HK1147275B

HK1147275B - Axl antibodies

Info

Publication number: HK1147275B
Application number: HK11101502.6A
Authority: HK
Inventors: T‧海特曼; J‧尼韦纳; J‧鲁赫; P‧沃特斯; K‧泽勒; E‧茨维克-瓦拉施; M‧罗瑟
Original assignee: U3制药有限公司
Priority date: 2007-11-12
Filing date: 2008-11-12
Publication date: 2015-03-06

Description

AXL antibodies

Description of the invention

The present invention relates to antibodies, in particular monoclonal antibodies, that bind to the extracellular domain of the AXL receptor tyrosine kinase and at least partially inhibit AXL activity.

Background

The AXL (Ark, UFO, Tyro-7) receptor tyrosine kinase is a member of the Tyro-3 family of kinases, the other members of which are Mer (Eyk, Nyk, Tyro-12) and Sky (Rse, Tyro-3, Dtk, Etk, Brt, Tif). It can be activated by the binding of the heterophil ligand Gas6 (a 70-kDa protein homologous to anticoagulant protein S). AXL tyrosine phosphorylation is also induced by homophilic binding (homotrophic binding), in contrast to other receptor tyrosine kinases. AXL activation results in signal transduction through PI-3-kinase/Akt (Franke et al, Oncogene 22: 8983-8998, 2003) and other major pathways such as Ras/Erk and β -catenin/TCF (Goruppi et al, mol.cell biol.21: 902-915, 2001).

AXL is weakly expressed in organ capsules and connective tissue of many normal tissues including brain, heart, skeletal muscle, several other organs, and in monocytes but not lymphocytes. Akt phosphorylation induced by AXL has been described in the survival of fibroblasts (Goruppi et al, Mol Cell Biol 17: 4442-44531997), endothelial cells (Hasanbasic et al, Am J Physiol Heart Circuit Physiol, 2004), vascular smooth muscle cells (Melragno et al, J.mol.cell Cardiol.37: 881-887, 2004) and neurons (Allen et al, mol.Endocrinol.13: 191-2011999). In addition, AXL plays an important role in cell adhesion and chemotaxis. The knockout of AXL demonstrated impaired platelet aggregation stabilization and thrombosis (due to reduced activation of platelet integrin IIb 3).

Has been described in various Cancer types such as breast Cancer (Meric et al, Clin. Cancer Res.8: 361-367, 2002; Bercalaz et al, Ann. Oncol.12: 819-824, 2001), colon Cancer (Chen et al, int. J. Cancer 83: 579-584, 1999; Craven et al, int. J. Cancer 60: 791-797, 1995), prostate Cancer (Jacob et al, Cancer Detect. Prev.23: 325-332, 1999), lung Cancer (Wimmel et al, EurJ Cancer 37: 2264-2274, 2001), stomach Cancer (Wu et al, Anticancer Res 22: 1071-1078, 2002), ovarian Cancer (Sun et al, Oncolog 66: 450-457, 2004), endometrial Cancer (Angin et al, Ancinar 533, 2002; Thyroid Cancer 978, 2003-975; Cell DNA, Tsao et al, 2003, Australia Japanese Cancer, 975; Tsao et al, Australia Cancer, Australine Cancer, Skoncol et al, 975, Australine Cancer (Tsao, Skoemn et al, Skoncol, Skoch et al, Skoncol, 2003, 2000, Skoncol, Skovic, 2000, Skovic, 2003, 2000, Skovic, thyroid 9: 563-567, 1999) and esophageal cancer (Nemoto et al, 1997), in addition to CML (Janssen et al, A novel reactive tyrosine receptor with environmental potential. oncogene, 6: 2113-212O, 1991; braunger et al, Oncogene 14: 2619-; o' Bryan et al, Mol Cell Biol 11: 5016-: 1352-1358, 1999), osteosarcoma (Nakano et al, j.biol.chem.270: 5702-: 128-134, 2004) and in head and neck squamous cell carcinoma (Green et al, Br J cancer. 200694: 1446-5, 2006) confirmed the overexpression of AXL.

Furthermore, AXL was identified as a metastasis associated gene that is up-regulated in aggressive breast cancer cell lines compared to non-aggressive cells. In vitro, AXL activity was found to be essential for migration and invasion, and this activity could be inhibited by antibody treatment (WO 04008147). Similarly, the elimination of AXL activity in vivo, either by expression of the dominant negative form of AXL (dominant negative version) (Vajkoczy, P., et al, Proc. Natl.Acad. science U.S.A.103: 5799-5804.2005) or by siRNA-mediated down-regulation of AXL (Holland et al, Cancer Res.65: 9294-.

To date, two antibodies have been described that bind AXL and have biological activity. One antibody was able to reduce AXL mediated cell invasion (WO04008147), while the other antibody reportedly reduced AXL/ligand interaction. However, both antibodies are polyclonal antibodies, which makes them unsuitable for therapeutic administration.

Thus, in view of the therapeutic potential of AXL, there is a high need for monoclonal AXL antibodies, antibody fragments or derivatives thereof that effectively and specifically block AXL-mediated signal transduction and are suitable for therapeutic treatment.

A first aspect of the invention therefore relates to a monoclonal antibody, including fragments or derivatives thereof, that binds to the extracellular domain of AXL, in particular human AXL, and at least partially inhibits AXL activity.

Preferably, the antibodies of the invention also have at least one or more of the following properties: reducing or blocking the ability of AXL-mediated signal transduction, reducing or blocking the ability of AXL phosphorylation, reducing or blocking the ability of cells to proliferate, reducing or blocking the ability of angiogenesis, reducing or blocking the ability of cells to migrate, reducing or blocking the ability of tumor metastasis, reducing or blocking the ability of AXL-mediated PI3K signaling, and reducing or blocking the ability of AXL-mediated anti-apoptosis, thereby increasing the sensitivity of, for example, cells to treatment with an anti-tumor agent. Furthermore, the antibodies of the invention may exhibit high specificity for AXL, in particular human AXL and do not significantly recognize other Tyro-3 family members, such as MER and/or SKY and/or mammalian non-primate AXL, such as murine AXL. Antibody specificity can be determined by measuring cross-reactivity, as described in the examples.

The term "activity" refers to the biological function of AXL that affects the phenotype of a cell, particularly but not limited to cancer phenotype such as escape of apoptosis, self-sufficiency of growth signals, cell proliferation, tissue invasion and/or metastasis, insensitivity against growth signals (anti-apoptosis) and/or sustained angiogenesis.

The term "AXL-mediated signal transduction" means the activation of second messenger pathways triggered by the direct or indirect interaction of AXL with second messenger molecules.

The term "AXL phosphorylation" refers to the phosphorylation of an amino acid residue, preferably a tyrosine residue, by a second AXL protein (transphosphorylation) or by another protein having protein kinase activity.

The term "cell proliferation" refers to all processes involving AXL that cause the proliferation of human cells, particularly but not limited to human cancer cells. They contribute to or cause the replication of cellular DNA, the separation of the replicated DNA into two comparably sized genomes, and the physical division of the whole cell (called cytokinesis), and are stimulated or mediated by the non-catalytic or catalytic activity of AXL, preferably including AXL phosphorylation and/or AXL mediated signal transduction.

The term "angiogenesis" refers to all processes involving AXL that contribute to the growth of new blood vessels from existing vessels, particularly but not limited to new tumor supply vessels. These processes include multiple cellular events such as proliferation, survival, migration and sprouting (sprouting) of vascular endothelial cells, attraction and migration of pericytes and secretion of angiogenic factors of basal membrane formation, vascular perfusion, or stromal or neoplastic cells for vascular stabilization, and are stimulated or mediated by the non-catalytic or catalytic activity of AXL (preferably including AXL phosphorylation and/or AXL mediated signaling).

The term "metastasis" refers to all processes involved in AXL that support the spread of cancer cells from a primary tumor, infiltration into lymphatic and/or blood vessels, circulation through the bloodstream, and growth in distant foci (metastases) in normal tissues in other parts of the body. In particular, it refers to a cellular event of the tumor cell such as proliferation, migration, anchorage independence, escape of apoptosis or secretion of angiogenic factors that causes metastasis and is stimulated or mediated by non-catalytic or catalytic activity of AXL (preferably including AXL phosphorylation and/or AXL mediated signal transduction).

The term "AXL-mediated anti-apoptosis" refers to all processes involving AXL that prevent programmed cell death (apoptosis) in human cells, preferably but not limited to human cancer cells. In particular, it refers to processes that protect human cells, preferably but not limited to human cancer cells, from apoptosis induced by the withdrawal of growth factors, hypoxia, exposure to chemotherapeutic agents or radiation, or initiation of Fas/Apo-1 receptor mediated signal transduction, and are stimulated or mediated by the non-catalytic or catalytic activity of AXL (preferably including AXL phosphorylation and/or AXL mediated signal transduction).

In addition, the invention includes binding activities to AXL of KD ═ 10^-5M or less, preferably KD ═ 10^-7M or less, most preferably KD ═ 10^-9M or lower. The binding activity of the antibody of the present invention to AXL is not KD-10^-5M or lower can be determined by methods known to those skilled in the art. For example, activity can be determined using Biacore using surface plasmon resonance and/or by ELISA (enzyme linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody techniques such as FACS.

In a second aspect, the antibody may have at least one antigen binding site, e.g., 1 or 2 antigen binding sites. Furthermore, the antibody preferably comprises at least one immunoglobulin heavy chain and at least one immunoglobulin light chain. Immunoglobulin chains comprise variable domains and optionally constant domains. The variable domain may comprise Complementarity Determining Regions (CDRs), such as the CDR1, CDR2, and/or CDR3 regions, as well as framework regions. The term "complementarity determining regions" (CDRs) are well defined in the art (see, e.g., Harlow and Lane, "Antibodies, a laboratory Manual", CSH Press, Cold Spring harbor, 1988), which refer to a segment of amino acids within the variable region of an antibody that predominantly contacts an antigen.

A further aspect of the invention relates to an antibody, including fragments or derivatives thereof, which binds to the extracellular domain of AXL, said antibody comprising at least one heavy chain amino acid sequence comprising at least one CDR selected from the group consisting of:

(a) SEQ ID NOs: 16, 22, 28, or a CDRH1 sequence differing by 1 or 2 amino acids therefrom,

(b) SEQ ID NOs: CDRH2 as shown in 17, 23, 29, or a CDRH2 sequence which differs from it by 1 or 2 amino acids, and

(c) SEQ ID NOs: 18, 24, 30, or a CDRH3 sequence differing by 1 or 2 amino acids therefrom,

and/or at least:

a light chain amino acid sequence comprising at least one CDR selected from the group consisting of:

(d) SHQ ID NOs: 13, 19, 25, or a CDRL1 sequence differing by 1 or 2 amino acids therefrom,

(e) SEQ ID NOs: CDRL2 shown in 14, 20, 26, or a CDRL2 sequence which differs therefrom by 1 or 2 amino acids, and

(f) SEQ ID NOs: 15, 21, 27, or a CDRL3 sequence differing by 1 or 2 amino acids therefrom,

or a monoclonal antibody recognizing the same epitope on the extracellular domain of AXL.

In a preferred embodiment, the antibody comprises a heavy chain comprising at least one CDR selected from the group consisting of:

(a) SEQ ID NO: 16, or a CDRH1 sequence differing by 1 or 2 amino acids therefrom,

(b) SEQ ID NO: CDRH2 shown in 17, or a CDRH2 sequence which differs from it by 1 or 2 amino acids, and

(c) SEQ ID NO: 18, or a CDRH3 sequence differing by 1 or 2 amino acids therefrom,

and/or a light chain comprising at least one CDR selected from:

(d) SEQ ID NO: CDRL1 shown in 13, or a CDRL1 sequence which differs therefrom by 1 or 2 amino acids,

(e) SEQ ID NO: CDRL2 shown in 14, or a CDRL2 sequence which differs therefrom by 1 or 2 amino acids, and

(f) SEQ ID NO: 15, or a CDRL3 sequence differing therefrom by 1 or 2 amino acids,

In a further preferred embodiment, the antibody comprises a heavy chain comprising at least one CDR selected from the group consisting of:

(a) SEQ ID NO: 22, or a CDRH1 sequence differing by 1 or 2 amino acids therefrom,

(b) SEQ ID NO: CDRH2 shown in 23, or a CDRH2 sequence which differs by 1 or 2 amino acids, and

(c) SEQ ID NO: CDRH3 shown in 24, or a CDRH3 sequence which differs from it by 1 or 2 amino acids,

and/or a light chain comprising at least one CDR selected from:

(d) SEQ ID NO: 19, or a CDRL1 sequence which differs therefrom by 1 or 2 amino acids,

(e) SEQ ID NO: CDRL2 shown in 20, or a CDRL2 sequence which differs therefrom by 1 or 2 amino acids, and

(f) SEQ ID NO: 21, or a CDRL3 sequence which differs therefrom by 1 or 2 amino acids,

(a) SEQ ID NO: 28, or a CDRH1 sequence differing by 1 or 2 amino acids therefrom,

(b) SEQ ID NO: 29, or a CDRH2 sequence which differs from it by 1 or 2 amino acids, and

(c) SEQ ID NO: 30, or a CDRH3 sequence differing by 1 or 2 amino acids therefrom,

and/or a light chain comprising at least one CDR selected from:

(d) SEQ ID NO: CDRL1 shown in 25, or a CDRL1 sequence which differs therefrom by 1 or 2 amino acids,

(e) SEQ ID NO: CDRL2 shown in 26, or a CDRL2 sequence which differs therefrom by 1 or 2 amino acids, and

(f) SEQ ID NO: CDRL3 as shown in 27, or a CDRL3 sequence which differs therefrom by 1 or 2 amino acids,

In another embodiment, the invention relates to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10, 12 or at least the variable domain thereof or an amino acid sequence having at least 90% sequence identity thereto and/or a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11 or at least the variable domain thereof or an amino acid sequence having at least 90% sequence identity thereto or an antibody recognizing the same epitope on the extracellular domain of AXL.

As used herein, "sequence identity" between two polypeptide sequences refers to the percentage of amino acids that are identical between the sequences. Preferred polypeptide sequences of the invention have at least 90% sequence identity.

In a particularly preferred embodiment, the antibody is selected from 11B7, 11D5, 10D12 or an antibody recognizing the same epitope on the extracellular domain of AXL.

The antibody may be any antibody of natural and/or synthetic origin, for example an antibody of mammalian origin. Preferably, the constant domain, if present, is a human constant domain. The variable domain is preferably a mammalian variable domain, e.g. a humanized or human variable domain. More preferably, the antibody is a chimeric, humanized or human antibody.

The antibodies of the invention may be of the IgA, IgD, IgE, IgG or IgM class, preferably of the IgG or IgM class, including but not limited to the IgG1, IgG2, IgG3, IgG4, IgM1 and IgM2 class. In the most preferred embodiments, the antibody is of the human IgG1, IgG2, or IgG4 type.

As discussed (supra), there are many isotypes of antibodies. It will be appreciated that the antibody produced need not initially have such an isotype, rather, the antibody produced may have any isotype and may be isotype switched by using conventional molecular biology techniques well known in the art to apply molecularly cloned V region genes or cloned constant region genes or cdnas to a suitable expression vector and then expressing the antibody in a host cell using techniques known in the art.

The term antibody includes "fragments" or "derivatives" having at least one antigen binding site of an antibody. Antibody fragments include Fab fragments, Fab 'fragments, F (ab')₂Fragments and Fv fragments. Derivatives of antibodies include single chain antibodies, nanobodies (nanobodies) and diabodies (diabodies). Derivatives of antibodies also include scaffold proteins having antibody-like binding activity to AXL.

In the description of the present invention, the term "scaffold protein", as used herein, means a polypeptide or protein having an exposed surface region in which amino acid insertions, substitutions or deletions are highly tolerated. Examples of scaffold proteins which can be used according to the invention are protein A from Staphylococcus aureus (Staphylococcus aureus), the binding protein of the bile pigments or other lipocalins (lipocalins) from Pieris brassicae (Pieris brassiccus), ankyrin repeat protein (ankyrin repeat protein) and human fibronectin (reviewed in Binz and Pluckthun, Curr Opin Biotechnol, 16: 459-69, 2005). Engineering of scaffold proteins can be thought of as grafting or integrating affinity functions into or into the structural framework of a stably folded protein. Affinity function according to the invention refers to protein binding affinity. The scaffold may be structurally separated from the amino acid sequence conferring binding specificity. In general, proteins that exhibit suitability for the development of such artificial affinity reagents can be obtained by rational, or most commonly, combinatorial protein engineering techniques such as panning for AXL (purified protein or protein displayed on the surface of cells) for binding agents in vitro displayed artificial scaffold libraries (techniques known in the art) (Skerra, J.mol.Recog., Biochim Biophyacta 1482: 337-350, 2000; Binz and Pluckthun, Curr Opin Biotechnol, 16: 459-69, 2005). In addition, a scaffold protein having antibody-like binding activity can be derived from a scaffold domain-containing acceptor polypeptide (to which the binding domain of a donor polypeptide can be grafted to confer binding specificity of the donor polypeptide to the scaffold domain-containing acceptor polypeptide). The inserted binding domain may comprise, for example, at least one CDR of an anti-AXL antibody, preferably at least one CDR selected from SEQ ID NOs: 13-30. Insertion can be accomplished by a variety of methods known to those skilled in the art, including, for example, polypeptide synthesis, synthesis of nucleic acids encoding amino acids, and by various forms of recombinant methods well known to those skilled in the art.

As already indicated above, the specificity of an antibody, antibody fragment or derivative thereof lies in the amino acid sequence of the CDRs. The variable domains of antibodies (heavy chain VH and light chain VL) preferably comprise 3 complementarity determining regions (sometimes referred to as hypervariable regions) flanked by 4 relatively conserved framework regions or "FRs". Typically, the specificity of an antibody is determined by or primarily by a CDR, e.g., a CDR or CDRs of a VH chain. Those skilled in the art will readily understand that the variable domains of antibodies, antibody fragments or derivatives thereof having the CDRs described above, can be used in the construction of antibodies with further improved specificity and biological function. Accordingly, the present invention includes antibodies, antibody fragments or derivatives thereof comprising at least one CDR of the above-described variable domains and advantageously having substantially the same, similar or improved binding properties as the antibodies described in the accompanying examples. By starting with an antibody comprising at least one CDR recited in the attached sequence listing and required for an embodiment of the invention, one skilled in the art can combine additional CDRs from the originally identified monoclonal antibody or a different antibody for enhanced specificity and/or affinity. CDR grafting is well known in the art and may also be used to fine tune the specific affinity and other properties of the antibodies, fragments or derivatives thereof of the invention, as long as the original specificity is maintained. Advantageously the antibody, fragment or derivative thereof comprises at least 2, more preferably at least 3, more preferably at least 4 or at least 5 and particularly preferably all 6 CDRs of the original donor antibody. In a further alternative embodiment of the invention, CDRs from different originally identified monoclonal antibodies may be combined in a new antibody entity. In these cases, it is preferred that the 3 CDRs of the heavy chain are derived from the same antibody, while the 3 CDRs of the light chain are all derived from different antibodies (but all derived from the same antibody). The antibodies of the invention or their corresponding immunoglobulin chains may also be modified using conventional techniques known in the art, e.g., by using amino acid deletions, insertions, substitutions, additions and/or recombinations, either alone or in combination, and/or any other modification known in the art. Methods for introducing such modifications into the DNA sequence encoding the amino acid sequence of an immunoglobulin chain are well known to those skilled in the art; see, e.g., Sambrook, Molecular Cloning Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.

The antibody, antibody fragment or derivative thereof is optionally deimmunized (deimmunized) for therapeutic purposes. Deimmunized antibodies are proteins that have no or reduced epitopes recognized by T helper lymphocytes. An example of a method for identifying such epitopes is shown in Tangri et al, (J Immunol.174: 3187-96, 2005). The preparation of deimmunized antibody fragments or derivatives thereof can be performed as described in U.S. Pat. nos. 6,054,297, 5,886,152 and 5,877,293.

In one embodiment, the antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). The production of chimeric antibodies is described, for example, in WO 89/09622.

Preferably, the present invention relates to a chimeric antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 39, 41, 42 or at least the variable domain thereof or an amino acid sequence having at least 90% sequence identity thereto and/or an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 40 or at least a variable domain thereof or an amino acid sequence having at least 90% sequence identity thereto.

In additional embodiments, the antibodies of the invention are humanized antibodies or fully human antibodies. Humanized forms of the antibodies can be generated according to methods known in the art, e.g., chimerization or CDR grafting. Alternative methods for the production of humanized antibodies are well known in the art and are described, for example, in EP-A10239400 and WO 90/07861. Typically, humanized antibodies have one or more amino acid residues introduced into them that are derived from a non-human source. Such non-human amino acid residues are often referred to as "import" residues, which are typically obtained from an "import" variable domain. Humanization can be performed, for example, by replacing the corresponding sequence of a human antibody with a CDR or CDR sequence of non-human origin according to the method of Winter and coworkers (Jones et al, Nature, 321: 522-525 (1986); Riechmann et al, Nature, 332: 323-327 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988)). Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an intact human variable domain is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous sites in nonhuman antibodies.

