OA16895A

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

Info

Publication number: OA16895A
Application number: OA1201400176
Authority: OA
Inventors: Charlotte Beau-Larvor; Liliane Goetsch; Nicolas Boute
Original assignee: Pierre Fabre Medicament
Priority date: 2011-11-03
Filing date: 2012-11-05
Publication date: 2016-01-18

Abstract

The present invention relates to an antigen binding protein, in particular a monoclonal antibody, capable of binding specifically to the protein Axl as well as the amino and nucleic acid sequences coding for said protein. From one aspect, the invention relates to an antigen binding protein, or antigen binding fragments, capable of binding specifically to Axl and, by inducing internalization of Axl, being internalized into the cell. The invention also comprises the use of said antigen binding protein as an addressing product in conjugation with other anti- cancer compounds, such as toxins, radio - elements or drugs, and the use of same for the treatment of certain cancers.

Description

ANTIGEN BINDING PROTEIN AND ITS USE AS AD DRE S S ING

PRODUCT FOR THE TREATMENT OF CANCER

The présent invention relates to a novel antigen binding protein, in particular a monoctonal antibody, capable of binding specifically to the protein Axl as well as the amino and nucleic acid sequences coding for said protein. From one aspect, the invention relates to a novel antigen binding protein, or antigen binding fragments, 5 capable of binding specifically to Axl and, by inducing intemalization of Axl, being intemalized into the cell. The invention also comprises the use of said antigen binding protein as an addressing product in conjugation with other anti-cancer compounds, such as toxins, radio-elements or drugs, and the use of same for the treatment of certain cancers.

Axl” (also referred to as “Ufo”, “Ark” or *Tyro7”) was cloned from patients with chronic mycloid Icukemia as an oncogene triggering the transformation when overexpressed by mouse NIH3T3. It bclongs to a family of receptor tyrosine kinases (RTKs) called the TAM (Tyro3, Axl, Mer) family, which includes Tyro3 (Rsc, Sky, Dtk, Etk, Brt, Tif), Axl, and Mer (Eyk, Nyk, Tyro-12) [Lemke G. Nat. Rev. Immunol. (2008).8, 15 327-336].

The human protein Axl is a 894 amino acids protein which sequence is represented in the sequence listing as SEQ ID NO. 29. Amino acids 1-25 corresponding to the signal peptide, the human protein Axl, without the said peptide signal, is represented in the sequence listing as SEQ ID NO. 30.

Gas6, originally isolated as growth arrest-specîfic gene, is the common ligand for the members of the TAM femily [Vamum B.C. et al Nature (1995).373, 623-626]. Gas6 exhibits the highest afimity for Axl, followed by Tyro3 and finally by Mer [Nagata K. et al J. BioL Chem. (1996).271, 30022-30027]. Gas6 consista in a γcarboxyglutamate (Gta)-rich domain that médiates binding to phospholipîd membranes, four epidermal growth factor-like domains, and two laminin G-tike (LG) domains [Manfioletti G., Brancolini,C., Avanzi.G. & Schneider,C. Mol. Cell BioL ¢1993).13,

4976-4985]. As many other RTKs, ligand binding results in receptor dimerization and autophosphorylation of tyrosine residues (tyrosine residues 779, 821 and 866 for the receptor Axl) which serve as docking sites for a variety of intracellular signaling

molécules [Linger R.M. Adv. Cancer Res. (2008).100, 35-83]. Moreover, the Axl receptor can be activatcd through a ligand-independent process. This activation can occur when the Axl receptor îs overexpressed.

Gas6/Axl signaling has been shown to regulate various cellular processes including cell prolifération, adhesion, migration and survival in a large variety of cells in vitro [Hafizi S. & Dahlback.B. FEBS J. (2006).273, 5231-5244]. In addition, the TAM receptors are involved in the control of înnatc immunîty; they inhibit the inflammatory responses to pathogens in dendritic cells (DCs) and macrophages. They also drive phagocytosis of apoptotic cells by these immune cells and they are required 10 for the maturation and killing activity of natural killer (NK) cells [Lemke G. Nat. Rev.

Immunol. (2008).8,327-336].

Weakly expressed on normal cells, it is predominantly observed in fibroblasts, myeloid progenitor cells, macrophages, neural tissues, cardiac and skeletal muscle where it supports mainty cell survival The Gas6/Axl system plays an important rôle in 15 vascular biology by regulating vascular smooth muscle cell homeostasis [Korshunov V.A., Mohan, A.M., Georger, M.A. & Berk, B.C. Cire. Rcs. (2006).98, 1446-1452; Korshunov V.A., Daul, M., Massett, M.P. & Berk, B.C. Hypertension (2007).50, 10571062].

In tumor cells, Axl plays an important rote in regulating cellular invasion and 20 migration. O ver-express ion of Axl is associated not only with poor prognosis but also wîth inercased invasiveness of various human cancers as reported for breast, colon, esophagcal carcinoma, hepatocellular, gastric, glioma, lung, melanoma, osteosarcoma, ovarian, prostate, rhabdomyosarcoma, rénal, thyroid and utérine endométrial cancer [Linger R.M. Adv. Cancer Res. (2008).100, 35-83 and Verma A. Mot. Cancer Ther. 25 (2011).10, 1763-1773, for reviews]. In breast cancer, Axl appears to be a strong effector of the Epithelial-to-mesenchymal transition (EMT); EMT program contributes actively to migration and dissémination of cancer cells in the organism [Thiery J.P. Curr. Opin. Cell Biol. (2003).15, 740-746].

Axl has also been shown to regulate angiogenesis. Indeed knockdown of Axl in 30 endothélial cells impaired tube formation and migration [Holland S.J. et al. Cancer Res.

(2005).65, 9294-9303] as well as disturbed spécifie angiogenîc signaling pathways [Li Y. et at. Oncogene (2009).28,3442-3455].

More rccently scveral studics on a range of cellular models described the involvement of an Axl overexpression in drug résistance phenomena. The following table 1 summarized these studies.

Table 1

Référencé	Cancer type	Therapeutic agent	Cellular model
Macleod et al., 2005	Ovarian cancer	Clsplatln	PE01/PE01CDDP
Mahadevan et al., 2007	G1ST	Imatinlb Inhibitor of ckit/PDGFR	GIST882 >GIST-R
Lay et al., 2007	NSCLC	Doxorubldn	CL-1 clones CL1-5F4 clones
Hong et al., 2008	AML	Dcjxotu bldn/dsplati n	U937
Uu et al., 2009	Breast Cancer	Lapatlnib (HER1 and HER2 inhibitor)	HER2 (+) BT474 (34)
Keating étal., 2010	Astrocytoma	Temozolomide Ca rboplatin Vlncristin	G12 A172
Ye et al. ,2010	NSCLC	Ertatinib	HOC827

Complété référencés cited tn table 1 above are as follow:

- 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

In such a context the Ax! RTK is considered as an interesting target in oncology.

Severa! groups already developed anti-tumoral strategies targeting the gas6/Axl axis, either using naked monoclonal antibodies or targeted small molécules [Verma A. Mol.

Cancer Ther. (2011).10, 1763-1773].

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In a first embodiment, the invention relates to an antigen binding protein, or an antigen binding fragment thereof, which i) specifically binds to the human protein Axl, and ΐί) is intemalized following its binding to said human protein Axl.

More generally, the invention relates to the use of the protein Axl for the sélection of an antigen binding protein, or an antigen binding fragment thereof, capable of being intemalized following its binding to the said target Axl. More particularly, the said target is the extraccllular domain of Axl.

In thîs particular aspect, the présent invention is thus directed to an in vitro method for the screening of a compound, or a binding fragment thereof, capable of delivering or intemalizing a molécule of interest into mammalian cells, said molécule of interest being covalently linked to said compound, wherein said method comprises the following steps of:

a) selecting a compound which is capable of specifically binding the Ax! protein, or the extracellular domain (ECD) thereof, or an epitope thereof ;

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

c) contacting said compound selected in step a), or said complex obtained in step b), with a mammal cell, preferably viable cell, expressing at its surface the Axl protein, or a functional fragment thereof;

d) determîning whether said compound, or said molécule of interest or said complex, has been intraccllularly delivered or intemalized into said mammalian cell; and

e) selecting said compound as a compound capable of delivering or intemalizing a molécule of interest into a viable mammalian cell.

In a preferred embodiment, said compound capable of delivering or intemalizing a molécule of interest into a viable mammalian cell is a protein (also designated herein polypeptide or peptide) or a protein-like compound comprising a peptidic structure, particularly an amino-acid sequence of at least 5, 10, 15 or more amino acids residues, said amino-acid rcsiduc(s) can be glycosylated.

When said compound capable of delivering or intemalizing a molécule of interest into a viable mammalian cell is a protein or a protein-like compound, said i . <

compound is also called herein an “antigen binding protein”, said antigen binding protein, or binding fragment thereof, can:

- i) specifically binds to the protein Axl, preferably the human Ax! protein, and

- ii) is intemalized into a mammalian cell following its binding to said protein Axl when said Axl protein is expressed at the surface of said mammalian cell.

In a preferred embodiment, said mammalian viable cell is a human cell, preferably a cell naturally expressing the Axl protein receptor.

In a particular embodiment, the mammalian viable cells in step c) are mammalian cells which express recombinant Axl protein(s) at their surface.

In an also preferred embodiment, said molécule of interest is a cytotoxic molécule (also designated herein as cytoxic or cytostatic agent).

In an also preferred embodiment, said molécule of interest is covalently linked to said compound capable of binding the Axl protein using a linker, more preferably a peptidic linker, more preferably a cleavable peptidic linker, more preferably a linker which can be clcaved by naturel intracellular compounds contained in the mammalian cell, partîcularly in the cytosol of said mammalian cell.

In an also preferred embodiment, said compound capable of binding the Axl protein is an antibody, or fiinctional binding fragment thereof, which is specifically directed against the Ax! protein, or against an épitope thereof located into the Ax! EDC domain.

The sélection step of e) can be realized by any method known by the person skilled in the art for the évaluation of the intracellular delivering or intemalization. Assay or test capable of demonstrating or evaluating the presence, absence, or the activity of said compound capable of specifically binding the Axl protein, or of said complex formed by said compound and said molécule of interest, or of said molécule of interest which is covalently linked to said compound, are well known by the skilled person (see some examples of such test or assay disclosed hereinafter, without limiting these tests to these following test examples).

More partîcularly, these tests or assays can be realized by FACS, Immunofluorescence, flow cytometry, westem-blot, cytotoxicity/cytostatic évaluations, etc...

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In this aspect, the présent invention is also directed to an in vitro method for the préparation of a cytotoxic or cytostatic complex capable of delivering a cytotoxic compound into a mammalian cell, preferably a viable cell, said method comprising the step of:

- covalcntly linked a cytotoxic agent to a compound which is:

- i) capable of specifically binding the Axl protein, preferably the human Axl protein, and

- ii) is intemalizcd into a mammalian cell following its binding to said protein Axl when said Axl protein is expressed at the surface ofsaid mammalian cell.

Preferably said compound is a protcin-likc protein, more preferably an antibody which is specifically directed against the Ax! protein, or against an epitope thereof located into the Axl EDC domain, or a functional binding fragment of said antibody.

In preferred embodiment, said cytotoxic agent is covalently linked to the said anti-Axl antibody or functional fragment thereof, using a linker, more preferably a peptidic linker, more preferably a cleavablc peptidic linker, more preferably a linker which can be cleaved, as non limitative example by natural intracellular compounds.

Like the other members of the TAM family, the Axl extracellular domain (ECD) has an organization closcd to those of ce!! adhesion molécules. Axl ECD is characterized by a combination of two immunoglobulin-like domains foilowed by two adjacent fibronectin type 111-üke domains [O'Bryan J.P. et al. Mol. Cell Biol. (1991).11, 5016-5031]. The two N-tcrmïnal immunoglobulin-like domains are sufficient for Gas6 ligand binding [Sasaki T. et al. EMBO J. (2006).25, 80-87].

The ECD of the human protein Axl is a 451 amino acids fragment, corresponding to amino acids 1-451 of the sequence SEQ ID NO. 29, which sequence is represented in the sequence listing as SEQ ID NO. 31. Amino acids 1-25 corresponding to the signa! peptide, the ECD of the human protein Axl without the signal peptide corresponds to the amino acids 26-451 of the sequence SEQ ID NO.29, represented by the sequence SEQ ID NO. 32.

To date different modes of internalization hâve been identified. They orientate the becoming the intemalized proteins or proteic complex in the cell. After cndocytosis, most membranes proteins or lipids retums to the cel! surface (recycling), but some membrane components are delivered to late endosomes or the Golgi [Maxfield F.R. & McGraw, T.E. Nat. Rev. Mol. Cell Biol. (2004).5, 121-132].

In a preferred embodiment, the invention relates to an antigen binding protein, or an antigen binding fragment thereof, which i) specifically binds to the human protein Axl, and ii) is intemalized following its binding to said human protein Axl, said antigen binding protein comprising at least an amino acid sequence selected from the group consisting of SEQ ID NOs. 1 to 14, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NOs. 1 to 14.

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

i) specifically binds to the human protein Axl, preferably having the sequence SEQ ID NO. 29 or 30 or natural variant sequence thereof, and ii) is intemalized following its binding to said human protein Axl, said antigen binding protein comprising at least an amino acid sequence selected from the group consisting of SEQ ID NOs. I to 14.

A “binding protein” or “antigen binding protein” is a peptidic chain having a spécifie or general afTmity with another protein or molécule (generally referred as antigen). Proteins are brought into contact and form a complex when binding is possible. The antigen binding protein of the invention can preferably be, without limitation, an antibody, a fragment or dérivative of an antibody, a protein or a peptide.

By “antigen binding fragment” of an antigen binding protein according to the invention, it is întended to indicate any peptide, polypeptide, or protein retaining the ability to specifically bind to the target (also generallyreferred as antigen) ofthe antigen binding protein and comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous 175 amino acid residues, at least contiguous 200 amino acid residues, or at least contiguous 250 amino acid residues ofthe amino acid sequence ofthe antigen binding protein.

In a preferred embodiment wherein the antigen binding protein is an antibody, such “antigen binding fragments” arc selected in the group consisting of Fv, scFv (se for single chain), Fab, F(ab’)2, Fab’, scFv-Fc fragments or diabodies, or any fragment of which the half-lîfe time would hâve been increased by chemical modification, such as the addition of poly(alkylene) glycol such as poly(ethylene) glycol (“PEGylation”) (pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab’)2-PEG or Fab’-PEG) (“PEG” for Poly(EthyIene) Glycol), or by incorporation in a liposome, said fragments having at least one of the characteristic CDRs of the antibody according to the invention. Preferably, said “antigen binding fragments” will be constituted or will comprise a partial sequence of the heavy or light variable chain of the antibody from which they are derived, said partial sequence being suffïcient to retain the same specificity of binding as the antibody from which it is descended and a suffïcient affinity, preferably at least equal to 1/100, in a more preferred manner to at least 1/10, of the affinity of the antibody from which it is descended, with respect to the target. Such a functional fragment will contain at the minimum 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the sequence of the antibody from which it is descended.

The term epitope is a région of an antigen that is bound by an antigen binding protein, including antibodies. Epitopes may be defined as structural or functional. Functional épitopes are generally a subset of the structural épitopes and hâve those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include déterminants that are chemically active surface groupings of molécules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may hâve spécifie threc-dimensional structural characteristics, and/or spécifie charge characteristics.

In the présent application, the epitope is localizcd into the extracellular domain ofthe human protein Axl.

According to a preferred embodiment of the invention, the antigen binding protein, or an antigen binding fragment thereof, spccifïcally binds to an epitope localized into the human protein Axl extracellular domain, preferably having the sequence SEQ ID NO. 31 or 32 or natural variant sequence thereof.

By “specifically binding”, “specifically binds”, or the like, it is intended that the antigen binding protein, or antigen-binding fragment thereof, forms a complex with an antigen that is relatively stable under physiologie conditions. Spécifie binding can be characterized by an equilibrium dissociation constant of at least about 1,10^-6 M or less. Methods for determining whether two molécules specifically bind arc well known in the art and include, for example, equilibrium dialysis, surface plasmon résonance, and the like. For the avoidance of doubt, it does not mean that the said antigen binding fragment could not bind or interfère, at a low level, to another antigen. Nevertheless, as a preferred embodiment, the said antigen binding fragment binds only to the said antigen.

In this sense, “EC50 refers to 50% effective concentration. More precisely the term half maximal effective concentration (EC50) corresponds to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum after some specifïed exposure time. It is commonîy used as a measure of drug's potency. The EC50 of a graded dose response curve therefore represents the concentration of a compound where 50% of its maximal effect is observed. The EC50 of a quanta! dose response curve represents the concentration of a compound where 50% of the population exhibits a response, after specifïed exposure duration. Concentration measures typically follow a sîgmoidal curve, increasing rapidly over a relatively small change in concentration. This can be determined mathematically by dérivation of the best-fit line.

As a preferred embodiment, the EC50 determined in the présent invention characterized the potency of antibody binding on the Axl ECD exposed on human tumor cells. The EC50 parameter is determined using FACS analysis. The EC50 parameter reflects the antibody concentration for which 50% of the maximal binding on the human Axl expressed on human tumor cells is obtained. Each EC50 value was calculated as the midpoint of the dose response curve using a four-parameter régression curve fitting program (Prism Software). This parameter has been selected as to be représentative of physiological/pathological conditions.

