FIELD OF THE INVENTION

The present invention concerns new humanized monoclonal anti-VEGFC antibodies and their uses, in particular in the prevention and treatment of cancers and disorders characterized by undesirable lymphatic endothelial cell migration and/or proliferation (lymphangiogenesis) and/or autocrine proliferation loops mediated by overexpression of VEGFC by tumor cells expressing their receptors/co-receptors. BACKGROUND OF THE INVENTION

In the last decades, angiogenesis, the formation of new blood vessels from a pre existing vasculature, has emerged as a major target in age-related pathologies (including cancer, retinopathies, psoriasis, arthritis, and rheumatoid arthritis). Several therapeutic agents targeting the VEGFA165, the main pro-angiogenic factor and its associated receptors have been approved for cancer treatment (e.g. Bevacizumab (BVZ, Avastin®), a recombinant humanized monoclonal antibody, or sunitinib (Sutent®), a small-sized kinase inhibitor which targets specific VEGF receptors (Flt-1, Flk-1, Flt-4 as well as PDGF-R, CSFR1, c-KIT...). For the patients, these conventional drugs lead to an indisputable initial period of clinical benefit, however they fail to definitively cure cancers. The treated primary tumors relapse and the remaining malignant cells disseminate to distant healthy tissues inducing metastases.

Therefore, new therapeutics are needed. This invention aims at developing a new strategy to tackle concomitantly the proangiogenic and the pro-lymphangiogenic pathways that are involved in tumor growth and metastatic dissemination.

To date, the mechanisms of tumor resistance are not fully understood (Giuliano et al, 2013). In cancer, inflammation and angiogenesis are two closely integrated processes. Moreover, lymphangiogenesis is crucial since the lymphatic network is one of the main routes of metastatic dissemination. VEGFC is one of the major growth factors of lymphatic endothelial cells (Alitalo et al., 2005). Several reports indicate that VEGFC expression in cancer cells correlates with accelerated tumor progression and a poor clinical outcome (Alitalo et al. 2012). VEGFC overexpression in breast cancers has been shown to correlate with lymphangiogenesis and metastasis (Wang et al. 2012). In preclinical models of renal cell carcinoma (RCC), endothelial cells chronically exposed to an anti-VEGFA antibody proliferate in response to VEGFC stimulation whereas naive endothelial cells are unable to do so. In particular, it is demonstrated that VEGFC expression is induced following administration of bevacizumab or sunitinib in experimental models of RCC in mice (Dufies et al. 2017; Grepin et al. 2012). The direct exposure of tumor cells to different anti- angiogenic compounds stimulates expression of VEGFC by tumor cells. Moreover, after irradiation by X Ray, head and neck tumor cells overexpress VEGFC (Lupu-Plesu et al Oncogenesis 2017). Therefore, the expression of VEGFC can be considered as a factor of response to treatment-induced stresses.

RCC, also known as hypernephroma, is a renal cancer that originates in the lining of the proximal convoluted tubule, the very small tubes in the kidney that filter the blood and remove waste products. Clear cell metastatic renal cell carcinoma (ccRCC) is the most common type of kidney cancer in adults, responsible for approximately 80% of cases. Initial treatment is most commonly a radical or partial nephrectomy and remains the mainstay of curative treatment. When the tumor is confined to the renal parenchyma, the 5-year survival rate is 60-70%, but this is lowered considerably where metastases have spread. It is resistant to radiation therapy and chemotherapy, but some cases respond to “old-fashion “ immunotherapies including interferon alpha and interleukin 2. Anti-angiogenic therapies such as sunitinib, axitinib, lenvatinib, pazopanib, temsirolimus, bevacizumab, , and sorafenib have improved the outlook for RCC (progression-free survival), although they have not yet demonstrated improved survival.

Anti-angiogenic therapies have been used in clear cell renal cancers (ccRCC, the most common renal cancer) with mitigated results (Escudier et al., 2010). Although these treatments increase progression-free survival of patients, they do not impact the overall survival except in some extremely rare cases. One hypothesis that could explain this semi failure is the production by the tumor cells or cells of the tumor microenvironment of angiogenesis factors redundant to VEGFA.

