HK1199460B - Antigen binding protein and its use as addressing product for the treatment of cancer - Google Patents

Description

Antigen binding proteins and their use as targeted products for treating cancer

Technical Field

The present invention relates to a novel antigen-binding protein (particularly a monoclonal antibody) that specifically binds to the protein Ax1, as well as amino acid and nucleic acid sequences encoding the protein. According to one aspect, the present invention relates to an antigen-binding protein or antigen-binding fragment that specifically binds to Ax1 and is internalized into cells by inducing its internalization. The present invention also includes the use of the antigen-binding protein as an addressing product in combination with other anti-cancer compounds (e.g., toxins, radioactive elements, or drugs), and its use in treating certain cancers.

Background Art

"Ax1" (also known as "Ufo," "Ark," or "Tyro7") was cloned from a patient with chronic myeloid leukemia and, when overexpressed in murine NIH3T3 cells, acts as an oncogene, inducing transformation. It belongs to a family of receptor tyrosine kinases (RTKs), known as the TAM (Tyro3, Ax1, Mer) family, which includes Tyro3 (Rse, Sky, Dtk, Etk, Brt, Tif), Ax1, and Mer (Eyk, Nyk, Tyro-12) (Lemke G. Nat. Rev. Immunol. (2008). 8, 327-336).

Human protein Ax1 is a protein of 894 amino acids, and its sequence is shown in SEQ ID NO. 29. Amino acids 1 to 25 correspond to the signal peptide, and human protein Ax1 without the signal peptide is shown in SEQ ID NO. 30.

Gas6 was originally isolated as a growth arrest-specific gene and is a common ligand for TAM family members (Varnum B.C. et al. Nature (1995). 373, 623-626). Gas6 shows the highest affinity for Ax1, followed by Tyro3 and finally Mer (Nagata K et al. J. Biol. Chem. (1996). 271, 30022-30027). Gas6 is composed of a domain rich in gamma-carboxyglutamic acid (Gla) that mediates binding to phospholipid membranes, four epidermal growth factor-like domains, and two laminin G-like (LG) domains (Manfioletti G., Brancolini, C, Avanzi, G. & Schneider, C. Mol. Cell Biol. (1993). 13, 4976-4985). For many other RTKs, ligand binding leads to receptor dimerization and autophosphorylation of tyrosine residues (for the Ax1 receptor, tyrosine residues 779, 821, and 866), which serve as docking sites for various intracellular signaling molecules (Linger R.M. Adv. Cancer Res. (2008). 100, 35-83). In addition, the Ax1 receptor can be activated by a ligand-independent process. This activation can occur when the Ax1 receptor is overexpressed.

Gas6/Ax1 signaling has been shown to regulate various cellular processes, including in vitro cell proliferation, adhesion, migration, and survival of a wide variety of cells (Hafizi S. & Dahlback, B. FEBS J. (2006). 273, 5231-5244). In addition, TAM receptors are involved in the control of innate immunity; they inhibit the inflammatory response to pathogens in dendritic cells (DCs) and macrophages. They also drive the phagocytosis of apoptotic cells by these immune cells and are required for the maturation and killing activity of natural killer (NK) cells (Lemke G. Nat. Rev. Immunol. (2008). 8, 327-336).

It is weakly expressed in normal cells, primarily in fibroblasts, myeloid progenitor cells, macrophages, neural tissue, myocardium, and skeletal muscle, where it primarily supports cell survival. The Gas6/Ax1 system plays a crucial role in vascular biology by regulating the homeostasis of vascular smooth muscle cells (Korshunov V.A., Mohan, A.M., George, M.A. & 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).

In tumor cells, Ax1 plays an important role in regulating the invasion and migration of cells. The overexpression of Ax1 is not only related to poor prognosis, but also to the invasion increase of various human cancers reported in breast, colon, esophageal cancer, hepatocellular carcinoma, stomach, glioma, lung, melanoma, osteosarcoma, ovary, prostate, rhabdomyosarcoma, kidney, thyroid and endometrial cancer (Linger R.M.Adv.Cancer Res. (2008). 100, 35-83 and Verma A.Mol.Cancer Ther. (2011). 10, 1763-1773, review). In breast cancer, Ax1 appears to be a powerful effector of epithelial to mesenchymal transition (EMT); EMT process actively promotes the migration and propagation of cancer cells in organisms (Thiery J.P.Curr.Opin.Cell Biol. (2003). 15, 740-746).

Ax1 has also been shown to regulate angiogenesis. Knockout of Ax1 in endothelial cells indeed impairs tube formation and migration (Holland S.J. et al. Cancer Res. (2005). 65, 9294-9303) and interferes with specific angiogenic signaling pathways (Li Y. et al. Oncogene (2009). 28, 3442-3455).

A number of recent studies across a range of cell models have described the involvement of Ax1 overexpression in drug resistance. Table 1 summarizes these studies.

Table 1

The complete literature cited in Table 1 above is as follows:

-Macleod, K et al. Cancer Res. (2005). 65, 6789-6800

- Mahadevan D et al. Oncogene (2007). 26, 3909-3919

-Lay J.D. et al. Cancer Res. (2007). 67, 3878-3887

-Hong C.C et al. Cancer Lett. (2008). 268, 314-324

-Liu L et al. Cancer Res. (2009). 69, 6871-6878

-Keating A.K. et al. Mol. Cancer Ther. (2010). 9, 1298-1307

- Ye X. et al. Oncogene (2010). 29, 5254-5264

Among these, Ax1 RTK is considered an interesting target in oncology. Several groups have developed anti-tumor strategies targeting the gas6/Ax1 axis using naked monoclonal antibodies or targeted small molecules (Verma A. Mol. Cancer Ther. (2011). 10, 1763-1773).

Summary of the Invention

In a first embodiment, the present invention relates to an antigen binding protein, or an antigen binding fragment thereof, which i) specifically binds to the human protein Ax1 and ii) is internalized after binding to the human protein Ax1.

More generally, the present invention relates to the use of protein Ax1 for selecting antigen binding proteins, or antigen binding fragments thereof, wherein the antigen binding proteins, or antigen binding fragments thereof, are capable of being internalized after binding to the target Ax1. More specifically, the target is the extracellular domain of Ax1.

In this particular aspect, the present invention therefore relates to an in vitro method for screening compounds, or binding fragments thereof, which are capable of delivering or internalizing a molecule of interest into mammalian cells, said molecule of interest being covalently linked to said compound, wherein said method comprises the following steps:

a) selecting a compound that can specifically bind to the Ax1 protein, or its extracellular domain (ECD), or its epitope;

b) optionally, covalently linking the molecule of interest or control molecule to the compound selected in step a) to form a complex;

c) contacting the compound selected in step a) or the complex obtained in step b) with a mammalian cell, preferably a living cell, expressing an Ax1 protein or a functional fragment thereof on its surface;

d) determining whether the compound or the molecule of interest or the complex has been intracellularly delivered or internalized into the mammalian cell; and

e) selecting the compound as being capable of delivering or internalizing the molecule of interest into living mammalian cells.

In a preferred embodiment, the compound capable of delivering or internalizing a molecule of interest into living mammalian cells is a protein (also referred to herein as a polypeptide or peptide) or a protein-like compound containing a peptide structure, in particular an amino acid sequence of at least 5, 10, 15 or more amino acid residues, which may be glycosylated.

When the compound capable of delivering or internalizing a molecule of interest into living mammalian cells is a protein or protein-like compound, the compound is also referred to herein as an "antigen binding protein," and the antigen binding protein or binding fragment thereof can:

-i) specifically binding to protein Ax1, preferably human Ax1 protein, and

-ii) When the Ax1 protein is expressed on the surface of the mammalian cell, it binds to the Ax1 protein and is then internalized into the mammalian cell.

In a preferred embodiment, the living mammalian cells are human cells, preferably cells that naturally express the Ax1 protein receptor.

In a specific embodiment, the living mammalian cells in step c) are mammalian cells expressing recombinant Ax1 protein on their surface.

In yet another preferred embodiment, the molecule of interest is a cytotoxic molecule (also referred to herein as a cytotoxic agent or cytostatic agent).

In another preferred embodiment, the molecule of interest is covalently linked to the compound capable of binding to the Ax1 protein using a linker, more preferably a peptide linker, more preferably a cleavable peptide linker, more preferably a linker that can be cleaved by a natural intracellular compound contained in a mammalian cell (particularly in the cytoplasm of the mammalian cell).

In another preferred embodiment, the compound capable of binding to the Ax1 protein is an antibody or a functional binding fragment thereof that is specific to the Ax1 protein or to an epitope thereof located in the Ax1 EDC domain.

The selection step e) can be accomplished by any method known to those skilled in the art for evaluating intracellular delivery or internalization. Assays or tests capable of demonstrating or evaluating the presence, absence, or activity of the compound capable of specifically binding to the Ax1 protein, or the complex formed between the compound and the molecule of interest, or the molecule of interest covalently linked to the compound, are well known to those skilled in the art (see, for example, some examples of such assays or analyses disclosed hereinafter, which are not limited to the examples of such assays).

More specifically, these tests or analyses can be achieved by FACS, immunofluorescence, flow cytometry, Western blotting, cytotoxicity/cytostasis assessment, and the like.

In this respect, the present invention also relates to an in vitro method for preparing a cytotoxic or cytostatic complex capable of delivering a cytotoxic compound into mammalian cells, preferably living cells, comprising the steps of:

- Covalently linking the cytotoxic agent to a compound:

-i) a compound capable of specifically binding to Ax1 protein, preferably human Ax1 protein, and

-ii) When the Ax1 protein is expressed on the surface of the mammalian cell, the compound binds to the Ax1 protein and is then internalized into the mammalian cell.

Preferably, the compound is an albumin-like protein, more preferably an antibody specific for the Ax1 protein or an epitope thereof located in the Ax1 EDC domain, or a functional binding fragment of the antibody.

In a preferred embodiment, the cytotoxic agent is covalently linked to the anti-Ax1 antibody or a functional fragment thereof using a linker, more preferably a peptide linker, more preferably a cleavable peptide linker, more preferably a linker that can be cleaved by a natural intracellular compound as a non-limiting example.

Like other members of the TAM family, the Ax1 extracellular domain (ECD) has a structure close to that of cell adhesion molecules. The Ax1 ECD is characterized by a combination of two immunoglobulin-like domains, followed by two adjacent type III fibronectin-like domains (O'Bryan J.P. et al., Mol. Cell Biol. (1991). 11, 5016-5031). The two N-terminal immunoglobulin-like domains are sufficient to bind to the Gas6 ligand (Sasaki T et al., EMBO J. (2006). 25, 80-87).

The ECD of the human Ax1 protein is a 451-amino acid fragment corresponding to amino acids 1-451 of SEQ ID No. 29, and its sequence is represented in the sequence listing as SEQ ID No. 31. Amino acids 1-25 correspond to the signal peptide. The ECD of the human Ax1 protein without the signal peptide corresponds to amino acids 26-451 of SEQ ID No. 29, and is represented by SEQ ID No. 32.

Different internalization modes have been identified to date. They direct proteins or protein complexes to be internalized in the cell. After endocytosis, most membrane proteins or lipids return to the cell surface (recycling), but some membrane components are delivered to late endosomes or the Golgi apparatus (Maxfield F.R. & McGraw, T.E. Nat. Rev. Mol. Cell Biol. (2004). 5, 121-132).

In a preferred embodiment, the present invention relates to an antigen-binding protein or an antigen-binding fragment thereof, which i) specifically binds to the human protein Ax1, and ii) is internalized after binding to the human protein Ax1, wherein the antigen-binding protein comprises at least an amino acid sequence selected from SEQ ID NO.1 to 14, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO.1 to 14.

In a most preferred embodiment, the present invention relates to an antigen binding protein or an antigen binding fragment thereof,

i) specifically binds to human protein Ax1, preferably having the sequence of SEQ ID NO. 29 or 30 or a natural variant thereof, and

ii) binds to the human protein Ax1 and is internalized,

The antigen binding protein comprises at least an amino acid sequence selected from SEQ ID NO. 1 to 14.

"Binding protein" or "antigen binding protein" refers to a peptide chain that has a specific or general affinity for another protein or molecule (usually referred to as an antigen). When binding is possible, the proteins are brought into contact and a complex is formed. The antigen binding protein of the present invention may preferably be, but is not limited to, an antibody; a fragment or derivative of an antibody, protein, or peptide.

According to the present invention, an "antigen-binding fragment" of an antigen-binding protein is intended to mean any peptide, polypeptide or protein that retains the ability of the antigen-binding protein to specifically bind to a target (also commonly referred to as an antigen), and comprises an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues of the amino acid sequence of the antigen-binding protein.

In a preferred embodiment, wherein the antigen-binding protein is an antibody, such an "antigen-binding fragment" is selected from Fv, scFv (sc means single chain), Fab, (Fab') ₂ , Fab', scFv-Fc fragment or bispecific antibody, or any fragment modified by chemical modification, such as the addition of poly (alkylene) glycols such as polyethylene glycol ("PEGylated") (PEGylated fragments are referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab') ₂ -PEG or Fab'-PEG) ("PEG" means polyethylene glycol), or by incorporation into liposomes to extend its half-life, said fragment having at least one characteristic CDR of an antibody according to the present invention. Preferably, said "antigen-binding fragment" will consist of or include a partial sequence of the variable heavy chain or light chain of the antibody from which it is derived, said partial sequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably said sufficient affinity being at least 1/100, more preferably at least 1/10, of the affinity of the antibody from which it is derived for the target. Such functional fragments contain at least 5 amino acids of the antibody sequence from which they are derived, preferably 10, 15, 25, 50 or 100 consecutive amino acids.