Preferably, the invention relates to a humanized antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 46 or at least the variable domain thereof or an amino acid sequence having at least 90% sequence identity thereto and/or a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 45 or at least the variable domain thereof or an amino acid sequence having at least 90% sequence identity thereto.

Method for producing fully human antibodiesThe method is using mice that have been engineered to contain germline configured fragments (germline configured fragments) of human heavy chain loci and kappa light chain loci up to but below 1000kb in sizeAnd (5) strain. See Mendez et al (Nature Genetics 15: 146-.Strains are available from AMGEN, Inc (formerly ABGENIX, Fremont, CA).

Of miceThe generation of strains is discussed and described in US patent application series 07/466,008 filed on 12.1.1990, 07/610,515 filed on 8.11.1990, 07/919,297 filed on 24.7.1992, 07/922,649 filed on 30.7.1992, 08/031,801 filed on 15.3.1993, 08/112,848 filed on 27.8.1993, 08/234,145 filed on 28.4.1994, 08/376,279 filed on 20.1.1995, 08/430,938 filed on 27.4.1995, 08/464,584 filed on 5.6.1995, 08/464,582 filed on 5.6.6.1995, 08/463,191 filed on 5.6.1995, 08/462,837 filed on 5.6.5.1995, 08/486,853 filed on 5.6.5.1995, 08/486,857 filed on 5.6.5.6.1995, 08/486,859 filed on 5.6.5.6, 08/462,513 filed on 5.6.1995, 08/724,752 filed on 2.10.1996, 08/759,620 filed on 3.12.1996, U.S. publication 2003/0093820 and U.S. Pat. nos. 6,162, 963, 6,150,584, 6,114,598, 6,075,181 and 5,939,598 filed on 30.11.2001, and japanese patents 3068180B 2, 3068506B 2 and 3068507B 2. See also European patent EP 0463151B 1, granted on 12.6.1996, International patent application W09402602, published on 3.2.1994, International patent application WO9634096, published on 31.10.31.1996, WO9824893, published on 11.6.6.1998, and public 21.12.2000WO 0076310. The disclosures of each of the above-mentioned patents, applications, and references are incorporated herein by reference in their entirety.

In an alternative approach, other companies, including GenPharm International, inc, have used the "minilocus" approach. In the minilocus approach, the foreign Ig locus is mimicked by the inclusion of fragments (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a μ constant region, and generally a second constant region (preferably a γ constant region) are composed into a construct for insertion into an animal. The method is described in U.S. Pat. No. 5,545,807 to Surani et al and U.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 to Lonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023,010 to Krimpen and Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Berns et al, and U.S. Pat. No. 5,643,763 to Choi and Dunn, and Genpharm International U.S. patent application series 07/574,748 filed on 8/29/1990, 07/575,962 filed on 31/1990, 07/810,279 filed on 17/1991, 463/18/1992, 07/904,068 filed on 6/23/1992, 07/990,860/16/1993, 634/26/1993, 08/053,131/18/1993, 593/1993, 599/1993, 08/165,699 filed on 12/10/1993 and 08/209,741 filed on 3/9/1994, the disclosures of which are incorporated herein by reference. See also european patent 0546073B 1, international patent applications WO9203918, WO9222645, WO9222647, WO9222670, WO9312227, WO9400569, WO9425585, WO9614436, WO9713852 and WO9824884, and us 5,981,175, the disclosures of which are incorporated herein by reference in their entirety.

Kirin has also been shown to produce human antibodies from mice into which large fragments of chromosomes or complete chromosomes have been introduced by minicell fusion. See, european patent applications 773288 and 843961, the disclosures of which are incorporated herein by reference. In addition, KMTM mice have been generated as a result of cross-breeding Kirin's Tc mice with Metarx's minimus (Humab) mice. Such mice have human IgH transchromosomes of Kirin mice and kappa chain transgenes of Genpharm mice (Ishida et al, Cloning Stem Cells 4: 91-102, 2002).

Human antibodies can also be produced by in vitro methods. Suitable examples include, but are not limited to, phage display (CAT, Morphosys, Dyax, Biosite/Metarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (CAT), yeast display, and the like.

For therapeutic purposes, the antibody may be conjugated with a therapeutic effector group, such as a radioactive group or a cytotoxic group (cytoxic group).

For diagnostic purposes, the antibody may be labeled. Suitable labels include radioactive labels, fluorescent labels or enzymatic labels.

Further antibodies used according to the invention are the so-called xenogeneic antibodies (xenogeneic antibodies). The general principles for the production of xenogenous antibodies, e.g., human antibodies, in mice are described, for example, in WO9110741, WO 9402602, WO9634096 and WO 9633735.

As discussed above, in addition to intact antibodies, the antibodies of the invention may exist in a variety of forms, including, for example, Fv, Fab 'and F (ab')₂And in single stranded form; see, e.g., W08809344.

If desired, the antibodies of the invention can be mutated in the variable domains of the heavy and/or light chains to alter the binding properties of the antibody. For example, mutations can be made in one or more CDR regions to increase or decrease the Kd of an antibody for AXL or to alter the binding specificity of an antibody. Site-directed mutagenesis techniques are well known in the art. See, e.g., Sambrook et al and Ausubel et al, supra. Furthermore, amino acid residues known to be altered (compared to germline) can be mutated in the variable region of the AXL antibody. In another aspect, mutations can be introduced into one or more framework regions. Mutations may be made in the framework regions or constant domains to increase the half-life of the AXL antibody. See, for example, WO 0009560. Mutations may also be made in the framework or constant domains to alter the immunogenicity of the antibody, to provide sites for covalent or non-covalent binding to another molecule, or to alter properties such as complement fixation. Mutations can be made in each of the framework, constant domain and variable regions of a single mutant antibody. Alternatively, mutations may be made in only one of the framework, variable or constant domains of a single mutant antibody.

In a further aspect, the antibody may have a constant domain possessing effector function, whereby AXL-expressing cells that have bound the antibody, antibody fragment or derivative thereof on the cell surface may be attacked by immune system function. For example, antibodies may be capable of binding complement and involved in Complement Dependent Cytotoxicity (CDC). In addition, antibodies may be capable of binding Fc receptors on effector cells, such as monocytes and Natural Killer (NK) cells, and participate in antibody-dependent cellular cytotoxicity (ADCC).

In another aspect, the antibodies of the invention are useful in therapeutic treatment, preferably for the treatment of hyperproliferative diseases, cardiovascular diseases, in particular atherosclerosis and thrombosis, diabetes related diseases, in particular glomerular hypertrophy or diabetic nephropathy, and in particular disorders associated with, concomitant with or caused by AXL expression, overexpression or hyperactivity. The hyperproliferative disease is preferably selected from disorders associated with, accompanied by or caused by AXL expression, overexpression or hyperactivity, such as cancer, e.g. breast, colon, lung, kidney, follicular lymphoma, myeloid leukemia, skin/melanoma, glioblastoma, ovarian, prostate, pancreatic, Barrett's esophagus and esophageal, gastric, bladder, cervical, liver, thyroid and head and neck cancers, or proliferative and neoplastic diseases or other hyperproliferative diseases expressing or overexpressing AXL.

In another aspect, the antibodies of the invention can be used for co-administration with an anti-tumor agent to treat one of the above-mentioned conditions.

As used herein, co-administration includes administration of the antibodies of the invention with an anti-neoplastic agent, preferably an anti-neoplastic agent that induces apoptosis. The term co-administration also encompasses the administration of the antibody of the invention and an anti-neoplastic agent, preferably an anti-neoplastic agent that induces apoptosis, either in the form of a single composition or in the form of two or more different compositions. Co-administration includes administering the antibody of the invention simultaneously (i.e., at the same time) or sequentially (i.e., at certain intervals) with an anti-neoplastic agent, preferably an anti-neoplastic agent that induces apoptosis.

The invention also relates to nucleic acid molecules encoding the antibodies, antibody fragments or derivatives thereof of the invention. The nucleic acid molecule of the invention encoding the above-described antibody, antibody fragment or derivative thereof may be, for example, DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule (which comprises any such nucleic acid molecule, alone or in combination). The nucleic acid molecule may also be genomic DNA corresponding to the complete gene or a large part thereof or to fragments and derivatives thereof. The nucleotide sequence may correspond to a naturally occurring nucleotide sequence or may comprise single or multiple nucleotide substitutions, deletions or additions. In a particularly preferred embodiment of the invention, the nucleic acid molecule is a cDNA molecule.

Preferably, the present invention relates to an isolated nucleic acid molecule selected from the group consisting of:

(a) encoding SEQ ID NOs: 7-12, 13-30, 37-42, 43-46

(b) SEQ ID NOs: 1-6, 31-36, nucleic acid sequences shown in

(c) A nucleic acid complementary to any of the sequences in (a) or (b); and

(d) a nucleic acid sequence capable of hybridizing under stringent conditions to (a), (b) or (c).

The term "hybridize under stringent conditions" means that two nucleic acid fragments hybridize under conditions such as, for example, in Sambrook et al, "Expression of connected genes in E.coll" in molecular cloning: a laboratory manual (1989), Cold Spring harbor laboratory Press, New York, USA under standard hybridization conditions. Such conditions are for example hybridisation in 6.0XSSC at about 45 ℃ followed by a wash step using 2.0XSSC at 50 ℃, preferably 2.0XSSC at 65 ℃, or 0.2XSSC at 50 ℃, preferably 0.2XSSC at 65 ℃.

The invention also relates to vectors comprising the nucleic acid molecules of the invention. The vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication deficient vectors. In the latter case, viral propagation usually occurs only in complementing host cells (complementing host cells).

The nucleic acid molecules of the invention may be ligated to a vector comprising a selectable marker for amplification in a host. Typically, the plasmid vector is introduced in the form of a precipitate, e.g., a calcium phosphate precipitate or a rubidium chloride precipitate, or in the form of a complex with a charged lipid or in the form of a carbon-based cluster, e.g., a fullerene (fullerene). If the vector is a virus, it may be packaged in vitro using a suitable packaging cell line prior to application to a host cell.

Preferably, the vectors of the invention are expression vectors wherein the nucleic acid molecule is operably linked to one or more control sequences allowing transcription and optionally expression in prokaryotic and/or eukaryotic host cells. Expression of the nucleic acid molecule includes transcription of the nucleic acid molecule (preferably to a translatable mRNA). Regulatory elements which ensure expression in eukaryotic, preferably mammalian, cells are well known to the person skilled in the art. They generally comprise regulatory sequences which ensure the initiation of transcription and optionally poly-A signals which ensure the termination of transcription and the stabilization of the transcript. Additional regulatory elements may include transcriptional and translational enhancers. Possible regulatory elements which allow expression in prokaryotic host cells include the lac, trp or tac promoter in e.coli (e.coli), examples of regulatory elements which allow expression in eukaryotic host cells are the AOXI or GAL1 promoter in yeast or the CMV promoter, SV40 promoter, RSV (rous sarcoma virus) promoter, CMV enhancer, SV40 enhancer or globin intron in mammalian and other animal cells. In addition to the elements responsible for transcription initiation, such regulatory elements may also include transcription termination signals downstream of the polynucleotide, such as the SV40-poly-A site or tk-poly-A site. In the present specification, suitable expression vectors are known in the art, for example, the Okayama-Berg cDNA expression vector pcDV1(Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3(Invitrogen) or pSPORTI (GIBCO BRL). Preferably, the vector is an expression vector and/or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses may be used to deliver the polynucleotides or vectors of the invention into targeted cell populations. Methods well known to those skilled in the art can be used to construct recombinant viral vectors; see, e.g., techniques described in Sambrook, Molecular cloning A Laboratory Manual, Cold Spring Harbor Laboratory (2001, 3 rd edition) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). Alternatively, the nucleic acid molecules of the invention can be reconstituted into liposomes for delivery to target cells.

The invention also relates to a host comprising the vector of the invention. The host may be a prokaryotic or eukaryotic cell or a non-human transgenic animal. The polynucleotide or vector of the invention present in the host may be integrated into the genome of the host or it may be maintained extrachromosomally. In this respect, it is also understood that the nucleic acid molecules of the invention may be used for "gene targeting" and/or "gene replacement" to restore a mutated gene or to produce a mutated gene by homologous recombination; see, e.g., Mouellic, Proc. nat! Acad.Sci.USA, 87(1990), 4712-; joyner, GeneTargeting, A Practical Approach, Oxford University Press.

The host may be any prokaryotic or eukaryotic cell, such as a bacterial cell, an insect cell, a fungal cell, a plant cell, an animal cell, a mammalian cell or preferably, a human cell. Preferred fungal cells are e.g. fungal cells of the genus Saccharomyces (Saccharomyces), in particular of the species Saccharomyces cerevisiae (s. The term "prokaryotic" is intended to include all bacteria that can be transformed or transfected with polynucleotides to express the variant polypeptides of the present invention. Prokaryotic hosts may include gram-negative as well as gram-positive bacteria, such as escherichia coli, salmonella typhimurium (s.typhimurium), Serratia marcescens (Serratia marcescens), and Bacillus subtilis. Polynucleotides encoding mutant forms of the variant polypeptides of the invention can be used to transform or transfect a host using any technique known to those skilled in the art. The genetic constructs and methods described herein can be used to express the variant antibodies of the invention, antibody fragments or derivatives thereof, for example, in a prokaryotic host An antibody fragment or derivative thereof. The antibodies, antibody fragments or derivatives thereof of the present invention expressed by a microorganism or other organism may be isolated and purified by any conventional method such as preparative chromatographic separation and immunological separation such as those involving the use of monoclonal or polyclonal antibodies.

In a preferred embodiment of the invention, the host is a bacterial, fungal, plant, amphibian or animal cell. Preferred animal cells include, but are not limited to, Chinese Hamster Ovary (CHO) cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), 3T3 cells, NSO cells, and many other cell lines including human cells, e.g., per.c 6. In another preferred embodiment, the animal cell is an insect cell. Preferred insect cells include, but are not limited to, SF9 cell line cells.

In a more preferred embodiment of the invention, the host is a human cell or a human cell line. The human cells include, but are not limited to, human embryonic kidney cells (HEK293, 293T, 293 freestyle). In addition, the human cell lines include, but are not limited to, HeLa cells, human hepatocellular carcinoma cells (e.g., Hep G2), a549 cells.

The invention also provides transgenic non-human animals comprising one or more nucleic acid molecules of the invention that can be used to produce antibodies of the invention. Antibodies can be produced and recovered in tissues or body fluids such as milk, blood or urine of goats, cows, horses, pigs, rats, mice, rabbits, hamsters or other mammals. See, for example, U.S. patent 5,827,690; 5,756,687, respectively; 5,750,172, respectively; and 5,741,957. As described above, a non-human transgenic animal comprising a human immunoglobulin locus may be produced by immunization with AXL or a portion thereof.

The invention also relates to a method for producing an antibody comprising culturing a host of the invention under conditions allowing synthesis of said antibody and recovering said antibody from said culture.

The transformed host may be grown and cultured in a fermentor according to techniques known in the art to obtain optimal cell growth. After expression, the intact antibodies of the invention, their dimers, individual light and heavy chains or other immunoglobulin forms, may be purified according to standard methods in the art including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like; see, Scopes, "Protein Purification", Springer-Yerlag, N.Y. (1982). The antibodies of the invention or their corresponding immunoglobulin chains can then be isolated from the growth medium, cell lysate or cell membrane fraction. Antibodies or immunoglobulin chains of the invention, e.g., expressed by a microorganism, can be isolated and purified by any conventional method, e.g., preparative chromatographic separation and immunological separation, such as those involving the use of monoclonal or polyclonal antibodies against, e.g., the constant regions of antibodies of the invention.

It will be apparent to those skilled in the art that the antibodies of the invention may also be conjugated to other moieties for, for example, drug targeting and imaging applications. Conjugation to the attachment site can be performed chemically after expression of the antibody or antigen, or the conjugation product can be engineered into the antibody or antigen of the invention at the DNA level. The DNA is then expressed in a suitable host system and, if desired, the expressed protein is collected and renatured.

In a preferred embodiment of the invention, the antibody is conjugated to an effector such as a radioisotope or a toxic chemotherapeutic agent. Preferably, such antibody conjugates are used to target AXL-expressing cells, such as cancer cells, to eliminate them. The linking of the antibodies/antibody fragments of the invention with a radioisotope provides an advantageous aspect, for example, in the treatment of tumours. Unlike chemotherapy and other forms of cancer therapy, administration of radioimmunotherapy or radioisotope-antibody combinations targets cancer cells directly with minimal damage to surrounding normal, healthy tissue. Preferred radioisotopes include, for example³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I。

Furthermore, the antibodies of the invention can be used for the treatment of cancer when conjugated with toxic chemotherapeutic drugs such as geldanamycin (Mandler et al, J.Natl.cancer Inst., 92(19), 1549-51(2000)) and maytansinoids such as maytansinoid (maytansinoid) drug DM1(Liu et al, Proc.Natl.Acad.Sci.U.S.A.93: 8618-8623(1996) and auristatin-E or monomethyylaristin-E (Doronina et al, Nat.Biotechnol.21: 778-784(2003) or calicheamicin (calicheamicin)) and different linkers that release the drug under acidic or reducing conditions or when exposed to specific proteases are used for this technique.

The invention also relates to pharmaceutical compositions comprising the antibodies, nucleic acid molecules, vectors, hosts of the invention or antibodies obtained by the methods of the invention.

The term "composition", as used herein, comprises at least one compound of the invention. Preferably, such a composition is a pharmaceutical composition or a diagnostic composition.

Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or diluent. The pharmaceutical compositions disclosed herein are useful, in part, for the treatment of disorders associated with, accompanied by or caused by AXL expression, overexpression or hyperactivity, such as hyperproliferative diseases, cardiovascular diseases, in particular atherosclerosis and thrombosis, diabetes related diseases, in particular glomerular hypertrophy or diabetic nephropathy. Such disorders include, but are not limited to, cancers such as breast, colon, lung, kidney, follicular lymphoma, myeloid leukemia, skin/melanoma, glioblastoma, ovarian, prostate, pancreatic, barrett's esophagus and esophagus, stomach, bladder, cervical, liver, thyroid and head and neck, or other proliferative or neoplastic diseases or other AXL expressing or over-expressing diseases.

The term "hyperactive" refers herein to uncontrolled AXL signaling that may result from a lack of negative regulation and/or dysfunction. For example, negative regulation includes dephosphorylation, degradation, and/or endocytosis of the protein. Furthermore, uncontrolled AXL signaling may be the result of genetic alterations (somatic or germline) that result in changes in the AXL amino acid sequence.

Examples of suitable pharmaceutical carriers, excipients, and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions comprising such carriers may be formulated by well-known conventional methods. Such pharmaceutical compositions may be administered to a subject in a suitable dose. Administration of a suitable composition can be carried out by different methods, for example by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The compositions of the invention may also be administered directly to a target site, for example by gene gun (biolistic) delivery to an external or internal target site such as the brain. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage for any one patient depends on many factors, including the size of the patient, the body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The proteinaceous pharmaceutical active substance may be present in an amount of 1 μ g to 100mg/kg body weight per dose; however, it is contemplated to use dosages below or above this exemplary range, particularly in view of the above factors. If the regimen is a continuous infusion, it should also be in the range of 1pg to 100mg/kg body weight/min.

Progress can be monitored by periodic evaluation. The compositions of the present invention may be administered locally or systemically. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the present invention may further comprise additional agents depending on the intended use of the pharmaceutical composition. Particularly preferably, the pharmaceutical composition comprises an additional active agent such as, for example, an additional anti-tumor agent, a small molecule inhibitor, an anti-tumor agent or a chemotherapeutic agent.

The invention also relates to a pharmaceutical composition comprising an anti-AXL antibody, preferably an antibody of the invention in combination with at least one additional anti-neoplastic agent. The combination is effective, for example, in inhibiting abnormal cell growth.

Many antineoplastic agents are currently known in the art. In general, the term includes all agents capable of preventing, alleviating and/or treating a hyperproliferative disorder. In one embodiment, the antineoplastic agent is selected from therapeutic proteins, including but not limited to antibodies or immunomodulatory proteins. In another embodiment, the antineoplastic agent is selected from the group consisting of a small molecule inhibitor or chemotherapeutic agent consisting of mitotic inhibitors, kinase inhibitors, alkylating agents, antimetabolites, intercalating antibiotics (intercalating antibiotics), growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, histone deacetylase inhibitors, anti-survival agents (anti-survivability agents), biological response modifiers, anti-hormones such as antiandrogens, and anti-angiogenic agents.

