ES2712736T3 - Anti-Axl antibodies and their uses - Google Patents

Abstract

A monoclonal antibody having specificity for Axl or an antigen-binding fragment thereof, comprising a variable region of the heavy chain comprising SEQ ID NO: 2 in the H-CDR1 region, SEQ ID NO: 3 in the H-CDR2 region and SEQ ID NO: 4 in the H-CDR3 region; and a variable region of the light chain comprising SEQ ID NO: 6 in the L-CDR1 region, SEQ ID NO: 7 in the L-CDR2 region and SEQ ID NO: 8 in the L-CDR3 region, in wherein said antibody binds to the extracellular domain of Axl in the amino acid sequence set forth as SEQ ID NO: 9, in the amino acid sequence set forth as SEQ ID NO: 10 and in the amino acid sequence set forth as SEQ ID NO: 11.

Description

DESCRIPTION

Anti-AxI antibodies and their uses

Field of the invention

The present invention relates to anti-AxI antibodies and their uses in diagnostic and therapeutic methods.

Background of the invention

Axl belongs to the TAM subfamily of tyrosine kinase receptor (RTK) that also include Tyro3 and Mer. TAM receptors are characterized by a combination of two immunoglobulin-like domains and double repeats of fibronectin type III in the extracellular region and a cytoplasmic kinase domain. The ligands for the TAM receptors are Gas6 (gene 6 specific to the arrest of growth) and the protein S, two vitamin K-dependent proteins that show 43% amino acid sequence identity and share similar domain structures. Each protein has an N-terminal Gla domain that contains 11 g-carboxyglutamic acid residues, followed by four epidermal growth factor (EGF) type modules, and a C-terminal sex hormone-binding globulin (SHBG) -type structure. in two G domains of tandem laminin. The SHBG domain is both necessary and sufficient for the binding and activation of the TAM receptor, while the Gla domain binds negatively charged membrane phospholipids and plays an important role in TAM-mediated phagocytosis of apoptotic cells. Activation and signaling of TAM has been implicated in multiple cellular responses including survival, proliferation, migration and cellular adhesion.

The deregulation of Axl or its ligand Gas6 is implicated in the pathogenesis of a variety of human cancers. It has been published that overexpression of Axl in a wide series of human cancers (lung, prostate, breast, gastric, pancreatic, ovarian, thyroid, blood cancers, renal cell carcinoma as well as glioblastoma ...) and is associated with the invasiveness, metastasis and negative prognosis. These findings suggest that Axl may be involved in the regulation of multiple aspects of tumorigenesis including growth, invasion and tumor angiogenesis and, therefore, represents an objective for therapeutic intervention in cancer especially for the development of metastatic and anti-cancer therapy. for another treatment of multiple cancer including the treatment of drug resistance.

Accordingly, anti-Axl monoclonal antibodies have been described for use in the treatment of cancers. For example, publications in relation to anti-Axl antibodies include WO2009 / 063965, WO2009 / 062690 and WO2011 / 014457.

Other roles of Axl dependent or not on its ligands such as the inhibition of immune functions, the activation of platelet aggregation and the inducers of infection vmca (as an example, the absorption of Ebola and Lassa virus is promoted by Axl) highlight the potential of Axl as a therapeutic object for other applications other than oncology.

Compendium of the invention

The present invention is defined in the claims. The present invention relates to a monoclonal antibody having specificity for Axl, comprising a variable region of the heavy chain comprising SEQ ID NO: 2 in the H-CDR1 region, SEQ ID NO: 3 in the H-CDR2 region and SEQ ID NO: 4 in the H-CDR3 region; and a variable region of the light chain comprising SEQ ID NO: 6 in the L-CDR1 region, SEQ ID NO: 7 in the L-CDR2 region and SEQ ID NO: 8 in the L-CDR3 region. Said monoclonal antibody binds to the extracellular domain of Axl by SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

Detailed description of the invention

Definitions:

The term "Axl" has its general meaning in the art and refers to the human Axl. Axl is also known as "Ark", "Tyro-7", "ufo" or "jtk11".

The term "anti-Axl antibody" refers to an antibody directed to Axl.

According to the present invention, "antibody" or "immunoglobulin" has the same meaning, and will also be used in the present invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that immunospecifically binds to an antigen. By definition, the term "antibody" encompasses not only complete antibody molecules, but also fragments of the antibody as well as variants (including derivatives) of the antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to one light chain for a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains different sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine the recognition and specificity of antigen binding. The domains of the constant region of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable parts of a light chain and a heavy chain. The specificity of the antibody lies in the structural complementarity between the combination site to the antibody and the antigenic determinant. The sites of combination with the antibody are composed of residues that are mainly hypervariable regions or determinants of complementarity (CDR). Occasionally, the remains of the non-hypervariable or structural (FR) regions influence the complete domain structure and, therefore, the combination site. The complementarity determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen binding site, therefore, includes six CDRs, comprising the set of CDRs from each of region V of a heavy and a light chain. The structural regions (FR) refer to amino acid sequences interposed between the CDRs.

The term "chimeric antibody" refers to an antibody comprising a VH domain and a VL domain of an antibody derived from the 20G7-D9 antibody, and a CH domain and a CL domain of a human antibody.

According to the invention, the term "humanized antibody" refers to an antibody having the variable structural region and constant regions of a human antibody but retaining the CDRs of the 20G7-D9 antibody.

The term "Fab" indicates an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about one half of the N-terminal end of the H chain and the entire L chain, between the fragments obtained treating IgG with a protease, papama, bind together through a disulfide bond.

The term "F (ab ') 2" refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly greater than the Fab linked by a disulfide bond of the hinge region, among the fragments obtained by treating IgG with a protease, pepsin.

The term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond from the hinge region of F (ab ') 2.

A single chain Fv polypeptide ("scFv") is a covalently linked VH :: VL heterodrimer that is normally expressed from a gene fusion that includes genes encoding VH and VL linked by a peptide-linker coding. "DsFv" is a VH :: VL heterodrimer stabilized by a disulfide bond. The divalent and multivalent antibody fragments can be formed either spontaneously by monovalent scFv association, or they can be generated by coupling monovalent scFvs by a linker peptide, such as divalent sc (Fv) 2.

The term "diabodies" refers to small antibody fragments with two antigen binding sites, such fragments comprising a variable domain of the heavy chain (VH) connected to a variable domain of the light chain (VL) in the same chain of polypeptide (VH-VL). Using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites.

By "purified" and "isolated" is meant, when referring to an antibody according to the invention or to a nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified" as used herein preferably means that at least 75% by weight, more preferably at least 85% by weight, more preferably at least 95% by weight, and most preferably at least 95% by weight is present. minus 98% by weight, of the biological macromolecules of the same type. An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide; however, the molecule may include some additional bases or moieties that do not detrimentally affect the basic characteristics of the composition.

Antibodies of the invention:

The present invention provides isolated anti-Axl antibodies or their fragments. In particular, the inventors have raised a murine anti-AxI antibody (20G7-D9) that produces hybridoma. The inventors have cloned and characterized the variable domain of the light and heavy chains of said mAb 20G7-D9 and, therefore, have determined the domain of the regions determining the complementarity (CDR) of said antibody as described in Table 1 :

Therefore, the invention relates to a monoclonal antibody having specificity for Axl, comprising a heavy chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO: 2 for H-CDR1, SEQ ID NO: 3 for H-CDR2 and SEQ ID NO: 4 for H-CDR3.

The invention also relates to a monoclonal antibody having specificity for Axl, comprising a light chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO: 6 for L-CDR1 , SEQ ID NO: 7 for L-CDR2 and SEQ ID NO: 8 for L-CDR3. The monoclonal antibody of the invention can comprise a heavy chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO: 2 for H-CDR1, SEQ ID NO: 3 for H-CDR2 and SEQ ID NO: 4 for H-CDR3 and a light chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO: 6 for L-CDR1, the SEQ ID NO: 7 for L-CDR2 and SEQ ID NO: 8 for L-CDR3.

In particular, the invention provides an anti-Axl monoclonal antibody comprising:

- a variable region of the heavy chain comprising SEQ ID NO: 2 in the H-CDR1 region, SEQ ID NO: 3 in the H-CDR2 region and SEQ ID NO: 4 in the H-CDR3 region; Y

- a variable region of the light chain comprising SEQ ID NO: 6 in the L-CDR1 region, SEQ ID NO: 7 in the L-CDR2 region and SEQ ID NO: 8 in the L-CDR3 region.

In a particular embodiment, the variable region of the heavy chain of said antibody has the amino acid sequence set as SEQ ID NO: 1 and / or the variable region of the light chain has the amino acid sequence set forth as SEQ ID NO: 5.

