(19) TZZ 58Z9A_T (11) EP 2 589 609 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.05.2013 Bulletin 2013/19 C07K 16/28 (2006.01) (21) Application number: 11306416.6 (22) Date of filing: 03.11.2011 (84) Designated Contracting States: (72) Inventors: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB •Beau-Larvor, Charlotte GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO 74520 Jonzier Epagny (FR) PL PT RO RS SE SI SK SM TR • Goetsch, Liliane Designated Extension States: 74130 Ayze (FR) BA ME (74) Representative: Regimbeau (83) Declaration under Rule 32(1) EPC (expert 20, rue de Chazelles solution) 75847 Paris Cedex 17 (FR) (71) Applicant: PIERRE FABRE MEDICAMENT 92100 Boulogne-Billancourt (FR) (54) Antigen binding protein and its use as addressing product for the treatment of cancer (57) The present invention relates to an antigen bind- of Axl, being internalized into the cell. The invention also ing protein, in particular a monoclonal antibody, capable comprises the use of said antigen binding protein as an of binding specifically to the protein Axl as well as the addressing product in conjugation with other anti- cancer amino and nucleic acid sequences coding for said pro- compounds,such as toxins, radio- elements ordrugs, and tein. From one aspect, the invention relates to an antigen the use of same for the treatment of certain cancers. binding protein, or antigen binding fragments, capable of binding specifically to Axl and, by inducing internalization EP 2 589 609 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 589 609 A1 Description [0001] The present invention relates to a novel 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 5 one aspect, the invention relates to a novel 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. [0002] "Axl" (also referred to as "Ufo", "Ark" or "Tyro7") was cloned from patients with chronic myeloid leukemia as 10 an oncogene triggering the transformation when overexpressed by mouse NIH3T3. It belongs to a family of receptor tyrosine kinases (RTKs) called the TAM (Tyro3, Axl, Mer) family, which includes Tyro3 (Rse, Sky, Dtk, Etk, Brt, Tif), Axl, and Mer (Eyk, Nyk, Tyro-12) [Lemke G. Nat. Rev. Immunol. (2008).8, 327-336]. [0003] 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 15 signal, is represented in the sequence listing as SEQ ID NO. 30. [0004] Gas6, originally isolated as growth arrest-specific gene, is the common ligand for the members of the TAM family [Varnum B.C. et al. Nature (1995). 373, 623-626]. Gas6 exhibits the highest affinity for Axl, followed by Tyro3 and finally by Mer [Nagata K. et al. J. Biol. Chem. (1996). 271, 30022-30027]. Gas6 consists in a γ-carboxyglutamate (Gla)-rich domain that mediates binding to phospholipid membranes, four epidermal growth factor-like domains, and two laminin 20 G-like (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 molecules [Linger R.M. Adv. Cancer Res. (2008).100, 35-83]. Moreover, the Axl receptor can be activated through a ligand-independent process. This activation can occur when the Axl receptor is overexpressed. 25 [0005] Gas6/Axl signaling has been shown to regulate various cellular processes including cell proliferation, 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 innate immunity; 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 for the maturation and killing activity of natural killer (NK) cells [Lemke G. Nat. Rev. Immunol. 30 (2008).8, 327-336]. [0006] Weakly expressed on normal cells, it is predominantly observed in fibroblasts, myeloid progenitor cells, mac- rophages, neural tissues, cardiac and skeletal muscle where it supports mainly cell survival. The Gas6/Axl system plays an important role in vascular biology by regulating vascular smooth muscle cell homeostasis [Korshunov V.A., Mohan, A.M., Georger, M.A. & Berk, B.C. Circ. Res. (2006).98, 1446-1452; Korshunov V.A., Daul, M., Massett, M.P. & Berk, 35 B.C. Hypertension (2007).50, 1057-1062]. [0007] In tumor cells, Axl plays an important role in regulating cellular invasion and migration. Over-expression of Axl is associated not only with poor prognosis but also with increased invasiveness of various human cancers as reported for breast, colon, esophageal carcinoma, hepatocellular, gastric, glioma, lung, melanoma, osteosarcoma, ovarian, pros- tate, rhabdomyosarcoma, renal, thyroid and uterine endometrial cancer [Linger R.M. Adv. Cancer Res. (2008).100, 40 35-83 and Verma A. Mol. Cancer Ther. (2011). 