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In an embodiment of the invention, the antigen binding protein, or an antigen binding fragment thereof, binds to its epitope with an ECso of at least 10’⁹ M, preferentially between IO'⁹ and 10¹² M.

Another embodiment of the invention is a process or method for the sélection of an antigen binding protein, or an antigen binding fragment thereof, capable of being intracellularly interna!izing into a mammalian cell, preferably into a human cell, preferably a viable cell, comprising the steps of:

- i) selecting antigen binding protein which specifically binds to Axl, preferably to its EDC domain or to an epitope thereof; and

- ii) selecting said antigen binding protein from previous step i) which is intemalized into a mammalian cell following their binding to an Axl protein expressed at the surface of said mammalian cell.

In a particular embodiment, said mammalian cell naturally expresses the Axl protein receptor at their surface or are mammalian cells which express recombinant Axl 15 protein at their surface, preferably human cells.

Such method or process can comprise the steps of i) selecting antigen binding protein which specifically bind to Axl with an ECso of at least 10'⁹ M and ii) selecting antigen binding protein from previous step which arc intemalized following their binding to Axl. The sélection step of ii) can be realized by any method known by the 2 0 person skilled in the art for the évaluation of the intemalization. More particularly, tests can be realized by FACS, Immunofluorescence, flow cytometry, westem-blot, cytotoxicity évaluations, etc...

Another characteristic of the antigen binding protein according to the invention is that it does not hâve any significant activity on the prolifération of tumor cells. More 25 particularly, as illustrated in the following examples, the antigen binding protein according to the invention does not hâve any significant in vitro activity on the prolifération SN12C model.

In oncology, there are multiple mechanisms by which mAbs can exert therapeutic efficacy, but often their activity is not sufficient to produce a lasting benefit.

Hence several strategies hâve been employed to enhancc their activity particularly by combining them with drugs as chcmothcrapcutic agents. As an efficient alternative to combination protocols, immunotoxins become a novel therapeutic option for treatïng n

cancer [Beck A. et al. Discov. Med. (2010).10, 329-339; Altey S.C. et at. J. Pharmacol. Exp. Ther. (2009).330, 932-938]. Antibody-drug conjugates (ADCs) represent one approach where the abîlity to hamess mAbs specificity and target the detïvery of a cytotoxic agent to the tumor may significantly enhance both mAbs and drug activîties.

Ideally the mAb will specifïcally bind to an antigen with substantial expression on tumor cells but limited expression on normal cells.

The présent invention focused on a spécifie antî-AxI binding protein, and more particularly on a spécifie anti-Axl antibody, presenting a high abîlity to bc intemalized following Axl binding. Such antigen binding protein is interesting as one of the 10 immuno-drug-conjugate components, so it addrcsscs the linked cytotoxic into the targeted cancer cells. Once intemalized the cytotoxic triggers cancer cell death.

Important keys to succcss with immunoconjugate therapy are thought to be the target antigen specificity and the intemalization of the antigen-binding protein complexes into the cancer cells. Obviousty non-intemalizing antigens are less effective 15 than intcmalizîng antigens to delivers cytotoxic agents. Intemalization processes are variable across antigens and dépend on multiple parameters that can bc influcnced by binding proteins. Cetl-surface RTKs constitute an interesting antigens family to investîgate for such an approach.

In the biomolecule, the cytotoxic brings the cytotoxic activity and the used 20 antigen binding protein brings its specificity against cancer cells, as well as a vector for entering within the cells to corrcctly address the cytotoxic.

Thus to improve the immunoconjugate molécule, the carrier-binding protein must exhibit high abîlity to intemalize into the targeted cancer cells. The efficiency with which the binding proteins mediated internalisation differs significantly depending on 2 5 the épitope targeted. Sélection of potent intemalizing anti-Axl binding proteins rcquîrcs various experimental data studying not only Axl downrcgulation but also following anti-Axl binding proteins becoming into the cells.

In a preferred embodiment, the intemalization of the antigen binding protein according to the invention can be evaluated preferably by immunofluorescence (as exemplified hereinafter in the présent application) or any method or proccss known by the person skilled in the art spécifie for the intemalization mechanism.

»

In another preferred embodiment, as the complex Axl-antigen binding protein, according to the invention, is intemalized after the binding of the binding protein of the invention to the ECD of said Axl, a réduction in the quantity of Axl at the surface ofthe cells is induced. This réduction can be quantified by any method known by the person skilled in the art (westem-blot, FACS, immunofluorescence, etc...).

In an embodiment of the invention, this réduction, thus reflecting the intemalization, can be preferably measured by FACS and expressed as the différence or delta between the Mean Fluorescence Intensity (MFI) measured on untreated cells with the MFI measured with cells treated with the antigen binding protein according to the invention.

As non limitative example of the présent invention, this delta is determined based on MFIs obtained with untreated cells and cells treated with the antigen binding protein of the invention as described in example 9 using i) human rénal tumor SN12C cells after a 24 hour incubation period with the antigen binding protein of the invention and ii) a secondary antibody labclled with Alexa488. This parameter is defined as calculated with the following formula:

Δ (MFI₂4h untreated cells - MFI₂4h antigen binding protein treated cells)

This différence between MFIs reflects the Axl downregulation as MFIs are proportional of Axl expressed on the cell-surface.

In a more preferred and advantageous aspect, the antigen binding protein, or an antigen binding fragment thereof, ofthe invention consiste of a monoclonal antibody, preferably an isolated Mab, triggering a Δ (MFI₂4h untreated cells - MFI₂4h treated cells) of at least 200, preferably of at least 300.

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

In more details, the above mentioned delta can be measured according to the following process, which must be considered as an illustrative and non limitative example:

a) Treating and incubating tumoral cells of interest with the antigen binding protein of the invention;

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

c) Measuring the MFI (représentative of the quantity of Axl présent at the surface) for the treated and the non treated cells with a secondary labeled antibody capable of binding to the antigen binding protein, and

d) Calculating the delta as the subtraction of the MFI obtained with the treated cells from the MFI obtained with the non treated cells.

The terms antibody, “antibodies” or immunoglobulin are used interchangeably in the broadest sense and include monoclonal antibodies, preferably isolated Mab, (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies or multispecific antibodies (e.g., bispecifîc antibodies so long as they exhibit the desired biological activity).

More particularly, such molécule consists of a glycoprotcin comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable région (or domain) (abbreviated herein as HCVR or VH) and a heavy chain constant région. The heavy chain constant région comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable région (abbreviated herein as LCVR or VL) and a light chain constant région. The light chain constant région comprises one domain, CL. The VH and VL régions can be further subdivided into régions of hypcrvariability, termed complementarity determining régions (CDR), interspersed with régions that are more conserved, termed framework régions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-tcrminus in the following order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable régions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant régions of the antibodies may médiate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune System (e.g. effector cells) and the first component (Clq) of the classical complément system.

Antibodies in the sense of the invention also include certain antibody fragments, thereof. The said antibody fragments exhibit the desired binding specificity and affinity, regardless of the source or immunoglobulin type (i.e., IgG, IgE, IgM, IgA, etc.), i.e., they arc capable of binding specifically the Axl protein with an affinity comparable to 5 the full-length antibodies of the invention.

In general, for the préparation of monoclonal antibodies or their functional fragments, espccially of murine origin, it is possible to refer to techniques which are described în particular in the manual “Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 10 1988) or to the technique of préparation from hybridomas described by Kohler and

Milstein (Nature, 256:495-497, 1975).

The term monoclonal antibody or Mab as used herein refers to an antibody molceule that is directed against a spécifié antigen and which may be produced by a single clone of B cells or hybridoma. Monoclonal antibodies may also be recombinant, 15 i.e. produced by protein engineering. In addition, in contrast with préparations of polyclonal antibodies which typicaliy include various antibodies directed against various déterminants, or epitopes, each monoclonal antibody is directed against a single epitope of the antigen. The invention relates to antibodies isolated or obtained by purification from naturel sources or obtained by genetic recombination or chemical 2 0 synthesis.

A preferred embodiment of the invention is an antigen binding protein, or an antigen binding fragment thereof, comprising or consisting of an antibody, said antibody comprising the three light chain CDRs comprising the sequences SEQ ID NOs. 1, 2 and 3, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% 25 and 98% Îdentîty with SEQ ID NOs. 1, 2 and 3; and the three heavy chain CDRs comprising the sequences SEQ ID NOs. 4, 5 and 6, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NOs. 4, 5 and 6.

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

* ln a preferred aspect, by CDR régions or CDR(s), it is intended to indicate the hypervariable régions of the heavy and light chains of the immunoglobulins as defined by IMGT. Without any contradictory mention, the CDRs will be defined in the présent spécification according to the IMGT numbcring system.

The IMGT unique numbcring has been defined to compare the variable domains whatever the antigen receptor, the chain type, or the spccies [Lefranc M.-P., lmmunology 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., Thouvcnin-Contet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT unique numbering, the conserved amino acids always hâve the same position, for instance cystein 23 (lst-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobie amino acid 89, cystein 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or JTRP). The IMGT unique numbering provides a standardized délimitation of the framework régions (FR1-IMGT: positions 1 to 26, FR2-1MGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining régions: CDR 1-IMGT: 27 to 38, CDR2-1MGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps represent unoccupicd positions, the CDR-1MGT lengths (shown between brackets and separated by dots, e.g. [8.8.13]) become crucial information. The IMGT unique numbering is used in 2D graphical représentations, designated as IMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q. and Lefranc, M.-P., Current Bioinfbrmatics, 2, 21-30 (2007)], and in 3D structures in IMGT/3Dstructure-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 that, without contradictory spécification in the présent spécification, complcmentarity-determining régions or CDRs, mean the hypervariable régions of the heavy and light chains of immunoglobulins as defined according to the IMGT numbering System.

Nevertheless, CDRs can also be defined according to the Kabat numbering system (Kabat et al., Sequences of proteins of immunological interest, 5^Ü Ed., U.S. Department of Health and Human Services, NIH, 1991, and later éditions). There are three heavy-chain CDRs and three light-chain CDRs. Here, the terms “CDR” and “CDRs” are used to indicate, depending on the case, one or more, or even ail, of the régions containing the majority of the amino acid residues responsiblc for the antibody’s binding affinity for the antigen or epitope it rccognizes.

According to the Kabat numbering system, the présent invention relates to an antigen binding protein, or an antigen binding fragment thereof, consisting of an antibody, said antibody comprising the three light chain CDRs, as defined according to Kabat numbering system, comprising the sequences SEQ ID NOs. 9, 10 and 11, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NOs. 9, 10 and 11; and the three heavy chain CDRs, as defined according to Kabat numbering system, comprising the sequences SEQ ID NOs. 12, 13 and 14, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NOs. 12,13 and 14.

In the sense of the présent invention, the “pcrcentage identity between two sequences of nucieic acids or amino acids means the perccntage of identical nucléotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the différences between the two sequences being distributed randomly along their length. The comparison of two nucieic acid or amino acid sequences is traditionally carried out by comparing the sequences after having optîmally aligned them, said comparison being able to be conducted by segment or by using an “alignment window. Optimal alignment of the sequences for comparison can be carried out, in addition to comparison by hand, by means of the local homology algorithm of Smith and Watcrman (1981) [Ad. App. Math. 2:482], by means of the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol. 48:443], by means of the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444] or by means of computer software using these algorithme (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by the comparison software BLAST NR or BLAST P).

The percentage identity between two nucieic acid or amino acid sequences is determined by comparing the two optimally-aligned sequences in which the nucieic acid or amino acid sequence to compare can hâve additions or délétions compared to the refcrencc sequence for optimal alignment between the two sequences. Percentage identity is calculated by determining the number of positions at which the amino acid nucléotide or residue is identical between the two sequences, preferably between the two complété sequences, dividing the number of identical positions by the total number of positions in the alignment window and multiplying the resuit by 100 to obtain the percentage identity between the two sequences.

For exemple, the BLAST program, “BLAST 2 sequences” (Tatusova et al., “Blast 2 sequences - a new tool for comparing protein and nucléotide sequences”, FEMS Microbiol., 1999, Lett. 174:247-250) available on the site http://www.ncbi.nlm.nih.gov/gorf/bl2.html, can be used with the default parameters (notably for the parameters “open gap penalty”: 5, and “extension gap penalty”: 2; the 10 selected matrix being for example the “BLOSUM 62” matrix proposed by the program);

the percentage identity between the two sequences to compare is calculated directly by the program.

For the amino acid sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with a reference amino acid sequence, preferred exemples include 15 those containing the reference sequence, certain modifications, notably a délétion, addition or substitution of at least one amino acid, troncation or extension. In the case of substitution of one or more consecutive or non-consecutive amino acids, substitutions are preferred in which the substituted amino acids arc replaced by “équivalent” amino acids. Here, the expression “équivalent amino acids” is meant to indicate any amino 20 acids likely to be substituted for one of the structural amino acids without however modifying the biological activités of the corresponding antibodies and of those spécifie examples defined below.

Equivalent amino acids can be determined either on their structural homology with the amino acids for which they are substituted or on the results of comparative tests 25 of biological activity between the various antigen binding proteins likely to be gcncratcd.

As a non-limiting example, table 2 below summarizes the possible substitutions likely to bc carried out without resulting in a signifïcant modification of the biological activity of the corresponding modified antigen binding protein; inverse substitutions are

0 naturally possible under the same conditions.

Table 2

Original residue	Substitution(s)
Ata (A)	Val, Gly, Pro
Arg (R)	Lys, His
Asn (N)	Gin
Asp (D)	Glu
Cys(C)	Ser
Otn(Q)	Asn
Glu (E)	Asp
Gty (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

An embodiment of the invention relates to an antigen binding protein, or an antigen binding fragment thereof, comprising a light chain variable domain of sequence

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

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

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

Another embodiment of the invention relates to an antigen binding protein, or an antigen binding fragment thereof, comprising the three light chain CDRs comprising the sequences SEQ ID NOs. 1, 2 and 3, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NOs.l, 2 and 3; and a heavy chain variable domain of sequence SEQ ID NO. 8, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 8.

According to a preferred embodiment ofthe invention, the antigen binding protein, or an antigen binding fragment thereof comprises the three light chain CDRs comprising the sequences SEQ ID NOs. 1,2 and 3; and a heavy chain variable domain of sequence SEQ ID NO. 8, or any sequence exhibiting at least 80% identity with SEQ IDNO.8.

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

Another embodiment of the invention relates to an antigen binding protein, or an antigen binding fragment thereof, comprising a light chain variable domain of sequence SEQ IDNO. 7, or any sequence exhibiting 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 exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 8.

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

For more clarity, table 3a below summarizes the various amino acid sequences corresponding to the antigen binding protein ofthe invention (with Mu. = murine).

Table 3a

	CDR numbering	Heavy chain	Light chain	SEQ IDNO.
1613F12	1MGT		CDR-L1	1
	CDR-L2	2
	CDR-L3	3
CDR-H1		4
CDR-H2		5
CDR-H3		6
Rabat		CDR-L1	9
	CDR-L2	10
	CDR-L3	11
CDR-H1		12
CDR-H2		13
CDR-H3		14
		Mu. variable domain	7
Mil variable domain		8

A spécifie aspect of the présent invention relates to a murine antibody, or its derived compounds or antigen binding fragments, characterized in that said antibody also comprises light-chain and heavy-chain constant régions derived from an antibody of a species hctcrologous with the mouse, notably man.

Another spécifie aspect of the présent invention relates to a chimeric antibody, or its derived compounds or antigen binding fragments, characterized in that said antibody also comprises light-chain and heavy-chain constant régions derived from an antibody of a species heterologous with the mouse, notably human.

Yet another spécifie aspect of the présent invention relates to a humanized antibody, or its derived compounds or antigen binding fragments, characterized in that the constant régions of the light-chain and the heavy-chain derived from human antibody are, respectively, the lambda or kappa région and the gamma-1, gamma-2 or gamma-4 région.

Another aspect of the invention is an antigen binding protein consisting of the monoclonal antibody I613F12 derived from the hybridoma 1-4505 deposited at the CNCM, Institut Pasteur, France, on the 28 July 2011, or an antigen binding fragment thereof.

According to another aspect, the invention relates to a murine hybridoma capable of secreting an antigen binding protein according to the invention, notably the hybridoma of murine origin filed with the Frcnch collection for microorganism cultures (CNCM, Pasteur Institute, Paris, France) on July 28, 2011, under number 1-4505. Said hybridoma was obtained by the fusion of Balb/C immunized mice splcnocytcs/lymphocytes and cells of the mycloma Sp 2/O-Ag 14 cell line.

According to another aspect, the invention relates to a murine hybridoma capable of secreting an antibody comprising the three light chain CDRs comprising the sequences SEQ ID NOs. 1, 2 and 3; and the three heavy chain CDRs comprising the sequences SEQ ID NOs. 4, 5 and 6, said hybridoma being filed at the CNCM, Pasteur Institute, Paris, France, on July 28, 2011, under number 1-4505. Said hybridoma was obtained by the fusion of Balb/C immunized mice splenocytes/lymphocytes and cells of the myeloma Sp 2/O-Ag 14 cell line.

An object of the invention is the murine hybridoma 1-4505 deposited at the CNCM, Institut Pasteur, France, on the 28 July 2011.

The antigen binding protein of the invention also comprises chimeric or humanized antibodies.

A chimeric antibody is one containing a natural variable région (light chain and heavy chain) derived from an antibody of a given species in combination with constant régions of the light chain and the heavy chain of an antibody of a species heterologous to said given species.