In another embodiment, the monoclonal antibody of the invention is a chimeric antibody, preferably a mouse / human chimeric antibody. In particular, said mouse / human chimeric antibody can comprise the variable domains of the 20G7-D9 antibody as defined above.

In another embodiment, the monoclonal of the invention is a humanized antibody. In particular, in said humanized antibody, the variable domain comprises human acceptor structural regions, and optionally the human constant domain where it is present, and non-human donor CDRs, such as mouse CDRs as defined above.

The invention further provides anti-Axl fragments directed to Axl of said antibodies including but not limited to Fv, Fab, F (ab ') 2, Fab', dsFv, scFv, sc (Fv) 2 and diabodies.

The invention further provides anti-AxI antibody or fragmented that binds to the amino acid sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 in the extracellular part of Axl.

In another aspect, the invention relates to a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 ; SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8.

Methods of production of the antibodies of the invention:

The Anti-Axl antibodies of the invention can be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.

By knowing the amino acid sequence of the desired sequence, one skilled in the art can easily produce said antibodies, by standard techniques for the production of the polypeptides. For example, they can be synthesized using the well-known method of the solid phase, preferably using a commercially available peptide synthesis apparatus (such as that produced by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, the antibodies of the invention can be synthesized by recombinant DNA techniques well known in the art. For example, antibodies can be obtained as DNA expression products following the incorporation of DNA sequences encoding the antibodies into the expression vectors and the introduction of such vectors into suitable eukaryotic and prokaryotic hosts that express the desired antibodies, from which they can be isolated later using well-known techniques.

Accordingly, a further objective of the invention relates to a sequence of nucleic acids encoding an antibody according to the invention. More particularly, the nucleic acid sequence encodes a heavy chain or a light chain of an antibody of the invention.

Generally, said nucleic acid is a DNA or RNA molecule, which can be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a vector vmco.

The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (eg, a foreign gene) can be introduced into a host cell, to transform the host and promoting the expression (eg, transcription and translation) of the introduced sequence.

Thus, a further objective of the invention relates to a vector comprising a nucleic acid of the invention. Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to elicit or direct the expression of said antibody upon administration to a subject. Examples of promoters and enhancers used in the expression vector for the animal cell include promoter and early enhancer of SV40 (Mizukami T. et al 1987), LTR promoter and enhancer of the Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason JO et al, 1985) and enhancer (Gillies SD et al 1983) of the H chain of immunoglobulin and the like.

Any expression vector can be used for the animal cell, provided that a gene encoding the C region of the human antibody can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al.

1990), pAGE103 (Mizukami T et al 1987), pHSG274 (Brady G et al 1984), pKCR (O'Hare K et al 1981), pSG1 beta d2-4- (Miyaji H et al 1990) and Similar.

Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for example pUC, pcDNA, pBR and the like.

Other examples of vector vmco include adenovirus, retrovirus, herpesvirus vectors and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, such as by transfection of packaging cells or by transient transfection with plasmids or helper viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv + cells, 293 cells, etc. Detailed protocols for the production of such recombinant replication-deficient viruses can be found, for example, in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278. 056 and WO 94/19478.

A further objective of the present invention relates to a host cell that has been transfected, infected or transformed by a nucleic acid and / or a vector according to the invention.

The term "transformation" means the introduction of a "foreign" (ie, extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, such that the host cell expresses the gene or sequence introduced to produce a substance desired, generally a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed".

The nucleic acids of the invention can be used to produce an antibody of the invention in a suitable expression system. The term "expression system" means a host cell and compatible vector under suitable conditions, for example, for the expression of a protein encoded by foreign DNA carried by the vector and introduced into the host cell.

Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and host cells and mammalian vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeasts, cell lines mammals (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts). , fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse cell SP2 / 0-Ag14 (ATCC CRL1581), mouse cell P3X63-Ag8.653 (ATC ^c CRL1580), CHO cell in which a dehydrofolate reductase gene (hereinafter referred to as "gene DHFR ") is defective (Urlaub G et al., 1980), rat cell YB2 / 3HL.P2.G11.16Ag.20 (ATCC CRL1662, hereinafter referred to as" cell YB2 / 0 "), and the like.

The present invention also relates to a method of producing a recombinant host cell expressing an antibody according to the invention, said method comprising the steps of: (i) introducing a recombinant nucleic acid or a vector as in vitro or ex vivo. described above in a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells that express and / or secrete said antibody. Such recombinant host cells can be used for the production of antibodies of the invention.

In another particular embodiment, the method comprises the steps of:

(i) culturing the 20G7-D9 hybridoma under suitable conditions to allow expression of the 20G7-D9 antibody; Y

(ii) recover the expressed antibody.

The antibodies of the invention are suitably separated from the culture medium by conventional immunoglobulin purification methods such as, for example, Protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

In a particular embodiment, the human chimeric antibody of the present invention can be produced by obtaining nucleic sequences encoding the VL and VH domains as previously described, constructing an expression vector of human chimeric antibody by inserting them into an expression vector for the cell animal having genes encoding Ch of the human antibody and CL of the human antibody, and expressing the coding sequence by introducing the expression vector into an animal cell.

As the CH domain of a human chimeric antibody, it can be any region that belongs to the human immunoglobulin, but those of the IgG class are suitable and any one of the subclasses belonging to the IgG class, such as IgG1, IgG2, can also be used. , IgG3 and IgG4. Also, as CL of a human chimeric antibody, it can be any region belonging to Ig, and those of the kappa or lambda class can be used.

Methods for the production of chimeric antibodies involve conventional recombinant DNA and the techniques of genetic transfection are well known in the art (Vease Morrison SL et al (1984) and patent documents US5,202,238 and US5,204,244).

The humanized antibody of the present invention can be produced by obtaining nucleic acid sequences encoding the CDR domains, as previously described, by constructing a humanized antibody expression vector by inserting them into an expression vector for the animal cell having genes that encode (i) a constant region of the heavy chain identical to that of a human antibody and (ii) a constant region of the light chain identical to that of a human antibody, and expressing the genes by introducing the expression vector into an animal cell .

The expression vector of the humanized antibody can be either of a type in which a gene encoding a heavy chain of the antibody and a gene encoding a light chain of the antibody exists in separate vectors or of a type in which both genes exist in the same vector (tandem type). Regarding the ease of construction of a humanized antibody expression vector, the ease of introduction into animal cells, and the balance between the expression levels of the H and L chains of the antibody in animal cells, the expression vector of the humanized antibody of the animal is preferred. tandem type (Shitara K et al., 1994). Examples of the expression vector of the humanized tandem antibody include pKANTEX93 (WO 97/10354), pEE18 and the like.

Methods for the production of humanized antibodies based on conventional recombinant DNA and the techniques of genetic transfection are well known in the art (See, for example, Riechmann L. et al 1988, Neuberger MS et al, 1985). The antibodies can be humanized using a variety of techniques known in the art including, for example, CDR grafting (EP 239,400, PCT Publication WO91 / 09967, US Patent Nos. 5,225,539, 5,530,101, and 5,585,089; ), coating or coating (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA., et al. (1994)), and chain exchange (US Patent No. 5,565). .332). Also known is general recombinant DNA technology for the preparation of such antibodies (see European patent application EP 125023 and international patent application WO 96/02576).

The Fab of the present invention can be obtained by treating an antibody that specifically reacts with Axl with a protease, papama. Also, Fab can be produced by inserting DNA encoding antibody Fab into a vector for the prokaryotic expression system, or for the eukaryotic expression system, and introducing the vector into a prokaryote or eukaryotic (as appropriate) to express the Fab. .

The F (ab ') 2 of the present invention can be obtained by treating an antibody that specifically reacts with Axl with a protease, pepsin. Also, F (ab ') 2 can be produced by joining the Fab' described below by a thioether bond or a disulfide bond.

The Fab 'of the present invention can be obtained by treating F (ab') 2 which specifically reacts with Axl with a reducing agent, dithiothreitol. Also, Fab 'can be produced by inserting DNA encoding the Fab' fragment of the antibody into an expression vector for prokaryote, or an expression vector for eukaryote, and introducing the vector into a prokaryotic or eukaryotic (as appropriate) to carry out your expression.

The scFv of the present invention can be produced by obtaining cDNA encoding the VH and VL domains as previously described, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote, or an expression vector for eukaryote and , then, introducing the expression vector into a prokaryote or eukaryote (as appropriate) to express the scFv. To generate a humanized scFv fragment, a well known technology called CDR grafting can be used, which involves selecting the complementarity determining regions (CDRs) from a donor scFv fragment, and grafting them onto a structural region of the human scFv fragment. of known three-dimensional structure (see, for example, WO98 / 45322; WO 87/02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).