10, 1763-1773, for reviews]. In breast cancer, Axl appears to be a strong effector of the Epithelial-to-mesenchymal transition (EMT); EMT program contributes actively to migration and dissem- ination of cancer cells in the organism [Thiery J.P. Curr. Opin. Cell Biol. (2003). 15, 740-746]. [0008] Axl has also been shown to regulate angiogenesis. Indeed knockdown of Axl in endothelial cells impaired tube formation and migration [Holland S.J. et al. Cancer Res. (2005). 65, 9294-9303] as well as disturbed specific angiogenic 45 signaling pathways [Li Y. et al. Oncogene (2009).28, 3442-3455]. [0009] More recently several studies on a range of cellular models described the involvement of an Axl overexpression in drug resistance phenomena. The following table 1 summarized these studies. Table 1 50 Reference Cancer type Therapeutic agent Cellular model Macleod et al., 2005 Ovarian cancer Cisplatin PE01/PEO1CDDP Mahadevan et al., Mahadevan Imatinib inhibitor of c-kit/ GIST GIST882 >GIST-R et al., 2007 PDGFR 55 Lay et al., 2007 NSCLC Doxorubicin CL-1 clones CL1-5F4 clones Hong et al., 2008 AML Doxorubicin/Cisplatin U937 2 EP 2 589 609 A1 (continued) Reference Cancer type Therapeutic agent Cellular model Lapatinib (HER1 and HER2 Liu et al., 2009 Breast Cancer HER2 (+) BT474 (J4) 5 inhibitor) Temozolomide Carboplatin Keating et al., 2010 Astrocytoma G12 A172 Vincristin Ye et al., 2010 NSCLC Erlotinib HCC827 10 [0010] Complete references cited in table 1 above are as follow: - Macleod, K. et al. Cancer Res. (2005).65, 6789-6800 15 - 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 20 - Liu L. et al. Cancer Res. (2009).69, 6871-6878 - Keating A.K. et al. Mol. Cancer Ther. (2010).9, 1298-1307 25 - Ye X. et al. Oncogene (2010).29, 5254-5264 [0011] In such a context the Axl RTK is considered as an interesting target in oncology. Several groups already developed anti-tumoral strategies targeting the gas6/Axl axis, either using naked monoclonal antibodies or targeted small molecules [Verma A. Mol. Cancer Ther. (2011).10, 1763-1773]. 30 [0012] 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 ii) is internalized following its binding to said human protein Axl. [0013] More generally, the invention relates to the use of the protein Axl for the selection of an antigen binding protein, or an antigen binding fragment thereof, capable of being internalized following its binding to the said target Axl. More particularly, the said target is the extracellular domain of Axl. 35 [0014] In this particular aspect, the present invention is thus directed to anin vitro method for the screening of a compound, or a binding fragment thereof, capable of delivering or internalizing a molecule of interest into mammalian cells, said molecule of interest being covalently linked to said compound, wherein said method comprises the following steps of: 40 a) selecting a compound which is capable of specifically binding the Axl protein, or the extracellular domain (ECD) thereof, or an epitope thereof ; b) optionally, covalently linking said molecule of interest, or a control molecule, 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 45 viable cell, expressing at its surface the Axl protein, or a functional fragment thereof; d) determining whether said compound, or said molecule of interest or said complex, has been intracellularly delivered or internalized into said mammalian cell; and e) selecting said compound as a compound capable of delivering or internalizing a molecule of interest into a viable mammalian cell . 50 [0015] In a preferred embodiment, said compound capable of delivering or internalizing a molecule 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 residue(s) can be glycosylated.