The antibodies, or chimeric fragments of same, can be prepared by using the techniques of recombinant genetics. For example, the chimeric antibody could be produced by ctoning recombinant DNA containing a promoter and a sequence coding for the variable région of a nonhuman monoclonal antibody ofthe invention, notably murine, and a sequence coding for the human antibody constant région. A chimeric antibody according to the invention coded by one such recombinant gene could be, for example, a mouse-human chimcra, the specificity of this antibody being determined by the variable région derived from the murine DNA and its isotype determined by the constant région derived from human DNA. Refer to Verhoeyn et al. (BioEssays, 8:74, 1988) for methods for preparing chimeric antibodies.

In another aspect, the invention describes a binding protein which consists of a chimeric antibody.

In a particular preferred embodiment, the chimeric antibody, or an antigen binding fragment of same, of the invention comprises a light chain variable domain sequence comprising the amino acid sequence SEQ ID NO. 7, and in that it comprises a heavy chain variable domain sequence comprising the amino acid sequence SEQ ID NO. 8.

In another aspect, the invention describes a binding protein which consists of a humanized antibody.

“Humanized antibodies” means an antibody that contains CDR régions derived from an antibody of nonhuman origin, the other parts of the antibody molécule being derived from one (or several) human antibodies. In addition, some of the skeleton segment residues (called FR) can be modified to préservé binding afïinity (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 ofthe invention or fragments of same can be prepared by techniques known to a person skilled in the art (such as, for example, those described in the documents Singer et al., J. Immun., 150:2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10:1-142, 1992; and Bebbington et al., Bîo/Technology, 10:169-175, 1992). Such humanized antibodies are preferred for their use in methods involving in vitro diagnoses or préventive and/or therapeutic treatment in vivo. Other humanization techniques, also known to a person skilled in the art, such as, for example, the “CDR grafting” technique described by PDL in patents EP 0 451 261, EP 0 682 040, EP 0 939 127, EP 0 566 647 or US 5,530,101, US 6,180,370, US 5,585,089 and US 5,693,761. US patents 5,639,641 or 6,054,297, 5,886,152 and 5,877,293 can also be cited.

In addition, the invention also relates to humanized antibodies arising from the murine antibodies described above.

In a preferred manner, constant régions of the light-chain and the heavy-chain derived from human antibody are, respectively, the lambda or kappa and the gamma-1, gamma-2 or gamma-4 région.

In a preferred embodiment, the invention relates to an antigen binding protein consisting of a humanized antibody, or an antigen binding fragment, which comprises a light chain variable domain comprising the sequence SEQ ID NO. 36, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 36; and the three heavy chain CDRs comprising the sequences SEQ ID NO. 4,5 and 6.

Another embodiment of the invention relates to an antigen binding protein, or an antigen binding fragment thereof, comprising a light chain variable domain of sequence 5 selected in the group consisting of SEQ ID NO. 37 to 47, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 37 to 47; and the three heavy chain CDRs comprising the sequences SEQ ID NOs. 4,5 and 6.

By “any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 36 or 37 to 47”, its is intended to designate the sequences 10 exhibiting the three light chain CDRs SEQ ID NOs. I, 2 and 3 and, in addition, exhibiting at least 80%, preferably 85%, 90%, 95% and 98% , identity with the full sequence SEQ ID NO. 36 or 37 to 47 outsidc the sequences corresponding to the CDRs (Le. SEQIDNO. 1,2 and 3).

For more clarity, table 3b below summarizes the various amino acid sequences 15 corresponding to the humanized antigen binding protein light chain (VL) of the invention (with Hz. = humanized)

Table 3b

In an embodiment of the invention, the antigen binding protein, or an antigen binding fragment thereof, comprises a light chain variable domain selected in the group consisting of:

i) a light chain variable domain of sequence SEQ ID NO. 7 or any sequence exhîbiting at least 80% identity with SEQ ID NO.7, ii) a light chain variable domain of sequence SEQ ID NO. 36 or any sequence exhîbiting at least 80% identity with SEQ ID NO. 36; and iii) a light chain variable domain of sequence SEQ ID NO. 37 to 47 or any sequence exhîbiting at least 80% identity with SEQ ID NO. 37 to 47.

In a preferred embodiment, the invention relates to an antigen binding protein consisting of a humanized antibody, or an antigen binding fragment, which comprises a heavy chain variable domain comprising the sequence SEQ ID NO. 48, or any sequence exhîbiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 48; and the three light chain CDRs comprising the sequences SEQ ID NO. 1,2 and 3.

Another embodiment of the invention relates to an antigen binding protein, or an antigen binding fragment thereof, comprising a heavy chain variable domain of sequence selected in the group consisting of SEQ ID NO. 49 to 68, or any sequence exhîbiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 49 to 68; and the three light chain CDRs comprising the sequences SEQ ID NOs. 1, 2 and 3.

By “any sequence exhîbiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 48 and 49 to 68”, its is intended to designate the sequences exhîbiting the three heavy chain CDRs SEQ ID NOs. 4, 5 and 6 and, in addition, exhîbiting at least 80%, preferably 85%, 90%, 95% and 98% , identity with the full sequence SEQ ID NO. 48 and 49 to 68 outside the sequences corresponding to the CDRs (i.e. SEQ ID NO. 4,5 and 6).

For more clarity, table 3c below summarizes the various amino acid sequences corresponding to the humanized antigen binding protein heavy chain (VH) of the invention (with Hz. = humanized)

Table 3c

Hzl613F12 VH	Version	SEQ ID NO.
consensus	48
VH1	49
VH1 M39I	50
VH1 W55RN66K	51
VH1 184S	52
VHI S85N	53
VH1 184N S85N	54
VH2.1	55
VH2.1 Q3H	56
VH2.1 W55R	57
VH2.1 N66K	58
VH2.1 W55RN66K	59
VH2.1 R80S	60
VH2.I N66K.R80S	61
VH2.2	62
VH2.2 M89L	63
VH2.3	64
VH2.3 W55R	65
VH2.3 Q3H W55R	66
VH2.4	67
VH3	68

In an embodiment of the invention, the antigen binding protein, or an antigen binding fragment thereof, comprises a heavy chain variable domain selected in the group consisting of:

i) a heavy ehain variable domain of sequence SEQ ID NO. 8 or any sequence exhibiting at least 80% identity with SEQ ID NO.8;

ii) a heavy chain variable domain of sequence SEQ ID NO. 48 or any sequence exhibiting at least 80% identity with SEQ ID NO. 48; and iii) a heavy chain variable domaine of sequence SEQ ID NO. 49 to 68 or any sequence exhibiting at leat 80% identity with SEQ ID NO. 49 to 68.

In an embodiment of the invention, the antigen binding protein, or an antigen binding fragment thereof, comprises a light chain variable domain of sequence SEQ ID NO. 36, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 36; and a heavy chain variable domain of sequence SEQ ID NO. 48, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 48.

In another embodiment of the invention, the antigen binding protein, or an antigen binding fragment thereof, comprises a light chain variable domain of sequence selected in the group consisting of SEQ ID NO. 37 to 47, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 37 to 47; and a heavy chain variable domain of sequence selected in the group consisting of SEQ ID

NO. 49 to 68, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 49 to 68.

In an embodiment of the invention, the antigen binding protein, or an antigen binding fragment thereof, comprises:

i) a light chain variable domain of sequence SEQ ID NO. 7, 36 or 37 to 47 or

0 any sequence exhibiting at least 80% identity with SEQ ID NO.7, 36 or 37 to 47; and ii) a heavy chain variable domain of sequence SEQ ID NO. 8, 48 or 49 to 68 or any sequence exhibiting at least 80% identity with SEQ ID NO.8,48 or 49 to 68.

A novel aspect of the présent invention relates to an isolated nucleic acid characterized in that it is selected among the following nucleic acids (including any 25 degenerate genetic code):

a) a nucleic acid codîng for an antigen binding protein, or for an antigen binding fragment of same, according to the invention;

b) a nucleic acid comprising:

- a nucleic acid sequence selected from the group consisting of SEQ ID NOs.

15 to 28 and 69 to 99, or

- a nucleic acid sequence comprising the 6 nucleic acid sequences SEQ ID

NOs.: I5to20,or

- a nucleic acid sequence comprising the two nucleic acid sequences SEQ ID NOs.: 21, 22, or the two nucleic acid sequences selected from one part from SEQ ID NOs.: 69 to 79 and for the other part from SEQID NOs: 80 to 99;

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

d) a nucleic acid, preferably having at least 18 nucléotides, capable of hybridizîng under highly stringent conditions with a nucleic acid sequence as defined in part a) or b), or with a sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alïgnment with a nucleic acid sequence as defined in part a) or b),.

Table 4a below summarizes the various nucléotide sequences concerning the 10 binding protein of the invention (with Mu. - Murine).

Table 4a

	CDR numbering	Heavy chain	Light chain	SEQID NO.
1613F12	1MGT		CDR-L1	15
	CDR-L2	16
	CDR-L3	17
CDR-H1		18
CDR-H2		19
CDR-H3		20
Rabat		CDR-L1	23
	CDR-L2	24
	CDR-L3	25
CDR-H1		26
CDR-H2		27
CDR-H3		28
		Mu. variable domain	21
Mu. variable domain		22

For more clarity, table 4b below summarizes the various nucléotide sequences corresponding to the humanized antigen binding protein light chain (VL) ofthe invention (with Hz. = humanized)

Table 4b

HzI613F12VL	Version	SEQ ID NO.
VL1	69
VL1 12V	70
VL1 M4I	71
VL2.I	72
VL2.I V49T	73
VL2.I P50N	74
VL2.2	75
VL2.2 V49T	76
VL2.2 P50N	77
VL2.3	78
VL3	79

For more clarîty, table 4c below summarizcs the various nucléotide sequences corresponding to the humanizcd antigen binding protein heavy chain (VH) of the invention (with Hz. = humanized)

Table 4c

HZ1613F12 VH	Version	SEQ ID NO.
VH1	80
VH1 M39I	81
VH1 W55RN66K	82
VH1 184S	83
VH1 S85N	84
VH1 I84N S85N	85
VH2.1	86
VH2.1 Q3H	87
VH2.1 W55R	88
VH2.1 N66K	89
VH2.1 W55RN66K	90
VH2.1 R80S	91
VH2.1 N66KR80S	92
VH2.2	93
VH2.2 M89L	94
VH2.3	95
VH2.3 W55R	96
VH2.3 Q3H W55R	97
VH2.4	98
VH3	99

The terms “nucleic acid”, “nucleic sequence”, “nucleic acid sequence”, “polynucleotide”, “oligonucleotide”, “polynucleotide sequence” and “nucléotide sequence”, used interchangeably in the présent description, mean a précisé sequence of nuclcotides, modified or not, deftning a fragment or a région of a nucleic acid, containing unnatural nuclcotides or not, and being either a doublc-strand DNA, a single* strand DNA or transcription products of said DNAs.

The sequences of the présent invention hâve been isolated and/or purified, i.e., they were sampled directly or indirectly, for example by a copy, their environment having been at least partially modified. Isolated nucleic acids obtained by recombinant genetics, by means, for example, of host cells, or obtained by chemical synthesis should also be mentioned here.

“Nucleîc sequences exhibiting a percentage identity of at least 80%, preferably 85%, 90%, 95% and 98%, after optimal alîgnment with a preferred sequence” means nucleic sequences exhibiting, with respect to the reference nucleic sequence, certain modifications such as, in particular, a délétion, a troncation, an extension, a chimeric fusion and/or a substitution, notably punctual. Preferably, these are sequences which code for the same amino acid sequences as the reference sequence, this being related to the degeneration of the genetic code, or complementarity sequences that are likely to hybridize specifically with the reference sequences, preferably under highly stringent conditions, notably those defined below.

Hybridization under highly stringent conditions means that conditions related to température and ionic strength are selected in such a way that they allow hybridization to be maintained between two complementarity DNA fragments. On a purely illustrative basis, the highly stringent conditions of the hybridization step for the purpose of defïning the polynucleotide fragments described above are advantageously as follows.

DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42°C for three hours in phosphate buffer (20 mM, pH 7.5) containing 5X SSC (IX SSC corresponds to a solution of 0.15 M NaCl + 0.015 M sodium citrate), 50% formamide, 7% sodium dodccyl sulfate (SDS), 10X Dcnhardt’s, 5% dextran sulfate and 1% salmon sperm DNA; (2) primary hybridization for 20 hours at a température depending on the length of the probe (i.e.: 42°C for a probe >100 nucléotides in length) followed by two 20-minute washings at 20°C in 2X SSC + 2% SDS, one 20-minute washing at 20°C in 0.1 X SSC + 0.1% SDS. The last washing is carried out in 0.1 X SSC + 0.1% SDS for 30 minutes at 60°C for a probe >100 nucléotides in length. The highly stringent hybridization conditions described above for a polynucleotide of defined size can be adapted by a person skilled in the art for longer or shorter oligonucleotides, according to the procedures described in Sambrook, et al. (Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory; 3rd édition, 2001).

The invention also relates to a vector comprising a nucleic acid as described in the invention.

The invention notably targets cloning and/or expression vectors that contain such a nucléotide sequence.

The vectors of the invention preferably contain éléments which allow the expression and/or the sécrétion of nucléotide sequences in a given host cell. The vector thus must contain a promoter, translation initiation and termïnation signais, as well as suitable transcription régulation régions. It must be able to be maintained in a stable manner in the host cell and may optionally hâve spécifie signais which specify sécrétion 10 of the translated protein. These various cléments are selected and optimized by a person skillcd in the art according to the host cell used. For this purposc, the nucléotide sequences can be inserted in sclf-replicating vectors within the chosen host or be intégrative vectors of the chosen host.

Such vectors arc prepared by methods typically used by a person skilled in the 15 art and the resulting clones can be introduced into a suitable host by standard methods such as lipofection, electroporation, heat shock or chemical methods.

The vectors arc, for example, vectors of plasmid or viral origin. They are used to transform host cells in order to clone or express the nucléotide sequences ofthe invention.

The invention also comprises isolated host cells transformed by or comprising a vector as described in the présent invention.

The host cell can be selected among prokaryotic or eukaryotic Systems such as bacterial cells, for examplc, but also yeast cells or animal cells, notably mammal cells (with the exception of human). Insect or plant cells can also be used.

The invention also relates to animais, other than human, that hâve a transformed cell according to the invention.

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

a) the culture in a medium with the suitable culture conditions for a host cell according to the invention; and

b) the recovcry of 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 invention are of use in methods for the préparation of recombinant antigen binding proteins according to the invention. Methods for the préparation of antigen binding proteins according to the invention in recombinant form, characterized in that said methods use a vector and/or a cell transformed by a vector according to the invention, are also comprised in the présent invention. Preferably, a cell transformed by a vector according to the invention is cultured under conditions that allow the expression of the aforesaid antigen binding protein and rccovery of said recombinant protein.

As already mentioned, the host cell can be selected among prokaryotic or cukaryotic Systems. In particular, it is possible to identify the nucléotide sequences of the invention that facilitate sécrétion in such a prokaryotic or eukaryotîc system. A vector according to the invention carrying such a sequence can thus be used advantageously for the production of recombinant proteins to be secreted. Indecd, the purification of these recombinant proteins of interest will be facilitated by the fact that they are présent in the supematant ofthe cellular culture rather than inside host cells.

The antigen binding protein of the invention can also be prepared by chemical synthesis. One such method of préparation is also an object of the invention. A person skilled in the art knows methods for chemical synthesis, such as solid-phase techniques (see notably Steward et ai, 1984, Solid phase peptides synthesis, Pierce Chem. Company, Rockford, 111, 2nd ed., pp 71-95) or partial solid-phase techniques, by condensation of fragments or by conventional synthesis in solution. Polypeptides obtained by chemical synthesis and capable of containing corresponding unnatural amino acids are also comprised in the invention.

The antigen binding protein, or the antigen binding fragments of same, likely to be obtained by the method ofthe invention are also comprised in the présent invention.

According to a particular aspect, the invention concems an antigen binding protein, or an antigen binding fragment thereof, as above described for use as an addressing product for detivering a cytotoxic agent at a host target site, said host target site consisting of an epitope localized into the protein Axl extracellular domain, preferably the human protein Axl extracellular domain, more preferably the human protein Axl cxtraccllular domain having the sequence SEQ ID NO. 31 or 32, or naturel variant sequence thereof.

In a preferred embodiment, said host target site is a target site of a mammalian cell, more preferably of a human cell, more preferably ceils which naturally or by way 5 of genetical recombination, express the Axl protein.

The invention relates to an immunoconjugate comprising the antigen binding protein as described in the présent spécification conjugated to a cytotoxic agent.

In the sense of the présent invention, the expression “immunoconjugate” or “immuno-conjugatc” refers generally to a compound comprising at least an addressing 10 product physically linked with a one or more therapeutic agent(s), thus creating a highly targeted compound.

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

By “cytotoxic agent” or “cytotoxic, it is intended an agent which, when administered to a subjcct, treats or prevents the development of cell prolifération, 15 preferably the development of cancer in the subject's body, by inhibiting or preventing a cellular fonction and/or causing cell dcath.