The new standard of care for ccRCC is a combination of immunotherapies (nivolumab ipilimumab) or a combination of anti-angiogenic drugs plus an immunotherapies

(pebrolizumab, axitinib, bevacizumab atezolizumab see Motzer et al NEJM 2018, 2019, Rini et al NEJM 2019). However, despite an impressive efficacy, these treatments are effective in only 20-30% of patients. New treatments are needed to further improve overall survival of these patients. Therefore, targeting a factor involved in the mechanisms of resistance to anti-angiogenics should be efficient in the second line or in the first line as a triple combination.

The invention now provides new humanized monoclonal antibodies directed against the part of VEGFC that specifically binds to and activates the VEGF receptor 3 (VEGF-R3), to specifically target the development of lymphatic vessels that occur following anti- angiogenic therapy. By inactivating VEGFC, these antibodies also inhibited the autocrine proliferation loop occurring on tumor cells expressing VEGFC and their receptors/co receptors neuropilin 1 and 2 (Dumond A et al JECCR 2021). In particular, the inventors previously demonstrated that murine anti-VEGFC antibodies inhibit the growth of experimental kidney tumors in nude mice. These murine antibodies inhibited the activation of VEGFR3 signaling and therefore the proliferation and the migration of VEGFC-stimulated endothelial cells. Moreover, they inhibited the proliferation of VEGFC-expressing renal cancer cells through NRP2 signaling (Dumond A et al JECCR 2021 and Dumond A Cells 2021). Whereas anti-VEGFA antibodies have no effect or even favor experimental tumor growth in this model, the combination of an anti-VEGFA and an anti-VEGFC have an additive effect on the inhibition of tumor growth. Therefore, these new antibodies may constitute a new therapeutic strategy for cancers and disorders characterized by undesirable lymphatic endothelial cell migration and/or proliferation, in particular a new therapeutic strategy for RCC or for tumors characterized by an overexpression of VEGFC by tumors cells and cells of the microenvironment. Moreover, aggressive RCC are characterized by enhanced fibrosis (Ambrosetti et al BJU 2021). Since VEGFC is a strong inducer of fibrosis (Kinashi et al., Int J Mol Sci. 2018), inhibiting such fibrosis with our antibody would counteract another hallmark of cancer aggressiveness. So the inventors now developed new humanized monoclonal antibodies derived from these murine antibodies.

SUMMARY OF THE INVENTION

A first object of the present invention is a humanized anti-vascular endothelial growth factor- C (VEGFC) antibody or a functional fragment thereof, said antibody comprising:

(i) the three heavy-chain CDRs of sequences SEC ID NO: 6, SEC ID NO: 7 and SEC ID NO: 8 under Kabat nomenclature,

(ii) the three light-chain CDRs of sequences SEC ID NO: 9, SEC ID NO: 10 and SEC ID NO: 11 under Kabat nomenclature,

(iii) a heavy chain variable domain of sequence selected in the group consisting of SEC ID NO: 15 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEC ID NO: 13, and SEC ID NO: 101 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 99, with the proviso that sequence SEQ ID NO: 4 is excluded and

(iv) a light chain variable domain of sequence SEQ ID NO: 130 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID 128, with the proviso that SEQ ID NO:5 is excluded. Another object of the present invention is a humanized monoclonal anti-vascular endothelial growth factor-C (VEGFC) antibody or a functional fragment thereof, comprising

(i) the three heavy-chain CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 under Kabat (ii) the three light-chain CDRs of sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 under Kabat,

(iii) a heavy chain variable domain of sequence selected in the group consisting of consensus sequence SEQ ID NO: 144 QX1QLVQX2GAEVKKPGASVKVSCKASGYTFX3SYWMHWVRQAPGQGLEWX4GEINP S N G RTN YN E KF KS RX5TX6TVDX7SX8STX9YM E LSX10 LRSX11 DTAVYYCASTSLFYYA M N YWGQGTLVTVSS wherein X1: A or V X2: S or P

X3: I or T X4: I or M X5: V or A X6: L or M X7: K or R

X8: I or T X9: A or V X10: R or S X11 : D or E consensus sequence SEQ ID NO: 145

QX1QLQX2X3GX4GLX5KPSX6TLSLTCAAYGYTFX7SYWMHWX8RQPPGKGLEWIG EIN PSNGRTNYNEKX9KSRVTX10SVDRSKNQASLKLSSVTAADTAVYYCASTSLFYYAMNY WGQGTLVTVSS Wherein X1: V or A

X2: E or Q X3: P or S X4: 1 or P X5: V or L X6: E or Q

X7: S or I X8: I or V X9: F or L X10: I or L, and consensus sequence SEQ ID NO: 147

