numbering in the framework and in the CDR sequences can be determined by the convention of Kabat (Kabat, e.a. et al, US dept.health and Human Services, US Goverment Printing Offices, 1991).

The full-length amino acid sequence of an antibody or the amino acid sequence of a variable region can be determined according to methods known to those skilled in the art.

An antibody having the same CDR sequence as a certain antibody may be an antibody having the same sequence in at least 1 CDR among 6 CDRs present in the antibody, preferably in all 3 CDRs present in the heavy chain, or an antibody having the same sequence in all 3 CDRs present in the light chain, and further preferably an antibody having the same sequence in all 6 CDRs present in the antibody.

Antibodies having CDR sequences identical to those of a certain antibody include chimeric antibodies and humanized antibodies. The chimeric antibody and the humanized antibody are as described below.

Another preferred embodiment of the anti-AXL antibody of the present invention may be: an anti-AXL antibody having antagonistic activity against AXL. An anti-AXL antibody having antagonistic activity against AXL means an antibody having an activity of inhibiting AXL-mediated phosphorylation reaction, particularly tyrosine phosphorylation reaction, induced by binding of an AXL ligand (for example, Gas6 or the like) to AXL. The inhibition of the phosphorylation reaction can be performed by inhibiting the binding of AXL ligand to AXL, or can be performed by other methods. The target to which the phosphorylation reaction is inhibited by an anti-AXL antibody having an antagonistic activity is not particularly limited, and examples thereof include autophosphorylation of AXL.

Whether an anti-AXL antibody has an antagonistic activity can be determined by a method known to those skilled in the art, and for example, the following method can be used. The test antibody is contacted with cells expressing AXL (e.g., Calu-1, MDA-MB-231, DU-145, etc.) together with an AXL ligand, and then AXL is extracted from the cells. It was confirmed that no phosphotyrosine was detected in AXL extracted with an anti-phosphotyrosine antibody. More specifically, whether an anti-AXL antibody has an antagonistic activity can be confirmed according to the method described in the examples.

Examples of the anti-AXL antibody having an antagonistic activity include the following antibodies (a) to (d):

(a) an antibody produced by the hybridoma deposited under accession number FERM BP-10850(Ax 7);

(b) an antibody produced by the hybridoma deposited under accession number FERM BP-10851(Ax 51);

(c) an antibody that recognizes the same epitope as the epitope recognized by the antibodies of (a) to (b);

(d) an antibody having the same CDR sequence as any of the antibodies (a) to (c).

The obtaining of antibodies recognizing the same epitope and the obtaining of antibodies having the same CDR sequences can be performed according to the methods described above.

The antibody having antagonistic activity is effective for inhibiting angiogenesis, inhibiting cell proliferation, or the like.

Another preferred mode of the antibody of the present invention may be: an antibody having an activity of reducing the expression level of AXL. In the present invention, the expression level of AXL may be decreased by decomposing AXL or the like to decrease the amount of AXL existing in the past, or by suppressing the expression of AXL to decrease the amount of AXL newly expressed. Whether or not the expression amount of AXL is reduced can be confirmed by a method known to those skilled in the art. For example, the following method can be used. The anti-AXL antibody to be tested is contacted with cells expressing AXL (e.g., Calu-1, MDA-MB-231, DU-145, etc.), and the amount of AXL present in the cells is detected by immunoblotting, etc. The amount of AXL detected when the test antibody is contacted is compared with the amount of AXL detected when the test antibody is not contacted. More specifically, the confirmation may be performed in accordance with the method described in the embodiment.

Examples of anti-AXL antibodies having an activity of reducing the expression level of AXL include the following antibodies (a) to (j):

(a) an antibody produced by the hybridoma deposited under accession number FERM BP-10858(Ax 285);

(b) an antibody produced by the hybridoma deposited under accession number FERM BP-10852(Ax 96);

(c) an antibody produced by the hybridoma deposited under accession number FERM BP-10856(Ax 258);

(d) an antibody produced by the hybridoma deposited under accession number FERM BP-10850(Ax 7);

(e) an antibody produced by the hybridoma deposited under accession number FERM BP-10859(Ax 292);

(f) an antibody (Ax223) produced by the hybridoma deposited under accession number FERM BP-10853;

(g) an antibody produced by the hybridoma deposited under accession number FERM BP-10854(Ax 225);

(h) an antibody produced by the hybridoma deposited under accession number FERM BP-10857(Ax 284);

(i) an antibody that recognizes the same epitope as the epitope recognized by the antibodies of (a) to (h);

(j) an antibody having a CDR sequence identical to that of any of the antibodies (a) to (i).

The obtaining of antibodies recognizing the same epitope and the obtaining of antibodies having the same CDR sequences can be performed according to the methods described above.

An antibody having an activity of reducing the expression level of AXL is effective for inhibiting angiogenesis, inhibiting cell proliferation, or the like.

One of the preferred other modes of the antibody of the present invention may be: an antibody having an angiogenesis inhibitory effect. In the present invention, the angiogenesis inhibiting action is not particularly limited as long as it is an action of inhibiting angiogenesis, and examples thereof include an action of inhibiting migration activity of vascular endothelial cells, an action of inducing apoptosis of vascular endothelial cells, and an action of inhibiting formation of blood vessel morphology of vascular endothelial cells. A preferable example of the antibody having an angiogenesis inhibitory effect is an antibody having an angiogenesis inhibitory effect in a tumor tissue. The tumor tissue is not particularly limited, and examples thereof include: pancreatic cancer tissue (pancreatic cancer tissue, etc.), gastric cancer tissue, lung cancer tissue (small cell lung cancer, non-small cell lung cancer tissue, etc.), osteosarcoma tissue, colorectal cancer tissue, prostate cancer tissue, melanoma tissue, endometrial cancer tissue, ovarian cancer tissue, uterine leiomyoma tissue, thyroid cancer tissue, stem cell cancer tissue, breast cancer tissue, bladder cancer tissue, kidney cancer tissue, glioma tissue, neuroblastoma tissue, esophageal cancer tissue, etc. Further preferred are glioma tissue, gastric cancer tissue, endometrial cancer tissue, non-small cell lung cancer tissue, pancreatic cancer tissue, and breast cancer tissue. Pancreatic cancer tissue and breast cancer tissue are particularly preferable.

Whether or not an antibody has an angiogenesis inhibitory effect can be confirmed by a method known to those skilled in the art, and for example, a commercially available angiogenesis kit can be used. More specifically, confirmation can be performed by the method described in the embodiment, or the like.

Specific examples of the antibody having an angiogenesis inhibitory activity include the above-mentioned antibodies.

One of the preferred modes of the antibody of the present invention may be: an antibody having cell proliferation inhibitory activity.

The cells whose proliferation is inhibited by the anti-AXL antibody are not particularly limited, and cells associated with diseases are preferable, and cancer cells are particularly preferable. When the cell is a cancer cell, the type of cancer is not particularly limited, and examples thereof include: pancreatic cancer (pancreatic cancer, etc.), gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer, etc.), osteosarcoma, colorectal cancer, prostate cancer, melanoma, endometrial cancer, ovarian cancer, uterine leiomyoma, thyroid cancer, stem cell cancer, breast cancer, bladder cancer, renal cancer, glioma, neuroblastoma, esophageal cancer, etc. More preferably glioma, gastric cancer, endometrial cancer, non-small cell lung cancer, pancreatic cancer, and breast cancer. Pancreatic cancer and breast cancer are particularly preferable.

The mechanism for inhibiting cell proliferation by the antibody of the present invention is not particularly limited, and cell proliferation can be inhibited by any mechanism such as inhibition of angiogenesis, inhibition of phosphorylation, induction of phosphorylation, and reduction of AXL expression level.

The following method is preferably used as a method for evaluating or measuring the cell growth inhibitory effect of the anti-AXL antibody.

The method for evaluating or measuring the cell growth inhibitory activity in vitro may employ the following methods: measuring the amount of [ 2], [ added ] to the medium taken up by the living cells³H]Marked with thymidine as a markerIs an indicator of DNA replication ability. A more convenient method is a dye exclusion method in which the ability to exclude dyes such as trypan blue from cells is measured under a microscope, or an MTT method. The latter is a methyl using living cells to convert tetrazolium salt, namely MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide) into blue(formalzan) product capacity. More specifically, the antibody to be detected is added to a culture solution of the cell to be detected together with the ligand, and after a lapse of a certain period of time, an MTT solution is added to the culture solution and allowed to stand for a certain period of time, whereby MTT is taken into the cell. As a result, succinate dehydrogenase in the cell mitochondria reduces the yellow compound MTT to a blue compound. The blue product was dissolved and developed, and then the absorbance thereof was measured, and this was used as an index of the number of living cells. Reagents such as MTS, XTT, WST-1 and WST-8 are also commercially available (nacalai tesque, etc.) in addition to MTT, and can be preferably used. Control antibodies can be used in determining activity.

The method for evaluating or measuring the cell proliferation inhibitory activity in vivo can employ a tumor-bearing murine model. For example, AXL-expressing cancer cells are transplanted into the skin or subcutaneous of a test animal, and then the test antibody is administered intravenously or intraperitoneally every day or every several days from the day or the next day. The size of the tumor is measured at different times, whereby the cell proliferation inhibitory activity can be evaluated. When the control antibody was administered in the same manner as in the in-vitro evaluation, the size of the tumor in the anti-AXL antibody-administered group was significantly smaller than that of the tumor in the control antibody-administered group, and thus the cell proliferation inhibitory activity could be determined. When a mouse is used as the non-human test animal, a nude (nu/nu) mouse with a genetically deficient thymus or a loss of T lymphocyte function can be preferably used. By using such a mouse, T lymphocytes can be excluded from the test animal when evaluating and measuring the cell growth inhibitory activity caused by the administered antibody.

Examples of the anti-AXL antibody having a cell growth inhibitory effect include the above-mentioned antibodies.

The anti-AXL monoclonal antibody of the present invention can be obtained by a known method. The anti-AXL antibody of the invention is particularly preferably a monoclonal antibody from a mammal. The monoclonal antibody derived from a mammal includes an antibody produced by a hybridoma, an antibody produced by a host transformed with an expression vector containing an antibody gene according to a genetic engineering method, and the like.

Hybridomas producing monoclonal antibodies can be prepared by known techniques, for example, as follows. First, the AXL protein was used as a sensitizing antigen, and it was immunized according to a conventional immunization method. The hybridoma is obtained by fusing an immune cell obtained from an immunized animal with a known parent cell according to a conventional cell fusion method. Further, by screening the hybridomas for cells producing the target antibody by a conventional screening method, hybridomas producing an anti-AXL antibody can be selected.

More specifically, the monoclonal antibody is produced, for example, as follows. First, an AXL protein can be obtained by expressing the AXL gene, and the AXL protein is used as a sensitizing antigen for obtaining an antibody. The nucleotide sequence of the human AXL gene is already known (GenBank Accession No. m 76125). That is, a gene sequence encoding AXL is inserted into a known expression vector, an appropriate host cell is transformed, and then the target human AXL protein is purified from the host cell or culture supernatant by a known method. Purified native AXL protein may also be used as such. Purification can be carried out by combining a plurality of types of chromatography such as ordinary ion chromatography or affinity chromatography in a single step or a plurality of steps or by using them alone. Fusion proteins obtained by fusing a desired partial polypeptide of the AXL protein with a different polypeptide can also be used as immunogens. For the preparation of the fusion protein as an immunogen, for example, an Fc fragment of an antibody, a peptide tag, or the like can be used. A vector for expressing the fusion protein can be prepared by fusing the genes encoding the desired two or more polypeptide fragments in frame, inserting the fused genes into an expression vector as described above. Preparation of fusion proteins is described in Molecular Cloning 2nd ed (Sambrook, J.et al, Molecular Cloning 2^nd ed.，9.47-958, Cold Spring Harbor Lab.Press, 1989). The purified AXL protein described above can be used as a sensitizing antigen for the immunization of a mammal.

Partial peptides of AXL may also be used as sensitizing antigens. Examples of partial peptides of AXL include: a peptide obtained by chemical synthesis from the amino acid sequence of human AXL; a peptide obtained by integrating a part of the human AXL gene into an expression vector and expressing the same; peptides obtained by decomposing the human AXL protein with a protease, and the like. The region used as the partial peptide is not particularly limited, and an extracellular region of AXL or the like can be used.

And a partial peptide may preferably use a peptide having all or at least 5 or more consecutive amino acid sequences of FND1 of AXL. The sequence of at least 5 or more consecutive amino acids means a sequence of preferably 6 or more, and more preferably 8 or more consecutive amino acids. At least 5 or more consecutive amino acid sequences refer to an amino acid sequence having antigenicity.