The term "epitope" refers to the region of an antigen that is bound by antigen binding proteins (including antibodies). Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and have those residues that contribute directly to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope can include a determinant, which is a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments, can have specific three-dimensional structural characteristics, and/or specific charge characteristics.

In the present application, the epitope is located in the extracellular domain of the human protein Ax1.

According to a preferred embodiment of the present invention, the antigen binding protein or its antigen binding fragment specifically binds to an epitope located in the extracellular domain of human protein Ax1, preferably having the sequence SEQ ID NO. 31 or 32 or a natural variant sequence thereof.

"Specific binding,""specificbinding," and the like are intended to indicate that an antigen-binding protein or antigen-binding fragment thereof forms a relatively stable complex with an antigen under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1.10 ^<-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 to or interfere with another antigen at a low level. In any case, as a preferred embodiment, the antigen-binding fragment binds only to the antigen.

In this sense, " _EC50 " refers to the 50% effective concentration. More precisely, the term half-maximal effective concentration ( _EC50 ) corresponds to the concentration of a drug, antibody, or toxicant that induces a response halfway between baseline and maximum after a specific exposure time. It is often used as a measure of drug potency. Thus, the _EC50 of a stepwise dose-response curve represents the concentration of a compound at which 50% of its maximal effect is observed. The _EC50 of a quantal dose-response curve represents the compound concentration at which 50% of the population exhibits a response after a specific period of exposure. Concentration measurements typically follow a sigmoidal curve, increasing rapidly with relatively small changes in concentration. This can be determined mathematically by derivation of a best-fit line.

As a preferred embodiment, the _EC50 value determined in the present invention characterizes the potency of an antibody for binding to the Ax1 ECD exposed on human tumor cells. The _EC50 parameter is determined using FACS analysis. The _EC50 parameter reflects the antibody concentration that achieves 50% maximal binding to human Ax1 expressed on human tumor cells. Each _EC50 value is calculated as the midpoint of the dose-response curve using a four-parameter regression curve fitting program (Prism software). This parameter has been selected to be representative of physiological/pathological conditions.

In one embodiment of the invention, the antigen binding protein, or antigen binding fragment thereof, binds its epitope with an _EC50 of at least 10 ^" 9M, preferably between 10 ^"9 and 10 ^" 12M.

Another embodiment of the present invention is a process or method for selecting an antigen-binding protein or antigen-binding fragment thereof that can be internalized into mammalian cells, preferably human cells, preferably living cells, comprising the steps of:

-i) selecting an antigen binding protein that specifically binds to Ax1, preferably specifically binds to its ECD domain or an epitope thereof; and

-ii) selecting the antigen-binding protein from the previous step i), wherein the antigen-binding protein binds to the Ax1 protein expressed on the surface of a mammalian cell and is then internalized into the mammalian cell.

In a specific embodiment, the mammalian cell naturally expresses an Ax1 protein receptor on its surface, or is a mammalian cell expressing a recombinant Ax1 protein on its surface, preferably a human cell.

Such a method or process may include the following steps: i) selecting an antigen-binding protein that specifically binds to Ax1 with an ^EC50 of at least 10 ^{^-9} M, and ii) selecting an antigen-binding protein from the previous step that is internalized after binding to Ax1. The selection step ii) can be performed by any method known to those skilled in the art for assessing internalization. More specifically, testing can be performed by FACS, immunofluorescence, flow cytometry, western blotting, cytotoxicity assessment, and the like.

Another feature of the antigen-binding protein according to the present invention is that it does not have any significant activity against the proliferation of tumor cells. More specifically, as shown in the examples below, the antigen-binding protein according to the present invention does not have any significant in vitro activity against the proliferation SN12C model.

In oncology, there are multiple mechanisms by which mAbs can exert therapeutic effects, but their activity is generally insufficient to produce lasting benefits. Therefore, some strategies have been used to enhance their activity, particularly by combining them with drugs as chemotherapeutic agents. As an effective alternative to combination regimens, immunotoxins have become a new therapeutic option for treating cancer (Beck A. et al. Discov. Med. (2010). 10, 329-339; Alley S.C. et al. J. Pharmacol. Exp. Ther. (2009). 330, 932-938). Antibody drug conjugates (ADCs) represent one such approach, in which the activity of mAbs and drugs can be significantly enhanced by utilizing mAb specificity and the ability to target cytotoxic agents to tumors. Ideally, mAbs will specifically bind to antigens that are expressed in large quantities on tumor cells but are limited in expression on normal cells.

The present invention concerns specific anti-Ax1 binding proteins, and more specifically, specific anti-Ax1 antibodies, that exhibit a high ability to internalize after Ax1 binding. Such antigen-binding proteins are of interest as components of immunodrug conjugates, as they deliver linked cytotoxins to targeted cancer cells. Once internalized, the cytotoxins trigger cancer cell death.

The key to the success of immunoconjugate therapy is believed to be the specificity of the target antigen and the internalization of the antigen-binding protein complex into cancer cells. Obviously, compared with internalized antigens, non-internalized antigens cannot effectively deliver cytotoxic agents. The process of internalization is variable depending on the different antigens and various parameters affected by the binding protein. Cell surface RTKs constitute an interesting family of antigens for studying this process.

Among the biomolecules, the cytotoxins provide the cytotoxic activity, the antigen binding proteins used provide the specificity for cancer cells, and the carriers are used to enter the cells to correctly target the cytotoxins.

Therefore, to improve immunoconjugate molecules, carrier binding proteins must demonstrate a high ability to internalize into target cancer cells. The efficiency of binding protein-mediated internalization varies significantly depending on the targeted epitope. Selecting potent internalizing anti-Ax1 binding proteins requires not only studying Ax1 downregulation but also various experimental data after the anti-Ax1 binding proteins enter cells.

In a preferred embodiment, internalization of the antigen binding proteins according to the invention may be assessed, preferably by immunofluorescence (as exemplified below in this application) or by any method or process specific for the internalization mechanism known to those skilled in the art.

In another preferred embodiment, the Ax1-antigen binding protein complex according to the present invention is internalized after the binding protein of the present invention binds to the ECD of Ax1, thereby inducing a decrease in the amount of Ax1 on the cell surface. This decrease can be quantified by any method known to those skilled in the art (western blotting, FACS, immunofluorescence, etc.).

In an embodiment of the invention, this reduction in internalization is thus manifested, preferably measurable by FACS and expressed as the difference or delta between the mean fluorescence intensity (MFI) measured on untreated cells and the MFI measured on cells treated with an antigen binding protein according to the invention.

As a non-limiting example of the present invention, this Δ is determined based on the MFI obtained for untreated cells and cells treated with the antigen binding protein of the invention, as described in Example 9, using i) human renal tumor SN12C cells after a 24-hour incubation period with the antigen binding protein of the invention, and ii) a secondary antibody labeled with Alexa 488. This parameter is defined as calculated using the following formula:

Since MFI is proportional to Ax1 expressed on the cell surface, this difference between MFIs reflects downregulation of Ax1.

In a more preferred and advantageous aspect, the antigen binding protein or antigen binding fragment thereof of the invention consists of a monoclonal antibody, preferably an isolated Mab, which elicits a Δ(MFI _24h untreated cells - MFI _24h treated cells) of at least 200, preferably at least 300.

The antigen binding protein according to the present invention, or an antigen binding fragment thereof, induces a reduction in MFI of at least 200.

In more detail, the above-mentioned Δ can be measured according to the following method, which must be regarded as an illustrative and non-limiting example:

a) treating and incubating tumor cells of interest with the antigen binding protein of the present invention;

b) treating the cells treated in step a) and untreated cells in parallel with the antigen binding protein of the invention,

c) measuring the MFI (representing the amount of Ax1 present on the surface) of treated and untreated cells using a second labeled antibody that binds to the antigen binding protein, and

d) Δ was calculated as the difference between the MFI obtained for untreated cells minus the MFI obtained for treated cells.

In the broadest sense, the terms "antibody" or "immunoglobulin" are used interchangeably and include monoclonal antibodies, preferably isolated Mabs (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, or multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity).

More specifically, such molecules are composed of glycoproteins that contain at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (or domain) (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first complement (Clq) of the classical complement system.

Antibodies within the meaning of the present invention also include certain antibody fragments thereof. Such antibody fragments exhibit the desired binding specificity and affinity, regardless of their origin or immunoglobulin class (i.e., IgG, IgE, IgM, IgA, etc.), that is, they are able to specifically bind to the Ax1 protein with an affinity comparable to that of the full-length antibodies of the present invention.

In general, for the preparation of monoclonal antibodies or functional fragments thereof, in particular of murine origin, it is possible to involve, inter alia, the techniques described in the handbook "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or the technique for preparing hybridomas described by Kohler and Milstein (Nature, 256: 495-497, 1975).

As used herein, the term "monoclonal antibody" or "Mab" refers to an antibody molecule that is directed against a specific antigen and is produced by a single clone of a B cell or hybridoma. Monoclonal antibodies can also be recombinant, i.e., produced by protein engineering. In addition, unlike polyclonal antibody preparations that typically include various antibodies directed against different determinants or epitopes, each monoclonal antibody is directed against a single epitope of an antigen. The present invention relates to antibodies that have been isolated by purification or obtained from a natural source, or obtained by genetic recombination or chemical synthesis.

A preferred embodiment of the present invention is an antigen-binding protein or an antigen-binding fragment thereof, which comprises or consists of an antibody, wherein the antibody comprises three light chain CDRs and three heavy chain CDRs; the three light chain CDRs comprise the sequences of SEQ ID NO.1, 2 and 3, or any sequences that exhibit at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO.1, 2 and 3; the three heavy chain CDRs comprise the sequences of SEQ ID NO.4, 5 and 6, or any sequences that exhibit at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO.4, 5 and 6.

In a more preferred embodiment of the present invention, the antigen binding protein or its antigen binding fragment consists of an antibody comprising three light chain CDRs containing sequences of SEQ ID NOs. 1, 2 and 3; and three heavy chain CDRs containing sequences of SEQ ID NOs. 4, 5 and 6.

In a preferred aspect, the CDR region or CDRs are intended to represent the hypervariable regions of immunoglobulin heavy and light chains as defined by IMGT. In the absence of any contradictory description, CDRs are defined in this specification according to the IMGT numbering system.

The IMGT unique numbering designation is used to compare variable domains regardless of 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., Pommié, 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, such as cysteine 23 (first CYS), tryptophan 41 (conserved TRP), hydrophobic amino acid 89, cysteine 104 (second CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides standardized delimitations for the framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104, and FR4-IMGT: 118 to 128) and the complementarity determining regions (CDR1-IMGT: positions 27 to 38, CDR2-IMGT: 56 to 65, and CDR3-IMGT: 105 to 117). Since gaps represent unoccupied positions, the CDR-IMGT length (shown between brackets and separated by dots, e.g., [8.8.13]) becomes key information. IMGT unique numbering, designated IMGT Colliers de Perles (Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)), is used in 2D diagrams and in 3D structures in the IMGT/3D structure DB (Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)).

It must be understood, and there is no contradictory description in this specification, that by complementarity determining regions or CDRs are meant the hypervariable regions of immunoglobulin heavy and light chains as defined according to the IMGT numbering system.

In any case, CDRs can also be defined according to the Kabat numbering system (Kabat et al., Sequences of proteins of immunological interest, 5th ed., U.S. Department of Health and Human Services, NIH, 1991 and later versions). There are three heavy chain CDRs and three light chain CDRs. Here, depending on the context, the term "CDR" is used to refer to one or more, or even all, regions comprising the majority of amino acid residues that are responsible for the antibody's binding affinity to the antigen or epitope it recognizes.

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 comprising three light chain CDRs defined according to the Kabat numbering system and three heavy chain CDRs defined according to the Kabat numbering system; the three light chain CDRs comprise sequences of SEQ ID NO.9, 10 and 11, or any sequences that exhibit at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO.9, 10 and 11; the three heavy chain CDRs comprise sequences of SEQ ID NO.12, 13 and 14, or any sequences that exhibit at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO.12, 13 and 14.

In the meaning of the present invention, "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, which is obtained after optimal alignment, this percentage being purely statistical, the differences between the two sequences being randomly distributed along their lengths. The comparison of two nucleic acid or amino acid sequences is conventionally performed by comparing the sequences after their optimal alignment, which comparison can be performed by segment or by using an "alignment window". In addition to manual comparisons, optimal alignment of the sequences to be compared can be performed by the local homology algorithm of Smith and Waterman (1981) (Ad. App. Math. 2:482), by the local homology algorithm of Neddleman and Wunsch (1970) (J. Mol. Biol. 48:443), by the search for similarity method of Pearson and Lipman (1988) (Proc. Natl. Acad. Sci. USA 85:2444), or by computer software that uses these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computing Group, 575 Science Dr., Madison, WI, or by the comparison software BLAST NR or BLAST P).

