Humanized antibody, or an antigen-binding fragment thereof, immunoconjugate and pharmaceutical compo

专利摘要:
ANTIGEN-BINDING PROTEIN AND USE THEREOF AS A TARGETED PRODUCT FOR THE TREATMENT OF CANCER. The present invention relates to an antigen-binding protein, in particular a monoclonal antibody, capable of specifically binding the Axl protein, as well as the amino acid and nucleic acid sequences that encode said protein. In one aspect, the invention relates to an antigen-binding protein, or antigen-binding fragments, capable of specifically binding Axl and, upon inducing internalization of Axl, being internalized into the cell. The invention also comprises the use of said antigen-binding protein, as a targeting product in conjunction with other anticancer compounds, such as toxins, radioelements or drugs, as well as their use for the treatment of certain cancers.
公开号:BR112014010383B1
申请号:R112014010383-6
申请日:2012-11-05
公开日:2022-02-01
发明作者:Charlotte BEAU-LARVOR；Liliane Goetsch；Nicolas Boute
申请人:Pierre Fabre Medicament；
IPC主号:

专利说明:

[001] The present invention relates to a novel antigen-binding protein, in particular, a monoclonal antibody, capable of specifically binding the Axl protein, as well as the amino acid and nucleic acid sequences that encode the protein. In one aspect, the invention relates to a novel antigen-binding protein, or antigen-binding fragments, capable of specifically binding Axl and, by inducing internalization of Axl, being internalized into the cell. The invention also comprises the use of this antigen-binding protein as a targeting product in conjunction with other anti-cancer compounds, such as toxins, radioelements or drugs, and its use for the treatment of certain cancers.
[002] "Axl" (also referred to as "Ufo", "Ark" or "Tyro7") was cloned from patients with chronic myeloid leukemia as an oncogene that triggers transformation when overexpressed by NIH3T3 mouse. It belongs to a family of receptor tyrosine kinases (RTQs) called the TAM family (Tyro3, Axl, Mer), which includes Tyro3 (Rse, Sky, Dtk, Etk, Brt, Tif), Axl and Mer (Eyk, Nyk, Tyro- 12) [Lemke G. Nat. Rev. Immunol. (2008).8, 327-336].
[003] The human Axl protein is an 894 amino acid protein whose sequence is represented in the sequence listing as SEQ ID NO: 29. Amino acids 1-25 which correspond to the signal peptide, the human Axl protein, without the signal peptide , are represented in the sequence listing as SEQ ID NO: 30.
[004] Gas6, initially isolated as a specific growth arrest gene, is the common ligand for members of the TAM family [Varnum B.C. et al., Nature (1995).373, 623626 ]. Gas6 exhibits the highest affinity for Axl, followed by Tyro3 and finally Mer [Nagata K. et al., J. Biol. Chem. (1996). 271,30022-30027]. Gas6 consists of a Y-carboxyglutamate (Gla)-rich domain that mediates binding to phospholipid membranes, four epidermal growth factor-like domains, and two laminin G (LG)-like domains [Manfioletti G., Brancolini, C, Avanzi, G. & Schneider, C. Mol. Cell Biol. (1993).13, 4976-4985]. Like many other RTQs, ligand binding results in receptor dimerization and auto-phosphorylation of tyrosine residues (tyrosine residues 779, 821, and 866 for the Axl receptor) that serve as docking sites for a variety of intracellular signaling molecules [Linger , RM, Adv. Cancer Res. (2008).100, 35-83]. Furthermore, the Axl receptor can be activated by a ligand-independent process. This activation can occur when the Axl receptor is overexpressed.
[005] Gas6/Axl signaling has been shown to regulate various cellular processes including proliferation, adhesion, migration and cell survival in a wide variety of cells in vitro [Hafizi, S. & Dahlback, B. FEBS J. (2006) ).273, 52315244]. Furthermore, TAM receptors are involved in the control of innate immunity; they inhibit inflammatory responses to pathogens in dendritic cells (DCs) and macrophages. They also drive the phagocytosis of apoptotic cells via these immune cells and are required for the maturation and killing activity of natural killer cells (NAs) [Lemke, G. Nat. Rev. Immunol. (2008).8, 327-336].
[006] Weakly expressed in normal cells, it is seen predominantly in fibroblasts, myeloid progenitor cells, macrophages, neural tissues, cardiac and skeletal muscle, where it primarily supports cell survival. The Gas6/Axl system plays an important role in vascular biology by regulating vascular smooth muscle cell homeostasis [Korshunov, V.A., Mohan, A.M., Georger, M.A. and Berk, B.C., Circ. Res. (2006).98, 1446-1452; Korshunov, V.A., Daul, M., Massett, M.P. & Berk, B.C., Hypertension (2007).50, 1057-1062].
[007] In tumor cells, Axl plays an important role in regulating cell invasion and migration. Axl overexpression is associated not only with poor prognosis, but also with increased invasiveness of various human cancers as reported for breast, colon, esophageal, hepatocellular, gastric, glioma, lung, melanoma, osteosarcoma, ovarian cancer, prostate, rhabdomyosarcoma, renal, thyroid, and uterine endometrium [Linger, RM, Adv. Cancer Res. (2008).100, 35-83, and Verma A., Mol. Cancer Ther. (2011).10, 1763-1773, for reviews]. In breast cancer, Axl appears to be a strong effector of the epithelial-mesenchymal transition (EMT); the TEM program actively contributes to the migration and spread of cancer cells in the body [Thiery, J.P., Curr. opinion Cell Biol. (2003).15, 740-746].
[008] It has also been shown that Axl regulates angiogenesis. Indeed, silencing of Axl in endothelial cells impaired tube formation and migration [Holland, S.J. and collaborators. Cancer Res. (2005).65, 9294-9303], as well as disturbed specific angiogenic signaling pathways [Li, Y. et al. Oncogene (2009).28, 3442-3455].
[009] More recently, several studies on a range of cellular models have described the involvement of an overexpression of Axl in drug resistance phenomena. Table 1 below summarizes these studies. Table 1

[0010] Complete references cited in table 1 are as follows: - MacLeod, K. et al. Cancer Res. (2005).65, 6789-6800 - Mahadevan, D. et al. Oncogene (2007),26, 3909-3919 - Lay, J.D. and collaborators. Cancer Res. (2007).67, 3878-3887 - Hong, C.C. and collaborators. Cancer Lett. (2008).268, 314-324 - Liu, L. et al. Cancer Res. (2009).69, 6871-6878 - Keating, A.K. and collaborators. Mol. Cancer Ther. (2010).9, 1298-1307 - Ye, X. et al. Oncogene (2010).29, 5254-5264
[0011] In this context, Axl RTQ is considered as a target of interest in oncology. Several groups have already developed antitumor strategies targeting the gas6/Axl axis, using naked monoclonal antibodies or small target molecules [Verma, A. Mol. Cancer Ther. (2011).10, 1763-1773].
[0012] In a first embodiment, the invention relates to an antigen-binding protein, or a fragment of such a protein which binds to antigen, which: i) specifically binds to the human protein Axl, and ii) it is internalized after its binding to the human protein Axl.
[0013] More generally, the invention relates to the use of the Axl protein for the selection of an antigen-binding protein, or an antigen-binding fragment thereof, capable of being internalized after its binding to the Axl target. More particularly, the target is the extracellular domain of Axl.
[0014] In this particular aspect, the present invention is thus directed to an in vitro method for screening a compound, or a binding fragment of such a compound, capable of transporting or internalizing a molecule of interest in mammalian cells, that molecule of interest by covalently binding to the compound, wherein the method comprises the following steps: selecting a compound that is capable of specifically binding to the Axl protein, or to the extracellular domain (ECD) thereof, or to an epitope thereof ; optionally, covalently linking to the molecule of interest, or a control molecule, in the compound selected in step a) to form a complex; contacting the compound selected in step a), or the complex obtained in step b), with a mammalian cell, preferably viable cells, which expresses the Axl protein on its surface, or a functional fragment of these proteins; determining whether the compound, or molecule of interest or complex, has been transported or internalized intracellularly in the mammalian cell; and selecting the compound as a compound capable of transporting or internalizing a molecule of interest in a viable mammalian cell.
[0015] In a preferred embodiment, the compound capable of transporting or internalizing a molecule of interest into a viable mammalian cell is a protein (also referred to herein as a polypeptide or peptide) or a protein-like compound comprising a peptide backbone, particularly an amino acid sequence of at least 5, 10, 15 or more amino acid residues; the amino acid residue(s) may be glycosylated.
[0016] When the compound capable of transporting or internalizing a molecule of interest into a viable mammalian cell is a protein or a protein-like compound, the compound is also referred to herein as an "antigen-binding protein"; the antigen-binding protein, or an antigen-binding fragment thereof, can: - i) specifically bind to the Axl protein, in preference to the human Axl protein, and - ii) be internalized into a mammalian cell after its binding to the Axl protein, when the Axl protein is expressed on the surface of the mammalian cell.
[0017] In a preferred embodiment, the viable mammalian cell is a human cell, preferably a cell that naturally expresses the Axl protein receptor.
[0018] In a particular embodiment, the viable mammalian cells in step c) are mammalian cells that express recombinant Axl protein(s) on their surface.
[0019] In an also preferred embodiment, the molecule of interest is a cytotoxic molecule (also called cytotoxic or cytostatic agent herein).
[0020] In an also preferred embodiment, said molecule of interest covalently binds to the compound capable of binding the Axl protein using a linker, more preferably a peptide linker, more preferably a cleavable peptide linker, most preferably a linker which can be cleaved by naturally occurring intracellular compounds contained in the mammalian cell, particularly in the cytosol of the mammalian cell.
[0021] In an also preferred embodiment, the compound capable of binding to the Axl protein is an antibody, or a functional binding fragment of that antibody, which is specifically directed against the Axl protein or against an epitope thereof located in the DEC domain axl.
[0022] Selection step e) can be performed by any method known to the person skilled in the art for the assessment of intracellular transport or internalization. Assay or test capable of demonstrating or evaluating the presence, absence or activity of the compound capable of binding specifically to the Axl protein, or of the complex formed by the compound and the molecule of interest, or of the molecule of interest that covalently binds to the compound, are well known to the person skilled in the art (see some examples of such a test or assay described below, without limiting such tests to the following test examples).
[0023] More particularly, these tests or assays can be performed by FACS, immunofluorescence, flow cytometry, Western blot, cytotoxicity/cytostatic assessments, etc.
[0024] In this aspect, the present invention is also directed to an in vitro method for the preparation of a complex cytotoxic or cytostatic agent capable of transporting a cytotoxic compound into a mammalian cell, preferably a viable cell, the method comprising the step of: - Covalently binding a cytotoxic agent to a compound that is: - i) capable of specifically binding to the Axl protein, preferably the human Axl protein, and - ii) internalizing into mammalian cells after binding to the protein Axl, when the Axl protein is expressed on the surface of the mammalian cell.
[0025] Preferably, the compound is a protein-like protein, more preferably an antibody that is specifically directed against the Axl protein, or against an epitope of that protein located in the ECD Axl domain, or a functional binding fragment of the antibody.
[0026] In the preferred embodiment, the cytotoxic agent so binds the anti-Axl antibody or a functional fragment of that antibody using a linker, more preferably a peptide linker, more preferably a cleavable peptide linker, most preferably a linker which can be cleaved, by way of non-limiting example, by naturally occurring intracellular compounds.
[0027] Like the other members of the TAM family, the extracellular domain of Axl (DEC) has an organization closed to those of cell adhesion molecules. DEC Axl is characterized by a combination of two immunoglobulin-like domains, followed by two adjacent type III fibronectin-like domains [O'Bryan, J.P. and collaborators. Mol. Cell Biol. (1991).11, 5016-5031]. The two N-terminus immunoglobulin-like domains are sufficient for binding to the Gas6 ligand [Sasaki, T. et al. EMBO J. (2006).25, 80-87].
[0028] The ECD of the human Axl protein is a fragment of 451 amino acids, corresponding to amino acids 1-451 of the sequence of SEQ ID NO:29, the sequence of which is represented in the sequence listing as SEQ ID NO:31. 1-25 corresponding to the signal peptide, the ECD of the human Axl protein lacking the signal peptide corresponds to amino acids 26-451 of the sequence SEQ ID NO:29, represented by the sequence SEQ ID NO:32.
[0029] So far, different modes of internalization have been identified. They direct the proteins or protein complex to become internalized in the cell. After endocytosis, most membrane proteins or lipids return to the cell surface (recycling), but some membrane components are transported to late endosomes or the Golgi [Maxfield, F.R. & McGraw, T.E. nat. Rev. Mol. Cell Biol. (2004).5, 121-132].
[0030] In a preferred embodiment, the invention relates to an antigen-binding protein, or an antigen-binding fragment thereof, which: i) specifically binds to human Axl protein, and ii) is internalized after its binding to human Axl protein, the antigen-binding protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 14, or any sequence that exhibits at least 80%, preferably 85%, 90 %, 95% and 98% identity to SEQ ID NOs: 1 to 14.
[0031] In a more preferred embodiment, the invention relates to an antigen-binding protein, or an antigen-binding fragment thereof, which specifically binds to the human protein Axl, preferably having the sequence SEQ ID NO: 29 or 30 or natural variant sequence thereof, and is internalized after its binding to the human protein Axl, the antigen-binding protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 14.
[0032] A "binding protein" or "antigen-binding protein" is a peptide chain with a specific or general affinity for another protein or molecule (often referred to as an antigen). Proteins are brought into contact and form a complex when binding is possible. The antigen-binding protein of the invention may preferably be, without limitation, an antibody, a fragment or derivative of an antibody, a protein or a peptide.
[0033] By "antigen-binding fragment" of an antigen-binding protein in accordance with the invention is meant any peptide, polypeptide or protein that retains the ability to specifically bind to the target (also generally referred to as antigen) of the antigen-binding protein and comprises an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least o at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the antigen-binding protein amino acid sequence.
[0034] In a preferred embodiment where the antigen-binding protein is an antibody, such "antigen-binding fragments" are selected from the group consisting of Fv, scFv (sc for single chain), Fab, F( ab')2, Fab', scFv-Fc fragments or diabodies, or any fragment whose half-life would have been increased by chemical modification, such as the addition of poly(alkylene)glycol, such as poly(ethylene) glycol ("PEGylation") (pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG" from Poly(Ethylene)Glycol), or by incorporation into a liposome, the fragments having at least one of the characteristic CDRs of the antibody according to the invention. Preferably, the "antigen binding fragments" will consist of or comprise a partial sequence of the variable heavy or light chain of the antibody from which they are derived, the partial sequence being sufficient to maintain the same binding specificity of the antibody from which it is descended, and a sufficient affinity, preferably at least equal to 1/100, more preferably at least 1/10, of the affinity of the antibody from which it is descended, for the target. Such a functional fragment will contain at least 5 amino acids, preferably 10, 15, 25, 50 and 100 amino acids, consecutive from the sequence of the antibody from which it is descended.
[0035] The term "epitope" is a region of an antigen that is bound by an antigen-binding protein, including antibodies. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. The epitopes can also be conformational, that is, composed of non-linear amino acids. In certain embodiments, the epitopes may include determinants that are chemically active groupings of surface molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may exhibit specific three-dimensional structural features and /or specific load characteristics.
[0036] In the present application, the epitope is located in the extracellular domain of the human protein Axl.
[0037] In accordance with a preferred embodiment of the invention, the antigen-binding protein, or an antigen-binding fragment thereof, specifically binds to an epitope located in the extracellular domain of the human Axl protein, preferably having the sequence SEQ ID NO: 31 or 32 or natural variant sequence thereof.
[0038] By "specifically binding", "specifically binding" or the like, it is intended that the antigen-binding protein, or antigen-binding fragment thereof, forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 x 10 -6 M or less. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance and the like. For the avoidance of doubt, this does not mean that the antigen-binding fragment cannot bind or interfere, at a low level, with another antigen. However, as a preferred embodiment, the antigen-binding fragment only binds to said antigen.
[0039] In this sense, "EC50" refers to 50% effective concentration. More precisely, the term mean maximum effective concentration (EC 50) corresponds to the concentration of a drug, antibody or toxic agent that induces a response midway between the reference and the maximum after a specified exposure time. The term is commonly used as a measure of drug potency. The EC50 of a graduated dose-response curve, therefore, represents the concentration of a compound at which 50% of its maximum effect is observed. The EC50 of a quantum dose-response curve represents the concentration of a compound at which 50% of the population exhibits a response, after a specified duration of exposure. Concentration measurements normally follow a sigmoidal curve, increasing rapidly with a relatively small change in concentration. This can be mathematically determined by deriving the best-fit line.
[0040] As a preferred embodiment, the EC50 value determined in the present invention characterized the binding potency of the antibody on Axl ECD exposed in human tumor cells. The EC50 parameter is determined using SCAF analysis. The EC50 parameter reflects the antibody concentration at which 50% of maximal binding is obtained in human Axl expressed in human tumor cells. Each EC50 value was calculated as the midpoint of the dose-response curve using a four-parameter regression curve-fitting program (Prism Software). This parameter was selected as being representative of physiological/pathological conditions.
[0041] In one embodiment of the invention, the antigen-binding protein, or an antigen-binding fragment thereof, binds to its epitope with an EC50 of at least 10-9 M, preferably between 10- 9 and 10-12 M.
[0042] Another embodiment of the invention is a process or method for selecting an antigen-binding protein, or an antigen-binding fragment thereof, capable of being intracellularly internalizable in a mammalian cell, preferably in a human cell, preferably a viable cell, comprising the steps of: - i) selecting an antigen-binding protein that specifically binds to Axl, preferably to its DEC domain or an epitope thereof; and - ii) selecting the antigen binding protein of the previous step i) which is internalized in a mammalian cell after its binding to an Axl protein expressed on the surface of the mammalian cell.
[0043] In a particular embodiment, the mammalian cell naturally expresses the Axl protein receptor on its surface or are mammalian cells that express recombinant Axl protein on its surface, preferably human cells.
[0044] This method or process may comprise the steps of 1) selecting antigen-binding protein that specifically binds to Axl with an EC50 of at least 10-9 M and ii) selecting antigen-binding protein from the previous step, the which are internalized after their attachment to Axl. Selection step ii) can be carried out by any method known to a person skilled in the art for the evaluation of internalization. More particularly, tests can be performed by FACS, immunofluorescence, flow cytometry, Western blot, cytotoxicity assessments, etc.
[0045] Another feature of the antigen binding protein according to the invention is that it does not have any significant activity in the proliferation of tumor cells. More particularly, as illustrated in the examples below, the antigen binding protein according to the invention does not have any significant in vitro activity on the SN12C proliferation model.
[0046] In oncology, there are multiple mechanisms by which mAbs can exert therapeutic efficacy, but often their activity is not sufficient to produce a lasting benefit. Thus, several strategies have been employed to improve their activity, particularly by combining them with drugs such as chemotherapeutic agents. As an efficient alternative to combination protocols, immunotoxins become a new therapeutic option to treat cancer [Beck, A. et al. Discovery Med. (2010).10, 329339; Alley, S.C. and collaborators. J. Pharmacol. Exp. The R. (2009).330, 932-938]. Antibody-drug conjugates (CAFs) represent an approach where the ability to harness the specificity of mAbs and direct the transport of a cytotoxic agent to the tumor can significantly elevate both mAb activity and drug activity. Ideally, the mAb will specifically bind to an antigen with substantial expression on tumor cells, but limited expression on normal cells.
[0047] The present invention focused on a specific anti-Axl binding protein, and more particularly on a specific anti-Axl antibody, showing a high capacity to be internalized after binding of Axl. Such an antigen-binding protein is of interest as one of the components of the immunodrug conjugate, as it targets the cytotoxic agent bound to specific cancer cells. Once internalized, the cytotoxic agent causes cancer cell death.
[0048] Important keys to success with immunoconjugate therapy are thought to be target antigen specificity and internalization of antigen-binding protein complexes into cancer cells. Obviously non-internalizable antigens are less effective than internalizable antigens in transporting cytotoxic agents. Internalization processes are variable across antigens and depend on several parameters that can be influenced by binding proteins. Cell surface RTQs constitute a family of antigens of interest to investigate such an approach.
[0049] In the biomolecule, the cytotoxic leads to cytotoxic activity and the antigen binding protein used causes its specificity against cancer cells as well as a vector for insertion into the cells to correctly target the cytotoxic agent.
[0050] Thus, to improve the immunoconjugated molecule, the vehicle binding protein must exhibit a high ability to internalize into specific cancer cells. The efficiency with which binding proteins mediated internalization differs significantly depending on the target epitope. Selection of potent internalizing anti-Axl binding proteins requires several experimental data that not only study Axl inactivation, but also track anti-Axl binding proteins that are constituted within cells.
[0051] In a preferred embodiment, the internalization of the antigen-binding protein according to the invention may preferably be evaluated by immunofluorescence (as exemplified below in the present patent application) or any method or process known to the person skilled in the art. from the specific technique to the internalization mechanism.
[0052] In another preferred embodiment, as the Axl-antigen complex binding protein according to the invention is internalized after binding of the binding protein of the invention to the ECD of Axl, a reduction in the amount of Axl in the cell surface is induced. This reduction can be quantified by any method known to a person skilled in the art (Western blot, SCAF, immunofluorescence, etc.).
[0053] In one embodiment of the invention, this reduction, which thus reflects internalization, may preferably be measured by FACS and expressed as the difference or delta or the Mean Fluorescence Intensity (MFI) measured in cells not treated with the IMF measured with cells treated with the antigen binding protein according to the invention.
[0054] As a non-limiting example of the present invention, this delta is determined based on IFMs obtained with untreated cells and cells treated with the antigen binding protein of the present invention, as described in example 9, using i) SN12C cells from human kidney tumor after a 24 hour incubation period with the antigen binding protein of the invention and ii) a secondary antibody labeled with Alexa488. This parameter is defined as calculated with the following formula:

[0055] This difference between the IMFs reflects the inactivation of Axl as IFMs are proportional to the Axl expressed on the cell surface.
[0056] In a more preferred and advantageous aspect, the antigen-binding protein, or an antigen-binding fragment thereof, of the invention consists of a monoclonal antibody, preferably an isolated mAb, producing a Δ(IMF24h from untreated cells - IMF24h of treated cells) of at least 200, preferably at least 300.
[0057] The antigen-binding protein, or an antigen-binding fragment thereof, according to the invention, induces an IMP reduction of at least 200.
[0058] In more detail, the aforementioned delta can be measured according to the following procedure, which should be considered as an illustrative and non-limiting example: treating and incubating tumor cells of interest with the antigen-binding protein of the present invention ; treating the treated cells of step a) and, in parallel, untreated cells with the antigen-binding protein of the present invention; measure the IMF (representative of the amount of Axl present on the surface) of treated and untreated cells with a labeled secondary antibody capable of binding to the antigen-binding protein, and calculate the delta as the subtraction of the IMF obtained with the treated cells of the IMF obtained with the untreated cells.
[0059] The terms "antibody", "antibodies" or "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies, preferably isolated mAbs (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies or multispecific antibodies (eg, bispecific antibodies as long as they exhibit the desired biological activity).
[0060] More particularly, this molecule consists of a glycoprotein comprising at least two heavy (H) and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (or domain) (herein abbreviated as RVCP or VP) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as RVCL or VL) and a light chain constant region. The light chain constant region comprises a CL domain. The VP and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (REs). Each VP and VL is composed of three CDRs and four REs, arranged from amino terminus to carboxyl terminus in the following order: RE1, RDC1, RE2, RDC2, RE3, RDC3, RE4. The variable regions of the light and heavy chains contain a binding domain that interacts with an antigen. Constant regions of antibodies can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
[0061] Antibodies within the meaning of the invention also include certain antibody fragments. Said antibody fragments exhibit the desired binding specificity and affinity, irrespective of the immunoglobulin origin or type (i.e. IgG, IgE, IgM, IgA etc.), i.e. they are capable of specifically binding the Axl protein with a affinity comparable to that of the full-length antibodies of the invention.
[0062] In general, for the preparation of monoclonal antibodies or their functional fragments, especially of murine origin, it is possible to refer to techniques that are described in particular in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 726, 1988) or the hybridoma preparation technique described by Kohler and Milstein (Nature, 256:495-497, 1975).
[0063] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule that is directed against a specific antigen and that can be produced by a single B cell clone or hybridoma. Monoclonal antibodies can also be recombinant, i.e. produced by protein engineering. Furthermore, in contrast to polyclonal antibody preparations which typically include multiple antibodies directed against multiple determinants or epitopes, each monoclonal antibody is directed against a single epitope of the antigen. The invention relates to antibodies isolated or obtained by purification from natural sources or obtained by genetic recombination or chemical synthesis.
[0064] A preferred embodiment of the invention is an antigen-binding protein, or an antigen-binding fragment thereof, comprising or consisting of an antibody, the antibody comprising the three light chain CDRs comprising the sequences SEQ ID NOs: 1, 2 and 3, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NOs: 1, 2 and 3; and the three heavy chain CDRs comprising the sequences SEQ ID NOs: 4, 5 and 6, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NOs : 4, 5 and 6.
[0065] In a more preferred embodiment of the invention, the antigen-binding protein, or an antigen-binding fragment thereof, comprises an antibody, that antibody comprising the three light chain CDRs comprising the sequences SEQ ID NOs: 1, 2 and 3; and the three heavy chain CDRs comprising the sequences SEQ ID NOs: 4, 5 and 6.
[0066] In a preferred aspect, by CDR regions or CDRs is meant the hypervariable regions of immunoglobulin heavy and light chains, as defined by IMGT. Without any contradictory mention, the RDCs will be defined in this descriptive report according to the IMGT numbering system.
[0067] IMGT unique numbering was defined to compare the variable domains of whatever antigen receptor, chain type or species [Lefranc, M.-P., Immunology Today 18, 509 (1997)/Lefranc, M.-P., The Immunologist, 7, 132-136 (1999)/Lefranc, M.-P., Pommie, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L. , Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT unique numbering, conserved amino acids always have the same position, e.g. cysteine 23 (1st CYS), tryptophan 41 (TRP CONSERVED), hydrophobic amino acid 89, cysteine 104 (2nd CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). The unique IMGT numbering provides a standardized delineation of framework regions (RE1-IMGT: positions 1 to 26, RE2-IMGT: 39-55, RE3-IMGT: 66-104 and RE4-IMGT: 118-128) and determinant regions of complementarity: RDC1-IMGT: 27 to 38, RDC2-IMGT: 56 to 65 years and RDC3-IMGT: 105 to 117. As gaps represent unoccupied positions, the RDC-IMGT lengths (shown in square brackets and separated by dots, for example [8.8.13]) becomes crucial information. The unique IMGT numbering is used in 2D graphical representations, designated as IMGT Pearl Colliers [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002)/Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)], and on 3D structures in IMGT/3D-DB Structure [Kaas, Q., Ruiz, M. and Lefranc, M. -P., T cell receptor and MHC structural data. Nucl. Acids Res., 32, D208-D210 (2004)].
[0068] It should be understood that, without contradictory specification in the present specification, complementarity determining regions or CDRs mean the hypervariable regions of immunoglobulin heavy and light chains, as defined according to the IMGT numbering system.
[0069] However, RDCs can also be defined according to the Kabat numbering system (Kabat et al., Sequences of proteins of immunological interest, 5th ed., US Department of Health and Human Services, NIH, 1991 and later editions ). There are three heavy chain CDRs and three light chain CDRs. Here, the terms "CDRs" and "CDRs" are used to indicate, as the case may be, one or more, or even all, of the regions that contain the majority of the amino acid residues responsible for the binding affinity of the antibody for the antigen. or epitope it recognizes.
[0070] According to the Kabat numbering system, the present invention relates to an antigen-binding protein, or an antigen-binding fragment thereof, which consists of an antibody, the antibody comprising the three CDRs of light chains, as defined according to the Kabat numbering system, comprising the sequences SEQ ID NOs: 9, 10 and 11, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98 %, identity to SEQ ID NOs: 9, 10 and 11; and the three heavy chain CDRs, as defined according to the Kabat numbering system, comprising the sequences SEQ ID NOs: 12, 13 and 14, or any sequence that exhibits at least 80%, preferably 85%, 90% , 95% and 98%, identity to SEQ ID NOs:12, 13 and 14.
[0071] For the purposes of the present invention, the "percent identity" between two nucleic acid or amino acid sequences means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the differences between the two sequences being randomly distributed along their length. Comparison of two nucleic acid or amino acid sequences is traditionally performed by comparing the sequences after they have been optimally aligned, such comparison being able to be performed per segment or using an "alignment window". Optimal alignment of sequences so that comparison can be performed, in addition to manual comparison, using the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2:482], using the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol. 48:443], using the similarity search method of Pearson and Lipman (1988) [Proc. natl. academy Sci. USA 85:2444] or through computer programs that use these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or through the BLAST NR comparison software or BLAST P).
[0072] The percent identity between two nucleic acid or amino acid sequences is determined by comparing the two optimally aligned sequences, wherein the nucleic acid or amino acid sequences of comparison may have additions or deletions, compared to the sequences of reference for optimal alignment between the two sequences. Percent identity is calculated by determining the number of positions at which the nucleotide or amino acid residue is identical between the two sequences, preferably between the two complete sequences, by dividing the number of identical positions by the total number of positions in the alignment window and multiplying the result by 100 to obtain the percent identity between the two sequences.
[0073] For example, the BLAST program, "Blast 2 sequences" (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol., 1999, Lett. 174:247- 250), available at http://www.ncbi. nlm.nih.gov/gorf/bl2.html, can be used with the default parameters (particularly in the case of the parameters "open gap penalty": 5, and "length gap penalty": 2; the selected matrix being, for example, the matrix "BLOSUM 62" proposed by the program); the percentage of identity between the two sequences to be compared is calculated directly by the program.
[0074] For the amino acid sequence that has at least 80%, preferably 85%, 90%, 95% and 98% identity to a reference amino acid sequence, preferred examples include those amino acids that contain the reference sequence, certain modifications, particularly a deletion, addition or substitution of at least one amino acid, truncation or extension. In the case of substitution of one or more consecutive or non-consecutive amino acids, substitutions in which the substituted amino acids are replaced by "equivalent" amino acids are preferred. Here, the term "equivalent amino acid acids" is used to indicate any acids susceptible of being substituted by one of the structural amino acids, without, however, modifying the biological activities of the corresponding antibodies and the specific examples defined below.
[0075] Equivalent amino acids can be determined either on their structural homology to the amino acids by which they are substituted or on the results of comparative tests of biological activity between the various antigen-binding proteins that may be generated.
[0076] As a non-limiting example, Table 2 summarizes the possible substitutions that can be performed without resulting in a significant modification of the biological activity of the corresponding modified antigen-binding protein; inverse substitutions are of course possible under the same conditions. Table 2

[0077] An embodiment of the invention relates to an antigen-binding protein, or an antigen-binding fragment thereof, which comprises a light chain variable domain of sequence SEQ ID NO: 7, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NO: 7; and the three heavy chain CDRs comprising the sequences SEQ ID NOs: 4, 5 and 6, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NOS: 4, 5 and 6.
[0078] In accordance with a preferred embodiment of the invention, the antigen-binding protein, or an antigen-binding fragment thereof, comprises a light chain variable domain of sequence SEQ ID NO: 7, or any sequence which exhibit at least 80% identity with SEQ ID NO:7; and the three heavy chain CDRs comprising the sequences of SEQ ID NOs: 4, 5 and 6.
[0079] In accordance with another preferred embodiment of the invention, the antigen-binding protein, or an antigen-binding fragment thereof, comprises a light chain variable domain of sequence SEQ ID NO: 7, or any sequence that exhibit at least 80% identity with SEQ ID NO:7.
[0080] Another embodiment of the invention relates to an antigen-binding protein, or an antigen-binding fragment thereof, which comprises the three light chain CDRs comprising the sequences SEQ ID NOs: 1, 2 and 3, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NOs: 1, 2 and 3; and a heavy chain variable domain of sequence SEQ ID NO: 8, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NO: 8.
[0081] According to a preferred embodiment of the invention, the antigen-binding protein, or an antigen-binding fragment thereof, comprises the three light chain CDRs comprising the sequences SEQ ID NOs: 1, 2 and 3; and a heavy chain variable domain of sequence SEQ ID NO:8, or any sequence that exhibits at least 80% identity to SEQ ID NO:8.
[0082] In accordance with another preferred embodiment of the invention, the antigen-binding protein, or an antigen-binding fragment thereof, comprises a heavy chain variable domain of sequence SEQ ID NO: 8, or any sequence that exhibit at least 80% identity with SEQ ID NO:8.
[0083] Another embodiment of the invention relates to an antigen-binding protein, or an antigen-binding fragment thereof, which comprises a light chain variable domain of sequence SEQ ID NO: 7, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98% identity to SEQ ID NO: 7; and a heavy chain variable domain of sequence SEQ ID NO: 8, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98% identity to SEQ ID NO: 8.
[0084] In accordance with a preferred embodiment of the invention, the antigen-binding protein, or an antigen-binding fragment thereof, comprises a light chain variable domain of the sequence SEQ ID NO: 7, or any sequence that exhibits at least 80% identity to SEQ ID NO: 7, and a heavy chain variable domain of sequence SEQ ID NO: 8, or any sequence exhibiting at least 80% identity to SEQ ID NO: 8.
[0085] For clarity, Table 3a below summarizes the various amino acid sequences corresponding to the antigen-binding protein of the invention (with Mu. = murine). Table 3a