Specific examples of antineoplastic agents that can be used in combination with the antibodies provided herein include, for example, gefitinib, lapatinib, sunitinib, pemetrexed, bevacizumab, cetuximab, imatinib, trastuzumab, alemtuzumab, rituximab, erlotinib, bortezomib, and the like. Other specific antineoplastic agents for use in the compositions described and claimed herein include, for example, chemotherapeutic agents such as capecitabine, daunorubicin, daunomycin, actinomycin D, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, macsfamide, ifosfamide, cytarabine, dicloethylnitrosurea, busulfan, mitomycin C, actinomycin D, plicamycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, mechlorethamine, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, Cytarabine (CA), 5-azacytidine, hydroxyurea, desoxymestranilicin, 4-hydroxyperoxycyclophosphamide, doxycycline, daunorubicin, doxycycline, doxyc, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), Methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin, and Diethylstilbestrol (DES). See generally, The Merck Manual of Diagnosis and Therapy, 15 th edition 1987, pp.1206-1228, Berkow et al, eds., Rahway, N.J. Particularly preferred are such antineoplastic agents that induce apoptosis.

When used with the AXL antibody, such antineoplastic agents can be used alone (e.g., 5-FU and antibody), sequentially (e.g., 5-FU and antibody for a period of time, followed by administration of MTX and antibody), or in combination with one or more other such antineoplastic agents (e.g., 5-FU, MTX and antibody, or 5-FU, radiotherapy and antibody).

The term antineoplastic agent may also include methods of treatment, such as radiation or radiation therapy.

The pharmaceutical compositions of the invention are useful in human medicine, but also for veterinary purposes.

Furthermore, the present invention relates to the use of an antibody of the invention, a nucleic acid molecule of the invention, a vector, a host or an antibody obtained by a method of the invention for the preparation of a pharmaceutical composition for the diagnosis, prevention or treatment of hyperproliferative diseases, cardiovascular diseases, in particular atherosclerosis and thrombosis, diabetes related diseases, in particular glomerular hypertrophy or diabetic nephropathy, and in particular disorders associated with, accompanied by or caused by AXL expression, overexpression or hyperactivity.

Such hyperproliferative diseases include any neoplasia, i.e. any abnormal and/or uncontrolled new tissue growth. The term "uncontrolled new tissue growth", as used herein, may rely on dysfunction and/or loss of growth regulation. Hyperproliferative diseases include neoplastic diseases and/or cancers, such as metastatic or invasive cancers.

In a preferred embodiment of the use of the invention, the hyperproliferative disease is in particular breast cancer, colon cancer, lung cancer, kidney cancer, follicular lymphoma, myeloid leukemia, skin cancer/melanoma, glioblastoma, ovarian cancer, prostate cancer, pancreatic cancer, barrett's esophagus and esophagus cancer, stomach cancer, bladder cancer, cervical cancer, liver cancer, thyroid cancer and head and neck cancer, or a proliferative or neoplastic disease or other hyperproliferative disease expressing or overexpressing AXL.

In another embodiment, the invention relates to the use of an anti-AXL antibody, preferably an antibody of the invention, for the preparation of a medicament for co-administration with an anti-tumour agent for the treatment of one of the above mentioned conditions.

According to a further preferred embodiment, the invention relates to the use of an anti-AXL antibody for the manufacture of a pharmaceutical composition for the treatment of drug resistant cancer. In a particularly preferred embodiment, the anti-AXL antibody is a monoclonal antibody as defined in claims 1 to 22.

The invention also relates to a diagnostic composition comprising an antibody of the invention, a nucleic acid molecule, vector, host of the invention or an antibody obtained by a method of the invention and optionally a pharmaceutically acceptable carrier.

The diagnostic compositions of the invention may be used to detect undesired expression, overexpression or hyperactivity of mammalian AXL in different cells, tissues or another suitable sample, comprising contacting the sample with an antibody of the invention, and detecting the presence of AXL in the sample. Thus, the diagnostic compositions of the present invention can be used to assess the onset or disease state of hyperproliferative diseases.

In addition, malignant cells such as AXL-expressing cancer cells can be targeted with the antibodies of the invention. Cells that have bound the antibodies of the invention may therefore be attacked by immune system functions such as the complement system or by cell-mediated cytotoxicity, thereby reducing the number of or eradicating cancer cells. These considerations apply equally to the treatment of metastatic and recurrent tumors.

In another aspect of the invention, the antibody of the invention is conjugated to a labeling group. Such antibodies are particularly suitable for diagnostic applications. As used herein, the term "labeling group" refers to a detectable marker, such as a radiolabeled amino acid or a biotin moiety that is detectable by labeled avidin. For labelling polypeptides orVarious methods of glycoproteins, such as antibodies, are known in the art and can be used to carry out the present invention. Examples of suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g. of the type³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) A fluorescent group (e.g., FITC, rhodamine, lanthanide), phosphor), an enzymatic group (e.g., horseradish peroxidase,. beta. -galactosidase, luciferase, alkaline phosphatase), a chemiluminescent group, a biotin group, or a predetermined polypeptide epitope (e.g., leucine zipper pair sequence, binding site for a secondary antibody, metal binding domain, epitope tag) recognized by a second reporter.

In certain aspects, it may be desirable to attach the labeling groups through spacer arms of different lengths to reduce potential steric hindrance.

In another embodiment, the invention relates to a method of assessing the presence of cells expressing AXL comprising contacting an antibody of the invention with cells or tissues suspected of having AXL on their/its surface. A suitable method for detecting AXL expression in a sample may be enzyme linked immunosorbent assay (ELISA) or Immunohistochemistry (IHC).

The ELISA assay can be performed in a microtiter plate format, wherein for example the wells of the microtiter plate are adsorbed with the AXL antibody. The plate is rinsed and treated with a blocking agent such as milk protein or albumin to prevent non-specific adsorption of the analyte. The wells were then treated with the test samples. After rinsing away the test sample or standard, the wells are treated with a labeled second AXL antibody (e.g., by biotin conjugation). After washing away excess secondary antibody, the label is detected with, for example, avidin-conjugated horseradish peroxidase (HRP) and a suitable chromogenic substrate. The concentration of AXL antigen in the test sample is determined by comparison to a standard curve generated from a standard sample.

For IHC, paraffin embedded tissue may be used, where the tissue is first dewaxed in xylene, for example, then dehydrated with ethanol, for example, and rinsed in distilled water. Epitopes masked by formaldehyde fixation and paraffin embedding can be exposed by epitope exposure (epitope unmasking), enzymatic degradation, or saponin. For epitope exposure, paraffin sections can be heated in an epitope retrieval solution (e.g., 2N HCl solution (pH 1.0) in a steam generator, water bath, or microwave oven for 20 to 40 minutes. In case of enzymatic degradation, the tissue sections can be incubated in different enzyme solutions, e.g. proteinase K, trypsin, pronase, pepsin, etc. at 37 ℃ for 10 to 30 minutes.

After rinsing away the epitope repair solution or excess enzyme, the tissue sections were treated with blocking buffer to prevent non-specific interactions. The first AXL antibody is added at a suitable concentration. Excess primary antibody was rinsed off and the sections were incubated for 10 minutes at room temperature in peroxidase lock solution. After performing another washing step, the tissue section is incubated with a second labeled antibody (e.g., labeled with a group that can serve as an anchor for the enzyme). Thus, an example is a biotin-labeled secondary antibody that is recognized by streptavidin coupled to horseradish peroxidase. Detection of the antibody/enzyme complex is carried out by incubation with a suitable chromogenic substrate.

In a further embodiment, the invention relates to a method of blocking AXL function comprising contacting an antibody of the invention with a cell or tissue suspected to have AXL on their/their surface under conditions wherein the antibody is capable of blocking AXL function. The contacting may be in vitro or in vivo.

The present invention also relates to a method of treating hyperproliferative diseases, cardiovascular diseases, in particular atherosclerosis and thrombosis, diabetes related diseases, in particular glomerular hypertrophy or diabetic nephropathy, comprising administering a suitable dose of an antibody or antibody fragment or derivative thereof of the invention to a patient in need thereof. The hyperproliferative disease is preferably selected from disorders associated with, accompanied by or caused by AXL expression, overexpression or hyperactivity, such as cancer, e.g. breast, colon, lung, kidney, follicular lymphoma, myeloid leukemia, skin/melanoma, glioblastoma, ovarian, prostate, pancreatic, barrett's esophagus and esophagus, stomach, bladder, cervical, liver, thyroid and head and neck cancer or a proliferative or neoplastic disease or other hyperproliferative diseases expressing or overexpressing AXL.

According to another preferred embodiment of the present invention, the cancer to be treated is a drug-resistant cancer.

The invention also relates to a method of treating a disease, wherein an antibody of the invention is administered to a mammal and wherein the disease is directly or indirectly associated with expression or activity of abnormal levels of AXL.

Finally, the invention relates to a kit comprising an anti-AXL antibody, preferably an antibody, antibody fragment or derivative thereof of the invention, a nucleic acid molecule encoding said component and/or a vector of the invention.

All embodiments including the compounds disclosed herein can be used as single compounds or in combination for the preparation of medicaments.

Brief description of the drawings

FIG. 1 flow cytometric analysis of cell surface AXL in Ratl-mock and Ratl-AXL c1.2 fibroblasts. Polyclonal Ratl-mock and clonal Ratl-AXL c1.2 cells generated by infection of Ratl fibroblasts with pLXSN and pLXSN-hAXL ecotropic virus (ecotropic virus), respectively, were collected and stained with 3. mu.g/ml of mouse control antibody 72A1 (left panel) or mouse anti-AXL MAB154 primary antibody (right panel) and PE-conjugated anti-mouse secondary antibody. For details see the text. Staining of Ratl-AXL c1.2 cells resulted in 3 orders of magnitude shift and demonstrated that AXL was overexpressed on the surface of these cells.

FIG. 2 flow cytometry analysis of cell surface AXL in NIH3T 3-mock and NIH3T3-AXL c1.7 fibroblasts. Polyclonal NIH3T 3-mock and clonal NIH3T3-AXL c1.7 cells generated by infection of NIH3T3 fibroblasts with pLXSN and pLXSN-AXL ecotropic virus, respectively, were collected and stained with 3 μ g/ml of mouse control antibody 72A1 (left panel) or mouse anti-AXL MAB154 primary antibody (right panel) and PE-conjugated anti-mouse secondary antibody. For details see the text. Staining of NIH3T3-AXL c1.7 cells resulted in a shift of 2 orders of magnitude and demonstrated that AXL was overexpressed on the surface of these cells.

Figure 3 flow cytometric analysis of the cross-reactivity of rat anti-AXL antibodies with mouse and cynomolgus AXL as well as Mer and Sky. HEK293T fibroblasts were transiently transfected with pcDNA3, pcDNA3-hAXL, pcDNA3mAXL, pcDNA3-cyAXL, pcDNA3-hMer or pcDNA 3-hSky. Cells were harvested and then stained with 10 μ g/ml anti-AXL 1D5, 11D5, 11B7, 10D12, 6E7, 2a1, 11D7, or 12B7 primary and/or PE-conjugated donkey-anti-rat secondary antibody or PE-conjugated donkey-anti-mouse secondary antibody (used only for control). For details see the text. No anti-AXL antibodies cross-reacted with these molecules except 12B7, which showed moderate cross-reactivity with mouse AXL and human Mer and Sky. In contrast, all anti-AXL antibodies tested cross-reacted with cynomolgus monkey AXL.

FIG. 4 ELISA assay to study the effect of rat anti-AXL antibodies on the phosphorylation of AXL receptors. Starved NIH3T3-AXL c1.7 fibroblasts (A) and NCI-H292 lung cancer cells (B), pre-incubated with 10. mu.g/ml of the mouse control antibody 72A1 and the rat anti-AXL antibody 2A1, 11D7, 11D5, 11B7, 6E7 or 10D12, with or without 400ng/ml of mGas6, and then lysed. Lysates were transferred to Maxi-Sorp 96 well plates coated with anti-phospho-tyrosine antibody 4G10, the plates were then washed and incubated with 0.5G/ml biotinylated rat anti-AXL antibody 12B7, AP conjugated streptavidin and AttoPhos substrate solution to collect fluorescence intensity. For details see the text. Rat anti-AXL antibodies 11B7, 11D5, 6E7, and 10D12 are capable of blocking or reducing ligand-mediated AXL activation (as shown by reduced phosphorylation) and are therefore considered to be antagonistic anti-AXL antibodies. In contrast, rat anti-AXL antibodies 2a1 and 11D7 stimulated basal AXL activation (as shown by increased phosphorylation), did not significantly reduce ligand-mediated AXL activation, and were therefore considered agonistic anti-AXL antibodies.

FIG. 5 ELISA assay to investigate the effect of rat anti-AXL antibodies on p42/p44MAP kinase phosphorylation. Starved CaSki cervical cancer cells were preincubated with 10. mu.g/ml of isotype control antibody 1D5 and rat anti-AXL antibody 11D5, 11B7 or 2A1, treated with or without 400ng/ml mGas6, and then fixed with formaldehyde. Cells were washed, quenched and then incubated with anti-phospho-p 44/p42MAP kinase (Thr202/Tyr204) primary, HRP-conjugated anti-rabbit secondary and tetramethylbenzidine solution to measure absorbance intensity. For details see the text. Rat anti-AXL antibodies 11B7 and 11D5 were able to reduce ligand-mediated p42/p44MAP kinase activation, as shown by reduced phosphorylation, and were thus considered antagonistic anti-AXL antibodies. In contrast, rat anti-AXL antibody 2a1 stimulated basal p42/p44MAP kinase activation (as shown by increased phosphorylation), without decreasing ligand-mediated p42/p44MAP kinase activation, and was considered an agonistic anti-AXL antibody.

FIG. 6 ELISA assay to study the effect of rat anti-AXL antibody on Akt kinase phosphorylation. Starved NIH3T3-AXL c1.7 fibroblasts (A) and CaLu-1 lung carcinoma cells (B) were preincubated with 10g/ml of isotype control antibody 1D5 and rat anti-AXL antibodies 11D5, 11B7 or 2A1, with or without 400ng/ml of mGas6, and then fixed with formaldehyde. Cells were washed, quenched and then incubated with anti-phospho-Akt (Ser473) primary antibody, HRP-conjugated anti-rabbit secondary antibody, and tetramethylbenzidine solution to measure absorbance intensity. For details see the text. Rat anti-AXL antibodies 11B7 and 11D5 were able to block or reduce ligand-mediated Akt kinase activation, as shown by reduced phosphorylation, and were thus considered antagonistic anti-AXL antibodies. In contrast, rat anti-AXL antibody 2a1 stimulates basal Akt-kinase activation (as shown by increased phosphorylation), does not decrease ligand-mediated Akt kinase activation, and is therefore considered an agonistic anti-AXL antibody.

FIG. 7 ELISA assay comparing the effect of rat and chimeric anti-AXL antibodies on Akt kinase phosphorylation. Starved NIH3T3-AXL c1.7 fibroblasts were preincubated with rat anti-AXL antibody 11B7 or chimeric anti-AXL antibody ch11B7 and 50ng/ml, 100ng/ml, 300ng/ml, 500ng/ml, 1g/ml, 5 μ g/ml and 10 μ g/ml of rat anti-AXL antibody 11D5 or chimeric anti-AXL antibody ch11D5 with or without 400ng/ml mGas6 and then fixed with formaldehyde. Cells were washed, quenched, and incubated with anti-phospho-Akt (Ser473) primary antibody, HRP-conjugated anti-rabbit secondary antibody, and tetramethylbenzidine solution to measure absorbance intensity. For details see the text. Rat anti-AXL antibody 11B7 and chimeric anti-AXL antibody ch11B7 as well as rat anti-AXL antibody 11D5 or chimeric anti-AXL antibody ch11D5 were able to inhibit ligand-mediated activation of Akt kinase to a similar extent, as shown by reduced phosphorylation. Thus, the chimeric anti-AXL antibodies ch11B7 and ch11D5 retained activity compared to their respective rat counterparts.

FIG. 8 competitive ELISA experiments to study the binding properties of rat anti-AXL antibodies. 96-well Maxi-Sorp plates were coated with 1. mu.g/ml human AXL-ECD and then pre-incubated with 10. mu.g/ml of the non-biotinylated isotype control antibody 1D5 or the rat anti-AXL antibody 11B7, 11D5, 6E7, 10D12, 11D7 or 2A 1. After incubation with 0.5 μ g/ml biotinylated isotype control antibody 1D5 or biotinylated rat anti-AXL antibody 11B7, 11D5, 6E7, 10D12, 11D7, or 2a1, AP-conjugated streptavidin and AttoPhos substrate solution were added and fluorescence was collected to measure bound biotinylated antibody. For details see the text. Control antibody 1D5 did not bind AXL-ECD. Antagonistic anti-AXL antibodies 11B7, 11D5, 6E7 and 10D12 compete with each other for the same or structurally adjacent epitopes. Agonistic antibodies 11D7 and 2a1 recognize different epitopes and do not compete with antagonistic antibodies for binding to AXL-ECD.

Figure 9 wound healing/scratch assay to study the effect of rat and chimeric anti-AXL antibodies on cell migration and proliferation. After growth to confluence, NCI-H292 lung cancer cells were starved and the cells were scratched with a pipette tip. Cells were allowed to re-enter the cleared area in the presence of 10 μ g/ml of isotype control antibody 1D5, antagonistic rat anti-AXL antibody 11D5, 11B7, 6E7 or 10D12, chimeric anti-AXL antibodies chn11D5 IgG2 and chn11B7IgG2, agonistic rat anti-AXL antibodies 2a1 and 11D7, and 10 μ g/ml of Erbitux or 5 μ M Sutent. After 24 hours, the cells were fixed and stained, and a photograph of the wound was taken. For details see the text. Antagonistic rat anti-AXL antibodies 11D5, 11B7, 6E7 and 10D12 and chimeric anti-AXL antibodies chn11D5 IgG2 and chn11B7IgG2 reduced re-entry into the cleared area compared to isotype control antibody 1D5, whereas agonistic rat anti-AXL antibodies 2a1 and 11D7 resulted in complete wound closure.

FIG. 10. Boyden chamber/transwell assay to study the effect of rat anti-AXL antibodies on directed cell migration. Serum-starved NIH3T3-AXL c1.7 fibroblasts were preincubated with 10 μ g/ml of rat anti-AXL antibody 4a6, 11B7, or 2a1, plated on top of collagen I coated fluoroblock inserts, and then exposed to serum-free medium with or without Gas6 in a lower compartment. After 7 hours, the migrated cells were stained with calcein-AM and the fluorescence of each well was measured. For details see the text. Antagonistic anti-AXL antibody 11B7 reduced basal and Gas 6-induced migration of NIH3T3-AXL c1.7 fibroblasts, whereas agonistic rat anti-AXL antibody 2a1 increased ligand-induced migration and specifically basal migration of NIH3T3-AXL c 1.7. Antibody 4a6 did not affect directional cell migration.

FIG. 11 AlamarBlue to study the effect of rat anti-AXL antibodies on Gas 6-induced cell proliferation^TMAnd (4) measuring. Serum-starved NIH3T3-AXL c1.7 fibroblasts were pre-incubated with 20 μ g/ml of mouse control antibody 72A1, rat antagonist anti-AXL antibodies 11D5 and 11B7, and agonist anti-AXL antibody 2A1, and then grown in the absence or presence of 400ng/ml Gas 6. After 4 days, AlamarBlue was added to the cells^TMThen, the absorbance was measured. For details see the text. Antagonistic anti-AXL antibodies 11D5 and 11B7 inhibited Gas 6-induced proliferation of NIH3T3-AXL c1.7 fibroblasts, whereas agonistic rat anti-AXL antibody 2a1 increased ligand-induced proliferation and in particular basal proliferation of NIH3T3-AXL c1.7 cells.

FIG. 12 caspase-3/7 assay to study the effect of rat anti-AXL antibodies on Gas6 mediated anti-apoptosis. Serum-starved NIH3T3-AXL c1.7 fibroblasts were pre-incubated with 10. mu.g/ml of isotype control antibody 1D5, antagonistic rat anti-AXL antibodies 11B7 and 11D5, or agonistic rat anti-AXL antibodies 11D7 and 2A1, and then treated with or without Gas 6. Apo-ONE substrate solution was added and fluorescence was collected to measure caspase-3/7 activity. For details see the text. Antagonistic rat anti-AXL antibodies 11B7 and 11D5 reduced Gas 6-mediated anti-apoptosis of serum-starved NIH3T3-AXL c1.7 fibroblasts compared to isotype control antibodies, thereby inducing apoptosis. In contrast, the agonistic rat anti-AXL antibodies 2a1 and 11D7 induced anti-apoptosis of serum-starved NIH3T3-AXL c1.7 cells, regardless of the presence or absence of Gas6, thereby inhibiting apoptosis.