MKHLWFFLLLVAAPRWVLSQAQLX1QX2GX3X4X5X6KPX7X8X9X10X11 X12X13CX14AX15GYTFISYWM HWVX16QX17PGX18GLEWIGEI N PSNG RTNYNEKX19KSKX20TLX21VDRSX22X23X24AX25MX26LSSX27X28X2 9X30DX31AVYYCASTSLFYYAMNYWGQGTX32VTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK Wherein

X1= R or V or Q X2= P or S X3= A or P X4= E or G X5= L or V

X6= V or K X7= G or S X8= A or Q X9= S or T X10= V or L

X11 = K or S X12= L or V X13= S or T X14= K or A X15= S ou Y

X16= K or R X17= R or A or P X18= Q or K X19= F or L X20= A or V

X21 = T or S X22= S or T or K X23= S or N X24= T or Q X25= Y or S

X26= Q or K X27= L or V X28= T or R X29= S or A X30= E or A

X31 = S or T

(iv) X32= S or L and a light chain variable domain of consensus sequence SEQ ID NO: 146

DX1VMTQX2PX3SLPVX4X5GX6PASISCRSSQSLVHSNGNTYLHWX7X8QX9PGQS PX1 0LLIYKVFNRLSGVPDRFSGSGSGX11DFTLKISX12VEAEDVGVYX13CSQNTHVPWTF G QGTKLEIK wherein X1: V or I X2: T or S X3: P or L X4: N or T

X5: P or L X6: E or Q X7: Y or F X8: L or Q X9: K or R

X10: Q or R X11: T or S X12: R or W X13: Y or F.

Another object is a polynucleotide encoding a variable heavy chain (VH) for a humanized monoclonal anti-VEGFC antibody according to the invention, a variable light chain (VL) for a humanized monoclonal anti- VEGFC antibody according to the invention, or both a variable heavy chain (VH) and a variable light chain (VL) for a humanized monoclonal anti- VEGFC antibody according to the invention. The invention also relates to a combination of two polynucleotides encoding a variable heavy chain (VH) and a variable light chain (VL) for a humanized monoclonal anti- VEGFC antibody according to the invention.

The present invention also concerns an expression vector comprising the polynucleotide or combination of polynucleotides of the invention, a non-human host cell transformed with a polynucleotide or a combination of two polynucleotides suitable for expressing an humanized monoclonal anti-VEGFC according to the invention, and a method of producing an humanized monoclonal anti-VEGFC antibody of the invention comprising: culturing the non-human host cell transformed with a polynucleotide according to the invention or a combination of two polynucleotides suitable for expressing an anti-VEGFC according to the invention, in particular non-human fibroblasts, under suitable conditions and recovering the anti-VEGFC antibody from the culture medium or from the said cultured cells.

Another object of the present invention is a combination of a humanized monoclonal antibody anti-VEGFC according to the invention and an anti-angiogenic compound, in particular selected from the group consisting antibodies anti-VEGF distinct from the anti- VEGFC antibody of the invention, inhibitors of receptors involve in angiogenesis including VEGFR1, 2, 3, CSFR, PDGFR, inhibitors of m-TOR, preferably an anti-VEGF antibody distinct from the anti-VEGFC antibody of the invention. The invention also concerns a pharmaceutical composition comprising a humanized monoclonal antibody according to the invention or a combination according to the invention and an excipient, carrier, and/or diluent.

The present invention also relates to a humanized monoclonal antibody as defined in the invention, or a combination of the invention or a pharmaceutical composition of the invention, for use as a medicament.

The present invention also relates to a humanized monoclonal antibody as defined in the invention, or a combination of the invention or a pharmaceutical composition of the invention, for use in therapy or diagnosis, in particular in the treatment of cancers or disorders characterized by undesirable lymphatic endothelial cell migration and/or proliferation, or in an in vitro method for diagnosing or predicting the risk to relapse and/or to develop a tumor metastasis in a subject treated by its conventional treatment comprising an anti-angiogenic compound. In a particular embodiment, the said humanized monoclonal antibody or the said combination or the said pharmaceutical composition are used in the treatment of cancers or disorders characterized by undesirable lymphatic endothelial cell migration and/or proliferation, in particular renal cancer carcinoma (RCC), breast cancer, head and neck cancers, retinopathies, arthritis or rheumatoid arthritis, preferably renal cancer carcinoma (RCC).