[0086] A specific aspect of the present invention relates to a murine antibody, or its derivative compounds or antigen-binding fragments, characterized in that the antibody also comprises light chain and heavy chain constant regions derived from an antibody. of a heterologous species with the mouse, particularly man.
[0087] Another specific aspect of the present invention relates to a chimeric antibody, or its derivative compounds or antigen-binding fragments, characterized in that the binding antibody also comprises light chain constant regions and heavy chains derived from a antibody from a species heterologous to the mouse, particularly human.
[0088] Yet another specific aspect of the present invention relates to a humanized antibody, or its derivative compounds or antigen-binding fragments, characterized in that the constant regions of human antibody-derived light chains and heavy chains are, respectively, the lambda or kappa region and the gamma-1, gamma-2 or gamma-4 region.
[0089] Another aspect of the invention is an antigen-binding protein consisting of the hybridoma-derived monoclonal antibody 1613F12 I-4505 deposited at the CNCM, Institut Pasteur, France, on July 28, 2011, or an antigen-binding fragment thereof .
[0090] According to another aspect, the invention relates to a murine hybridoma capable of secreting an antigen-binding protein according to the invention, particularly the hybridoma of murine origin deposited with the French collection of microorganism cultures (CNCM , Institut Pasteur, Paris, France) on July 28, 2011, under number I-4505. This hybridoma was obtained by fusing splenocytes/lymphocytes from immunized Balb/C mice and cells of the myeloma cell line Sp 2/O-Ag 14.
[0091] In another aspect, the invention relates to a murine hybridoma capable of secreting an antibody comprising the three light chain CDRs comprising the sequences SEQ ID NOs: 1, 2 and 3; and the three heavy chain CDRs comprising the sequences SEQ ID NOs: 4, 5 and 6, the hybridoma being deposited at CNCM, Institut Pasteur, Paris, France, on July 28, 2011, under number I-4505. This hybridoma was obtained by fusion of splenocytes/lymphocytes from immunized Balb/C mice and cells of the Sp 2/O-Ag 14 myeloma cell line.
[0092] An object of the invention is the murine hybridoma I-4505 deposited at the CNCM, Institut Pasteur, France, on July 28, 2011.
[0093] The antigen binding protein of the invention also comprises chimeric or humanized antibodies.
[0094] A chimeric antibody is an antibody that contains a naturally occurring variable region (light chain and heavy chain) derived from an antibody of a given species in combination with light chain and heavy chain constant regions of an antibody of a species heterologous to the species. given species.
[0095] Antibodies, or chimeric fragments thereof, can be prepared using recombinant genetic techniques. For example, the chimeric antibody can be produced by cloning recombinant DNA containing a promoter and sequence encoding the variable region of a non-human, particularly murine, monoclonal antibody of the invention, and a sequence encoding the human constant region antibody. A chimeric antibody according to the invention encoded by such a recombinant gene can be, for example, a mouse-human chimera, the specificity of that antibody being determined by the variable region derived from murine DNA and its isotype, determined by the constant region derived from human DNA. See Verhoeyn et al (BioEssays, 8:74, 1988) for methods of preparing chimeric antibodies.
[0096] In another aspect, the invention describes a binding protein consisting of a chimeric antibody.
[0097] In a particular preferred embodiment, the chimeric antibody, or an antigen-binding fragment thereof, of the present invention comprises a light chain variable domain sequence comprising the amino acid sequence SEQ ID NO: 7, as well as a heavy chain variable domain sequence comprising the amino acid sequence SEQ ID NO: 8.
[0098] In another aspect, the invention describes a binding protein consisting of a humanized antibody.
[0099] "Humanized antibody" means an antibody which contains the CDR regions derived from an antibody of non-human origin, the other parts of the antibody molecule being derived from one (or more) human antibodies. In addition, some of the framework segment residues (called RE) can be modified to preserve binding affinity (Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al., Science, 239:15341536, 1988; Riechmann et al., Nature, 332:323-327, 1988).
[00100] The humanized antibodies of the invention, or fragments thereof, can be prepared by techniques known to a person skilled in the art (such as, for example, those described in Singer et al., J. Immun., 150:2844 -2857, 1992; Monte et al., Biotechnol. Genet. Eng. Rev., 10:1-142, 1992; and Bebbington et al., Bio/Technology, 10:169-175, 1992). Such humanized antibodies are preferred for their use in methods involving in vitro diagnostics or in vivo preventive and/or therapeutic treatment. Other humanization techniques, also known to a person skilled in the art, such as, for example, the "CDR grafting" technique, described by PDL in patents EP 0 451 261, EP 0 682 040, EP 0 939 127, EP 0,566,647 or US 5,530,101, US 6,180,370, US 5,585,089 and US 5,693,761. US patents 5,639,641 or 6,054,297, 5,886,152 and 5,877,293 may also be cited.
[00101] Furthermore, the invention also relates to humanized antibodies resulting from the above-described murine antibodies.
[00102] Preferably, light chain and heavy chain constant regions derived from a human antibody are, respectively, the lambda or kappa region and the gamma-1, gamma-2 or gamma-4 region.
[00103] In a preferred embodiment, the invention relates to an antigen-binding protein consisting of a humanized antibody, or an antigen-binding fragment thereof, which comprises a light chain variable domain comprising the sequence SEQ ID NO:36, or any sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity to SEQ ID NO:36; and the three heavy chain CDRs comprising the sequences of SEQ ID NOs: 4, 5 and 6.
[00104] Another embodiment of the invention relates to an antigen-binding protein, or an antigen-binding fragment thereof, which comprises a light chain variable domain of a sequence selected from the group consisting of SEQ ID NOs: 37 to 47, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NOs: 37 to 47; and the three heavy chain CDRs comprising the sequences SEQ ID NOs: 4, 5 and 6.
[00105] By "any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NO: 36 or 37 to 47", it is intended to denote those sequences which exhibit the three light chain CDRs SEQ ID NOs: 1, 2 and 3 and which, in addition, exhibit at least 80%, preferably 85%, 90%, 95% and 98%, identity to the complete sequence SEQ ID NO: 36 or 37 to 47 external to the sequences corresponding to the CDRs (i.e., SEQ ID NOs: 1, 2 and 3).
[00106] For clarity, Table 3b below summarizes the various amino acid sequences corresponding to the light chain (VL) of the humanized antigen-binding protein of the invention (with Hz. = humanized). Table 3b

[00107] In one embodiment of the invention, the antigen-binding protein, or an antigen-binding fragment thereof, comprises a light chain variable domain selected from the group consisting of: a sequence light chain variable domain SEQ ID NO:7 or any sequence that exhibits at least 80% identity to SEQ ID NO:7; a light chain variable domain of sequence SEQ ID NO: 36 or any sequence that exhibits at least 80% identity to SEQ ID NO: 36; and a light chain variable domain of sequence SEQ ID NO: 37 to 47, or any sequence that exhibits at least 80% identity to SEQ ID NO: 37 to 47.
[00108] In a preferred embodiment, the invention relates to an antigen-binding protein which consists of a humanized antibody, or an antigen-binding fragment thereof, which comprises a heavy chain variable domain comprising the sequence SEQ ID NO: 48, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NO: 48; and the three light chain CDRs comprising the sequences SEQ ID NOs: 1, 2 and 3.
[00109] Another embodiment of the invention relates to an antigen-binding protein, or an antigen-binding fragment thereof, which comprises a heavy chain variable domain of sequence selected from the group consisting of SEQ ID NOs : 49 to 68, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NOs: 49 to 68; and the three light chain CDRs comprising the sequences of SEQ ID NOs: 1, 2 and 3.
[00110] By "any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NOs: 48 and 49 to 68", it is intended to denote those sequences that exhibit the three heavy chain CDRs SEQ ID NOs: 4, 5 and 6, and further that exhibit at least 80%, preferably 85%, 90%, 95%, and 98% identity to the full-length SEQ ID NOs: 48 and 49 to 68 external to the sequences corresponding to the CDRs (i.e., SEQ ID NOs: 4, 5 and 6).
[00111] For clarity, Table 3c below summarizes the various amino acid sequences corresponding to the humanized antigen-binding protein heavy chain (VP) of the invention (with Hz. = humanized) Table 3c

[00112] In one embodiment of the invention, the antigen-binding protein, or an antigen-binding fragment thereof, comprises a heavy chain variable domain selected from the group consisting of: a sequence heavy chain variable domain SEQ ID NO: 8 or any sequence that exhibits at least 80% identity with SEQ ID NO: 8; a heavy chain variable domain of sequence SEQ ID NO: 48 or any sequence that exhibits at least 80% identity to SEQ ID NO: 48; and a heavy chain variable domain of sequence SEQ ID NOs: 49 to 68 or any sequence that exhibits at least 80% identity with SEQ ID NOs: 49 to 68.
[00113] In one embodiment of the invention, the antigen-binding protein, or an antigen-binding fragment thereof, comprises a light chain variable domain of sequence SEQ ID NO: 36, or any sequence that exhibits at least at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NO: 36; and a heavy chain variable domain of sequence SEQ ID NO: 48, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NO: 48.
[00114] In another embodiment of the invention, the antigen-binding protein, or an antigen-binding fragment thereof, comprises a light chain variable domain of sequence selected from the group consisting of SEQ ID NOs: 37 to 47 , or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98%, identity to SEQ ID NOs: 37 to 47; and a heavy chain variable domain of a sequence selected from the group consisting of SEQ ID NOs: 49 to 68, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95%, and 98%, identity to SEQ ID NOs: 49 to 68.
[00115] In one embodiment of the invention, the antigen-binding protein, or an antigen-binding fragment thereof, comprises: a light chain variable domain of sequence SEQ ID NOs: 7, 36 or 37 to 47, or any sequence that exhibits at least 80% identity to SEQ ID NOs: 7, 36, or 37 to 47; and a heavy chain variable domain of sequence SEQ ID NOs: 8, 48, or 49 to 68 or any sequence that exhibits at least 80% identity to SEQ ID NOs: 8, 48, or 49 to 68.
[00116] A novel aspect of the present invention relates to an isolated nucleic acid characterized in that it is selected from the following nucleic acids (including any degenerate genetic code): a nucleic acid encoding an antigen-binding protein, or an antigen-binding fragment thereof, in accordance with the invention; a nucleic acid comprising: - a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 to 28 and 69 to 99, or - a nucleic acid sequence comprising the six nucleic acid sequences SEQ ID NOs: 15 to 20, or - a nucleic acid sequence comprising the two nucleic acid sequences SEQ ID NOs: 21, 22 or the two nucleic acid sequences selected from one part of SEQ ID NOs: 69 to 79 and the other part of SEQ ID NOs: 80 to 99; a nucleic acid complementary to a nucleic acid as defined in a) or b); and a nucleic acid, preferably having at least 18 nucleotides, capable of hybridizing under highly stringent conditions to a nucleic acid sequence as defined in a) or b), or to a sequence of at least 80%, preferably 85 %, 90%, 95% and 98% identity after optimal alignment with a nucleic acid sequence as defined in a) or b).
[00117] Table 4a below summarizes the various nucleotide sequences with respect to the binding protein of the invention (with Mu. = murine). Table 4a

[00118] For clarity, Table 4b below summarizes the various nucleotide sequences corresponding to the light chain (VL) of the humanized antigen-binding protein of the invention (with Hz. = humanized) Table 4b

[00119] For clarity, Table 4c below summarizes the various nucleotide sequences corresponding to the heavy chain (VP) of the humanized antigen-binding protein of the invention (with Hz. = humanized) Table 4c