The percent identity between two nucleic acid or amino acid sequences is determined by comparing two optimally aligned sequences, wherein the nucleic acid or amino acid sequence to be compared may have additions or deletions compared to a reference sequence used for optimal alignment between the two sequences. The percent identity is calculated by determining the number of positions at which the two sequences (preferably the two complete sequences) have identical amino acid nucleotides or residues, dividing the number of identical positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percent identity between the two sequences.

For example, the BLAST program, "BLAST2 sequences" (Tatusova et al., "Blast2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol., 1999, Lett. 174: 247-250), available on the website http://www.ncbi.nlm.nih.gov/gorf/bl2.html, can be used with the default parameters (in particular the parameters "open gap penalty": 5, and "extend gap penalty": 2; the matrix selected is, for example, the "BLOSUM62" matrix proposed by the program); the percentage identity between the two sequences to be compared can be calculated directly using the program.

For amino acid sequences showing at least 80%, preferably 85%, 90%, 95% and 98% identity to a reference amino acid sequence, preferred examples include those comprising the reference sequence, certain modifications (particularly deletions, insertions or substitutions of at least one amino acid, truncations or extensions). In the case where one or more consecutive or discontinuous amino acids are substituted, it is preferred that the substituted amino acids in the substitution are replaced by "equivalent" amino acids. Here, the expression "equivalent amino acid" means any amino acid that can replace a structural amino acid without changing the biological activity of the corresponding antibody, as well as any amino acid of those specific examples defined below.

Equivalent amino acids can be determined based on their structural homology to the amino acid they replace, or based on the results of comparative biological activity testing between various antigen binding proteins that may be generated.

As non-limiting examples, Table 2 below summarizes possible substitutions that can be made without resulting in a significant change in the biological activity of the corresponding modified antigen binding protein; the opposite substitutions can naturally be made under the same circumstances.

Table 2

Original residue replace Ala(A) Val,Gly,Pro Arg(R) Lys,His Asn(N) Gln Asp(D) Glu Cys(C) Ser Gln(Q) Asn Glu(E) Asp Gly(G) Ala His(H) Arg

Ile(I) Leu Leu(L) Ile,Val,Met Lys(K) Arg Met(M) Leu Phe(F) Tyr Pro(P) Ala Ser(S) Thr,Cys Thr(T) Ser Trp(W) Tyr Tyr(Y) Phe,Trp Val(V) Leu,Ala

One embodiment of the present invention relates to an antigen binding protein or an antigen binding fragment thereof, which comprises three light chain CDRs, the three light chain CDRs containing sequences SEQ ID NO.1, 2 and 3, or any sequences that show at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO.1, 2 and 3; and a heavy chain variable domain of sequence SEQ ID NO.8, or any sequence that shows at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO.8.

According to a preferred embodiment of the present invention, the antigen binding protein or its antigen binding fragment comprises three light chain CDRs, the three light chain CDRs contain sequences of SEQ ID NO.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 with SEQ ID NO.8.

According to another preferred embodiment of the present invention, the antigen binding protein or antigen binding fragment thereof comprises a heavy chain variable domain of SEQ ID NO. 8, or any sequence showing at least 80% identity with SEQ ID NO. 8.

“Any sequence that exhibits at least 80% identity with SEQ ID NO. 8” is used to refer to sequences presenting the three heavy chain CDRs SEQ ID NOs. 4, 5 and 6, as well as sequences that exhibit at least 80% identity with the full-length sequence SEQ ID NO. 8 excluding the sequences corresponding to the CDRs (i.e., SEQ ID NOs. 4, 5 and 6).

Another embodiment of the present 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 with SEQ ID NO.7; and three heavy chain CDRs comprising sequences SEQ ID NO.4, 5 and 6, or any sequence that exhibits at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO.4, 5 and 6.

According to a preferred embodiment of the present invention, the antigen binding protein or its antigen binding fragment comprises a light chain variable domain of sequence SEQ ID NO.7, or any sequence showing at least 80% identity with SEQ ID NO.7; and three heavy chain CDRs comprising sequences SEQ ID NO.4, 5 and 6.

According to another preferred embodiment of the present invention, the antigen binding protein or antigen binding fragment thereof comprises a light chain variable domain of SEQ ID NO. 7, or any sequence showing at least 80% identity with SEQ ID NO. 7.

"Any sequence that exhibits at least 80% identity with SEQ ID NO. 7" is also used to refer to sequences that present the three light chain CDRs SEQ ID NOs. 1, 2 and 3, as well as sequences that exhibit at least 80% identity with the full-length sequence SEQ ID NO. 7 excluding the sequences corresponding to the CDRs (i.e., SEQ ID NOs. 1, 2 and 3).

Another embodiment of the present 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 with 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 with SEQ ID NO.8.

According to a preferred embodiment of the present invention, the antigen binding protein or its antigen binding fragment comprises a light chain variable domain of sequence SEQ ID NO.7, or any sequence that shows at least 80% identity with SEQ ID NO.7, and a heavy chain variable domain of sequence SEQ ID NO.8, or any sequence that shows at least 80% identity with SEQ ID NO.8.

For greater clarity, Table 3 below summarizes various amino acid sequences corresponding to antigen binding proteins of the present invention (wherein Mu. = murine).

Table 3

A specific aspect of the present invention relates to a murine antibody, or a compound or antigen-binding fragment derived therefrom, characterized in that the antibody further comprises light and heavy chain constant regions of an antibody derived from a species heterologous to the murine, in particular human.

Another specific aspect of the present invention relates to a chimeric antibody, or a compound or antigen-binding fragment derived therefrom, characterized in that the antibody further comprises light chain and heavy chain constant regions derived from an antibody of a species heterologous to the mouse, in particular human.

Yet another specific aspect of the present invention relates to a humanized antibody, or a compound or antigen-binding fragment thereof, characterized in that the constant regions of the light chain and heavy chain are derived from the λ or κ region and the γ1, γ2 or γ4 region, respectively, of a human antibody.

Another aspect of the present invention is an antigen binding protein or antigen binding fragment thereof consisting of the monoclonal antibody 1613F12, which is derived from hybridoma I-4505 deposited on July 28, 2011 at the Pasteur Institute CNCM, France.

According to another aspect, the present invention relates to a murine hybridoma capable of secreting an antigen-binding protein according to the invention, in particular a murine hybridoma submitted to the French National Collection of Cultures and Microorganisms (CNCM, Institut Pasteur, Paris, France) under the reference number I-4505 on July 28, 2011. Said hybridoma was obtained by fusion of Balb/C immunized murine spleen cells/lymphocytes and cells of the myeloma Sp2/O-Ag14 cell line.

According to another aspect, the present invention relates to a murine hybridoma capable of secreting an antibody comprising three light chain CDRs comprising the sequences of SEQ ID NOs. 1, 2, and 3; and three heavy chain CDRs comprising the sequences of SEQ ID NOs. 4, 5, and 6. The hybridoma was submitted to the Institut Pasteur (CNCM), Paris, France, under reference number I-4505 on July 28, 2011. The hybridoma was obtained by fusing spleen cells/lymphocytes from a Balb/C-immunized mouse with cells of the myeloma Sp2/O-Ag14 cell line.

A subject of the present invention is the murine hybridoma I-4505, deposited on July 28, 2011 at the Institut Pasteur CNCM, France.

The antigen binding proteins of the present invention also include chimeric or humanized antibodies.

A chimeric antibody is an antibody that comprises the naturally occurring variable regions (light and heavy chains) of an antibody from a given species combined with the constant regions of the light and heavy chains of an antibody from a species foreign to the given species.

Antibodies or chimeric fragments thereof can be prepared using recombinant genetic techniques. For example, chimeric antibodies can be produced by cloning recombinant DNA containing a promoter and sequences encoding the variable regions of a non-human (particularly murine) monoclonal antibody of the present invention, and sequences encoding the constant regions of a human antibody. A chimeric antibody according to the present invention encoded by such a recombinant gene can be, for example, a mouse-human chimera, with the variable regions from the mouse DNA determining the specificity of the antibody and the constant regions from the human DNA determining its isotype. Reference is made to methods for preparing chimeric antibodies by Verhoeyn et al. (BioEssays, 8:74, 1988).

In another aspect, the invention features binding proteins composed of chimeric antibodies.

In a specific preferred embodiment, the chimeric antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable domain sequence comprising the amino acid sequence of SEQ ID NO.7, and a heavy chain variable domain sequence comprising the amino acid sequence of SEQ ID NO.8.

In another aspect, the invention features binding proteins composed of humanized antibodies.

"Humanized antibody" refers to an antibody containing CDR regions derived from an antibody of non-human origin, with the remainder of the antibody molecule derived from one (or several) human antibodies. In addition, certain framework segment residues (called FRs) may be modified to maintain binding affinity (Jones et al., Nature, 321: 522-525, 1986; Verhoeyen et al., Science, 239: 1534-1536, 1988; Riechmann et al., Nature, 332: 323-327, 1988).

The humanized antibodies of the present invention or fragments thereof can be prepared by techniques known to those skilled in the art (e.g., those described in Singer et al., J. Immun., 150: 2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10: 1-142, 1992; and Bebbington et al., Bio/Technology, 10: 169-175, 1992). Such humanized antibodies are preferably used in methods involving in vitro diagnosis or in vivo prophylactic and/or therapeutic treatment. Other humanization techniques known to those skilled in the art are, for example, the "CDR grafting" technique described in PDL patents EP 0 451 216, 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. U.S. Patents 5,639,641 or 6,054,297, 5,886,152 and 5,877,293 can also be cited.

In addition, the present invention also relates to humanized antibodies generated from the above murine antibodies.

Preferably, the light chain and heavy chain constant regions from human antibodies are λ or κ and γ1, γ2 or γ4 regions, respectively.

A novel aspect of the present invention relates to an isolated nucleic acid characterized in that it is selected from the group consisting of the following nucleic acids (including any degenerate genetic code):

a) a nucleic acid encoding an antigen-binding protein or an antigen-binding fragment thereof according to the present invention;

b) a nucleic acid comprising:

- a nucleic acid sequence selected from SEQ ID No. 15 to 28, or

- a nucleic acid sequence comprising six nucleic acid sequences SEQ ID No. 15 to 20, or

- a nucleic acid sequence comprising two nucleic acid sequences SEQ ID No. 21 and 22;

c) a complementary nucleic acid to a nucleic acid as defined in a) or b); and

d) a nucleic acid, preferably having at least 18 nucleotides, which is capable of hybridizing under highly stringent conditions to a nucleic acid sequence as defined in part a) or b), or to a sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity to a nucleic acid sequence as defined in part a) or b) after optimal alignment.

Table 4 below summarizes various nucleotide sequences for the binding proteins of the present invention (wherein Mu. = mouse).

Table 4

The terms "nucleic acid," "nucleic acid sequence," "nucleic acid sequence," "polynucleotide," "oligonucleotide," "polynucleotide sequence," and "nucleotide sequence" are used interchangeably in this specification to refer to the precise nucleotide sequence of a defined nucleic acid segment or region, whether modified or not, with or without non-natural nucleotides, and whether double-stranded DNA, single-stranded DNA, or the transcription product of said DNA.

The sequences of the present invention have been isolated and/or purified, i.e. they have been sampled (e.g. in a copy) directly or indirectly, their environment has been at least partially altered. Also mentioned here are isolated nucleic acids obtained by recombinant genetics (e.g. in a host cell) or by chemical synthesis.

By "nuclear sequences showing at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with a preferred sequence" is meant a nuclear sequence which shows certain modifications (especially site-specific modifications) relative to a reference nuclear sequence, such as, in particular, deletions, truncations, extensions, chimeric fusions and/or substitutions. Preferably, these sequences encode an amino acid sequence identical to the reference sequence (this relates to the degeneracy of the genetic code) or a complementary sequence which is capable of specifically hybridizing to the reference sequence, preferably under highly stringent conditions, in particular as defined below.

Hybridization under highly stringent conditions means that the conditions concerning 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 for the hybridization step for the purpose of defining polynucleotide fragments described above are advantageously as follows.

DNA-DNA or DNA-RNA hybridization was performed in two steps: (1) prehybridization for three hours at 42°C in phosphate buffer (20 mM, pH 7.5, containing 5×SSC (1×SSC corresponds to a solution of 0.15 M NaCl + 0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10×Denhardt's, 5% dextran sulfate, and 1% salmon sperm DNA); (2) initial hybridization for 20 hours at a temperature dependent on the probe length (i.e., 42°C for probes greater than 100 nucleotides long), followed by two 20-minute washes at 20°C in 2×SSC + 2% SDS and one 20-minute wash at 20°C in 0.1×SSC + 0.1% SDS. For probes greater than 100 nucleotides long, a final wash was performed at 60°C in 0.1×SSC + 0.1% SDS for 30 minutes. For longer or shorter oligonucleotides, one skilled in the art can adjust the highly stringent hybridization conditions for polynucleotides of a specific size according to the procedure described by Sambrook et al. (Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory; 3rd edition, 2001).

The present invention also relates to a vector comprising the nucleic acid of the present invention.

The present invention particularly targets cloning and/or expression vectors comprising such nucleotide sequences.

The vector of the present invention preferably contains elements that allow the nucleotide sequence to be expressed and/or secreted in a given host cell. Thus, the vector must contain a promoter, translation initiation and termination signals, and an appropriate transcriptional regulatory region. It must be able to be maintained in a stable manner in the host cell and optionally have specific signals that direct the secretion of the translated protein. Depending on the host cell used, those skilled in the art can select and optimize these different elements. For this purpose, the nucleotide sequence can be inserted into an autonomously replicating vector in the selected host, or an integration vector of the selected host.