Many cytotoxic agents hâve been isolated or synthesized and make it possible to inhibit the ceils prolifération, or to destroy or rcducc, if not definitively, at least signifïcantly the tumour ceils. However, the toxic activity of these agents is not limited 20 to tumour ceils, and the non-tumour ceils arc also cffccted and can be destroyed. More particularly, side effects arc observed on rapidly renewing ceils, such as haematopoietic ceils or ceils of the epithelium, in particular of the mucous membranes. By way of illustration, the ceils of the gastrointestinal tract are largely efïccted by the use of such cytotoxic agents.

5 One of the aims of the présent invention is also to be able to providc a cytotoxic agent which makes it possible to limit the side effects on normal ceils while at the same time conserving a high cytotoxicity on tumour ceils.

More particularly, the cytotoxic agent may preferably consist of, without limitation, a drug (i.e “antibody-drug conjugale”), a toxin (i.e. “immunotoxin” or 30 “antibody-toxin conjugate), a radioisotope (i.e. “radioimmunoconjugate” or “antibodyradioisotope conjugate”), etc.

In a first preferred embodiment of the invention, the immunoconjugate consists of a binding protein linked to at least a drug or a médicament. Such an immunoconjugate is referred as an antibody-drug conjugate (or “ADC”) when the binding protein is an antibody, or an antigen binding fragment thereof.

In a first embodiment, such drugs can be described regarding their mode of action. As non limitative example, it can be mentioned alkylating agents such as nitrogen mustard, alkyle-sulfonates, nitrosourea, oxazophorins, aziridines or imineethylenes, anti-métabolites, anti-tumor antibiotics, mitotic inhibitors, chromatin function inhibitors, anti-angiogenesis agents, anti-estrogens, anti-androgens, chelating agents, Iron absorption stimulant, Cyclooxygenase inhibitors, Phosphodîesterase inhibitors, DNA inhibitors, DNA synthetis inhibitors, Apopstotis stimulants, Thymidylate inhibitors, T cell inhibitors, Interferon agonists, Ribonucleoside triphosphate reductase inhibitors, Aromatase inhibitors, Estrogen receptor antagoniste, Tyrosine kinase inhibitors, Cell cycle inhibitors, Taxane, Tubulin inhibitors, angiogcnesis inhibitors, macrophage stimulants, Neurokinin receptor antagoniste, Cannabînoid receptor agonists, Dopamine receptor agonsists, granulocytes stimulating factor agonists, Erythropoietin receptor agonists, somatostatin receptor agonists, LHRH agonists, Calcium sensitizers, VEGF receptor antagoniste, interlcukin receptor antagoniste, osteoclast inhibitors, radical formation stimulants, endothelin receptor antagonists, Vînca alkaloid, anti-hormone or immunomodulators or any other new drug that fullfills the activity criteria of a cytotoxic or a toxin.

Such drugs are, for example, citcd in the VIDAL 2010, on the page devoted to the compounds attached to the cancerology and hematology column “Cytotoxics”, these cytotoxic compounds cited with reference to this document are cited here as preferred cytotoxic agents.

More particularly, without limitation, the following drugs are preferred according to the invention : mcchlorethamine, chlorambucol, melphalen, chlorydratc, pipobromen, prednimustin, disodic-phosphate, estramustine, cyclophosphamide, altretamine, trofosfamidc, sulfofosfamide, ifosfamide, thiotepa, triethylcnaminc, altctramine, carmustine, streptozocin, fotemustin, lomustine, busulfan, treosulfan, improsulfan, dacarbazine, cis-platinum, oxaliplatin, lobaplatin, heptaplatin, miriplatin hydrate, carboplatin, méthotrexate, pemetrexed, 5-fluoruracil, floxuridine, 516895 fluorodcoxyuridine, capecitabine, cytarabine, fludarabine, cytosine arabinoside, 6mercaptopurine (6-MP), nelarabine, 6-thioguanine (6-TG), chlorodesoxyadenosine, 5azacytidinc, gemcitabine, cladribine, deoxycoformycin, tcgafur, pentostatin, doxorubicin, daunorubicin, idarubicin, valrubicin, mitoxantrone, dactinomycin, 5 mithramycin, plicamycin, mitomycin C, bleomycin, procarbazine, paclitaxel, docetaxel,

Vinblastine, vincristine, vindesine, vinorelbine, topotecan, irinotecan, etoposide, valrubicin, amrubicin hydrochloride, pirarubicin, elliptinium acetate, zorubicin, epirubicin, idarubicin and teniposide, razoxin, marimastat, batimastat, prinomastat, tanomastat, ilomastat, CGS-27023A, halofùginon, COL-3, neovastat, thalidomide, 10 CDC 501, DMXAA, L-651582, squalamïne, endostatin, SU54I6, SU6668, interferonalpha, EMD121974, interleukin-12, IM862, angiostatin, tamoxifen, toremifene, raîoxifene, droîoxifene, iodoxyfene, anastrozole, letrozole, exemestane, flutamide, nilutamide, sprironolactone, cyproterone acetate, finasteride, cimitidine, bortezomid, Velcade, bicalutamide, cyproterone, flutamide, fulvestran, exemestane, dasatinîb, 15 erlotinib, gcfitïnib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib, retînoid, rexinoid, methoxsalene, methylaminolevulinate, aides leu kine, OCT-43, denileukin diflitox, interleukin-2, tasonermine, lentinan, sizofîlan, roquïnimex, pidotimod, pegademase, thymopcntïne, poîy I:C, procodazol, Tic BCG, coryncbacterium parvum, NOV-002, ukrain, levamisole, 1311-chTNT, H-101, celmoleukin, interferon alfa2a, interferon 20 alfa2b, interferon gamma la, interleukin-2, mobenakin, Rexin-G, teceîeukin, aclarubïcin, actinomycin, arglabin, asparaginase, carzinophilin, chromomycin, daunomycin, leucovorin, masoprocol, neocarzinostatin, peplomycin, sarkomycin, solamargine, trabectedin, streptozocin, testosterone, kunecatechins, sinecatechins, alitretinoin, belotecan hydrocholoride, calusterone, dromostanolone, elliptinium acetate, ethinyl 25 estradiol, etoposide, fluoxymestcrone, formestane, fosfctrol, goserelin acetate, hexyl aminolevulinate, histrelin, hydroxyprogesteronc, ixabepilone, leuprolide, medroxyprogesterone acetate, megesterol acetate, méthylprednisolone, methyltestosterone, miltefosine, mitobronitol, nadrolone phenylpropionate, norethindrone acetate, prednisolone, prednisone, tcmsirrolimus, testolactone, 30 triamconolonc, triptorelin, vapreotide acetate, zinostatin stimalamcr, amsacrine, arsenic trioxide, bisantrene hydrochloride, chlorambucil, chlortrianisene, cisdiamminedichloroplatinium, cyclophosphamide, diethyIstilbestro 1, hexamethylmelamine, hydroxyurea, lenalidomide, lonidamine, mechlorethanamine, mitotane, nedaplatin, nimustine hydrochloride, pamidronate, pipobroman, porfimer sodium, ranimustinc, razoxane, semustine, sobuzoxane, mesylate, triethylenemelamine, zoledronic acid, camostat mesylate, fadrozole HCl, nafoxidine, aminoglutéthimide, 5 carmofur, clofarabine, cytosine arabinoside, decitabine, doxifluridine, enocitabine, fludarabne phosphate, fluorouracil, fiora fur, uracil mus tard, abarelix, bexarotene, raltiterxed, tamibarotene, temozolomide, vorinostat, megastrol, clodronate disodium, levamisole, ferumoxytol, iron isomaltoside, celccoxib, ibudilast, bendamustine, altrctamine, mitolactol, temsirolimus, pralatrcxate, TS-1, decitabine, bicalutamîdc, 10 flutamide, letrozole, clodronate disodium, degarelix, toremifene citrate, histamine dihydrochloride, DW-166HC, nitracrine, decitabine, irinoteacn hydrochloride, amsacrine, romidepsin, tretinoîn, cabazitaxel, vandetanib, lenalidomide, ibandronic acid, mîltefosine, vitespen, mifamurtide, nadroparin, granisetron, ondansetron, tropisetron, alizapride, ramosetron, dolasctron mesilate, fosaprepitant dimeglumine, 15 nabilone, aprepitant, dronabinol, TY-10721, lisuride hydrogen maleate, epiceram, defibrotide, dabigatran etexilate, fïlgrastim, pegfilgrastim, reditux, epoetin, molgramostim, oprelvekin, sipuleucel-T, M-Vax, acetyl L-camitine, donepezil hydrochloride, 5-aminolevulinic acid, methyl aminolevulinate, cetrorelix acetate, icodextrin, leuprorelin, metbylphenidate, octreotide, amlexanox, plerixafor, 20 menatetrenone, anethole dithiolethione, doxercalciferol, cinacalcet hydrochloride, alefaccpt, romiplostïm, thymoglobulin, thymalfasin, ubenimex, imiquimod, everolimus, sirolimus, H-101, lasofoxifene, trilostane, incadronate, gangliosides, pegaptanib octasodium, vertoporfin, minodronîc acid, zoledronic acid, gallium nitrate, alendronate sodium, étidronate disodium, disodium pamidronate, dutasteride, sodium 25 stibogluconate, armodafinil, dexrazoxane, amifostine, WF-10, temoporfin, darbepoctin alfa, ancestim, sargramostim, palifermin, R-744, nepidermin, oprelvekin, denileukin diftitox, crisantaspase, buserelin, deslorclin, lanreotide, octreotide, pilocarpine, bosentan, calicheamicin, maytansinoids and ciclonicate.

For more detail, the person skilled in the art could refer to the manual edited by the “Association Française des Enseignants de Chimie Thérapeutique’’ and entitled “traité de chimie thérapeutique, vol. 6, Médicaments antitumoraux et perspectives dans le traitement des cancers, édition TEC & DOC, 2003”.

In a second preferred embodiment of the invention, the immunoconjugate consists of a binding protein linked to at least a radioisotope. Such an immunoconjugate is referred as an antibody-radioisotope conjugale (or “ARC”) when the binding protein is an antibody, or an antigen binding fragment thereof.

For sélective destruction of the tumor, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of ARC such as, without limitation, At²¹¹, C¹³, N¹⁵, O¹⁷, Fl¹⁹, I¹²³, I¹³¹, I^I2Î, In¹¹¹, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm^lî3, tc⁹⁹m, Bi²¹², P³², Pb²¹², radioactive isotopes of Lu, gadolinium, manganèse or iron.

Any methods or processes known by the person skilled in the art can be used to incorporatc such radioisotope in the ARC (see, for example “Monoclonal Antibodies in Immunoscintigraphy”, Chatal, CRC Press 1989). As non limitative example, tcm or 1 , Re , Re and In can be attached via a cysteine residue. Y can be attached via a lysine residue. I¹²³ can be attached using the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Rcs. Commun. 80:49-57).

Scveral examples can bc mentioned to illustrate the knowledge of the person skilled in the art in the field of ARC sueh as Zevalin® which is an ARC composed of an anti-CD20 monoclonal antibody and In¹¹¹ or Y⁹⁰ radioisotope bound by a thiourea linker-chelator (Wiseman et at (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et at (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin. Oncol. 20(15):3262-69) ; or Mylotarg® which is composed of an anti-CD33 antibody linked to calicheamicin, (US 4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116; 5,767,285; 5,773,001). More recently, it can also be mentioned the ADC referred as Adcetris (corresponding to the Brentuxîmab vedotin) which has been recently accepted by the FDA in the treatment of Hodgkin’s lymphoma (Nature, vol 476, pp380-381,25 August 2011).

In a third preferred embodiment ofthe invention, the immunoconjugate consists of a binding protein linked to at least a toxin. Such an immunoconjugate is referred as an antibody-toxin conjugale (or “ATC”) when the binding protein is an antibody, or an antigen binding fragment thereof.

Toxins are effective and spécifie poisons produced by living organisme. They usually consîst of an amino acid chain which can vary in molecular weight between a couple of hundred (peptides) and one hundred thousand (proteins). They may also be low-molecular organic compounds. Toxins are produced by numerous organisme, e.g., bacteria, fungi, algae and plants. Many of them are extremely poisonous, with a toxicity that is scvcral orders of magnitude greater than the nerve agents.

Toxins used in ATC can include, without limitation, ail kind of toxins which may exert their cytotoxic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alphasarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPI1, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria ofïïcinalis inhibitor, gelonin, mitogellîn, restrictocin, phenomycin, enomycin, and the tricotheccnes.

Small molécule toxins, such as dolastatins, auristatins, a trichothecene, and CC1065, and the dérivatives of these toxins that hâve toxin activity, are also contemplated herein. Dolastatins and auristatins hâve been shown to interfère with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division and hâve anticancer and antifungal activity.

Linker, Linker Unit, or link means a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches a binding protein to at least one cytotoxic agent.

Linkers may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(Nmaleimidomcthyl)cyclohcxanc-I-carboxylate (SMCC), iminothiolane (IT), bifunctional dérivatives of imidoesters (such as dimethyi adipimidate HCl), active esters (such as disuccînimidyl suberatç), aldéhydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobcnzoyl) hexanediamine), bis-diazonium dérivatives (such as bis(p-diazoniumbcnzoyl)-ethylcnediamme), diisocyanatçs (such as toluene 2,6diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4dinitrobenzene). Carbon-14-labcled l-isothiocyanatobcnzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of cyctotoxic agents to the addressing system. Other cross-linker reagents may 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 (succinîmidyl-(4-vinylsulfone)benzoate) which arc commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, 111., U.S.A).

The linker may be a “non cleavable” or “cleavable”.

In a preferred embodiment, it consists în a cleavable linker facilitating release ofthe cytotoxic agent in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker may bc used. The linker is, in a preferred embodiment, cleavable under intracellular conditions, such that cleavage of the linker releases the cytotoxic agent from the binding protein in the intracellular environment.

For example, in some embodiments, the linker is cleavable by a cleaving agent that is présent in the intracellular environment (e.g., within a lysosome or endosome or cavcolea). The linker can be, for example, a peptidyl linker that is cleavcd by an intracellular peptidasc or protease enzyme, including, but not limited to, a lysosomal or cndosomal protease. Typically, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents can inciude cathepsins B and D and plasmin, ail of which arc known to hydrolyze dipeptide drug dérivatives resulting in the release of active drug inside target cells. For example, a peptidyl linker that is cleavable by the thiol-depcndent protease cathepsin-B, which is highly expressed in canccrous tissue, can be used (e.g., a Phc-Leu or a Gly-Phe-Lcu-Gly linker). In spécifie embodiments, 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 typically attenuated when conjugatcd and the sérum stabilities of the conjugates are typically high.

In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolyzablc under acidic conditions. For example, an acid-labile linker that is hydrolyzablc in the lysosome (e.g., a hydrazonc, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used. Such linkers are relatively stable under ncutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linkcr (such as, e.g., a thiocther attached to the therapcutic agent via an acylhydrazone bond.

In yet other embodiments, the linker is cleavable under rcducing conditions (e.g., a disulfîdc linker). A variety of 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-(2pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-mcthyl-alpha(2-pyridyl-dithio)toluene)-, SPDB and SMPT.

As non limitative example of non-clcavable or “non réductible” linkers, it can be mentioned the immunoconjugate Trastuzumab-DM 1 (TDM 1) which combines trastuzumab with a linkcd chemotherapy agent, maytansine (Cancer Rescarch 2008; 68: (22). November 15,2008).

In a preferred embodiment, the immunoconjugate of the invention may be prepared by any method known by the person skilled in the art such as, without limitation, i) reaction of a nucleophilîc group of the antigen binding protein with a bivalent linker reagent followed by réaction with the cytotoxic agent or ii) réaction of a nucleophilîc group of a cytotoxic agent with a bivalent linker rcagent followed by reaction with the nucleophilîc group of the antigen binding protein.

Nucleophilîc groups on antigen binding protein include, without limitation, Nterminal amine groups, side chain amine groups, e.g. lysine, side chain thiol groups, and sugar hydroxyl or amino groups when the antigen binding protein is glycosylated. Amine, thiol, and hydroxyl groups arc nucleophilîc and capable of reacting to form covalent bonds with electrophilic groups on linker moîeties and linker reagents including, without limitation, active esters such as NHS esters, HOBt esters, haloformates, and acid halides; alkyl and benzyl halides such as haloacetamides; aldéhydes, ketoncs, carboxyl, and maleimide groups. The antigen binding protein may hâve rcducible interchain disulfïdcs, i.e. cysteine bridges. The antigen binding proteins may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two réactive thiol nucleophilcs. Additional nucleophilîc groups can be introduced into the antigen binding protein through any reaction known by the person skilled in the art. As non limitative example, reactive thiol groups may be introduced into the antigen binding protein by introducing one or more cysteine residues.

Immunoconjugates may also be produced by modification of the antigen binding protein to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or cytotoxic agent. The sugars of glycosylated antigen binding protein may be oxidized to form aldéhyde or ketone groups which may react with the amine group of linker reagents or cytotoxic agent. The resulting imine Schiff base groups may form a stable linkage, or may be reduced to form stable amine linkages. In one embodiment, réaction of the carbohydrate portion of a glycosylated antigen binding protein with either galactose oxidase or sodium meta-periodatc may yield carbonyl (aldéhyde and ketone) groups in the protein that can react with appropriate groups on the drug. In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldéhyde in place of the first amino acid.

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

-Ta-Ww-Yywhercin:

-T- is a stretcher unit;

aisOor 1;

-W- is an amino acid unit;

w is independently an integer ranging from 1 to 12;

-Y- is a spacer unit;

y is 0, 1 or 2.