[00120] The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", used interchangeably in the present specification, means an exact sequence of nucleotides, modified or not, defining a fragment or region of a nucleic acid, whether or not containing unnatural nucleotides, and being double-stranded DNA, single-stranded DNA or transcription products of the DNAs.
[00121] The sequences of the present invention have been isolated and/or purified, that is, they have been sampled directly or indirectly, for example by a copy, their environment having been at least partially modified. Isolated nucleic acids obtained by recombinant genetics, by means of, for example, host cells, or obtained by chemical synthesis, should also be mentioned herein.
[00122] "Nucleic sequences that show a percent identity of at least 80%, preferably 85%, 90%, 95% and 98%, after optimal alignment with a preferred sequence" means nucleic sequences that exhibit, with respect to the sequence reference nucleic acid, certain modifications such as, in particular, a deletion, a truncation, an extension, a chimeric fusion and/or a particularly point substitution. Preferably, these are sequences that encode the same amino acid sequences as the reference sequence, the latter being related to the degeneracy of the genetic code, or complementarity sequences that are capable of specifically hybridizing to the reference sequences, preferably under highly stringent, particularly those defined below.
[00123] Hybridization under highly stringent conditions means that conditions related to temperature and ionic strength are selected in such a way as to allow hybridization to be maintained between two complementary DNA fragments. On a purely illustrative basis, the highly stringent conditions of the annealing step in order to define the above-described polynucleotide fragments are advantageously as follows.
[00124] DNA-DNA or DNA-RNA hybridization is performed in two steps: (1) pre-hybridization at 42°C for three hours in phosphate buffer (20 mM, pH 7.5) containing 5X SSC (IX SSC corresponds to a solution of 0.15 M NaCl + 0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10X Denhardt's solution, 5% dextran sulfate and salmon sperm DNA at 1%; (2) primary hybridization for 20 hours at a temperature dependent on probe length (i.e.: 42°C for a probe > 100 nucleotides in length), followed by two 20-minute washes at 20°C in 2X SSC + SDS 2%, a 20 minute wash at 20°C in 0.1X SSC + 0.1% SDS. The last wash is performed in 0.1X SSC + 0.1% SDS for 30 minutes at 60°C for a probe >100 nucleotides in length. The highly stringent hybridization conditions described above for a polynucleotide of defined size can be adapted by one skilled in the art to longer or shorter oligonucleotides, according to the procedures described in Sambrook et al. (Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory; 3rd edition, 2001).
[00125] The invention also relates to a vector comprising a nucleic acid as described in the invention.
[00126] The invention particularly aims at cloning and/or expression vectors that contain such a nucleotide sequence.
[00127] The vectors of the invention preferably contain elements that allow the expression and/or secretion of nucleotide sequences in a given host cell. The vector must therefore contain a promoter, translation initiation and termination signals, as well as suitable transcriptional regulatory regions. The vector must be able to maintain itself stably in the host cell and may optionally have specific signals that specify secretion of the translated protein. These various elements are selected and optimized by one skilled in the art according to the host cell used. For this purpose, the nucleotide sequences can be inserted into self-replicating vectors in the chosen host or be integrative vectors of the chosen host.
[00128] These vectors are prepared by methods normally used by one skilled in the art and the resulting clones can be introduced into an appropriate host by conventional methods such as lipofection, electroporation, heat shock or chemical methods.
[00129] The vectors are, for example, vectors of plasmid or viral origin. They are used to transform host cells in order to clone or express the nucleotide sequences of the invention.
[00130] The invention also comprises isolated host cells transformed by a vector or comprising a vector, as described in the present invention.
[00131] The host cell can be selected from prokaryotic or eukaryotic systems, such as bacterial cells, for example, but also yeast cells or animal cells, particularly mammalian cells (with the exception of humans). Insect or plant cells can also be used.
[00132] The invention also relates to animals, other than humans, that have a cell transformed according to the invention.
[00133] Another aspect of the invention relates to a method for producing an antigen-binding protein according to the invention, or an antigen-binding fragment thereof, characterized in that the method comprises the following steps : culturing in a medium with suitable culture conditions for a host cell according to the invention; and recovering the antigen-binding protein, or one of its antigen-binding fragments, so produced from the culture medium or the cultured cells.
[00134] Cells transformed according to the invention are of use in methods for preparing recombinant antigen-binding proteins according to the invention. Methods for the preparation of antigen-binding proteins according to the invention in recombinant form, characterized in that they use a vector and/or a cell transformed by a vector according to the invention, are also included in the present invention. Preferably, a cell transformed by a vector according to the invention is cultured under conditions which allow expression of the aforementioned antigen-binding protein and recovered from the recombinant protein.
[00135] As already mentioned, the host cell can be selected between prokaryotic or eukaryotic systems. In particular, it is possible to identify nucleotide sequences of the invention that facilitate secretion from such a prokaryotic or eukaryotic system. A vector according to the invention which carries such a sequence can thus be used advantageously for the production of recombinant proteins to be secreted. Indeed, the purification of these recombinant proteins of interest will be facilitated by the fact that they are present in the cell culture supernatant rather than inside host cells.
[00136] The antigen binding protein of the invention can also be prepared by chemical synthesis. A preparation method like this is also an object of the invention. One skilled in the art is aware of methods for chemical synthesis, such as solid phase techniques (see particularly Steward et al., 1984, Solid phase peptides synthesis, Pierce Chem. Company, Rockford, 111, 2nd ed., pp. 71-95). ) or partial solid phase techniques, by fragment condensation or by conventional solution synthesis. Polypeptides obtained by chemical synthesis and capable of containing corresponding unnatural amino acids are also included in the invention.
[00137] Antigen binding protein, or antigen binding fragments thereof, obtainable by the method of the invention are also included in the present invention.
[00138] In a particular aspect, the invention relates to an antigen-binding protein, or an antigen-binding fragment thereof, as described above for use as a targeting product for the delivery of a cytotoxic agent. at a host target site, the host target site consisting of an epitope located in the extracellular domain of the Axl protein, preferably the extracellular domain of the human Axl protein, more preferably the extracellular domain of the human Axl protein with the sequence SEQ ID NO: 31 or 32, or natural variant sequence thereof.
[00139] In a preferred embodiment, the target site of the host is a target site of a mammalian cell, more preferably of a human cell, more preferably cells that, naturally or through genetic recombination, express the protein axl.
[00140] The invention relates to an immunoconjugate comprising the antigen-binding protein as described herein, conjugated to a cytotoxic agent.
[00141] In the sense of the present invention, the term "immunoconjugate" or "immunoconjugate" generally refers to a compound that comprises at least one targeting product physically linked to one or more therapeutic agents, thereby creating a highly targeted compound.
[00142] In a preferred embodiment, such therapeutic agents consist of cytotoxic agents.
[00143] By "cytotoxic agent" or "cytotoxic" is meant an agent which, when administered to an individual, treats or prevents the development of cell proliferation, preferably the development of cancer in the individual's body, by inhibiting or preventing of cell function and/or leading to cell death.
[00144] Many cytotoxic agents have been isolated or synthesized and made possible to inhibit cell proliferation, or to destroy or reduce, if not definitively, at least significantly, tumor cells. However, the toxic activity of these agents is not limited to tumor cells, and non-tumor cells are also affected and can be destroyed. More particularly, side effects are seen in rapidly renewing cells, such as hematopoietic cells or epithelial cells, especially of the mucous membranes. By way of illustration, cells of the gastrointestinal tract are largely affected by the use of such cytotoxic agents.
[00145] One of the objects of the present invention is also to be able to provide a cytotoxic agent that makes it possible to limit the side effects on normal cells, while maintaining a high cytotoxicity on tumor cells.
[00146] More particularly, the cytotoxic agent may preferably consist of, without limitation, a drug (i.e., "antibody-drug conjugate"), a toxin (e.g., "immunotoxin" or "antibody-toxin conjugate"), a radioisotope (i.e. "radioimmunoconjugate" or "antibody-radioisotope conjugate") etc.
[00147] In a first preferred embodiment of the invention, the immunoconjugate comprises a binding protein bound to at least one drug or drug. Such an immunoconjugate is referred to as an antibody-drug conjugate (or "CAF") when the binding protein is an antibody, or an antigen-binding fragment thereof.
[00148] In a first embodiment, such drugs can be described in relation to their mode of action. As a non-limiting example, alkylating agents such as nitrogen mustard, alkylsulfonates, nitrosourea, oxazophorins, aziridines or imineethylenes, antimetabolites, antitumor antibiotics, mitosis inhibitors, inhibitors of chromatin function, antiangiogenesis agents, antiestrogens, antiandrogens, chelating agents, iron absorption stimulants, cyclooxygenase inhibitors, phosphodiesterase inhibitors, DNA inhibitors, DNA synthesis inhibitors, apoptosis stimulants, thymidylate inhibitors, T cell inhibitors, interferon agonists, ribonucleoside triphosphate inhibitors reductase, aromatase inhibitors, estrogen receptor antagonists, tyrosine kinase inhibitors, cell cycle inhibitors, taxane, tubulin inhibitors, angiogenesis inhibitors, macrophage stimulants, neurokinin receptor antagonists, cannabinoid receptor agonists, dopamine receptors, agonies granulocyte-stimulating factor agents, erythropoietin receptor agonists, somatostatin receptor agonists, LHRH agonists, calcium sensitizers, VEGF receptor antagonists, interleukin receptor antagonists, osteoclast inhibitors, radical stimulants, endothelin receptor antagonists, vinca alkaloid, antihormone or immunomodulators or any other new drug that fulfills the criteria for activity of a cytotoxic agent or toxin.
[00149] These drugs are, for example, mentioned in VIDAL 2010, on the page dedicated to compounds linked to cancerology and hematology in the column "Cytotoxic"; those cytotoxic compounds cited with reference to that document are cited herein as preferred cytotoxic agents.
[00150] More particularly, without limitation, the following drugs are particularly preferred according to the invention: mechlorethamine, chlorambucol, melphalene, hydrochloride, pipobromene, prednimustine, disodium phosphate, estramustine, cyclophosphamide, altretamine, trophosphamide, sulphophosphamide, ifosfamide, thiotepa, triethylenamine, altetramine, carmustine, streptozocin, fotemustine, lomustine, busulfan, treosulfan, improsulfan, dacarbazine, cis-platinum, oxaliplatin, lobaplatin, heptaplatin, myriplatin hydrate, carboplatin, methotrexate, pemetrexed, 5-fluorouracil, floxuridine, 5-fluordeoxyuridine, capecitabine, cytarabine, fludarabine, cytosine arabinoside, 6-mercaptopurine (6-MP), Nelerabine, 6-thioguanine (6-TG), chlordeoxyadenosine, 5-azacytidine, gemcitabine, cladribine, deoxycoformycin, tegafur, pentostatin, doxorubicin, daunorubicin, idarubicin , valrubicin, mitoxantrone, dactinomycin, mithramycin, plicamycin, mitomycin C, bleomycin, procarbazine, pac litaxel, docetaxel, vinblastine, vincristine, vindesine, vinorelbine, topotecan, irinotecan, etoposide, valrubicin, amrubicin hydrochloride, pirarubicin, elliptinium acetate, zorubicin, epirubicin, idarubicin and teniposide, razoxine, marimastat, batimastat, prinomastat, tanomastat, lomastat, CGS-27023, halofuginone, COL-3, neovastat, thalidomide, CDC 501, DMXAA, L-651582, squalamine, endostatin, SU5416, SU6668, interferon-alpha, EMD121974, interleukin-12, IM862, angiostatin, tamoxifen, toremifene, raloxifene , droloxifene, iodoxifene, anastrozole, letrozole, exemestane, flutamide, nilutamide, sprironolactone, cyproterone acetate, finasteride, cimitidine, bortezomide, Velcade, bicalutamide, cyproterone, flutamide, fulvestrane, exemestane, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib , sorafenib, sunitinib, retinoid, rexinoid, methoxsalen, methylaminolevulinate, aldesleukin, OCT-43, denileucine diflitox, interleukin-2, tasonermin, lentinan, sizofilan o, roquinimex, pidotimod, pegademase, thymopentin, poly I:C, procodazole, Tic BCG, Corynebacterium parvum, NOV-002, ukraine, levamisole, 1311-chTNT, H-101, celmoleucine, interferon alfa2a, interferon alfa2b, interferon gamma1a, interleukin-2, mobenacin, Rexin-G, teceleleucine, aclarrubicin, actinomycin, arglabin, asparaginase, carzinophylline, chromomycin, daunomycin, leucovorin, masoprocol, neocarzinostatin, peplomycin, sarkomycin, solamargine, trabectedin, streptozocin, testosterone, cunecatechins, sinecatechins, alitretinoin, belotecan hydrochloride, calusterone, dromostanolone, elliptinium acetate, ethinylestradiol, etoposide, fluoxymesterone, formestane, phosphatrol, goserelin acetate, hexylaminolevulinate, histrelin, hydroxyprogesterone, ixabepilone, leuprolide, medorxyprogesterone acetate, megesterol acetate, methylprednisolone, methyltestosterone, miltefosterone mitobronitol, nadrolone phenylpropionate, norethisterone acetate, prednisolone, prednisone, has sirrolim, testolactone, triamconolone, triptorelin, vapreotide acetate, zinostatin stimalamer, amsacrine, arsenic trioxide, bisanthrene hydrochloride, chlorambucil, chlortrianissen, cis-diaminodichloroplatin, cyclophosphamide, diethylstilbestrol, hexamethylmelamine, hydroxyurea, lenalidomide, lonidamine, methanochlorethanamine, mitothanochlorethanamine , nedaplatin, nimustine hydrochloride, pamidronate, pipobroman, porfimer sodium, ranimustine, razoxane, semustine, sobuzoxane, mesylate, triethylenemelamine, zoledronic acid, camostat mesylate, fadrozole HCl, nafoxine, aminoglutethimide, carmofur, clofarabine, cytosine arabinoside, decitabine, doxyfluridine, enocitabine, fludarabne phosphate, fluorouracil, phthorafur, uracilmustard, abarelix, bexarotene, raltiterxed, tamibarotene, temozolomide, vorinostat, megastrol, disodium clodronate, levamisole, ferumoxitol, isomaltoside iron, celecoxib, lbudilast, bendamustine, altretamine, temsirrolimo pralatrexate, TS-1, decitabine , bicalutamide, futamide, letrozole, disodium clodronate, degarelix, toremifene citrate, histamine dihydrochloride, DW-166HC, nitracrine, decitabine, irinotecan hydrochloride, amsacrine, romidepsin, tretinoin, cabazitaxel, vandetanib, lenalidomide, bandronic acid, miltefosine, vitespene , mifamurtide, nadroparin, granisetron, ondansetron, tropissetron, alizapride, ramosetron, dolassetron mesylate, fosaprepitant dimeglumine, nabilone, aprepitant, dronabinol, TY-10721, lissuride hydrogenmaleate, epiceram, defibrotide, dabigatran etexilate, filgrastim, pegfilgrastim, reditux, epoetin, molgramostim, oprelvecin, sipuleucel-T, M-Vax, acetyl-L-carnitine, donepezil hydrochloride, 5-aminolevulinic acid, methyl aminolevulinate, cetrorelix acetate, icodextrin, leuprorelin, methylphenidate, octreotide, amlexanox, plerixafor, menatetrenone , anethole dithiolethione, doxercalciferol, cinacalcet hydrochloride, alefacept, romiplostim, thymoglobulin, thymalfasin, ubenime x, imiquimod, everolimus, sirolimus, H-101, lassofoxifene, trilostane, incadronate, gangliosides, pegaptanib octasodium, vertoporfin, minodronic acid, zoledronic acid, gallium nitrate, sodium alendronate, disodium etidronate, disodium pamidronate, dutasteride, sodium stibogluconate, armodafinil, dexrazoxane, amifostine, WF-10, temoporfin, darbepoetin alfa, anceestima, sargramostim, palifermin, R-744, nepidermin, oprelvecine, denileucine diftitox, chrysantaspass, busserelin, deslorelin, lanreotide, octreotide, pilocarpine, bosentan, calicheamicin, and maytan cyclonicate.
[00151] For more details, the person skilled in the art may consult the manual edited by the "Association Française des Enseignants de Chimie Therapeutique" and entitled "Traité de chimie thérapeutique, vol. 6, Medicaments antitumoraux et perspectives dans le traitement des cancers, TEC & DOC edition, 2003".
[00152] In a second preferred embodiment of the invention, the immunoconjugate comprises a binding protein bound to at least one radioisotope. Such an immunoconjugate is referred to as an antibody-radioisotope conjugate (or "CAR") when the binding protein is an antibody, or an antigen-binding fragment thereof.
[00153] For selective tumor destruction, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of CAR, such as, without limitation, At211, C13, N15, O17, Fl19, I123, I131, I125, In111, Y90, Re186, Re188, Sm153, tc99m, Bi212, P32 , Pb212, radioactive isotopes of Lu, gadolinium, manganese or iron.
[00154] Any methods or processes known to the person skilled in the art can be used to incorporate such radioisotopes into CAR (see, for example, "Monoclonal Antibodies in Immunoscintigraphy", Chatal, CRC Press, 1989). As a non-limiting example, tc99m or I123, Re186, Re188 and In111 can be linked via a cysteine residue. Y90 can be linked via a lysine residue. I123 can be ligated using the IODOGEN method (Fraker et al. (1978), Biochem. Biophys. Res. Commun. 80:49-57).
[00155] Several examples can be cited to illustrate the knowledge of the person skilled in the art in the field of CAR, such as Zevalin®, which is a CAR compound of an anti-CD20 monoclonal antibody and radioisotope In111 or Y90 linked by a ligand-chelator of thiourea (Wiseman et al. (2000), Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al. (2002), Blood 99 (12):4336-42; Witzig et al. (2002) , J. Clin. Oncol. 20(10):2453-63; Witzig et al. (2002), J. Clin. Oncol. 20(15):3262-69 ); or Mylotarg®, which is composed of an anti-CD33 antibody linked to calicheamicin (US 4,970,198, 5,079,233, 5,585,089, 5,606,040, 5,693,762, 5,739,116, 5,767,285, 5,773,001) . More recently, CAF referred to as Adcetris (corresponding to brentuximab vedotin) which was recently accepted by the FDA in the treatment of Hodgkin's lymphoma (Nature, vol. 476, pp. 380-381, Aug. 25, 2011) can also be mentioned.
[00156] In a third preferred embodiment of the invention, the immunoconjugate comprises a binding protein bound to at least one toxin. Such an immunoconjugate is referred to as an antibody-toxin conjugate (or "ATV") when the binding protein is an antibody, or an antigen-binding fragment thereof.
[00157] Toxins are effective and specific poisons produced by living organisms. They generally consist of a chain of amino acids that can vary in molecular weight between a couple of hundred (peptides) and a hundred thousand (proteins). They may also be low molecular weight organic compounds. Toxins are produced by numerous organisms, for example bacteria, fungi, algae and plants. Many of them are extremely poisonous, with a toxicity that is several orders of magnitude greater than that of nerve agents.
[00158] Toxins used in ATC can include, without limitation, all types of toxins that can exert their cytotoxic effects by mechanisms that include tubulin binding, DNA binding, or topoisomerase inhibition.
[00159] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, active non-binding diphtheria toxin fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, A chain of modecin, alpha-sarcin, Aleurites fordii proteins, diantin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaoria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin and the trichothecenes.
[00160] Small molecule toxins, such as dolastatins, auristatins, a trichothecene, and CC1065, and derivatives of these toxins that have toxin activity, are also contemplated herein. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division, and have anticancer and antifungal activity.
[00161] "Linker", "Binding Unit" or "bond" means a chemical moiety that comprises a covalent bond or a chain of atoms that covalently links a binding protein to at least one cytotoxic agent.
[00162] Linkers can be produced using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane -1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as such as bis(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds ( such as 1,5-difluoro-2,4-dinitrobenzene). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene-triaminopentacetic acid (MX-DTPA) is an example of a chelating agent for the conjugation of cytotoxic agents in the targeting system. Other crosslinking reagents can be BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfono)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).
[00163] The linker may be an "uncleavable" or "cleavable" linker.
[00164] In a preferred embodiment, the linker consists of a "cleavable linker" that facilitates the release of the cytotoxic agent into the cell. For example, an acid-labile linker, peptidase sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker may be used. The linker is, in a preferred embodiment, cleavable under intracellular conditions, such that cleavage of the linker releases the cytotoxic agent from the binding protein into the intracellular medium.
[00165] For example, in some embodiments, the linker is cleavable by a cleavage agent that is present in the intracellular environment (e.g., within a lysosome or endosome or caveola). The linker can be, for example, a peptidyl linker that is cleaved by a peptidase enzyme or intracellular protease, including, but not limited to, a lysosomal or an endosomal protease. Typically, the peptidyl linker is at least two amino acids in length or at least three amino acids in length. Cleavage agents may include cathepsins B and D and plasmin, which are all known to hydrolyze derivatives of dipeptide drugs, resulting in the release of active drug into target cells. For example, a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin B, which is highly expressed in cancer tissue (e.g., a Phe-Leu linker or a Gly-Phe-Leu-Gly linker) can be used. In specific embodiments, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker. An advantage of using proteolytic intracellular delivery of the cytotoxic agent is that the agent is typically attenuated when conjugated and the stability of the conjugates with respect to serum is generally high.
[00166] In other embodiments, the cleavable linker is pH sensitive, i.e. sensitive to hydrolysis at certain pH values. Typically, the pH sensitive binder is hydrolysable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in the lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal or the like) can be used. Such ligands are relatively stable under neutral pH conditions, such as those in blood, but are unstable under pH less than 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolysable linker is a thioether linker (such as, for example, a thioether linked to the therapeutic agent via an acylhydrazone linkage.
[00167] In still other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide ligands are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) , SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)toluene)-, SPDB and SMPT.
[00168] As a non-limiting example of non-cleavable linkers or "non-reducible" linkers, the immunoconjugate trastuzumab-DM1 (TDM1) which combines trastuzumab with an associated chemotherapy agent, maytansine can be mentioned (Cancer Research 2008; 68:(22) . November 15, 2008).
[00169] In a preferred embodiment, the immunoconjugate of the invention may be prepared by any method known to the person skilled in the art, such as, without limitation, i) reacting a nucleophilic group of the antigen-binding protein with a bivalent binding reagent, followed by reaction with the cytotoxic agent, or ii) the reaction of a nucleophilic group of a cytotoxic agent with a bivalent binding reagent, followed by reaction with the nucleophilic group of the antigen-binding protein.
[00170] Nucleophilic groups under the antigen-binding protein include, without limitation, N-terminus amine groups, side chain amine groups, e.g. lysine, side chain thiol groups, and sugar hydroxyl or amino groups, when the protein binding to the antigen is glycosylated. Amine, thiol and hydroxyl groups are nucleophilic groups and capable of reacting to form covalent bonds with electrophilic groups in linking moieties and linking reagents, including, without limitation, active esters such as NHS esters, HOBt esters, haloformates and halides of acid; alkyl and benzyl halides such as haloacetamides; aldehydes, ketones, carboxyl and maleimide groups. The antigen-binding protein may have reducible interchain disulfides, that is, cysteine bridges. Antigen binding proteins can be made reactive for conjugation with binding reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antigen-binding protein by any reaction known to the person skilled in the art. As a non-limiting example, reactive thiol groups can be introduced into the antigen-binding protein by introducing one or more cysteine residues.
[00171] Immunoconjugates can also be produced by modifying the antigen-binding protein to introduce electrophilic moieties, which can react with nucleophilic substituents on the binding reagent or cytotoxic agent. The sugars of antigen-binding glycosylated protein can be oxidized to form aldehyde or ketone groups that can react with the amine group of binding reagents or cytotoxic agent. The resulting Schiff imine base groups can form a stable bond or can be reduced to form stable amine bonds. In one embodiment, reaction of the carbohydrate moiety of a glycosylated antigen-binding protein with galactose oxidase or sodium metaperidate can yield carbonyl groups (aldehyde and ketone) on the protein, which can react with suitable groups on the protein. drug. In another embodiment, proteins containing N-terminus serine or threonine residues can react with sodium metaperiodate, resulting in the production of an aldehyde in place of the first amino acid.
[00172] In certain preferred embodiments, the binding unit may have the following general formula: --Ta--Ww--Yy-- where: -T- is an extension unit; a is 0 or 1; -W- is an amino acid unit; w is independently an integer ranging from 1 to 12; -Y- is a spacer unit; y is 0, 1 or 2.
[00173] The extension unit (-T-), when present, binds the antigen-binding protein to an amino acid unit (-W-). Useful functional groups that may be present on the antigen-binding protein, either naturally or by chemical manipulation, include sulfhydryl, amino, hydroxyl, the anomeric hydroxyl group of a carbohydrate, and carboxyl. Suitable functional groups are sulfhydryl and amino. Sulphydryl groups can be generated by reducing the intramolecular disulfide bonds of the antigen-binding protein, if present. Alternatively, sulfhydryl groups can be generated by reacting the amino group of a lysine portion of the antigen-binding protein with 2-iminothiolane or other sulfhydryl-generating reagents. In specific embodiments, the antigen-binding protein is a recombinant antibody and is engineered to transport one or more lysines. More preferably, the antigen-binding protein can be engineered to transport one or more cysteines (cf. ThioMabs).
[00174] In certain specific embodiments, the extension unit forms a bond with a sulfur atom of the antigen-binding protein. The sulfur atom may be derived from a sulfhydryl (-SH) group of a reduced group of an antigen-binding protein.
[00175] In certain other specific embodiments, the extension unit binds to the antigen-binding protein via a disulfide bond between a sulfur atom of the antigen-binding protein and a sulfur atom of the extension unit.
[00176] In other specific embodiments, the reactive group of the extension unit contains a reactive site that can be reactive with an amino group of the antigen-binding protein. The amino group may be that of an arginine or a lysine. Suitable amine reactive sites include, but are not limited to, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates.
[00177] In yet another aspect, the reactive function of the extension unit contains a reactive site that is reactive with a modified carbohydrate group that may be present on the antigen-binding protein. In a specific embodiment, the antigen-binding protein is enzymatically glycosylated to provide a carbohydrate moiety (note that when the antigen-binding protein is an antibody, the antibody is usually naturally glycosylated). The carbohydrate may be lightly oxidized with a reagent such as sodium periodate and the resulting carbonyl unit of the oxidized carbohydrate may be condensed with an extension unit that contains a functionality such as a hydrazide, an oxime, a reactive amine, a hydrazine , a thiosemicarbazide, a hydrazine carboxylate or an aryl hydrazide.
[00178] The amino acid unit (-W-) links the extension unit (-T) to the spacer unit (-Y-), if the spacer unit is present, and links the extension unit to the cytotoxic agent, if the spacer unit is present. is absent.
[00179] As mentioned above, -Ww- can be a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit.
[00180] In some embodiments, the amino acid moiety may comprise amino acid residues such as, without limitation, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, acetyl- or formyl-protected lysine, arginine, arginine protected with tosyl or nitro groups, histidine, ornithine, ornithine protected with acetyl or formyl and citrulline. Exemplary amino acid binding components preferably include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
[00181] Exemplary dipeptides include: Val-Cit, Ala-Val, Lys-Lys, Cit-Cit, Val-Lys, Ala-Phe, Phe-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit , Trp-Cit, Phe-Ala, Phe-N9-tosyl-Arg, Phe-N9-Nitro-Arg.
[00182] Exemplary tripeptides include: Val-Ala-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn-Phe-Phe-Lys, Gly-Gly-Gly, D-Phe-Phe-Lys , Gly-Phe-Lys.
Exemplary tetrapeptides include: Gly-Phe-Leu-Gly (SEQ ID NO: 33), Ala-Leu-Ala-Leu (SEQ ID NO: 34).
[00184] Exemplary pentapeptides include: Pro-Val-Gly-Val-Val (SEQ ID NO: 35).
[00185] Amino acid residues that comprise an amino acid binding component include naturally occurring ones as well as minor amino acids and non-naturally occurring amino acid analogues such as citrulline. Amino acid binding components can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C, and D, or a plasmin protease.
[00186] The amino acid unit of the linker may be enzymatically cleaved by an enzyme that includes, but is not limited to, a tumor-associated protease to release the cytotoxic agent.
[00187] The amino acid unit can be designed and optimized in its selectivity for enzymatic cleavage by a particular tumor-associated protease. Suitable moieties are those whose cleavage is catalyzed by the proteases, cathepsin B, C and D, and plasmin.
[00188] The spacer unit (-Y-), when present, attaches an amino acid unit to the cytotoxic agent. Spacer units are of two general types: self-immolating and non-self-immolating. A non-autoimolative spacer unit is one in which part or all of the spacer unit remains attached to the cytotoxic agent after enzymatic cleavage of an amino acid unit of the immunoconjugate. Examples of a non-self-immolating spacing unit include, but are not limited to, a spacing unit (glycine-glycine) and a glycine spacing unit. To release the cytotoxic agent, an independent hydrolysis reaction must take place within the target cell to cleave the glycine-drug moiety bond.
[00189] In another embodiment, a non-self-immolating (-Y-) spacer unit is -Gly-.
[00190] In one embodiment, the immunoconjugate lacks a spacer unit (y = 0). Alternatively, an immunoconjugate containing an autoimmolative spacer unit can release the cytotoxic agent without the need for a separate hydrolysis step. In these embodiments, -Y- is a p-aminobenzyl alcohol (PAB) moiety that bonds to -Ww- via the nitrogen atom of the PAB group, and bonds directly to -D via a carbonate group, carbamate or ether.
[00191] Other examples of self-immolating spacers include, but are not limited to, aromatic compounds that are electronically equivalent to the PAB group, such as 2-aminoimidazole-5-methanol derivatives and ortho or para-aminobenzyl acetals. Spacers that undergo easy cyclization after hydrolysis of the amide bond may be used, such as substituted and unsubstituted 4-aminobutyric acid amides, suitably substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems and amides of 2-aminophenylpropionic acid.
[00192] In an alternative embodiment, the spacing unit is a branched bis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporate additional cytotoxic agents.
[00193] Finally, the invention relates to an immunoconjugate as described above for use in the treatment of cancer.
[00194] Cancers can be preferentially selected from Axl-related cancers, including tumor cells that express or overexpress all or part of the Axl protein on their surface.
[00195] More particularly, these cancers are breast, colon, esophageal carcinoma, hepatocellular, gastric, glioma, lung, melanoma, osteosarcoma, ovarian, prostate, rhabdomyosarcoma, renal, thyroid, endometrial cancer uterus and any drug resistance phenomenon. Another object of the invention is a pharmaceutical composition comprising the immunoconjugate as described in the specification.
[00196] More particularly, the invention relates to a pharmaceutical composition comprising the immunoconjugate of the invention with at least one pharmaceutically acceptable excipient and/or carrier.
[00197] In the present description, the expression "pharmaceutically acceptable carrier" or "excipient" is intended to indicate a compound or a combination of compounds that enter a pharmaceutical composition without causing side reactions and that allow, for example, to facilitate the administration of the active compound(s), an increase in its shelf life and/or its effectiveness in the body, an increase in its solubility in solution, or an improvement in its conservation. Such pharmaceutically acceptable carriers and excipients are well known and will be adapted by the person skilled in the art depending on the nature and mode of administration of the active compound(s) chosen.
[00198] Preferably, such immunoconjugates will be administered systemically, in particular intravenously, intramuscularly, intradermally, intraperitoneally or subcutaneously, or orally. More preferably, the composition comprising the immunoconjugates according to the invention will be administered several times, sequentially.
[00199] Its modes of administration, dosages and optimal pharmaceutical forms can be determined according to criteria generally taken into account when establishing a treatment adapted to a patient, such as, for example, the age or body weight of the patient, the severity of your general condition, treatment tolerance, and observed side effects.
[00200] Other features and advantages of the invention appear in the continuation of the description with the examples and figures whose legends are represented below. PICTURE'S DESCRIPTION
[00201] Figure 1: In vitro cytotoxicity assay using mAb-zap conjugated secondary antibody in SN12C cells.
[00202] Figures 2A, 2B and 2C: Binding specificity of 1613F12 on immobilized rhAxl-Fc protein (2A), rhDtk-Fc protein (2B) or rhMer-Fc protein (2C) by ELISA.
[00203] Figure 3: FACS analysis of 1613F12 binding in human tumor cells.
[00204] Figure 4: ELISA of immobilized protein rmAxl-Fc ("rm" from murine recombinant).
[00205] Figure 5: Binding of 1613F12 in COS7 cells as determined by indirect labeling protocol using flow cytometry method.
[00206] Figure 6: Gas6 binding ELISA competition using 1613F12.
[00207] Figure 7: Western blot epitope binding analysis using SN12C cell lysate. NH (no heat); NR (no reduction); H (heat); R (reduction). GAPDH detection attests to the correct sample loading on the gel.
[00208] Figures 8A and 8B: Study of Axl inhibition after binding of 1613F12 in SN12C cells by Western blot with Figure 8 - Western blot image representative of three independent experiments performed (Western blot analysis was performed after an incubation of 4 h and 24 h of 1613F12 in SN12C cells); and Figure 8B - Quantification of optical density of the presented film using the "QuantityOne" software.
[00209] Figures 9A, 9B and 9C: Immunofluorescence microscopy of SN12C cells after incubation with 1613F12. Figure 9A - Photographs of mIgG1 isotype control conditions for both membrane and intracellular staining. Figure 9B - Membrane staining. Figure 9C - Intracellular staining of both the Axl receptor using 1613F12 and the initial endosome EEA1 marker. Image overlays are shown below and visualized co-locations are indicated by arrows.
[00210] Figure 10: Effect of 1613F12 on in vitro proliferation of SN12C cells compared to the effect of mIgG1 isotype control antibody.
[00211] Figures 11A-11K: Direct cytotoxicity assays of the 1613F12-saporin immunoconjugate using various human tumor cell lines. A-SN12C, B-Calu-1, C-A172, D-A431, E-DU145, F-MDA-MB435S, G-MDA-MB231, H-PC3, I-NCI-H226, J-NCI-H125, K- Panc1.
[00212] Figure 12: ELISA experiments studying rhAxl-Fc protein binding of both m1613F12 and hz1613F12 antibodies.
[00213] Figure 13: Comparison of binding of murine, chimeric and humanized 1613F12 antibodies on SN12C cells.
[00214] Figure 14: Direct cytotoxicity assay in the presence of both the mouse and humanized immunoconjugate 1613F12-saporin and isotype controls using SN12C human renal tumor cell line.
[00215] Figure 15: Direct cytotoxicity assay in the presence of both the mouse and humanized immunoconjugate 1613F12-saporin and isotype controls using Calu-1 human lung carcinoma cell line. EXAMPLES
[00216] In the following examples, the terms 1613F12 antibody or m1613F12 antibody refer to a murine form of the 1613F12 antibody. Humanized forms of the 1613F12 antibody are called hz1613F12.
[00217] Likewise, the used isotype control antibody consists of a murine IgG1 referred to as 9G4. This means that, in the examples that follow, the control expressions of mIgG1 and 9G4 are similar. Example 1: Axl receptor internalization
[00218] As an immunoconjugate approach is more efficient when the target antigen is an internalizing protein, internalization of the Axl receptor was studied using mAb-Zap cytotoxicity assay in human tumor cell lines. More precisely, the mAb-Zap reagent is a chemical conjugate that includes an affinity purified goat anti-mouse IgG and the ribosome inactivating protein, saporin. If internalization of the immune complex occurs, saporin breaks away from the targeting agent and inactivates ribosomes, resulting in inhibition of protein synthesis and ultimately cell death. Determination of cell viability after 72 hours of incubation with the 1613F12 isotype control antibody or mIgG1 in Axl-positive cells allows us to conclude on the internalization of the Axl receptor induced by 1613F12.
[00219] For this example, cells highly positive for Axl were used, as determined using Qifikit reagent (Dako). The data are presented in table 5 below. Table 5