FIG. 13. Spheroid-based (Spheroid-based) cellular angiogenesis assay to study the effect of rat anti-AXL antibodies on VEGF-A induced endothelial cell sprouting. HUVEC spheroids were embedded in 3D collagen gel, stimulated with 25ng/ml VEGF-A, and then treated with the indicated concentrations of antagonistic rat anti-AXL antibodies 11B7(A) and 11D5(B) for 24 hours. The mean ± SEM of cumulative shoot length of 10 randomly selected spheroids was analyzed per data point to determine the relative inhibition of the antibody (right panel). IC with GraphPad Prism 4.03₅₀Fitting of curves and IC₅₀And (4) calculating a value. For details see the text. Antagonistic rat anti-AXL antibodies 11B7 and 11D5 inhibited VEGF-a stimulated HUVEC sprouting in a dose-dependent manner in a spheroid-based angiogenesis assay. Whereas treatment with the highest concentration of 11B7 reduced HUVEC sprouting to basal levels, inhibition with the highest concentration of 11D5 was not as effective as the former (left panel). HUVEC sprouting was inhibited, for 11B7 and 11D5, IC₅₀The values are respectively 9.8x10^-8M and 7.0x10^-7M (right panel).

Figure 14. orthotopic xenograft model investigating the effect of rat anti-AXL antibodies on human prostate cancer growth in nude mice. Orthotopic implantation of PC-3-LN prostate cancer cells into NMRI^-nu/nuProstate of mouse. Animals were randomized into 4 groups and received 25mg/kg of either isotype control antibody 1D5 or antagonistic rat anti-AXL antibody 11B7, and40mg/kg of Sutent or 12.5mg/kg of Taxotere. During treatment, the growth of orthotopic PC-3-LN tumors and peripheral metastases was monitored weekly by in vivo bioluminescence imaging on day 15, day 23, day 29 and day 34. For details see the text. The antagonistic rat anti-AXL antibody 11B7 reduced the overall growth of PC-3-LN prostate tumor in nude mice compared to isotype control antibody 1D 5.

Figure 15. orthotopic xenograft model investigating the effect of rat anti-AXL antibodies on human prostate cancer metastasis in nude mice. Orthotopic implantation of PC-3-LN prostate cancer cells into NMRI^-nu/nuProstate of mouse. Animals were randomized into 4 groups and received 25mg/kg of either isotype control antibody 1D5 or antagonistic rat anti-AXL antibody 11B7, and 40mg/kg Sutent or 12.5mg/kg Taxotere. After necropsy, selected organs (liver, spleen, lung, femur and part of lumbar spine) were collected and analyzed for the presence of metastases by bioluminescent imaging. For details see the text. The antagonistic rat anti-AXL antibody 11B7 of the invention reduced the occurrence of splenic metastases compared to the isotype control antibody 1D 5. Notably, 11B7 was more resistant to metastasis than Sutent in this experiment.

Figure 16 immunohistochemical analysis of AXL expression in different human malignancies. 17 human solid tumor types, each represented by paired tumor tissues and matched non-malignant tissues, were analyzed for AXL expression by immunohistochemistry. For details see the text. Results (a) are summarized, where an intensity of 1 means that there is weak staining in more than 25% of the cells observed. An example of the strongest staining observed in breast tumors and gastric signet ring cell carcinomas is shown (B).

Figure 17 ELISA experiments comparing the effect of rat and chimeric anti-AXL antibodies on Ax1 phosphorylation. Starved CaSki cervical cancer cells, cells pre-incubated with rat anti-AXL antibody 11B7(A) or chimeric anti-AXL antibody ch11B7(B) at 50ng/ml, 100ng/ml, 300ng/ml, 750ng/ml, 1. mu.g/ml and 10. mu.g/ml, treated with or without 400ng/ml mGas6 and then lysed. The lysates were transferred to Maxi-Sorp 96 well plates coated with anti-phospho-tyrosine antibody 4G 10. Plates were then washed and incubated with 0.5 μ g/ml biotinylated rat anti-AXL antibody 12B7, AP conjugated streptavidin and AttoPhos substrate solution to collect fluorescence intensity. For details see the text. As shown by the concentration-dependent decrease in the cervical cancer cell line CaSki relative to Ax1 phosphorylation, the rat anti-Ax 1 antibody 11B7(a) and the chimeric anti-Ax 1 antibody ch11B7(B) of the present invention were able to prevent ligand-mediated activation of the receptor tyrosine kinase Ax1 to a similar extent.

Figure 18 ELISA experiments comparing the effect of rat and chimeric anti-Ax 1 antibodies on Ax1 phosphorylation. Starved CaSki cervical cancer cells were preincubated with 50ng/ml, 100ng/ml, 300ng/ml, 750ng/ml, 1. mu.g/ml and 10. mu.g/ml rat anti-AXL antibody 11B7(A) or chimeric anti-Ax 1 antibody ch11B7(B), with or without treatment with 400ng/ml mGas6, and then fixed with formaldehyde. Cells were washed, quenched, and incubated with anti-phospho-p 44/p42MAP kinase (Thr202/Tyr204) primary antibody, HRP-conjugated anti-rabbit secondary antibody, and tetramethylbenzidine solution to measure absorbance intensity. For details see the text. The rat anti-Ax 1 antibody 11B7(a) and the chimeric anti-Ax 1 antibody ch11B7(B) of the present invention were able to prevent Gas 6-induced activation of p42/p44MAP kinase to a similar extent in cervical cancer cells, as shown by the concentration-dependent decrease in phosphorylation relative to p42/p44MAP kinase.

Figure 19. TUNEL staining to study the combined effect of rat anti-AXL antibody and chemotherapeutic agents to overcome drug resistance in human ovarian cancer cells. Human NCI/ADR-RES ovarian cancer cells were pre-incubated with 10. mu.g/ml of control antibody or antagonistic anti-Ax 1 antibody 11B7, and then co-incubated with doxorubicin at a final concentration of 100. mu.M, 150. mu.M or 200. mu.M. TUNEL staining was performed to visualize and determine apoptosis by using a commercially available kit. For details see the text. For NCI/ADR-RES ovarian cancer cells treated with 100 μ M doxorubicin, neither incubation of the cells with control antibody nor with antagonistic anti-Ax 1 antibody 11B7 observed TUNEL staining and thus no apoptosis (top panel). However, at a concentration of 150 μ M doxorubicin, only very weak apoptosis was detected in cells co-treated with the control antibody, whereas co-incubation with the antagonistic anti-Ax 1 antibody 11B7 resulted in a significant induction of apoptosis (middle panel). Also in the presence of 200 μ M doxorubicin, co-incubation of cells with 11B7 significantly increased the rate of apoptosis compared to cells incubated with control IgG antibody (lower panel), suggesting that even co-treatment of multi-drug resistant tumor cells with chemotherapeutic agents and the antagonistic anti-Ax 1 antibody of the invention may be useful to overcome drug resistance.

Figure 20 soft agar assay to study the combined effect of rat anti-AXL antibody and chemotherapeutic agents on human melanoma cells independent of anchored growth. Human C-8161 melanoma cells remained untreated or were treated with rat antagonist anti-AXL antibody 11B7 at a final concentration of 2.5 μ g/ml. Agar-embedded cells were grown on top of a 0.7% bottom agar layer by combining with the indicated concentration of cisplatin for 5 days. MTT staining was used and then the area of colonies was measured. For details see the text. Absolute numbers reflecting the overall area of colonies (a) and relative growth inhibition calculated based on these data (B) are shown. Incubation with cisplatin resulted in a dose-dependent manner in a retardation of colony growth when compared to untreated control cells. Consistent with the inhibition of 11B7 alone, in the range of 30%, the combination with the antagonistic anti-Ax 1 antibody 11B7 resulted in a significantly enhanced inhibition of soft agar growth of C-8161 melanoma cells by cisplatin, particularly at lower concentrations.

Further, the present invention will be explained by the following examples and drawings.

Examples

General notes

The following examples, including experiments conducted and results obtained, are for illustrative purposes only and are not to be construed as limiting the invention.

Example 1 production of AXL overexpressing Ratl fibroblasts as immunogen and AXL overexpressing NIH3T3 fibroblasts as experimental model system

The full length coding sequence of human receptor tyrosine kinase AXL transcript variant 1 according to the National Center for Biotechnology Information (NCBI) reference sequence (NM — 021913) was subcloned into pLXSN by flanking recognition elements of the restriction endonucleases EcoRI and BamHI to yield the retroviral expression vector pLXSN-hAXL.

To generate antibodies that specifically bind to the human receptor tyrosine kinase AXL, rall fibroblasts stably overexpressing human AXL were generated by retroviral gene transfer. Briefly, 3x10⁵One Phoenix-E cell was seeded in a 60mm dish and then transfected with 2. mu.g/ml of either pLXSN vector or pLXSN-hAXL using the calcium phosphate method. After 24 hours, the medium was replaced with fresh medium and the Phoenix-E cells were incubated therein for 4 hours. Supernatants of Phoenix-E cells releasing pLXSN or pLXSN-hAXL ecotropic virus were harvested and used to incubate subconfluent Ratl cells (2X 10) in the presence of polybrene (4 mg/ml; Aldrich)⁵Individual cells/6 cm dish) for 3 hours. At the same time, Phoenix-E cells were re-incubated with fresh medium and used for a second infection of Ratl fibroblasts in the presence of polybrene (4 mg/ml; Aldrich) after an additional 3 hours. Likewise, a third infection cycle was performed. After medium exchange, selection of Ratl cells with G418 was started. Typically, stable clones were selected 21 days after selection.

A stable set of clones was propagated and quantified for membrane-localized human AXL expression by FACS analysis. Specifically, 1 × 10 was harvested with 10mM EDTA in PBS⁵Cells were washed once with FACS buffer (PBs, 3% FCS, 0.4% sodium azide) and seeded in 96-well round bottom plates. The cells were centrifuged at 1,000rpm for 3 minutes to remove the supernatant, and then mouse anti-AXL-primary anti-MAB 154 (R)&D Systems, 3. mu.g/ml) were resuspended. The cell suspension was incubated on ice for 1 hour, washed 2 times with FACS buffer, and then resuspended at 100. mu.l/well in PE-conjugate diluted 1: 50 in FACS bufferPooled donkey anti-mouse secondary antibody (Jackson). The cell suspension was incubated on ice in the dark for 30 minutes, washed 2 times with FACS buffer and analyzed using an Epics XL-MCL flow cytometer (BeckmanCoulter).

FIG. 1 shows FACS analysis of a polyclonal Ratl-mock population stably infected with pLXSN empty vector and Ratl-AXL c1.2 stably infected with pLXSN-hAXL and demonstrates that AXL is overexpressed on the cell surface of this representative clone.

Furthermore, NIH3T3 fibroblasts stably overexpressing AXL were generated in a manner similar to that described for Ratl in order to generate a suitable cell model system for experimental purposes. Briefly, 3x10⁵One Phoenix-E cell was seeded in a 60mm dish and then transfected with 2. mu.g/ml of either pLXSN vector or pLXSN-AXL cDNA using the calcium phosphate method. After 24 hours, the medium was replaced with fresh medium and the Phoenix-E cells were incubated for 4 hours therein. Supernatants of Phoenix-E cells releasing pLXSN or pLXSN-hAXL ecotropic virus were harvested and used to incubate sub-confluent NIH3T3 cells (2X 10) in the presence of polybrene (4 mg/ml; Aldrich)⁵Individual cells/6 cm dish) for 3 hours. At the same time, Phoenix-E cells were re-incubated with fresh medium and used for a second infection of NIH3T3 fibroblasts in the presence of polybrene (4 mg/ml; Aldrich) after an additional 3 hours. Likewise, a third infection cycle was performed. After medium exchange, selection of NIH3T3 cells with G418 was started. Typically, stable clones were selected 21 days after selection.

A stable set of clones was propagated and quantified for membrane-localized AXL expression by FACS analysis. Specifically, 1 × 10 was harvested with 10mM EDTA in PBS⁵Cells were washed once with FACS buffer (PBS, 3% FCS, 0.4% sodium azide) and seeded in 96-well round bottom plates. The cells were centrifuged at 1,000rpm for 3 minutes to remove the supernatant, and then mouse anti-AXL-primary anti-MAB 154 (R)&D Systems, 3. mu.g/ml) were resuspended. The cell suspension was incubated on ice for 1 hour, washed 2 times with FACS buffer and then resuspended at 100. mu.l/well in 150 in PE-conjugated donkey anti-mouse secondary antibody (Jackson) diluted in FACS buffer. The cell suspension was incubated on ice in the dark for 30 minutes, washed 2 times with FACS buffer and analyzed using an Epics XL-MCL flow cytometer (BeckmanCoulter).

FIG. 2 shows FACS analysis of polyclonal NIH3T 3-mock populations stably infected with pLXSN empty vector and NIH3T3-AXL c1.7 stably infected with pLXSN-hAXL and demonstrates that AXL is overexpressed on the cell surface of this representative clone.

Example 2 Generation of rat anti-AXL monoclonal antibodies

By mixing about 10x10⁶A single frozen cell of Ratl-AXL C1.2 was injected (intraperitoneally and subcutaneously) into either Lou/C or Long Evans rats to generate monoclonal rat anti-AXL antibodies. After 8 week intervals, final boosts were given intraperitoneally and subcutaneously 3 days prior to fusion. The fusion of the myeloma cell line P3X63-Ag8.653 with rat immune spleen cells was performed according to standard procedures to generate 105 hybridomas. After 2 weeks, a first suspension from the hybridoma was collected and tested in a preliminary FACS screen for binding to NIH3T3-AXL c1.7 fibroblasts (versus NIH3T 3-mock control cells). Clones positive for AXL binding were further cultured. Antibodies were purified from the supernatant of 50ml of these clones and reanalyzed for specific binding to AXL on NIH3T3-AXL c1.7 fibroblasts (versus NIH3T 3-mock control cells). Purified antibodies that specifically bind NIH3T3-AXL c1.7 fibroblasts but not NIH3T 3-mock control cells were further detected in an Akt-kinase phosphorylation ELISA and isotype-determined ELISAs were performed. To purify the rat antibody, the supernatant was centrifuged at 5,000g for 20 minutes, followed by sterile filtration. Add 500 u l protein G agarose (sepharose) FF, at 4 ℃ in a rotating wheel (spinning wheel) temperature in at least 1 hours. The agarose was centrifuged, the supernatant discarded, and the protein G matrix was washed 2 times with PBS before protein elution using citrate buffer (100mM) pH 2.1. The eluted fractions were immediately re-buffered to neutral pH by addition of 1M Tris pH 8.0, and then dialyzed against PBS.

Of the oligoclonal antibodies tested, 91 specifically bound NIH3T3-AXL c1.7 fibroblasts but not NIH3T 3-mock control cells, 9 inhibited Gas 6-induced Akt phosphorylation in the same cells, whereas 71 stimulated Akt phosphorylation. Cryopreservation (kryoconsensus) and subcloning 4 antagonistic antibodies (I11B7, I10D12, I6E7 and III11D5, referred to in the following examples as 11B7, 10D12, 6E7 and 11D5, respectively), two agonistic antibodies (I11D7 and III2a 1; referred to in the following examples as 11D7 and 2a1) and 1 control antibody (III1D 5; referred to in the following examples as 1D 5).

Example 3 rat anti-AXL antibodies of the invention do not cross-react with mouse AXL or other members of the human AXL family, Mer and Sky

This example discusses the cross-reactivity of the rat anti-AXL antibodies of the invention with mouse and cynomolgus monkey AXL as well as with other members of the human AXL family, human Mer and human Sky. After subcloning the mouse and monkey AXL coding sequences and human Mer and Sky into pcDNA3, each expression construct was transfected into HEK293T fibroblasts. The ability of the rat anti-AXL antibodies of the invention to bind to such proteins was tested by FACS analysis.

Cloning of mouse AXL

In this study, the mouse AXL expression construct pcDNA3-mAXL was generated. The full-length coding sequence of mouse AXL was amplified by Polymerase Chain Reaction (PCR) using mouse heart cdna (ambion) as template and appropriate primers based on the National Center for Biotechnology Information (NCBI) reference sequence of mouse AXL (NM — 009465). The full-length sequence encoding mouse AXL is thus encompassed by two overlapping PCR fragments, the 5 '-fragment and the 3' -fragment. The primers used to amplify such fragments are as follows:

forward primer for 5' -fragment with EcoRI recognition sequence mosse 1:

5′-GCG AAT TCG CCA CCA TGG GCA GGG TCC CGC TGG CCT G-3′

reverse primer for 5' -fragment MOUSE 2:

5′-CAG CCG AGG TAT AGG CTG TCA CAG ACA CAG TCA G-3′

forward primer for 3' -fragment, MOUSE 3:

5′-GCA CCC TGT TAG GGT ACC GGC TGG CAT ATC-3′

reverse primer for 3' -fragment with NotI recognition sequence MOUSE 4:

5′-ATA AGA ATG CGG CCG CTC AGG CTC CGT CCT CCT GCC CTG-3′

EcoRI and BstEII were used to degrade the 5 '-fragment, BstEII and NotI were used to degrade the 3' -fragment, and EcoRI and NotI were used to cleave pcDNA 3. Factor 3 ligation of the isolated and purified fragments was performed and transformed into DH5 a bacterial cells. Individual colonies were picked and cultured in the presence of ampicillin. The mouse AXL expression vector pcDNA3-mAXL was purified using a commercially available plasmid purification kit (Qiagen) and the sequence was verified for subsequent transient transfection into HEK293T cells.

Cloning of cynomolgus monkey AXL

In this study, the cynomolgus monkey AXL expression construct pcDNA3-cyAXL was generated. The full-length coding sequence of cynomolgus monkey AXL was PCR amplified using cDNA prepared from cynomolgus monkey brain tissue as template. Since the nucleotide sequence of cynomolgus monkey AXL is not available, each primer was designed by assuming significant homology to human AXL. The full-length sequence encoding cynomolgus monkey AXL is thus covered by two overlapping PCR fragments, the 5 '-fragment and the 3' -fragment. The primers used to amplify such fragments are as follows:

forward primer CYNO1 with EcoRI recognition sequence for the 5' -fragment:

5′-CGG AAT TCG CCA CCA TGG CGT GGC GGT GCC CCA G-3′

reverse primer CYNO2 for 5' -fragment:

5′-CTC TGA CCT CGT GCA GAT GGC AAT CTT CAT C-3′

forward primer for the 3' -fragment CYNO 3:

5′-GTG GCC GCT GCC TGT GTC CTC ATC-3′

reverse primer CYNO4 with NotI recognition sequence for 3' -fragment:

5′-ATA AGA ATG C GG CCG CTC AGG CAC CAT CCT CCT GCC CTG-3′

EcoRI and DraIII were used to degrade the 5 '-fragment, DraIII and NotI were used to degrade the 3' -fragment, and EcoRI and NotI were used to cleave pcDNA 3. Factor 3 ligation of the isolated and purified fragments was performed and transformed into DH5 a bacterial cells. Individual colonies were picked and cultured in the presence of ampicillin. The cynomolgus monkey AXL expression vector pcDNA3-cyAXL was purified using a commercially available plasmid purification kit (Qiagen) and the sequence was verified for subsequent transient transfection into HEK293T cells. The nucleotide and amino acid sequences of cynomolgus monkeys are as follows:

the nucleotide sequence is as follows:

ATGGCGTGGCGGTGCCCCAGGATGGGCAGGGTCCCGCTGGCCTGGTG

CTTGGCGCTGTGCGGCTGGGTGTGCATGGCCCCCAGGGGCACACAGG

CTGAAGAAAGTCCTTTCGTGGGTAACCCAGGGAATATCACAGGTGCCC

GGGGACTCACGGGCACCCTTCGGTGTCAGCTCCAGGTTCAGGGAGAG

CCCCCCGAGGTACACTGGCTTCGGGACGGACAGATCCTGGAGCTCGC

GGACAGTACCCAGACCCAGGTGCCCCTGGGTGAAGATGAGCAGGATGA

CTGGATAGTGGTCAGCCAGCTCAGAATCGCCTCCCTACAGCTTTCCGAC

GCGGGACAGTACCAGTGTTTGGTGTTTCTGGGACATCAGAACTTCGTGT

CCCAGCCTGGCTACGTAGGGCTGGAGGGCTTACCTTACTTCCTGGAGG

AGCCTGAGGACAGGACTGTGGCCGCCAACACCCCCTTCAACCTGAGCT

GCCAAGCCCAGGGACCCCCAGAGCCCGTGGACCTACTCTGGCTCCAG

GATGCTGTCCCCCTGGCCACAGCTCCAGGTCATGGTCCCCAGCGCAAC

CTGCATGTTCCAGGGCTGAACAAGACATCCTCTTTCTCCTGCGAAGCCC

ATAACGCCAAGGGAGTCACCACATCCCGCACGGCCACCATCACAGTGC

TCCCCCAGCAGCCCCGTAACCTCCATCTGGTCTCCCGCCAACCCACGG

AGCTGGAGGTGGCTTGGACTCCAGGCCTGAGCGGCATCTACCCCCTGA

CCCACTGCACCCTGCAGGCTGTGCTGTCAGACGATGGGATGGGCATCC

AGGCGGGAGAACCAGACCCCCCAGAGGAGCCCCTCACCTTGCAAGCAT

CTGTGCCCCCCCACCAGCTTCGGCTGGGCAGCCTCCATCCTCACACCC

CTTATCACATCCGTGTGGCATGCACCAGCAGCCAGGGCCCCTCATCCT

GGACACACTGGCTTCCTGTGGAGACGCCGGAGGGAGTGCCCCTGGGC

CCCCCTGAGAACATTAGTGCCACGCGGAATGGGAGCCAGGCCTTCGTG

CATTGGCAGGAGCCCCGGGCGCCCCTGCAGGGTACCCTGTTAGGGTA

CCGGCTGGCGTATCAAGGCCAGGACACCCCAGAGGTGCTAATGGACAT

AGGGCTAAGGCAAGAGGTGACCCTGGAGCTGCAGGGGGACGGGTCTG

TGTCCAATCTGACAGTGTGTGTGGCAGCCTACACTGCTGCTGGGGATG

GACCCTGGAGCCTCCCAGTACCCCTGGAGGCCTGGCGCCCAGGGCAA

GCACAGCCAGTCCACCAGCTGGTGAAGGAAACTTCAGCTCCTGCCTTC

TCGTGGCCCTGGTGGTATATACTGCTAGGAGCAGTCGTGGCCGCTGCC

TGTGTCCTCATCTTGGCTCTCTTCCTTGTCCACCGGCGAAAGAAGGAGA

CCCGTTATGGAGAAGTGTTCGAGCCAACAGTGGAAAGAGGTGAACTGG

TAGTCAGGTACCGCGTGCGCAAGTCCTACAGTCGCCGGACCACTGAAG

CTACCTTGAACAGCCTGGGCATCAGTGAAGAGCTGAAGGAGAAGCTGC

GGGATGTGATGGTGGACCGGCACAAGGTGGCCCTGGGGAAGACTCTG

GGAGAAGGAGAGTTTGGAGCCGTGATGGAAGGCCAGCTCAACCAGGA

CGACTCCATCCTCAAGGTGGCTGTGAAGACAATGAAGATTGCCATCTGC

ACAAGGTCAGAGCTGGAGGATTTCCTGAGTGAAGCAGTCTGCATGAAG

GAATTCGACCATCCCAATGTCATGAGGCTCATCGGTGTCTGTTTCCAGG

GTTCTGAACGAGAGAGCTTTCCAGCACCTGTGGTCATCTTACCTTTCAT

GAAGCATGGAGACCTACACAGCTTCCTCCTCTATTCCCGGCTTGGGGA

CCAGCCAGTGTACCTGCCCACTCAGATGCTAGTGAAGTTCATGGCGGA

CATCGCCAGTGGCATGGAATATCTGAGTACCAAGAGATTCATACACCGG

GACCTGGCGGCCAGGAACTGCATGCTGAATGAGAACATGTCCGTGTGT

GTGGCGGACTTCGGGCTCTCCAAGAAGATCTACAACGGGGACTACTAC

CGCCAGGGACGTATCGCCAAGATGCCAGTCAAGTGGATTGCCATTGAG

AGTCTAGCTGACCGTGTCTACACGAGCAAGAGTGATGTGTGGTCCTTC

GGGGTGACAATGTGGGAGATTGCCACAAGAGGCCAAACCCCATATCCA

GGCGTGGAGAACAGCGAGATTTATGACTATCTGCGCCAGGGAAATCGC

CTGAAGCAGCCTGCGGACTGTCTGGATGGACTGTATGCCTTGATGTCG

CGGTGCTGGGAGCTAAATCCCCAGGACCGGCCAAGTTTTACAGAGCTG

CGGGAAGATTTGGAGAACACACTGAAGGCCTTGCCTCCTGCCCAGGAG

CCTGACGAAATCCTCTATGTCAACATGGATGAAGGTGGAGGTTATCCTG

AACCTCCCGGCGCTGCTGGAGGAGCTGACCCCCCAACCCAGCTAGACC

CTAAGGATTCCTGTAGCTGCCTCACTTCGGCTGAGGTCCATCCTGCTGG

ACGCTATGTCCTCTGCCCTTCCACAGCCCCTAGCCCCGCTCAGCCTGC

TGATAGGGGCTCCCCAGCAGCCCCAGGGCAGGAGGATGGTGCC

amino acid sequence:

MAWRCPRMGRVPLAWCLALCGWVCMAPRGTQAEESPFVGNPGNITGAR

GLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIV

VSQLRIASLQLSDAGQYQCLVFLGHQNFVSQPGYVGLEGLPYFLEEPEDRT

VAANTPFNLSCQAQGPPEPVDL LWLQDAVPLATAPGHGPQRNLHVPGLNK

TSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLS

GIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTLQASVPPHQLRLGSLHP

HTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFV

HWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSN

LTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKETSAPAFSWPW

WYILLGAWAAACVLILALFLVHRRKKETRYGEVFEPTVERGELWRYRVRK

SYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEG

QLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCF

QGSERESFPAPWILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADI

ASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQG

RIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEI

YDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLK

ALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQLDPKDSCSCLTSA

EVHPAGRYVLCPSTAPSPAQPADRGSPAAPGQEDGA

cloning of human Mer

In this study, the human Mer expression construct pcDNA3-hMer was generated. The full-length coding sequence of human Mer was obtained by cleaving the vector pCMV6-XL 4-human Mer (Origene # TC116132) with EcoRI and XbaI. After degradation of pcDNA3 with the same restriction endonuclease, the two fragments were ligated to generate pcDNA 3-hMer. To introduce the Kozak consensus sequence, the 5' region of the human Mer coding sequence in pcDNA3-hMer was PCR amplified using appropriate primers based on the NCBI reference sequence of human Mer (NM-006343). The primers used to amplify this fragment were as follows:

forward primer MER1 with EcoRI recognition sequence and Kozak consensus sequence:

5′-CGG AAT TCG CCA CCA TGG GGC CGG CCC CGC TGC CGC-3′

reverse primer MER2 for 5' -fragment:

5′-TCG GCT GCC ATT CTG GCC AAC TTC C-3′

the PCR product and pcDNA3-hMer were degraded using EcoRI and EcoRV and then ligated to yield pcDNA3-Kozak-hMer, where the full-length human Mer coding sequence was preceded by a Kozak consensus sequence. Cells were transformed into DH 5. alpha. bacteria, and individual colonies were selected and cultured in the presence of ampicillin. The pcDNA3-Kozak-hMer expression vector was purified using a commercially available plasmid purification kit (Qiagen) and the sequence was verified for subsequent transient transfection into HEK293T cells.

Cloning of human Sky

In this study, the human Sky expression construct pcDNA3-hSky was generated. The full-length coding sequence of human Sky was PCR amplified using the vector pCMV6-XL 4-human Sky (origin # MG1044_ A02) as template and appropriate primers based on the NCBI reference sequence of human Sky (NM-006293). The primers used for amplification were as follows:

forward primer SKY1 with EcoRI recognition sequence:

5′-CGG AAT TCG CCA CCA TGG CGC TGA GGC GGA GC-3′

reverse primer SKY2 with XhoI recognition sequence:

5′-GCC CTC GAG CTA ACA GCT ACT GTG TGG CAG TAG-3′

EcoRI and XhoI were used to degrade the PCR product and pcDNA3, which was then ligated to generate pcDNA3-hSky expression vector. Cells were transformed into DH 5. alpha. bacteria, and individual colonies were selected and cultured in the presence of ampicillin. The pcDNA3-hSky expression vector was purified using a commercially available plasmid purification kit (Qiagen) and the sequence was verified for subsequent transient transfection into HEK293T cells.

Transfection and expression of mouse AXL, cynomolgus monkey AXL, human Mer and human Sky

For transient expression of mouse AXL, cynomolgus monkey AXL, human Mer or human Sky HEK293T cells were transiently transfected with pcDNA3 empty vector, pcDNA3-hAXL, pcDNA3mAXL, pcDNA3-cyAXL, pcDNA3-hMer or pcDNA3-hSky using the calcium phosphate method. Briefly, 3x10 was added prior to transfection⁶HEK293T cells were plated in 16ml of medium onto 15cm tissue culture dishes at 7% CO₂And cultured at 37 ℃ for 30 hours. Mu.g of DNA of each expression construct or of the empty vector (in 720. mu.l of ddH)₂In O) with 2.5M CaCl₂And 2xBBS (pH 6.96) and held at room temperature for 10 minutes. Gently add solution to cell culture in 3% CO₂And incubated at 37 ℃ for 8 hours. The medium was then replaced with fresh medium and the cells were incubated at 7% CO₂And incubated at 37 ℃ for 24 hours.

FACS analysis for detection of rat anti-AXL antibodies for Cross-reactivity

For FACS analysis, 2X10 was harvested using 10mM EDTA in PBS⁵Cells were washed 1 time with FACS buffer (PBS, 3% FCS, 0.4% sodium azide) and then seeded on 96-well round bottom plates. To remove the supernatant, plates were centrifuged at 1000rpm for 3 minutes and cells were resuspended in 10 μ g/ml isotype control antibody 1D5 and anti-AXL 11D5, 11B7, 10D12, 6E7, 2a1, 11D7 and 12B7 primary antibody solutions (100 μ l/well). After 1 hour incubation on ice, cells were washed 2 times with frozen FACS buffer and cells were resuspended with PE-conjugated donkey anti-rat (Jackson) secondary antibody or PE-conjugated donkey anti-mouse secondary antibody (for control) diluted 1: 50 in FACS buffer (100 μ Ι/well). Cells were incubated on ice for 30 minutes without light, washed 2 times with FACS buffer, and then analyzed using an epicsx xl-MCL flow cytometer (Beckman Coulter).

Fig. 3 shows representative results of this experiment. None of the other anti-AXL antibodies of the invention cross-react with such molecules, except 12B7, which shows moderate cross-reactivity with mouse AXL and human Mer and Sky. In contrast, all tested rat anti-AXL antibodies of the invention cross-reacted with cynomolgus monkey AXL.

Example 4 rat anti-AXL antibodies of the invention inhibit ligand-induced AXL phosphorylation in vitro

An ELISA experiment was performed to investigate whether the rat anti-AXL antibodies of the invention could prevent ligand Gas 6-mediated activation of AXL. Gas 6-mediated AXL activation was detected by increased receptor tyrosine phosphorylation. Briefly, on day 1, 3x10 was added⁴Individual cells/well were seeded in flat bottom 96-well plates in normal growth medium. The following day, the growth medium was replaced with serum-free medium to starve the cells overnight for 24 hours. Likewise, black Maxi-Sorp 96-well plates (Nunc) were also coated overnight at 4 ℃ with 2. mu.g/ml of the mouse anti-phosphotyrosine antibody 4G10 in PBS. On day 3, the 4G10 antibody solution was removed and the Maxi-Sorp wells blocked with PBS, 0.5% BSA at room temperature for at least 4 hours. In parallel, cells were preincubated with 10. mu.g/ml of mouse control antibody 72A1 and rat anti-AXL antibodies 2A1, 11D7, 11D5, 11B7, 6E7 and 10D12 at 37 ℃ for 1 hour, with or without 400ng/ml Gas6 (R.sub.&D Systems) at 37 ℃ for 10 minutes. The medium was then discarded and supplemented with phosphatase and protease inhibitors (10mM Na) on ice₄P₂O₇1mM phenylmethylsulfonyl fluoride, 1mM orthovanadate, 1mM NaF and 0, 5% aprotinin) for 30 minutes in lysis buffer (50mM HEPES, pH7.5, 150mM NaCl, 1mM EDTA, 10% glycerol and 1% Triton X-100). At the same time the blocking buffer was removed and the Maxi-Sorp plates were washed 6 times with washing buffer (PBS, 0.05% Tween20) before transferring and incubating the lysates overnight at 4 ℃. After washing the plates 6 times with wash buffer on day 4, the wells were incubated with 0.5 μ g/ml of biotinylated rat anti-AXL antibody 12B7 in PBS for 2 hours at room temperature. The plates were washed 6 times with wash buffer, AP-conjugated streptavidin (Chemicon # SA110) diluted 1: 4,000 in PBS was added to each well, and incubated for 30 minutes at room temperature. Thereafter, the wells were washed 6 times with wash buffer and AttoPhos bottom was addedSolution (Roche # 11681982). Fluorescence was collected from each well at an excitation wavelength of 430nm and an emission wavelength of 580nm by using a Victor plate reader (Perkin Elmer).

FIG. 4 shows representative results of this experiment for NIH3T3-AXL c1.7 fibroblasts (A) and NCI-H292 lung cancer cells (B). The rat anti-AXL antibodies 11B7, 11D5, 6E7 and 10D12 of the present invention are capable of preventing or reducing ligand-mediated AXL activation, as shown by reduced phosphorylation, and are thus considered to be antagonistic anti-AXL antibodies. In contrast, the rat anti-AXL antibodies 2a1 and 11D7 of the present invention stimulate basal AXL activation (as shown by increased phosphorylation) without significantly reducing ligand-mediated AXL activation, and are thus considered agonistic anti-AXL antibodies. Similar effects were observed for the same group of antibodies in the lung cancer cell lines CaLu-1 and CaLu-6, the breast cancer cell lines Hs578T and MDA-MB-231, the prostate cancer cell line PC-3, the pancreatic cancer cell line PANC-1, the melanoma cell line C-8161, the ovarian cancer cell lines SkoV-3 and SkoV-8, the glioblastoma cell line SF-126, the cervical cancer cell line CaSki, and the gastric cancer cell lines Hs746T and MKN-1.

Example 5 rat anti-AXL antibodies of the invention inhibit ligand-induced phosphorylation of p42/p44 MAP-kinase in vitro

Then, an ELISA experiment was performed to investigate whether the rat anti-AXL antibodies of the invention could prevent ligand Gas6 mediated activation of p42/p44 MAP-kinase. Gas 6-mediated activation of p42/p44 MAP-kinase was detected by increased protein (Thr202/Tyr204) phosphorylation. Briefly, on day 1, 2x10 was added⁴Individual cells/well were seeded in flat bottom 96-well plates. The following day, the normal growth medium was replaced with serum-free medium to starve the cells for 36 hours. Thereafter, the cells were preincubated with 10. mu.g/ml of isotype control antibody 1D5 and rat anti-AXL antibodies 11D5, 11B7 and 2A1 at 37 ℃ for 1 hour, followed by incubation with or without 400ng/ml of Gas6 (R)&DSystems) was treated at 37 ℃ for 10 minutes. The medium was discarded and the cells were fixed with 4% formaldehyde in PBS (pH 7.5) for 30 minutes at room temperature. The formaldehyde solution was removed and the cells were washed 2 times with washing buffer (PBS, 0.1% Tween 20). With 1% H₂O₂，0.1％NaN₃The cells were quenched (in wash buffer) and incubated for 20 minutes at room temperature. After that, the quenching solution was removed, and the cells were washed 2 times with washing buffer, and then blocked with PBS, 0.5% BSA at 4 ℃ for 4 hours. Primary anti-phospho-p 42/p44MAP kinase (Thr202/Tyr204) antibody (polyclonal rabbit; CellSignaling #9101) diluted 1: 500 in PBS, 0.5% BSA, 5mM EDTA was added and left overnight at 4 ℃. On day 4, the antibody solution was removed and the plate was washed 3 times with wash buffer. HRP-conjugated anti-rabbit secondary antibody (Dianova #111-036-045) diluted 1: 2,500 in PBS, 0.5% BSA was then added to each well and incubated at room temperature for 1.5 hours. The plates were washed 3 times with wash buffer and 2 times with PBS for 5 minutes each. Tetramethylbenzidine (TMB, Calbiochem) was added and monitored at 620 nm. The reaction was stopped by adding 100. mu.l of 250nM HCl and the absorbance was read at 450nM (reference wavelength of 620 nM) using a Vmax plate reader (ThermoLab Systems).

Fig. 5 shows representative results of this experiment for the cervical cancer cell line CaSki. The rat anti-AXL antibodies 11B7 and 11D5 of the present invention were able to reduce ligand-mediated activation of p42/p44 MAP-kinase, as shown by reduced phosphorylation, and were thus considered antagonistic anti-AXL antibodies. In contrast, the rat anti-AXL antibody 2a1 of the present invention stimulates activation of basal p42/p44 MAP-kinase (as shown by increased phosphorylation), without reducing ligand-mediated activation of p42/p44MAP kinase, and is thus considered an agonistic anti-AXL antibody. Similar effects were observed for the same group of antibodies in the breast cancer cell line Hs578T and the lung cancer cell line NCI-H292.

Example 6 rat anti-AXL antibodies of the invention inhibit ligand-induced phosphorylation of Akt in vitro

In addition, an ELISA experiment was performed to investigate whether the rat anti-AXL antibodies of the invention could prevent ligand Gas6 mediated activation of Akt-kinase. Gas 6-mediated activation of Akt-kinase was detected by increased protein (Ser473) phosphorylation. Briefly, on day 1, 2x10 was added⁴Individual cells/well were seeded in flat bottom 96-well plates. The following day, low serum (for NIH) was used3T3-AXL c1.7 fibroblasts, cells were starved for 36 hours using DMEM, 0.5% FCS) or serum-free (for cancer cell lines) medium instead of normal growth medium. Thereafter, the cells were preincubated with 10g/ml of isotype control antibody 1D5 and rat anti-AXL antibodies 11D5, 11B7 and 2A1 at 37 ℃ for 1 hour, followed by incubation with or without 400ng/ml of Gas6 (R)&DSystems) was treated at 37 ℃ for 10 minutes. The medium was discarded and the cells were fixed with 4% formaldehyde in PBS (pH 7.5) for 30 minutes at room temperature. The formaldehyde solution was removed and the cells were washed 2 times with washing buffer (PBS, 0.1% Tween 20). With 1% H₂O₂，0.1％NaN₃The cells were quenched (in wash buffer) and incubated for 20 minutes at room temperature. After that, the quenching solution was removed, and the cells were washed 2 times with washing buffer, and then blocked with PBS, 0.5% BSA at 4 ℃ for 4 hours. Primary anti-phospho-Akt (Ser473) antibody (polyclonal rabbit; Cell Signaling #9271) diluted 1: 500 in PBS, 0.5% BSA, 5mM EDTA was added overnight at 4 ℃. On day 4, the antibody solution was removed and the plate was washed 3 times with wash buffer. HRP-conjugated anti-rabbit secondary antibody (Dianova #111-036-045) diluted 1: 2,500 in PBS, 0.5% BSA was then added to each well and incubated at room temperature for 1.5 hours. The plates were washed 3 times with wash buffer and 2 times with PBS for 5 minutes each. Tetramethylbenzidine (TMB, Calbiochem) was added and monitored at 620 nm. The reaction was stopped by adding 100. mu.l of 250nM HCl and the absorbance was read at 450nM (reference wavelength of 620 nM) using a Vmax plate reader (Thermo Lab Systems).

FIG. 6 shows representative results of this experiment for NIH3T3-AXL c1.7 fibroblasts (A) and CaLa-1 lung cancer cells (B). The rat anti-AXL antibodies 11B7 and 11D5 of the present invention are capable of preventing or reducing ligand-mediated activation of Akt-kinase, as shown by reduced phosphorylation, and are thus considered antagonistic anti-AXL antibodies. In contrast, the rat anti-AXL antibody 2a1 of the present invention stimulates activation of basal Akt-kinase (as shown by increased phosphorylation), without decreasing ligand-mediated activation of Akt-kinase, and is thus considered an agonistic anti-AXL antibody. Similar effects were observed for the same group of antibodies in the lung cancer cell line NCI-H292, the breast cancer cell lines Hs578T and MDA-MB-231, the prostate cancer cell line PC-3, the pancreatic cancer cell line PANC-1, the melanoma cell line C-8161, the ovarian cancer cell lines SkoV-3 and SkoV-8, the bladder cancer cell line TCC-Sup, and the fibrosarcoma cell line HT 1080.