The stretcher unit (-T-), when présent, links the antigen binding protein to an amino acid unit (-W-). Usefùl functional groups that can be présent on the antigen binding protein, either naturally or via chemical manipulation, include sulfhydryl, amino, hydroxyl, the anomeric hydroxyl group of a carbohydrate, and carboxyl. Suitable functional groups are sulfhydryl and amino. Sulfhydryl groups can be generated by réduction of the intramolecular disulfide bonds of the antigen binding protein, if présent. Altematively, sulfhydryl groups can be generated by reaction of an amino group of a lysine moiety of the antigen binding protein with 2-iminothiolane or other sulfhydryl generating reagents. In spécifie embodiments, 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 Cystéines (cf. ThioMabs).

In certain spécifie embodiments, the strctcher unit forms a bond with a sulfur atom of the antigen binding protein. The sulfur atom can be derived from a sulfhydryl (· -SH) group of a reduced antigen binding protein.

In certain other spécifie embodiments, the stretcher unit 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 unit.

In other spécifie embodiments, the reactive group of the stretcher contains a reactive site that can bc reactive to an amino group of the antigen binding protein. The amino group can be that of an arginine or a lysine. Suitable amine reactive sites include, but arc not limited to, activated esters such as succinimidc esters, 4-nitrophenyl esters, pentafluorophenyl esters, anhydrides, acid chlorides, sutfonyl chlorides, isocyanates and isothiocyanates.

In yet another aspect, the reactive function of the stretcher contains a réactive site that is réactive to a modified carbohydrate group that can be présent on the antigen binding protein. In a spécifie embodiment, the antigen binding protein is glycosylated cnzymatically to provide a carbohydrate moiety (to be noticed that, when the antigen binding protein is an antibody, said antibody is generally naturally glycosylated). The carbohydrate may be mildly oxidized with a reagent such as sodium periodatc and the resulting carbonyl unit of the oxidized carbohydrate can be condenscd with a stretcher that contains a functionality such as a hydrazide, an oxime, a reactive amine, a hydrazinc, a thiosemicarbazide, a hydrazine carboxylatc, or an arylhydrazide.

The amino acid unit (-W-) links the stretcher unit (-T-) to the Spacer unit (-Y-) if the spacer unit is présent, and links the stretcher unit to the cytotoxic agent if the spacer unit is absent.

As above mcntioned, -Ww- may be a dipeptide, tripeptîde, tetrapeptide, pentapeptide, hcxapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit

In some embodiments, the amino acid unit may comprise amino acid residues such as, without limitation, alanine, valine, leucine, isotcucine, méthionine, phenylalanine, tryptophan, proline, lysine protected with acety! or formyt, arginine, arginine protected with tosyl or nitro groups, histidine, omithine, omithine protected with acetyl or formyl and citrulline. Exemplary amino acid linker components include preferably a dipeptide, a tripeptîde, a tetrapeptide or a pentapeptide.

Exemplary dipeptides include: Vat-Cit, Ala-Val, Lys-Lys, Cit-Cit, Val-Lys, AlaPhe, Phe-Lys, Ala-Lys, Phe-Cit, Leu-Cit, lle-Cit, Trp-Cit, Phe-Ata, Phe-N⁹-tosy!-Arg, Phc-N⁹-Nitro-Arg.

Exemplary tripeptides include: Val-Ala-Val, Ala-Asn-Vat, Val-Leu-Lys, AlaAla-Asn, Phe-Phe-Lys, Gly-Gty-Gly, D-Phe-Phc-Lys, Gly-Phe-Lys.

Exemplary tetrapeptide include: Gly-Phe-Leu-Gly (SEQ ID NO. 33), Ala-LeuAla-Leu (SEQIDNO. 34).

Exemplary pentapeptide include: Pro-Val-Gly-Vat-Vat (SEQ IDNO. 35).

Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzyme, for example, a tumor-assocîated protease, cathepsin B, C and D, or a plasmin protease.

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

The amino acid unit can be designed and optimized in its selectivity for enzymatic cleavage by a particular tumor-assocîated protease. The suitable units are those whose cleavage is catalyzed by the proteases, cathepsin B, C and D, and plasmin.

The spacer unit (-Y-), when présent, links an amino acid unit to the cytotoxic agent. Spacer units are of two general types: sclf-immolative and non self-immolative. A non self-immolative spacer unit is one in which part or ail of the spacer unit remains bound to the cytotoxic agent after enzymatic cleavage of an amino acid unit from the immunoconjugate. Examples of a non self-immolative spacer unit include, but are not limited to a (glycine-glycine) spacer unit and a glycine spacer unit. To liberate the cytotoxic agent, an independent hydrolysis reaction should take place within the target cell to cleave the glycine-drug unit bond.

In another embodiment, a non sclf-immolative the spacer unit (-Y-) is -Gly-.

In one embodiment, the immunoconjugate lacks a spacer unit (y=0).

Altematively, an imunoconjugate containing a self-immolatîve 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 that is linked to -Ww- via the nitrogen atom of the PAB group, and connected directly to -D via a carbonate, 10 carbamate or ether group.

Other examples of sclf-immolative spacers include, but arc not limited to, aromatic compounds that are electronically équivalent to the PAB group such as 2aminoimidazol-5-mcthanol dérivatives and ortho or para-aminobenzylacetals. Spacers can be used that undergo facile cyclization upon amide bond hydrolysis, such as 15 substituted and unsubstituted 4-aminobutyric acid amides, appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring Systems and 2-aminophenylpropionic acid amides.

In an alternate embodiment, the spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporate additional 20 cytotoxic agents.

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

Cancers can be preferably selected through Axl-related cancers including tumoral cells expressing or over-expressing whole or part of the protein Axl at their

5 surface.

More particularly, said cancers are breast, colon, esophageal carcinoma, hepatocellular, gastric, glioma, lung, melanoma, osteosarcoma, ovarian, prostate, rhabdomyosarcoma, rénal, thyroid, uterine endométrial cancer and any drug résistance phenomena. Another object of the invention is a pharmaceutical composition

0 comprising the immunoconjugate as described in the spécification.

More particularly, the invention relates to a pharmaceutical composition comprising the immunoconjugate of the invention with at least an excipient and/or a pharmaceutical acceptable vehicle.

In the présent description, the expression “pharmaceutically acceptable vehicle” or “excipient” is intended to indicatc a compound or a combination of compounds entering into a pharmaceutical composition not provoking secondary reactions and which allows, for example, facilitation of the administration of the active compound(s), an increase in its lifespan and/or in its efficacy in the body, an increase in its solubility in solution or else an improvement in its conservation. These pharmaceutically 10 acceptable vehictes and excipients arc well known and will be adapted by the person skilled in the art as a function of the nature and of the mode of administration of the active compound(s) chosen.

Preferably, these immunoconjugates will be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal, intraperitoneal 15 or subeutaneous route, or by the oral route. In a more preferred manner, the composition comprising the immunoconjugates according to the invention will be administered several times, in a sequential manner.

Their modes of administration, dosages and optimum pharmaceutical forms can be determined according to the criteria generally taken into account in the establishment 20 of a treatment adapted to a patient such as, for example, the âge or the body weight of the patient, the scriousness of his/hcr general condition, the tolérance to the treatment and the secondary cffccts noted.

Other characteristics and advantages of the invention appear in the continuation of the description with the examples and the figures whose legends are represented 25 below.

FIGURE LEGENDS

Figure 1: in vitro cytotoxicity assay using Mab-zap conjugated secondary antibody on SN12C cells.

Figures 2A, 2B and 2C: Binding specificity of 1613F12 on the immobilized rhAxl-Fc protein (2A), rhDtk-Fc (2B) or rhMcr-Fc (2C) proteins by EL1SA. Figure 3: FACS analysis of the 1613F12 binding on human tumor cells Figure 4: EL1SA on the immobilized rmAxl-Fc protein (“rm” for murine recombinant).

Figure 5: 1613F12 binding on COS7 cells as determined by indirect labelling protocol using flow cytometry method.

Figure 6: Compétition ELISA of Gas6 binding using 1613F12.

Figure 7: Epitope binding analysis by western Blot using SN12C cell lysate. NH (no heat); NR (no réduction); H (heat); R (réduction). GAPDH détection attests to the correct sample loading on the geL

Figures 8A and 8B: Study of Axl downregulation after 1613F12 binding on

SN12C cells by Western Blot with Figure 8A· Western blot image représentative of the independent experiments performed (The western blot analysis was performed after a h and 24 h incubation of the 1613F12 on SN12C cells) ; and Figure 8B- Optical density quantification of the presented film using “QuantityOnc” software.

0 Figures 9A, 9B and 9C: Immunofluorescence microscopy of SN12C cells after incubation with the 1613F12 Figure 9A- Photographs of the mlgGl isotype control conditions both for the membrane and the intracellular staining. Figure 9B- Membrane staining. Figure 9C- Intracellular staining of both Axl receptor using the 1613F12 and of the early endosome marker EEA1. Image overlays are presented bellow and co· 25 localizations visualized are indicated by the arrows.

Figure 10: Effect of 1613F12 on in vitro SN12C cells prolifération compared to theeffect ofthemlgGl isotype control antibody.

Figures 11A-I1K: Direct cytotoxicity assays of the 1613F12-saporin immunoconjugate using various human tumor cell lincs. A- SN12C , B-Calu-1, C30 Al72, D-A431, E-DU145, F-MDA-MB435S, G-MDA-MB231, H-PC3,1-NCI-H226, JNCI-H125, K-Pancl.

Figure 12: ELIS A experiments studying binding on rhAxl-Fc protein of both ml6I3F12 and hz!613F12 antibodies.

Figure 13: Binding comparison of the murine, chimeric and humanized 1613F12 antibodies on SN12C cells.

Figure 14: Direct cytotoxicity assay in présence of both mouse and humanized 1613F12-saporin immunoconjugate and of the isotype controls using SN12C human rénal tumor cell line.

Figure 15: Direct cytotoxicity assay in presence of both mouse and humanized 1613F12-saporin immunoconjugate and of the isotype controls using Calu-1 human lung carcinoma cell line.

EXÀMPLES

In the following examples, the expressions 1613F12 or m!613F12 antibody refer to a murine form of the 1613F12 antibody. Humanized forms of the 1613F12 antibody are named hz!613F12.

In the same way, isotype control antibody used consists of a murine IgGl referred as 9G4. It means that, in the following examples, the expressions mlgGl control and 9G4 arc similar.

Example 1; Axl receptor Intemalization

As an immunoconjugate approach is more efficient when the targeted antigen is an intemalizing protein, Axl receptor intemalization using Mab-Zap cytotoxicity assay on human tumor cell lines was studied. More precisely, the Mab-Zap reagent is a chemical conjugate including an affinity purified goat anti-mouse IgG and the ribosome-inactivaiing protein, saporin. If intemalization of the immune complex occurs, saporin breaks away from the targeting agent and inactivâtes the ribosomes, resulting in protein synthesis inhibition and, ultimately, cell death. Cell viability détermination after 72 hours of incubation with the 1613F12 or with mlgGl isotype control antibody on Axl-positive cells allows concluding on the 1613F12 induced Axl receptor intemalization.

For this exemple highly Axl-positivc cells, as determined using Qifïkit reagent (Dako), were used. Data arc presented in the following table 5.

Table 5

Antigen binding capacity of the MAB154 commercial antibody determined for the human rénal cancer SN12C cells
RTK	AXL
Antibody Cell line	MAB154
SN12C	>100 000

In the following example, the SN12C cells were used as non limitative exemple. Any other cell line expressing appropriate level of Axl receptor on its cell surface could be used.

Concentration ranges of the I613F12 orthemlgGl isotype control antibody were pre-incubated with 100 ng of Mab-Zap (Advanced targeting Systems) secondary antibody in cell culture medium for 30 min at RT. These mixtures were loaded on subconfluent SN12C cells plated in white 96-well plate microplate. Plates were incubated for 72 h at 37°C in presence of 5% CO₂. Cell viabî lit y was determined using a Cell Titer Glo cell prolifération method according to the manufactureras instructions (Promcga). Several controls are performed: i) without any secondary îmmunoconjugate and ii) without primary antibody. In parallel, assays are performed with a mlgGI isotype control.

Obtained results are represented in the Figure I.

The 16I3F12 shows a maximal cytotoxic effect on the SNI2C cells of ~36 %. No cytotoxic effect wasobserved inpresenceofthe9G4 antibody, consideredas mlgGI isotype control in the experiment. No cytotoxicity was observed in wells containing only primary antibodies (data not shown). Thus the Axl receptor appears to be a convenient antigen to target for an îmmunoconjugate approach as the immune complcx comprising Axl-l613F12-MabZap triggers an effective cytotoxicity of the targeted cells.

Example 2: Génération of an antibody against rhAxl ECD.

To generate murine monoclonal antibodies (Mabs) against human extracellular domain (ECD) of the Axl receptor, 5 BALB/c mice were immunized 5-times s.c. with 15-20.10⁶ CHO-Axl cells and twice with 20 pg of the rh Axl ECD. The first immunization was perfonned in presence of Complété Freund Adjuvant (Sigma, St Louis, MD, USA). Incomplète Freund adjuvant (Sigma) was added for following immunizations.

Three days prior to the fusion, immunized mice were boosted with both 20.10⁶CHO-Axl cells and 20 pg of the rhAxl ECD with IFA.

To generate hybridomas, splénocytes and lymphocytes were prepared by perfusion of the spleen and by mincing of the proximal lymph nodes, respectively, harvested from 1 out of the 5 immunized mice (selected after sera titration) and fused to SP2/0-AgI4 myeloma cells (ATCC, Rockvillc, MD, USA). The fusion protocol is described by Kohler and Milstein (Nature, 256:495-497, 1975). Fused cells are then subjected to HAT sélection. In general, for the préparation of monoclonal antibodies or their functional fragments, espccially of murine origin, it is possible to refer to techniques which are described in particular in the manual “Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988).

Approximately 10 days after the fusion, colonies of hybrid cells were screened. For the primary screen, supematants of hybridomas were evaluated for the sécrétion of Mabs ratsed against the Axl ECD protein using an ELISA. In parallel, a FACS analysis was perfonned to select Mabs able to bind to the cellular form of Axl présent on the cell surface using both wt CHO and Axl expressing CHO cells (ATCC).

As soon as possible, selected hybridomas were cloned by limit dilution and subsequently screened for their reactivity against the Axl ECD protein. Cloned Mabs were then isotyped using an Isotyping kit (cat #5300.05, Southern Biotech, Birmingham, AL, USA). One clone obtained from each hybridoma was selected and expanded.

ELISA assays are performed as followed either using pure hybridoma supematant or, when IgG content in supematants was determined, titration was realized starting at 5pg/ml. Then a Ά serial dilution was perfonned in the following 11 rows.

Bricfly, 96-well ELISA plates (Costar 3690, Coming, NY, USA) were coated 50 μΐ/well of the rh Axl-Fc protein (R and D Systems, cat N° 154-AL) or rhAxl ECD at 2 pg/ml in PBS ovemight at 4°C. The plates were then blocked with PBS containing 0.5% gelatin (#22151, Serva Electrophoresis GmbH, Heidelberg, Germany) for 2 h at 37°C. Once the saturation buffer discarded by flicking plates, 50 μΐ of pure hybridoma cell supcmatants or 50 μΐ of a 5 pg/ml solution were added to the ELISA plates and incubated for 1 h at 37°C. After three washes, 50 μΐ horseradish peroxidase-conjugated polyclonal goat anti-mousc IgG (#115-035-164, Jackson Immuno-Research Laboratories, Inc., West Grave, PA, USA ) was added at a 1/5000 dilution in PBS containing 0.1% gelatin and 0.05% Tween 20 (w:w) for l h at 37°C. Then, ELISA plates were washed 3-times and the TMB (#UP664782, Uptima, Interchim, France) substrate was added. After a 10 min incubation time at room température, the reaction was stoppcd using 1 M sulfuric acid and the optical density at 450 nm was measured.

For the sélection by flow cytometry, 10⁵ cells (CHO wt or CHO-Axl) were plated in each well of a 96 wcll-plate in PBS containing 1% BSA and 0.01% sodium azide (FACS buffer) at 4°C. After a 2 min centrifugation at 2000 rpm, the buffer was removed and hybridoma supematants or purified Mabs (1 pg/ml) to be tested were added. After 20 min of incubation at 4°C, cells were washed twice and an Alexa 488conjugated goat anti-mouse antibody 1/500° diluted in FACS buffer (#Al 1017, Molecular Probes Inc., Eugene, USA) was added and incubated for 20 min at 4°C. After a final wash with FACS buffer, cells were analyzed by FACS (Facscalibur, BectonDickinson) after addition of propidium iodide to each tube at a final concentration of 40 pg/ml. Wells containing cells alone and cells incubated with the secondary Alexa 488conjugated antibody were included as négative contrais. Isotype contrais were used in each experiment (Sigma, ref M90351MG). At least 5000 cells were assessed to calculate the mean value of fluorescence intensity (MFI).

More prccisely, the fusion was performed with 300.10⁶ of harvested splénocytes and 300.10^e mycloma cells (1:1 ratio). Two hundred cells ofthe resulting cell suspension were then plated at 2.10⁶ cell/ml in 30 96-well plates.

A first screen (around Day 14 after fusion) both by ELISA on the rhAxl ECD protein and by FACS analysis using the both wt CHO and Axl expressing CHO cells allowed to select 10 hybridomas presenting optical densifies (ODs) above 1 on the rh

AxlECD coating and MFI bcllow 50 on wt CHO cells and above 200 on CHO-Axl cells.