The mammal immunized with the sensitizing antigen is not particularly limited. In order to obtain a monoclonal antibody by the cell fusion method, it is preferable to select an immunized animal in consideration of suitability to a parent cell for cell fusion. Usually, rodents are preferred as the immunized animals. Specifically, a mouse, rat, hamster, or rabbit may be used as the immunized animal. Furthermore, monkeys and the like can be used as the immunized animals.

The animal can be immunized with the sensitizing antigen according to a known method. For example, the conventional methods are: immunization of mammals can be carried out by intraperitoneal or subcutaneous injection of a sensitizing antigen. Specifically, the sensitizing antigen is administered to the mammal a plurality of times every 4 to 21 days. The sensitizing antigen is diluted with PBS (phosphate buffered saline) or physiological saline at an appropriate dilution ratio and used for immunization. Also, the sensitizing antigen can be administered with an adjuvant. For example, the sensitizing antigen can be prepared by mixing and emulsifying the sensitizing antigen with Freund's complete adjuvant. In addition, an appropriate carrier can be used for immunization with a sensitizing antigen. In particular, when a partial peptide having a small molecular weight is used as a sensitizing antigen, it is preferable to immunize the antigen by binding to a carrier protein such as albumin or keyhole limpet hemocyanin (keyhole limpet hemocyanin).

After the mammal is immunized in this manner and the increase in the amount of the desired antibody in the serum is confirmed, immune cells are collected from the mammal for cell fusion. Particularly preferably, spleen cells are used as immune cells.

The cells fused with the above immune cells are mammalian myeloma cells. Preferably, the myeloma cells are provided with appropriate selection markers for selection. Selectable markers refer to the property of being viable (or not) under specific culture conditions. Among the selection markers, hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency), thymidine kinase deficiency (hereinafter abbreviated as TK deficiency), and the like are known. HGPRT-or TK-deficient cells have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells do not synthesize DNA in HAT selective medium and die, but when fused with normal cells, they can continue DNA synthesis by a salvage pathway of normal cells, and therefore can proliferate in HAT selective medium.

HGPRT-deficient or TK-deficient cells can be selected in a medium containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG) or 5' -bromodeoxyuridine, respectively. The normal cells die by taking the pyrimidine analog into DNA, but the cells lacking the enzyme can survive in a selective medium because they do not take the pyrimidine analog. In addition, a selection marker called G418 resistance provides resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) due to a neomycin resistance gene. Various myeloma cells suitable for cell fusion are known. For example, it is possible to use: p3(P3x63Ag8.653) (J.Immunol. (1979)123, 1548-1550), P3x63Ag8U.1(Current Topics in Microbiology and Immunology (1978)81, 1-7), NS-1(Kohler.G.and Milstein, C.Eur.J.Immunol. (1976)6, 511-519), MPC-11(Margulies.D.H. et al, Cell (1976)8, 405-415), SP2/0(Shulman, M. et al, Nature (1978)276, 269-270), FO (de St.Groth, S.F.ethanol., J.Immunol.1980) 35, 1-21), S194 (Trowbridge.Exp.313), Nature (1978, 11-277) myeloma cells (Gal.R.11, 1979, 11-29, 11-1-415).

The above-mentioned cell fusion of the immunocytes with myeloma cells can be carried out by a known method, for example, the method of Kohler and Milstein et al (Kohler.G. and Milstein, C., Methods Enzymol (1981)73, 3 to 46).

More specifically, for example, the cell fusion can be carried out in the presence of a cell fusion promoter in a normal nutrient medium. For example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like can be used as the fusion promoter. In order to further improve the fusion efficiency, an auxiliary agent such as dimethyl sulfoxide may be added as necessary.

The ratio of the immune cells to the myeloma cells can be arbitrarily set. For example, it is preferable that the immune cells are 1 to 10 times as many as myeloma cells. The culture solution for the cell fusion can be, for example: RPMI1640 culture medium and MEM culture medium suitable for proliferation of the above myeloma cell line, and conventional culture medium for culturing the cells. Further, a serum replacement fluid such as Fetal Calf Serum (FCS) may be added to the culture fluid.

Cell fusion can be formed as follows: predetermined amounts of the above immune cells and myeloma cells are mixed well in the above culture solution, and then a PEG solution heated to about 37 ℃ in advance is mixed to form fused cells (hybridomas) of interest. In the cell fusion method, PEG having an average molecular weight of about 1000 to 6000, for example, can be added at a concentration of 30 to 60% (w/v). Then, the appropriate culture medium as exemplified above is added in order, centrifuged to remove the supernatant, and this operation is repeated to remove the cell fusion agent and the like which are not favorable for the growth of the hybridoma.

The hybridoma obtained in this manner can be selected by using a selection medium corresponding to a selection marker possessed by the hybridoma to be used for cell fusion. For example, cells deficient in HGPRT or TK can be selected by culturing in HAT medium (medium containing hypoxanthine, aminopterin and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have succeeded in cell fusion with normal cells can selectively proliferate in HAT culture medium. The culture is continued for a sufficient time using the HAT culture solution in order to cause the death of cells (non-fused cells) other than the target hybridoma. Specifically, the target hybridoma can be selected, usually by culturing for several days to several weeks. Then, by performing a conventional limiting dilution method, screening and monoclonality of a hybridoma producing the target antibody can be performed. Alternatively, an antibody recognizing AXL can also be prepared according to the method described in international publication WO 03/104453.

The screening and monoclonal antibody production of the target antibody can be preferably carried out according to a screening method based on a known antigen-antibody reaction. For example, the antigen is bound to beads made of polystyrene or the like or a carrier such as a commercially available 96-well microtiter plate, and then reacted with a culture supernatant of hybridoma. The carrier is then washed and then reacted with a secondary antibody or the like labeled with an enzyme. When the culture supernatant contains the target antibody that reacts with the sensitizing antigen, the secondary antibody binds to the carrier via the antibody. Finally, by detecting the secondary antibody bound to the carrier, the presence or absence of the antibody of interest in the culture supernatant can be determined. Hybridomas producing desired antibodies capable of binding to antigens can be cloned by limiting dilution or the like. In this case, not only an antigen used for immunization but also an AXL protein having substantially the same properties can be used as appropriate.

In addition to the method of obtaining the above-mentioned hybridoma by immunizing an animal other than a human with an antigen, the target antibody can be obtained by antigen-sensitizing human lymphocytes. Specifically, first, human lymphocytes are sensitized with AXL protein in vitro. The immunosensitized lymphocytes are then fused with the appropriate fusion partner. As the fusion partner, for example, a human-derived myeloma cell having a permanent division ability can be used (see Japanese patent publication No. Hei 1-59878). The anti-AXL antibody obtained by this method is a human antibody having binding activity to the AXL protein.

Furthermore, an anti-AXL antibody can be obtained by administering AXL protein as an antigen to a transgenic animal having all the components of a fully human antibody gene. Antibody-producing cells of an immunized animal can be immortalized by cell fusion with an appropriate fusion partner or by treatment such as EB virus infection. From the immortalized cells thus obtained, a human antibody against the AXL protein can be isolated (see International publications WO 94/25585, WO93/12227, WO 92/03918 and WO 94/02602). Furthermore, by cloning immortalized cells, cells producing antibodies specific to the desired reaction can also be cloned. When a transgenic animal is used as an immunized animal, the immune system of the animal recognizes human AXL as a foreign body. Therefore, a human antibody against human AXL can be easily obtained. The monoclonal antibody-producing hybridoma thus prepared can be subcultured in a conventional culture medium. The hybridomas can also be stored in liquid nitrogen for long periods of time.

The hybridoma is cultured according to a conventional method, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma may be administered to a mammal having a suitability for the hybridoma to proliferate the hybridoma, and the monoclonal antibody may be obtained as ascites thereof. The former method is suitable for obtaining an antibody of high purity.

In the present invention, an antibody encoded by an antibody gene cloned from an antibody-producing cell can also be used. The cloned antibody gene is integrated into an appropriate vector and then introduced into a host, so that it can be expressed in the form of an antibody. Methods for the isolation of antibody genes, the introduction of vectors, and the transformation of host cells have been established (see, e.g., Vandamme, a.m., et al, eur.j.biochem. (1990)192, 767-775).

For example, a cDNA encoding the variable region (V region) of an anti-AXL antibody can be obtained from a hybridoma cell producing the anti-AXL antibody. For this purpose, total RNA is usually first extracted from the hybridomas. Methods for extracting mRNA from cells can be, for example, guanidine ultracentrifugation (Chirgwin, J.M. et al, Biochemistry (1979)18, 5294-5299), AGPC (Chomczynski, P. et al, anal. biochem. (1987)162, 156-159), and the like.

The extracted mRNA can be purified using an mRNA purification kit (manufactured by GE Healthcare Bio-sciences) or the like. Alternatively, a kit for directly extracting total mRNA from cells, such as a QuickPrep mRNA purification kit (manufactured by GE Healthcare Bio-sciences), is also commercially available. Total mRNA can be obtained from the hybridoma using the kit described above. A cDNA encoding the V region of the antibody can be synthesized from the resulting mRNA using reverse transcriptase. The cDNA was synthesized using AMv reverse transcriptase first strand cDNA Synthesis kit (manufactured by Biochemical industries, Ltd.). For the synthesis and amplification of cDNA, the 5 '-Ampli FINDER kit (manufactured by Clontech) and the 5' -RACE method using PCR (Frohman, M.A. et al, Proc. Natl. Acad. Sci. USA (1988)85, 8998-9002, Belyavsky, A. et al, Nucleic Acids Res (1989)17, 2919-2932) can be used. In addition, in the process of synthesizing such cDNA, appropriate restriction enzyme sites described later can be introduced into both ends of the cDNA.

The target cDNA fragment was purified from the resulting PCR product, followed by ligation with vector DNA. The recombinant vector is prepared in this manner, and then introduced into E.coli or the like, and after the colony is selected, the desired recombinant vector is prepared from the E.coli in which the colony has been formed. Whether or not the recombinant vector has the nucleotide sequence of the target cDNA can be confirmed by a known method, for example, the dideoxynucleotide chain termination method.

In order to obtain a gene encoding a variable region, a PCR method using a primer for variable region gene amplification can be used. First, cDNA was synthesized using the extracted mRNA as a template, and a cDNA library was obtained. For the synthesis of cDNA libraries, commercially available kits are convenient. In fact, since only a small number of cells produce a very small amount of mRNA, direct purification of mRNA results in a low yield. Therefore, usually, vector RNA which is clearly free of antibody gene is added and then purified. Alternatively, when a certain amount of RNA can be extracted, even only RNA from antibody-producing cells can be extracted efficiently. For example, when RNA is extracted from antibody-producing cells of 10 or more, or 30 or more, preferably 50 or more, it may not be necessary to add carrier RNA.

The resulting cDNA library was used as a template, and the antibody gene was amplified by PCR. Primers for amplifying antibody genes by the PCR method are well known. For example, primers for amplifying human antibody genes can be designed according to the disclosure of the article (J.mol.biol. (1991)222, 581-597) and the like. These primers form different nucleotide sequences in each immunoglobulin subclass. Therefore, when a cDNA library of an unknown subclass is used as a template, the PCR method is performed in consideration of all the possibilities.

Specifically, for example, in order to obtain a gene encoding human IgG, primers capable of amplifying genes encoding γ 1 to γ 5 as heavy chains, and κ chain and λ chain as light chains can be used. For amplifying the variable region gene of IgG, a primer annealing to a portion corresponding to the hinge region is generally used as the 3' -side primer. In addition, primers corresponding to each subclass can be used as primers on the 5' side.

PCR products obtained from the primers for gene amplification of each subclass of heavy and light chains were made into independent libraries. Using the library so synthesized, immunoglobulins comprising a combination of heavy and light chains can be reconstituted. The antibody of interest can be screened by using the binding activity of the reconstituted immunoglobulin to AXL as an index.

After obtaining a cDNA encoding the V region of the target anti-AXL antibody, the cDNA is digested with restriction enzymes that recognize restriction sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest nucleotide sequences that are less likely to occur in the nucleotide sequences constituting the antibody genes. To further insert a single copy of the digested fragment into the vector in the correct orientation, it is preferred to provide a restriction enzyme with a sticky end. An antibody expression vector can be obtained by inserting the digested cDNA encoding the V region of the anti-AXL antibody into an appropriate expression vector. In this case, a chimeric antibody can be obtained by in-frame fusion of a gene encoding the constant region (C region) of an antibody and a gene encoding the above V region. A chimeric antibody herein means that the constant region and the variable region are derived from different sources. Therefore, in addition to mouse-human and the like heterochimeric antibodies, human-human homochimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector having a constant region in advance.