Example 7 rat and chimeric anti-AXL antibodies of the invention inhibit ligand-induced phosphorylation of Akt to a similar extent in vitro

As part of the present invention, chimeric derivatives of the rat anti-AXL antibodies 11B7 and 11D5 were generated (see below). To investigate whether the rat anti-AXL antibody of the invention and the corresponding chimeric anti-AXL antibody of the invention could prevent ligand Gas 6-mediated activation of Akt-kinase in NIH3T3-AXL c1.7 fibroblasts to a similar extent, ELISA experiments were performed. Antibody-mediated inhibition of Ak t-kinase was detected by reduced protein (Ser473) phosphorylation. Briefly, on day 1, 2x10 was added⁴Individual cells/well were seeded in flat bottom 96-well plates. The following day, the normal growth medium was replaced with low serum medium (DMEM, 0.5% FCS) to starve the cells for 36 hours. Thereafter, the cells were preincubated with 50ng/ml, 100ng/ml, 300ng/ml, 500ng/ml and 1. mu.g/ml of rat anti-AXL antibody 11B7 or chimeric anti-AXL antibody ch11B7 and 50ng/ml, 100ng/ml, 300ng/ml, 500ng/ml, 1. mu.g/ml, 5. mu.g/ml and 10. mu.g/ml of rat anti-AXL antibody 11D5 or chimeric anti-AXL antibody ch11D5 at 37 ℃ for 1 hour, and then with or without 400ng/ml of Gas6 (R/ml of Gas6 (R/ml)&D Systems) at 37 ℃ for 10 minutes. The medium was discarded and the cells were fixed with 4% formaldehyde in PBS (pH 7.5) for 30 minutes at room temperature. The formaldehyde solution was removed and the cells were washed 2 times with washing buffer (PBS, 0.1% Tween 20). With 1% H₂O₂，0.1％NaN₃The cells were quenched (in wash buffer) and incubated for 20 minutes at room temperature. After that, the quenching solution was removed, and the cells were washed 2 times with washing buffer, and then blocked with PBS, 0.5% BSA at 4 ℃ for 4 hours. Primary anti-phospho-Akt (Ser473) antibody (polyclonal rabbit; Cell Signaling #9271) diluted 1: 500 in PBS, 0.5% BSA, 5mM EDTA was added overnight at 4 ℃. On day 4, the antibody solution was removed and the plate was washed 3 times with wash buffer. Then adding into each holeHRP-conjugated anti-rabbit secondary antibody (Dianova #111-036-045) diluted 1: 2,500 in PBS, 0.5% BSA was incubated at room temperature for 1.5 hours. The plates were washed 3 times with wash buffer and 2 times with PBS for 5 minutes each. Tetramethylbenzidine (TMB, Calbiochem) was added and monitored at 620 nm. The reaction was stopped by adding 100. mu.l of 250nM HCl and the absorbance was read at 450nM (reference wavelength of 620 nM) using a Vmax plate reader (Thermo Lab Systems). Is there a

Figure 7 shows that the rat anti-AXL antibody 11B7 and the chimeric anti-AXL antibody ch11B7 of the invention and the rat anti-AXL antibody 11D5 and the chimeric anti-AXL antibody ch11D5 of the invention are able to inhibit ligand-mediated activation of Akt kinase to a similar extent, as shown by reduced phosphorylation. Thus, the chimeric anti-AXL antibodies ch11B7 and ch11D5 retained activity compared to their respective rat counterparts.

Example 8 antagonistic murine anti-AXL antibodies of the invention compete with each other for the same or structurally related epitopes and do not share a binding site with the agonistic murine anti-AXL antibodies of the invention

The anti-AXL antibodies of the invention were examined to determine whether they competed with each other for similar binding epitopes on the AXL-ECD domain. The binding of biotinylated anti-AXL antibody to AXL-ECD domain coated plates pre-incubated with anti-AXL antibody was thus determined in a competition ELISA. Briefly, 30. mu.g of isotype control antibody 1D5 and rat anti-AXL antibodies 11B7, 11D5, 6E7, 10D12, 11D7 and 2A1 were biotinylated with sulfo-NHS-biotin (Pierce #21217) according to the manufacturer's instructions and then purified using a Micro-BioSpin P6 column SSC (BIO-RAD # 732-. On day 1, black 96-well Maxi-Sorp plates (Nunc) were coated with 100. mu.l/well of 1. mu.g/ml human AXL-ECD (R & D Systems #154-AL) in PBS overnight at 4 ℃. On day 2, the coated Maxi-Sorp plates (250. mu.l/well) were blocked with blocking buffer (PBS, 1% BSA 0.05% TWEEN-20) at room temperature for 2 hours, followed by incubation with PBS or 10. mu.g/ml of the non-biotinylated isotype control antibody 1D5 in blocking buffer and the non-biotinylated rat anti-AXL antibodies 11B7, 11D5, 6E7, 10D12, 11D7 or 2A1 (100. mu.l/well) at room temperature for 1 hour. The antibody solution was discarded, without washing, PBS or 0.5 μ g/ml biotinylated isotype control antibody 1D5 in blocking buffer and biotinylated rat anti-AXL antibody 11B7, 11D5, 6E7, 10D12, 11D7 or 2a1 were added at 100 μ l/well and incubated at room temperature for 15 minutes. After washing 6 times with washing buffer (PBS, 0, 1% TWEEN-20), AP-conjugated streptavidin (Chemicon # SA110) diluted 1: 4,000 in blocking buffer was added at 80. mu.l/well, incubated at room temperature for 20 minutes, washed 6 times with washing buffer and finally washed 1 time with PBS. For detection, 100. mu.l/well of an Attophos substrate solution (Roche #11681982) was added. The fluorescence of each well was collected at an excitation wavelength of 430nm and an emission wavelength of 580nm using a Victor plate reader (Perkin Elmer).

Fig. 8 shows representative results of this analysis. The antagonistic anti-AXL antibodies of the invention 11B7, 11D5, 6E7 and 10D12 compete with each other for identical or structurally adjacent epitopes. The two agonistic antibodies 11D7 and 2a1 of the present invention each recognize different epitopes and thus are not mutually exclusive. Furthermore, 11D7 and 2a1 did not compete with antagonistic antibodies for binding to AXL-ECD. Control antibody 1D5 did not bind AXL-ECD.

Example 9 rat and chimeric anti-AXL antibodies of the invention inhibit migration and proliferation of lung cancer cells in vitro

In vitro wound healing/scratch assays have been used for many years in order to examine the migration and proliferation rates of different cells and culture conditions. Such assays typically involve first culturing a confluent cell monolayer. The small area is then destroyed, and a population of cells is destroyed or displaced by streaking through the layer with, for example, a pipette tip. The gaps were then examined microscopically over a period of time (cells migrated into and filled the damaged area ("healed")). Briefly, at 1.5x10⁶NCI-H292 lung cancer cells were seeded per well in 12-well culture plates and cultured in normal growth medium (RPMI, 10% FCS). After 8 hours, cells were rinsed with PBS and starved overnight in low serum medium (RPMI, 0.5% FCS) for 24 hours. Cross-over of pooled NCI-H29 Using a sterile 200. mu.l pipette tip2 cell monolayers, yielding 3 separate uniform wounds per well. The cells were gently rinsed with PBS and incubated for comparison with additive-free low serum medium (RPMI, 0.5% FCS) containing 10 μ g/ml of isotype control antibody 1D5, antagonistic rat anti-AXL antibody 11D5, 11B7, 6E7, or 10D12, chimeric anti-AXL antibodies chn11D5 IgG2 and chn11B7IgG2, agonistic rat anti-AXL antibodies 2a1 and 11D7, and 10 μ g/ml Erbitux or 5 μ M Sutent. Cells were allowed to migrate into the cleared area, for 24 hours, washed 1 time with PBS, and cells were fixed with ice-cold methanol (100%) at-20 ℃. After staining the cells with crystal violet (0.5% in 20% methanol), rinsed with water and then dried overnight, a photograph of the wound was taken.

FIG. 9 shows representative results of this experiment for NCI-H292 lung cancer cells. The antagonistic rat anti-AXL antibodies 11D5, 11B7, 6E7 and 10D12 of the invention and the chimeric anti-AXL antibodies chn11D5 IgG2 and chn11B7IgG2 of the invention reduced the re-entry of the cleared area compared to the isotype control antibody, whereas the agonistic rat anti-AXL antibodies 2a1 and 11D7 of the invention resulted in complete closure of the wound. Similar results were observed for the same group of antibodies in the ovarian cancer cell line SkOv-3 or the gastric cancer cell line MKN-1.

Example 10 in vitro inhibition of ligand-induced migration of NIH3T3-AXL c1.7 fibroblasts by the rat anti-AXL antibody of the invention

A migration experiment (transmission experiment) was performed to investigate whether the antibody of the present invention prevents cell migration. For this purpose, NIH3T3-AXL c1.7 cells were seeded on a 15cm dish in normal growth medium in the morning of day 1, and the medium was replaced with low serum medium (DMEM, 0.5% FCS) in the evening to starve the cells for 36 hours. Day 2, FluoroBlock 96-well plates (Becton Dickinson #351164, 8 μ M well size) were coated with 10 μ g collagen I/ml 0.1M acetic acid at 37 ℃ overnight. On day 3, the low serum medium (DMEM, 0.5% FCS) was replaced with serum-free medium (DMEM, 0% FCS, 0.1% BSA) for an additional 4 hours. Cells were harvested using 10mM EDTA in PBS, 4X10⁵Cell/mCell density of l and antibody concentration of 10. mu.g/ml cells were preincubated with rat anti-AXL antibody 4A6, 11B7 or 2A1 for 45 minutes. Then 50. mu.l cell suspension (20,000 cells)/well was placed in the top chamber of a FluoroBlock 96-well plate using 225. mu.l per well with or without 400ng/ml mouse Gas6 (R)&D Systems) (DMEM, 0% FCS, 0.1% BSA). Cells were allowed to migrate at 37 ℃ for 7 hours, after which time they were plated with 4.2. mu.M PBS, 1mM CaCl₂，1mM MgCl₂calcein-AM (Molecular Probes # C3099) in (E) was stained at 37 ℃ for 1 hour. The fluorescence of each well was measured at a wavelength of 530nm using a Victor plate reader (Perkin Elmer).

Figure 10 shows that the antagonistic anti-AXL antibody 11B7 of the invention reduced basal and Gas 6-induced migration of NIH3T3-AXL c1.7 fibroblasts, whereas the agonistic rat anti-AXL antibody 2a1 of the invention increased ligand-induced migration and specifically basal migration of NIH3T3-AXL c1.7 cells. Antibody 4a6 did not affect cell migration.

Example 11 in vitro inhibition of ligand-induced proliferation of NIH3T3-AXL c1.7 fibroblasts by the rat anti-AXL antibody of the invention

In vitro experiments were performed to determine the ability of rat anti-AXL antibodies of the invention to inhibit Gas 6-induced cell proliferation. For this purpose, NIH3T3-AXL c1.7 fibroblasts were seeded in a 96-well plate at 2,500 cells/well in medium containing FCS and allowed to stand overnight. On day 2, cells were starved for 10 hours in low serum medium (DMBM, 0.5% FCS) and then preincubated with 20 μ g/ml mouse control antibody 72a1, antagonistic rat anti-AXL antibodies 11D5 and 11B7, and agonistic antibody 2a1 in DMEM, 0.5% FCS for 1 hour at 37 ℃. By adding the ligand directly to the antibody solution, with or without 400ng/ml mouse Gas6 (R)&DSystems) and then allowed to grow for 96 hours. Adding AlamarBlue^TM(BIOSOURCE # DAL1100), incubated at 37 ℃ in the dark. The absorbance was measured at 590nm every 30 minutes. After adding AlamarBlue^TMData were collected 4 hours later.

Fig. 11 shows representative results of this experiment. Antagonistic anti-AXL antibodies 11D5 and 11B7 of the invention prevent Gas 6-induced proliferation of NIH3T3-AXL c1.7 fibroblasts, whereas agonistic rat anti-AXL antibody 2a1 of the invention increases ligand-induced proliferation and in particular basal proliferation of NIH3T3-AXL c1.7 cells.

Example 12 in vitro inhibition of ligand-mediated anti-apoptosis in serum-starved NIH3T3-AXL c1.7 fibroblasts by the rat anti-AXL antibodies of the invention

The induction of apoptosis and the activation of caspases can be caused by a variety of stimuli, including growth factor withdrawal, exposure to chemotherapeutic agents or radiation, or the initiation of Fas/Apo-1 receptor mediated cell death processes. The Gas6-AXL interaction has been shown to be involved in protecting many cell types from apoptosis, including serum-starved NIH3T3 fibroblasts (Goruppi et al, 1996, Oncogene 12, 471-. In this example, we examined whether the rat anti-AXL antibody of the invention interferes with the anti-apoptosis of Gas 6-mediated serum starved NIH3T3-AXL c1.7 fibroblasts, thereby inducing apoptosis. The rate of apoptosis was therefore determined by measuring the caspase-3/7 activity of the cells. For this purpose, NIH3T3-AXL c1.7 cells were treated at 1.5X10³The density of individual cells/well was seeded in black clear-bottom 96-well plates (100. mu.l/well). On day 2, the normal growth medium was replaced with low serum medium (DMEM, 0.5% FCS) to starve the cells overnight for 24 hours. The following day, antibody solutions of isotype control antibody ID5, antagonistic rat anti-AXL antibodies 11B7 and 11D5, and agonistic rat anti-AXL antibodies 11D7 and 2a1 were prepared in DMEM, 0% FCS, 0.01% BSA at 80 μ g/ml. Cells were washed with PBS, covered with 60. mu.l DMEM, 0% FCS, 0.01% BSA, and 10. mu.l of each antibody solution was added. After 1 hour incubation at 37 deg.C, 10. mu.l of mouse Gas6 (R) with or without 3.2. mu.g/ml was added&D Systems), 0% FCS, 0.01% BSA (final concentrations of antibody and Ga s6 of 10. mu.g/ml and 400ng/ml, respectively), the cells were incubated at 37 ℃ for a further 5 hours. The following procedure was performed for the determination of Apo-ONE Homogenwous caspase-3/7 (Promega, G7791)Shown in the figure. Briefly, plates were removed from the incubator and allowed to equilibrate at room temperature for 20 minutes. Mu.l of Apo-ONE substrate and 6ml of buffer were thawed, combined and then added to the sample (75. mu.l/well). The contents of the wells were gently shaken for 30 seconds and protected from light and incubated at room temperature for 1 hour. The fluorescence of each well was measured at an excitation wavelength of 485nm and an emission wavelength of 530nm using a Victor plate reader (Perkin Elmer).

Fig. 12 shows representative results of this experiment. The antagonistic rat anti-AXL antibodies 11B7 and 11D5 of the invention reduced the anti-apoptosis of Gas 6-mediated serum starved NIH3T3-AXL c1.7 fibroblasts compared to isotype control antibodies, thereby inducing apoptosis. In contrast, the agonistic rat anti-AXL antibodies 2a1 and 11D7 of the present invention strongly induce anti-apoptosis of serum-starved NIH3T3-AXL c1.7 cells regardless of the presence or absence of Gas6, thereby inhibiting apoptosis.

Example 13 rat anti-AXL antibodies of the invention inhibit spheroid-based cellular angiogenesis in vitro

AXL is a key regulator of a variety of angiogenic behaviors, including endothelial cell migration, proliferation and in vitro tube formation (Holland et al, Cancer Res: 65, 9294-9303, 2005). Thus, the rat anti-AXL monoclonal antibodies 11B7 and 11D5 of the present invention were tested for their inhibitory effect on VEGF-A induced vascular sprouting of HUVEC-spheroids. The experiments were carried out according to a modification of the originally published protocol (Korff and Augustin: J Cell Sci 112: 3249-58, 1999). Briefly, spheroids were prepared as described (Korff and Augustin: J Cell Biol 143: 1341-52, 1998) by transferring 500 Human Umbilical Vein Endothelial Cells (HUVEC) in hanging drops with a pipette onto plastic dishes and allowing them to spherically aggregate overnight. 50 HUVEC spheroids were then seeded in 0.9ml collagen solution (2mg/ml) and transferred to individual wells of a 24-well plate using a pipette to allow polymerization. Rat anti-AXL antibodies 11B7 and 11D5(1X 10) at decreasing concentrations were added before polymerization^-6M、1x10^-7M、1x10^-8M、1x 10^-9M、1x10^-10M) directly mixed in the glueIn the original solution, however after 30 minutes, the growth factor VEGF-A (final concentration 25ng/ml) was added by transferring 100. mu.l of 10-fold concentrated working dilution to the top of the polymerized gel with a pipette. The plates were incubated at 37 ℃ for 24 hours and then fixed by adding 4% paraformaldehyde. The budding intensity of HUVEC spheroids was quantified by an image Analysis system that measured the cumulative budding length per spheroid using an inverted microscope and digital imaging software Analysis 3.2(Soft imaging system, Munster, Germany). The average of the cumulative shoot length of 10 randomly selected spheroids was analyzed as a single data point.

Fig. 13 shows the results of this experiment. Antagonistic rat anti-AXL antibodies 11B7(a) and 11D5(B) of the invention inhibited VEGF-a stimulated HUVEC sprouting in a dose-dependent manner in a spheroid-based angiogenesis assay. Treatment with the highest concentration of 11B7 reduced HUVEC sprouting to basal levels, however inhibition with the highest concentration of 11D5 was not as effective as the former (left panel). 11B7 and 11D5 at 9.8X10, respectively^-8M and 7.0x10^-7IC of M₅₀Values inhibited HUVEC sprouting (right panel).

Example 14 rat anti-AXL antibodies of the invention reduce the growth of human prostate cancer in nude mice

The anti-tumor efficacy of therapeutic antibodies is often evaluated in human xenograft tumor studies. In such model systems, human tumors grow as xenografts in immunocompromised mice, and the efficacy of treatment is measured in terms of the degree of tumor growth inhibition. The objective of this study was to assess whether the antagonistic rat anti-AXL antibody 11B7 of the invention interferes with tumor growth of human prostate cancer cells in nude mice. Briefly, on day 0, 7 to 8 week old male NMRI was anesthetized with 1.5-2.0 volume percent isoflurane at a flow rate of oxygen of 21/min^-nu/nuMice (approximate weight after acclimatization: 30g) were treated with 1X10 in 25. mu.l PBS⁶Individual PC-3-LN cells were implanted orthotopically in the prostate. PC-3-LN cells were derived from a PC-3 prostate cancer cell line infected with a retrovirus encoding a luciferase-neomycin fusion protein. Initiation of tumor growthAnd tumor growth progression can thus be measured by in vivo bioluminescence imaging. For this purpose, luciferin was injected intraperitoneally (i.p.) into mice, and the emission of light was measured 10 minutes after injection using a NightOWL LB 981 bioluminescence imaging system (Berthold Technologies, Germany). Prior to the first treatment, mice were randomized and statistical tests were performed to ensure consistency in initial tumor volume (mean, median and standard deviation) among treatment groups (10 animals per group). On day 8, all treatments were started and continued until day 34, and necropsy was performed on day 35. Animals of groups 1 and 2 were administered intraperitoneally (i.p.) 3 times per week (monday, wednesday, friday) with 25mg/kg of isotype control antibody 1D5 and antagonistic rat anti-AXL antibody 11B7, respectively. Animals of group 3 received 40mg/kg of Sutent orally (p.o.) 1 time per day. Animals of group 4 received 3 intravenous (i.v.) injections (4 days apart from each other) using 12.5mg/kg of Taxotere. An overview of the treatment groups is given below.

Fig. 14 shows the results of this experiment. The antagonistic rat anti-AXL antibody 11B7 of the invention reduced the overall growth of PC-3-LN prostate tumors in nude mice compared to isotype control antibody 1D 5.

Example 15 rat anti-AXL antibodies of the invention inhibit metastasis of human prostate cancer

In the same experiment as described under "the rat anti-AXL antibody of the present invention reduces the growth of human prostate cancer in nude mice", the relocation (metastasis) of PC-3-LN tumor cells into other organs was analyzed after necropsy to evaluate the anti-metastatic effect of the antagonistic rat anti-AXL antibody 11B7 of the present invention. For this purpose, selected organs (liver, spleen, lung, femur, part of the lumbar spine) were collected after necropsy, homogenized and supplemented with luciferin. Subsequently, the emission of light was measured using a NightOWL LB 981 bioluminescence imaging system (Berthold Technologies, Germany).

Figure 15 shows the results of the analysis of the spleen for this experiment. The antagonistic rat anti-AXL antibody 11B7 of the invention reduced the occurrence of splenic metastases compared to the isotype control antibody 1D 5. Notably, the anti-metastatic effect of 11B7 was stronger than that of Sutent in this experiment. Similar observations were obtained for liver, lung, femoral and lumbar spinal metastases.

Example 16 AXL expression predominantly in tumors but not in adjacent normal tissue

In this study, AXL expression in 17 different human malignancies was immunohistochemically analyzed on formalin fixed paraffin embedded tissue in a tissue multi-array format. For each tumor type, pairs of tumor tissues and matching non-malignant tissues were examined. Briefly, tissues were fixed in 4% neutral buffered formalin for 16 to 20 hours and then embedded in paraffin. To construct a 60-nuclear Tissue Microarray (TMA), one perforated healthy tissue and one perforated corresponding tumor tissue for each case were selected by the pathologist. 96-core TMAs with normal control tissue perforations (3 per tissue type) were generated according to FDA guidelines. The diameter of each perforation was 1.5 mm.

Using a microtome, selected tissue blocks were cut into 2-4 μm sections, which were placed on silanized glass slides (Sigma), then dried at 60 ℃ for 30 minutes and at 38 ℃ overnight. Sections were dewaxed by incubation in a xylene bath for 5 minutes 2 times, in acetone for 5 minutes 2 times and finally in distilled water for 5 minutes. The thermal pretreatment of the sections was carried out in a steam generator in 10mM citrate buffer, pH 6.0, for 30 minutes, followed by washing in distilled water. By reacting freshly prepared 0.3% H in methanol at room temperature₂O₂The solution was incubated for 20 minutes to block endogenous peroxidase and then washed with distilled water and PBS for 5 minutes each. Sections were incubated with polyclonal goat anti-human AXL antibody (Santa CruzSC-1096) (diluted 1: 20 in TBST) at room temperature for 60 minutes. After 3 washes in TBST, biotinylated rabbit anti-goat secondary antibody was used(Dianova, diluted 1: 200 in TBST) sections were incubated for 45 min at room temperature. After washing as before, sections were incubated with streptavidin/HRP (DAKO, diluted 1: 300 in TBST) for 30 min at room temperature and then washed as before. Staining was performed with DAB solution (DAKO; 1: 50 dilution in substrate buffer) for 10 min at room temperature. Finally, the slides were rinsed with water, counterstained with Harris' hematoxylin, and then covered with slides. Control antibody with goat IgG (R)&D) But not anti-AXL-one anti-warm control sections.