These 10 hybridomas were expanded and cloned by limit dilution. One 96-well plate was prepared for each code. Nine days after plating, supematants from cloning plates were first screened by ELISA for their binding specifïcity for the extracellular domain of the rh AxlECD protein. Three clones of each code were expanded and isotyped. Once produced the anti-Axl antibodies were further studied for their ability to be intemalized following Axl binding on the ccll-surfacc.

Exemple 3: Axl binding specificity

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

Briefly, the recombinant human Axl-Fc (R and D Systems, cat N® 154AL/CF), rhDtk (R and D Systems, cat N® 859-DK) or rhMer-Fc (R and D Systems, cat N® 891 MR) proteins were coated ovemight at 4®C to Immulon II 96-well plates and, after a 1 h blocking step with a 0.5% gélatine solution, 1613F12 purified antibody was added for an additional 1 h at 37®C at starting concentration of 5 gg/mt (3.33 lO^M). Then Ά serial dilutions were donc over 12 columns. Plates were washed and a goat anti-mousc (Jackson) spécifie IgG-HRP was added for 1 h at 37®C. Reaction development was performed using the TMB substrate solution. The isotype control antibody mlgGl and the commercial anti-Axl Mab 154 antibody were also used in parallel. Coating controls were performed in présence of a goat anti-human IgG Fc polyclonal sérum labelled with HRP (Jackson, ref 109-035-098) and/or in presence of a HRP-coupled anti-Histidinc antibody (R and D Systems, ref : MAB050H). Results are represented in Figures 2A, 2B and 2C, respectively.

This example shows that the 1613F12 antibody only binds to the rhAxl-Fc protein and does not bind on the two other members of the TAM family, rhDtk or rhMer. No cross-specificity of binding of the 1613F12 antibody is observed between TAM members. No non spécifie binding was observed in absence of primary antibody (diluant). No binding was observed in presence of the isotype control antibody.

Example 4:1613F12 recognlzed the cellular form of Axl expressed on tumor cells.

Cell surface Axl expression level on human tumor cells was first established using a commercial Axl antibody (R and D Systems, ref: ΜΑΒΙ54) in parallel of 5 calibration bcads to allow the quantification of Axl expression level. Secondly, binding of the cell-surface Axl was studied using the 1613F12.

For cell surface binding studies, two fold serial dilutions ofa 10 pg/ml (6.66 10 M) primary antibody solution (1613F12, MAB154 antibody or mlgGl isotype control 9G4 Mab) are prepared and arc appiied on 2.10^s cells for 20 min at 4°C. After 3 washes 10 in phosphate-buffered saline (PBS) supplcmentcd with t% BSA and 0.01% NaNj, cells were incubatcd with secondary antibody Goat antî-mouse Alexa 488 (1/500° dilution) for 20 minutes at 4°C. After 3 additional washes in PBS supplemented with 1% BSA and 0.1% NaN₃, cells were analyzed by FACS (Facscalibur, Bccton-Dickinson). At least 5000 cells were assessed to calculate the mean value of fluorescence intensity.

For quantitative ABC détermination using MAB 154 antibody, Q1FIKIT® calibration bcads arc used. Then, the cells are incubated, in parallel with the QIFIKIT® bcads, with Polyclonal Goat Anti-Mousc Immunoglobulins/FITC, Goat F(ab*)2, at saturating concentration. The number of antigenic sites on the spécimen cells is then determined by interpolation of the calibration curve (the fluorescence intensity of the 2 0 individual bcad populations against the number of Mab molécules on the bcads.

4.1, Quantification of cell-surface Axl expression level

Axl expression level on the surface of human tumor cells was determined by flow cytometry using indirect immunofluorescence assay (QIFIKIT® method (Dako, 25 Denmark), a quantitative flow cytometry kit for assessing cell surface antigens. A comparison of the mean fluorescence intensity (MFI) of the known antigen levels of the bcads via a calibration graph permits détermination of the antibody binding capacity (ABC) of the cell lines.

Table 6 présents Axl expression level detected on the surface of various human tumor cell lines (SN12C, Calu-1, A172, A43I, DUI45, MDA-MB435S, MDA-MB231,

PC3, NCI-H226, NCI-H125, MCF7, Panel) (ATCC, NCI) as determined using

Q1FIKIT® using the commercial antibody ΜΑΒΙ54 (R and D Systems). Values are given as Antigen binding complex (ABC).

Table 6

	MCF7	NCI-H125	MDA-MB-435S	Panel	MDA-MB-231	Calu-l	SN12C
Tumor type/organ	Breast	NSCLC	Breast	Pancréas	Breast	Lung	Rénal
ABC (QlflHt)	71	5540	17814	36 809	61 186	>100 000	>100 000

	A172	A431	tXJ-145	PC3	NCI-H226
Tumor type/organ	gfioblastoma	Epidermoid carcinome	Prostate	prostate	NSCLC
ABC (Qrfiktt)	52421	3953	55268	8421	32142

Results obtained with a commercial Axl monoclonal antibody (ΜΑΒΙ 54) showed that Axl receptor is expressed at various levels depending of the considcred human tumor cell.

4.2. Axl détection bv 1613F12 on human tumor cells

More specifïcally, Axl binding was studied using the 1613F12.

1613F12 dose response curves were prepared. MFIs obtained using the various human tumor cells were then analysed with Prism software. Data are presented in Figure 3.

Data indicate that the I613F12 binds specifïcally to the membrane Axl receptor as attested by the saturation curve profiles. However different intensifies of labelling were observed, revealing variable levels of cell-surface Axl receptor on human tumor cells. No binding of Axl receptor was observed using MCF7 human breast tumor cell line.

\ ^? ï

Example 5:1613F12 Inter-species crosspecificîty

To address the species cross-spccificity of the 1613F12, two species were considered: mouse and monkey. First the binding on the recombinant mouse (rm) Axl receptor is studied by ELISA (Figure 4). Then, flow cytomctry experiments were 5 performed using monkey COS7 cells as these cells express the Axl receptor on their surface (Figure 5). The COS7 cell line was obtained by immortalizing a CV-1 cell line derived from kidney cells of the African green monkey with a version of the SV40 genome that can produce large T antigen but has a dcfect in genomie réplication.

rmAxl-Fc ELISA

Bricfly, the recombinant mouse Axl-Fc (R and D Systems, cat N° 854-AX /CF) proteins were coated ovemight at 4°C to Immulon II 96-well plates and, after a 1 h blocking step with a 0.5% gélatine solution, the 1613F12 purified antibody was added for one additional hour at 37°C at starting concentration of 5 pg/ml (3.33 10^-8 M). Then Vi serial dilutions were donc over 12 columns. Plates were then washed and a goat anti15 mouse (Jackson) spécifie IgG HRP was added for 1 h at 37°C. Réaction development was performed using the TMB substrate solution. The mlgGl isotype control and the commercial antibody Mab 154 are also used in parallel. Coating controls are performed in présence of a goat antî-human IgG Fc polyclonal sérum coupled with HRP (Jackson, ref 109-035-098) and/or in presence of a HRP-couplcd anti-Histidine antibody (R and D 2 0 Systems, ref : Μ AB050H).

Results are represented in Figure 4. This figure shows that the 1613F12 does not bind to the murine Axl ECD domain. No spécifie binding is observed in the absence of primary antibody (diluant).

FACS COS7

For 1613F12 cellular binding studies using COS7 cells, 2.10⁵ cells were incubated with an antibody concentration range prepared by ^lA serial dilution (12 points) of a 10 pg/ml (6,66 ΙΟ^-8 M) antibody solution of 1613F12 or mlgGl isotype control Mab for 20 min at 4°C. After 3 washes in phosphate-buffered saline (PBS) 30 supplemented with 1% BSA and 0.01% NaNj, cells were incubated with secondary antibody goat anti-mousc Alexa 488 (dilution 1/500) for 20 minutes at 4°C. After 3 additional washes in PBS supplemented with 1% BSA and 0.1% NaN_Jt cells were analyzed by FACS (Facscalibur, Bccton-Dick inson). At least 5000 cells were assesscd to calculatc the mean value of fluorescence intensity. Data are analyzed using Prism software.

Results are represented in Figure 5. The titration curve established on COS7 5 cells using 1613F12 confïrms that 1613F12 is able to recognize the monkey cellular form of the Axl receptor expressed on the surface of the COS7 cells. Plateau is reached for 1613F12 concentrations above 0.625 pg/ml (4.2 ΙΟ¹⁰ M). No binding is observed in prcsence of the mlgGl isotype control.

This example illustrâtes the fact that the 1613F12 does not cross-react with the 10 mouse Axl receptor. In contrast it strongly binds to the monkey Axl receptor expressed on the surface of COS7 cells.

Example 6: Gas6 Compétition experiments performed In presence of the 1613F12

To further charactcrize the 1613F12, Gas6 compétition assays were performed.

In this assay, the free rhAxl-Fc protein and the 1613F12 are incubated to form antigenantibody complex and then the complexes are loaded on Gas6-coated surface in the assay plate. The unbound antibody-antigen complexes are washed out before adding enzyme-linked secondary antibody against the human Fc portion of the rhAxl-Fc 2 0 protein. The substrate is then added and the antigen concentration can be determined by the signal strength elicitcd by the enzyme-substrate reaction.

Briefly réaction mixture comprising the rhAxl-Fc protein in the presence or not of the anti-Axl Mabs to be tested, are prepared on a separate saturated (0.5% gelatin in PBS IX) plate. Serial 1: 2 dilutions (starting from 80 pg/ml on 12 columns) of murine 25 anti-Axl antibodies are performed. Then 0.5 pg/ml of the rhAxl-Fc protein is added (R and D Systems, ref. 154AL/CF), except to the négative control line that contains only ELISA diluant (0.1% gelatin, 0.05% Tween 20 in PBS IX). After homogénisation, the compétition samples are loaded on Gas6-coated plates with a 6 pg/ml rhGasô solution tn PBS (R and D Systems cat N° 885-GS-CS / CF). After incubation and several 30 washes, bound rhAxl-Fc proteins are detected using a goat anti-Human IgG-HRP (Jackson, ref. 109-035-098). Once bound, the TMB substrate is added to the plates. The reaction is stopped by addition of IM H2SO4 acid solution and the obtained optical densities read at 450 nm using a microplate reader instrument.

This experiment (Figure 6) shows that the 1613F12 is able to compete with the rhAxI-Fc binding on its immobilizcd ligand. Compétition with Gas6 binding occurs in présence of 1613F12 antibody concentrations above 2.5 pg/ml (1.67 lff® M). No more binding of the rhAxl-Fc on the immobilizcd Gas6 is observed in presence of a 1613Fl2 concentration above 10 pg/ml (6.67 10^ M). The 1613F12 blocks Gas6 binding to rhAxl-Fc.

Example 7: Epitope récognition by Western Blot

To déterminé if the 1613F12 recognizes a linear or a conformational epitope, western blot analysis was done using SN12C cell lysâtes. Samples were differently treated to be in reducing or non reducing conditions. If a band is visualized with reduced sample, the tested antibody targets a linear epitope of the ECD domain; If not, it is raised against a conformation epitope of the Axl ECD.

SN12C ceils were sceded in RPMI + 10 % heat inactivated FBS + 2 mM Lglutamine at 5.I0⁴ ceils /cm² in T162 cm² flasks for 72h at 37°C in a 5% CO2 atmosphère. Then the ceils were washed twice with phosphate bufTercd saline (PBS) and iysed with 1.5 ml of ice-cold lysis buffer [50 mM Tris-HCI (pH7.5); 150 mM NaCl; 1% Nonidet P40; 0.5% deoxycholate; and 1 complété protease inhibitor cocktail tablet plus 1% antiphosphatases]. Cell lysâtes were shaken for 90 min at 4°C and cleared at 15 000 rpm for 10 min. Protein concentration was quantified using BCA. Various samples were loaded. First 10 pg of whole cell lysate (10 pg in 20 pl) were prepared in reducing conditions (lx sample buffer (B1ORAD) + lx reducing agent (BIORAD)) and loaded on a SDS-PAGE after 2 min incubation at 96°C. Secondly two other samples of 10 pg of whole cell lysate were prepared in non-reducing conditions (in lx sample buffer (BIORAD) only). Prior to be loaded on the SDS-PAGE gel, one of these two last samples is heated 2 min incubation at 96°C; the other one is kept on ice. After migration, the proteins are transferred to nitrocellulose membrane. Membranes were saturated for 1 h at RT with TBS-tween 20 0.1% (TBST), 5% non-fat milk and probed with the I613F12 at 10 pg/ml ovemight at 4°C. Antibodies were diluted in Trisbuffered saline-0.1% tween 20 (v/v) (TBST) with 5% non-fat dry milk. Then *

membranes were washed with TBST and incubated with pcroxydasc-conjugated secondary antibody (dilution 1/1000) for 1 h at RT. Immunoreactive proteins were visualized with ECL (Pierce #32209). After Axl visualization, membranes were washed once again with TBST and incubated for 1 h at RT with mousc anti-GAPDH antibody (dilution 1/200 000). Then membranes were washed in TBST and incubated with pcroxydase-conjugated secondary antibodies, for lh at RT. Membranes were washed and GAPDH was revealed using ECL.

Results are represented in Figure 7.

The 16I3FI2 mainly rccognizes a conformationa! epitope as a spécifie band is esscntially observed in non-reduced conditions. However a faint signal is detected in the denaturating migrâting condition of the SN12C cell lysate indicating 1613F12 is able to weakly bind to a linear epitope.

Example 8: Measurement of Axl down-regulatlon trlggered by the 1613F12 by Western Blot

In the following example, the human rénal cell carcinoma cell line SN12C (ATCC) was selected to address the activity of Axl antibodies on Axl receptor expression. The SNI2C cell line overexpresses the Axl receptor. The Axl downrcgulation was studied by Westem-Blot on whole cell extracts in Figures 8A-8B.

SN12C cells were seeded in RPMI + 10 % heat inactivated FBS + 2 mM Lglutaminc at 6.10⁴ cells/cm* in six-well plates for 48 h at 37°C in a 5% CO₂ atmosphère. After two washes with phosphate buffer saline (PBS), cells were serum-starved in a medium containing either 800 ng/ml recombinant mouse gas6 ligand (R and D Systems, ref: 986-GS/CF) or 10 pg/ml of a mlgGl isotype control antibody (9G4) or 10 pg/ml of the Axl antibody of the présent invention and incubated for 4 h or 24 additional hours. Then the medium was gently removed and cells washed twice with cold PBS. Cells were lysed with 200 μΐ of ice-cold lysis buffer [50 mM Tris-HCl (pH7.5); 150 mM NaCl; 1% Nonidct P40; 0.5% deoxycholate; and 1 complété protease inhibitor cocktail tablet plus 1% antiphosphatases]. Cell lysâtes were shaken for 90 min at 4°C and cleared at 15 000 rpm for 10 min. Protein concentration was quantified using BCA method. Whole cell lysâtes (10 pg in 20 μΐ) were separated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were saturated for 1 h at RT with \ <

TBS-Tween 20 0.1% (TBST), 5% non-fat milk and probcd with a commercial M02 Axl antibody at 0.5 pg/ml (AbNova H00000558-M02) ovemight at 4°C. Antibodies were diluted in Tris-buffered saline-0.1% tween 20 (v/v) (TBST) with 5% non-fat dry milk. Then membranes were washed with TBST and incubated with peroxydase-conjugated 5 secondary antibody (dilution 1/1000) for 1 h at RT. Immunoreactive proteins were visualized with ECL (Picrce #32209). After Axl visualization, membranes were washed once again with TBST and incubated for 1 h at RT with mouse anti-GAPDH antibody (dilution 1/200000). Then membranes were washed in TBST and incubated with peroxydase-conjugated secondary antibodies, for 1 h at RT. Membranes were washed 10 and GAPDH was rcvcalcd using ECL. Band intensity was quantifïed by densitometry.

Results presented in Figures 8A and 8B are représentative of 3 independent experiments and demonstrate that 1613F12 is able to down-regulate Axl in an Axloverexpressing human tumor cell line. At 4 h, the 1613F12 triggers a 66 % Axl downregulation, and up to 87 % after a 24 hour incubation with the 1613F12.

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

Flow cytometry technique allows labclling of cell-surface Axl receptor. The use of this technique can highlight the effect of antibodies on the membrane Axl expression. 20 Human rénal tumor SN12C cells that express high levels of Axl were used in this example.

SN12C tumor cell line was cultured în RMP11640 with 1% L-glutamine and 10% of FCS for 3 days before experiment. Cells were then detached using trypsin and plated in 6-multiwcll plate in RPMI1640 with 1% L-glutamine and 5% FBS. The next 25 day, antibodies of interest were added at 10 pg/ml. Untreated wells were also included.

The cells are incubated at 37°C, 5% CO₂. Twcnty four hours later, cells were washed with PBS, detached and incubated with the same antibodies of interest in FACS buffer (PBS, 1% BSA, 0.01% sodium azide). Untreated wells were also stained with the same antibody in order to compare the signal intensity obtained with the same Mab on the 30 treated and the non-treated cells. Cells were incubated for 20 minutes at 4°C and washed three times with FACS buffer. An Alexa 488-labcled goat antî-mouse IgG

antibody was incubated for 20 minutes and cells were washed three times before FACS analysis on propidium iodide négative cell population.