[00220] In the following example, cells SN12C were used as a non-limiting example. Any other cell line that expresses the appropriate level of Axl receptor on its cell surface can be used.
[00221] Control antibody concentration ranges of isotype 1613F12 or mIgG1 were pre-incubated with 100 ng of secondary antibody mAb-Zap (advanced targeting systems) in cell culture medium for 30 min at RT. These mixtures were loaded onto subconfluent SN12C cells plated in 96-well white plate microplates. The plates were incubated for 72 h at 37°C in the presence of 5% CO 2 . Cell viability was determined using a Cell Titer Glo cell proliferation method according to the manufacturer's instructions (Promega). Several controls are performed: i) without any secondary immunoconjugate and ii) without primary antibody. In parallel, assays are performed with a mIgG1 isotype control.
[00222] The results obtained are represented in Figure 1.
[00223] 1613F12 shows a maximal cytotoxic effect on SN12C cells of ~36%. No cytotoxic effect was observed in the presence of the 9G4 antibody, considered as a mIgG1 isotype control in the experiment. No cytotoxicity was observed in wells containing only primary antibodies (data not shown). Thus, the Axl receptor appears to be a convenient antigen to guide an immunoconjugate approach, as the immune complex comprising Axl-1613F12-mAbZap triggers effective cytotoxicity of target cells. Example 2: Generation of an antibody against rhAxl DEC.
[00224] To generate murine monoclonal antibodies (mAbs) against the human extracellular domain (ECD) of the Axl receptor, five BALB/c mice were immunized five times s.c. with 15-20 x 10 6 CHO-Axl cells and twice with 20 µg rhAxl DEC. The first immunization was performed in the presence of Complete Freund's Adjuvant (Sigma, St. Louis, MD, USA). Incomplete Freund's Adjuvant (Sigma) was added for later immunizations.
[00225] Three days prior to fusion, immunized mice were stimulated with both 20 x 10 6 CHO-Axl cells and 20 µg rhAxl ECD with IFA.
[00226] To generate hybridomas, splenocytes and lymphocytes were prepared by perfusing the spleen and mincing the proximal lymph nodes, respectively, harvested from 1 of 5 immunized mice (selected after titration of sera) and fused with Sp2/0-Agl4 myeloma cells (ATCC, Rockville, MD, USA). The fusion protocol is described by Kohler and Milstein (Nature, 256:495-497, 1975). The fused cells are then subjected to selection with HAT. In general, for the preparation of monoclonal antibodies or their functional fragments, especially of murine origin, it is possible to refer to techniques that are described in particular in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 726, 1988).
[00227] Approximately ten days after fusion, colonies of hybrid cells were screened. For the first screen, hybridoma supernatants were evaluated for secretion of mAbs raised against the DEC Axl protein using an ELISA. In parallel, a SCAF analysis was performed to select mAbs capable of binding to the cellular form of Axl present on the cell surface, using both wt CHO cells and CHO cells expressing Axl (ATCC).
[00228] As quickly as possible, selected hybridomas were cloned by limiting dilution and subsequently screened for their reactivity against the DEC Axl protein. Cloned mAbs were then isotyped using an isotyping kit (cat. No. 5300.05, Southern Biotech, Birmingham, AL, USA). One clone obtained from each hybridoma was selected and amplified.
[00229] ELISA assays were performed as follows using pure hybridoma supernatant, or, when the IgG content in the supernatants was determined, titration was performed from 5 µg/ml. Then, a serial dilution was performed on the next 11 rows.
[00230] Briefly, 96-well ELISA plates (Costar 3690, Corning, NI, USA) were coated with 50 µL/well of rhAxl-Fc protein (Systems R and D, Cat. No. 154-AL) or rhAxl DEC under 2 µg/ml in PBS overnight at 4°C. The plates were then blocked with PBS containing 0.5% gelatin (No. 22151, Serva Eletrophoresis GmbH, Heidelberg, Germany) for 2 h at 37°C. Once the saturation buffer was discarded by rapidly shaking the plates, 50 µL of pure hybridoma cell supernatants or 50 µL of a 5 µg/mL solution were added to the ELISA plates and incubated for 1 h at 37°C. After three washes, 50 µL of horseradish peroxidase-conjugated goat anti-mouse polyclonal IgG (No. 115-035-164, Jackson Immuno-Research Laboratories, Inc., West Grove, PA, USA) was added at a 1/2 dilution. 5,000 in PBS containing 0.1% gelatin and 0.05% (w:w) Tween 20 for 1 h at 37°C. Then, the ELISA plates were washed three times and TMB substrate (No. UP664782, Uptima, Interchim, France) was added. After an incubation time of 10 min at room temperature, the reaction was stopped using 1 M sulfuric acid and the optical density at 450 nm was measured.
[00231] For selection by flow cytometry, 105 cells (wt CHO or CHO-Axl) were plated in each well of a 96-well plate, in PBS containing 1% BSA and 0.01% sodium azide ( SCAF buffer) at 4°C. After two minutes of centrifugation at 2000 rpm, the buffer was removed and hybridoma supernatants or purified mAbs (1 µg/ml) to be tested were added. After 20 min incubation at 4°C, cells were washed twice and an Alexa 488-conjugated goat anti-mouse antibody diluted 1/500° in FACS buffer (No. A11017, Molecular Probes Inc., Eugene, USA) was added. and incubated for 20 minutes at 4°C. After a final wash with FACS buffer, cells were analyzed by FACS (FACSCalibur, Becton-Dickinson), after addition of propidium iodide to each tube, under a final concentration of 40 μg/mL. Wells containing isolated cells and cells incubated with the Alexa 488-conjugated secondary antibody were included as negative controls. Isotype controls were used in each experiment (Sigma, ref. M90351MG). At least 5,000 cells were evaluated to calculate the mean fluorescence intensity (MFI) value.
[00232] More precisely, the fusion was performed with 300 x 10 6 harvested splenocytes and 300 x 10 6 myeloma cells (ratio 1:1). Two hundred cells from the resulting cell suspension were then plated at 2 x 10 6 cells/ml in 30 96-well plates.
[00233] The first search (around the 14th day after fusion) both by ELISA on the rhAxl DEC protein and by SCAF analysis using both wt CHO cells and CHO cells expressing Axl allowed to select ten hybridomas with higher optical densities (ODs) at 1 on rhAxl DEC coating, IMF below 50 in wt CHO cells and above 200 in CHO-Axl cells.
[00234] These ten hybridomas were expanded and cloned by limiting dilution. A 96-well plate was prepared for each code. Nine days after plating, supernatants from cloning plates were first screened by ELISA for their binding specificity for the extracellular domain of the ECD rhAxl protein. Three clones of each code were expanded and isotyped. Once produced, anti-Axl antibodies were further studied for their ability to be internalized after binding of Axl to the cell surface. Example 3: Axl binding specificity
[00235] In this example, binding of 1613F12 was studied first with the rhAxl-Fc protein. Then, its binding with the other two members of the TAM family, rhDtk-Fc and rhMer-Fc, was studied.
[00236] Briefly, human recombinant proteins Axl-Fc (R and D systems, cat. No. 154AL/CF), rhDtk (R and D Systems, cat. No. 859-DK) or rhMer-Fc (R and D, Cat. No. 891-MR) were coated overnight at 4°C in 96-well Immulon II plates, and, after a 1 h blocking step with a 0.5% gelatin solution, purified antibody. 1613F12 was added for an additional 1 h at 37°C, under an initial concentration of 5 μg/mL (3.33 10-8 M). Then serial dilutions were performed on 12 columns. The plates were washed and a specific goat anti-mouse IgG-HRP (Jackson) was added for 1 hour at 37°C. The development of the reaction was carried out using the TMB substrate solution. The isotype control mIgG1 antibody and the commercial anti-Axl mAb 154 antibody were also used in parallel. Coating controls were performed in the presence of an HRP-labeled goat anti-human IgG Fc polyclonal serum (Jackson, ref. 109-035-098) and/or in the presence of an HRP-coupled anti-histidine antibody (R Systems and D, ref.: MAB050H). The results are shown in Figures 2A, 2B and 2C, respectively.
[00237] This example shows that the 1613F12 antibody only binds to the rhAxl-Fc protein and does not bind to the two other members of the TAM family, rhDtk or rhMer. No cross-binding specificity of the 1613F12 antibody is observed among the TAM members. No non-specific binding was observed in the absence of primary antibody (diluent). No binding was observed in the presence of the isotype control antibody. Example 4: 1613F12 recognized the cellular form of Axl expressed in tumor cells.
[00238] Axl expression level on the surface of human tumor cells was first established using a commercial Axl antibody (R and D Systems, ref: MAB154) in parallel with calibration beads to allow quantification of the Axl expression level. Second, cell surface binding of Axl was studied using 1613F12.
[00239] For cell surface binding studies, two double serial dilutions of 10 μg/mL primary antibody solution (6.66 x 10-8 M), (1613F12, MAB154 antibody or mIgG1 isotype control 9G4 mAb ) are prepared and applied to 2 x 105 cells for 20 min at 4°C. After three washes in phosphate-buffered saline (PBS) supplemented with 1% BSA and 0.01% NaN3, cells were incubated with Alexa 488 goat anti-mouse secondary antibody (1/500° dilution) for 20 minutes at 4°C. After three additional washes in PBS supplemented with 1% BSA and 0.1% NaN3, cells were analyzed by FACS (FACSCalibur, Becton-Dickinson). At least 5,000 cells were evaluated to calculate the mean value of fluorescence intensity.
[00240] For quantitative determination of ABC using MAB154 antibody, QIFIKIT® calibration beads are used. The cells are then incubated, in parallel with the QIFIKIT® beads, with goat anti-mouse/FITC, goat F(ab')2 polyclonal immunoglobulins at saturation concentration. The number of antigenic sites on the sampled cells is then determined by interpolation of the calibration curve (the fluorescence intensity of the individual bead populations versus the number of mAb molecules in the beads). 4.1. Quantification of Axl expression level on cell surface
[00241] Axl expression level on the surface of human tumor cells was determined by flow cytometry using indirect immunofluorescence assay (QIFIKIT® method (Dako, Denmark)), a quantitative flow cytometry kit for evaluating cell surface antigens. A comparison of the mean fluorescence intensity (MFI) of the beads' known antigen levels via a calibration plot allows the determination of the antibody binding capacity (ABC) of the cell lines.
[00242] Table 6 shows Axl expression levels detected on the surface of various human tumor cell lines (SN12C, Calu-1, A172, A431, DU145, MDA-MB435S, MDA-MB231, PC3, NCI-H226, NCI -H125, MCF7, Panc 1) (ATCC, NCI), as determined using QIFIKIT®, using the commercial antibody MAB154 (Systems R and D). Values are given as antigen binding complex (ABC). Table 6