Modification (s) of amino acid sequence of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. It is known that when a humanized antibody is produced by simply grafting only the CDRs in VH and VL of an antibody derived from a non-human animal in the FRs of the VH and VL of a human antibody, the antigen binding activity is reduced in comparison with that of the original antibody derived from a non-human animal. It is considered that various amino acid residues of the VH and VL of the non-human antibody, not only in CDR but in FR, are directly or indirectly associated with the antigen binding activity. Therefore, the substitution of these amino acid residues with different amino acid residues derived from the FRs of the VH and VL of the human antibody reduces the binding activity. To solve the problem, in the human CDR-grafted antibodies, attempts have to be made to identify, between the amino acid sequences of the VH and VL FR of the human antibodies, an amino acid residue which is directly associated with the binding to the antibody, or which interacts with a CDR amino acid residue, or which retains the three-dimensional structure of the antibody and which is directly associated with binding to the antigen. The reduced antigen binding activity could be increased by replacing the amino acids identified with the amino acid residues of the original antibody derived from a non-human animal.

Modifications and changes can be made in the structure of the antibodies of the present invention, and in the DNA sequences encoding them, and still obtain a functional molecule encoding an antibody with the desirable characteristics.

In the realization of the changes in the amino sequences, the hydropathic index of the amino acids can be considered. The importance of the hydropathic molecule of the amino acids in granting the interactive biological function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acids contributes to the secondary structure of the resulting protein, which gradually defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens and Similar. Each amino acid has been assigned a hydropathic index according to its hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valina (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); Alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). A further objective of the present invention also encompasses the conservative variants of the function of the antibodies of the present invention.

"Conservative variants of function" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the general conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one which has similar properties (such as, for example, polarity, hydrogen bonding potential, acids, basics, hydrophobic, aromatic, and the like). Amino acids other than those indicated as preserved may differ in a protein such that the percentage of protein similarity or sequence of amino acids between any of the two proteins of similar function may vary and may be, for example, from 70% to 99% determined according to an alignment scheme such as by the grouping method, where the similarity is based on the MEGALIGN algorithm. A "conservative variant of the function" also includes a polypeptide having at least 60% amino acid identity determined by the BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, and having the same properties or functions or basically similar as the native or parental protein with which it is compared.

Two amino acid sequences are "basically homologous" or "basically similar" when more than 80%, preferably more than 85%, preferably more than 90% of the amino acids are identical, or more than about 90%, preferably more than 95%, they are similar (functionally identical) throughout the length of the shortest sequence. Preferably, similar or homologous sequences are identified by alignment using, for example, the GCG "pileup" program (Genetics Computer Group, program manual for the GCG package, Version 7, Madison, Wisconsin), or any of the algorithms of sequence comparison such as BLAST, FASTA, etc.

For example, certain amino acids may be substituted with other amino acids in a protein structure without appreciable loss of activity. Since the interactive ability and the nature of a protein define the biological functional activity of the protein, certain amino acid substitutions can be made in a protein sequence, and, of course, in its DNA coding sequence, while still obtaining a protein with similar properties. Therefore, it is contemplated that various changes may be made in the antibody sequences of the invention, or the corresponding DNA sequences encoding said antibodies, without appreciable loss of their biological activity.

It is known in the art that certain amino acids can be substituted with other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a functionally equivalent biological protein.

As outlined above, amino acid substitutions, therefore, are generally based on the relative similarity of amino acid side chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size and the like. Illustrative substitutions which take various of the above characteristics into consideration are well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

Accordingly, the invention also provides an antibody comprising a heavy chain wherein the variable domain comprises:

- an H-CDR1 having at least 90% or 95% identity with the sequence set forth as SEQ ID NO: 2, - an H-CDR2 having at least 90% or 95% identity with the sequence set forth as SEQ ID NO: 3, - an H-CDR3 having at least 90% or 95% identity with the sequence set forth as SEQ ID NO: 4, - an L-CDR1 having at least 90% or 95% identity with the sequence set forth as SEQ ID NO: 6, - an L-CDR2 having at least 90% or 95% identity with the sequence set forth as SEQ ID NO: 7, - an L-CDR3 having at least 90% or 95% identity with the sequence set forth as SEQ ID NO: 8, and - specifically binding to Axl with basically the same affinity as an antibody comprising a heavy chain wherein the variable domain comprises SEQ ID NO: 2 for H-CDR1, the ^s E ^q ID NO: 3 for H-CDR2 and SEQ ID NO: 4 for HCDR3 and a light chain wherein the variable domain comprises SEQ ID NO: 6 for L-CDR1, SEQ ID NO: 7 for LCDR2 and SEQ ID NO: 8 for L-CDR3, and more p basically with the same affinity as the murine anti-Axl antibody 20G7-D9.

Accordingly, the invention also provides an antibody that binds immunoglobulin-like domain 2, FN3 domain 1 and FN3 domain 2 of the extracellular part of Axl (amino acid sequences of Axl SEQ ID NO: 9, SEQ ID NO. : 10 and SEQ ID NO: 11).

Said antibodies can be analyzed for the specific binding by any method known in the art. Many different competitive binding test format (s) can be used for pre- sorting (binning) of epftopo. Immunoassays that can be used include, but are not limited to, competitive assay systems that use techniques such as Western blots, radioimmunoassays, ELISA, sandwich immunoassays, immunoprecipitation assays, precipitin assays, diffusion precipitin assays, gel, immunoradiometric assays, fluorescent immunoassays, immunoassays with proterne A, and complementation fixation assays Such assays are routine and well known in the art (see, for example, Ausubel et al., eds, 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & sons, Inc., New York). For example, BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats that are used routinely for pre-sorting epitope panels of monoclonal antibodies. In addition, routine cross-block assays such as those described in "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988 can be performed.

The engineered antibodies of the invention include those in which modifications have been made to residues of the structural region within VH and / or VL, for example, to improve the properties of the antibody. Generally such modifications of the structural region are made to decrease the immunogenicity of the antibody. For example, one approach is to "retromue" one or more remains of the structural region to the corresponding germ line sequence. More specifically, an antibody that has undergone somatic mutation may contain residues of the structural region that differ from the germ line sequence from which the antibody is derived. Such residues can be identified by comparing the sequences of the structural region of the antibody with the sequences of the germ line from which the antibody is derived. In order to return the sequences of the structural region to their germline configuration, somatic mutations can be "retromutated" to the germ line sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such "retromutated" antibodies also claim to be encompassed by the invention. Another type of structural region modification involves the mutation of one or more residues within the structural region, or even within one or more CDR regions, to separate epitopes from the T lymphocyte to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in more detail in U.S. Patent Publication No. 20030153043 of Carr et al.

In addition to or alternatively to modifications made within the structural or CDR regions, the antibodies of the invention can be engineered to include modifications within the Fc region, generally to alter one or more functional properties of the antibody, such as half-life of the antibody. serum, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In addition, an antibody of the invention can be modified chemically (for example, one or more chemical residues can be bound to the antibody) or can be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments are described in more detail below. The numbering of the remains in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CHI is modified so that the number of cysteine residues in the hinge region is altered, for example, increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 to Bodmer et al. The number of cysteine residues in the hinge region of CHI is altered, for example, to facilitate the assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the interface region of the CH2-CH3 domain of the Fc hinge fragment so that the antibody has altered the binding of the A staphylococcal (SpA) protein in relation to the SpA domain binding hinge of Fc. This approach is described in more detail in U.S. Patent No. 6,165,745 to Ward et al.

In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254s, T256F, as described in US Pat. No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CL or CHI region to contain a wild-type receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to Presta et al.

In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the ability to bind to the antigen of the parent antibody. The effector ligand to which the affinity is altered can be, for example, an Fc receptor or the C1 component of the complement. This approach is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both to Winter et al.

In another embodiment, one or more amino acids selected from the amino acid moieties may be replaced with a different amino acid moiety such that the antibody has altered C1q binding and / or reduced or suppressed complement-dependent cytotoxicity (CDC). This approach is described in more detail in the U.S. Patent No. 6,194,551 to Idusogie et al.

In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to bind to the complemented This approach is further described in PCT publication WO 94/29351 of Bodmer et al.

In still another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fc receptor by modifying one or more amino acids. This approach is described more in PCT publication WO 00/42072 of Presta. In addition, binding sites on human IgGI for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with enhanced binding have been described (see Shields, RL et al., 2001 J. Biol. Chen. 276: 6,591.6604, WO2010106180).