Two parametcrs arc determined: (i) the différence of the fluorescent signal detected on the surface of untreated (no Ab) cells compared to the Ab-treated cells at ΊΓ24 h and (ii) the percentage of remaining Axl on the cell surface. The percentage of remaining Axl is calculated as follows:

% remaining Axl = (MF1 _Ab 24 h / MF1_B0 ab 24 h) x 100

Data from one représentative experiment are presented in Table 7. The results were reproduced in three independent experiments.

The différence of MFI between the staining of a Mab in the untreated cells and the treated condition with the same antibody reflects a down-regulation of the Axl protein on the surface of the cells due to the binding of the considered Mab. Conditions without antibody gave similar results to conditions in présence of the isotype control antibody (m9G4).

Table 7

UNÜrç ¹ * ' , 1 · i¹, J	1 r	MFlettMh Z / ,, . ,	À (MFI Ne MM II -MFt «4 M 4	Axt ’ ' . ...'-u'··.
1613F12	No Ab	938	514	45.2
1613F12	424
9G4	No Ab	11	2	117
9G4	13
MAB154	No Ab	950	ND	ND
9G4	ND

The data demonstratc that the mean fluorescence intensity detected on the surface ofthe cells treated with 1613F12 for 24 hours is reduced (-514) compared to the MFIs obtained with untreated cells labellcd with the 1613F12. After a 24 h incubation with the 16I3F12 antibody, 45.2 % of the cell-surface Axl receptor romains at the SN12C cell-surface.

» »

Example 10: 1613F12 Intemalization study using fluorescent

Immunocytochemlstry labelling.

Complementary intemalization results are obtained by confocal microscopy using indirect fluorescent labelling method.

Briefly, SN12C tumor cell line was cultured in RMPII640 with 1 % L-glutamine and 10 % of FCS for 3 days before experiment. Cells were then detached using trypsin and plated in 6-multiwell plate containing covcrslidc in RPMH640 with 1 % Lglutamine and 5 % FCS. The next day, the i6I3F12 was added at 10 gg/ml. Cells treated with an irrelevant antibody were also included. The cells were then incubated for 1 h and 2 h at 37°C, 5% CO2. For T 0 h, cells were incubated for 30 minutes at 4°C to détermine antibody binding on cell surface. Cells were washed with PBS and fixed with paraformaldéhyde for 15 minutes. Cells were rinsed and incubated with a goat antimouse IgG Alcxa 488 antibody for 60 minutes at 4°C to identify remainîng antibody on the cell surface. To follow antibody pénétration into the cells, cells were fixed and permeabilized with saponin. A goat anti-mouse IgG Alexa 488 (invitrogen) was used to stained both the membrane and the intracellular antibody. Early endosomes were identifïed using a rabbit polyclonal antibody against EEAi revealed with a goat antirabbit IgG-Alexa 555 antibody (Invitrogen). Cells were washed three times and nuclei were stained using Draq5. After staining, cells were mounted in Prolong Gold mounting medium (Invitrogen) and analyzed by using a Zeiss LSM 510 confocal microscope.

Photographs are presented in Figures 9A-9C.

Images were obtained by confocal microscopy. In presence of the mlgGl isotype control (9G4), neither membrane staining nor intracellular labelling is observed (Figure 9A). A progressive loss of the membrane anti-Axl labelling is observed as soon as after i h incubation of the SN12C cells with the I6I3FI2 (Figure 9B). Intracellular accumulation of the 1613F12 antibody is clcarly observed at 1 h and 2 h (Figure 9C). Intracellular antibody co-localizes with EEAI, an early endosome marker. These photographs confïrm the intemalization of the 1613F12 into SN12C cells.

> 1

Example 11: in vitro antl-AxI medlated antl-tumoral activity.

SN12C prolifération assay

Ten thousand SN12C cells per well were seeded in FCS-free medium on 96 well plates over night at 37°C in a 5% CO2 atmosphère. The next day, cells were preincubatcd with 10 pg/ml of each antibody for lh at 37°C. Cells were treated with or wîthout rmGasô (R and D Systems, cat N°986-GS/CF), by adding the ligand directly to the well, and then left to grown for 72h. Prolifération was measured following ³H thymidine incorporation.

Data are presented in figure 10. No effect was observed with the 1613F12 which is silent when added to SN12C cells.

Example 12; Cytotoxlclty potency of 1613F12-saporln immunoconjugate ln various human tumor cell lines

In the présent example, is documcnted the cytotoxicity potcncy of the saporin couplcd-1613F12. For this purposc direct in vitro cytotoxicity assays using a large panel of human tumor cell fines were perfonned (Figures 11A-11K). This tumor cell fine panel ofiers various cell surface Axl expressions,

Briefly, 5000 cells were seeded in 96 well culture plates in 100 μΐ of 5 % FBS adéquate culture medium (D0). After 24 hours incubation in a 5% CO2 atmosphère at 37°C, a range of concentration of the immunoconjugate (1613F12-saporin or 9G4saporin or the naked 1613F12 or 9G4) is applied to the cells. Culture plates are then incubatcd at 37°C in a humidified 5% CO2 incubator for 72 hours.

At D4, the cell viabilïty is assessed using the CellTiter-Glo® Luminescent Cell Viability kit (Promega Corp., Madison, Wis.) that allows determining the number of viable cells in culture based on quantification of the ATP présent, an indicator of metabolically active cells. Luminescent émissions are recorded by a luminometer device.

From luminescence output is calculatcd the perccntage of cytotoxicity using the following formula:

% cytotoxlclty = 100- [(RLU Ab-Mlp X 100)/RLU No Ah]

On Figures 11 A-l 1K. are put together graphs prcsenting cytotoxicity percentage in function of the îmmunoconjugate concentration obtained in distinct in vitro cell cytotoxicity assays with (A) SN12C, (B) Calu-1, (C) A172, (D) A431, (E) DU 145, (F) MDA-MB-435S, (G) MDA-MB-231, (H) PC3, (1) NC1-H226, (J) NCI-H125 or (K) Panel tumor cells treated with a range of 1613FI2-saporin îmmunoconjugate concentrations.

Figures 11A-11K. shows that the 1613F12-saporin îmmunoconjugate triggered cytotoxicity in these different human tumor cell lines. The potency of the resulting cytotoxicity effect dépends on the human tumor cell line.

Example 13: Humanlzation of the 1613F12 antibody variable domains

The use of mouse antibodies (Mabs) for therapeutic applications in humans generally results in a major adverse effect, patients raise a human anti-mouse antibody (HAMA) response, thereby reducing the efficacy of the treatment and preventing continued administration. One approach to overcome this problem is to humanize mouse Mabs by replacing mouse sequences by their human counterpart but without modifying the antigen binding activity. This can be achieved in two major ways: (i) by construction of mouse/human chimeric antibodies where the mouse variable régions arc joined to human constant régions (Boulianne et al., 1984) and (ii) by grafting the complementarity determining régions (CDRs) from the mouse variable régions into carefully selected human variable régions and then joining these re-shaped human variable régions to human constant régions (Ricchmann et al., 1988).

13.1. Design of humanized version of the 1613F12 antibody

13.1.1 Humanlzation of the light chain variable domain VL

As a prciiminary step, the nucieotide sequence of the 1613F12 VL was compared to the murine germline gene sequences part of the IMGT databasc (http://www.imgt.org). Murine IGK.VI6-104*01 andIGKJ5*01 germline genes were identified. In order to identify the best human candidate for the CDR grafting, the human germline gene displaying the best identity with the 1613F12 VL murine sequence has been searched. With the help of the IMGT database analyses tools, a possible acceptor human V régions for the murine 1613F12 VL CDRs was identified:

t * k

IGKV 1-27*01 and IGKJ4*02. In order to perform the humanization to the light chain variable domain each residue which is different between the human and mouse sequences was given a priority rank order. These priorités (1 -4) were used to create 11 different humanized variants ofthe light chain variable région with up to 14 5 backmutations.

	FR1-IMGT	CDR1-IMGT	FR2-IMGT	CD
1613F12VL		DVQITQSPSYLATSPGETITINCRAS	KSI...	...SKÏ	LAWYQEKPGKTNKLLIY	SG
Homsap IGKVl-27’01 Priority	DIQHTQSPSSLSASVGDRVTITCRAS V X ï AT P 1TI W 1 1 3 34 4 433 2	QGI...	...SNY	LAWÏQQKPGKVPKLLIÏ K TM 3 33	AA
hal613F12	(VL1)	DIQMTQS PS SLSASVGDRVTI TC RAS	KSI...	.. .SKÏ	LAWÏQQKPGKVPKLLIÏ	SG
hll613F12	(VL1I2V)	DVQMTQS PS SLSASVGDRVTITCRA S	KSI...	.. .SKÏ	LAWÏQQKPGKVPKLLIÏ	SG
hll613F12	(VL1M4I)	DIQXTQS PSSLSASVGDRVTITCRAS	KSI'	,SKY	LAWÏQQKPGKVPKLLIÏ	SG
hïl613F12	(VL2.1)	DVQITQS PS SLSASVGDRVTITCRAS	KSI...	...SKÏ	LAWÏQQKPGKVPKLLIÏ	SG
M11613F12	(VL2.1V49T)	DVQIT QS PS SLSASVGDRVTITCRAS	KSI. ..	. SKÏ	lawyqqkpgktpklliï	SG
MH613F12	(VL2.1P50N)	DVQITQS PS SLSASVGDRVTITCRAS	KSI...	...SKÏ	lawïqqkpgkvmklliï	SG
M11613F12	(VL2.2)	DVQITQS PS SLSASVGDRVTI ITCRAS	KSI'	SKÏ	LAWÏQQKPGKVPKLLIÏ	SG
hil613F12	(VL2.2V49T)	DVQITQSPS SLSASVGDRVTIMC RAS	KSI...	'..SKÏ	LAWYQQKPGKTPKLLIY	SG
hxl613F12	(VL2.2P50N)	DVQITQS PS SLSASVGDRVTI WCRAS	KSI...	...skï	LAWÏQQKPGKVMKLLIÏ	SG
hxl613F12	(VL2.3)	DVQITQSPSSLSASVGDRVTIifCRAS	KSI...	.SKÏ	LAWYQXK PGKTWKLLIY	SG
hi!613F12	(VL3)	DVQITQS PS YLAASVGDTI TI MCRAS	KSI...	...SKÏ	LAWÏQXKPGKTWK LLIY	SG

R2-IMGT

1613F12VL .......S

Homsap IGKVl-27‘01 .......S

Priority

FR3-IMGT

TLQSGVP.SRFSGSG..SGTDFTLTISSLEPEDFAMÏFC TLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYYC i t m r

4 4 2 hil«13F12 (VU) .......S hll613F12 (VL1I2V) .......3 hll613F12 (VL1M4I) .......S hil613F12 (VL2.1) .......S hil613F12 (VL2.1V49T) .......S hil613F12 (VL2.1P50N) .......S hxl613F12 (VL2.2) .......S hll£13F12 (VL2.2V49T) .......S hxl613F12 (VL2.2P50N) .......S hll613F12 (VL2.3) .......S hi!6l3F12 (VL3) .......S

TLQSGVP.SRFSGSG..S GTDFTLTISS LQPEDVATYYC TLQSGVP.SRFSGSG..SGTDFTLTI SSLQPEDVATYYC TLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYYC TLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYYC TLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYYC TLQSGVÎ'. SRFSGSG. .SGTDFTLTISSLQPEDVATYYC TLQSGVP.SRFSGSG,.SGTDFTLTISSLQPEDVATYTC TLQSGVP,SRFS GSG..SGTDFTLTIS S LQPE DVATÏfC TLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATÏXC TLQSGVP.SRFSGSG..SGTDFTLTISSLQPEDVATYTC TLQSGVP.SRFSGSG..S GTDFTLTISS LQPEDVATYTC

CDR3-IMGT FR4-IMGT

1613F12VL Homsap IGKJ4O2

Priority hil613F12 hll613F12 hxl«13F12 hxl613F12 hil613F12 hx!613F12 htl6l3F12 hil613F12 hxl613F12 hlI613F12 hxl613F12 (VL1) (VL1I2V) (VL1M4I) (VL2.1 ) (VL2.1V49T) (VL2.1P50N) (VL2.2) (VL2.2V49T) (VL2.2P5DN) (VL2.3) (VL3)

QQHHEÏPLT FGAGTELELK LT FGGGTKVEIK i 11 L 3 33 4

QQHHEÏPLT QQHHEÏPLT QQHHEÏPLT QQHHEÏPLT QQHHEÏPLT QQHHEÏPLT QQHHEÏPLT QQHHEÏPLT QQHHEÏPLT

FGGGTKVEIK FGGGTKVEIK FGGGTKVEIK FGGGTKVEIK FGGGTKVEIK FGGGTKVEIK FGGGTKVEIK FGGGTKVEIK FGGGTKVEIK

QQHHEÏPLT FGGGTKVEIK QQHHEÏPLT FGAGTXLEIK ' ‘ 16895

13.1.2 Humanizatlon of the heavy chain variable domain VH

In order to identify the best human candidate for the CDR graftïng, the mouse and human germline genes displaying the best identity with the I6I3F12 VH were searched. The nucléotide sequence of 1613F12 VH was aligned with both mouse and 5 human germline gene sequences by using the sequence alignment software “IMGT/VQUEST” which is part of the IMGT database. Alignments of amino acid sequences were also performed to verify the results of the nucléotide sequence alignment using the “Align X” software of the VectorNTI package. The alignment with mouse germline genes showed that the mouse germline V-gene IGHV 14-3*02 and J-gene IGHJ2*01 arc 10 the most homologue mouse germline genes. Using the IMGT database the mouse Dgenc germline IGHDl-l*01 was identified as ho n» logo us sequence. In order to select an appropriate human germline for the CDR graftïng, the human germline gene with the highest homology to the 1613F12 VH murine sequence was identified. With the help of IMGT databases and tools, the human IGHVI-2*02 germline gene and human 15 IGHJ5*01 J germline gene were selected as human acceptor sequences for the murine I613F12 VH CDRs. In order to perform the humanization to the heavy chain variable domain each residue which is different between the human and mouse sequences was given a priority rank order (1-4). These prioritics were used to create 20 different humanized variants of the heavy chain variable région with up to 18 backmutations, \ k

FR1-IMGT

CDR1-IMGT FR2-IMGT CD (1-26) (27-391 <33-553 (

1613F12

Homsap IGHV1-2*O2

Prlorlty hzl613F12 hzl6I3F12 hzl613F12 hzl613F12 hzl613FI2 hzl613F12 hzl«13F12 hzl613F12 hzl613F12 hzl613F12 hzl613F12 hzl613F12 hzl613F12 hzI6l3F12 hzl613F12 hzl613F12 hzl613F12 J1Z1613F12 hzl613F12 hzl613F12 (VH1) (VH1M39I) (VH1W55RN66K) (VH1I843) (VH1S85N) (VH1I84NS85N) (VH2.1) (VH2.1Q3H) (VH2.1W55R) (VH2.1N66K) (VH2.1W55RN66K) (VH2.1R80S) (VH2.1N66KR80S) (VH2.2) (VH2.2H89L) (VH2.3) (VH2.3H55R) (VH2.3Q3HW55R) (VH2.4) (VH3)

EVHLQQSGA.ELVKPGASVKLSCTAS QVQLVQSGA.EVKKPGASVKVSCKAS I I fi LV LT

2 3 33 3 3

QVQLVQSGA, EVKKPGASVKVSCKAS QVQLVQSGA .EVKKPGASVKVSCKAS QVQLVQSGA.EVKKPGASVKVSCKAS QVQLVQSGA. EVKK PGASVKVSCKAS QVQLVQSGA. EVKK PGASVKVSCKAS QVQLVQSGA.EVKK PGASVKVSCKAS QVQLVQSGA.EVKKPGASVKVSCKAS QVBLVQSGA.EVKK PGASVKVSCKAS QVQLVQSGA .EVKKPGASVKVSCKAS QVQLVQS GA. EVKK PGASVKVSCKAS QVQLVQSGA .EVKKPGASVKVSCKAS QVQLVQSGA .EVKKPGASVKVSCKAS QVQLVQSGA .EVKKPGASVKVSCKAS QVBLVQSGA. EVKKPGASVKVSCKAS QVBLVQSGA. EVKKPGASVKVSCKAS QVQLQQSGA. EVKKPGASVKLSCTAS QVQLQQSGA. EVKKPGASVKLSCTAS QVB LQQSGA. EVKKPGASVKLSCTAS QVQLQQSGA. EVKKPGASVKLSCTAS XVB LQQSGA. ELVKPGASVKLSCTAS

GFNI....RDTÏ IHWVKQRPEQGLEHIGR LD GYTF....TGYÏ MHWVRQAPGQGLEWMGW IN

I K R B IR

3 4 4 3 2

GFHI... .RDTÏ MHWVRQAPGQGLEWMGW LD GFNI....RDTÏ IHWVRQAPGQGLEWMGW LD GFNI....RDTÏ MHWVRQAPGQGLEWMGR LD GFNI....RDTÏ MHWVRQAPGQGLEWMGW LD GFNI....RDTY MHWVRQAPGQGLEWMGW LD GFNI....RDTY MHWVRQAPGQGLEWMGW LD GFNI....RDTÏ IHWVRQAPGQGLEWMGW LD GFNI....RDTÏ IHWVRQAPGQGLEWMGW LD GFNI....RDTÏ IHWVRQAPGQGLEWMGR LD GFNI....RDTY IHWVRQAPGQGLEWMGW LD GFNI....RDTÏ IHWVRQAPGQGLEWMGR LD GFNI....RDTY IHWVRQAPGQGLEWMGW LD GFNI....RDTY IHWVRQAPGQGLEWMGW LD GFNI....RDTÏ IHWVRQAPGQGLEWMGW LD GFNI....RDTY IHWVRQAPGQGLEWMGW LD GFNI....RDTY IHWVRQAPGQGLEWMGW LD GFNI....RDTY IHWVRQAPGQGLEWMGR LD GFNI....RDTY IHWVRQAPGQGLEWMGR LD GFNI....RDTÏ IHWVRQAPGQGLEWIGR LD GFNI....RDTY IHWVKQAPGQGLEWIGR LD