[00243] The results obtained with a commercial monoclonal antibody Axl (MAB154) showed that the Axl receptor is expressed at various levels depending on the human tumor cell considered. 4.2. Detection of Axl by 1613F12 in human tumor cells
[00244] More specifically, Axl binding was studied using 1613F12.
[00245] Dose-response curves of 1613F12 were prepared. IMFs obtained using the various human tumor cells were then analyzed with Prism software. The data are presented in Figure 3.
[00246] The data indicate that 1613F12 specifically binds to the membrane Axl receptor as evidenced by the saturation curve profiles. However, different labeling intensities were observed, revealing varying levels of Axl receptor on the surface of human tumor cells. No Axl receptor binding was observed using MCF7 human breast tumor cell line. Example 5: Cross-species specificity of 1613F12
[00247] To address the cross-species specificity of 1613F12, two species were considered: mouse and monkey. First, binding on the mouse recombinant Axl receptor (rm) is studied by ELISA (Figure 4). Then, flow cytometry experiments were performed using monkey COS7 cells, since these cells express the Axl receptor on their surface (Figure 5). The COS7 cell line was obtained by immortalization of a CV-1 cell line derived from African green monkey kidney cells, with a version of the SV40 genome that can produce the large T antigen but has a defect in genomic replication. rmAxl-Fc ELISA
[00248] Briefly, mouse recombinant Axl-Fc proteins (Systems R and D, Cat. No. 854-AX/CF) were coated overnight at 4°C in 96-well Immulon II plates and, after a After 1 h blocking with a 0.5% gelatin solution, the purified antibody 1613F12 was added for an additional hour at 37°C under an initial concentration of 5 mg/mL (3.33 x 10 -8 M). Then serial dilutions were performed on 12 columns. The plates were then washed and a specific goat anti-mouse IgG HRP (Jackson) was added for 1 hour at 37°C. The development of the reaction was carried out using the TMB substrate solution. The mIgG1 isotype control and the commercial antibody mAb 154 are also used in parallel. Coating controls are performed in the presence of an HRP-coupled goat anti-human IgG-Fc polyclonal serum (Jackson, ref. 109-035-098) and/or in the presence of an HRP-coupled anti-histidine antibody (Systems R and D, ref.: MAB050H).
[00249] The results are represented in Figure 4. This figure shows that 1613F12 does not bind to the murine DEC Axl domain. No specific binding is observed in the absence of primary antibody (diluent). COS7 SCAF
[00250] For cell binding studies of 1613F12 using COS7 cells, 2 x 10 5 cells were incubated with a range of antibody concentrations prepared by serial dilution ^ (12 points) of a 10 μg/mL antibody solution (6, 66 x 10 -8 M) of 1613F12 or mIgG1 isotype mAb control for 20 min at 4°C. After three washes in phosphate-buffered saline (PBS) supplemented with 1% BSA and 0.01% NaN3, cells were incubated with Alexa 488 goat anti-mouse secondary antibody (1/500 dilution) for 20 minutes at 4°C. . After three additional washes in PBS supplemented with 1% BSA and 0.1% NaN3, cells were analyzed by FACS (FACSCalibur, Becton-Dickinson). At least 5,000 cells were evaluated to calculate the mean value of fluorescence intensity. Data are analyzed using Prism software.
[00251] The results are represented in Figure 5. The titration curve established on COS7 cells using 1613F12 confirms that 1613F12 is able to recognize the monkey cell form of the Axl receptor expressed on the surface of COS7 cells. Plateau is reached by concentrations of 1613F12 above 0.625 μg/mL (4.2 x 10-10 M). No binding is observed in the presence of the mIgG1 isotype control.
[00252] This example illustrates the fact that 1613F12 does not cross-react with the rat Axl receptor. In contrast, it strongly binds to the monkey Axl receptor expressed on the surface of COS7 cells. Example 6: Gas6 competition experiments performed in the presence of 1613F12
[00253] To further characterize 1613F12, competition tests of Gas6 were performed. In this assay, free rhAxl-Fc protein and 1613F12 are incubated to form antigen-antibody complexes and then the complexes are loaded onto the surface of the Gas6 coated assay plate. Unbound antibody-antigen complexes are washed before addition of enzyme-linked secondary antibody against the human Fc portion of the rhAxl-Fc protein. Substrate is then added and the concentration of antigen can be determined by the strength of the signal caused by the enzyme-substrate reaction.
[00254] Briefly, reaction mixtures comprising the rhAxl-Fc protein in the presence or absence of the anti-Axl mAbs to be tested are prepared on a separate saturated plate (0.5% gelatin in PBS IX). Serial 1:2 dilutions (starting from 80 μg/mL in 12 columns) of murine anti-Axl antibodies are performed. Then, 0.5 μg/mL of the rhAxl-Fc protein is added (R and D systems, ref. 154AL/CF), except for the negative control strain which contains only ELISA diluent (0.1% gelatin, Tween 20 to 0.05% in 1X PBS). After homogenization, the competition samples are loaded onto Gas6 coated plates with a 6 µg/mL solution of rhGas6 in PBS (Systems R and D, Cat. No. 885-GM-CS/CF). After incubation and several washes, bound rhAxl-Fc proteins are detected using a goat anti-human IgG-HRP antibody (Jackson, ref. 109-035098). Once bound, TMB substrate is added to the plates. The reaction is stopped by adding 1M H2SO4 acid solution and the optical densities obtained read at 450 nm using a microplate reading instrument.
[00255] This experiment (Figure 6) shows that 1613F12 is able to compete with rhAxl-Fc binding on its immobilized ligand. Competition with Gas6 binding occurs in the presence of 1613F12 antibody concentrations above 2.5 μg/mL (1.67 x 10 -8 M). No further binding of rhAxl-Fc on immobilized Gas6 is observed in the presence of a concentration of 1613F12 above 10 µg/mL (6.67 x 10 -8 M). 1613F12 blocks binding of Gas6 to rhAxl-Fc. Example 7: Recognition of epitopes by Western blot
[00256] To determine whether 1613F12 recognizes a linear epitope or a conformational epitope, Western blot analysis was performed using SN12C cell lysates. Samples were treated differently to be in reducing or non-reducing conditions. If a band is visualized with reduced sample, the tested antibody reaches a linear epitope of the ECD domain; if not, it is fired against a conformation epitope of DEC Axl.
[00257] SN12C cells were seeded in RPMI medium + heat-inactivated 10% FBS + 2 mM L-glutamine under 5 x 104 cells/cm2 in T162 cm2 flasks for 72 hours at 37°C in a 5% CO2 atmosphere . Then, the cells were washed twice with phosphate-buffered saline (PBS) and lysed with 1.5 ml of ice-cold lysis buffer [50 mM Tris-HCl (pH 7.5); 150 mM NaCl; 1% Nonidet P40; 0.5% deoxycholate; and one tablet of complete protease inhibitor cocktail plus 1% antiphosphatases]. Cell lysates were shaken for 90 min at 4°C and cleaned at 15,000 rpm for 10 min. Protein concentration was quantified using BCA. Several samples were loaded. First, 10 μg of whole cell lysate (10 μg in 20 μL) were prepared under reducing conditions (1x sample buffer (BIORAD) + 1x reducing agent (BIORAD)) and loaded onto SDS-PAGE after 2 min incubation. at 96°C. Second, two other 10 μg samples of whole cell lysate were prepared under non-reducing conditions (in 1x sample buffer (BIORAD) only). Before being loaded onto the SDS-PAGE gel, one of these last two examples is heated under 2 min incubation at 96°C; the other is kept on ice. After migration, the proteins are transferred to a nitrocellulose membrane. Membranes were saturated for 1 h at RT with 0.1% TBS-Tween 20 (TBST), 5% non-fat milk and probed with 1613F12 at 10 µg/ml overnight at 4°C. Antibodies were diluted in 0.1% (v/v) Tris-Tween 20 buffered saline solution (TBST) with 5% fat-free powdered milk. Then, membranes were washed with TBST and incubated with peroxidase-conjugated secondary antibody (1/1000 dilution) for 1 h at RT. Immunoreactive proteins were visualized with ECL (Pierce No. 32209). After visualization of Axl, membranes were washed again with TBST and incubated for 1 h at RT with anti-mouse anti-GAPDH antibody (dilution 1/200,000). Then, the membranes were washed in TBST and incubated with peroxidase-conjugated secondary antibodies for 1 h at RT. Membranes were washed and GAPDH was developed using ECL.
[00258] The results are represented in Figure 7.
[00259] 1613F12 primarily recognizes a conformational epitope, as a specific band is essentially observed under non-reduced conditions. However, a weak signal is detected under the denaturing migration conditions of the SN12C cell lysate, indicating that 1613F12 is capable of weakly binding a linear epitope. Example 8: Measurement of Axl Inactivation Triggered by 1613F12 by Western Blot.
[00260] In the following example, the human renal cell carcinoma (ATCC) cell line SN12C was selected to direct Axl antibody activity on Axl receptor expression. The SN12C cell line overexpresses the Axl receptor. Axl inactivation was studied by Western blot on whole cell extracts in Figures 8A-8B.
[00261] SN12C cells were seeded in RPMI medium + 10% heat-inactivated FBS + 2 mM L-glutamine under 6 x 10 4 cells/cm2 in six-well plates for 48 h at 37°C in a 5% CO2 atmosphere. After two washes with physiological phosphate buffer solution (PBS), cells were serum starved in a medium containing 800 ng/mL of recombinant mouse Gas6 ligand (Systems R and D, ref: 986-GS/CF), or 10 μg/mL of an isotype control antibody mIgGI (9G4) or 10 μg/mL of the Axl antibody of the present invention, and incubated for an additional 4 hours or 24 hours. Then, the medium was gently removed and the cells washed twice with cold PBS. Cells were lysed with 200 µL of ice-cold lysis buffer [50 mM Tris-HCl (pH 7.5); 150 mM NaCl; 1% Nonidet P40; 0.5% deoxycholate; and one tablet of complete protease inhibitor cocktail plus 1% antiphosphatases]. Cell lysates were shaken for 90 min at 4°C and cleaned at 15,000 rpm for 10 min. Protein concentration was quantified using the BCA method. Whole cell lysates (10 μg in 20 μL) were prepared by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were saturated for 1 h at RT with 0.1% TBS-Tween 20 (TBST), 5% non-fat milk and probed with a commercial M02 Axl antibody at 0.5 μg/mL (AbNova H00000558-M02) for overnight at 4°C. Antibodies were diluted in 0.1% (v/v) Tris-Tween 20 buffered saline solution (TBST) with 5% fat-free powdered milk. Then, membranes were washed with TBST and incubated with peroxidase-conjugated secondary antibody (1/1000 dilution) for 1 h at RT. Immunoreactive proteins were visualized with ECL (Pierce No. 32209). After visualization of Axl, membranes were washed again with TBST and incubated for 1 h at RT with anti-mouse anti-GAPDH antibody (dilution 1/200,000). Then, the membranes were washed in TBST and incubated with peroxidase-conjugated secondary antibodies for 1 h at RT. Membranes were washed and GAPDH was developed using ECL. The intensity of the bands was quantified by densitometry.
[00262] The results presented in Figures 8A and 8B are representative of three independent experiments and demonstrate that 1613F12 is able to inactivate Axl in a human tumor cell line that overexpresses Axl. At 4 hours, 1613F12 triggers an inactivation of 66% of Axl, and up to 87% after a 24-hour incubation with 1613F12. Example 9: Flow cytometric study of the effect of 1613F12 on cell surface Axl expression
[00263] Flow cytometry technique allows labeling of Axl receptor on cell surface. The use of this technique can highlight the effect of antibodies on the expression of Axl in membrane. Human kidney tumor SN12C cells expressing high levels of Axl were used in this example.
[00264] Tumor cell line SN12C grown in RMPI1640 with 1% L-glutamine and 10% FCS for 3 days prior to the experiment. Cells were then separated using trypsin and plated in a 6-well multi-well plate in RPMI1640 with 1% L-glutamine and 5% FBS. The next day, antibodies of interest were added at 10 μg/mL. Untreated wells were also included. Cells are incubated at 37°C, 5% CO 2 . Twenty-four hours later, cells were washed with PBS, sorted and incubated with the same antibodies of interest in FACS buffer (PBS, 1% BSA, 0.01% sodium azide). Untreated wells were also stained with the same antibody in order to compare the signal intensity obtained with the same mAb in treated and untreated cells. Cells were incubated for 20 minutes at 4°C and washed three times with FACS buffer. An Alexa 488-labeled goat anti-mouse IgG antibody was incubated for 20 minutes and the cells were washed three times prior to FACS analysis on propidium iodide negative cell population.
[00265] Two parameters are determined: (i) the difference in fluorescent signal detected on the surface of untreated cells (without Ab) compared to Ab treated cells on T24 h and (ii) the percentage of Axl remaining on the cell surface . The percentage of Axl remaining is calculated as follows: % remaining Axl = (IMFAb 24h/IMF without Ab 24h) x 100
[00266] Data from a representative experiment are presented in Table 7. The results were reproduced in three independent experiments.
[00267] The IMF difference between staining a mAb in untreated cells and the condition treated with the same antibody reflects an inactivation of the Axl protein on the surface of the cells, due to binding of the considered mAb. Conditions without antibody gave similar results to the condition in the presence of the isotype control antibody (m9G4). Table 7