In yet another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be produced (ie, the antibody lacks glycosylation). The glycosylation can be altered, for example, to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made which result in the removal of one or more glycosylation sites from the variable structural region to thereby eliminate glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such an approach is described in more detail in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

In addition or alternatively, an antibody having an altered type of glycosylation, such as a hypofucosylated or non-fucosylated antibody having reduced amounts of or not having fucosyl residues or an antibody having increased bisection GIcNac structures can be produced. It has been shown that such altered glycosylation patterns increase the ADCC capacity of the antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with the altered glycosylation machinery. Cells with the altered glycosylation machinery have been described in the art and can be used as host cells in which recombinant antibodies of the invention are expressed to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 to Hang et al. describes a cell line with a functionally interrupted FUT8 gene, which encodes a fucosyl transferase, so that the antibodies expressed in such a cell line present hypofucosylation or are devoid of fucosyl residues. Therefore, in one embodiment, the antibodies of the invention can be produced by recombinant expression in a cell line which has a hypofucosylation or non-fucosylation pattern, for example, a maternal cell line with deficient expression of the FUT8 gene encoding fucosyltransferase. PCT Publication WO 03/035835 to Presta discloses a variant CHO cell line, Lecl3 cells, with reduced capacity to bind fucose to Asn-linked carbohydrates (297), also resulting in the hypophosphorylation of the antibodies expressed in that host cell (see , also, Shields, RL et al., 2002 J. Biol. Chem. 277: 26,733 26,740). PCT publication WO 99/54342 of Umana et al. describes engineered cell lines for expressing glycoprotein-modifying glycosyltransferases (eg, beta (1,4) -N-acetylglucosaminyltransferase III (GnTIII)) so that antibodies expressed in engineered cell lines have increased bisection GlcNac structures which result in increased ADC ^c activity of the antibodies (see also Umana et al., 1999 Nat. Biotech 17: 176-180). Eureka Therapeutics also describes genetically modified CHO maize cells capable of producing antibodies with glycosylation pattern of altered maize devoid of fucosyl residues (http://www.eurekainc.com/a&boutus/companyoverview.html). Alternatively, the antibodies of the invention can be produced in yeast or filamentous fungi modified by engineering for the maize type glycosylation pattern and capable of producing antibodies lacking fucose as the glycosylation pattern (see, for example, EP1297172B1) .

Another modification of the antibodies in the present document contemplated by the invention is pegylation. An antibody can be subjected to pegylation to, for example, increase the biological half-life (eg, serum) of the antibody. To subject an antibody to pegylation, the antibody, or fragment thereof, is generally reacted with polyethylene glycol (PEG), such as a reactive ester or PEG aldehyde derivative, under conditions in which one or more PEG groups become available. bound to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the PEG forms that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol -maleimida. In certain embodiments, the antibody to be subjected to pegylation is an anglicosylated antibody. Methods for protein pegylation are known in the art and can be applied to the antibodies of the invention. See, for example, EP O 154316 of Nishimura et al. and EP 0401 384 of Ishikawa et al.

Another modification of the antibodies contemplated by the invention is a protein conjugate or fusion of at least the antigen binding region of the antibody of the invention with whey protein, such as human serum albumin or a fragment thereof for increase the half-life of the resulting molecule. Such This approach is described, for example, in Ballance et al. EP0322094.

Another possibility is a fusion of at least the antigen binding region of the antibody of the invention with proteins capable of binding to serum proteins, such as human serum albumin to increase the half-life of the resulting molecule. Such an approach is described, for example, in Nygren et al., EP 0486525.

Immunoconjugates:

An antibody of the invention can be conjugated with a detectable label to form an anti-Axl immunoconjugate. Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzymatic label, a bioluminescent label, or colloidal gold. The methods of production and detection of such detectably labeled immunoconjugates are well known to those skilled in the art, and are described below in more detail.

The detectable marker can be a radioisotope that is detected by autoradiography. The isotopes that are particularly useful for the purpose of the present invention are 3 H, 125 I, 131 I, 35 S and 14 C.

The anti-Axl immunoconjugates can also be labeled with a fluorescent compound. The presence of a fluorescently labeled antibody is determined by exposing the immunoconjugate to light of the appropriate wavelength and detecting the resulting fluorescence. The fluorescent labeling compounds include fluorescema isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldelddo and fluorescamine.

Alternatively, the anti-Axl immunoconjugates can be detectably labeled by coupling an antibody to a chemiluminescent compound. The presence of the immunoconjugate with chemiluminescent label is determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label the anti-Axl immunoconjugates of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.

Alternatively, anti-Axl immunoconjugates can be detectably labeled by binding an anti-Axl monoclonal antibody to an enzyme. When the anti-Axl-enzyme conjugate is incubated in the presence of the appropriate substrate, the enzyme moiety reacts with the substrate to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric or visual means. Examples of enzymes that can be used to detectably label polypeptide immunoconjugates include p-galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.

Those skilled in the art will know other suitable markers that can be employed in accordance with the present invention. The binding of the marker moieties to anti-Axl monoclonal antibodies can be achieved using standard techniques known in the art. The typical methodology in this respect is described by Kennedy et al., Clin. Chim. Acta 70: 1, 1976; Schurs et al., Clin. Chim. Acta 81: 1, 1977; Shih et al., Int'l J. Cancer 46: 1,101, 1990; Stein et al., Cancer Res. 50: 1330, 1990; and Coligan, supra.

In addition, the convenience and versatility of immunochemical screening can be increased by using anti-Axl monoclonal antibodies that have been conjugated to avidin, streptavidin and biotin (see, for example, Wilchek et al (eds.), "Avidin-Biotin Technology. , "Methods In Enzymology (Vol. 184) (Academic Press 1990); Bayer et al.," Immunochemical Applications of Avidin-Biotin Technology, "in Methods in Molecular Biology (Vol. 10) 149-162 (Manson, ed., The Humana Press, Inc. 1992).)

Methods for carrying out immunoassays are well established (see, for example, Cook and Self, "Monoclonal Antibodies in Diagnostic Immunoassays," in Monoclonal Antibodies: Production, Engineering, and Clinical Application 180-208 (Ritter and Ladyman, eds., Cambridge University Press 1995), Perry, "The Role of Monoclonal Antibodies in the Advancement of Immunoassay Technology," in Monoclonal Antibodies: Principles and Applications 107-120 (Birch and Lennox, eds., Wiley-Liss, Inc. 1995); , Immunoassay (Academic Press, Inc. 1996).)

In another aspect, the present invention provides an anti-Axl-drug monoclonal antibody conjugate. A "conjugated anti-Axl-drug monoclonal antibody" as described herein refers to an anti-Axl monoclonal antibody according to the invention conjugated to a therapeutic agent. Such conjugates anti-Axl-drug monoclonal antibody produce clinically beneficial effects on cells expressing Axl when administered to a subject, such as, for example, a subject with a cancer expressing Axl, generally when administered alone but also in combination. with other therapeutic agents.

In typical embodiments, an anti-AxI monoclonal antibody is conjugated to a cytotoxic agent, so that the resulting antibody-drug conjugate exerts a cytotoxic or cytostatic effect on a cell expressing Axl (eg, a cancer cell expressing Axl) when it is absorbed or internalized by the cell. Particularly suitable remnants for conjugation with antibodies are chemotherapeutic agents, prodrug converting enzymes, radioactive isotopes or compounds, or toxins. For example, an anti-Axl monoclonal antibody can be conjugated to a cytotoxic agent such as a chemotherapeutic agent or a toxin (eg, a cytostatic or cytocidal agent such as, for example, abrin, ricin A, pseudomonas exotoxin, or toxin). of diphtheria.

Useful classes of cytotoxic agents include, for example, anti-tubulin agents, auristatins, minor groove DNA linkers, inhibitors of DNA replication, alkylating agents (eg, platinum complexes such as cis-platinum, mono (platinum), bis (platinum) and complexes of platinum trinuclear and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, sensitizers of chemotherapy, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinum, preformation compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.

Individual cytotoxic agents include, for example, an androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, butionine sulfoximine, captothecin, carboplatin, carmustine (BSNU), CC-1065 (Li et al., Cancer Res. 42: 999-1,004, 1982), chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, docetaxel, doxorubicin, an estrogen, 5-fluorodeoxyuridine , etoposide phosphate (VP-16), 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mitramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel , plicamycin, procarbizine, streptozotocin, tenoposide (VM-26), 6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine and vinorelbine.

Particularly suitable cytotoxic agents include, for example, dolastatins (e.g., auristatin E, AFP, MMAF, MMAE), minor groove DNA linkers (e.g., enediins and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel). ), puromycins, vinca alkaloids, c-1065, SN-38 (7-ethyl-10-hydroxycamptothein), topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, equinomycin, combretastatin, netropsin, epothilone A and B, estramustine , cryptophysins, cemadotine, maytansinoids, discodermolide, eleuterobina, and mitoxantrone.

In certain embodiments, a cytotoxic agent is a conventional chemotherapeutic compound such as, for example, doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C or etoposide. In addition, potent agents such as CC-1065 analogs, calicheamicin, maytansine, dolastatin 10 analogs, rhizoxin, and palitoxin may be linked to an anti-Axl expressing antibody.