Specifically, for example, a restriction enzyme recognition sequence of a restriction enzyme digesting the V region gene may be prepared in advance on the 5' side of an expression vector holding a DNA encoding a constant region (C region) of a desired antibody. The two were digested with the same combination of restriction enzymes and subjected to in-frame fusion to construct a chimeric antibody expression vector.

To produce the anti-AXL antibody of the present invention, the antibody gene may be integrated into an expression vector and expressed under the control of an expression control region. Expression control regions for expressing antibodies include, for example, enhancers and promoters. Subsequently, an appropriate host cell is transformed with the expression vector, whereby a recombinant cell expressing a DNA encoding an anti-AXL antibody can be obtained.

When the antibody gene is expressed, DNAs encoding the heavy chain (H chain) and light chain (L chain) of the antibody may be incorporated into different expression vectors. The vector having the H chain and the L chain incorporated therein is simultaneously transformed (co-transfected) into the same host cell, whereby the antibody molecule having the H chain and the L chain can be expressed. Alternatively, the host cell may be transformed by integrating the DNA encoding the H chain and the L chain into a single expression vector (see International publication WO 94/11523).

First, the antibody gene is isolated and introduced into an appropriate host, and many combinations of hosts and expression vectors for producing antibodies are known. These expression systems can be applied to the present invention. When eukaryotic cells are used as the host, animal cells, plant cells or fungal cells can be used. Specifically, as animal cells that can be used in the present invention, for example, there can be used: mammalian cells (CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, etc.), amphibian cells (Xenopus laevis oocytes, etc.), insect cells (sf9, sf21, Tn5, etc.), etc.

Alternatively, as plant cells, expression systems of antibody genes produced by cells of Nicotiana (Nicotiana) such as Nicotiana tabacum are known. In transformation of plant cells, cells cultured from callus may be used.

Further, as the fungal cell, the following cells can be used. Yeast: genus Saccharomyces such as Saccharomyces cerevisiae and genus Pichia such as methylotrophic yeast (Pichia pastoris); filamentous fungi: aspergillus (Aspergillus) such as Aspergillus niger.

Alternatively, expression systems using antibody genes from prokaryotic cells are also known. For example, when bacterial cells are used, bacterial cells such as Escherichia coli (E.coli) and Bacillus subtilis can be used in the present invention.

When mammalian cells are used, an expression vector can be constructed in which a polyA signal is functionally linked to a commonly used effective promoter, an antibody gene to be expressed, and the 3' -side downstream thereof. For example, the promoter/enhancer may be the human cytomegalovirus early promoter/enhancer.

In addition, other examples of promoters/enhancers that may be used to express the antibodies of the invention are: a viral promoter/enhancer, a promoter/enhancer derived from mammalian cells such as human elongation factor 1 α (HEF1 α), and the like. Examples of viruses that can use promoters and enhancers include: retroviruses, polyoma viruses, adenoviruses, simian virus 40(SV40), and the like.

When the SV40 promoter/enhancer is used, the method of Mulligan et al (Nature (1979)277, 108) can be used. In addition, the HEF1 α promoter/enhancer can be readily used for expression of a gene of interest using the method of Mizushima et al (Nucleic Acids Res. (1990)18, 5322).

In the case of E.coli, the gene can be expressed by functionally linking a commonly used effective promoter, a signal sequence for antibody secretion, and the antibody gene to be expressed. Examples of the promoter include lacZ promoter and araB promoter. When the lacZ promoter is used, the method of Ward et al (Nature (1989)341, 544 to 546; FASEBJ. (1992)6, 2422 to 2427) can be employed. Alternatively, the araB promoter can be used for expression of a target gene by the method of Better et al (Science (1988)240, 1041 to 1043).

When the signal sequence for antibody secretion is produced in the periplasm of E.coli, the pel B signal sequence may be used (Lei, S.P. et al, J.Bacteriol. (1987)169, 4379). Next, the antibody produced in the periplasm is isolated, and then the structure of the antibody can be refolded to have a desired binding activity by using a protein modifier such as guanidine hydrochloride of urea.

As the origin of replication to be inserted into the expression vector, those derived from SV40, polyoma virus, adenovirus, Bovine Papilloma Virus (BPV) and the like can be used. Furthermore, in order to amplify the gene copy number in the host cell system, a selection marker may be inserted into the expression vector. Specifically, the following selection markers can be used: an Aminoglycoside Phosphotransferase (APH) gene; a Thymidine Kinase (TK) gene; e.coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene; dihydrofolate reductase (dhfr) gene, and the like.

These expression vectors are introduced into host cells, and then the transformed host cells are cultured in vitro or in vivo to produce the target antibody. The host cell can be cultured according to a known method. For example, DMEM, MEM, RPMI1640, IMDM may be used as the culture medium, and a serum replacement solution such as Fetal Calf Serum (FCS) may be used in combination.

The antibody expressed and produced as described above can be purified by using known methods used in conventional protein purification, either alone or in an appropriate combination. For example, Antibodies can be isolated and purified by appropriately selecting and combining affinity columns such as protein A columns, chromatography columns, filters, ultrafiltration, salting out, dialysis, and the like (Antibodies A Laboratory Manual. Ed Harbor, David Lane, Cold Spring Harbor Laboratory, 1988).

In addition, in the production of recombinant antibodies, transgenic animals may be used in addition to the above-described host cells. That is, a gene encoding an antibody of interest is introduced into an animal, and the antibody can be obtained from the animal. For example, fusion genes can be constructed by inserting antibody genes in-frame into genes encoding proteins produced by traits in milk. As the protein secreted in milk, for example, goat β casein or the like can be used. A DNA fragment containing a fusion gene into which an antibody gene has been inserted is injected into a goat embryo, and the injected embryo is introduced into a female goat. The goat which received the embryo gives rise to a transgenic goat from which the desired antibody can be obtained as a fusion protein with a milk protein in the milk produced by the transgenic goat (or its offspring). In addition, in order to increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones may be suitably used for the transgenic goat (Ebert, K.M. et al, Bio/Technology (1994)12, 699-702). The C region of the recombinant antibody of the present invention may be the C region of an animal antibody. For example, the H chain C region of a mouse antibody may be C.gamma.1, C.gamma.2 a, C.gamma.2 b, C.gamma.3, C.mu.C.delta.C.alpha.1, C.alpha.2, C.epsilon.C.and the L chain C region may be C.kappa.C.lambda.C. As the animal antibody other than mouse, an animal antibody of rat, rabbit, goat, sheep, camel, monkey, or the like can be used. Their sequences are well known. The C region may be modified in order to improve the stability of the antibody or its production. In the present invention, when the antibody is administered to a human, a genetically modified recombinant antibody can be prepared in order to reduce the antigenicity of the human. Recombinant antibodies include, for example: chimeric antibodies, humanized antibodies, and the like.

These modified antibodies can be prepared by a known method. A chimeric antibody is an antibody in which variable regions and constant regions of different origins are linked to each other. For example, an antibody comprising the variable regions of the heavy and light chains of a mouse antibody and the constant regions of the heavy and light chains of a human antibody is a mouse-human-xenochimeric antibody. A recombinant vector for expressing a chimeric antibody can be prepared by ligating a DNA encoding a variable region of a mouse antibody with a DNA encoding a constant region of a human antibody and integrating them into an expression vector. The chimeric antibody produced in the culture can be obtained by culturing the recombinant cell transformed with the vector to express the integrated DNA. The C region of a human antibody is used for the C regions of the chimeric antibody and the humanized antibody. For example, in the H chain, C.gamma.1, C.gamma.2, C.gamma.3, C.gamma.4, C.mu.C.delta.C.alpha.1, C.alpha.2 and C.epsilon.can be used as the C region. In the L chain, C.kappa.and C.lambda.can be used as the C region. The amino acid sequence of the above-mentioned C region and the nucleotide sequence encoding the amino acid sequence are known. To improve the stability of the antibody itself or of the antibody production, the C region of the human antibody may be modified.

Generally, a chimeric antibody is composed of a V region derived from an antibody derived from an animal other than a human and a C region derived from a human antibody. In contrast, a humanized antibody is composed of Complementarity Determining Regions (CDRs) of an antibody derived from an animal other than a human, Framework Regions (FRs) derived from a human antibody, and a C region derived from a human antibody. The humanized antibody has reduced antigenicity in humans and is therefore useful as an active ingredient of the therapeutic agent of the present invention.

Antibody variable regions are typically composed of 3 Complementarity Determining Regions (CDRs) sandwiched between 4 Framework Regions (FRs). CDRs are essentially the regions that determine the binding specificity of an antibody. The amino acid sequences of the CDRs are rich in diversity. On the one hand, the amino acid sequences constituting the FRs often show high homology even among antibodies having different binding specificities. Thus, the binding specificity of an antibody can be grafted to other antibodies, usually by grafting CDRs.

Humanized antibodies are also known as reshaped (reshaped) human antibodies. Specifically, humanized antibodies and the like in which CDRs of an animal other than a human, for example, a mouse antibody are grafted onto a human antibody are known. Conventional genetic recombination methods for obtaining humanized antibodies are also known.

Specifically, as a method for grafting CDRs of a mouse antibody into human FRs, for example, overlap Extension pcr (overlap Extension pcr) is known. In the overlap sequence extension PCR, the grafted nucleotide sequence encoding the CDR of the mouse antibody is added to primers for synthesizing the FR of the human antibody. Primers for 4 FRs were prepared. In general, when a mouse CDR is grafted to a human FR, it is advantageous to select a human FR having high homology with the mouse FR in order to maintain the function of the CDR. That is, it is generally preferable to use a human FR comprising an amino acid sequence having high amino acid sequence homology with the FR adjacent to the grafted mouse CDR.

In addition, the linked nucleotide sequences are designed to be linked in frame with each other. Human FRs can be synthesized separately by specific primer sets. As a result, a product in which DNA encoding the mouse CDR was added to each FR was obtained. The nucleotide sequences encoding the mouse CDRs of each product were designed to overlap each other. Subsequently, the overlapping CDR portions of the product synthesized using the human antibody gene as a template are annealed to each other, and a complementary strand synthesis reaction is performed. By this reaction, human FRs are linked via mouse CDR sequences.

Finally, the full-length V region gene to which 3 CDRs and 4 FRs are ligated is amplified using primers that anneal to their 5 'and 3' ends and are appended with appropriate restriction enzyme recognition sequences. The DNA obtained as described above and a DNA encoding a human antibody C region are inserted into an expression vector and fused in frame, thereby preparing a human antibody expression vector. The humanized antibody is produced in a culture of the cultured cells by introducing the recombinant vector into a host to create recombinant cells, and then culturing the recombinant cells to express a DNA encoding the humanized antibody (see European patent publication EP 239400 and International publication WO 96/02576).

By qualitatively or quantitatively measuring and evaluating the binding activity of the humanized antibody prepared as described above to an antigen, the FR of a human antibody in which the CDR forms a good antigen-binding site when linked via the CDR can be appropriately selected. If necessary, the amino acid residues of the FR may be substituted so that the CDRs of the reshaped human antibody form an appropriate antigen-binding site. For example, a mutation of an amino acid sequence can be introduced into an FR by using a PCR method for grafting a mouse CDR into a human FR. Specifically, a mutation of a part of the nucleotide sequence may be introduced into a primer annealing to FR. The FR synthesized by using such a primer has a mutation in the nucleotide sequence introduced therein. By measuring and evaluating the binding activity of the mutant antibody in which the amino acid has been substituted to the antigen by the above-described method, a mutant FR sequence having desired properties can be selected (Sato, K. et al, Cancer Res, 1993, 53, 851-856).

In addition, methods for obtaining human antibodies are also known. For example, human lymphocytes are sensitized in vitro with a desired antigen or cells expressing a desired antigen. Subsequently, the sensitized lymphocytes are fused with human myeloma cells to obtain a desired human antibody having an antigen-binding activity (see Japanese examined patent publication (Kokoku) No. 1-59878). For example, U266 or the like can be used for human myeloma cells as a fusion partner.

The desired human antibody can be obtained by immunizing a transgenic animal having all the components of a fully human antibody gene with the desired antigen (see International publications WO93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735). Also, a technique for obtaining a human antibody by panning using a human antibody library is known. For example, phage display can be used to express a human antibody V region as a single chain antibody (scFv) on the surface of a phage, thereby selecting a phage that binds to an antigen. By analyzing the genes of the selected phage, the DNA sequence encoding the V region of a human antibody that binds to the antigen can be determined. After the DNA sequence of the scFv that binds to the antigen has been determined, the V region sequence is fused in frame with the desired human antibody C region sequence, and then inserted into an appropriate expression vector, thereby preparing an expression vector. The human antibody can be obtained by introducing the expression vector into the above-exemplified preferred expression cells and expressing the gene encoding the human antibody. These methods are already known (International publications WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388).