In a particular and preferred embodiment, the said humanized monoclonal antibody or the said combination or the said pharmaceutical composition are used in a subject who is relapsed from or refractory to its conventional treatment, and/or who is developing or is at risk of developing tumor metastasis.

Another object of the invention is an in vitro method for diagnosing or predicting the risk to relapse and/or to develop a tumor metastasis in a subject treated by its conventional treatment comprising an anti-angiogenic compound, characterized in that said method comprises a step of contacting a biological sample of said subject with a humanized monoclonal anti-VEGFC antibody of the invention, it being possible for said antibody to be, if necessary, labelled. The present invention also relates to a kit for carrying out the in vitro method for diagnosing or predicting the risk to relapse and/or to develop a tumor metastasis in a subject treated by its conventional treatment comprising an anti-angiogenic compound, the said kit comprising at least one humanized monoclonal anti-VEGFC antibody and/or a functional fragment thereof according to the invention and optionally at least one reagent for detecting said anti-VEGFC antibody.

DETAILED DESCRIPTION OF THE INVENTION

New humanized anti-VEGFC antibody or a functional fragment

The protein VEGFC (Vascular endothelial growth factor C) encoded by the human gene (Gene ID: 7424, RefSeq mRNA NM_005429) is a member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family. The encoded protein promotes endothelial cell proliferation and angiogenesis, and can also affect the permeability of blood vessels. It can also promote the proliferation of lymphatic endothelial cells and lymphangiogenesis. The proprotein is further cleaved into a fully processed form that can bind and activate VEGFR-2 and VEGFR-3 receptors.

VEGFC (Uniprot P49767, RefSeq protein NP_005420, amino-acid sequence represented by SEQ ID NO: 12) is proteolytically matured along a complex mechanism. The entire VEGFC molecule can stimulate both the VEGF receptor 2 (KDR) and VEGF receptor 3 (FLT 4) whereas the proteolytically cleaved form of VEGFC stimulates only VEGFR3 (Joukov et al. 1996).

To specifically target the lymphatic pathway, the inventors immunized mice with the VEGFC form that only stimulates VEGFR3 according to the protocol described in Figure 1. They obtained two monoclonal antibodies with a high specificity to VEGFC. The variable regions of these antibodies were sequenced (heavy and light chains as illustrated in Figure 7). The CDR of these two antibodies were fused to human lgG1 light and heavy chains to obtain chimeric antibodies that can be used in the clinic. Expression vectors were transfected in CHO cells to obtain stable clones expressing the chimeric antibodies. The chimeric antibodies recognized with a high affinity the human VEGFC. As illustrated in the examples below, they inhibit the growth of experimental kidney tumors in nude mice. Whereas anti- VEGF antibodies have no effect or even favor experimental tumor growth in this model, the combination of anti VEGF and anti-VEGFC of the invention have an additive effect on the inhibition of tumor growth.

So, a first object of the invention concerns a humanized anti-vascular endothelial growth factor-C (VEGFC) antibody or a functional fragment thereof, said antibody comprising: (i) the three heavy-chain CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 under Kabat nomenclature,

(ii) the three light-chain CDRs of sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 under Kabat nomenclature, (iii) a heavy chain variable domain of sequence selected in the group consisting of SEQ ID NO: 15 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 13, and SEQ ID NO: 101 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 99, with the proviso that sequence SEQ ID NO: 4 is excluded and (iv) a light chain variable domain of sequence SEQ ID NO: 130 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID 128, with the proviso that SEQ ID NO:5 is excluded.

The murine antibody was obtained by using an immunogen comprising a peptide antigen having an amino acid sequence corresponding to SEQ ID NO: 1 (Figure 1).

In a particular embodiment, the humanized antibody of the invention is able to compete with or inhibit the binding of VEGFC to VEGF-R3.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one skilled in the relevant art.

For convenience, the meaning of certain term and phrases employed in the specification and claims are provided.