Such vectors are prepared by methods commonly used by those skilled in the art, and the resulting clones can be introduced into appropriate hosts by standard methods such as lipofection, electroporation, heat shock or chemical methods.

The vector is, for example, of plasmid or viral origin and is used to transform host cells to clone or express the nucleotide sequence of the present invention.

The present invention also includes isolated host cells transformed with or containing the vectors of the present invention.

The host cell can be selected from prokaryotic or eukaryotic systems, such as bacterial cells, for example, but also yeast cells or animal cells, in particular mammalian cells (except human). Insect or plant cells can also be used.

The present invention also relates to animals (other than humans) having a transformed cell according to the present invention.

Another aspect of the present invention relates to a method for producing an antigen-binding protein according to the present invention, or an antigen-binding fragment thereof, characterized in that the method comprises the following steps:

a) cultivating the host cell according to the present invention in a culture medium under appropriate culture conditions; and

b) recovering the antigen binding protein, or one of its antigen binding fragments, thus produced from the culture medium or from said cultured cells.

The transformed cells according to the present invention can be used in methods for producing recombinant antigen-binding proteins according to the present invention. The present invention also encompasses methods for producing recombinant antigen-binding proteins according to the present invention, characterized in that the methods utilize vectors according to the present invention and/or cells transformed with the vectors according to the present invention. Preferably, cells transformed with the vectors according to the present invention are cultured under conditions that allow for expression of the antigen-binding protein and recovery of the recombinant protein.

As mentioned, host cells can be selected from prokaryotic or eukaryotic systems. In particular, it is possible to identify nucleotide sequences of the present invention that are conducive to secretion in this prokaryotic or eukaryotic system. Therefore, vectors according to the present invention that carry such sequences can be advantageously used to produce recombinant proteins to be secreted. In fact, the fact that proteins are present in the cell culture supernatant rather than in the host cell will be conducive to the purification of these recombinant proteins of interest.

The antigen-binding proteins of the present invention can also be prepared by chemical synthesis. A method of such preparation is also an object of the present invention. Those skilled in the art are aware of methods for chemical synthesis, such as by concentrated fragments or by solid phase techniques for traditional synthesis in solution (see in particular Steward et al., 1984, Solid phase peptides synthesis, Pierce Chem. Company, Rockford, 111, 2nd edition pp71-95) or partial solid phase techniques. The invention also includes polypeptides obtained by chemical synthesis and capable of containing the corresponding non-natural amino acids.

The present invention also includes the antigen binding protein or antigen binding fragment thereof obtainable by the method of the present invention.

According to a specific aspect, the present invention relates to the above-mentioned antigen binding protein or its antigen binding fragment, which is used as a positioning product for delivering cytotoxic agents to host target sites, wherein the host target site is composed of an epitope located in the extracellular domain of protein Ax1, preferably the extracellular domain of human protein Ax1, more preferably the extracellular domain of human protein Ax1 having the sequence SEQ ID NO.31 or 32, or its natural variant sequence.

In a preferred embodiment, the host target site is a target site of a mammalian cell, more preferably a human cell, and more preferably a cell that expresses the Ax1 protein naturally or by genetic recombination.

The present invention relates to an immunoconjugate comprising an antigen binding protein as described herein conjugated to a cytotoxic agent.

In the sense of the present invention, the expression "immunoconjugate" or "immuno-conjugate" generally refers to a compound comprising at least one targeting product, wherein the targeting product is physically linked to one or more therapeutic agents, thereby resulting in a highly targeted compound.

In a preferred embodiment, such therapeutic agents consist of cytotoxic agents.

"Cytotoxic agent" or "cytotoxin" refers to an agent that, when administered to a subject, treats or prevents the development of cell proliferation, preferably cancer, in the subject's body by inhibiting or preventing cellular function and/or causing cell death.

Many cytotoxic agents have been isolated or synthesized and are capable of inhibiting cell proliferation or, if not specifically defined, at least significantly destroying or reducing tumor cells. However, the toxic activity of these agents is not limited to tumor cells; non-tumor cells are also affected and can be destroyed. More specifically, side effects have been observed in rapidly renewing cells, such as hematopoietic cells or epithelial cells, particularly in the mucosa. By way of example, cells of the gastrointestinal tract are particularly affected by the use of such cytotoxic agents.

One of the objects of the present invention is also to provide a cytotoxic agent which makes it possible to limit side effects on normal cells while retaining a high cytotoxicity towards tumor cells.

More specifically, the cytotoxic agent may preferably be composed of, but not limited to, drugs (i.e., "antibody-drug conjugates"), toxins (i.e., "immunotoxins" or "antibody-toxin conjugates"), radioisotopes (i.e., "radioimmunoconjugates" or "antibody-radioisotope conjugates"), and the like.

In a first preferred embodiment of the present invention, the immunoconjugate is composed of a binding protein linked to at least one drug or pharmaceutical. When the binding protein is an antibody, or an antigen-binding fragment thereof, such an immunoconjugate is referred to as an antibody drug conjugate (or "ADC").

In a first embodiment, such drugs can be described according to their mode of action. As non-limiting examples, there can be mentioned alkylating agents (such as nitrogen mustards, alkyl sulfonates, nitrosoureas, oxazophorins (no corresponding Chinese translation), aziridines or ethyleneimines), antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, anti-angiogenic 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 reductase inhibitors, aromatase inhibitors, estrogen receptor antagonists, Tyrosine kinase inhibitors, cell cycle inhibitors, taxanes, tubulin inhibitors, angiogenesis inhibitors, macrophage stimulators, neurokinin receptor antagonists, cannabinoid receptor agonists, dopamine receptor agonists, granulocyte stimulating factor agonists, erythropoietin receptor agonists, somatostatin receptor agonists, LHRH agonists, calcium sensitizers, VEGF receptor antagonists, interleukin receptor antagonists, osteoclast inhibitors, free radical formation stimulators, endothelin receptor antagonists, vinca alkaloids, antihormonal or immunomodulatory agents, or any other new drug that meets the criteria for cytotoxic or toxin activity.

For example, VIDAL 2010 cites such drugs in the pages concerning compounds attached to the "Cytotoxin" section of Oncology and Hematology, and these cytotoxic compounds cited with reference to this document are cited herein as preferred cytotoxic agents.

More specifically, but not limited to, the following drugs are preferred according to the present invention: dichloromethane, chlorambucol, melphalan, chlorydrate, pipobromen, prednimustine, disodic-phosphate, estramustine, cyclophosphamide, altretamine, trofosfamide, sulfofosfamide, ifosfamide, thiotepa, triethylamine, altetramine (no corresponding Chinese translation), carmustine, streptozotocin, fotiam, lomustine, busulfan, treosulfan , improsulfan, dacarbazine, cisplatin, oxaliplatin, lobaplatin, heptaplatin, miriplatin hydrate, carboplatin, methotrexate, pemetrexed, 5-fluorouracil, floxuridine, 5-fluorodeoxyuridine, capecitabine, cytarabine, fludarabine, cytosine arabinoside, 6-mercaptopurine (6-MP), nelarabine, 6-mercaptoguanine (6-TG), chlorodeoxyadenosine, 5-azacytidine, gemcitabine, cladribine, deoxycoformycin, tegafur, pentostatin, doxorubicin, daunorubicin, idarubicin, valrubicin, mitoxantrone, dactinomycin, mithramycin, plicamycin , mitomycin C, bleomycin, procarbazine, paclitaxel, docetaxel, vinblastine, vincristine, vindesine, vinorelbine, topotecan, irinotecan, etoposide, valrubicin, amrubicin hydrochloride, pirarubicin, elliptonium acetate, daunorubicin, epirubicin, idarubicin and teniposide, razoxane, marimastat, batimastat, prinomastat, tanomastat, ilomastat, CGS-27023A, halofuginone, COL-3, neivastat, thalidomide, CDC501, DMXAA, L-651582, squalamine, endostatin, SU5416, SU6668, interferon-α, EMD121974, interleukin-12, IM862, angiostatic factor, tamoxifen, tori Omidyfen, raloxifene, droloxifene, iodoxyfene (no corresponding Chinese translation), anastrozole, letrozole, exemestane, flutamide, nilutamide, spironolactone, cyproterone acetate, finasteride, cimetidine, bortezomib, Velcade, bicalutamide, cyproterone acetate, flutamide, fulvestran, exemestane, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib, retinoid, rexinoid (no corresponding Chinese translation), methoxsalene (no corresponding Chinese translation), methyl aminolevulinate, aldesleukine (no corresponding Chinese translation), OCT-43, denileukin diflitox), interleukin-2, tasonermine, lentinan, sizoran, roquine, pidotimod, pegastatin, thymopentine, poly I: C, procodazol (no corresponding Chinese translation), TicBCG, corynebacterium parvum), NOV-002, ukrain (no corresponding Chinese translation), levamisole, 1311-chTNT, H-101, simoleukin, interferon alpha 2a, interferon alpha 2b, interferon gamma 1a, interleukin-2, mobeleukin, Rexin-G, tesileukin, aclarubicin, actinomycin, arglabin (no corresponding Chinese translation), asparaginase, carzinophilin (no corresponding Chinese translation), chromomycin, daunomycin, folinic acid, masoprofen, neocarzinostatin, peplomycin, sarcopeniain, solanine, trabectedin, streptozocin, testosterone, kunenecatechin, sinecatechins (no corresponding Chinese translation), alitretinoin, belotec hydrocholoride (no corresponding Chinese translation), caprotestosterone, drostanolone, elliptonium acetate, ethinyl estradiol, etoposide, fluoxymesterone, formestrol, fosfetrol (no corresponding Chinese translation), goserelin acetate, hexylaminolevulinic acid, histrelin, hydroxyprogesterone, ixabepilone, leuprorelin, medroxyprogesterone acetate, megestrol acetate, methylprednisolone, methyltestosterone, miltefosine, dibromomannitol, nandrolone phenylpropionate, norethindrone acetate, prednisolone, prednisone, temsirolimus, testolactone, triamcinolone hydroxybenzoate, triptorelin, vapreotide acetate, zenastatin, amsacrine, arsenic trioxide, bisantrene hydrochloride, chlorambucil, chlortrianisene, cisplatin, cyclophosphamide, diethylstilbestrol, hexamethylmelamine, hydroxyurea , lenalidomide, lonidamine, mechlorethamine (no corresponding Chinese translation), mitotane, nedaplatin, nimustine hydrochloride, pamidronate, pipobroman, porfimer sodium, ranimustine, razoxane, semustine, sobuzoxane, mesylate, triethylene melamine, zoledronic acid, camostat mesylate, fadrozole HCl, nafoxidine, aminoglutethimide, carmofur, clofarabine, cytosine arabinoside, decitabine, doxifluridine, enocitabine, fludarabinephosphate (no corresponding Chinese translation), fluorouracil, tegafur, uracil nitrogen mustard, abarelix, bexarotene, raltiterxed (no corresponding Chinese translation), tamibarotene, temozolomide, vorinostat, megestrol acetate, clodronate disodium, levamisole, nano-iron oxide, iron isomaltoside (no corresponding Chinese translation), celecoxib, ibudilast, bendamustine, hexamethylmelamine, dibromodulcitol, sirolimus, pralatrexate, TS-1, decitabine, bicalutamide, flutamide, letrozole, clodronate disodium, degarelix, toremifene citrate, histamine dihydrochloride, DW-166HC, diamine nitrazepam, decitabine, irinoteacn hydrochloride (no corresponding Chinese translation), amsacrine, romidepsin, tretinoin, cabazitaxel, vandetanib, lenalidomide, ibandronic acid, miltefosine, vitespen (no corresponding Chinese translation), mifatide, nadroparin, granisetron, ondansetron, tropisetron, alizapril, ramosetron, dolasetron mesylate, fosaprepitant meglumine, nabilone, aprepitant, dronabinol, TY-10721, lisuride maleate, epiceram (no corresponding Chinese translation), defibrotide , dabigatran etexilate, filgrastim, pegfilgrastim, reditux (no corresponding Chinese translation), erythropoietin, molaspagrin, oprelleukin, sipuleucel-T (no corresponding Chinese translation), M-Vax, acetyl-L-carnitine, donepezil hydrochloride, 5-aminolevulinic acid, methyl aminolevulinate, cetrorelix acetate, icodextrin, leuprorelin, metbylphenidate (no corresponding Chinese translation), octreotide, amlexanox, plerixafor, menatetrenone, anethole dithiolethione (no corresponding Chinese translation), doxorcalciferol, cinacalcet hydrochloride, alefacept, lindane, thyroglobulin, thymosin, ubenimex, imiquimod, everolimus, sirolimus, H-101, lasofoxifene, trilostane, incadronic acid, ganglioside, pegaptanib sodium, vertoporfin (no corresponding Chinese translation), minodronic acid, zoledronic acid, gallium nitrate, alendronate sodium, etidronate disodium, pamidronate disodium, dutasteride, sodium stibogluconate, armodafinil, dexrazoxane, amifostine, WF-10, temoporphin, darbepoetin alfa, ancilastin, sargramostim, palifermin, R-744, nepidramine, oprelvekin, denileukin diftitox (no corresponding Chinese translation), crisantaspase, buserelin, deserelin, lanreotide, octreotide, pilocarpine, bosentan, calicheamicin, maytansine, and cyclonicotinamide.