The antibody of the present invention may bind to the AXL protein, and includes not only a bivalent antibody represented by IgG but also a monovalent antibody or a multivalent antibody represented by IgM. Multivalent antibodies of the invention include: multivalent antibodies with identical antigen binding sites, or multivalent antibodies with a portion or completely different antigen binding sites. The antibody of the present invention is not limited to the full length molecule of the antibody, and may be a small molecule antibody or a modified product thereof as long as it binds to the AXL protein.

Small molecule antibodies include antibody fragments in which a portion of a full length antibody (e.g., full length IgG, etc.) is deleted. As long as it has binding ability to the AXL antigen, partial deletion of the antibody molecule is allowed. The antibody fragment of the present invention preferably contains a heavy chain variable region (VH) and/or a light chain variable region (VL). The amino acid sequence of VH or VL may include substitutions, deletions, additions and/or insertions. Furthermore, VH and/or VL may be partially deleted as long as they have a binding ability to the AXL antigen. In addition, the variable region may be chimeric or humanized. Specific examples of the antibody fragment include: fab, Fab ', F (ab') 2, Fv, etc. Specific examples of the small molecule antibody include: fab, Fab ', F (ab') 2, Fv, scFv (single chain Fv), diabody, sc (Fv)2 (single chain (Fv)2), and the like. Multimers (e.g., dimers, trimers, tetramers, multimers) of these antibodies are also encompassed by the small molecule antibodies of the invention.

The antibody fragment can be obtained by treating an antibody with an enzyme to produce an antibody fragment. Enzymes for producing antibody fragments, such as papain, pepsin, and plasmin, are known. Alternatively, genes encoding these antibody fragments are constructed, introduced into expression vectors, and then expressed in appropriate host cells (see, e.g., Co, M.S. et al, J.Immunol. (1994)152, 2968-2976, Better, M. and Horwitz, A.H.methods in Enzymology (1989)178, 476-496, Plcuethroughout, A. and Skerra, A.methods in Enzymology (1989)178, 476-496, Lamoyi, E. Methods in Enzymology (1989)121, 652-663, Rousseaux, J. et al, Methods in Enzymology (1989)121, 663-669, Bird, R.E. et al, TIBTECH (1991)9, 137).

The digestion enzyme cleaves a specific site of the antibody fragment to obtain an antibody fragment of the following specific structure. When the antibody fragment obtained by the above enzymatic hydrolysis is subjected to a genetic engineering method, any part of the antibody can be deleted.

And (3) papain digestion: f (ab)2 or Fab;

and (3) pepsin digestion: f (ab ') 2 or Fab';

plasmin digestion: facb.

Diabodies (diabodies) refer to bivalent antibody fragments constructed by gene fusion (Holliger P et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP404,097; WO 93/11161 et al). Diabodies are dimers consisting of two polypeptide chains. Typically, the polypeptide chains that make up the dimer, each VL and VH in the same chain, are joined by a linker. The linker in diabodies is typically so short that VL and VH cannot bind to each other. Specifically, the amino acid residues constituting the linker are, for example, about 5 residues. Thus, VL and VH encoded on the same polypeptide chain cannot form a single chain variable segment, but rather form a dimer with another single chain variable segment. As a result, the diabody has two antigen binding sites.

scFv can be obtained by linking the H chain V region and L chain V region of an antibody. In scFv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, J.S. et al, Proc. Natl.Acid.Sci.U.S.A.1988, 85, 5879-5883). The H chain V region and the L chain V region in the scFv may be derived from any of the antibodies described in the present specification. The peptide linker connecting the V regions is not particularly limited. For example, any single-chain peptide containing about 3 to 25 residues can be used as a linker. The V region can be ligated by, for example, the PCR method described above. In order to perform V region ligation by the PCR method, first, a DNA encoding all or a desired partial amino acid sequence of the DNA sequence encoding the H chain or H chain V region of the above-mentioned antibody and the DNA sequence encoding the L chain or L chain V region of the above-mentioned antibody is used as a template.

The DNAs encoding the V regions of the H chain and the L chain are amplified by PCR using a pair of primers having sequences corresponding to the sequences at both ends of the DNA to be amplified. Next, a DNA encoding the peptide linker moiety is prepared. DNA encoding a peptide linker can also be synthesized using PCR. The primer used at this time is added with a nucleotide sequence that can be ligated to the amplification product of each V region that is additionally synthesized, on the 5' side. Then, PCR reaction was carried out using each of the DNAs [ H chain V region DNA ] - [ peptide linker DNA ] - [ L chain V region DNA ] and a primer for assembly PCR. The primer for assembly PCR comprises a combination of a primer that anneals to the 5 '-side of [ H chain V region DNA ] and a primer that anneals to the 3' -side of [ L chain V region DNA ]. That is, the assembly of PCR primers is a primer set capable of amplifying DNA encoding the full-length sequence of scFv to be synthesized. On the other hand, [ peptide linker DNA ] is added with a nucleotide sequence that can be ligated to each V region DNA. As a result, the DNAs are ligated together, and PCR primers are assembled to finally produce scFv in full length as an amplified product. DNA encoding scFv is prepared once, and an expression vector containing the DNA and a recombinant cell transformed with the expression vector can be obtained according to a conventional method. As a result, the obtained recombinant cell is cultured to express DNA encoding the scFv, thereby obtaining the scFv.

sc (fv)2 is a small molecule antibody in which two VH and two VL are connected by a linker or the like to form a single chain (Hudson et al, J immunol. methods 1999; 231: 177-189). sc (fv)2 can be prepared, for example, by linking an scFv with a linker.

Preferred are antibodies having the following characteristics: the 2 VH and 2 VL are arranged in the order of VH, VL, VH and VL ([ VH ] -linker- [ VL ] -linker- [ VH ] -linker- [ VL ]) with the N-terminal side of the single-chain polypeptide as the base.

As the linker for linking the antibody variable region, any peptide linker that can be introduced by genetic Engineering, chemically synthesized linker (for example, the linkers disclosed in Protein Engineering, 9(3), 299 to 305, 1996) and the like can be used. Peptide linkers are preferred in the present invention. The length of the peptide linker is not particularly limited, and may be appropriately selected by those skilled in the art according to the purpose. The amino acid residue constituting the peptide linker is usually 1 to 100 amino acids, preferably 3 to 50 amino acids, more preferably 5 to 30 amino acids, and particularly preferably 12 to 18 amino acids (for example, 15 amino acids).

The amino acid sequence constituting the peptide linker may be any sequence as long as it does not inhibit the binding action of scFv.

Alternatively, chemically synthesized linkers (chemical crosslinkers) can also be used to link the V regions. In the present invention, a crosslinking agent generally used for crosslinking a peptide compound or the like can be used. For example, N-hydroxysuccinimide (NHS); disuccinimidyl suberate (DSS); bis (sulfosuccinimidyl) suberate (BS 3); dithiobis (succinimidyl propionate) (DSP); dithiobis (sulfosuccinimidyl propionate) (DTSSP); ethylene glycol bis (succinimidyl succinate) (EGS); ethylene glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS); disuccinimidyl tartrate (DST); disulfosuccinimidyl tartrate (sulfo-DST); bis [2- (succinimidyloxycarbonyloxy) ethyl ] sulfone (BSOCOES); and bis [2- (sulfosuccinimidyloxycarbonyloxy) ethyl ] sulfone (sulfo-BSOCOES), and the like.

To link 4 antibody variable regions, typically 3 linkers are required. The same or different linkers may be used for the plurality of linkers. In the present invention, the preferred small molecule antibody is a diabody or sc (fv) 2. In order to obtain such a small molecule antibody, the antibody may be treated with an enzyme such as papain, pepsin, or the like to produce an antibody fragment; alternatively, DNAs encoding these antibody fragments can be constructed, introduced into expression vectors, and then expressed in appropriate host cells (see, for example, Co, M.S. et al, J.Immunol. (1994)152, 2968-2976; Better, M. and Horwitz, A.H., Methods Enzymol. (1989)178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989)178, 497-515; Lamori, E., Methods Enzymol. (1986)121, 652-663; Rousseaux, J. et al, Methods Enzymol. (1986)121, 663-669; 137, R.E. and Bilker, B.W., Waldnol. (1991).

The antibody of the present invention can be further used as an antibody-modified product to which various molecules such as polyethylene glycol (PEG) and cytotoxic substances are bonded. The modified antibody can be obtained by chemically modifying the antibody of the present invention. Methods for modifying antibodies are well established in the art.

Also, the antibody of the invention may be a bispecific antibody. Bispecific antibodies are antibodies that have variable regions within the same antibody molecule that recognize different epitopes, although the epitopes can be present in different molecules or can be present in the same molecule. That is, in the present invention, the bispecific antibody may have an antigen binding site that recognizes a different epitope on the AXL molecule. Alternatively, the antibody may be a bispecific antibody in which one recognition site recognizes AXL and the other recognition site recognizes a cytotoxic substance. The "antibody" in the present invention also encompasses these antibodies.

In the present invention, bispecific antibodies recognizing antigens other than AXL may be combined. For example, a bispecific antibody that recognizes an antigen that is specifically expressed on the cell surface of a cancer cell that targets the same as AXL but is different from AXL may be combined.

Methods for making bispecific antibodies are well known. For example, a bispecific antibody can be prepared by binding two antibodies recognizing different antigens. The antibody to be bound may be 1/2 molecules each having an H chain and an L chain, or 1/4 molecules containing only an H chain. Alternatively, a fusion cell producing a bispecific antibody can be prepared by fusing hybridomas producing different monoclonal antibodies. Also, bispecific antibodies can be prepared by genetic engineering methods.

Binding Activity of antibodies

The antigen binding activity of an antibody can be measured by a known method (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). For example, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), or fluoroimmunoassay can be used. The method for measuring the binding activity between an antibody expressed in a cell and an antigen is, for example, the method described on pages 395 to 420 of the aforementioned Antibodies A Laboratory Manual.

The method of measuring the binding between an antigen expressed on the cell surface and an antibody against the antigen suspended in a buffer or the like is particularly preferably a method using a flow cytometer. Examples of flow cytometers used include: FACSCANTO^TM II、FACSAria^TM、FACSArray^TM、FACSVantage^TM SE、FACSCalibur^TM(from BD Biosciences, supra), or EPICS ALTRA HyPerSort, Cytomics FC 500, EPICS XL-MCL ADC, EPICS XL ADC, Cell Lab Quanta/Cell Lab Quanta SC (from Beckman Coulter, supra), and the like.

An example of a preferred method for measuring the binding activity of a test AXL antibody to an antigen is: a method comprising staining with a FITC-labeled secondary antibody that recognizes an antibody to be detected that reacts with CELLs expressing AXL, measuring the stained secondary antibody with FACS Calibur (BD Co.), and analyzing the fluorescence intensity using CELL QUEST software (BD Co.).

Hybridoma cell

The present invention further provides the hybridoma deposited under accession number FERM BP-10858(AX285), FERM BP-10859(AX292), FERM BP-10853(AX223), FERM BP-10852(AX96), FERM BP-10856(AX258), FERM BP-10857(AX284), FERM BP-10850(Ax7), FERM BP-10851(Ax51), FERM BP-10854(Ax225), FERM BP-10855(Ax 232). The hybridoma produces an anti-AXL antibody having agonistic activity, an anti-AXL antibody having antagonistic activity, an anti-AXL antibody having activity of reducing the expression level of AXL, an anti-AXL antibody having angiogenesis inhibitory activity, and/or an anti-AXL antibody having cell proliferation inhibitory activity.

Angiogenesis inhibitors

The present invention further provides an angiogenesis inhibitor comprising an anti-AXL antibody. The mechanism for inhibiting angiogenesis is not particularly limited, and examples thereof include: inhibition of migration activity of vascular endothelial cells, induction of apoptosis of vascular endothelial cells, inhibition of vascular morphogenesis of vascular endothelial cells, and the like. Preferably, the angiogenesis inhibitor of the present invention inhibits angiogenesis in tumor tissue. The tumor tissue is not particularly limited, and examples thereof include: pancreatic cancer tissue (pancreatic cancer tissue, etc.), gastric cancer tissue, lung cancer tissue (small cell lung cancer, non-small cell lung cancer tissue, etc.), osteosarcoma tissue, colorectal cancer tissue, prostate cancer tissue, melanoma tissue, endometrial cancer tissue, ovarian cancer tissue, uterine leiomyoma tissue, thyroid cancer tissue, stem cell cancer tissue, breast cancer tissue, bladder cancer tissue, kidney cancer tissue, glioma tissue, neuroblastoma tissue, esophageal cancer tissue, etc. Further preferred are glioma tissue, gastric cancer tissue, endometrial cancer tissue, non-small cell lung cancer tissue, pancreatic cancer tissue, and breast cancer tissue. Pancreatic cancer tissue and breast cancer tissue are particularly preferable.