In specific variations, the cytotoxic or cytostatic agent is auristatin E (also known in the art as dolastatin-10) or a derivative thereof. Generally, the auristatin derivative E is, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with para-acetylbenzoic acid or bazoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatin derivatives include AFP (dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediamine), MMAF (dovaline-valine-dolaisoleunin-dolaproine-phenylalanine), and MAE (monomethyl auristatin E). The synthesis and structure of auristatin E and its derivatives are described in the publication of the American patent application No. 20030083263; International Patent Publications No. WO 2002/088172 and WO 2004/010957; and U.S. Patent Nos. 6,884,869; 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414.

In other variations, the cytotoxic agent is a minor DNA groove binding agent (see, for example, US Pat. No. 6,130,237). For example, in certain embodiments, the minor groove binding agent is a CBI compound. In other embodiments, the minor groove binding agent is an enedin (eg, calicheamicin).

In certain embodiments, an antibody-drug conjugate comprises an anti-tubulin agent. Examples of anti-tubulin agents include, for example, taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), and dolastatins (for example, auristatin E, AFP, MMAF, m Ma E, AEB, AEVB). Other anti-tubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimide, estramustine, cryptophysins, cemadotine, maytansinoids, combretastatins, discodermolide, and eleuterobine. In some embodiments, the cytotoxic agent is a maytansinoid, another group of anti-tubulin agents. For example, in specific embodiments, maytansinoid is maytansine or DM-1 (ImmunoGen, Inc., see also Chari et al., Cancer Res. 52: 127-131, 1992).

In other embodiments, the cytotoxic agent is an antimetabolite. The antimetabolite can be, for example, a purine antagonist (eg, azothioprine or mycophenolate mofetil), an inhibitor of dihydrofolate reductase (eg, methotrexate), acyclovir, gangciclovir, zidovudine, vidarabine, ribavarin, azidothymidine, cytosine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine, postcarnet, or trifluridine.

In other embodiments, an anti-Axl monoclonal antibody is conjugated to a prodrug-converting enzyme. The profarmaco converting enzyme can be recombinantly fused with the antibody or chemically conjugated thereto using known methods. Exemplary prodrug converting enzymes are carboxypeptidase G2, p-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, p-lactamase, p-glucosidase, nitroreductase and carboxypeptidase A.

Techniques for conjugating the therapeutic agents with the proteins, and in particular with the antibodies, are well known (see, for example, Arnon et al., "Monoclonal Antibodies for Immunotherapy of Drugs in Cancer Therapy," in Monoclonal Antibodies and Cancer Therapy (Reisfeld et al., Alan R. Liss, Inc., 1985), Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (Robinson et al., Marcel Deiker, Inc., 2 ° ed.

1987); Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological and Clinical Applications (Pinchera et al., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy," in Monoclonal Antibodies for Cancer Detection and Therapy (Baldwin et al., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62: 119-58. See also, PCT publication WO 89/12624.)

Diagnostic uses:

A further objective of the invention relates to an anti-Axl antibody of the invention for diagnosing and / or monitoring a cancerous disease and other diseases in which the Axl levels are modified (increase or decrease).

In a preferred embodiment, the antibodies of the invention can be labeled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or other markers, any known in the art described above. For example, an antibody of the invention can be labeled with a radioactive molecule by any method known in the art. For example, radioactive molecules include, but are not limited to, radioactive atom for scintigraphic studies such as 1123, 1124, In111, Re186, Re188. The antibodies of the invention can also be labeled with an espm marker for nuclear magnetic resonance imaging (NMR) (also known as magnetic resonance imaging, irm), such as iodine-123, iodine-131, indium-111, fluor-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. After the administration of the antibody, the distribution of the antibody within the patient can be detected. Methods for detecting the distribution of any specific marker are known to those skilled in the art and any suitable method can be used. Some non-limiting examples include computerized tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescence, and sonography.

The antibodies of the invention can be useful for diagnosing and determining the phase of cancer diseases associated with Axl overexpression (eg, in radioimaging). Cancer diseases associated with Axl overexpression generally include but are not limited to squamous cell cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, tissue cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, kidney cancer, cancer prostata, vulval cancer, thyroid cancer, hepatic carcinoma, sarcomas, hematologic cancers (leukemias), astrocytomas, and various types of head and neck cancer or other hyperproliferative diseases that express or overexpress Axl. The antibodies of the invention may be useful for diagnosing diseases other than cancers for which the expression of Axl (soluble or cellular form of Axl) is increased or decreased.

Generally, said diagnostic methods involve the use of a biological sample obtained from the patient. As used herein, the term "biological sample" encompasses a variety of sample types obtained from a subject and can be used in a diagnostic or follow-up assay. Biological samples include but are not limited to samples of blood and other fluid of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and progeny thereof. For example, biological samples include cells obtained from a tissue sample collected from an individual suspected of having a cancer disease associated with Axl overexpression, and in a preferred embodiment of glioma, gastric, lung, pancreatic, breast, prostate , renal, hepatic and endometrial. Therefore, the biological samples include clinical samples, cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid and tissue samples.

In a particular embodiment, the invention is a method of diagnosing a cancerous disease associated with overexpression of Axl in a subject by detecting Axl on cells of the subject using the antibody of the invention. In particular, said method of diagnosis may comprise the steps consisting of:

(a) contacting a biological sample of a subject likely to suffer from a cancerous disease associated with overexpression of Axl with an antibody according to the invention under conditions sufficient for the antibody to complex with the cells of the biological sample expressing Axl;

(b) detecting and / or quantifying said complexes, thereby detecting said complexes is indicative of a cancerous disease associated with overexpression of Axl.

To monitor the cancer disease, the diagnostic method according to the invention can be repeated at different time intervals, to determine if the binding of the antibody to the samples increases or decreases, thereby determining if the cancer disease progresses or backs up

In a particular embodiment, the invention is a method of diagnosing a disease associated with the expression or overexpression of Axl or the decrease or increase of the soluble form of Axl, such as human immune disorders, thrombotic diseases (thrombosis and atherothrombosis), and cardiovascular diseases can also be diagnosed by the anti-Axl antibody of the invention.

Therapeutic uses:

The antibodies, fragments or immunoconjugates of the invention can be useful for treating any disease associated with the expression of Axl, preferably cancers. The antibodies of the invention can be used alone or in combination with any suitable agent.

1) The anti-Axl antibody of the description can be used as a treatment of hyperproliferative diseases associated with the expression, overexpression or activation of Axl and Gas6. There are no particular limitations on tumor tissues, and examples include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cancer cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial carcinoma, carcinoma of the salivary gland, kidney cancer, kidney cancer, prostate cancer, cancer vulval, thyroid cancer, hepatic carcinoma, sarcomas, hematologic cancers (leukemias), astrocytomas, and various types of head and neck cancer. More preferable cancers are glioma, gastric, lung, pancreatic, breast, prostate, renal, hepatic and endometrial.

2) The anti-Axl antibody of the description are potential activators of the innate immune response and can be used in the treatment of human immune diseases, such as sepsis, can be used as adjuvants for immunization such as for vaccine and can be used as anti-infective agents (against bacteria, viruses, parasites).

3) The anti-Axl antibody of the invention can protect or treat thrombotic diseases such as venous and arterial thrombosis and atherothrombosis.

4) The anti-Axl antibody of the description can protect, prevent or treat cardiovascular diseases.

5) The anti-Axl antibody of the disclosure can prevent or inhibit the entry of viruses such as Lassa and Ebola viruses and can be used to treat viral infections.

In each of the embodiments of the treatment methods described herein, the anti-Axl monoclonal antibody or the anti-Axl-drug monoclonal antibody conjugate is released in a manner consistent with conventional methodologies associated with the management of the disease. or disorder for which treatment is sought. According to the description herein, an effective amount of the antibody or antibody-drug conjugate is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.

Therefore, an objective of the disclosure relates to a method for treating a disease associated with the expression of Axl which comprises administering a subject in need thereof with a therapeutically effective amount of an antibody, fragment or immunoconjugate of the invention.

In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term is applied, one or more symptoms of such disorder or condition.

According to the invention, the term "patient" or "patient in need thereof" is intended for an affected or probable human affected with disease associated with the over expression of Axl.