The antibodies described herein can be in the form of full-length antibodies, multiple chain or single chain antibodies, antigen-binding fragments of such antibodies also named ‘functional fragments’, including but not limited to Fab, Fab', (Fab')2, Fv, and scFv), single domain antibodies, humanized antibodies, camelid single-domain antibodies and the like. More particularly, an antibody (or “immunoglobulin”) consists of a glycoprotein 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 region (or domain) (abbreviated herein as VH) and a heavy chain constant region (hereafter CH). Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. The heavy chain constant region of the immunoglobulin IgG, IgD, and IgA (g, d and a chains respectively) comprises three domains (CH1, CH2, and CH3) and a hinge region for added flexibility, and the heavy chain constant region of the immunoglobulin IgM and IgE contains 4 domains (CH1, CH2, CH3, and CH4). The antibody of the invention can be of the IgG, IgM, IgA, IgD, and IgE isotype, depending on the structure of its heavy chain. However, in a preferred embodiment, the antibody of the invention is of the IgG isotype, i.e. , its heavy chain is of the gamma (y) type.

IgG antibodies are classified in four distinct subtypes, named lgG1, lgG2, lgG3 and lgG4 in order of their abundance in serum (lgG1 being the most abundant). The structure of the hinge regions in the g chain gives each of these subtypes its unique biological profile (even though there is about 95% similarity between their Fc regions, the structure of the hinge regions is relatively different).

The antibody of the invention can be of the lgG1, lgG2, lgG3 or lgG4 subtype. However, in a preferred embodiment, the antibody of the invention is of the lgG1 subtype or of the lgG2 subtype, preferably of the lgG1 subtype.

Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region comprising only one domain, CL. There are two types of light chain in mammals: the kappa (K) chain, encoded by the immunoglobulin kappa locus on chromosome 2, and the lambda (l) chain, encoded by the immunoglobulin lambda locus on chromosome 22. In a preferred embodiment, the antibody of the invention has a Kappa light chain.

The V _H and V _L regions can be further subdivided into regions of hypervariability, termed “Complementarity Determining Regions” (CDR), which are primarily responsible for binding an epitope of an antigen, and which are interspersed with regions that are more conserved, termed “Framework Regions” (FR). Each V _H and V _L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acid sequences to each domain is in accordance with well-known conventions (Chothia et al. 1989; Chothia et al. 1987). The functional ability of the antibody to bind a particular antigen depends on the variable regions of each light/heavy chain pair and is largely determined by the CDRs. The variable region of the heavy chain differs in antibodies produced by different B cells but is the same for all antibodies produced by a single B cell or B cell clone (or hybridome).

As used herein, a “CDR” refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH b-sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL b-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al. (1977) J. Biol. Chem. 252:6609-6616; Kabat (1978) Adv. Prot. Chem. 32:1-75). The Kabat CDRs are based on sequence variability and are the most commonly used (Kabat eta/. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD). Chothia refers instead to the location of the structural loops (Chothia and Lesk (1987) J Mol. Biol. 196:901-917). CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved b- sheet framework, and thus are able to adopt different conformations (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). Both terminologies are well recognised in the art. CDR region sequences have also been defined by AbM, Contact and IMGT. The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modelling software. The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. Recently, a universal numbering system has been developed and widely adopted, ImMunoGeneTics (IMGT) Information System ^® (Lefranc et al. (2003) Dev. Comp. Immunol. 27(1):55-77). The IMGT universal numbering has been defined to compare the variable domains whatever the antigen receptor, the chain type, or the species [Lefranc M.-P. (1997) Immunol. Today 18: 509; Lefranc M.-P. (1999) The Immunologist 7: 132-136] In the IMGT universal numbering, the conserved amino acids always have the same position, for instance cysteine 23 (1st-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). The IMGT universal numbering provides a standardised delimitation of the framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining regions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps represent unoccupied positions, the CDR-IMGT lengths (shown between brackets and separated by dots, e.g. [8.8.13]) become crucial information. The IMGT universal numbering is used in 2D graphical representations, designated as IMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53: 857-883 (2002); Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2: 21-30 (2007)], 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)]. The positions of CDRs within a canonical antibody variable domain have been determined by comparison of numerous structures (Al-Lazikani et ai, J. Mol. Biol. 273:927-948 (1997); Morea et ai, Methods 20:267-279 (2000)). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997)). Such nomenclature is similarly well known to those skilled in the art.

Mention may be made also to software that determine CDRs under different nomenclature such as the AbYsis platform accessible on: http://www.abysis.org/abysis/sequence input/key annotation/key annotation. cgi.

In the context of the present invention for the design of humanized antibodies, the CDR and framework regions in the murine sequences were identified using the Kabat method. Table 1 lists the amino acid sequences of the murine CDR under Kabat method of the antibodies according to the invention

By contrast, the constant regions of the antibodies mediate 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 complement system.