The antibody used in the angiogenesis inhibitor of the present invention is not particularly limited as long as it has an angiogenesis inhibitory effect, and for example, the above-mentioned antibody (an antibody having an agonistic activity, an antibody having an antagonistic activity, an antibody having an activity of reducing the expression level of AXL, and the like) can be used.

The angiogenesis inhibitor containing an anti-AXL antibody of the present invention is also expressed as a method for inhibiting angiogenesis using an anti-AXL antibody. The angiogenesis inhibitor containing an anti-AXL antibody of the present invention also represents the use of an anti-AXL antibody for the production of an angiogenesis inhibitor.

Cell proliferation inhibitor

The present invention further provides a cell proliferation inhibitor containing an anti-AXL antibody. The mechanism for inhibiting cell growth is not particularly limited, and may be, for example: mechanisms based on angiogenesis inhibition, on cytotoxic activity of antibodies, or on cytotoxic substances bound to antibodies, and the like, mechanisms based on angiogenesis inhibition are preferred.

The cell whose proliferation is inhibited by the anti-AXL antibody is not particularly limited, and is preferably a cell associated with a disease, and more preferably a cancer cell. Therefore, a preferred embodiment of the cell growth inhibitor of the present invention is an anticancer agent containing an anti-AXL antibody. When the cell is a cancer cell, the type of cancer is not particularly limited, and examples thereof include: pancreatic cancer (pancreatic cancer, etc.), gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer, etc.), osteosarcoma, colorectal cancer, prostate cancer, melanoma, endometrial cancer, ovarian cancer, uterine leiomyoma, thyroid cancer, stem cell cancer, breast cancer, bladder cancer, renal cancer, glioma, neuroblastoma, esophageal cancer, etc. More preferably glioma, gastric cancer, endometrial cancer, non-small cell lung cancer, pancreatic cancer, and breast cancer. Pancreatic cancer and breast cancer are particularly preferable.

The antibody used in the cell growth inhibitor of the present invention is not particularly limited as long as it has a cell growth inhibitory activity, and for example, the above-mentioned antibody (an antibody having an agonistic activity, an antibody having an antagonistic activity, an antibody having an activity of reducing the expression level of AXL, and the like) can be used.

The cell growth inhibitor containing an anti-AXL antibody of the present invention may be expressed as a method for inhibiting cell growth using an anti-AXL antibody. When the cells whose proliferation is inhibited are cancer cells, the anti-cancer agent containing an anti-AXL antibody of the present invention can also be used as a method for treating and/or preventing cancer using an anti-AXL antibody. The cell growth inhibitor containing an anti-AXL antibody of the present invention may be used for the preparation of a cell growth inhibitor containing an anti-AXL antibody. When the cell whose proliferation is inhibited is a cancer cell, the use of an anti-AXL antibody for the production of an anti-cancer agent is also expressed.

Phosphorylation inducer

The present invention further provides a phosphorylation-inducing agent comprising an anti-AXL antibody. The phosphorylation inducer of the present invention generally induces phosphorylation in cells expressing AXL. The subject to which phosphorylation is induced is not particularly limited, but is usually a polypeptide having tyrosine, preferably AXL.

The antibody used in the phosphorylation inducer of the present invention is not particularly limited, and for example, the above-described antibody having an agonistic activity can be used.

The phosphorylation-inducing agent containing an anti-AXL antibody of the present invention is also expressed as a method for inducing phosphorylation using an anti-AXL antibody. The phosphorylation inducer containing an anti-AXL antibody of the present invention is also expressed as an application of an anti-AXL antibody in the preparation of a phosphorylation inducer.

Phosphorylation inhibitors

The present invention further provides phosphorylation inhibitors comprising anti-AXL antibodies. The phosphorylation inhibitor of the present invention generally inhibits phosphorylation induced by binding of AXL ligand (for example, Gas6 or the like) to AXL. The target to be inhibited from phosphorylation is not particularly limited, but is usually a polypeptide having tyrosine, preferably AXL.

The antibody used in the phosphorylation inhibitor of the present invention is not particularly limited, and for example, the above-mentioned antibody having an antagonistic activity can be used.

The phosphorylation inhibitor containing an anti-AXL antibody of the present invention is also expressed as a method for inhibiting phosphorylation using an anti-AXL antibody. The phosphorylation inducer containing an anti-AXL antibody of the present invention is also indicated as an application of an anti-AXL antibody in the preparation of a phosphorylation inhibitor.

AXL expression level-reducing agent

The present invention further provides an agent for reducing the amount of AXL expression, which comprises an anti-AXL antibody. The agent for reducing the expression level of AXL is an agent which reduces the expression level of AXL in cells expressing AXL. The cells expressing AXL are not particularly limited, and examples thereof include cancer cells (Calu-1, MDA-MB-231, DU-145, etc.).

The decrease in the amount of AXL expression may be a decrease in the amount of AXL already present due to decomposition of AXL or the like, or a decrease in the amount of AXL newly expressed by inhibiting the expression of AXL.

The agent for reducing the expression level of AXL comprising an anti-AXL antibody of the present invention is also expressed as a method for reducing the expression level of AXL using an anti-AXL antibody. The AXL expression level reducing agent containing the anti-AXL antibody of the present invention is also expressed as an application of the anti-AXL antibody to the preparation of an AXL expression level reducing agent.

Pharmaceutical composition

The method of administering the angiogenesis inhibitor, the cell proliferation inhibitor, the phosphorylation inducer, the phosphorylation inhibitor or the AXL expression level lowering agent of the present invention may be carried out by any of oral administration and parenteral administration. Particular preference is given to methods of administration by parenteral administration. The administration method specifically comprises the following steps: injection, nasal administration, pulmonary administration, transdermal administration, etc. Examples of administration by injection are: for example, the pharmaceutical composition of the present invention can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. The appropriate administration method can also be selected according to the age and symptoms of the patient. The dose can be selected, for example, from 0.0001mg to 1000mg per kg body weight per time. Alternatively, the dose may be selected, for example, from 0.001mg to 100000mg per patient. However, the pharmaceutical composition of the present invention is not limited to the above-mentioned dosage.

The angiogenesis inhibitor, cell proliferation inhibitor, phosphorylation inducer, phosphorylation inhibitor or AXL expression level reducing agent of the present invention can be prepared into a preparation according to a conventional method (for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S. A), and may contain a pharmaceutically acceptable carrier or additive. Examples include: surfactants, excipients, colorants, fragrances, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flowability enhancers, flavoring agents, and the like. The carrier is not limited to these, and other commonly used carriers may be suitably used. The method specifically comprises the following steps: light silicic anhydride, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, and the like.

It is to be noted that all the prior art documents cited in the present specification are incorporated herein by reference.

Examples

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1

1-1 antigen preparation

A gene of a fusion protein (hAXL-ECD-mIgG2aFc) expression vector fusing the extracellular domain of human AXL and the Fc region of mouse IgG2a was introduced into hamster ovary cells (CHO (dhfr-) cells), and a CHO cell line producing hAXL-ECD-mIgG2aFc protein was cloned by the G418 screening method. The culture supernatant of the recovered CHO cell line producing hAXL-ECD-mIgG2aFc Protein was added to a Protein G column (HiTrap Protein GHP, GE Healthcare) equilibrated with a binding buffer (20mM phosphate buffer, pH7.0) using a serum-free medium (CHO-S-SFM II, GIBCO). After washing the unbound protein with the binding buffer, the fractions of hAXL-ECD-mIgG2aFc protein were collected in a tube into which the neutralizing buffer (1M Tris-HCl, pH9.0) had been dispensed, using an elution buffer (100mM Glycine-HCl, pH2.7), and the purified protein was concentrated by buffer-replacing the buffer for the purified protein with phosphate buffered saline (pH 7.35-7.65, Takara Bio) using an ultrafiltration kit (Centricon (registered trademark), Millipore) having a cut-off of 10 kDa. As per c.n.pace et al, prof.sci.4: 2411 to 2423, (1995) calculating molar absorption coefficient, and using this coefficient, the concentration of the purified protein was calculated from the absorbance at 280 nm.

1-2 preparation of hybridomas producing anti-AXL antibody

The antigen prepared in the previous paragraph (hAXL-ECD-mIgG2aFc protein) was immunized as follows against 4 BALB/c mice (male, 6 weeks old at the start of immunization, Charles River Japan) and 2 MRL/lpr mice (male, 6 weeks old at the start of immunization, Charles River Japan). The primary immunization was performed by administering the antigen emulsified with FCA (Freund's complete adjuvant H37Ra (Difco laboratories)) subcutaneously at 40. mu.g/head. After 2 weeks, antigen emulsified with FIA (Freund's incomplete adjuvant) was administered subcutaneously at 40. mu.g/head. Additional immunizations were performed 3 times at 1 week intervals thereafter. The increase in serum antibody titer to the antigen was confirmed by ELISA (enzyme-linked immunosorbent assay) shown in the following item, and the antigen diluted with phosphate-buffered physiological saline (phosphate buffer solution (PBS (-)) containing no calcium ion or magnesium ion, daily pharmacy) was intravenously administered at 10. mu.g/head as the final immunization. 3 days after the final immunization, spleen cells of mice and mouse myeloma cells P3X63Ag8U.1 (designated as P3U1, ATCC CRL-1597) were subjected to cell fusion according to a conventional method using PEG1500(Roche Diagnostics). The fused cells were cultured in RPMI1640 medium (Invitrogen) containing 10% FBS (Invitrogen) (hereinafter referred to as 10% FBS/RPMI 1640). The day after the fusion, the fused cells were suspended in a semi-flow medium (StemCells) and hybridoma was selectively cultured and simultaneously the hybridoma was cloned. On day 9 or 10 after the fusion, colonies of hybridomas were picked and inoculated into 96-well plates to which HAT selection medium (10% FBS/RPMI1640, 2 vol% HAT 50X concentrate (Dainippon pharmaceutical), 5 vol% BM-conditioned H1(Roche Diagnostics)) was added, at 1 colony per well. After 3 to 4 days of culture, culture supernatants from the wells were collected, and the binding activity to the antigen and a control protein fused to the Fc region of mouse IgG2a was measured by ELISA as described below, thereby selecting hybridomas having a binding activity to the extracellular region of human AXL.

The binding activity of the selected hybridoma supernatants is shown in table 1.

[ Table 1]

AXL	2nd SC Abs	2nd SC Abs	2nd SC Abs	2nd SC Abs	IgG
						Clone No.	AXL-mFc	FGFR2-mFc	AbsΔ	AXL-His	Bonding of
7	2.053	0.057	1.996	1.118	0.66
						51	1.844	0.058	1.786	0.538	0.55

[0316]

232	1.353	0.061	1.292	1.204	0.575
						96	2.122	0.058	2.064	1.554	0.635
119	2.208	0.063	2.145	1.527	0.668
						223	2.076	0.071	2.005	1.542	0.339
225	0.629	0.055	0.574	0.642	0.859
						258	2.005	0.078	1.927	1.028	0.74
284	0.619	0.064	0.555	0.124	0.857
						285	1.804	0.058	1.746	0.914	0.965
292	1.877	0.069	1.808	1.234	1.052

The hybridoma selected by the present inventors is deposited in the International patent organism depositary, national institute of advanced Industrial science and technology. The contents of the specific deposit are described below.