For more details, those skilled in the art can refer to the manual edited by the “Association des Enseignants de Chimie Thérapeutique” and entitled “Traité de chimie thérapeutique, Volume 6, Médicaments antitumoraux et perspectives dans le traitement des cancers, TEC&DOC Edition, 2003”.

In a second preferred embodiment of the present invention, the immunoconjugate consists of a binding protein linked to at least a radioisotope. When the binding protein is an antibody or an antigen-binding fragment thereof, this immunoconjugate is called an antibody radioisotope conjugate (or "ARC").

In order to selectively destroy tumors, antibodies can include highly radioactive atoms. Various radioactive isotopes can be used to produce ARCs, such as, but not limited to, At ²¹¹ , C ¹³ , N ¹⁵ , O ¹⁷ , Fl ¹⁹ , I ¹²³ , I ¹³¹ , I ¹²⁵ , In ¹¹¹ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , tc ⁹⁹ m , Bi ²¹² , P ³² , Pb ²¹² , radioactive isotopes of Lu, gadolinium, manganese or iron.

Any method or process known to those skilled in the art can be used to incorporate such radioisotopes into ARCs (see, for example, "Monoclonal Antibodies in Immunoscintigraphy" Chatal, CRC Press 1989). As non-limiting examples, tc ⁹⁹ m or I ¹²³ , Re ¹⁸⁶ , Re ¹⁸⁸ , and In ¹¹¹ can be attached via a cysteine residue. Y ⁹⁰ can be attached via a lysine residue. I ¹²³ can be attached using the IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80:49-57).

Several examples can be mentioned to illustrate the common knowledge of those skilled in the art in the field of ARC, such as an anti-CD20 monoclonal antibody and an In ¹¹¹ or Y 111 conjugated chelator via a thiourea linker. ⁹⁰ radioisotope (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 it consists of an anti-CD33 antibody linked to calicheamicin (U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116; 5,767,285; 5,773,001). Recently, we can also mention the ADC called Adcetris (corresponding to Brentuximab vedotin (no corresponding Chinese translation)), which has recently been accepted by the FDA for the treatment of Hodgkin's lymphoma (Nature, volume 476, pp380-381, August 25, 2011).

In a third preferred embodiment of the present invention, the immunoconjugate consists of a binding protein linked to at least one toxin. When the binding protein is an antibody or an antigen-binding fragment thereof, such an immunoconjugate is called an antibody-toxin conjugate (or "ATC").

Toxins are potent and specific poisons produced by living organisms. They are typically composed of chains of amino acids, which can vary in molecular weight from a few hundred (peptides) to hundreds of thousands (proteins). They can also be low-molecular organic compounds. Toxins are produced by many organisms, such as bacteria, fungi, algae, and plants. Many of these are extremely toxic, possessing toxicity several orders of magnitude greater than that of nerve agents.

Toxins used in ATC may include, but are not limited to, all classes of toxins that can exert their cytotoxic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

Useful enzymatically active toxins and fragments thereof include diphtheria toxin A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthus proteins, Phytolaca americana proteins (no corresponding Chinese translation) (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotonin, sapaonaria officinalis inhibitor, gelonin, mitogellin (no corresponding Chinese translation), restrictocin, phenomycin, enomycin and tricothecene (no corresponding Chinese translation).

Small molecule toxins such as dolastatin, auristatin, trichothecenes and CC1065, and derivatives of these toxins with toxin activity are also contemplated herein. Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division and have anticancer and antifungal activity.

"Linker," "Linker unit," or "Link" refers to a chemical moiety comprising a covalent bond or chain of atoms that covalently attaches a binding protein to at least one cytotoxic agent.

Linkers can be prepared 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 bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-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-methylethyltriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating cytotoxic agents to targeting systems. Other cross-linker 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-vinylsulfone)benzoate), which can be commercially obtained (e.g., from Pierce Biotechnology, Inc., Rockford, 111, USA).

Linkers can be "non-cleavable" or "cleavable."

In preferred embodiments, it includes a "cleavable linker" to facilitate intracellular release of the cytotoxic agent. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used. In preferred embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the cytotoxic agent from the binding protein in the intracellular environment.

For example, in some embodiments, the linker is cleavable by a cleavage agent present in the intracellular environment (e.g., within a lysosome or endosome or caveolae). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease (including but not limited to a lysosomal or endosomal protease). Typically, the peptidyl linker is at least 2 amino acids long, or at least 3 amino acids long. The cleavage agent can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drug within the target cell. For example, a peptidyl linker (such as a Phe-Leu or Gly-Phe-Leu-Gly linker) that is cleavable by the thiol-dependent protease cathepsin B, which is highly expressed in cancer tissue, can be used. In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker. One advantage of using intracellular proteolytic release of the cytotoxic agent is that the agent is generally attenuated upon conjugation, and the serum stability of the conjugate is generally high.

In other embodiments, the cleavable joint is pH sensitive, i.e. sensitive to hydrolysis under a certain pH value. Typically, the pH sensitive joint is hydrolyzable under acidic conditions. For example, an acid unstable joint (such as hydrazone, semicarbazone, thiosemicarbazone, cis-aconitamide, orthoester, acetal, ketal, etc.) that can be hydrolyzed in lysosome can be used. Such joints are relatively stable under neutral pH conditions, such as those in blood, but are unstable at pH 5.5 or 5.0 or less (approximate lysosomal pH). In some embodiments, the hydrolyzable joint is a thioether joint (such as, for example, attached to the thioether of the therapeutic agent by an acylhydrazone bond).

In yet other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). Various disulfide linkers 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-α-methyl-α-(2-pyridyl-dithio) toluene), SPDB, and SMPT.

As a non-limiting example of a non-cleavable or "non-reducible" linker, one can mention the immunoconjugate trastuzumab-DM1 (TDM1), which combines trastuzumab with the linked chemotherapeutic agent maytansine (Cancer Research 2008; 68:(22). November 15, 2008).

In a preferred embodiment, the immunoconjugates of the present invention can be prepared by any method known to those skilled in the art, such as, but not limited to, i) reacting a nucleophilic group of an antigen binding protein with a divalent linker reagent followed by reaction with a cytotoxic agent or ii) reacting a nucleophilic group of a cytotoxic agent with a divalent linker reagent followed by reaction with a nucleophilic group of an antigen binding protein.

When the antigen-binding protein is glycosylated, the nucleophilic groups on the antigen-binding protein include, but are not limited to, the N-terminal amino group, side chain amino groups such as lysine, side chain thiol groups, and hydroxyl or amino groups of sugars. Amines, thiols, and hydroxyls are nucleophilic and can react with electrophilic groups on linker moieties to form covalent bonds. Linker reagents include, but are not limited to, active esters such as NHS esters, HOBt esters, haloformates, and acid halides; alkyl and benzyl halides such as haloacetamides; aldehydes, ketones, carboxyl groups, and maleimide groups. The antigen-binding protein may have reducible interchain disulfide bonds, i.e., cysteine bridges. By treating with a reducing agent such as DTT (dithiothreitol), the antigen-binding protein can be made reactive for conjugation with the linker reagent. Thus, each cysteine bridge will theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antigen-binding protein by any reaction known to those 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.

Immunoconjugates can also be produced by modifying the antigen-binding protein to introduce an electrophilic moiety that can react with the nucleophilic substituents of the linker reagent or cytotoxic agent. The sugars of the glycosylated antigen-binding protein can be oxidized to form aldehyde or ketone groups, which can react with the amino groups of the linker reagent or cytotoxic agent. The resulting imine Schiff base group can form a stable bond or can be reduced to form a stable amine bond. In one embodiment, the carbohydrate portion of the glycosylated antigen-binding protein is reacted with galactose oxidase or sodium periodate to produce carbonyl groups (aldehydes and ketones) in the protein, wherein the carbonyl group can react with the appropriate group on the drug. In another embodiment, a protein containing an N-terminal serine or threonine residue can be reacted with sodium periodate to produce an aldehyde that replaces the first amino acid.

In certain preferred embodiments, the linker unit may have the following general formula:

--Ta--Ww--Yy--

in:

-T- is the stretcher unit;

a is 0 or 1;

-W- is an amino acid unit;

w is independently an integer from 1 to 12;

-Y- is a spacer unit;

y is 0, 1, or 2.

When the extension unit (-T-) is present, the antigen-binding protein is connected to the amino acid unit (-W-). Useful functional groups that may be present in the antigen-binding protein, whether natural or chemically treated, include sulfhydryl, amino, hydroxyl, anomeric hydroxyl of carbohydrates, and carboxyl. Suitable functional groups are sulfhydryl and amino. If present, sulfhydryl groups can be generated by reducing the intermolecular disulfide bonds of the antigen-binding protein. Alternatively, sulfhydryl groups can be generated by reacting the amino group of the lysine portion of the antigen-binding protein with 2-iminothiolane or other sulfhydryl generating reagents. In a specific embodiment, the antigen-binding protein is a recombinant antibody and is engineered to carry one or more lysines. More preferably, the antigen-binding protein can be engineered to carry one or more cysteines (see ThioMabs).

In certain embodiments, the Stretcher forms a bond with a sulfur atom of an antigen binding protein. The sulfur atom may be derived from a reduced sulfhydryl (-SH) group of the antigen binding protein.

In certain other embodiments, the Stretcher is linked to the antigen binding protein via a disulfide bond between a sulfur atom of the antigen binding protein and a sulfur atom of the Stretcher.

In other embodiments, the reactive group of the stretcher comprises a reactive site that reacts with an amino group of the antigen-binding protein. The amino group can be an arginine or lysine amino group. Suitable amine-reactive sites include, but are not limited to, active esters, such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters, anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

In yet another aspect, the reactive group of the stretch comprises a reactive site that can react with a modified carbohydrate group present on the antigen binding protein. In one embodiment, the antigen binding protein is enzymatically glycosylated to provide a carbohydrate moiety (it should be noted that when the antigen binding protein is an antibody, the antibody is typically naturally glycosylated). The carbohydrate can be mildly oxidized with a reagent such as sodium periodate, and the carbonyl unit of the resulting oxidized carbohydrate can be condensed with a stretch containing a functional group (e.g., a hydrazine, an oxime, a reactive amine, a hydrazine, a thiosemicarbazide, a hydrazine carboxylate, or an aryl hydrazide).

The Amino Acid unit (-W-) links the Stretcher unit (-T-) to the Spacer unit (-Y-) if the Spacer unit is present and links the Stretcher unit to the cytotoxic agent if the Spacer unit is absent.

As previously mentioned, -Ww- can be a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit.

In some embodiments, the amino acid unit may comprise amino acid residues such as, but not limited to, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, lysine, arginine, arginine protected by tosyl or nitro groups, histidine, ornithine, ornithine protected by acetyl or formyl groups, and citrulline. Exemplary amino acid linker components preferably include dipeptides, tripeptides, tetrapeptides, or pentapeptides.

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.

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).

Exemplary pentapeptides include: Pro-Val-Gly-Val-Val (SEQ ID NO. 35).

The amino acid residues comprising the amino acid linker component include those naturally occurring, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. The amino acid linker component can be designed and optimized for enzymatic cleavage selectivity for specific enzymes, such as tumor-associated proteases, cathepsins B, C and D, or plasmin proteases.

The amino acid unit of the linker can be enzymatically cleaved by an enzyme, including but not limited to a tumor-associated protease, to release the cytotoxic agent.

The amino acid units can be designed and optimized for their selectivity for enzymatic cleavage by specific tumor-associated proteases. Suitable units are those whose cleavage is catalyzed by proteases, cathepsins B, C and D, and plasmin.

When a Spacer unit (-Y-) is present, it links the Amino Acid unit to the cytotoxic agent. There are generally two types of Spacers: self-immolative and non-self-immolative. A non-self-immolative Spacer unit is one in which, after enzymatic cleavage of the Amino Acid unit from the immunoconjugate, some or all of the Spacer unit remains bound to the cytotoxic agent. Examples of non-self-immolative Spacers include, but are not limited to, (Glycine-Glycine) Spacers and Glycine Spacers. In order to release the cytotoxic agent, an independent hydrolysis reaction should be performed within the target cell to cleave the bond of the Glycine-Drug unit.

In another embodiment, the non-self-immolative Spacer unit (-Y-) is -Gly-.

In one embodiment, the immunoconjugate lacks a spacer unit (y = 0). Alternatively, an immunoconjugate containing a self-removable 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) unit linked to -Ww- via the nitrogen atom of the PAB group and directly linked to -D via a carbonate, carbamate or ether group.

Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically equivalent to PAB groups, such as 2-aminoimidazole-5-methanol derivatives and ortho- or para-aminobenzyl acetals. Spacers that readily undergo cyclization upon amide bond hydrolysis can be used, such as substituted and unsubstituted 4-aminobutyric acid amides, appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems, and 2-aminophenylpropionic acid amide.

In an alternative embodiment, the spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporate additional cytotoxic agents.

Finally, the present invention relates to immunoconjugates as described above for use in the treatment of cancer.

The cancer may preferably be selected from Ax1-related cancers comprising tumor cells expressing or overexpressing the whole or part of the protein Ax1 on their surface.

More specifically, the cancer is breast cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, gastric cancer, glioma, lung cancer, melanoma, osteosarcoma, ovarian cancer, prostate cancer, rhabdomyosarcoma, kidney cancer, thyroid cancer, endometrial cancer and any drug resistance phenomenon.Another object of the present invention is a pharmaceutical composition comprising the immunoconjugate described in the description.