[00268] The data demonstrate that the average fluorescence intensity detected on the surface of 1613F12-treated cells over 24 hours is reduced (-514) compared to the IMFs obtained with untreated 1613F12-labeled cells. After 24 h of incubation with the 1613F12 antibody, 45.2% of the cell surface Axl receptor remains on the surface of SN12C cells. Example 10: Internalization Study of 1613F12 Using Fluorescent Immunocytochemical Labeling
[00269] Complementary internalization results are obtained by confocal microscopy using indirect fluorescent labeling method.
[00270] Briefly, tumor cell line SN12C was cultured in RMPI1640 with 1% L-glutamine and 10% FCS for three days prior to the experiment. Cells were then separated using trypsin and plated in a six-well multi-well plate containing RPMI 1640 with 1% L-glutamine and 5% FCS. The next day, 1613F12 was added at 10 μg/mL. Cells treated with an irrelevant antibody were also included. Cells were then incubated for 1 h and 2 h at 37°C, 5% CO 2 . For T 0 h, cells were incubated for 30 minutes at 4°C for the determination of antibody binding to the cell surface. Cells were washed with PBS and fixed with paraformaldehyde for 15 minutes. Cells were washed lightly and incubated with a goat anti-mouse IgG Alexa 488 antibody for 60 minutes at 4°C to identify remaining antibody on the cell surface. To follow the penetration of antibody into cells, cells were fixed and permeabilized with saponin. A goat anti-mouse IgG Alexa 488 antibody (Invitrogen) was used to stain both the membrane and the intracellular antibody. Early endosomes were identified using a polyclonal rabbit antibody against EEA1 stained with a goat anti-rabbit IgG-Alexa 555 antibody (Invitrogen). Cells were washed three times and nuclei were stained with Draq5. After staining, cells were mounted in Prolong Gold mounting medium (Invitrogen) and analyzed using a Zeiss LSM 510 confocal microscope.
[00271] The photographs are shown in Figures 9A-9C.
[00272] Images were obtained by confocal microscopy. In the presence of the I isotype control (9G4), neither membrane staining nor intracellular staining is observed (Figure 9A). Progressive loss of anti-Axl membrane labeling is observed shortly after 1 h of incubation of SN12C cells with 1613F12 (Figure 9B). Intracellular accumulation of the 1613F12 antibody is clearly observed at 1 h and 2 h (Figure 9C). Intracellular antibody colocalizes with EEA1, an early endosome marker. These photographs confirm the internalization of 1613F12 into SN12C cells. Example 11: Anti-Axl-mediated in vitro antitumor activity SN12C Proliferation Assay
[00273] Ten thousand SN12C cells per well were seeded in medium without FCS in 96-well plates overnight at 37°C in a 5% CO2 atmosphere. The next day, cells were pre-incubated with 10 µg/ml of each antibody for 1 h at 37°C. Cells were treated with or without rmGas6 (Systems R and D, Cat. No. 986-GS/CF), by adding the ligand directly into the well, and then allowed to grow for 72 h. Proliferation was measured after incorporation of 3H thymidine.
[00274] Data are shown in Figure 10. No effect was observed with 1613F12 which is silent when added to SN12C cells. Example 12: Potency of 1613F12-saporin immunoconjugate cytotoxicity on various human tumor cell lines
[00275] In the present example, the potency of saporin-coupled 1613F12 cytotoxicity is documented. For this purpose, direct in vitro cytotoxicity assays were performed using a large panel of human tumor cell lines (Figures 11A-11K). This tumor cell lineage panel offers multiple cell surface expressions of Axl.
[00276] Briefly, 5,000 cells were seeded in 96-well culture plates in 100 µL of suitable culture medium (OD) 5% FBS. After 24 hours of incubation in a 5% CO2 atmosphere at 37°C, a range of immunoconjugate concentrations (1613F12-saporin or 9G4-saporin or 1613F12 or 9G4 nude) is applied to the cells. The culture plates are then incubated at 37°C in a humidified atmosphere incubator with 5% CO 2 for 72 hours.
[00277] At D4, cell viability is assessed using the CellTiter-Glo® Luminescent Cell Viability kit (Promega Corp., Madison, Wisconsin) which allows to determine the number of viable cells in culture based on the quantification of ATP present, an indicator of metabolically active cells. Luminescent emissions are recorded by a luminometer device.
[00278] From the luminescence output, the percentage of cytotoxicity is calculated using the following formula: % cytotoxicity = 100 - [(RLU Ab-sap x 100)/RLU No Ab]
[00279] Figures 11A-11K show graphs showing percentage cytotoxicity as a function of immunoconjugate concentration obtained in different in vitro cell cytotoxicity assays with (A) SN12C, (B) Calu-1, (C) A 172 , (D) A431, (E) DU145, (F) MDA-MB-435S, (G) MDA-MB-231, (H) PC3, (I) NCI-H226, (J) NCI-H125 or (K ) Panc1 tumor cells treated with a range of concentrations of 1613F12-saporin immunoconjugate.
[00280] Figures 11A-11K show that the 1613F12-saporin immunoconjugate triggered cytotoxicity in these different human tumor cell lines. The potency of the resulting cytotoxic effect depends on the human tumor cell line. Example 13: Humanization of 1613F12 Antibody Variable Domains
[00281] The use of mouse antibodies (mAbs) for therapeutic applications in humans often results in a major adverse effect, giving patients a human anti-mouse antibody (HAMA) response, thus reducing the effectiveness of treatment and preventing continued administration . One approach to overcome this problem is to humanize mouse mAbs, replacing mouse sequences with their human counterparts, but without altering antigen-binding activity. This can be accomplished in two main ways: (i) by constructing chimeric mouse/human antibodies, in which mouse variable regions are joined to human constant regions (Boulianne et al., 1984) and (ii) by grafting the complementarity determining regions ( CDRs) from mouse variable regions into carefully selected human variable regions and then joining these "reconfigured human" variable regions to human constant regions (Riechmann et al., 1988). 13.1. Humanized version of the 1613F12 antibody project 13.1.1 Humanization of the VL light chain variable domain
[00282] As a preliminary step, the 1613F12 VL nucleotide sequence was compared with the murine germline gene sequence part of the IMGT database (http://www.imgt.org). Murine germline genes GKV16-104*01 and IGKJ5*1 were identified. In order to identify the best human candidate for engraftment of CDRs, the human germline gene showing the best identity to the murine 1613F12 VL sequence was searched. With the help of the analysis tools of the IMGT database, a possible receptor of human V regions of the murine 1613F12 VL CDRs was identified: IGKV1-27*01 and IGKJ4*02. In order to carry out the humanization of the light chain variable domain, each residue that is different between the human and mouse sequences was established in a priority ranking order. These priorities (1-4) were used to create 11 different humanized variants of the light chain variable region with up to 14 backmutations.
13.1.2 Humanization of the VP heavy chain variable domain
[00283] In order to identify the best human candidate for CDR grafting, the mouse and human germline genes showing the best identity with 1613F12 VP were screened. The 1613F12 VP nucleotide sequence was aligned with both mouse and human germline gene sequences using the "IMGT/V-QUEST" sequence alignment software which is part of the IMGT database. Amino acid sequence alignments were also performed to verify the results of nucleotide sequence alignment using the "Align X" software of the VectorNTI package. Alignment with rat germline genes showed that the IGHV14-3*02 V gene and the IGHJ2*01 J gene are the most homologous rat germline genes. Using the IMGT database, the mouse D gene germline IGHD1 - 1*01 was identified as a homologous sequence. In order to select an appropriate human germline for engraftment of CDRs, the human germline gene with the greatest homology to the murine sequence 1613F12 VP was identified. With the help of the IMGT database and tools, the human germline gene IGHV1-2*02 and the human germline gene IGHJ5*01J were selected as human receptor sequences for the murine 1613F12 VP CDRs. In order to perform the humanization of the heavy chain variable domain of each residue which is different between the human and mouse sequences a priority ranking order (1-4) was established. These priorities were used to create 20 different humanized variants of the heavy chain variable region, with up to 18 backmutations,
3.2. Validation of hz1613F12 versus m1613F12
[00284] In order to determine whether humanized 1613F12 was comparable to its murine form 1613F12, binding experiments were performed both by ELISA, using rhAxl-Fc protein assays, and by FACS using SN12C cells. In addition, direct in vitro cytotoxicity assays were performed using SN12C human kidney tumor cells and Calu-1 human lung carcinoma cell line.
[00285] First, ELISA experiments were performed. In the assay, 96-well plates (Immulon II, Thermo Fisher) were coated with 5 µg/ml of 1613F12 solution in 1x PBS overnight at 4°C. After a saturation step, a range of rhAxl-Fc protein concentrations (R and D Systems, ref.: 154-AL) (from 5 μg/mL to 0.02 μg/mL) is incubated for 1 hour at 37° C on the coated plates. For the development step, a biotinylated antibody Axl (self product) was added at 0.85 μg/ml for 1 hour at 37°C. This Axl antibody belongs to a distinct epitope group. Then, a 1/2000° solution of avidin-horseradish peroxidase in dilution buffer is added to the wells. Then the TMB substrate solution is added for 5 min. After addition of the peroxidase stop solution, the absorbance at 405 nm was measured with a microplate reader.
[00286] Figure 12 shows that both murine and humanized 1613F12 antibodies similarly bind to the rhAxl-Fc protein.
[00287] For FACS analysis, SN12C cells were cultured in RPMI 1640 + 2 mM L-glutamine + 10% serum. Cells were detached using trypsin and the cell concentration was adjusted to 1 x 10 6 cells/ml in FACS buffer. A 100 μL volume of cell suspension was incubated with increasing concentrations of isotype controls or anti-Axl antibodies for 20 min. at 4°C. Cells were then washed three times with FACS buffer and incubated for 20 min. using an Alexa488 anti-mouse IgG secondary antibody or Alexa488 anti-human IgG secondary antibody at 4°C in the dark. Cells were washed three times with FACS buffer and resuspended with 100 µL FACS buffer before addition of propidium iodide.
[00288] Cells were incubated with increasing concentrations of either isotype control or anti-Axl antibodies. m1613F12 corresponds to murine 1613F12, c1613F12 corresponds to chimeric 1613F12, and hz1613F12 corresponds to the humanized antibody. EC50s were determined using Prism software.
[00289] As illustrated in Figure 13, the humanized form of 1613F12 bound to SN12C cells with EC50 equivalent to the chimeric and murine form of 1613F12. These results indicate that hz1613F12 recognized the Axl antigen with binding properties similar to those of murine 1613F12.
[00290] Experimental procedures of the direct in vitro cytotoxicity assay were previously described in Example 12. In the present example, four saporin immunoconjugates were prepared, m9G4-saporin, ch9G4-saporin, 1613F12-saporin and hzl613F12-saporin, and tested in two cellular models (SN12C human kidney tumor cells and Calu-1 human lung carcinoma cells).
[00291] Figure 14 shows that both m9G4-saporin and ch9G4-saporin isotype controls were silent and that the humanized antibody Axl 1613F12-saporin triggers similar cytotoxic effects on SN12C cells in contrast to the mouse immunoconjugate 1613F12-saporin.
[00292] Figure 15 shows that the humanized immunoconjugate 1613F12-saporin triggers similar cytotoxic effects on Calu-1 cells unlike the mouse immunoconjugate 1613F12-saporin. In contrast, both m9G4-saporin and ch9G4-saporin isotype controls showed weak activity (~10% maximal cytotoxicity) at antibody concentrations above 109 M. Example 14: Binding Kinetics of 1613F12 to Human ECD Axl
[00293] Affinity measurement of 1613F12 was then determined using Biacore. A Biacore X is used to measure the binding kinetics of 1613F12 on human Axl DEC.
[00294] The instrument based on the optical phenomenon of surface plasmon resonance (RPS) used by Biacore systems allows the detection and measurement of protein-protein interactions in real time, without the use of markers.
[00295] Briefly, the experiments were performed using a CM5 sensor chip as a biosensor. Rabbit IgGs were immobilized on flow cells 1 and 2 (FC1 and FC2) of a CM5 sensor chip at a level of 9,300-10,000 response units (RU) using amine coupling chemistry to capture antibodies.
[00296] Binding is evaluated using multiple cycles. Each measurement cycle is performed using a flow rate of 30 μL/min in an HBS-EP buffer. Then the Axl antibody for testing is captured on the chip for 1 min in FC2 just to reach a mean capture value of 311.8 RU (SD = 5.1 RU) for 1613F12. The analyte (DEC Axl antigen) is injected starting at 200 nM and using two-fold serial dilutions to measure raw ka and kd in real time.
[00297] At the end of each cycle, surfaces are regenerated by injection of a 10 mM glycine hydrochloride solution, pH 1.5, to eliminate antibody-antigen complexes and capture antibody as well. The signal considered corresponds to the difference of the observed signals between FC1 and FC2 (FC2-FC1). Association rates (ka) and dissociation rates (kd) were calculated using a Langmuir one-to-one binding model. The equilibrium dissociation constant (KD) is determined as the ratio ka/kd. Experimental values were analyzed in Biaevaluation software version 3.0. A x2 analysis will be performed to assess the accuracy of the data.
[00298] The data are summarized in the following Table 8. Table 8

[00299] To produce the human extracellular domain (ECD) of Axl, human cDNAs encoding the soluble human AXL receptor were first cloned into the pCEP4 expression vector by PCR. The purified product was then digested with restriction enzymes HindIII and BamHI and ligated into the pCEP4 expression vector that had been pre-cut with the same enzymes. Finally, the identified recombinant plasmid pCEP[AXL]His6 was further confirmed by DNA sequencing.
[00300] Then, suspension-adapted HEK293E cells were cultured in Ex-cell 293 medium (SAFC Biosciences) with 4 mM glutamine. All transfections were performed using 25 kDa linear polyethyleneimine (PEI). The transfected cells were kept at 37°C in a shaker incubator with 5% CO2 and shaking at 120 rpm for six days. Cells were collected by centrifugation, and the supernatant containing the recombinant His-tagged protein was treated for purification on a Ni-NTA agarose column.