(a) Name and address of depository

Name: patent biological collection center of institute for Integrated Industrial and technology research of independent administrative Law

Address: 1-Dimo 1-Fan 1 central 6 (zip code 305-8566)

(b) Preservation day: 2007, 7 and 5 days

(c) The preservation number is as follows:

AXL No.7#070402(Ax7) (accession number FERM BP-10850)

AXL No.51#070406(Ax51) (accession number FERM BP-10851)

AXL No.232#070406(Ax232) (accession number FERM BP-10855)

AXL No.96#070402(Ax96) (accession number FERM BP-10852)

AXL No.223#070402(Ax223) (accession number FERM BP-10853)

AXL No.225#070402(Ax225) (accession number FERM BP-10854)

AXL No.258#070402(Ax258) (accession number FERM BP-10856)

AXL No.284#070402(Ax284) (accession number FERM BP-10857)

AXL No.285#070402(Ax285) (accession number FERM BP-10858)

AXL No.292#070411(Ax292) (accession number FERM BP-10859)

1-3 binding Activity to human AXL

Antigen (hAXL-ECD-mIgG2aFc protein) diluted to 1. mu.g/mL with coating buffer (100mM sodium bicarbonate, pH9.6, 0.02% sodium azide), orThe control protein fused with the Fc region of mouse IgG2a was dispensed into 96-well plates (Nunc-Immuno) at 80. mu.L/well^TM 96Micro Well^TM plates MaxiSorp^TM(Nalge Nunc International)), and then incubated at 4 ℃ for more than one night. The plate was washed 3 times with phosphate buffered saline (tPBS (-)) containing 0.05 vol% Tween (registered trade mark) 20, and then blocked with 1/5 dilution buffer of dilution buffer ((BlockingOne, Nacalai Tesque)) at 4 ℃ overnight or more. After removing the buffer, 80. mu.L/well of mouse antiserum or hybridoma culture supernatant diluted with a dilution buffer was added to the plate, and the plate was incubated at room temperature for 1 hour. The plate was washed 3 times with tPBS (-) and then 80 μ L/well of HRP-labeled anti-mouse IgG antibody (Stressgen) diluted to 1/5000 with dilution buffer was added and incubated at room temperature for 1 hour. The plate was washed 5 times with tPBS (-), and then 80. mu.L/well chromogenic Substrate-Peroxidase Substrate (Kirkegaad) was added&Perry Laboratories), incubated at room temperature for 20 minutes. Add 8. mu.L/well Peroxidase Stop Solution (Peroxidase Stop Solution) (Kirkegaad)&Perry Laboratories), absorbance at 405nm was measured using a Microplate Reader (Microplate Reader) Model 3550(Bio-Rad Laboratories).

1-4 purification of antibodies from hybridoma culture supernatants

The hybridomas obtained above were cultured with HAT selection medium using natural low IgG fetal bovine serum (low IgG FBS) (InVitrogen) as FBS. To 20 to 50mL of the culture supernatant, 50. mu.L of protein G beads (Pharmacia) in which the solvent was replaced with a washing buffer (20mM sodium acetate buffer, pH5.0) was added in a proportion of 10mL of the culture supernatant, and the mixture was mixed overnight at 4 ℃ by inversion. After recovery of the protein G beads, they were washed with a washing buffer, and then the antibody was eluted with an elution buffer (50mM sodium acetate buffer, pH3.3) and immediately neutralized with a neutralization buffer (Tris-hydrochloric acid buffer, pH 7.8). The purified antibody was concentrated using an ultrafiltration kit (Amicon (registered trademark), Millipore) having a molecular weight cut-off of 10kDa, in which the buffer was replaced with phosphate-buffered saline (pH 7.35-7.65; Nishui pharmaceutical), and sterilized with a 0.22 μm sterilizing filter (Millipore GV Millipore).

Example 2 analysis of antibody induced phosphorylation

The ability of the anti-AXL monoclonal antibody obtained in example 1 to induce AXL phosphorylation in cancer cells was tested. Cells (human non-small cell lung cancer cell line Calu-1, human breast cancer cell line MDA-MB-231, human prostate cancer cell line DU-145) are cultured at 4 × 10⁵The density of individual cells/well was seeded in 6-well plates, and 24 hours later, the plates were replaced with serum-removed medium (serum-starved), and further cultured overnight. Next, the anti-AXL monoclonal antibody prepared as described above was added to a concentration of 2. mu.g/mL, and recombinant GAS6 (R) was added&D) As a positive control, the cells were incubated at 37 ℃ for 30 minutes at a concentration of 200 ng/mL. Next, the cells were washed with PBS (-) and lysed with lysis buffer (137mM NaCl/20mM Tris-HCl, pH8.0, 10% glycerol, 2mM EDTA, 1mM sodium vanadate, 1 vol% NP-40, 1mM phenylmethylsulfonyl fluoride (PMSF), 10. mu.g/mL aprotinin, 10. mu.g/mL leupeptin, 10. mu.g/mL pepstatin) on ice for 30 minutes. The cell solution mixture was disrupted with an ultrasonic disrupter (Tomy finisher) and then centrifuged (20,000 Xg) at 4 ℃ for 10 minutes. The supernatant of the cell solution mixture was mixed with 0.05 volume of protein G-agarose (Roche Diagnostics) for 30 minutes. Centrifugation (2,300 Xg) was carried out at 4 ℃ for 1 minute, and then 1.2. mu.g of anti-AXL monoclonal antibody (R) was added to the supernatant&G) After shaking at 4 ℃ for 1 hour, 10. mu.L of protein G-agarose was added, and the solution was further shaken at 4 ℃ for 1 hour. The immunoprecipitates were centrifuged at 4 ℃ for 1 min (2,300 Xg), washed, suspended in NuPAGE-LDS sample buffer (Invitrogen) and heated at 70 ℃ for 10 min. The immunoprecipitates were electrophoresed at 150V for 1 hour using 7% NuPAGE (Invitrogen).

Immunoprecipitated, 7% NuPAGE-electrophoresed proteins were electrophoretically transferred onto a 0.45 μm polyvinylidene fluoride filter (Immobilon-FL, Millipore) at 30mA for 1 hour using NuPAGE-transfer buffer (Invitrogen) and this buffer containing 20 vol% methanol. The filters were washed with TBS (50mM Tris-HCl, pH7.6, 150mM NaCl) and incubated with ODYSSEY blocking buffer (Li-COR)And then, closing is carried out. The filters were washed 4 times for 5 minutes each with TBST (TBS containing 0.05 vol% Tween (registered trade Mark) 20), biotinylated 4G10 anti-phosphotyrosine antibody (TBST diluted 1: 1,000, Upstate), and anti-AXL antibody (TBST diluted 1: 15,000, Santa Cruz)^TM) Incubate at room temperature for 2 hours. The filters were washed 4 times 5 minutes each with TBST and incubated for 1 hour with Alexa 680-labeled streptavidin (Invitrogen) diluted 1: 10,000 with TBST and IRDye 800-labeled anti-goat secondary antibody (Rockland) diluted 1: 10,000 with TBST. After washing 3 times with TBST for 5 minutes each and further washing 1 time with TBS (5 minutes), the filter was scanned using an infrared imaging system, ODYSSEY (Li-COR).

The enhancement of AXL tyrosine phosphorylation bands was observed by the overlap of the band obtained by immunoblotting of the immunoprecipitated intracellular AXL with the anti-AXL antibody and the band obtained by immunoblotting with the anti-phosphotyrosine antibody, which was observed after the addition of the anti-AXL monoclonal antibodies Ax285, Ax292, Ax223, Ax96, Ax258 and the addition of the recombinant GAS6 as a positive control (fig. 1a, b, c, d, e). Thus, enhanced tyrosine phosphorylation of AXL was observed by the addition of the anti-AXL monoclonal antibody obtained by the present inventors. These anti-AXL monoclonal antibodies can induce phosphorylation of the kinase domain of AXL.

Example 3 analysis of antibody inhibition of ligand-dependent phosphorylation

The ability of anti-AXL monoclonal antibodies to inhibit ligand-dependent phosphorylation in cancer cells was tested. The cells (human non-small cell lung cancer cell line Calu-1 or human breast cancer cell line MDA-MB-231 or human prostate cancer cell line DU-145) are 4 × 10⁵The density of individual cells/well was plated in 6-well plates and 24 hours later, the plates were replaced with serum-removed medium (serum starvation) and cultured overnight. The anti-AXL monoclonal antibody prepared in example 1 was added to a concentration of 2. mu.g/mL, and recombinant GAS6 (R) was added&D) The cells were incubated at 37 ℃ for 30 minutes at a concentration of 200 ng/mL. Subsequently, the cells were washed with PBS (-) and the cells were usedThe lysis buffer extracts the protein from the cells. Commercially available anti-AXL antibodies (Santa Cruz) will be used via 7% NuPAGE (Invitrogen)^TM) Immunoprecipitated cell lysates were isolated and immunoblotted by western blot and tyrosine phosphorylation analysis as described above. The immunoprecipitated intracellular AXL was treated with ligand GAS6 and blotted with anti-phosphotyrosine antibody, but the blotting of anti-phosphotyrosine antibody by anti-AXL monoclonal antibodies of Ax7, Ax51 was reduced (fig. 2a, b). This confirmed that tyrosine phosphorylation of ligand-dependent AXL was inhibited by using the anti-AXL monoclonal antibody obtained by the present inventors. These anti-AXL monoclonal antibodies can inhibit ligand-dependent phosphorylation of the kinase domain of AXL.

Example 4 analysis of the induction of AXL proteolysis by antibodies

The ability of anti-AXL monoclonal antibodies to induce AXL breakdown in cancer cells was tested. The cells (human non-small cell lung cancer cell line Calu-1 or human breast cancer cell line MDA-MB-231 or human prostate cancer cell line DU-145) are cultured at 4 × 10⁵The density of individual cells/well was plated in 6-well plates and 24 hours later, the plates were replaced with serum-removed medium (serum starvation) and cultured overnight. Next, the anti-AXL monoclonal antibody prepared as described above was added to a concentration of 2. mu.g/mL, and recombinant GAS6 (R) was added&D) As a positive control, the cells were incubated at 37 ℃ for 24 hours at a concentration of 200 ng/mL. The cells were then washed with PBS (-) and the proteins were extracted from the cells using the above cell lysis buffer. Commercially available anti-AXL antibodies (Santa Cruz) will be used via 7% NuPAGE (Invitrogen)^TM) Immunoprecipitated cell lysates were isolated and immunoblotted by western blot and tyrosine phosphorylation analysis as described above.

Mu.g of each protein solution was suspended in NuPAGE-LDS sample buffer (Invitrogen), heated at 70 ℃ for 10 minutes, and electrophoresed at 150V for 1 hour using 7% NuPAGE (Invitrogen). The gel obtained by electrophoresis was electrophoretically transferred to a 0.45 μm polyvinylidene fluoride filter (Immobilon-FL, Millipore) at 30mA for 1 hour using NuPAGE-transfer buffer (Invitrogen) and this buffer containing 20% vol methanol. The filters were washed with TBS (50mM Tris-HCl, pH7.6, 150mM NaCl) and incubated overnight with ODYSSEY blocking buffer (Li-COR), thereby blocking. The filters were washed 4 times with TBST for 5 minutes each, and then incubated with anti-AXL antibody (1: 15,000 in TBST, Santa Cruz) and anti-actin antibody (1: 5,000 in TBST) for 2 hours at room temperature. The filters were washed 4 times 5 minutes each with TBST. The cells were incubated for 1 hour with Alexa 680-labeled anti-rabbit secondary antibody (Invitrogen) diluted 1: 10,000 with TBST and IRDye 800-labeled anti-goat secondary antibody (Rockland) diluted 1: 10,000 with TBST. The filter was washed 3 times with TBST for 5 minutes each, then 1 time with TBS (5 minutes), and then scanned using an infrared imaging system ODYSSEY (Li-COR).

It can be observed that: the blotting of AXL was reduced after using anti-AXL monoclonal antibodies to Ax285, Ax292, Ax223, Ax96, Ax258, Ax284, Ax7, Ax225 (fig. 3a, b, c, d, e, f, g, h). That is, these anti-AXL monoclonal antibodies can induce the degradation of AXL protein.

Example 5 in vitro angiogenesis inhibiting Activity of anti-AXL antibodies

The activity of the anti-AXL antibody to inhibit luminal formation of human umbilical cord vascular endothelial cells (HUVECs) was determined using the angiogenesis kit sold by Kurabo. The experimental sequence was performed according to the protocol attached to the kit. The outline thereof is shown below. HUVEC and fibroblasts were CO-cultured, and 24-well plates (attached to kit) containing cells in the proliferation state at the early stage of tube lumen formation were incubated at 37 ℃ with 5% CO₂And the incubator humidified with air was left for 3 hours. The lids of 3 bottles (attached to the kit) containing 25mL of the special medium were released and incubated at 37 ℃ with 5% CO₂The incubator was left for about 30 minutes in a humidified air incubator. The plate was removed from the incubator and the well plate removed. The lid of the plate was replaced with a new one (kit attached). The cells were observed under a microscope to confirm the presence or absence of abnormalities. Media warmed to 37 ℃ (12 mL/plate) was dispensed into Falcon tubes, VEGF-A (2. mu.g/mL) was added to the media and diluted 200-fold to a final concentration of 10 ng/mL. For dispensing into tubesThe anti-AXL antibody prepared as described above was added to the medium to a final concentration of 10. mu.g/mL. The negative control was the addition of PBS (-) instead of antibody. The medium in the wells of the 24-well plate was removed by static suction, and 500. mu.L of the medium to which the drug was added statically. The state of the cells was observed under a microscope and returned to the incubator. The media exchange was performed in the same manner on day 1 of the addition of the antibody and on days 4, 7 and 9.