By a "therapeutically effective amount" of the antibody of the invention is meant a sufficient amount of the antibody to treat said cancer, at a reasonable benefit / risk ratio applicable to any medical treatment. However, it will be understood that the total daily use of the antibodies and the compositions herein invention will be decided by the attending physician within the scope of the medical criteria. The therapeutically effective dose level is specific for any particular patient will depend on a variety of factors including the disorder to be treated and the severity of the disorder; the activity of the specific antibody used; the specific composition used, age, body weight, general health, sex and diet of the patient; the administration time, the route of administration and the rate of excretion of the specific antibody used; the duration of the treatment; the drugs used in combination or simultaneous with the specific antibody used; and similar factors well known in the medical arts. For example, it is well known in the art to begin the dose of the compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

In certain embodiments, an anti-Axl monoclonal antibody or antibody-drug conjugate is used in combination with a second agent for the treatment of a disease or disorder. When used to treat cancer, an anti-Axl monoclonal antibody or antibody-drug conjugate of the present invention can be used in combination with conventional cancer therapies such as, for example, surgery, radiotherapy, chemotherapy or combinations thereof. In certain aspects, other therapeutic agents useful for the therapy of cancer in combination with an anti-Axl antibody or antibody-drug conjugate according to the present invention include anti-angiogenic agents. In some aspects, an antibody or antibody-drug conjugate according to the present invention is co-administered with a cytokine (eg, a cytokine that stimulates an immune response to a tumor).

In some embodiments, an anti-Axl monoclonal antibody or antibody-drug conjugate as described herein is used in combination with a tyrosine kinase inhibitor (TKI).

In some embodiments, an anti-Axl monoclonal antibody or antibody-drug conjugate as described herein is used in combination with another therapeutic monoclonal antibody (mAb). Trastuzumab (Herceptin, Roche), Bevacizumab (Avastin, Roche) and Cetuximab (Erbitux, Merck) are three such mAbs that have been approved. Another mAb includes, but is not limited to: Infliximab (Remicade, Johnson & Johnson), Rituximab (Rituxan, Roche), Adalimumab (Humira, Abbott) and Natalizumab (Tysabri, Biogen).

Pharmaceutical compositions:

For administration, the anti-Axl monoclonal antibody or antibody-drug conjugate is formulated as a pharmaceutical composition. A pharmaceutical composition comprising an anti-Axl monoclonal antibody or antibody-drug conjugate can be formulated according to known methods to prepare pharmaceutically useful compositions, thereby the therapeutic molecule is combined in a mixture with a pharmaceutically acceptable vedculo. A composition is said to be a "pharmaceutically acceptable vedculo" if its administration can be tolerated by a recipient patient. The sterile phosphate buffered saline solution is an example of a pharmaceutically acceptable vedculo. Other suitable vedicles are well known to those skilled in the art (See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed., 1995)). The formulations may also include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent the loss of protein on the living surfaces, etc. The form of the pharmaceutical compositions, the route of administration, the dose and the regimen naturally depends on the condition to be treated, the severity of the disease, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.

Preferably, the pharmaceutical compositions contain vedicles that are pharmaceutically acceptable for a formulation capable of being injected. These can be, in particular, isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry compositions, especially freeze-dried, which after addition, depending on the case, of sterilized water or physiological saline solution, allow the constitution of injectable solutions.

The doses used for administration can be adapted according to various parameters, and in particular depending on the mode of administration used, on the relevant pathology, or alternatively on the desired duration of treatment.

To prepare the pharmaceutical compositions, an effective amount of the antibody can be dissolved or dispersed in a pharmaceutically acceptable vedculo or aqueous medium.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations that include sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the improvised preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the point that there is the ability to be easily injected by syringe. It must be stable under the conditions of manufacture and storage and must be protected against the action contaminant of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. The dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

An antibody of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric , mandelico and similar. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.

The vehicle can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the particle size required in the case of dispersion and by the use of surfactants. The prevention of the action of the microorganisms can be caused by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. The prolonged absorption of the injectable compositions can be caused by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients listed above, as required, followed by filtered sterilization. In general, the dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the other ingredients required from those enumerated above. In the case of sterile powders for the preparation of the sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization techniques which produce a powder of the active ingredient plus any additional desired ingredient of a previously sterile filtered solution thereof.

Also contemplated is the preparation of more, or highly concentrated solutions for direct injection, where the use of DMSO as a solvent is expected to result in extremely rapid penetration, release of high concentrations of the active agents to a small tumor area.

After the formulation, the solutions will be administered in a manner compatible with the pharmaceutical form and in such amount that it is therapeutically effective. The formulations are easily administered in a variety of pharmaceutical forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be adequately buffered if necessary and the liquid diluent first becomes isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous media that can be employed will be known to those skilled in the art in view of the present disclosure. For example, a dose could be dissolved in 1 ml of isotonic NaCl solution and either added to 1,000 ml of hypodermoclysis fluid or injected into the proposed site of the infusion (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1,035-1,038 and 1,570-1,580). Some variation in the dose will necessarily occur depending on the affection of the subject to be treated. The person responsible for the administration will determine, in any case, the appropriate dose for the individual.

The antibodies of the invention can be formulated into a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose. more or less. Multiple doses can also be administered. In addition to compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; the prolonged release capsules; and another form currently used.

In certain embodiments, the use of liposomes and / or nanoparticles for the introduction of antibodies into the host cells is contemplated. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art.

Nanocapsules can generally trap compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymeric overload, such ultrafine particles (size around 0.1 μm) are generally designed using polymers capable of degrading in vivo. Biodegradable polyalkyl cyanoacrylate nanoparticles are contemplated which meet these requirements for use in the present invention, and such particles can be easily produced.

Liposomes are formed from phosphoKids that are dispersed in an aqueous medium and spontaneously form multilamellar bilayer concentric vesicles (also called multilamellar vesicles (MLV)). MLV in general have diameters from 25 nm to 4 pm. The sonication of MLV results in the formation of small unilamellar vesicles (SUV) with diameters in the range of 200 to 500 A, containing an aqueous solution in the center. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

Kits:

Finally, the invention also provides kits comprising at least one antibody of the invention. The kits containing antibodies of the invention find use in the detection of Axl expression (increase or decrease), or in therapeutic or diagnostic assays. The kits of the invention may contain an antibody coupled to a solid support, for example, a tissue culture plate or beads (eg sepharose beads). Antibody-containing kits can be provided for the detection and quantification of Axl in vitro, for example, in an ELISA or a Western blot. Such a useful antibody can be provided for detection with a label such as a fluorescent or a radiolabel.

The invention will also be illustrated by the following figures and examples. However, these examples and figures are not to be construed in any way as limiting the scope of the present invention.

Legends of the figure

Figure 1: ELISA experiments to investigate the affinity and specificity of mouse monoclonal antibodies against hAxl. Plates coated with human Axl-Fc (h-Axl), Axl-Fc mouse (m-Axl) or Mer-Fc (h-Mer) human, Tyro-3-Fc (h-Tyro-3) with antibodies were incubated monoclonal anti-Axl (mAbl, mAb2, mAb3, mAb4 or 20G7-D9). After washing, anti-mouse IgG conjugated with HRP was added. 20G7-D9 does not cross-react with h-Tyro-3 or h-Mer or m-Axl.

Figure 2: Analysis of flow cytometry of cell surface Axl in A549. A549 was stained with monoclonal anti-Axl antibodies (mAbl, mAb2, mAb3, mAb4 or 20G7-D9) and fluorescein-conjugated anti-mouse IgG. Staining with 20G7-D9 results in a displacement by an order of magnitude and demonstrates the overexpression of Axl on the surface of these cells.

Figure 3: Measurement of the affinity of 20G7-D9 in the presence or absence of Gas6 using BIAcore. (A) Without Gas6, the association rates (ka) and the dissociation rates (kd) were calculated using a simple one-to-one Langmuir union model. The equilibrium dissociation constant (K ^d ) is derived as the ratio ka / kd. 20G7-D9 binds to human Axl with high affinity, with a Kd of approximately 53 nM. (B) 20G7-D9 does not block the binding of ligand Gas6 to Axl.

Figure 4: ELISA experiments to investigate the effects of mAb 20G7-D9 on the phosphorylation of the Axl receptor. Pancreatic cancer cells BXPC3, Capan-1, PANC1 and MIAPaCa-2 were deprived of serum, pre-incubated with anti-Axl mouse antibodies and treated with Gas6 ligand. Cell lysates were transferred to PathScan® sandwich ELISA plates Phospho-Axl (PanTyr) (RD Systems, Minneapolis, MN). In comparison with other antibodies. 20G7-D9 was able to block or significantly reduce the activation of Ax6 mediated by Gas6 in the four cell lines as indicated by the decreased levels of Axl phosphorylation in cells stimulated by Gas6.

Figure 5: Healing wound test to investigate the effects of anti-Axl mouse antibodies on migration and cell proliferation. After being grown to confluence, the A549 cells were deprived of food and injured with a pipette tip. The Mouse Anti-Axl 20G7-D9 reduced the repopulation of the cleared area more significantly than the mAbl, although the cells were treated with Gas6. Figure 6: Cell viability assay to investigate the antiproliferative efficacy of anti-Axl 20G7-D9. Capan-1, PANC1 and MIAPaCa-2 pancreatic cancer cells were grown in medium and treated at the indicated concentrations of mAbl or 20G7-D9 for 5 days. Cell viability was measured by MTS. 20G7-D9 further inhibits the growth of all cell lines tested than the mAbl and the percentage inhibition is dependent on the concentration.