Figure 16 summarizes the results (a) of this analysis on AXL expression in 17 different human solid tumors and corresponding non-malignant tissues. In all cases screened for each indication, no significant expression was detected in follicular lymphoma, prostate cancer (except single cells) and in renal cancer. Melanoma and Merkel cell tumors show very low AXL expression. Faint expression was observed in a few lung tumors, mainly adenocarcinoma. Esophageal and barrett tumors, ovarian, colon and pancreatic tumors, and liver tumors (hepatocellular carcinoma) show faint staining in about 30% of cases. Head and neck tumors show weak to moderate staining in approximately 40% of tumors. Faint to moderate staining was detected in 60% to 100% of breast, cervical, bladder, thyroid and stomach tumors analyzed. The strongest staining was observed in breast tumors and gastric signet ring cell carcinomas (B). Non-malignant tissues mostly show no specific staining except for the renal tubules which sometimes show weak staining above background.

Example 17: structure and features of anti-AXL antibodies

Nucleotide sequence of rat antibody variable domain

The rat anti-AXL antibody variable domains were cloned from hybridoma cells. RNA was prepared using the RNA extraction kit RNeasy (RNeasy midi-kit, Qiagen). cDNA encoding the antibody gene was prepared using the 5 'RACE kit (Invitrogen) according to the manufacturer's instructions.

Briefly, gene-specific G was usedSP1 primer and Superscript^TMII reverse transcriptase synthesizes first strand cDNA from total RNA. After first strand cDNA synthesis, the original mRNA template was removed by treatment with RNase Mix (RNase Mix). A homopolymer tail was then added to the 3' end of the cDNA. PCR amplification was performed using Taq DNA polymerase, nested gene specific primers (GSP2) that anneal to sites located within the cDNA molecule, and the anchor primers provided with the kit. After amplification, the 5' RACE product was cloned into the pLXSN-ESK vector for sequencing. To facilitate cloning, the Anchor Primer (AP) contains the recognition sequence of Sal I and the GSP2 primer contains the Xho I site.

GSP1 primer:

kappa_GSP1：GATGGATGCATTGGTGCAGC

new_kappa_GSP1：ATAGATACAGTTGGTGCAGC

heavy_GSP1：CAGGGTCACCATGGAGTTA

GSP2 primer:

XhoI-hGSP2：CCGCTCGAGCGGGCCAGTGGATAGACAGATGG

XhoI-kGSP2：CCGCTCGAGCGGCCGTTTCAGCTCCAGCTTGG

GSP primers were used for rat anti-AXL Mab cloning:

11B7：kappa GSP1；XhoI-kGSP2

heavy GSP1；XhoI-hGSP2

10D12：kappa_GSP1，new_kappa_GSP1；XhoI-kGSP2

heavy GSP1；XhoI-hGSP2

11D5：new_kappa_GSP1；XhoI-kGSP2

heavy GSP1；XhoI-hGSP2

amino acid sequence of rat anti-AXL antibody variable domain

The rat antibody variable domain sequence was translated from a sequenced gene cloned into the pLXSN-ESK vector. The amino acid sequence given starts at position 1 of the variable domain. Complementarity Determining Regions (CDRs) required for an antibody to specifically bind its target are defined according to Kabat (Kabat et al Sequences of Proteins of Immunological Interest, 5 th edition NIH Publication No.91-3242, 1991). The Kabat definition is based on sequence variability within the variable domain. The anti-AXL specific CDR regions of the antibody are listed in SEQ ID NO: 13-30. Individual CDRs included the following sites:

CDR-L1：24-34

CDR-L2：50-56

CDR-L3：89-97

CDR-H1：31-35b

CDR-H2：50-65

CDR-H3：95-102

expression and purification of 17C rat antibody:

DMEM (containing 4.5g/L glucose; 1% glutamine, 1% pyruvate, 1% penicillin/streptomycin) was used in a Celline CL 1000 bioreactor (Integra Biosciences) at 37 ℃ with 5-7% CO₂The hybridomas were cultured under the conditions described above. FCS supplements were 1% FCS for nutrient chamber and 5% low IgG FCS for cell chamber. Harvest and medium change were performed 2 times per week. Cell division 1/1->1/3 depend on cell growth. Productivity was monitored 1 time per week by SDS-PAGE analysis. The supernatant was stored at-20 ℃ until purification. Mycoplasma (Mycoplasma) examination of the working cultures was performed 1 time per week.

By passingThe Explorer 100 system (GE-Healthcare) purified the antibody using protein A or G Sepharose FF (GE-Healthcare). For each purification, the column was packed separately. The column size was adjusted to the expected production rate and size of each batch (typically 50-500 mg). The protein-containing solution was kept on ice or at 4 ℃ where possible. Sterile buffer is used in the whole processLiquid and double distilled water.

The supernatant was thawed, buffered with 50mM TRIS pH8.5, centrifuged, filtered through a 0.22 μm membrane, and then loaded onto a column. 50mM PO in 8 Column Volumes (CV)₄After washing at pH8.5, the antibody was eluted in 10CV 100mM glycine, pH 3.3. The eluted fractions were immediately re-buffered to neutral pH by addition of 1/51M Tris pH 8.0(1ml Tris/4ml eluted fraction) and then analyzed by rSDS-PAGE. Fractions containing pure antibody were mixed, dialyzed against PBS at 4 ℃ and then sterile filtered.

The requirements of the buffer system are adjusted to the individual properties of each antibody. Specifically, rat IgG2a antibody 11D5 was bound to ProteinG 4FF matrix (GE-Healthcare) and washed under high salt (2M NaCl) conditions. The rat antibody IgG 111B 7 was purified by rProteinA (GE-Healthcare) under high salt conditions according to 11D 5. Antibody elution was performed at pH 5.5. The flow rate of rat antibody purification must be kept low to increase binding efficiency.

As a second purification step, ion exchange chromatography (under separate, suitable conditions) or preparative size exclusion chromatography (PBS, pH 7.4) can be carried out.

Standard protocols for quality control of purified antibodies include:

● rSDS-PAGE gel analysis; coomassie staining or silver staining

● BCA Assay (Pierce #23227 BCA Protein Assay Kit; rat IgG Standard #31233)

● analytical size exclusion (Superdex 200 Tricorn 10/300 GL, 250mg in 250. mu.l; 0.5 ml/min,Explorer 100)

● endotoxin test (LAL, Cambrex)Chromogenic LALEndpoint Assay # US50-648U)

● cell-based Activity assay (FACS binding; pAkt; pAXL)

Depending on their stability, purified antibodies were stored in PBS, pH7.4, under sterile conditions at 4 ℃ or-20 ℃.

Antibody affinity determination by FACS scatchard

NIH3T3 cells overexpressing human AXL were harvested by incubation with 10mM EDTA in PBS and resuspended in FACS buffer (PBS pH7.4, 3% FCS, 0.1% NaN) at 6 million cells/ml₃) In (1). In round bottom microtiter plates, 100. mu.l of the cell suspension was added to 100. mu.l of an antibody solution containing the antibodies 11B7, 11D5, ch11B7-IgG2 or ch11D5-IgG2 in FACS buffer at a concentration of 40 to 0.002. mu.g/ml (266 to 0.01 nM). Antibody binding was performed on ice for 2 hours. The cells were then washed 2 times with 250. mu.l FACS buffer/well and resuspended in 200. mu.l secondary antibody (anti-rat-PE; Jackson) diluted 1: 50 in FACS buffer. After incubation for 45 minutes, cells were washed 2 more times in FACS buffer and then resuspended in 500ml PBS for FACS analysis. The analysis was performed on a Beckman-Coulter FACS FC 500. To determine the apparent affinity constant K_DappAverage fluorescence values are directed to the average fluorescence and the corresponding antibody concentration ([ M)]) The ratios of (A) to (B) are plotted. K calculated from the inverse slope (inverse slope) of the line_DappListed below:

cloning	K_DValue (nM)
		11B7	0.38
Ch11B7-IgG2	0.6

11D5	0.81
		Ch11D5-IgG2	0.9

18. Chimerization (polymerization) of rat anti-AXL antibody:

human kappa light and heavy chain IgG 1/2 genes were cloned from Peripheral Blood Mononuclear Cells (PBMCs) of human volunteers as follows:

PBMCs were prepared from whole blood. Blood was diluted 1/2, 5 in PBS/2mM EDTA with 10U/ml heparin at room temperature in 15ml Biocoll solution (35 ml/tube) covered by diaphragm (diaphragm) [ Biocoll from Biochrom # L6115 [)]The layers above (a) are layered. The sample was centrifuged at 400Xg for 30 minutes at room temperature and the serum (approximately 15ml) was discarded. The interface containing PBMCs was carefully recovered using a Pasteur pipette. PBMCs were washed 2 times (first 100ml wash, second 50ml wash) in PBS/2mM EDTA and centrifuged at 300Xg for 10 minutes. The cell pellet was resuspended in RPMI/10% FCS (25ml) to yield 5.5X10⁷And (5) PBMCs.

RNA was prepared from PBMCs using RNeasy kit from Qiagen (#75142) according to the manufacturer's instructions. Purified RNA (30. mu.g) was stored in aliquots at-80 ℃.

cDNA for the antibodies IgG γ 1 and 2 and the kappa chain were prepared from the isolated RNA by RT-PCR using the following primers according to the manufacturer's instructions using SuperScript III reverse transcriptase (invitfogen # 18080-93):

1)RT-γ：GCG TGT AGT GGT TGT GCA GAG

2)RT-γ2：GGG CTT GCC GGC CGT G

3)RT-κ：TGG AAC TGA GGA GCA GGT GG

4)5′Blp：AGA TAA GCT TTG CTC AGC GTC CAC CAA GGG CCC ATCGGT

5)3′Bam(GAG)：AGA TGG ATC CTC ATT TAC CCG GAG ACA GGG AGAG

6)5′Bsi：AGA TAA GCT TCG TAC GGT GGC TGC ACC ATC TGT CTTCAT

7)3′Bam(CTT)：AGA TGG ATC CCT AAC ACT CTC CCC TGT TGA AGCTCT

the primers were dissolved at 100. mu.M. RT-PCR reactions were performed using 2pmol oligo RT γ and RT κ, respectively, adding 1 μ g RNA, 10mM dNTP mix and heating for 5 min to 65 ℃. Mu.l of first strand buffer, 1. mu.l of 0.1M DTT, 1. mu.l of RNase inhibitor (40U/. mu.l of Fermentas # E00311) and 2. mu.l of Superscript IIIRT were added, mixed, and then incubated at 50 ℃ for 1 hour, followed by a heat inactivation step at 70 ℃ for 15 minutes.

Mu.l of the first strand reaction was used for the second step PCR using Taq polymerase (Eurochrom # EME010001) to generate double stranded DNA of the antibody constant domain. Primers 5 'Blp and 3' bam (gag) were used to amplify the γ -strand, and 5 'Bsi and 3' bam (ctt) were used to amplify the κ -strand constant region using the following PCR setup:

kappa-chain amplification:

94 ℃ for 120 seconds

94 ℃ for 30 seconds

55 ℃ for 30 seconds

45 seconds at 72 ℃ and 35 cycles

10 minutes at 72 DEG C

Gamma-strand amplification:

94 ℃ for 120 seconds

94 ℃ for 30 seconds

30 seconds at 45 DEG C

60 seconds at 72 ℃ and 5 cycles

94 ℃ for 30 seconds

30 seconds at 50 DEG C

60 seconds at 72 ℃ and 35 cycles

10 minutes at 72 DEG C

PCR products were analyzed on a TAE buffered 2% agarose gel. A single band of approximately 350bp (for kappa light chains) and a single band of approximately 1000bp (for heavy chains. gamma.1 and. gamma.2) was found. The PCR product was purified using Qiagen gel extraction kit (QIAGEN, #28784) according to the manufacturer's instructions. To clone the PCR fragment into the multiple cloning site of the pcDNA3 vector (Invitrogen), the pcDNA3 vector and the PCR fragment were digested with HindIII (5 ') and BamHI (3') restriction endonucleases. Restriction sites were encoded within the PCR primers. The degraded fragments were purified using Qiagen PCR purification kit (Qiagen, 28104) and DNA encoding γ 1, γ 2 and κ strands were ligated into pcDNA3 vector using T4DNA ligase at 16 ℃ overnight. The ligase was inactivated at 65 ℃ for 10 minutes. Ligation of DNA plasmids directly into CaCl Using Standard protocols₂Competent E.coli were plated on LB plates containing ampicillin. After incubation at 37 ℃ overnight, individual colonies were picked and suspended in 10. mu. l H₂O and the colonies were verified by PCR (5 μ Ι of suspended cells, Taq polymerase, primers 5Blp and 3bam (gag) γ 1/γ 2 and 5Bsi and 3bam (ctt) (for κ colonies)) on plasmids containing the respective antibody chains:

94 ℃ for 120 seconds

94 ℃ for 30 seconds

55 ℃ for 30 seconds

60 seconds at 72 ℃ and 35 cycles

10 minutes at 72 DEG C

Samples were analyzed on a 1.5% agarose gel for PCR products. Colonies containing the antibody genes were selected to inoculate 5ml LB/ampicillin media. After incubation at 37 ℃ overnight, E.coli was harvested and DNA was prepared using Qiagen miniprep kit (QIAGEN, # 12123). The control degradants (HindIII, BamHI) showed all kappa and gamma chain gene inserts at the expected size; the sequence was verified by sequencing the DNA on mediganomix.

Rat variable domains were amplified by PCR from the pLXSN-ESK vector and then cloned into the g1/g2 and k pcDNA3 vectors to generate chimeric full-length antibodies. The variable VL domain was amplified using the following primers (which contained HindIII and BsmI sites at the 5 'end and BsiWI sites at the 3' end):

VL-11B7-5′：AGA TAA GCT TGT GCA TTC CGA CAT CCA

GAT GAC CCA GGC TCC

VL-11B7-3′：AGA TCG TAC GTT TCA GCT CCA GCT TGG

TGC CTC

VL-11D5-5′：AGA TAA GCT TGT GCA TTC CGA CAT CCA

GAT GAC CCA GTC TCC ATC

VL-11D5-3′：AGA TCG TAC GTT TCA GCT TGG TCC CAG

the variable VH domains were amplified using the following primers (which contained HindIII and BsmI sites at the 5 'end and BlpI sites at the 3' end):

VH-11B7/11D5-5′：AGA TAA GCT TGT GCA TTC CGA GGT GCA

GCT TCA GGA GTC AGG

VH-11B7/11D5-3′：AGA TGC TGA GCT GAC AGT GAC CAT GAG

TCC TTG GCC

BsiWI of the light chain and BlpI of the heavy chain are single sites on the 5 'end of the constant region that enable direct fusion to the 3' end of the variable domain gene.

Fusion to the leader sequence SEQ ID No: 69 into the pCEP vector system for recombinant expression. The light chain gene was cloned between NheI (5 ') and XhoI (3') into pCEP4(Invitrogen), and the heavy chain gene was cloned between KpnI (5 ') and XhoI (3') into pCEP-Pu (Kohfeld FEBS Vol.414; (3)557ff, 1997).

Using Standard CaPO₄Transfection, HEK293 cells seeded in 20x20cm plates were co-transfected with 1 μ g/ml of each plasmid encoding the light and heavy chain genes for transient expression. The culture conditions were 37 ℃ and 5% CO₂In DMEM/F12 high glucose medium (containing 5% low IgG FCS, 1% pyruvate, 1% glutamine, 1% penicillin/streptomycin). 24 hours after transfection, the medium was replaced with fresh medium. Supernatants were collected every 2 to 3 days for approximately 3 weeks. Washing with 50mM PO under standard buffer conditions (loading: 50mM Tris; pH 8.5; wash: 50mM PO) as described for rat antibody purification₄(ii) a pH8.5, elution: 100mM glycine; pH3, 3) chimeric antibody was purified from approximately 600ml supernatant using 1ml Hitrap protein a column (GE-Healthcare).

Example 19 humanization of rat anti-AXL antibody variable domains

Search of immunoglobulin domains by BLAST for chimeric antibodies at the protein levelRat variable regions were compared to human antibody germline sequences. The closest human counterpart within the V gene was identified and in addition it had the same CDR loop length. In a similar manner, from the V-BASE database based on their homology to rat sequences (http://vbase.mrc-cpe.cam.ac.uk/) Relevant D and J segments are selected.

For the rat variable domains of the 11B7 and 11D5 antibodies, the following best-fit human germline sequences (V, D and J segments) were found and defined as human frameworks:

VL11B7hum：Vκ1-O12+Jk1

VH11B7 hum: VH4-59+ D4-4 (reading frame 3) + JH4

VL11D5hum：Vκ1-L1+Jκ4

VH11D5 hum: VH4-59+ D4-4 (reading frame 3) + JH4

The leader sequence of the humanized variable domain employs the relevant germline V-gene sequence of choice. For anti-AXL specificity, CDR residues of a rat anti-AXL antibody as defined according to Kabat (Kabat et al, Sequences of proteins of Immunological Interest, 5 th edition. NIH Publication No.91-3242, 1991) were grafted into a human germline framework to obtain the final humanized forms hum11B7 and hum11D5 of the anti-AXL antibody.

The protein sequences of the humanized anti-AXL antibodies hum11B7 and hum11D5 are as follows:

the protein sequence is translated back into a DNA sequence. The DNA sequence was Codon optimized for recombinant expression in mammalian cells using the Kazusa-Codon-Usage database. The DNA sequence of the resulting humanized anti-AXL antibody is as follows:

an optimized DNA sequence encoding a humanized anti-AXL antibody was synthesized by PCR based on overlapping oligonucleotides.

The VL gene was cloned into the pCEP4 vector using a plasmid of the chimeric antibody construct pCEP4_ ch11B7k 1. The cloning sites are NheI (5 ') and BsiWI (3'), which are also included in the synthetic genes of the humanized antibodies. The VH genes were cloned into the corresponding chimeric heavy chain vector pCEP-PU _ ch11B7g1 using KpnI (5 ') and BlpI (3') as restriction sites. DNA optimization, gene synthesis, cloning and sequence verification were performed on Eurofins Medeigenmix GmbH, Martinsried, Germany.

Example 20 rat and chimeric anti-Ax 1 antibodies of the invention inhibit ligand-induced Ax1 phosphorylation to a similar extent in vitro

As part of the invention, chimeric derivatives of the rat anti-Ax 1 antibodies 11B7 and 11D5 were generated (see below). To investigate whether the rat anti-Ax 1 antibody of the invention and the corresponding chimeric anti-Ax 1 antibody of the invention were able to inhibit Ax1 activation mediated by ligand Gas6 in vitro to a similar extent, ELISA experiments were performed on CaSki cervical cancer cells. Thereby detecting Gas 6-mediated Ax1 activation through increased receptor tyrosine phosphorylation. Briefly, on day 1, 3x10 was added⁴Individual cells/well were seeded in flat bottom 96-well plates in normal growth medium. The following day, the growth medium was replaced with serum-free medium to starve the cells overnight for 24 hours. Similarly, Maxi-Sorp 96-well plates (Nunc) were coated with 2. mu.g/ml of the mouse anti-phospho-tyrosine antibody 4G10 in PBS at 4 ℃ overnight. On day 3, the 4G10 antibody solution was removed and the Maxi-Sorp wells blocked with PBS, 0.5% BSA at room temperature for at least 4 hours. In parallel, cells were preincubated with rat anti-Ax 1 antibody 11B7 or chimeric anti-Ax 1 antibody ch11B7 at 37 ℃ for 1 hour with 50ng/ml, 100ng/ml, 300ng/ml, 750ng/ml, 1. mu.g/ml and 10. mu.g/ml, with or without 400ng/ml Gas6 (R)&D Systems) at 37 ℃ for 10 minutes. The medium was then discarded and supplemented with phosphatase and protease inhibitors (10mM Na) on ice₄P₂O₇1mM phenylmethylsulfonyl fluoride, 1mM orthovanadate, 1mM NaF and 0.5% aprotinin) for 30 minutes in lysis buffer (50mM HEPES, pH7.5, 150mM NaCl, 1mM EDTA, 10% glycerol and 1% Triton X-100). At the same time the blocking buffer was removed and the Maxi-Sorp plates were washed 6 times with washing buffer (PBS, 0.05% Tween20) before transferring and incubating the lysates overnight at 4 ℃. Wash plates 6 times with Wash buffer on day 4Thereafter, wells were incubated with 0.5 μ g/ml of biotinylated rat anti-Ax 1 antibody 12B7 in PBS for 2 hours at room temperature. The plates were washed 6 times with wash buffer, AP-conjugated streptavidin (Chemicon # SA110) diluted 1: 4,000 in PBS was added to each well and incubated for 30 minutes at room temperature. Thereafter, the wells were washed 6 times with wash buffer and AttoPhos substrate solution (Roche #11681982) was added. The fluorescence of each well was collected at an excitation wavelength of 430nm and an emission wavelength of 580nm using a Victor plate reader (Perkin Elmer).