On day 11 of antibody addition, cell layer fixation and staining procedures were performed using a lumen staining kit (Kurabo). The sequence was performed according to the protocol attached to the kit. The outline thereof is shown below. Cells were observed under a microscope, then the medium was removed by aspiration, and 1mL of washing buffer (PBS (-), pH7.4, SIGMA) was added to each well to wash the well, followed by removal of the washing buffer by aspiration. 1mL of ice-cooled fixative (70% ethanol) was added to each well, and the mixture was allowed to stand at room temperature for 30 minutes. The fixative solution was removed, 1mL of blocking solution was added to each well, washed, and removed by suction. 0.5mL of the diluted primary antibody attached to the kit was added to each well according to the instructions, and incubated at 37 ℃ for 1 hour. The primary antibody was removed by aspiration, and each well was washed 3 times with 1mL of a blocking solution (1% BSA in PBS (-), pH7.4, SIGMA). 0.5mL of the diluted secondary antibody attached to the kit was added to each well according to the instructions, and incubated at 37 ℃ for 1 hour. The secondary antibody was removed by suction, and each well was washed 3 times with 1mL of distilled water. 0.5mL of the substrate solution attached to the kit was added to each well, and incubated at 37 ℃ for 10 to 30 minutes until the lumen became dark purple. The substrate solution was removed by suction, and each well was washed 3 times with 1mL of distilled water and dried naturally. Microscope images of each well immobilized at 5 spots were taken with a CCD camera (NIKON DIGITAL CAMERA dxm1200), and the area of the blood vessel was calculated with angiogenesis quantification software (Ver1.0 KURABO).

The rate of decrease in the area of the blood vessels with lumen in the well to which the anti-AXL antibody was added, relative to the area of the blood vessels with lumen in the well to which the negative control PBS (-) was added, was used as an index of the antibody inhibitory activity, and Ax232, Ax292, Ax285, Ax284 showed inhibitory activity (fig. 4).

Example 6 binding Activity of anti-AXL antibodies to mouse AXL

Mouse AXL extracellular region (R)&Company D, hereinafter referred to as mAXL-ECD), was diluted to 2. mu.g/mL with a coating buffer (100mM sodium bicarbonate buffer, pH9.6), and 100. mu.L of each was dispensed into a 96-well plate (Nunc-Immuno)^TM 96 MicroWell^TM plates MaxiSorp^TM(Nalge Nunc International)). After standing overnight in a refrigerator, the antibody solution in the plate was removed, and 200. mu.L/well of a dilution buffer (BlockingOne, Nacalai Tesque) was dispensed and blocked at room temperature for 2 hours. After removing the dilution buffer, 100. mu.L/well of the anti-AXL antibody prepared above diluted to 3. mu.g/mL with the dilution buffer was dispensed, and the mixture was left at room temperature for 1.5 hours. The antibody solution was removed and then washed 3 times with tPBS (-). 100. mu.L/well of a labeled antibody cocktail prepared by diluting alkaline phosphatase-labeled goat anti-mouse IgG1 antibody, alkaline phosphatase-labeled goat anti-mouse IgG2a antibody, and alkaline phosphatase-labeled goat anti-mouse IgG2b antibody (Southern Biotech) to 1/2250: 1/4000: 1/4000 at the final dilution was dispensed and allowed to stand at room temperature for 1 hour. After removal of the antibody solution, the cells were washed 3 times with tPBS (-). Each 100. mu.L/well of a chromogenic Substrate solution for alkaline Phosphatase (BluePhos Microwell Phosphatase Substrate System, Kirkegaad)&Perrylaboratories), developed at room temperature. The absorbance at 650nm was measured with a microplate reader (Emax, manufactured by Molecular Devices).

Binding of Ax96, Ax119, Ax223, Ax225, Ax284 to mouse AXL was confirmed.

Example 7 in vitro tumor cell proliferation inhibitory Activity of anti-AXL antibody

Evaluation was carried out using HCT-116(CCL-247), Calu-1(HTB-54), DU145(HTB-81), T-47D (HTB-133) purchased from ATCC and AsPC-1, MDA-MB-231, PANC-1 purchased from Dainippon Kogyo. The cells were maintained under the conditions recommended by each cell provider. The anti-AXL antibody prepared above was diluted with 10% FBS/RPMI1640 and 20% of the diluted antibody was addedmu.L was dispensed into a 96-well plate (flat bottom). Preparing cell suspension of HCT-116, Calu-1, DU145, T-47D, AsPC-1, MDA-MB-231, PANC-1 into 2000, 3000, 2000, 5000, 3000, 5000 cells per well, adding 180. mu.L cell suspension into each well, and adding 5% CO at 37 deg.C₂Culturing in an incubator. After 4 days, 10. mu.L of WST-8 (cell counting kit-8, manufactured by NIKO CHEMICAL CO., LTD.) was added to each well, and absorbance at 450nm was measured by a microplate reader (Model 3550-UV, manufactured by BIO-RAD) according to the protocol attached to the kit. The cell inhibitory activity (%) of the anti-AXL antibody was calculated by using the measured value without the test substance as 0% inhibition and the measured value without the test substance and the cell as 100% inhibition.

Ax51 showed more than 30% CGI activity on HCT116 cells.

[ Table 2]

Example 8 determination of antitumor Effect of anti-AXL antibody on human pancreatic cancer transplantation mouse model

1. Preparation of mouse model for human pancreatic cancer transplantation

Human pancreatic cancer cell line PANC-1 obtained from Dainippon pharmaceutical Co., Ltd (now Dainibito pharmaceutical Co., Ltd.) was prepared to 5X 10 with HBSS⁷Individual cells/mL. 200. mu.L (1X 10) of the above cell suspension was added⁷Individual cells/mouse) were transplanted to cann. cg-Foxn 1 purchased from Charles River corporation, japan<nu>The inguinal region of the/CrlCrlj nu/nu (BALB-nu/nu) mice was subcutaneous. Mean value in tumor volume was about 210mm³Mice were supplied to the experiment.

2. Antibody preparation and administration

The antibody of Table 1 was prepared at 2mg/mL with PBS and administered to the intraperitoneal cavity of a human pancreatic cancer transplant mouse at 2 times per week, 2 weeks, and 20 mg/kg. As a negative control, PBS was administered in the same manner. As a positive control, Gemzar (Gemzar) (Eli Lilly, Japan) was prepared at 12mg/mL in physiological saline and administered intraperitoneally at 120mg/kg 2 times a week for 2 weeks.

3. Evaluation of antitumor Effect

The antitumor effect in the human pancreatic cancer transplantation mouse model was calculated as a tumor proliferation inhibitory effect by comparing the tumor proliferation amount with that of the negative control group 4 days after the final administration (fig. 5).

Tumor growth inhibitory effect (%) (amount of tumor growth in 1-antibody-treated group/amount of tumor growth in control group) × 100

4. Statistical processing

Tumor volumes are expressed as mean ± standard deviation. The statistical analysis was performed by comparing the control group with the treatment group by the LSD method using SAS preliminary Package Version 5.0. It was significant with a 95% confidence (; p < 0.05).

5. Results

Any antibody inhibited tumor proliferation with anti-tumor effect (figure 5).

Example 9 determination of antitumor Effect of anti-AXL antibody on human pancreatic cancer transplantation mouse model (2)

1. Preparation of mouse model for human pancreatic cancer transplantation

Human pancreatic cancer cell line PANC-1 obtained from Dainippon pharmaceutical Co., Ltd (now Dainippon Sumitomo pharmaceutical Co., Ltd.) was prepared at 5X 10 with HBSS⁷Individual cells/mL. 200. mu.L (1X 10) of the above cell suspension was added⁷Individual cells/mouse) were transplanted to cann. cg-Foxn 1 purchased from Charles River corporation, japan<nu>The inguinal region of the/CrlCrlj nu/nu (BALB-nu/nu) mice was subcutaneous. Swelling in the middleAverage tumor volume was about 270mm³Mice were supplied to the experiment.

2. Antibody preparation and administration

The anti-AXL antibody was prepared at 2mg/mL using PBS and administered to the intraperitoneal cavity of a human pancreatic cancer transplant mouse at 2, 2 and 20mg/kg weekly. As a negative control, PBS was administered in the same manner. As a positive control, Gemzar (Gemzar) (Eli Lilly, Japan) was prepared at 12mg/mL in physiological saline and administered intraperitoneally at 120mg/kg 2 times a week for 2 weeks.

3. Evaluation of antitumor Effect

The antitumor effect in the human pancreatic cancer transplantation mouse model was calculated as a tumor proliferation inhibitory effect by comparison with the tumor proliferation amount of the negative control group 4 days after the final administration.

Tumor growth inhibitory effect (%) (amount of tumor growth in 1-antibody-treated group/amount of tumor growth in control group) × 100

4. Results

The tumor proliferation inhibitory effect is shown in fig. 6. The tumor growth inhibitory effect (%) is "-", when 30% or more is "+", and when 60% or more is "+". FIG. 6 also shows the results of analysis of the inhibition of ligand-dependent phosphorylation by the antibody of example 3.

For the antibody bound to FND-1, even the average value of the tumor volume was about 270mm³Administration started and showed more than 60% TGI activity. The anti-AXL antibody bound to FND-1 had such a remarkable anti-tumor effect in vivo, which was newly found in the present application and was completely unexpected.

With anti-AXL antibodies that bind to IgD2, the following effects were present: it was found that the compounds had phosphorylation inhibitory effects and showed antitumor effects in vivo as described in examples 3, 8 and 9, and this is completely unexpected.

Example 10 binding Activity with human AXL-FND1, human AXL-IgD2

1. Binding activity to human AXL-FND1 and human AXL-IgD2

The ability of anti-AXL monoclonal antibodies to bind to AXL-fibronectin type III domain 1(AXL-FND1) and AXL immunoglobulin family domain 2(AXL-IgD2) was tested.

2. Preparation of expression vectors of human recombinant AXL-FND1 and human recombinant AXL-IgD2

Human recombinant AXL-FND1 was constructed by amplifying a region corresponding to amino acids No.225 to No. 331 from the full-length human AXL cDNA (O' Bryan et al, mol.cell.biol.1991; 11: 5016 to 5031) (GenBank # NM-021913) by PCR, and cloning the amplified product into pET-41a (+) (Novagen) to express a fusion protein with GST-tag, thereby constructing pET-AXL-FND 1. For the other domains, the region AXL-IgD2 corresponding to amino acids 137 to 224 was amplified by PCR, and the amplified product was cloned into pET-41a (+) in order to express a fusion protein with GST-tag.

Each of the prepared vectors (5. mu.L) was transformed into DH 5. alpha. (Cat # DNA-903) by heat shock method, cultured in SOC medium, and then cultured overnight at 37 ℃ on LB plate containing kanamycin, followed by selection of colonies.

3. Purification of human recombinant AXL-FN, D1, human recombinant AXL-IgD2

Each of the prepared colonies was precultured overnight at 37 ℃ in 20mL of LB medium containing kanamycin, transferred to 500mL of medium, and cultured until A₆₀₀IPTG was added to a concentration of 0.5mM ± 0.05. After culturing at 37 ℃ for 1 hour, the bacterial cells were collected, suspended in buffer A (50mM Tris-HCl, pH8.0, 1mM EDTA, 0.5mM PMSF, 1mM DTT), and then thawed by repeated 2 times with liquid nitrogen. Adding NP-40 to a concentration of 0.5%, and crushing with an ultrasonic crusherThe cells were disrupted (30 seconds. times.5), and then centrifuged at 204,000 XG for 30 minutes to recover the supernatant.

Using the resulting supernatant, human recombinant AXL-FND1 was purified as follows. Mixing soluble Escherichia coli supernatant with glutathione agarose^TM(Glutathione Sepharose^TM)4Fast Flow (GE Healthcare) and stirring with a rotator (rotator) at 4 ℃ for 1 hour. Centrifugation at 500 XG for 5 minutes, discarding the supernatant, addition of buffer A, and washing of glutathione Sepharose^TM4B. This washing operation was repeated 3 times. From washed glutathione agarose^TM4Fast Flow transfer to Mini-column (mini-column), human recombinant AXL-FND1 was prepared from glutathione Sepharose with 50mM Tris-HCl, pH7.5, 25mM glutathione^TM4Fast Flow, and eluting. The other AXL domains were expressed, isolated and eluted in the same manner.