权利要求:
Claims (13)
[0001]
1. Humanized antibody, or an antigen-binding fragment thereof, characterized in that: i) it specifically binds to a human Axl protein, and ii) is internalized after its binding to said human Axl protein, wherein said antibody or fragment comprises the three light chains of the complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 of the sequences SEQ ID NOs: 1, 2 and 3, respectively; and the three heavy chains of the complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 of the sequences SEQ ID NOs: 4, 5 and 6, respectively, wherein the light chain and antibody-derived heavy chain constant regions are respectively the lambda or kappa region of the gamma-1, gamma-2 or gamma-4 region, and wherein said fragment is a Fab or scFv fragment.
[0002]
2. Antibody, or antigen-binding fragment thereof, according to claim 1, characterized in that it induces a reduction in the mean fluorescence intensity (MFI) measured by fluorescence activated cell sorting (FACS) of at least 200 .
[0003]
3. Antibody, or antigen-binding fragment thereof, according to claim 1 or 2, characterized in that the antibody is a monoclonal antibody.
[0004]
4. Antibody, or antigen-binding fragment thereof, according to claim 1, characterized in that the antibody or fragment comprises a light chain variable domain selected from the group consisting of: i) a chain variable domain light chain of sequence SEQ ID NO: 7, ii) a light chain variable domain of sequence SEQ ID NO: 36; and iii) a light chain variable domain of sequence SEQ ID NOs: 37 to 47.
[0005]
5. Antibody, or antigen-binding fragment thereof, according to claim 1, characterized in that the antibody or fragment comprises a heavy chain variable domain selected from the group consisting of: i) a heavy chain variable domain heavy sequence SEQ ID NO: 8; ii) a heavy chain variable domain of sequence SEQ ID NO: 48; and iii) a heavy chain variable domain of sequence SEQ ID NO: 49 to 68.
[0006]
6. Antibody, or antigen-binding fragment thereof, according to claim 1, characterized in that the antibody or fragment comprises: i) a light chain variable domain of sequence SEQ ID NOs: 7, 36 or 37 at 47; and ii) a heavy chain variable domain of sequence SEQ ID NOs: 8, 48 or 49 to 68.
[0007]
7. Antibody, according to claim 1, characterized in that it consists of the monoclonal antibody 1613F12 produced by the hybridoma 1-4505 deposited at the CNCM, Instituto Pasteur, France, on July 28, 2011.
[0008]
8. Murine hybridoma, characterized by the fact that it is hybridoma 1-4505 deposited at CNCM, Institut Pasteur, France, on July 28, 2011.
[0009]
9. Antibody, or antigen-binding fragment thereof, according to claim 1, characterized in that the human protein Axl, as defined in claim 1, has the sequence of SEQ ID NOs: 29 or 30, or of a natural variant sequence thereof.
[0010]
10. Antibody, or antigen-binding fragment thereof, according to claim 1, characterized in that the antibody or antigen-binding fragment specifically binds to an epitope located in the extracellular domain of human Axl protein presenting the sequence of SEQ ID NOs: 31 or 32, or a natural sequence variant thereof.
[0011]
11. Antibody, or antigen-binding fragment thereof, according to claim 1, characterized in that the antibody or antigen-binding fragment comprises a light chain variable domain of sequence SEQ ID NO: 47 and a domain heavy chain variable of SEQ ID NO: 68 sequence.
[0012]
12. Immunoconjugate, characterized in that it comprises the antibody, or antigen-binding fragment thereof, as defined in any one of claims 1 to 7 and 11, conjugated to a cytotoxic agent.
[0013]
13. Pharmaceutical composition, characterized in that it comprises the immunoconjugate, as defined in claim 12, and at least one pharmaceutically acceptable excipient and/or vehicle.

类似技术:

---

公开号 | 公开日 | 专利标题

---

JP6685270B2|2020-04-22|Antigen binding proteins and their use as addressing products for the treatment of cancer

---

ES2871815T3|2021-11-02|New antigen-binding proteins and their use as a targeting product for the treatment of cancer

---

ES2841249T3|2021-07-07|IGF-1R antibody and its use as a targeting vehicle for cancer treatment

---

NZ624989B2|2016-11-29|Antigen binding protein and its use as addressing product for the treatment cancer

---

OA16895A|2016-01-18|Antigen binding protein and its use as addressing product for the treatment of cancer.

同族专利:

---

公开号 | 公开日

---

US20140141023A1|2014-05-22|

---

AU2017219091B2|2019-05-02|

---

BR112014010383A2|2017-04-25|

---

JP6685270B2|2020-04-22|

---

CA2851941C|2021-01-12|

---

CN104039829A|2014-09-10|

---

HK1197073A1|2015-01-02|

---

EP2589609A1|2013-05-08|

---

US9689862B2|2017-06-27|

---

AU2012331069A1|2014-06-05|

---

KR20140094589A|2014-07-30|

---

US9173962B2|2015-11-03|

---

AU2017219091A1|2017-09-21|

---

US20150037340A1|2015-02-05|

---

RU2659094C2|2018-06-28|

---

IL232342A|2021-03-25|

---

HK1199460A1|2015-07-03|

---

UA119226C2|2019-05-27|

---

MA35659B1|2014-11-01|

---

RU2650771C1|2018-04-17|

---

JP2018052970A|2018-04-05|

---

KR102089114B1|2020-04-14|

---

KR102033805B1|2019-10-17|

---

BR112014010591A2|2017-05-02|

---

CN104039829B|2021-01-08|

---

RU2014120629A|2015-12-10|

---

JP2014532687A|2014-12-08|

---

US20160131639A1|2016-05-12|

---

TN2014000148A1|2015-09-30|

---

CA2851941A1|2013-05-10|

---

NZ624989A|2016-08-26|

---

EP2773663A1|2014-09-10|

---

US10161930B2|2018-12-25|

---

ZA201403141B|2015-04-29|

---

MY174259A|2020-04-01|

---

MX2014005244A|2015-03-23|

---

CN103998468A|2014-08-20|

---

CN103998468B|2017-04-19|

---

AU2012331069B2|2017-01-05|

---

EP2773666A1|2014-09-10|

---

IL232342D0|2014-06-30|

---

KR20140091035A|2014-07-18|

---

MX2014005240A|2014-08-22|

---

AU2012331068A1|2014-05-22|

---

WO2013064684A1|2013-05-10|

---

WO2013064685A1|2013-05-10|

---

CA2854126A1|2013-05-10|

---

JP2015504302A|2015-02-12|

---

JP6345595B2|2018-06-20|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US4970198A|1985-10-17|1990-11-13|American Cyanamid Company|Antitumor antibiotics |

---

US5079233A|1987-01-30|1992-01-07|American Cyanamid Company|N-acyl derivatives of the LL-E33288 antitumor antibiotics, composition and methods for using the same|

---

US5606040A|1987-10-30|1997-02-25|American Cyanamid Company|Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methyl-trithio group|

---

US5530101A|1988-12-28|1996-06-25|Protein Design Labs, Inc.|Humanized immunoglobulins|

---

IL162181A|1988-12-28|2006-04-10|Pdl Biopharma Inc|A method of producing humanized immunoglubulin, and polynucleotides encoding the same|

---

GB8924581D0|1989-11-01|1989-12-20|Pa Consulting Services|Bleaching of hair|

---

GB9014932D0|1990-07-05|1990-08-22|Celltech Ltd|Recombinant dna product and method|

---

WO1994004679A1|1991-06-14|1994-03-03|Genentech, Inc.|Method for making humanized antibodies|

---

JPH05244982A|1991-12-06|1993-09-24|Sumitomo Chem Co Ltd|Humanized b-b10|

---

US5639641A|1992-09-09|1997-06-17|Immunogen Inc.|Resurfacing of rodent antibodies|

---

US5773001A|1994-06-03|1998-06-30|American Cyanamid Company|Conjugates of methyltrithio antitumor agents and intermediates for their synthesis|

---

US20110045005A1|2001-10-19|2011-02-24|Craig Crowley|Compositions and methods for the treatment of tumor of hematopoietic origin|

---

US20040202666A1|2003-01-24|2004-10-14|Immunomedics, Inc.|Anti-cancer anthracycline drug-antibody conjugates|

---

JP4462964B2|2003-03-10|2010-05-12|第一三共株式会社|Antibodies targeting cancer-specific antigens|

---

JP5234734B2|2004-06-01|2013-07-10|ジェネンテック，インコーポレイテッド|Antibody-drug conjugates and methods|

---

NZ556055A|2004-12-21|2009-12-24|Viventia Biotech Inc|Cancer specific antibody and cell surface proteins|

---

AR064610A1|2006-12-22|2009-04-15|Schering Corp|ANTIBODIES AGAINST CD200R|

---

US8546546B2|2007-07-04|2013-10-01|Forerunner Pharma Research Co., Ltd.|Anti-Muc 17 antibody|

---

AU2008323206B2|2007-11-12|2014-08-14|U3 Pharma Gmbh|AXL antibodies|

---

KR101568051B1|2007-11-15|2015-11-10|추가이 세이야쿠 가부시키가이샤|Monoclonal antibody capable of binding to Anexelekto, and use thereof|

---

WO2009080831A1|2007-12-26|2009-07-02|Biotest Ag|Method of decreasing cytotoxic side-effects and improving efficacy of immunoconjugates|

---

US20120230991A1|2008-07-29|2012-09-13|Douglas Kim Graham|Methods and compounds for enhancing anti-cancer therapy|

---

EP2270053A1|2009-05-11|2011-01-05|U3 Pharma GmbH|Humanized AXL antibodies|

---

JP5909442B2|2009-05-11|2016-04-26|ユー３・ファーマ・ゲーエムベーハー|Humanized AXL antibody|

---

WO2011014457A1|2009-07-27|2011-02-03|Genentech, Inc.|Combination treatments|

---

CN102574917A|2009-08-17|2012-07-11|株式会社未来创药研究所|Pharmaceutical composition containing anti-HB-EGF antibody as active ingredient|

---

US8926976B2|2009-09-25|2015-01-06|Xoma Technology Ltd.|Modulators|

---

CA3056999A1|2010-01-22|2011-07-28|Amato J. Giaccia|Inhibition of axl signaling in anti-metastatic therapy|

---

KR20140104944A|2011-06-22|2014-08-29|인쎄름 |Anti-axl antibodies and uses thereof|

---

EP2589609A1|2011-11-03|2013-05-08|Pierre Fabre Medicament|Antigen binding protein and its use as addressing product for the treatment of cancer|

---

JP2016518382A|2013-04-26|2016-06-23|ピエール、ファーブル、メディカマン|Axl antibody drug conjugate and use thereof for the treatment of cancer|EP2589609A1|2011-11-03|2013-05-08|Pierre Fabre Medicament|Antigen binding protein and its use as addressing product for the treatment of cancer|

---

KR102134088B1|2012-08-24|2020-07-14|더 리젠츠 오브 더 유니버시티 오브 캘리포니아|Antibodies and vaccines for use in treating ror1 cancers and inhibiting metastasis|

---

WO2014068139A1|2012-11-05|2014-05-08|Pierre Fabre Medicament|Novel antigen binding proteins and their use as addressing product for the treatment of cancer|

---

AU2014293013A1|2013-07-26|2016-03-17|Race Oncology Ltd.|Combinatorial methods to improve the therapeutic benefit of bisantrene|

---

CN106470708B|2014-04-25|2019-12-31|皮埃尔法布雷医药公司|Antibody-drug-conjugates and their use for the treatment of cancer|

---

GB201410826D0|2014-06-18|2014-07-30|Bergenbio As|Anti-axl antibodies|

---

GB201410825D0|2014-06-18|2014-07-30|Bergenbio As|Anti-axl antibodies|

---

SG10202006715SA|2014-07-11|2020-08-28|Genmab As|Antibodies binding axl|

---

KR20180033523A|2015-07-10|2018-04-03|젠맵 에이/에스|AXL-specific antibody-drug conjugates for cancer treatment|

---

WO2016091891A1|2014-12-09|2016-06-16|INSERM |Human monoclonal antibodies against axl|

---

AU2015366213B2|2014-12-18|2021-10-07|Bergenbio Asa|Anti-Axl antagonistic antibodies|

---

GB201506411D0|2015-04-15|2015-05-27|Bergenbio As|Humanized anti-axl antibodies|

---

US9669106B2|2015-10-26|2017-06-06|Pierre Fabre Medicament|Conjugate of monomethyl auristatin F and trastuzumab and its use for the treatment of cancer|

---

WO2017121867A1|2016-01-13|2017-07-20|Genmab A/S|Formulation for antibody and drug conjugate thereof|

---

JP6943872B2|2016-03-25|2021-10-06|ジェネンテック， インコーポレイテッド|Multiple whole antibody and antibody complex drug quantification assay|

---

CN109476757A|2016-05-20|2019-03-15|新加坡科技研究局|Anti- axl receptor tyrosine-kinase enzyme antibody and application thereof|

---

GB201610902D0|2016-06-22|2016-08-03|Bergen Teknologioverforing As And Bergenbio As|Anti-Axl Antagonistic Antibodies|

---

EP3474854A4|2016-06-27|2020-02-19|The Regents of The University of California|Cancer treatment combinations|

---

KR101909955B1|2016-11-28|2018-10-19|재단법인 목암생명과학연구소|Novel Antibody Against AXL and Pharmaceutical Composition Comprising the Same|

---

CN108456251A|2017-02-21|2018-08-28|上海君实生物医药科技股份有限公司|Anti- PD-L1 antibody and its application|

---

EP3454063A1|2017-09-06|2019-03-13|AVA Lifescience GmbH|Flow cytometry measuring method and kit for its implementation|

---

US20210070869A1|2018-04-10|2021-03-11|Genmab A/S|Axl-specific antibodies for cancer treatment|

---

CN110540592A|2018-05-29|2019-12-06|杭州尚健生物技术有限公司|Antibodies that bind to AXL protein and uses thereof|

---

WO2021013746A1|2019-07-19|2021-01-28|Genmab A/S|Axl antibody-drug conjugates for use in treating cancer|

---

GB201912059D0|2019-08-22|2019-10-09|Bergenbio As|Combaination therapy of a patient subgroup|

---

GB202004189D0|2020-03-23|2020-05-06|Bergenbio As|Combination therapy|

---

WO2021204713A1|2020-04-08|2021-10-14|Bergenbio Asa|Axl inhibitors for antiviral therapy|

---

GB202006072D0|2020-04-24|2020-06-10|Bergenbio Asa|Method of selecting patients for treatment with cmbination therapy|

---

CN112194726A|2020-09-02|2021-01-08|沣潮医药科技（上海）有限公司|Chimeric antigen receptor for pathological protein aggregate clearance and application thereof|

法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2019-07-09| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NAO 10196/2001, QUE MODIFICOU A LEI NAO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUAANCIA PRA VIA DA ANVISA. CONSIDERANDO A APROVAA AO DOS TERMOS DO PARECER NAO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NAO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDAANCIAS CABA-VEIS. |

---

2020-10-06| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

---

2020-10-27| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2021-07-13| B350| Update of information on the portal [chapter 15.35 patent gazette]|

---

2021-08-10| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

---

2021-11-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2022-02-01| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 05/11/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

---

申请号 | 申请日 | 专利标题

---

EP11306416.6|2011-11-03|

---

EP11306416.6A|EP2589609A1|2011-11-03|2011-11-03|Antigen binding protein and its use as addressing product for the treatment of cancer|

---

PCT/EP2012/071833|WO2013064685A1|2011-11-03|2012-11-05|Antigen binding protein and its use as addressing product for the treatment cancer|

---