More specifically, the present invention relates to a pharmaceutical composition comprising the immunoconjugate of the present invention and at least one excipient and/or pharmaceutically acceptable carrier.

In this specification, the expression "pharmaceutically acceptable carrier" or "excipient" is intended to mean a compound or combination of compounds that enters into a pharmaceutical composition without causing secondary reactions and allows, for example, to facilitate administration of the active compound, increase its lifespan and/or efficacy in the body, increase its solubility in solution, or improve its preservation. These pharmaceutically acceptable carriers and excipients are well known and are adjusted by those skilled in the art according to the properties of the selected active compound and the mode of administration.

Preferably, these immunoconjugates are administered systemically, in particular intravenously, intramuscularly, intradermally, intraperitoneally or subcutaneously, or orally. In a more preferred embodiment, the composition comprising the immunoconjugate according to the invention is administered several times in a sequential manner.

The mode of administration, dosage and optimal pharmaceutical form thereof can be determined according to criteria which are usually taken into account when establishing a treatment suitable for a patient, such as, for example, the patient's age or weight, the severity of his/her general condition, tolerance to treatment and noted side effects.

Other features and advantages of the present invention are further presented in the following description using examples and figures, the legends of which are shown below.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: In vitro cytotoxicity analysis on SN12C cells using Mab-zap conjugated secondary antibodies.

Figures 2A, 2B and 2C: Binding specificity of 1613F12 to immobilized rhAx1-Fc protein (2A), rhDtk-Fc (2B) or rhMer-Fc (2C) protein by ELISA.

Figure 3: FACS analysis of 1613F12 binding on human tumor cells.

Figure 4: ELISA on immobilized rmAx1-Fc protein ("rm" stands for mouse recombinant).

Figure 5: 1613F12 binding on COS7 cells determined using flow cytometry via an indirect labeling procedure.

Figure 6: Competition ELISA for Gas6 binding using 1613F12.

Figure 7: Epitope binding analysis by western blot using SN12C cell lysate. NH (no heat); NR (no reduction); H (heat); R (reduction). GAPDH detection confirmed correct loading on the gel.

Figures 8A and 8B: Study of Ax1 downregulation after 1613F12 binding on SN12C cells by Western blotting, wherein Figure 8A - Western blot images are representative of three independently performed experiments (western blot analysis performed after 4 hours and 24 hours of incubation with 1613F12 on SN12C cells); and Figure 8B - Optical density quantification of the membrane presented using "QuantityOne" software.

Figures 9A, 9B, and 9C: Immunofluorescence microscopy of SN12C cells after incubation with 1613F12. Figure 9A - Photograph of mIgG1 isotype control condition for membrane and intracellular staining. Figure 9B - Membrane staining. Figure 9C - Intracellular staining of Ax1 receptors with 1613F12 and the early endosomal marker EEA1. Image overlays are shown below, with arrows indicating visualized co-localization.

Figure 10: Effect of 1613F12 on SN12C cell proliferation in vitro compared to the effect of a mIgG1 isotype control antibody.

Figures 11A-11K: Direct cytotoxicity analysis of 1613F12-saponin immunoconjugates 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.

DETAILED DESCRIPTION

Example

Example 1: Ax1 receptor internalization

Because the immunoconjugate process is more efficient when the target antigen is an internalizing protein, the Mab-Zap cytotoxicity assay was used to study Ax1 receptor internalization in human tumor cell lines. Specifically, the Mab-Zap reagent is a chemical conjugate consisting of affinity-purified goat anti-mouse IgG and the ribosome-inactivating protein, saponin. If internalization of the immune complex occurs, saponin dissociates from the target agent and inactivates ribosomes, leading to inhibition of protein synthesis and ultimately cell death. Cell viability was determined after 72 hours of incubation with anti-Ax1 antibodies or a mIgG1 isotype control antibody on Ax1-positive cells, allowing for 1613F12-induced Ax1 receptor internalization.

For this example, cells that were highly Ax1 positive as determined using Qifikit reagent (Dako) were used. The data are listed in Table 5 below.

Table 5

In the following examples, SN12C cells are used as a non-limiting example. Any other cell line that expresses appropriate levels of Ax1 receptors on its cell surface may be used.

A range of concentrations of 1613F12 or mIgG1 isotype control antibodies were preincubated with 100 ng of Mab-Zap (Advanced Targeting Systems) secondary antibody in cell culture medium for 30 minutes at room temperature. These mixtures were loaded onto subconfluent SN12C cells plated on white 96-well microplates. Plates were incubated at 37°C in the presence of 5% _CO2 for 72 hours. Cell viability was determined using the Cell Titer Glo cell proliferation assay according to the manufacturer's instructions (Promega). Several controls were performed: i) without any secondary immunoconjugate and ii) without primary antibody. In parallel, an mIgG1 isotype control was analyzed.

The obtained results are shown in FIG1 .

1613F12 showed a maximum cytotoxic effect of approximately 36% against SN12C cells. No cytotoxic effect was observed in the presence of the 9G4 antibody (considered as an mIgG1 isotype control in this experiment). No cytotoxicity was observed in wells containing only the primary antibody (data not shown). Thus, the Ax1 receptor appears to be a convenient antigen for targeting immunoconjugates, as immune complexes containing Ax1-1613F12-MabZap elicited potent target cell cytotoxicity.

Example 2: Generation of antibodies against rhAx1 ECD

To generate mouse monoclonal antibodies (MAbs) against the extracellular domain (ECD) of the human Ax1 receptor, five BALB/c mice were immunized subcutaneously five times with 15-20.10 ^CHO -Ax1 cells, twice with 20 μg of rhAx1 ECD. The first immunization was performed in complete Freund's adjuvant (Sigma, St. Louis, MD, USA). Subsequent immunizations were supplemented with incomplete Freund's adjuvant (Sigma).

Three days before fusion, immunized mice were boosted with both 20.10 ⁶ CHO-Ax1 cells and 20 μg of rhAx1 ECD and IFA.

To generate hybridomas, spleen perfusion and mincing of proximal lymph nodes were performed, harvested from 1 of 5 immune mice (selected after serum titration), and fused to SP2/0-Ag14 myeloma cells (ATCC, Rockville, MD, USA) to prepare splenocytes and lymphocytes, respectively. The fusion protocol is described by Kohler and Milstein (Nature, 256:495-497, 1975). The fused cells were then subjected to HAT selection. Generally, in order to prepare monoclonal antibodies or their functional fragments, particularly those of mouse origin, it is possible to relate to those techniques described in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988).

Approximately 10 days after fusion, hybridoma colonies were screened. For the primary screen, hybridoma supernatants were evaluated for secretion of mAbs produced against the Ax1 ECD protein using ELISA. In parallel, FACS analysis was performed using wt CHO and Ax1-expressing CHO cells (ATCC) to select mAbs capable of binding to cellular forms of Ax1 present on the cell surface.

Selected hybridomas were cloned as quickly as possible by limiting dilution and subsequently screened for reactivity to the Ax1 ECD protein. The cloned mAbs were then isotyped using an Isotyping Kit (cat# 5300.05, Southern Biotech, Birmingham, AL, USA). Clones obtained from each hybridoma were selected and expanded.

ELISA analysis was performed using pure hybridoma supernatants as follows, or when IgG content was determined in the supernatant, titration was performed starting at 5 μg/ml. Serial dilutions were then performed in 11 rows as follows. Briefly, 96-well ELISA plates (Costar 3690, Corning, NY, USA) were coated with 50 μl/well of rhAx1-Fc protein (R and D Systems, cat No. 154-AL) or 2 μg/ml of rhAx1 ECD in PBS at 4°C overnight. The plates were then blocked with PBS containing 0.5% gelatin (#22151, Serva Electrophoresis GmbH, Heidelberg, Germany) for 2 hours at 37°C. After knocking the plates to discard the saturation buffer, 50 μl of pure hybridoma supernatant or 50 μl of the 5 μg/ml solution was added to the ELISA plates and incubated at 37°C for 1 hour. After three washes, 50 μl of horseradish peroxidase-conjugated monoclonal goat anti-mouse IgG (#115-035-164, Jackson Immuno-Research Laboratories, Inc., West Grove, PA, USA) diluted 1/5000 in PBS containing 0.1% gelatin and 0.05% Tween 20 (w:w) was added for 1 hour at 37°C. The ELISA plate was then washed three times, and TMB (#UP664782, Uptima, Interchim, France) substrate was added. After a 10-minute incubation at room temperature, the reaction was terminated with 1 M sulfuric acid, and the optical density was measured at 450 nm.

For selection by flow cytometry, ¹⁰ cells (CHO wt or CHO-Ax1) were plated in each well of a 96-well plate containing PBS containing 1% BSA and 0.01% sodium azide (FACS buffer) at 4°C. After centrifugation at 2000 rpm for 2 minutes, the buffer was removed and the hybridoma supernatant or purified mAb to be tested (1 μg/ml) was added. After incubation at 4°C for 20 minutes, the cells were washed twice and incubated with Alexa 488-conjugated goat anti-mouse antibody (#A11017, Molecular Probes Inc., Eugene, USA) diluted 1/500 in FACS buffer for 20 minutes at 4°C. After a final wash with FACS buffer, propidium iodide was added to each tube at a final concentration of 40 μg/ml and the cells were analyzed by FACS (Facscalibur, Becton-Dickinson). Wells containing cells alone and cells incubated with a secondary Alexa488-conjugated antibody were included as negative controls. An isotype control (Sigma, ref M90351MG) was used in each experiment. At least 5000 cells were evaluated to calculate the mean fluorescence intensity (MFI).

More precisely, 300.10 ⁶ harvested spleen cells and 300.10 ⁶ myeloma cells (1:1 ratio) were fused. The resulting cell suspension of 200 cells was then plated in 30 96-well plates at 2.10 ⁶ cells/ml.

A first screening (approximately 14 days after fusion) was performed using Wt CHO and Ax1-expressing CHO cells by ELISA on rhAx1 ECD protein and by FACS analysis, allowing the selection of 10 hybridomas showing an optical density (OD) greater than 1 on rhAx1 ECD coating and an MFI lower than 50 on wt CHO cells and greater than 200 on CHO-Ax1 cells.

These 10 hybridomas were expanded and cloned by limiting dilution. A 96-well plate was prepared for each code. Nine days after plating, supernatants from the cloning plates were first screened by ELISA for binding specificity to the extracellular domain of the rhAx1-ECD protein. Three clones per code were expanded and isotyped. Once anti-Ax1 antibodies were generated, their ability to internalize after binding to Ax1 on the cell surface was further investigated.

Example 3: Binding specificity of Ax1

In this example, the binding of 1613F12 was first investigated on the rhAx1-Fc protein. Then, its binding was investigated on two other members of the TAM family (rhDtk-Fc and rhMer-Fc).

Briefly, recombinant human Ax1-Fc (R and D Systems, cat No. 154AL/CF), rhDtk (R and D Systems, cat No. 859-DK), or rhMer-Fc (R and D Systems, cat No. 891-MR) proteins were coated onto Immulon II 96-well plates overnight at 4°C. After a 1-hour blocking step with 0.5% gelatin solution, purified 1613F12 antibody was added at a starting concentration of 5 μg/ml ( ^3.3310-8 M) and incubated at 37°C for an additional 1 hour. Serial 1/2 dilutions were then performed across 12 columns. The plates were washed, and goat anti-mouse (Jackson) IgG-HRP was added and incubated at 37°C for 1 hour. The reaction was developed using TMB substrate solution. The commercially available anti-Ax1 Mab154 antibody was also used in parallel (data not shown). Coating controls were performed in the presence of HRP-labeled goat anti-human IgG Fc polyclonal serum (Jackson, ref: 109-035-098) and/or HRP-conjugated anti-histidine antibody (R and D Systems, ref: MAB050H). No nonspecific binding was observed in the absence of the primary antibody (diluent). The results are shown in Figures 2A, 2B, and 2C, respectively.

This example shows that the 1613F12 antibody binds only to the rhAx1-Fc protein and not to rhDtk or rhMer, two other members of the TAM family. No cross-specificity of binding of the 1613F12 antibody was observed between TAM members.

Example 4: 1613F12 recognizes the cellular form Ax1 expressed on tumor cells

First, we used a commercially available Ax1 antibody (R and D Systems, ref: MAB154) to establish cell surface Ax1 expression levels on human tumor cells. This was performed in parallel with calibration beads to allow for quantification of Ax1 expression levels. Next, we used the Ax11613F12 antibody to investigate cell surface Ax1 binding. In both cases, the experimental conditions are briefly described below.

For cell surface binding studies, a 2-fold serial dilution of a 10 μg/ml ( ^6.66 M) primary antibody solution (1613F12, MAB154Ax1 commercial antibody, or the mIgG1 isotype control 9G4 mAb) was prepared at 10 points and applied to ^2.10 cells for 20 minutes at 4°C. After washing _three times in phosphate-buffered saline (PBS) supplemented with 1% BSA and 0.01% NaN₃, cells were incubated with a secondary goat anti-mouse Alexa 488 antibody (1/500 dilution) for 20 minutes at 4°C. After an additional three 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 average fluorescence intensity.