Figure 7: The anti-Axl monoclonal antibody 20G7-D9 induces a rapid downregulation of the receptor of Axl and inhibits the Akt route. Cells were incubated in a panel with 100 μg / ml mAb 20G7-D9 for different time. The cells were lysed and the total protein was used to detect by western blotting. As shown in Figure 7A, mAb 20G7-D9 rapidly downregulates the expression of the Axl receptor in Pancl cells. After one hour of incubation with mAb 20G7-D9, the cells were incubated 30 minutes with Gas6 and the presence of phosphorylation of the Axl receptor on tyrosine 702 (activation of Axl) and phosphorylation of Akt on serine 473 (activation of Akt) was analyzed. ) by Western transfer. As shown in Figure 7B, incubation of mAb 20G7-D9 leads to a decrease in Gas6-induced phosphorylation of Axl and Akt proteins.

Figure 8: Xenograft models to investigate the effects of mouse anti-axi on human breast cancer triple negative and human pancreatic cancer in nude mice (nude). MDA-MB-231 (triple negative breast cancer cells) or MIAPaca-2, BXPC3 (pancreatic cancer cells) were implanted in the right side of nude nude mice. The animals received 300 jg / injection of anti-Axl mouse antibodies. During the treatment, tumor growth was monitored once a week with a calibrator. 20G7-D9 further reduced the overall growth of pancreatic and triple negative tumors in nude mice than mAbl (A, B, C) and significantly increased overall survival compared to the vehicle or Gemcitabine in mice xeroinjeted with pancreatic BXPC3 (D). On the explanted MIAPaca-2 xenograft tumors that received two injections, a drastic down expression of the Axl receptor by mAb 20G7-D9 (E) is also observed.

Figure 9: hAxl-hFc sequence and location / sequence of the epitope of the anti-Axl mAb 20G7-D9. The epitope of the anti-Axl 20G7-D9 antibody was identified by limited proteolysis assays using either Trypsin or GluC proteases and MALDI mass spectrometry analysis. The figure shows the composition of the antigen (hAxl-hFc) used in this experiment which is composed of amino acids 33 to 440 of the extracellular domain of Axl fused to the Fc part of human IgG1 and Histidine tag. Each of the immunoglobulin-like domains and the fibronectin 3 domains of the Axl protein are indicated in the sequence. mAb 20G7-D9 binds to the three peptides (conformational epitope) located in the immunoglobulin-like domain 2 and in the first and second fibronectin domains (the sequences are framed in the protein sequence and are detailed in the table).

Figure 10: Representation of an ectodomain model of human Axl and location of the epitope of the anti-Axl mouse monoclonal antibody 20G7-D9 and the Gas6 binding domain.

Figure 10A shows a vineta type representation of the complete extracellular domain model of human Axl with all four tagged domains. In Figure 10B, the fragment of amino acids 305 to 315 of Gas6 was added as a light gray p-sheet, illustrating the binding domain of Gas6 within domain 1 immunoglobulin type of Axl. Finally, Figure 10C presents the epitope 20G7-D9 within the domain 2 type immunoglobulin and the fibronectin domains type III 1 and 2 as gray surfaces. Confirm first that the three parts of the epitope are located on the outer surface of each domain. Second, Figure 10C also illustrates that the Gas6 interaction site and the epitope are located away from each other in the human Axl ectodomain.

Example

Example 1: Generation of anti-Axl mouse monoclonal antibody

The monoclonal antibodies against Axl were developed by sequential immunization of Balb / c mice. The Balb / c mice were hyperimmunized with the extracellular domain of human Axl (hAx1ECD) fused with the human Fc domain (hAxl-hFc protein, R & D system). Balb / c mice were injected subcutaneously with 10 jg of soluble hAxl-hFc on days 0, 14 and 28 in the presence of adjuvant, complete Freund (first injection) or incomplete (second and third injection). Spleen cells from the mice were fused with mouse myeloma cells (PX63.Ag8.653; ATCC, Rockville, MD) using a previously described protocol (Salhi et al., Biochem.

2004). The cells were plated (105 per well) with HAT media for hybridoma selection. After 12 days, the supernatants were collected and screened for the binding specificity of Axl (hAxl-hFc or hFc alone) by enzyme-linked immunosorbent assay (ELISA). Eight positive clones, which showed the highest immunounion after the second round of subcloning by limiting dilution, were expanded for large-scale in vitro production of mAb. The conditioned supernatants were purified by protein G affinity chromatography.

Example 2: Anti-Axl mouse monoclonal antibodies do not cross-react with mouse Axl or other members of the human TAM receptor family

Example 2.1: Anti-Axl mouse monoclonal antibodies do not cross-react with mouse Axl or other members of the human TAM receptor family as determined by ELISA

Briefly, plates coated with hAxl-hFc were saturated with bovine serum albumin (BSA) 1% PBS, 0.1% Tween 20 (PBST).

For the cross-reaction assay, plates coated with human Axl-Fc (h-AxI), Axl-Fc (m-AxI) from mouse or Mer-Fc (h-Mer) human, Tyro-3-Fc ( h-Tyro-3) for 1 hour at 37 ° C and washed four times in PBST. The plates were incubated with anti-Axl mAb (2 hours at 37 ° C) and washed four times in PBST. The plates were incubated with anti-mouse IgG conjugated with HRP (Sigma) at a dilution of 1: 2,000 in PBST, 1% BSA (1 hour at 37 ° C). Finally, an ortho-phenylenediamine solution (Sigma) was added for 30 min at room temperature in the dark and the absorbance at 450 nm was measured.

The specificity against h-Axl was demonstrated, in a manner specific to dose, of the ten selected anti-hAxlECD mAbs (Figure 1).

Example 2.2: The anti-Axl mouse monoclonal antibody binds specifically to cells expressing Axl as determined by FACS

The ability of the anti-Axl mouse monoclonal antibodies of the invention to specifically recognize Axl expressing cells was determined by FACS using standard techniques. Briefly, A549 cells (ATCC number: CCL-185) were collected, stained with purified anti-Axl mouse monoclonal antibodies of the invention at 4 ° C for 1 hour, washed three times in PBS-0.1 BSA. % and then stained with anti-mouse IgG conjugated with fluorescein (1:50) (Sigma) at 4 ° C in the dark for 45 min. Samples were analyzed in EPICS flow cytometer (Beckman-Coulter, Fullerton, CA). As shown in Figure 2, the anti-Axl mouse monoclonal antibodies of the invention specifically bound to A549 cells expressing Axl.

Example 2.3: Measurement of the affinity of the anti-Axl mouse monoclonal antibody evaluated by BIACore For the determination of the binding affinity of the anti-Axl antibodies, a resonance measurement by surface plasmons was used with a BIAcore-3000 instrument (BlACORE AB, Uppsala, Switzerland). The experiments were carried out in the facilities of the "Proteomic Imaging and Molecular Interactions" program (M. Pugniere) located in the laboratory. To measure the affinity between anti-Axl and hAxl-hFc antibodies, anti-Axl mouse monoclonal antibodies were captured by CM5 biosensing chips coated with hAxl-hFc (using an amine coupling kit (BIAcore AB)). For the measurement of the kinetics, various concentrations of anti-Axl mAb (from 2 to 133 nM) were injected in 10 mM HePeS, 150 mM NaCl, pH 7.4, 0.005% surfactant buffer P20 at 25 ° C with a flow rate of of 50 pl / min. The association rates (ka) and the dissociation rates (kd) were calculated using a single-to-one Langmuir union model (BIAcore Evaluation version 3.2 software). The equilibrium dissociation constant (K ^d ) was calculated as the ratio ka / kd. As indicated (Figure 3A), 20G7-D9 showed a K ^d of 5.3x10'8 M.

Example 3: Anti-Axl mouse monoclonal antibody does not block Gas6 binding

For the competition study, a saturating concentration of Gas6 (625 nM) was injected onto the CM5 biosensing chips with hAxl-hFc (using an amine coupling kit (BIAcore AB)). The anti-Axl mouse monoclonal antibody (666 nM) was injected without removing Gas6. The same experiment was carried out by first injecting the anti-Axl mouse monoclonal antibody (666 nM) and in second place Gas6 (625 nM). The results showed that 20G7-D9 did not compete with the ligand Gas6 for hAxlECD (Figure 3B).