R2-IHGT

56-651

1613F12 Homiap IGHVl-2’02	PA..NGHT PN..SGGT
Prorlty
hzl613F12 (VH1)	PA..NGHT
hzl613F12 (VH1M39I)	PA..NGHT
hzl613F12 (VHIH55RN66K)	PA..NGHT
hzl613F12 (VH1I84S)	PA. .NGHT
hzl613F12 (VH1S85N)	PA..NGHT
hzl613F12 (VH1I84NS85N)	PA..NGHT
hzl613F12 (VH2.1)	PA..NGHT
hZ1613F12 (VH2.1Q3H)	PA. .NGHT
hzl613F!2 (VH2.1W55R)	PA..NGHT
hzl613F12 (VH2.1N66K)	PA..NGHT
hzl613F12 (VH2.IW55RN66K)	PA..NGHT
H1I613F12 (VH2.1R8DS)	PA..NGHT
hzl613F12 (VH2.1N66KR80S)	PA..NGHT
hzl613F12 (VH2.2)	PA..NGHT
hzl613F12 (VH2.2M89L)	PA..NGHT
hzl613F12 (VH2.3)	PA,.NGHT
hzl613F12 (VH2.3W55R)	PA..NGHT
hzl613FI2 (VH2.3Q3HW55R)	PA..NGHT
hzl613F12 (VH2.4)	PA..NGHT
hzl613F12 (VH3)	PA..NGHT

FR3-IHGT (66-104)

KÏGPNFQ.GRATMTSDTSSNTAÏLQLSSLTSEDTAVÏÏC NYAQKFQ.GRVTMTRDTSISTAYMELSRLRSDDTAVYYC R GPN λ B SM LQ S T B

344 4 2 11 33 4 4 4

NYAQKFQ. GRVTMT RDTSISTAYME LSRLRSDDT AVYYC NYAQKFQ. GRVTHTRDTSI STAÏME LSRLRSDDT AVYYC XYAQKFQ. GRVTHTRDTSI STAÏME LSRLRSDDT AVYYC Nï AQKFQ.GR VT MTRDTSSSTAÏMELSRLRSDDTAVYÏC NYAQKFQ. GRVTMTRDTS IMTA YME LSRLR SDDT AVYYC NYAQKFQ.GRVTMTRDTSSMTAYMELSRLRSDDTAVÏYC NYAQKFQ. GRVTMTRDTSSMTAÏMELSRLRSDDT AVYYC NYAQKFQ. GRVTMTRDTSSMTA YMELSRLRSDDTAVÏYC NYAQKFQ. GRVTMTRDTSSMTAÏMELSRLRSDDT AVYYC XYAQKFQ. GRVTMTRDTS SMTA YMELSRLRSDDTAVÏYC XYAQKFQ.GRVTMTRDTSSMTAYMELSRLRSDDTAVYYC NYAQKFQ. GRVTMTSDTSSMTA YMELSRLRSDDTAVÏYC XYAQKFQ. GRVTMTSDTSBXTA YMELSRLRSDDT AVYYC XYAQKFQ. GRVTMTSDTSSMTAYMELSRLRSDDTAVYÏC XYAQKFQ. GRVTMTSDTSSMTAYLELSRLRSDDTAVYYC XYAQKFQ.GRVTMTSDTSSMTAÏMELSR LRSDDTAVYYC XYAQKFQ. GRVTMTSDTSSMTA YMELSRLRSDDT AVYYC XYAQKFQ. GRVTMTSDTSSMTA YMELSRLRSDDTAVÏYC XYAQKFQ. GRVTHTSDTSSMTAÏLELSR LRSDDT AVYYC XÏGQKFQ. GRVTMTSDTSSMTA YLQLSRLRSDDT AVYYC

CDR3-IMGT FR4-IMGT

1613F12VH Homsap IGHJ5«01

Prorlty hzl613F12 hzl613F12 hzl613F12 hZ1613F12 hzl613F12 hZ1613F12 hzl613F12 hZ1613F12 hzl613F12 hzl613F12 hzl613F12 hzl613F12 hzl613F12 hzl613FI2 hzl613F12 hzl613F12 hzl613F12 hzl613FI2 hzl613FI2 hzl613F12 (VH1) (VH1M39I) (VH1H55RN66K) (VH1I84S) (VH1S85N) (VH1I84NS85N) (VH2.1) (VH2.1Q3H) (VH2.1W55R) (VH2.1N66K) (VH2.1W55RN66K) (VH2.1RB0S) (VH2.1N66KR80S) (VH2.2) (VH2.2 M89L) (VH2.3) (VH2.3W55R) (VH2.3Q3HN55R) (VH2.4) (VH 3)

ARGAÏÏÏGSSGLFÏFDÏ WGQGTTLSVSS HGQGTLVTVSS

TLS

4

ARGAÏYYGSSGLFÏFDÏ HGQGTLVTVSS ARGAÏYYGSSGLFÏFDÏ WGQGTLVTVSS ARGAÏYYGSSGLFÏFDÏ NGQGTLVTVSS ARGAÏYYGSSGLFÏFDÏ WGQGTLVTVSS ARGAÏÏÏGSSGLFÏFDÏ NGQGTLVTVSS ARGAÏÏÏGSSGLFÏFDÏ NGQGTLVTVSS ARGAÏÏÏGSSGLFÏFDÏ WGQGTLVTVSS ARGAÏYYGSSGLFÏFDÏ NGQGTLVTVSS ARGAÏYYGSSGLFÏFDÏ NGQGTLVTVSS ARGAÏYYGSSGLFÏFDÏ HGQGTLVTVSS ARGAÏYYGSSGLFÏFDÏ WGQGTLVTVSS ARGAYÏÏGSSGLFYFDÏ WGQGTLVTVSS ARGAYYYGSSGLFÏFDY HGQGTLVTVSS ARGAÏÏÏGSSGLFÏFDÏ WGQGTLVTVSS ARGAÏYYGSSGLFÏFDÏ WGQGTLVTVSS ARGAÏÏÏGSSGLFÏFDÏ WGQGTLVTVSS ARGAÏÏÏGSSGLFÏFDÏ WGQGTLVTVSS ARGAÏÏÏGSSGLFÏFDÏ WGQGTLVTVSS ARGAÏÏÏGSSGLFÏFDÏ WGQGTLVTVSS ARGAÏÏÏGSSGLFÏFDÏ WGQGTLVTVSS < J k.

13.2. Validation ofthe hz1613F12 vs. m!613F12

In order to establish whether the humanized 1613FI2 was comparable to its murine 1613F12 form, binding experiments were performed both by ELISA using rhAxl-Fc protein assays and by FACS using SNI2C cells. In complément, direct in vitro 5 cytotoxicity assays were performed using SNI2C human rénal tumor cells and Calu-1 human lung carcinoma cell line.

First ELISA experiments were rcalized. In the assay, 96 well plates (Immulon II, Thcrmo Fisher) were coated with a 5 pg/ml of the 1613F12 solution in lx PBS, ovemight at 4°C. After a saturation step, a range of rh Axl-Fc protein (R and D 10 Systems, ref: I54-AL) concentration (from 5 pg/ml to 0.02 pg/ml) is incubated for I hour at 37°C on the coated plates. For the révélation step, a biotinylated-Axl antibody (in house product) was added at 0.85 pg/ml for 1 hour at 37°C. This Axl antibody bclongs to a distinct epitopic group. Then an avidin-horseradish pcroxidasc solution at 1/2000° in diluent buffer is added to the wells. Then the TMB substrate solution is 15 added for 5 min. After addition of the peroxydase stop solution, the absorbance at 405 nm was measured with a microplate reader.

Figure 12 shows that both murine and humanized 1613FI2 antibodies binds similarly the rhAxl-Fc protein.

For FACS analysis, SNI2C cells were cultured in RPMI 1640 + 2 mM L2 0 glutamine + 10% sérum. Cells were detachcd using trypsin and cell concentration was adjusted at 1 x 10⁶ cells / ml in FACS buffer. A volume of 100 pL of cell suspension was incubated with increasing concentrations of either isotype controls or anti-Axl antibodies for 20 min. at 4°C. Cells were then washed three times with FACS buffer and incubated for 20 min. more using either anti-mouse IgG Alexa488 secondary antibody 25 or anti-human IgG Alexa488 secondary antibody at 4°C in the dark. Cells were washed three times with FACS buffer and resuspended with 100 pl of FACS buffer before adding propidium iodidc.

Cells were incubated with increasing concentration of either isotype control or anti-Axl antibodies. mI613FI2 corresponds to the murine I613FI2, CI613FI2, corresponds to the chimeric I6I3FI2 and the hzl613FI2 corresponds to the humanized antibody. ECsos were determined using Prism software.

K *r _1

As illustrated in Figure 13, the humanized form of the 1613F12 bound SN12C cells with équivalent EC50 to the chimeric and the murine form of the 1613F12. Those results indicated that the hz 1613F12 recognizcd Axl antigen with similar binding properties to the murine 1613F12.

Experimental procedures of the direct in vitro cytotoxicity assay were previously described in example 12. In the présent example, four saporin-immunoconjugates were prepared: m9G4-saporin, ch9G4-saporin, 1613F12-saporin and hz!613F12-saporinand tested in two cellular modcls (human SN12C rénal tumor cells and human Calu-1 lung carcinoma cells).

Figure 14 shows that both m9G4-saporin and ch9G4-saporin isotype controls were silent, and that the humanized Axl 1613F12-saporin antibody triggers similar cytotoxic effects on SN12C cells than the mouse 1613F12-saporin immunoconjugate.

Figure 15 shows that the humanized 1613F12-saporin immunoconjugate triggers similar cytotoxic effects on Calu-1 cells than the mouse 1613F12-saporin 15 immunoconjugate. In contrast, both m9G4-saporin and ch9G4-saporin isotype controls showed weak activity (-10% max cytotoxicity) for antibody concentrations above 10'⁹M.

Example 14: Binding klnetlcs of 1613F12 to human Axl ECD

Afïïnity measurement of 1613F12 was then determined using Biacore. A

Biacore X is used to measure the binding kinetics of 1613F12 on human Axl ECD.

The instrument based on the optical phenomenon of surface plasmon résonance (SPR) used by Biacore Systems enables the détection and measurement of proteinprotein interactions in real time, without the use of labels.

5 Bricfly, the experiments were realized using a sensor chip CM5 as the biosensor.

Rabbit IgGs were tmmobilized on the flow cells 1 and 2 (FC1 and FC2) of a CM5 sensor chip at a level of 9300-10000 response units (RU) using amine coupling chemistry to capture antibodies.

Binding is evaluated using multiple cycles. Each cycle of measure is performed using a flow rate of 30pl/min in a HBS-EP buffer. Then the Axl antibody to test is captured on the chip for 1 min on FC2 only to reach a mean capture value of 311.8 RU (SD=5.1 RU) for the I613F12. The analytc (Axl ECD antigen) is injected starting at 200 nM and using two-fold serial dilutions to mcasure rough ka and kd in real time.

At the end of each cycle, the surfaces are regenerated by injecting a lOmM glycine hydrochloride pH1.5 solution to eliminate the antibody-antigen complexes and 5 the capture antibody as well. The considered signal corresponds to the différence of the signais observed between FC1 and FC2 (FC2-FC1). Association rates (ka) and dissociation rates (kd) were calculated using a onc-to-one Langmuir binding mode). The equilibrium dissociation constant (KD) is determined as the ka/kd ratio. The experimental values were analyzed in the Biaevaluation software version 3.0. A %2 10 analysis will be performed to assess the accuracy of the data.

Data are summarized in the following Table 8.

Table 8

Antibody	ka (1/Ms)	kd(l/s)	KD (M)	ChF
1613FI2	1.06 10’	2.42 I0	2.29 I0’⁹	0.71 (0.6%)

To produce the human extraccllular domain (ECD) of Axl, the human cDNAs coding for the human soluble AXL receptor was first cloned into the pCEP4 expression vector by PCR. The purified product was then digested with restriction enzymes HindllI and BamHI and ligatcd into pCEP4 expression vector which had been precut with the 2 0 same enzymes. Finally, the identified recombinant plasmid pCEP[AXL]Hîs<5 was further confirmed by DNA sequencing.

Then suspension adapted cells HEK293E were cultivated in Ex-cell 293 (SAFC Biosciences) medium with 4 mM glutamine. Ail transfections were performed using linear 25 kDa polyethylenciminc (PEI). The transfected cells were maintained at 37°C 25 in an incubateur shaker with 5% CO2 and with agitation at 120 rpm for 6 days. The cells were collected by centrifugation, and the supematant containing the recombinant Histagged protein was treated for purification on a Ni-NTA agarose column.

Claims

1. An antigen binding protein, or an antigen binding fragment thereof, which:

i) specifically binds to the human protein Axl, preferably having the sequence SEQ ID NOS. 29 or 30 or natural variant sequence thereof, and ii) is intemalized following its binding to said human protein Axl, said antigen binding protein being characterized in that it comprises at least an amino acid sequence selected from the group consisting of SEQ ID NOs. 1 to 14 or 36 to 68.

2. The antigen binding protein, or an antigen binding fragment thereof, according to claim I, characterized in that it specifically binds to an epitope localized into the human protein Axl extracellular domain, preferably having the sequence SEQ ID NO. 31 or 32 or natural variant sequence thereof.

3. The antigen binding protein, or an antigen binding fragment thereof, according to claim 1, characterized in that it induecs a réduction ofMFI ofat least 200.

4 · ·

i) a light chain variable domain of sequence SEQ ID NO. 7 or any sequence exhibiting at least 80% identity with SEQ IDNO.7, ii) a light chain variable domain of sequence SEQ ID NO. 36 or any sequence exhibiting at least 80% identity with SEQ ID NO. 36; and

4. The antigen binding protein, or an antigen binding fragment thereof, according any of the preceding claims, characterized in that it consists of a monoclonal antibody.

5 extracellular domain, more preferably the human protein Axl extracellular domain having the sequence SEQ ID NO. 31 or 32 or natural variant sequence thereof.

5 iii) a light chain variable domain of sequence SEQ ID NO. 37 to 47 or any sequence exhibiting at least 80% identity with SEQ ID NO. 37 to 47.

5. An antigen binding protein, or an antigen binding fragment thereof, characterized in that it consists of an antibody, said antibody comprising the three light chain CDRs comprising the sequences SEQ ID NOs. 1, 2 and 3; and the three heavy chain CDRs comprising the sequences SEQ ID NOs. 4, 5 and 6,

6. The antigen binding protein, or an antigen binding fragment thereof, according to claim 5, characterized in that it comprises a light chain variable domain selected in the group consisting of:

7. The antigen binding protein, or an antigen binding fragment thereof, according to claim 5, characterized in that comprises a heavy chain variable domain

8. The antigen binding protein, or an antigen binding fragment thereof, according to claim 5, 6 or 7, characterized in that it comprises:

20 i) a light chain variable domain of sequence SEQ ID NO. 7, 36 or 37 to 47 or any sequence exhibiting at least 80% identity with SEQ ID NO.7,36 or 37 to 47; and ii) a heavy chain variable domain of sequence SEQ ID NO. 8,48 or 49 to 68 or any sequence exhibiting at least 80% identity with SEQ ID NO.8,48 or 49 to 68.

25

9. The antigen binding protein according to claim 5, characterized in that it consists of the monoclonal antibody 1613F12 derived from the hybridoma 1-4505 deposited at the CNCM, Institut Pasteur, France, on the 28 July 2011, or an antigen binding fragment thereof.

30 10. The murine hybridoma 1-4505 deposited at the CNCM, Institut Pasteur,

France, on the 28 July 2011.

10 conjugated to a cytotoxic agent.

10 selected in the group consisting of:

i) a heavy chain variable domain of sequence SEQ ID NO. 8 or any sequence exhibiting at least 80% identity with SEQ ID NO.8;

ii) a heavy chain variable domain of sequence SEQ ID NO. 48 or any sequence exhibiting at least 80% identity with SEQ ID NO. 48; and

15 iii) a heavy chain variable domaine of sequence SEQ ID NO. 49 to 68 or any sequence exhibiting at leat 80% identity with SEQ ID NO. 49 to 68.

11. The antigen binding protein, or an antigen binding fragment thereof, according to any of the daims 1 to 9, for use as an addressing product for delivering a cytotoxic agent at a host target site, said host target site consisting of an epitope localized into the protein Axl extracellular domain, preferably the human protein Axl

12. An immunoconjugate comprising the antigen binding protein, or an antigen binding fragment thereof, according to any of the daims 1 to 9 and 11

13. The immunoconjugate of claim 12 for use in the treatment of cancer.

14. Pharmaceutical composition comprising the immunoconjugate of daim

15 13 and at least an excipient and/or a pharmaceutical acceptable vehicle.