To determine quantitative ABC using the MAB154Ax1 antibody, calibration beads were used. Cells were then incubated in parallel with the beads and polyclonal goat anti-mouse immunoglobulin/FITC (goat F(ab') ₂ ) at saturating concentrations. The number of antigenic sites on the investigated cells was then determined by interpolation of the calibration curve (fluorescence intensity of each bead population relative to the number of MAb molecules on the beads).

Quantification of cell surface Ax1 expression levels

Ax1 expression levels on the surface of human tumor cells were determined by flow cytometry using an indirect immunofluorescence assay (DAKO, Denmark). A quantitative flow cytometry kit was used to assess cell surface antigens. Comparison of the mean fluorescence intensity (MFI) of beads with known antigen levels via a calibration plot allowed determination of the antibody binding capacity (ABC) of the cell lines.

Table 6 shows the expression levels of Ax1 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, Pancl) (ATCC, NCI) as determined using the commercially available Ax1 antibody MAB154 (R and D Systems). Values are given as antigen-bound complex (ABC).

Table 6

The results obtained with a commercially available Ax1 monoclonal antibody (MAB154) showed that the Ax1 receptor is expressed at various levels depending on the human tumor cells considered.

4.2. Ax1 detection by 1613F12 on human tumor cells

More specifically, Ax1 binding was studied using 1613F12.

A 1613F12 dose-response curve was run. The MFI obtained using various human tumor cells was then analyzed using Prism software. The data are shown in Figure 3.

The data indicate that 1613F12 binds specifically to membrane Ax1 receptors as demonstrated by the saturation curve spectrum.

Example 5: Cross-specificity of 1613F12 between species

To understand the cross-species specificity of 1613F12, two species were considered: mouse and monkey. First, binding of recombinant mouse (rm) Ax1 receptor was investigated by ELISA (Figure 4). Next, flow cytometry experiments were performed using monkey COS7 cells, as these cells express the Ax1 receptor on their surface (Figure 5). The COS7 cell line was derived by immortalizing the CV-1 cell line, derived from African green monkey kidney cells, with a version of the SV40 genome that produces large T antigens but is defective in genome replication.

rmAx1-Fc ELISA

Briefly, recombinant murine Ax1-Fc (R and D Systems, cat No. 854-AX/CF) protein was coated overnight at 4°C onto Immulon II 96-well plates. After a 1-hour blocking step with 0.5% gelatin solution, purified 1613F12 antibody was added at a starting concentration of 5 μg/ml ( ^3.33 M) and incubated at 37°C for an additional 1 hour. Serial dilutions of 1/2 were then performed across 12 columns. The plates were then washed, and goat anti-mouse (Jackson) IgG-specific HRP was added for 1 hour at 37°C. The reaction was developed using TMB substrate solution. Commercially available murine anti-Ax1 Mab154 antibody was also used in parallel. Coating controls were performed in the presence of HRP-conjugated goat anti-human IgG Fc polyclonal serum (Jackson, ref. 109-035-098) and/or HRP-conjugated anti-histidine antibody (R and D Systems, ref: MAB050H). In the absence of primary antibody (diluent), no specific binding was observed.

The results are shown in Figure 4. This figure shows that the 1613F12 Mab described in the present invention does not bind to the murine Ax1 ECD domain.

FACS COS7

For 1613F12 cell binding studies using COS7 cells, 2.10 cells were incubated at 4°C for 20 minutes with a concentration range of antibodies (12 points) prepared by 1/2 serial dilution of 10 ^μg /ml ( ^6.6610-8 M) 1613F12 or m9G4 (mIgG1 isotype control mAb). After washing three times in phosphate-buffered saline (PBS) supplemented with 1% BSA and 0.01% _NaN3 , the cells were incubated with the secondary antibody goat anti-mouse Alexa 488 (1/500 dilution) for 20 minutes at 4°C. After an additional three washes in PBS supplemented with 1% BSA and 0.1% _NaN3 , the cells were analyzed by FACS (Facscalibur, Becton-Dickinson). At least 5000 cells were evaluated to calculate the mean value of fluorescence intensity. Data were analyzed using Prism software.

The results are shown in Figure 5. Titration curves established in COS7 cells using 1613F12 or an mIgG1 isotype control demonstrate that 1613F12 recognizes the monkey cell form of the Ax1 receptor expressed on the COS7 cell surface. A plateau was reached at 1613F12 concentrations above 0.625 μg/ml (4.2 ^μM ). As expected, no binding was observed in the presence of the mIgG1 isotype control.

This example illustrates the fact that 1613F12 does not cross-react with the murine Ax1 receptor. In contrast, it strongly binds to the monkey Ax1 receptor expressed on the surface of COS7 cells.

Example 6: Gas6 competition experiment in the presence of 1613F12

To further characterize the anti-Ax1 mAb, a Gas6 competition assay was performed. In this assay, free rhAx1-Fc protein and anti-Ax1 antibody were incubated to form an antigen-antibody complex, which was then loaded onto the Gas6-coated surface of an assay plate. Unbound antibody-antigen complexes were washed away before adding an enzyme-linked secondary antibody raised against the human Fc portion of the rhAx1-Fc protein. Substrate was then added, and the intensity of the signal resulting from the enzyme-substrate reaction was used to determine the antigen concentration.

Briefly, a reaction mixture containing rhAx1-Fc protein was prepared on separate saturated (0.5% gelatin in 1X PBS) plates in the presence or absence of the anti-Ax1 mAb to be tested. A serial 1:2 dilution of the mouse anti-Ax1 antibody was performed (starting at 80 μg/ml in 12 columns). 0.5 μg/ml of rhAx1-Fc protein (R and D Systems, ref. 154AL/CF) was then added, with the exception of a negative control containing only ELISA diluent (0.1% gelatin, 0.05% Tween 20 in 1X PBS). After homogenization, the competition sample was loaded onto a Gas6-coated plate (R and D Systems, cat. No. 885-GS-CS/CF) containing a 6 μg/ml rhGas6 solution in PBS. After incubation and several washes, bound rhAx1-Fc protein was detected using goat anti-human IgG-HRP (Jackson, ref. 109-035-098). Once bound, TMB substrate was added to the plate. The reaction was stopped by adding 1M _{H2SO4 acid} solution and the optical density was read at 450 nm using a microplate reader.

This experiment (Figure 6) demonstrates that 1613F12 can compete with rhAx1-Fc for binding to the immobilized ligand. Competition for Gas6 binding occurs in the presence of 1613F12 antibody concentrations above 2.5 μg/ml ( ^1.6710-8 M). At 1613F12 concentrations above 10 μg/ml ( ^6.6710-8 M), rhAx1-Fc binding to immobilized Gas6 is no longer observed. 1613F12 blocks Gas6 binding to rhAx1-Fc.

Example 7: Epitope identification by Western blotting

To determine whether 1613F12 recognizes a linear or conformational epitope, western blot analysis was performed using SN12C cell lysates. Samples were processed under reducing or non-reducing conditions. If a band is visible in the reduced sample, the tested antibody targets a linear epitope in the ECD domain; if not, it is directed against a conformational epitope in the Ax1 ECD.

SN12C cells were seeded at 5.10 ⁴ cells / cm ² in a T162 cm ² flask in RPMI + 10% heat-inactivated FBS + 2mM L-glutamine at 37 ° C in a 5% CO ₂ atmosphere for 72 hours. The cells were then washed twice with phosphate-buffered saline (PBS) and lysed with 1.5 ml of ice-cold lysis buffer (50mM Tris-HCl (pH 7.5); 150mM NaCl; 1% Nonidet P40; 0.5% sodium deoxycholate; and 1 complete protease inhibitor cocktail tablet, plus 1% anti-phosphatase). The cell lysate was shaken at 4 ° C for 90 minutes and clarified at 15000 rpm for 10 minutes. Protein concentration was quantified using BCA. Various samples were loaded. The first 10 μg of whole-cell lysate (10 μg in 20 μl) was prepared under reducing conditions (1× sample buffer (BIORAD) + 1× reducing agent (BIORAD)) and loaded on SDS-PAGE after incubation at 96°C for 2 minutes. Secondly, two additional 10 μg samples of whole-cell lysate were prepared under non-reducing conditions (1× sample buffer (BIORAD) only). Before loading on the SDS-PAGE gel, one of these two samples was heated in a 96°C incubation for 2 minutes; the other was kept on ice. After migration, the proteins were transferred to a nitrocellulose membrane. The membrane was saturated with TBS-Tween 20 0.1% (TBST), 5% skim milk at room temperature for 1 hour and probed with 10 μg/ml 1613F12 overnight at 4°C. The antibody was diluted in Tris-buffered saline-0.1% Tween 20 (v/v) (TBST) containing 5% skim milk. The membrane was then washed with TBST and incubated with a peroxidase-conjugated secondary antibody (dilution 1/1000) for 1 hour at room temperature. Immunoreactive proteins were visualized using ECL (Pierce #32209). After visualization of Ax1, the membrane was washed again with TBST and incubated with a mouse anti-GAPDH antibody (dilution 1/200,000) for 1 hour at room temperature. The membrane was then washed in TBST and incubated with a peroxidase-conjugated secondary antibody for 1 hour at room temperature. The membrane was washed and GAPDH was visualized using ECL.

The results are shown in FIG7 .

1613F12 primarily recognizes the conformational epitope, as specific bands were observed primarily under non-reducing conditions. However, a weak signal was detected under denaturing migration conditions in SN12C cell lysates, indicating that 1613F12 can weakly bind to the linear epitope.

Example 8: Measurement of Ax1 downregulation induced by 1613F12 by Western blotting

In the following examples, the human renal cell carcinoma cell line SN12C (ATCC) was selected to investigate the activity of Ax1 antibodies against Ax1 receptor expression. The SN12C cell line overexpresses the Ax1 receptor. In Figures 8A-8B, downregulation of Ax1 was investigated by Western blotting on whole cell extracts.

SN12C cells were seeded at ^6.10 cells/ ^cm in 6-well plates in RPMI + 10% heat-inactivated FBS + 2mM L-glutamine in a 5% _CO atmosphere at 37°C for 48 hours. After washing twice with phosphate-buffered saline (PBS), cells were serum-starved in medium containing 800ng/ml recombinant murine gas6 ligand (R and D Systems, ref: 986-GS/CF) or 10μg/ml mIgG1 isotype control antibody (9G4) or 10μg/ml Ax1 antibody of the present invention and incubated for an additional 4 hours or 24 hours. The medium was then gently removed and the cells were washed twice with cold PBS. The cells were lysed with 200μl of ice-cold lysis buffer (50mM Tris-HCl (pH 7.5); 150mM NaCl; 1% Nonidet P40; 0.5% sodium deoxycholate; and 1 complete protease inhibitor cocktail tablet plus 1% anti-phosphatase). The cell lysates were shaken at 4°C for 90 minutes and clarified at 15000rpm for 10 minutes. Protein concentration was quantified using the BCA method. Whole cell lysates (10 μg in 20 μl) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was saturated for 1 hour at room temperature with TBS-Tween 200.1% (TBST), 5% skim milk, and probe hybridization was performed overnight at 4°C with 0.5 μg/ml commercially available M02Ax1 antibody (AbNova H00000558-M02). The antibody was diluted in Tris-buffered saline-0.1% Tween 20 (v/v) (TBST) containing 5% skim milk. The membrane was then washed with TBST and incubated for 1 hour at RT with a peroxidase-conjugated secondary antibody (dilution 1/1000). Immunoreactive proteins were visualized using ECL (Pierce#32209). After Ax1 visualization, the membrane was washed again with TBST and incubated with mouse anti-GAPDH antibody (dilution 1/200,000) for 1 hour at room temperature. The membrane was then washed in TBST and incubated with a peroxidase-conjugated secondary antibody for 1 hour at room temperature. The membrane was washed and GAPDH was visualized using ECL. Band intensities were quantified by densitometry.

The results presented in Figures 8A and 8B are representative of three independent experiments and demonstrate that 1613F12 is able to downregulate Ax1 in human tumor cell lines that overexpress Ax1. At 4 hours, 1613F12 triggered 66% downregulation of Ax1, and up to 87% after 24 hours of incubation with 1613F12.

Example 9: Flow cytometry study of the effect of 1613F12 on cell surface Ax1 expression

Flow cytometry allows labeling of Ax1 receptors on the cell surface. Using this technique, it is possible to highlight the effects of antibodies on membrane Ax1 expression. In this example, human renal tumor SN12C cells, which express high levels of Ax1, were used.

Three days before the experiment, the SN12C tumor cell line was cultured in RMPI1640 containing 1% L-glutamine and 10% FCS. The cells were then detached with trypsin and plated in RMPI1640 containing 1% L-glutamine and 5% FBS in 6-well plates. The next day, the antibody of interest was added at 10 μg/ml. Untreated wells were also included. The cells were incubated at 37°C, 5% _CO2 . Twenty-four hours later, the cells were washed with PBS, detached and incubated with the same antibody of interest in FACS buffer (PBS, 1% BSA, 0.01% sodium azide). Untreated wells were also stained with the same antibody to compare the signal intensity obtained with the same Mab on treated and untreated cells. The cells were incubated at 4°C for 20 minutes and washed three times with FACS buffer. Alexa488-labeled goat anti-mouse IgG antibody was incubated for 20 minutes and the cells were washed three times before FACS analysis on the propidium iodide-negative cell population.