Example 4: The anti-Axl mouse monoclonal antibody of the invention inhibits the ligand induced by Axl phosphorylation in vitro

ELISA experiments were performed to investigate whether the anti-Axl mouse monoclonal antibody of the invention was capable of blocking the phosphorylation of Axl induced by Ligand Gas6. In summary, cells were seeded BXPC3 (ATCC number: CRL-1687), Capan-1 (ATCC number: HTB-79), PANC1 (ATCC number: CRL-1469) and MIAPaCa-2 (ATCC number: CRL-1420) in Normal growth medium in flat bottom 6-well plates. On the following day, the growth medium was replaced by serum-free medium to deprive the cells of food overnight for 24 hours. The cells were preincubated with 100 pg / l of purified mouse monoclonal anti-Axl of the invention and then treated with or without 250 ng / ml of Gas6 incubated with Gas6 for 30 min at 37 ° C. Then, the medium was separated, the cells were lysed in 50 μl of lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1.5 mM MgCl ² , 1 mM EDTA, Triton X-100 (v / v) 0.1%, 10% glycerol (v / v), 100 mM sodium fluoride, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate (Sigma)) supplemented with phosphatase and protease inhibitors (Roche Diagnostics , Meylan, France) for 30 min. The cell debris was separated by centrifugation and the protein concentrations were determined by the Bradford colorimetric reaction. The PathScan® Phospho-Axl sandwich ELISA kit (PanTyr) (RD Systems, Minneapolis, MN) was used as described by the manufacturer for the detection of the phospho-Axl level by measuring the absorbance at 450 nm in a colorimetric assay.

As shown in Figure 4, mAb 20G7-D9 strongly inhibited the phosphorylation of Axl induced by ligand Gas6 in all cell lines of pancreatic cancer. Other mAbs did not inhibit or slightly the phosphorylation of Axl induced by Gas6 ligand in all cell lines of pancreatic cancer.

Example 5: The anti-AxI mouse monoclonal antibody of the invention inhibits cell migration

A wound healing trial was performed observing the healing process in which the cells from the edges of the artificial wound migrate towards the wound area. Cells A549 were grown to confluence or near confluence (> 90%) in 24-well plates. A wound field was created in the center of the well using a sterile pipette tip. The migratory cells are able to extend protrusions and finally invade and close the field of the wound. The cells were rinsed very gently with PBS and incubated with purified anti-Axl mouse monoclonal antibodies of the invention (100 pg / ml) with or without 100 pg / ml Gas6. The cell migration rate was determined 24 hours after the treatment using microscopic image formation. As shown in Figure 5, mAb 20G7-D9 strongly inhibited cell migration since the healing area was still visible unlike cells treated with mAbl or Gas6.

Example 6: The anti-Axl mouse monoclonal antibody of the invention inhibits cell proliferation

Pancreatic cancer cells MIAPaca-2, Capan-1 and PANC1 were seeded at 4,000 cells / well in 96-well plates and treated with anti-Axl mouse monoclonal antibodies (25, 50 or 100 pg / ml) for 5 days. Cell proliferation assays were carried out using the MST assay (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium). MST is reduced by the cells in a formazan product that is soluble in tissue culture medium. The absorbance of the formazan at 490 nm was measured using a spectrophotometer. As shown in Figure 6, mAb 20G7-D9 strongly inhibited the proliferation of pancreatic cells while a slight inhibition was observed with the other antibody specific to Axl (mAbl).

Example 7: The anti-human Axl mouse monoclonal antibody of the invention down-regulates Axl expression and inhibits the Akt pathway

To decipher the mechanism involved in the inhibition of cell migration and the phosphorylation of Axl by mAb 20G7-D9, we analyzed its direct effect on the Axl receptor and downstream signaling pathways (it has been reported that the receptor activation of Axl by ligand Gas6 induces several key signaling cascades particularly the Akt route). The down regulation of the Axl receptor and the phosphorylation of the Axl and Akt receptor were analyzed in a pancreatic cancer cell line (Panc-1 cells) treated with 20G7-D9 mAb by western blot.

Panc-1 cells, pancreatic cancer cell line, were placed in 6-well plate (1x10 6 cells per well) and incubated with 100 pg / ml of 20G7-D9 at 37 ° C. Cell lines collected at different times were lysed with buffer (150 nM NaCl, 10 mM TRIS pH 7.4, 1 mM EDTA, 1% TRITONX100) containing 2 mM finylmethylsulfonyl fluoride, 100 mM sodium fluoride, sodium orthovanadate 10 mM, and a complete protease inhibitor mixture tablet (Sigma St Louis, MO). After a resolution on 8% or 10% SDS-PAGE under reducing conditions, the proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA), which were then saturated in PBS containing Tween 20 at 0 ° C. , 1% and skimmed milk powder at 5%. The membranes were incubated overnight at 4 ° C with appropriate dilutions of human anti-Axl (R & D systems), anti-phospho (Y702) Axl or anti-phospho (S473) Akt from Cell Signaling Technology. Immunoblots were normalized using an antibody directed to GAPDH (Millipore). The membranes were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) and processed for ECL detection (Amersham) and analysis with G: BOX jChemi (Syngene).

When the cells were treated with mAb 20G7-D9, the expression of Axl decreased rapidly after 90 minutes and is almost undetectable after 24 hours (Figure 7A). An induction of the amount of phospho-Axl and phospho-Akt was observed when the cells were stimulated with Gas6. Both signals were drastically inhibited by pretreatment with mAb 20G7-D9 (Figure 7B).

Example 8: The anti-human Axl mouse monoclonal antibody of the invention reduces the in vivo growth of triple negative breast cancer and human pancreatic cancer associated with down regulation of the Axl receptor.

All in vivo experiments were performed in accordance with the French guidelines for experimental animal studies (Agreement No. C34-172-27). Female nude athemic mice of six weeks of Harlan's life were purchased. Triple-negative breast cancer cells (5x106, MDA-MB-231, ATCC number: HTB-26) or pancreatic carcinoma cells (3.5x106, BXPC3, 5x106, MIAPaCa-2) were implanted in the right side of the mice Atheistic nudes. The tumor-bearing mice were randomized into different treatment groups when the tumors reached a volume of approximately 100 mm3. Mice were treated by intraperitoneal injections with vehicle (0.9% NaCl) or anti-Axl mouse monoclonal antibodies of the invention alone at 300 pg / injection (twice a week for 4 consecutive weeks) or with gemcitabine (GEMZAR). ). The tumor volume was measured weekly with a calibrator. The results for BXPC3 were also expressed by a modified Kaplan-Meier survival curve, using the time taken by the tumor to

Claims (14)

A monoclonal antibody having specificity for Axl or an antigen-binding fragment thereof, comprising a variable region of the heavy chain comprising SEQ ID NO: 2 in the H-CDR1 region, SEQ ID NO: 3 in the H-CDR2 region and SEQ ID ⁿ O: 4 in the H-CDR3 region; and a variable region of the light chain comprising SEQ ID NO: 6 in the L-CDR1 region, SEQ ID NO: 7 in the L-CDR2 region and SEQ ID NO: 8 in the L-CDR3 region, in wherein said antibody binds to the extracellular domain of Axl in the amino acid sequence set forth as SEQ ID NO: 9, in the amino acid sequence set forth as SEQ ID NO: 10 and in the amino acid sequence set forth as SEQ ID NO: 11. 2. The monoclonal antibody or its antigen-binding fragment according to claim 1 wherein the variable region of the heavy chain of said antibody has the amino acid sequence set forth as SEQ ID NO: 1 3. The monoclonal antibody or its antigen binding fragment according to claim 1 wherein the variable region of the light chain has the amino acid sequence set forth as SEQ ID NO: 5. 4. The monoclonal antibody or its antigen binding fragment according to claims 2 and 3 wherein the variable region of the heavy chain of said antibody has the amino acid sequence set forth as SEQ ID NO: 1 and the variable region of the light chain has the amino acid sequence set forth as SEQ ID NO: 5. 5. The monoclonal antibody according to claim 4 which is a chimeric antibody, preferably a mouse / human chimeric antibody. 6. The monoclonal antibody according to claim 1 which is a humanized antibody. 7. A fragment of a monoclonal antibody according to any of claims 1 to 6 which is selected from the group consisting of Fv, Fab, F (ab ') 2, Fab', dsFv, scFv, sc (Fv) 2 and diabodies. 8. A nucleic acid sequence encoding a heavy chain and light chain of a monoclonal antibody or its antigen binding fragment according to any of claims 1 to 7. 9. A vector comprising a nucleic acid according to claim 8. 10. A host cell comprising a nucleic acid according to claim 8 or a vector according to claim 9. 11. A pharmaceutical composition comprising an antibody or its antigen binding fragment according to any of claims 1 to 7. The antibody or its antigen binding fragment according to any of claims 1 to 7 for use as a drug. The antibody or its antigen binding fragment according to any one of claims 1 to 7, for use in the treatment of cancer diseases associated with the over expression of Axl. The antibody or its antigen binding fragment according to any one of claims 1 to 7, for use in the diagnosis of cancer diseases associated with the over expression of Axl.