4. Evaluation of binding Activity of anti-AXL antibody to AXL-FND1 by Western blotting

To extract from glutathione agarose^TMHuman recombinant AXL-FND1 separated and eluted from 4Fast Flow is a representative, AXL-IgD1, AXL-IgD2, AXL-FND2, AXL-IgD1+ IgD2, AXL-IgD2+ FND1, AXL-FND1+ FND2 were subjected to Protein quantification using BIO-RAD Dc Protein Assay, 1. mu.g of the mixture was mixed with NuPAGE (registered trademark) sample buffer (Invitrogen), and subjected to electrophoresis on NuPAGE (registered trademark) 10% Bis-Tris gel. Electrophoretic gel transfer to Immobilon^TM-FL (Millipore) PVDF membrane. The PVDF membrane containing the transferred protein was blocked with Odyssey (registered trademark) blocking buffer (LI-COR), then soaked in a primary antibody solution in which the anti-AXL antibody was diluted to 5. mu.g/mL, and incubated overnight at 4 ℃. The PVDF membrane containing the transferred protein immersed in the primary antibody solution was treated with 0.1% TBS-T [ TBS (Tris-buffered saline, TaKaRa) containing 0.1% Tween-20)]Washing was carried out 4 times for 5 minutes each. The PVDF membrane impregnated with the anti-AXL antibody was immersed in a secondary antibody solution of Alexa Fluor (registered trademark) 680 goat anti-mouse IgG (H + L) (Invitrogen) diluted to 80ng/mL and incubated at room temperature for 1 hour. PVDF membrane impregnated with secondary antibody solution was washed with 0.1% TBS-T3 times for 5 minutes each, followed by 5 minutes in TBS-T containing 0.01% SDS and 5 minutes in TBS. The washed PVDF membrane was scanned with a far infrared ray imaging system Odyssey (registered trademark) to evaluate the binding property.

5. Results

The evaluation results are shown in fig. 6.

It is clear that: the anti-AXL antibody (Ax225) produced by the hybridoma deposited under accession number FERM BP-10854 recognizes FND1 of AXL (fig. 6). Consider that: the anti-AXL antibody (Ax284) produced by the hybridoma deposited under accession number FERM BP-10857 recognizes FND1 and IgD2 of AXL (fig. 6). It is clear that: the anti-AXL antibody (Ax7) produced by the hybridoma deposited under the deposition number FERM BP-10850 and the anti-AXL antibody (Ax51) produced by the hybridoma deposited under the deposition number FERM BP-10851 recognize the IgD2 of AXL (FIG. 6).

Example 11 determination of antitumor Effect of anti-AXL antibody on human Breast cancer transplantation mouse model

1. Preparation of human breast cancer transplantation mouse model

Human breast cancer cell line MDA-MB-435S obtained from ATCC and prepared into 5 x 10 by HBSS⁷Individual cells/mL. 200. mu.L (1X 10) of the above cell suspension was added⁷Individual cells/mouse) were transplanted to cann. cg-Foxn 1 purchased from Charles River corporation, japan<nu>The inguinal region of the/CrlCrljnu/nu (BALB-nu/nu) mice was subcutaneous. In a tumor volume of about 200mm³Mice were supplied to the experiment.

2. Antibody preparation and administration

The anti-AXL antibody was prepared at 2mg/mL with PBS and administered 2 times per week, 2 weeks per week, 20mg/kg into the abdominal cavity of human breast cancer-transplanted mice. As a negative control, PBS was administered in the same manner.

3. Evaluation of antitumor Effect

The antitumor effect in the human breast cancer transplantation mouse model was calculated as a tumor proliferation inhibitory effect by comparing the tumor proliferation amount with that of the negative control group 4 days after the final administration.

Tumor growth inhibitory effect (%) (amount of tumor growth in 1-antibody-treated group/amount of tumor growth in control group) × 100

4. Statistical processing

Tumor volumes are expressed as mean ± standard deviation. The statistical analysis was performed by comparing the control group with the treatment group by the LSD method using SAS preliminary Package Version 5.0. It was significant with a 95% confidence (; p < 0.05).

5. Results

The anti-AXL antibody used inhibited tumor proliferation and had an anti-tumor effect (fig. 7). Therefore, the anti-AXL antibody bound to FND1 can be expected to have an antitumor effect on various tumors.

EXAMPLE 12 sequence analysis of antibody cDNA

1. Preparation of chimeric antibody expression vector

Total RNA was extracted from the hybridoma cells (Ax225) deposited under the accession number FERM BP-10854 using RNeasy Mini kit (QIAGEN), and cDNA was synthesized using SMART RACE cDNA amplification kit (BD Biosciences). The variable region genes of the antibody were isolated by PCR using 10X Universal Primer A Mix attached to SMART RACE cDNA amplification kit (BD Biosciences) and the following primers (H chain: MHCg1, L chain MLCk) set for each constant region of the antibody using PrimeSTAR HS DNA polymerase (TaKaRa).

MHCg1：5’-GGGCCAGTGGATAGACAGATG-3’(SEQ ID NO：1)

MLCk：5’-GCTCACTGGATGGTGGGAAGATG-3’(SEQ ID NO：2)

The nucleotide sequence of each isolated DNA fragment was determined by using BigDye end cycle sequencing kit (Applied Biosystems), by using a DNA sequencer (ABI PRISM 3730XL DNA sequencer or ABI PRISM 3700DNA sequencer) (Applied Biosystems) according to the method described in the attached specification.

2. Results

The heavy chain variable region in the amino acid sequence of the resulting mouse antibody to AXL225 is set forth in SEQ ID NO: 3, the CDR1 of this region is as shown in SEQ ID NO: 4, CDR2 is as shown in SEQ ID NO: 5, CDR3 is set forth in SEQ ID NO: and 6. The variable region of the light chain in the amino acid sequence of the obtained mouse antibody of AXL225 is shown in SEQ ID NO: 7, the CDR1 of this region is set forth in SEQ ID NO: 8, CDR2 is set forth in SEQ ID NO: 9, CDR3 is as shown in SEQ ID NO: shown at 10.

Industrial applicability

The present inventors have found for the first time that an anti-AXL antibody has an angiogenesis inhibitory effect and a cancer inhibitory effect. The anti-AXL antibody of the present invention is useful as an angiogenesis inhibitor and a cell proliferation inhibitor. By using the antibody of the present invention, the phosphorylation of AXL can be induced or inhibited. Further, the use of the antibody of the present invention can reduce the expression level of AXL.

Claims (46)

1.A monoclonal antibody that binds to AXL.

2. The antibody of claim 1, wherein the antibody has cell proliferation inhibitory activity.

3. The antibody of claim 1, wherein the antibody inhibits the proliferation of cancer cells.

4. The antibody of any one of claims 1 to 3 which binds to FND 1.

5. An antibody produced by using a peptide having all or at least 5 or more consecutive amino acid sequences of FND1 as an immunogen.

6. The antibody of any one of claims 1 to 5, which has agonist activity against AXL.

7. The antibody of any one of claims 1 to 5, which has antagonistic activity against AXL.

8. The antibody of claim 7, which is obtained by: the cells expressing AXL were contacted with the AXL ligand, and antibodies in which no phosphotyrosine was detected in AXL were selected.

9. The antibody according to any one of claims 1 to 8, which has an activity of reducing the expression level of AXL.

10. The antibody of any one of claims 1 to 9, which has angiogenesis inhibiting activity.

11. An antibody according to any one of the following (a) to (j):

(a) antibody Ax285 as produced by the hybridoma deposited under accession number FERM BP-10858;

(b) antibody Ax292 produced by the hybridoma deposited under accession number FERM BP-10859;

(c) the antibody Ax223 produced by the hybridoma deposited under accession number FERM BP-10853;

(d) the antibody Ax96 produced by the hybridoma deposited under accession number FERM BP-10852;

(e) antibody Ax258 produced by the hybridoma deposited under accession number FERM BP-10856;

(f) antibody Ax284 produced by the hybridoma deposited under accession number FERM BP-10857;

(g) antibody Ax7 produced by the hybridoma deposited under accession number FERM BP-10850;

(h) the antibody Ax51 produced by the hybridoma deposited under accession number FERM BP-10851;

(i) the antibody Ax225 produced by the hybridoma deposited under accession number FERM BP-10854;

(j) the antibody Ax232 produced by the hybridoma deposited under accession number FERM BP-10855.

12. An antibody that binds to the same epitope as the antibody of claim 11.

13. An antibody having the same CDR sequences as any of the antibodies of claim 11.

14. An antibody, wherein the sequence of the heavy chain CDR1, 2,3 is SEQ ID NO: 4. 5 and 6.

15. An antibody having a heavy chain CDR comprising: an antibody according to claim 14, wherein the heavy chain CDR amino acid sequence has an amino acid sequence obtained by substitution, deletion, insertion and/or addition of 1 or more amino acid sequences.

16. An antibody, wherein the light chain CDR1, 2,3 has the sequence of SEQ ID NO: 8. 9 and 10.

17. An antibody having a light chain CDR comprising: the light chain CDR amino acid sequence of the antibody of claim 16, which has an amino acid sequence obtained by substitution, deletion, insertion and/or addition of 1 or more amino acid sequences.

18. The antibody of any one of claims 13 to 17 which is a chimeric antibody.

19. The antibody of any one of claims 13 to 17 which is a humanized antibody.

20. A hybridoma described in any one of the following (a) to (j):

(a) hybridoma Ax285 as deposited under accession number FERM BP-10858;

(b) the hybridoma Ax292 deposited under accession number FERM BP-10859;

(c) the hybridoma Ax223 deposited under accession number FERM BP-10853;

(d) hybridoma Ax96 deposited under accession number FERM BP-10852;

(e) hybridoma Ax258 deposited under accession number FERM BP-10856;

(f) hybridoma Ax284 deposited under accession number FERM BP-10857;

(g) the hybridoma Ax7 deposited under accession number FERM BP-10850;

(h) hybridoma Ax51 deposited under accession number FERM BP-10851;

(i) the hybridoma Ax225 deposited under accession number FERM BP-10854;

(j) the hybridoma Ax232 deposited under accession number FERM BP-10855.

21. An angiogenesis inhibitor comprising an anti-AXL antibody as an active ingredient.

22. The angiogenesis inhibitor according to claim 21, wherein the antibody is the antibody according to any one of claims 1 to 19.

23. A cell growth inhibitor comprising an anti-AXL antibody as an active ingredient.

24. The inhibitor of claim 23, wherein the cell is a cancer cell.

25. The inhibitor of claim 23, wherein the antibody is an antibody of any one of claims 1 to 19.

26. The inhibitor of claim 23, wherein the anti-AXL antibody is an antibody that binds to FND 1.

27. The inhibitor according to claim 23, which comprises an antibody that binds to IgD2 and has an inhibitory activity on phosphorylation as an active ingredient.

An agent for inducing the phosphorylation activity of AXL, which comprises an anti-AXL antibody as an active ingredient.

29. The induction agent of claim 28, wherein the anti-AXL antibody is an antibody that binds to IgD.

30. The induction agent of claim 28, wherein the antibody is the antibody of claim 6.

An inhibitor of the phosphorylation activity of AXL, which comprises an anti-AXL antibody as an active ingredient.

32. The inhibitor of claim 31, wherein the anti-AXL antibody is an antibody that binds to IgD 2.

33. The inhibitor of claim 31, wherein the antibody is an antibody of any one of claims 7 or 8.

An agent for reducing the expression level of AXL, which comprises an anti-AXL antibody as an active ingredient.

35. The expression level-reducing agent of claim 34, wherein the anti-AXL antibody is an antibody that binds to FND 1.

36. The agent for reducing an expression level according to claim 34, wherein the antibody is the antibody according to claim 9.

37. A method of inducing phosphorylation of AXL using an anti-AXL antibody.

38. A method for reducing the expression level of AXL by using an anti-AXL antibody.

39. Methods of inhibiting the phosphorylation of AXL using an anti-AXL antibody.

40. An anticancer agent comprising an anti-AXL antibody as an active ingredient.

41. The anticancer agent according to claim 40, wherein the antibody is an antibody according to any one of claims 1 to 19.

42. The anticancer agent according to claim 40, which comprises an antibody having a phosphorylation activity inhibitory activity, which binds to IgD2, as an active ingredient.

43. The anticancer agent according to claim 40, wherein the cancer is pancreatic cancer, gastric cancer, lung cancer, osteosarcoma, large intestine cancer, prostate cancer, melanoma, endometrial cancer, ovarian cancer, uterine leiomyoma, thyroid cancer, stem cell cancer, breast cancer, bladder cancer, renal cancer, glioma, neuroblastoma, or esophageal cancer.

44. The anticancer agent according to claim 42, wherein the cancer is glioma, gastric cancer, endometrial cancer, non-small cell lung cancer, pancreatic cancer or breast cancer.

45. The anticancer agent according to claim 43, wherein the cancer is pancreatic cancer or breast cancer.

46. The antibody of claim 1, which inhibits AXL phosphorylation.