Two parameters were determined: (i) the difference in fluorescence signal detected on the surface of untreated (no Ab) cells compared to Ab-treated cells at T24h, and (ii) the percentage of Ax1 remaining on the cell surface. The percentage of Ax1 remaining was calculated as follows:

% Remaining Ax1 = (MFI _Ab24h / MFI _{no Ab24h} ) × 100

Data from one representative experiment (of 3) are listed in Table 7. Results were reproducible in three independent experiments.

The difference in MFI between Mab staining in untreated cells and in treatment with the same antibody reflects the downregulation of Ax1 protein on the cell surface of the cells due to the binding of the Mab in question. The condition without antibody gave similar results to the condition in the presence of isotype control antibody (m9G4).

Table 7

The data showed that the mean fluorescence intensity detected on the surface of cells treated with 1613F12 for 24 hours was reduced (-514) compared to the MFI obtained for untreated cells labeled with 1613F12. After incubation with 1613F12 antibody for 24 hours, 45.2% of the cell surface Ax1 receptors remained on the surface of SN12C cells.

Example 10: Study of 1613F12 internalization using fluorescent immunocytochemistry

Complementary internalization results were obtained by confocal microscopy using an indirect fluorescent labeling method.

Briefly, SN12C tumor cell lines were cultured in RMPI1640 containing 1% L-glutamine and 10% FCS for 3 days prior to the experiment. The cells were then detached with trypsin and plated in RMPI1640 containing 1% L-glutamine and 5% FCS on 6-well plates containing coverslips. The next day, 1613F12 was added at 10 μg/ml. Cells treated with irrelevant antibodies were also included. The cells were incubated at 37°C, 5% _CO2 for 1 h and 2 h. For T0h, cells were incubated at 4°C for 30 min to determine antibody binding on the cell surface. The cells were washed with PBS and fixed with paraformaldehyde for 15 min. The cells were rinsed and incubated with goat anti-mouse IgG Alexa 488 antibody for 60 min at 4°C to determine the remaining antibody on the cell surface. To track antibody penetration into the cells, the cells were fixed and permeabilized with saponin. Membranes and intracellular antibodies were stained with goat anti-mouse IgG Alexa 488 (Invitrogen). Early endosomes were identified using a rabbit polyclonal antibody against EEA1 and visualized with a goat anti-rabbit IgG-Alexa 555 antibody (Invitrogen). Cells were washed three times and nuclear staining was performed with Draq5. After staining, cells were mounted on Prolong Gold loading matrix (Invitrogen) and analyzed using a Zeiss LSM510 confocal microscope.

Photographs are shown in Figures 9A-9C.

Images were obtained by confocal microscopy. In the presence of the mIgG1 isotype control (9G4), neither membrane staining nor intracellular labeling was observed (Figure 9A). After 1 hour of incubation of SN12C cells with 1613F12, a gradual disappearance of membrane anti-Ax1 labeling was observed (Figure 9B). At 1 hour and 2 hours, intracellular accumulation of the 1613F12Ax1 antibody was clearly observed (Figure 9C). The intracellular antibody co-localized with EEA1 (an early endosomal marker). These images confirm the internalization of 1613F12 into SN12C cells.

Example 11: In vitro anti-Ax1-mediated anti-tumor activity

SN12C proliferation assay

10,000 SN12C cells were seeded per well of a 96-well plate in FCS-free medium and incubated overnight at 37°C in a 5% _CO₂ atmosphere. The following day, cells were preincubated with 10 μg/ml of each antibody for 1 hour at 37°C. Cells were treated with or without rmGas6 (R and D Systems, cat No. 986-GS/CF) by adding ligand directly to the wells and then grown for 72 hours. Proliferation was measured after ^3H thymidine incorporation.

The data are shown in Figure 10. When 1613F12 was added to SN12C cells, they remained silent and no effect was observed with 1613F12.

Example 12: Cytotoxic efficacy of 1613F12-saponin immunoconjugates in various human tumor cell lines

In this example, the cytotoxic efficacy of saponin-coupled 1613F12 was documented. To this end, direct in vitro cytotoxicity analysis was performed using a large panel of human tumor cell lines (Figures 11A-11K). The panel of tumor cell lines provides a variety of Ax1 expression.

Briefly, 5000 cells were seeded in 100 μl of 5% FBS complete medium on a 96-well culture plate. After incubation for 24 hours at 37°C in a 5% _CO2 atmosphere, a range of immunoconjugate concentrations (1613F12-saponin or 9G4-saponin or naked 1613F12 or 9G4) were applied to the cells. The culture plates were then incubated for 72 hours at 37°C in a humidified 5% _CO2 incubator.

At D4, cell viability was assessed using a luminescent cell viability kit (Promega Corp., Madison, Wis.), which allows determination of the number of viable cells in culture based on the quantification of the presence of ATP (an indicator of metabolically active cells). Luminescent emission was recorded by a luminometer device.

The percentage of cytotoxicity was calculated based on the luminescence output using the following formula:

Cytotoxicity % = 100 - [(RLU _Ab-sap × 100) / RLU _{no Ab} ]

In Figures 11A-11K, graphs showing the percentage of cytotoxicity as a function of immunoconjugate concentration, obtained from different in vitro cytotoxicity assays of (A) SN12C, (B) Calu-1, (C) A172, (D) A431, (E) DU145, (F) MDA-MB-435S, (G) MDA-MB-231, (H) PC3, (I) NCI-H226, (J) NCI-H125, or (K) Pancl tumor cells treated with a range of 1613F12-saponin immunoconjugate concentrations are grouped together.

Figures 11A-11K demonstrate that the 1613F12-saponin immunoconjugate elicits cytotoxicity in these different human tumor cell lines. The potency of the resulting cytotoxic effect depends on the human tumor cell line.

Example 13: Binding kinetics of Ax1 antibody to human Ax1 ECD

Affinity measurements of 1613F12 were then determined using Biacore.

Biacore X was used to measure the binding kinetics of Ax1 antibodies to human Ax1 ECD.

The Biacore system, an instrument based on the optical phenomenon of surface plasmon resonance (SPR), allows the detection and measurement of protein-protein interactions in real time without the use of labels.

Briefly, experiments were performed using a CM5 sensor chip as a biosensor. Rabbit IgG was immobilized on flow cells 1 and 2 (FC1 and FC2) of the CM5 sensor chip at a level of 9,300-10,000 response units (RU), and the antibody was captured using amine coupling chemistry.

Binding was assessed using multiple cycles. Each measurement cycle was performed in HBS-EP buffer at a flow rate of 30 μl/min. The Ax1 antibody to be tested was then captured on the chip for 1 minute on the FC2, achieving an average capture value of 311.8 RU (SD = 5.1 RU) for the 1613F12 antibody. Analyte (Ax1 ECD antigen) was injected starting at 200 nM, and crude ka and kd were measured in real time using two-fold serial dilutions.

At the end of each cycle, the surface was regenerated by injecting 10 mM glycine hydrochloride, pH 1.5, to eliminate the antibody-antigen complex and captured antibody. The signal considered corresponds to the difference in signal observed between FC1 and FC2 (FC2-FC1). The association rate (ka) and dissociation rate (kd) were calculated using a one-to-one Langmuir binding model. The equilibrium dissociation constant (KD) was determined as the ratio of ka/kd. Experimental values were analyzed in Biaevaluation software version 3.0. Chi- ^square analysis was performed to evaluate the accuracy of the data.

The data are summarized in Table 8 below.

Table 8. Binding kinetics and affinity of 1613F12 for human Ax1 ECD

Antibody ka(1/Ms) kd(1/s) KD(M) 1613F12 0.71 (0.6%)

To generate the human extracellular domain (ECD) of Ax1, human cDNA encoding the human soluble AXL receptor was first cloned into the pCEP4 expression vector by PCR. The purified product was then digested with the restriction enzymes HindIII and BamHI and ligated into the pCEP4 expression vector, which had been pre-cleaved with the same enzymes. Finally, the recombinant plasmid, pCEP[Ax1]His ₆ , was further confirmed by DNA sequencing.

HEK293E cells adapted for suspension were then cultured in Ex-Cell293 (SAFC Biosciences) medium containing 4 mM glutamine. All transfections were performed using linear 25 kDa polyethyleneimine (PEI). Transfected cultures were maintained at 37°C in a shaker incubator containing 5% CO2 and agitated at 120 rpm for 6 days. Cells were harvested by centrifugation, and the supernatant containing the recombinant His-tagged protein was processed for purification on a Ni-NTA agarose column.

Claims (17)

1. An in vitro method for screening monoclonal antibodies or their binding fragments, said monoclonal antibody or its binding fragment being capable of delivering or internalizing a molecule of interest into mammalian cells expressing Axl protein on their surface, said molecule of interest being covalently linked to said monoclonal antibody, characterized in that it comprises the following steps: a) Select monoclonal antibodies that can i) bind to the extracellular ECD of human protein Axl at an _EC50 of at least ^10⁻⁹ M; and ii) induce a decrease in average fluorescence intensity of at least 200. b) Covalently link the molecule of interest to the monoclonal antibody selected in step a) to form a complex; c) Contact the complex obtained in step b) with mammalian cells on which Axl protein is expressed; d) Determine whether the complex has been intracellularly delivered or internalized into the mammalian cells expressing the Axl protein on their surface; and e) Select the monoclonal antibody as a compound capable of delivering or internalizing the molecule of interest into mammalian cells that express the Axl protein on their surface. 2. The method according to claim 1, characterized in that the monoclonal antibody selected in step a) is capable of binding human protein Axl. 3. The method according to claim 2, characterized in that the monoclonal antibody selected in step a) is capable of binding to the extracellular ECD of human protein Axl as shown in sequence SEQ ID No. 31 or 32. 4. The method according to claim 1 or 2, characterized in that the monoclonal antibody selected in step a) is capable of binding human protein Axl extracellular ECD at an _EC50 between ^10⁻⁹ M and ^10⁻¹² M. 5. The method according to any one of claims 1 or 2, characterized in that the selection in step e) can be achieved by a method selected from immunofluorescence, flow cytometry, Western blotting, and cytotoxicity/cell inhibition evaluation methods. 6. An in vitro method for preparing a cytotoxic or cell-inhibiting complex, said cytotoxic or cell-inhibiting complex being capable of delivering a cytotoxic compound into mammalian cells expressing Axl protein on their surface, said method comprising the step of covalently linking a cytotoxic agent or cell inhibitor to a monoclonal antibody, said monoclonal antibody being characterized in that: i) Extracellular ECD of human protein Axl bound with at least ^10⁻⁹ M _EC₅₀ . ii) Induces at least a 200% decrease in average fluorescence intensity, and iii) When the Axl protein is expressed on the surface of the mammalian cell, it is internalized into the mammalian cell after binding to the Axl protein. 7. An in vitro method for preparing a cytotoxic or cell-inhibiting complex capable of delivering a cytotoxic compound into mammalian cells, the method comprising the step of covalently linking a cytotoxic agent to a compound, wherein the compound: i) Extracellular ECD of human protein Axl bound with at least ^10⁻⁹ M EC ₅₀ ; ii) Induces at least a 200% decrease in average fluorescence intensity, and iii) When the Axl protein is expressed on the surface of the mammalian cell, it is internalized into the mammalian cell after binding to the Axl protein. 8. A monoclonal antibody or an antigen-binding fragment thereof, used as a targeting product for delivering a cytotoxic agent to a host target site, said host target site comprising an epitope located in the extracellular domain of protein Axl, said monoclonal antibody comprising: The three light chain CDRs as shown in SEQ ID NO. 1, 2 and 3 respectively; and The three heavy chain CDRs are shown as SEQ ID NO.4, 5 and 6 respectively. 9. The monoclonal antibody or its antigen-binding fragment according to claim 8, characterized in that the monoclonal antibody: i) binds to the epitope in the extracellular ECD of human protein Axl with at least ^10⁻⁹ M EC ₅₀ ; ii) Induces at least a 200% decrease in average fluorescence intensity, and iii) It is internalized after binding with the human protein Axl. 10. The monoclonal antibody or its antigen-binding fragment according to any one of claims 8 or 9, characterized in that the extracellular domain of protein Axl is composed of the extracellular domain of human protein Axl as shown in sequence SEQ ID NO. 31 or 32. 11. The monoclonal antibody or its antigen-binding fragment according to claim 8 or 9, characterized in that it is capable of specifically binding to the epitope in the extracellular ECD of human protein Axl at an _EC50 between ^10⁻⁹ M and ^10⁻¹² M. 12. The monoclonal antibody or its antigen-binding fragment according to claim 8, characterized in that the monoclonal antibody elicits at least 200 Δ(MFI _24h untreated cells - MFI _24h treated cells). 13. The monoclonal antibody or its antigen-binding fragment according to claim 12, characterized in that the monoclonal antibody elicits at least 300 Δ(MFI _24h untreated cells - MFI _24h treated cells). 14. The monoclonal antibody or antigen-binding fragment thereof according to claim 8 or 9, wherein the monoclonal antibody was produced from hybridoma I-4505 deposited at the Pasteur Institute CNCM in France on July 28, 2011. 15. An immunoconjugate comprising a monoclonal antibody or an antigen-binding fragment thereof conjugated to a cytotoxic agent according to any one of claims 8 to 14. 16. The immunoconjugate according to claim 15, for the treatment of cancer. 17. A pharmaceutical composition comprising the immunoconjugate of claim 15 or 16 and at least one excipient and/or a pharmaceutically acceptable carrier.