Fig. 17 shows representative results of this experiment for the cervical cancer cell line CaSki. As demonstrated by the concentration-dependent reduction in phosphorylation relative to Ax1, the rat anti-Ax 1 antibody 11B7(a) and the chimeric anti-Ax 1 antibody ch11B7(B) of the invention were able to prevent ligand-induced activation of the receptor tyrosine kinase Ax1 to a similar extent. For melanoma cell line C-8161, comparable effects were observed using the same experimental setup.

Example 21 rat and chimeric anti-Ax 1 antibodies of the invention inhibit ligand-induced phosphorylation of p42/p44 MAP-kinase in vitro to a similar extent

To additionally verify whether the rat anti-Ax 1 antibody of the invention and the corresponding chimeric anti-Ax 1 antibody of the invention are also able to inhibit Gas 6-induced activation of p42/p44 MAP-kinase to a similar extent in CaSki cervical cancer cells, ELISA experiments were performed. Here, the activation of p42/p44 MAP-kinase induced by Gas6 was detected by increased protein (Thr202/Tyr204) phosphorylation. Briefly, on day 1, 2x10 was added⁴Individual cells/well were seeded in flat bottom 96-well plates. The following day, the normal growth medium was replaced with serum-free medium to starve the cells for 24 hours. Thereafter, cells were pre-incubated with 50ng/ml, 100ng/ml, 300ng/ml, 750ng/ml, 1. mu.g/ml and 10. mu.g/ml of either the rat anti-Ax 1 antibody 11B7 or the chimeric anti-Ax 1 antibody ch11B7 for 1 hour at 37 ℃ with or without 400ng/ml Gas6 (R)&D Systems) at 37 ℃ for 10 minutes. The medium was discarded and the cells were fixed with 4% formaldehyde in PBS (pH 7.5) for 30 minutes at room temperature. The formaldehyde solution was removed and the cells were washed 2 times with washing buffer (PBS, 0.1% Tween 20). By using1% H in Wash buffer₂O₂，0.1％NaN₃The cells were quenched and incubated at room temperature for 20 minutes. After that, the quenching solution was removed, and the cells were washed 2 times with washing buffer, and then blocked with PBS, 0.5% BSA at room temperature for 4 hours. Anti-phospho-p 42/p44MAP kinase (Thr202/Tyr204) primary antibody (polyclonal rabbit; CellSignaling #9101) diluted 1: 1,000 in PBS, 0.5% BSA, 0.05% Tween20, 5mM EDTA was added overnight at 4 ℃. On day 4, the antibody solution was removed and the plate was washed 3 times with wash buffer. HRP-conjugated anti-rabbit secondary antibody (Dianova #111-036-045) diluted 1: 2,500 in PBS, 0.5% BSA, 0.05% Tween20, 5mM EDTA was then added to each well and incubated at room temperature for 1.5 hours. The plates were washed 3 times for 5 minutes each with wash buffer. Tetramethylbenzidine (TMB, Calbiochem) was added and monitored at 620 nm. The reaction was stopped by adding 100. mu.l of 250nM HCl and the absorbance was read at 450nM (reference wavelength of 620 nM) using a Vmax plate reader (Thermo Lab Systems).

Fig. 18 shows representative results of this experiment. The rat anti-Ax 1 antibody 11B7(a) and the chimeric anti-Ax 1 antibody ch11B7(B) of the present invention were able to prevent Gas 6-induced activation of p42/p44 MAP-kinase in CaSki cervical cancer cells to a similar extent, as shown by the concentration-dependent decrease in phosphorylation relative to p42/p44 MAP-kinase.

Example 22 rat anti-Ax 1 antibodies of the invention act synergistically with chemotherapeutic agents to overcome drug resistance in vitro

The problem arises because the rat anti-Ax 1 antibody of the invention itself interferes with the anti-apoptosis of Gas 6-mediated serum starved NIH3T3-Ax1 c1.7 fibroblasts: whether antagonistic anti-Ax 1 antibodies synergistically induce apoptosis with chemotherapeutic agents facilitates overcoming drug resistance. In this example, NCI/ADR-RES (formerly MCF-7/AdrR) cells- -ovarian Cancer cells that exhibit high levels of resistance to several agents, including doxorubicin (Fairchild et al, 1987, Cancer Research, 47, 5141-Cell lines (Liscovitch and Ravid, 2007, Cancer Letters, 245, 350-. Briefly, at 3x10⁴Cells/well NCI/ADR-RES cells were seeded in normal growth medium on 8-chamber culture slides (BD Falcon, cat # 354118) preincubated with the same medium for 1 hour at 37 ℃. Day 2 morning, the normal growth medium was removed, and the cells were washed with low serum (0.5% FCS) medium and cultured therein. In the evening, isotype control antibody 1D5 or antagonistic anti-AXL antibody 11B7 were added at a final concentration of 10 μ g/ml each. On the morning of day 3, Robixin was added at final concentrations of 100. mu.M, 150. mu.M or 200. mu.M and the cells were incubated at 37 ℃. After 24 hours, the cells were rinsed 1 time with PBS, fixed with 4% formaldehyde in PBS (pH 7.5) at room temperature for 20 minutes, air-dried for 5 minutes, and then stored at-20 ℃. Using commercially available Fluorescein-FragEL^TMKit (Oncogene, cat # QIA39, currently distributed by Merck-Calbiochem), according to the instructions of the supplier's manual (section' Fluorescein-FragEL)^TMof cell precursors fixed on slides', page 10) were subjected to TUNEL staining. Cells were analyzed and photographed using a fluorescence microscope.

Fig. 19 shows representative results of this experiment. For NCI/ADR-RES ovarian cancer cells treated with 100 μ M doxorubicin, neither incubation of the cells with control antibody nor with antagonistic anti-Ax 1 antibody 11B7 (top panel) resulted in no TUNEL staining and thus no apoptosis. However, at a concentration of 150 μ M doxorubicin, only very weak apoptosis was detected in cells co-treated with the control antibody, whereas co-incubation with the antagonistic anti-Ax 1 antibody 11B7 resulted in a significant induction of apoptosis (middle panel). Similarly, co-incubation of cells with 11B7 also significantly increased the rate of apoptosis in the presence of 200 μ M doxorubicin compared to cells incubated with control IgG antibody (lower panel), suggesting that co-treatment with chemotherapeutic agents and the antagonistic anti-Ax 1 antibodies of the invention may be useful to overcome drug resistance even in multi-resistant cells.

Example 23 rat anti-Ax 1 antibodies of the invention synergize with chemotherapeutic agents in vitro to reduce the growth of anchorage-independent colonies

A soft agar assay was performed to investigate the ability of the anti-Ax 1 antibodies of the invention to inhibit the growth of anchorage-independent cells, alone or in combination with chemotherapeutic agents. The soft agar colony formation assay is a standard in vitro assay to detect transformed cells, since only transformed cells are able to grow on soft agar.

Briefly, 750C-8161 melanoma cells were left untreated or preincubated with 15 μ g/ml of antagonistic rat anti-Ax 1 antibody 11B7 in IMDM medium (Gibco) for 30 minutes at 37 ℃. The cells were then combined with Difco Nobel agar solution to produce 50. mu.l of a top layer agar cell suspension at concentrations of agar, FCS and 11B7 of 0.35%, 0.2% and 7.5. mu.g/ml, respectively. The cell suspension was plated on top of a 50. mu.l 0.7% agarose base containing 20% FCS and finally covered with another 50. mu.l of trophoblast solution containing 0.2% FCS and the corresponding concentration of cisplatin. The final concentrations of 11B7 and cisplatin were 2.5. mu.g/ml and 1.5. mu.M, 1.0. mu.M, 0.75. mu.M, 0.5. mu.M or 0.25. mu.M, respectively, in a total of 150. mu.l/sample. Colonies were allowed to form for 5 days and then stained with 50. mu.l MTT (Sigma, 1mg/ml in PBS) for 3 hours at 37 ℃. The effect of the antagonistic rat anti-Ax 1 antibody 11B7 in the absence or presence of cisplatin was analysed in triplicate using a Scanalyzer HTS camera system in combination with HTS Bonit colony formation software (Lemnatec, Wuerselen).

Fig. 20 shows representative results of this experiment. The data provided are the total area of colonies and reflect the absolute number measured (a) and the relative growth inhibition by cisplatin and/or antagonistic rat anti-Ax 1 antibody 11B7 (B). Incubation with cisplatin resulted in a dose-dependent retardation of colony growth compared to untreated control cells. Consistent with the inhibition of 11B7 alone in the range of 30%, the combination with the antagonistic anti-Ax 1 antibody 11B7 resulted in a significantly enhanced inhibition of soft agar growth of C-8161 melanoma cells by cisplatin, particularly at lower concentrations.

Example 24 murine anti-Ax 1 antibodies of the invention cooperate with an anti-tumor agent to reduce tumor-related events

In the foregoing examples, synergy of co-administration of the antagonistic anti-Ax 1 antibody of the invention with doxorubicin has been observed with respect to induction of apoptosis and overcoming drug resistance in multi-drug resistant cancer cells, such as the ovarian cancer cell line NCI/ADR-RES. In addition, the combined effect of antagonistic anti-Ax 1 antibodies of the invention with cisplatin in reducing anchorage-independent colony growth was detected using the melanoma cell line C-8161. Thus, when an antagonistic anti-Ax 1 antibody is combined with radiation and/or one or more additional anti-neoplastic agents to treat cancer cells or patients with a cancer disease, a synergistic effect is expected in inducing apoptosis and/or overcoming drug resistance in tumor cells, inhibiting tumor cell survival, inhibiting tumor cell growth and/or proliferation, reducing tumor cell migration, spread and metastasis, or attenuating tumor angiogenesis. In particular, when an antagonistic anti-Ax 1 antibody is used in combination with radiation and/or any additional antineoplastic agent (which is preferably, but not limited to, cisplatin, dacarbazine, temozolomide/temodal, warrior (murhoran)/fotemustine, paclitaxel or docetaxel) to treat melanoma cells or patients with melanoma, a synergistic effect is expected in inducing apoptosis and/or overcoming drug resistance in tumor cells, inhibiting tumor cell survival, inhibiting tumor cell growth and/or proliferation, reducing tumor cell migration, spreading and metastasis or impairing tumor angiogenesis. Furthermore, when an antagonistic anti-Ax 1 antibody is used in combination with radiation and/or any additional antineoplastic agent, which is preferably, but not limited to, doxorubicin, cisplatin, carboplatin, paclitaxel, docetaxel, melphalan, altretamine, topotecan, ifosfamide, etoposide, or 5-fluorouracil, to treat ovarian cancer cells or patients with ovarian cancer, a synergistic effect is expected in inducing apoptosis and/or overcoming resistance of tumor cells, inhibiting tumor cell survival, inhibiting tumor cell growth and/or proliferation, reducing tumor cell migration, spreading and metastasis, or impairing tumor angiogenesis. Furthermore, when an antagonistic anti-Ax 1 antibody is used in combination with radiation and/or any additional antineoplastic agent (which is preferably, but not limited to, mitoxantrone, doxorubicin, paclitaxel, docetaxel, or vinblastine) to treat prostate cancer cells or patients with prostate cancer, synergy is expected in inducing apoptosis and/or overcoming drug resistance of tumor cells, inhibiting tumor cell survival, inhibiting tumor cell growth and/or proliferation, reducing tumor cell migration, spread and metastasis, or impairing tumor angiogenesis. Furthermore, when an antagonistic anti-Ax 1 antibody is used in combination with radiation and/or any additional antineoplastic agent (which is preferably, but not limited to, 5-fluorouracil, mitomycin C, cisplatin, doxorubicin, methotrexate, etoposide, calcium folinate (leucovorin), epirubicin, paclitaxel, docetaxel, or irinotecan) to treat gastric/stomach cancer cells or patients with gastric/gastric cancer, a synergistic effect is expected in inducing apoptosis and/or overcoming resistance of tumor cells, inhibiting tumor cell survival, inhibiting tumor cell growth and/or proliferation, reducing tumor cell migration, diffusion, and metastasis, or impairing tumor angiogenesis. Furthermore, when an antagonistic anti-Ax 1 antibody is used in combination with radiation and/or any additional antineoplastic agent (which is preferably, but not limited to, doxorubicin, epirubicin, paclitaxel, docetaxel, cyclophosphamide, 5-fluorouracil, gemcitabine, capecitabine, vinorelbine, or trastuzumab) to treat breast cancer cells or patients with breast cancer, a synergistic effect is expected in inducing apoptosis and/or overcoming drug resistance of tumor cells, inhibiting tumor cell survival, inhibiting tumor cell growth and/or proliferation, reducing tumor cell migration, diffusion and metastasis, or impairing tumor angiogenesis. Furthermore, when an antagonistic anti-Ax 1 antibody is used in combination with radiation and/or any additional antineoplastic agent (which is preferably, but not limited to, cisplatin, ifosfamide, irinotecan, 5-fluorouracil, paclitaxel, docetaxel, gemcitabine, or topotecan) to treat cervical cancer cells or patients with cervical cancer, a synergistic effect is expected in inducing apoptosis and/or overcoming drug resistance in tumor cells, inhibiting tumor cell survival, inhibiting tumor cell growth and/or proliferation, reducing tumor cell migration, spread and metastasis, or attenuating tumor angiogenesis. Furthermore, when an antagonistic anti-Ax 1 antibody is used in combination with radiation and/or any additional antineoplastic agent (which is preferably, but not limited to, gemcitabine, capecitabine, or 5-fluorouracil) to treat pancreatic cancer cells or patients with pancreatic cancer, a synergistic effect is expected in inducing apoptosis and/or overcoming drug resistance of tumor cells, inhibiting tumor cell survival, inhibiting tumor cell growth and/or proliferation, reducing tumor cell migration, spread, and metastasis, or attenuating tumor angiogenesis. Finally, but not exclusively, other cancer types, when an antagonistic anti-Ax 1 antibody is used in combination with radiation and/or any additional antineoplastic agent (which is preferably, but not limited to, cisplatin, carboplatin, doxorubicin, paclitaxel, docetaxel, etoposide, vinorelbine, vincristine, ifosfamide, gemcitabine, methotrexate, cyclophosphamide, lomustine, or topotecan) to treat lung cancer cells or patients with lung cancer, a synergistic effect is expected in inducing apoptosis and/or overcoming drug resistance in tumor cells, inhibiting tumor cell survival, inhibiting tumor cell growth and/or proliferation, reducing tumor cell migration, diffusion and metastasis, or impairing tumor angiogenesis.

Claims

1. A monoclonal antibody that binds to the extracellular domain of AXL and at least partially inhibits AXL activity, the heavy chain of which comprises:

(a) SEQ ID NO: 22 of the CDRH1 shown in figure 22,

(b) SEQ ID NO: CDRH2 shown in 23, and

(c) SEQ ID NO: the CDRH3 shown in figure 24,

and the light chain thereof comprises:

(d) SEQ ID NO: the CDRL1 shown in figure 19,

(e) SEQ ID NO: CDRL2 shown in 20, and

(f) SEQ ID NO: CDRL3 shown in fig. 21.

2. The monoclonal antibody of claim 1, which reduces and/or blocks AXL-mediated signal transduction.

3. The monoclonal antibody of claim 1, which reduces and/or blocks AXL phosphorylation.

4. The monoclonal antibody of claim 1, which reduces and/or blocks cell proliferation.

5. The monoclonal antibody of claim 1, which reduces and/or blocks angiogenesis.

6. The monoclonal antibody of claim 1, which reduces and/or blocks cell migration.

7. The monoclonal antibody of claim 1, which reduces and/or blocks tumor metastasis.

8. The monoclonal antibody of claim 1, which reduces and/or blocks AXL-mediated anti-apoptosis.

9. The monoclonal antibody of claim 1, which reduces and/or blocks AXL-mediated PI3K signaling.

10. The monoclonal antibody of claim 1 which is a recombinant antibody.

11. The monoclonal antibody of claim 1 which is a humanized antibody, a chimeric antibody, a multispecific antibody or antibody fragment which is a Fab fragment, a Fab 'fragment, a F (ab')₂Fragments or Fv fragments.

12. The monoclonal antibody of claim 11, which is a chimeric antibody and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 42 and the heavy chain amino acid sequence of SEQ ID NO: 40, light chain amino acid sequence.

13. The monoclonal antibody of claim 11, which is a humanized antibody and comprises the amino acid sequence of SEQ id no: 46 and the heavy chain amino acid sequence of SEQ ID NO: 45, light chain amino acid sequence.

14. The monoclonal antibody of claim 1 which is a Fab fragment, a Fab 'fragment, a F (ab') fragment, an Fv fragment, a diabody, or a single chain antibody molecule.

15. The monoclonal antibody of claim 1 which is of the IgG1, IgG2, IgG3 or IgG4 type.

16. The monoclonal antibody of claim 1, which is coupled to a labeling group.

17. The monoclonal antibody of claim 1, which is coupled to an effector group.

18. The monoclonal antibody of claim 1, which is a scaffold protein.

19. The monoclonal antibody of claim 1, comprising the amino acid sequence of SEQ ID NO: 10 and the heavy chain amino acid sequence of SEQ ID NO: 9, light chain amino acid sequence.

20. An isolated nucleic acid molecule selected from the group consisting of:

a nucleic acid sequence encoding the monoclonal antibody, antibody fragment or derivative thereof of any one of claims 1 to 19, wherein the antibody fragment is a Fab fragment, Fab 'fragment, F (ab')₂A fragment or an FV fragment, and said derivative is a single chain antibody, a nanobody or a diabody.

21. A vector comprising the nucleic acid sequence of claim 20.

22. The vector of claim 21 which is an expression vector and the nucleic acid sequence is operably linked to a control sequence.

23. A host comprising the vector of claim 21 or 22, which is a prokaryotic or eukaryotic cell.

24. A method of preparing a monoclonal antibody according to any one of claims 1 to 19, comprising the step of obtaining the monoclonal antibody from the host of claim 23.

25. A pharmaceutical composition comprising the monoclonal antibody of any one of claims 1 to 19 or produced by the method of claim 24.

26. A pharmaceutical composition according to claim 25, comprising a pharmaceutically acceptable carrier, diluent and/or adjuvant.

27. The pharmaceutical composition of claim 25 or 26, comprising an additional active agent.

28. The pharmaceutical composition of claim 25 or 26 for use in the diagnosis, prevention or treatment of a hyperproliferative disease.

29. The pharmaceutical composition of claim 28, wherein the hyperproliferative disease is associated with AXL expression and/or hyperactivity.

30. The pharmaceutical composition of claim 29, wherein the hyperproliferative disease is associated with AXL overexpression and/or hyperactivity.

31. The pharmaceutical composition of claim 28, wherein the hyperproliferative disease is selected from the group consisting of breast cancer, lung cancer and other AXL expressing cancers and the formation of tumor metastases.

32. The pharmaceutical composition of claim 31, wherein the AXL-expressing cancer is an AXL-overexpressing cancer.

33. The monoclonal antibody of any one of claims 1 to 19 for use in the diagnosis, prevention or treatment of a hyperproliferative disease.

34. Use of a monoclonal antibody according to any one of claims 1 to 19 for the preparation of a pharmaceutical composition for the diagnosis, prevention or treatment of a hyperproliferative disease, wherein said hyperproliferative disease is a hyperproliferative disease as defined in any one of claims 29-32.

35. Use of a monoclonal antibody according to any one of claims 1 to 19 for the preparation of a medicament for the diagnosis of a disorder associated with the expression of AXL.

36. The use according to claim 35, wherein the disorder is a hyperproliferative disease as defined in any one of claims 29 to 32.

37. Use of a monoclonal antibody of any one of claims 1 to 19 in the manufacture of a medicament for preventing or treating a disorder associated with the expression of AXL in a patient.

38. The use according to claim 37, wherein the disorder is a hyperproliferative disease as defined in any one of claims 29 to 32.

39. The use of claim 37 or 38, wherein the patient is a mammalian patient.

40. The use of claim 39, wherein the patient is a human patient.

41. A kit comprising a monoclonal antibody according to any one of claims 1 to 19, a nucleic acid sequence according to claim 20 or a vector according to claim 21 or 22.

42. The kit of claim 41, further comprising an additional anti-neoplastic agent.

43. Use of an anti-AXL antibody for the preparation of a pharmaceutical composition for the treatment of a drug resistant cancer, wherein said anti-AXL antibody is a monoclonal antibody of any one of claims 1 to 19.

44. Use of an anti-AXL antibody for the manufacture of a medicament for co-administration with an anti-tumour agent for the treatment of a hyperproliferative disease, wherein the anti-AXL antibody is the monoclonal antibody of any one of claims 1 to 19.