The term "antibody fragment" refers to a molecule comprising only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C- terminus of the CH 1 domain; (iii) the Fd fragment having VH and CH 1 domains; (iv) the Fd' fragment having VH and CH 1 domains and one or more cysteine residues at the C-terminus of the CH 1 domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment which consists of a VH domain; (vii) isolated complementarity determining regions (CDRs); (viii) F(ab')2 fragments, a bivalent fragment including two Fab' fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g. single chain Fv; scFv); (x) "diabodies" with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain; (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-CH 1 -VH-CH 1 ) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Antibodies can be fragmented using conventional techniques. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.

In a particular embodiment, the humanized antibody or a functional fragment thereof, which is directed to the human proteolytically cleaved form of VEGFC, according to the invention comprises

(i) the three heavy-chain CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 under Kabat nomenclature,

(ii) the three light-chain CDRs of sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 under Kabat nomenclature,

(iii) a heavy chain variable domain of sequence selected in the group consisting of SEQ ID NO: 15 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 13, and SEQ ID NO: 101 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 99, with the proviso that sequence SEQ ID NO: 4 is excluded and

(iv) a light chain variable domain of sequence SEQ ID NO: 130 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID 128, with the proviso that SEQ ID NO:5 is excluded.

In another embodiment, the humanized monoclonal antibody comprises

(i) the three heavy-chain CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 under Kabat nomenclature,

(ii) the three light-chain CDRs of sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 under Kabat nomenclature,

(iii) a heavy chain variable domain of sequence selected in the group consisting of SEQ ID NO: 82 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 13, and SEQ ID NO: 111 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 109, with the proviso that sequence SEQ ID NO: 4 is excluded and (iv) a light chain variable domain of sequence SEQ ID NO: 130 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID 128, with the proviso that SEQ ID NO:5 is excluded.

In another embodiment, the humanized monoclonal antibody comprises

(i) the three heavy-chain CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 under Kabat nomenclature,

(ii) the three light-chain CDRs of sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 under Kabat nomenclature,

(iii) a heavy chain variable domain of sequence selected in the group consisting of SEQ ID NO: 82 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 13, and SEQ ID NO: 111 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 109, with the proviso that sequence SEQ ID NO: 4 is excluded and

(iv) a light chain variable domain of sequence SEQ ID NO: 136 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID 134, with the proviso that SEQ ID NO:5 is excluded.

In another embodiment, the humanized monoclonal antibody comprises

(i) the three heavy-chain CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 under Kabat nomenclature,

(ii) the three light-chain CDRs of sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 under Kabat nomenclature,

(iii) a heavy chain variable domain of sequence selected in the group consisting of SEQ ID NO: 15 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 13, and SEQ ID NO: 101 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 99, with the proviso that sequence SEQ ID NO: 4 is excluded and

(iv) a light chain variable domain of sequence SEQ ID NO: 136 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID 134, with the proviso that SEQ ID NO:5 is excluded.

The percentages of identity to which reference is made in the present invention are determined on the basis of a global alignment of sequences to be compared, that is to say, on an alignment of sequences taken in their entirety over their entire length, using any algorithm well-known to a person skilled in the art, for example the algorithm of Needleman and Wunsch 1970. This sequence comparison may be performed using any software well- known to a person skilled in the art, for example using the needle software by using the "Gap open" parameter equal to 10.0, the "Gap Extend" parameter equal to 0.5, and a "BLOSUM 62" matrix. The Needle software is for example available on the website ebi.ac.uk worldwide, under the name "Align". When the CDR or variable region of an humanized antibody according to the invention has an amino acid sequence that is not 100% identical to one of those described above and in the sequence listing (reference sequences) but that has at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with one such reference sequence, it may have insertions, deletions or substitutions with regard to the reference sequence. When it is a matter of substitutions, the substitution is preferably made with an “equivalent” amino acid, i.e., any amino acid whose structure is similar to that of the original amino acid and therefore unlikely to change the biological activity of the antibody. Such substitutions are also named ‘conservative mutation’. Examples of such substitutions are presented in Table 2 below:

Table 2. Substitutions with equivalent amino acids In a particular embodiment, the CDRs sequences of the heavy chain variable region and/or of the light chain variable region have 100% identity with the SEQ ID NO:6 to SEQ ID NO: 11.

In another particular embodiment, the CDRs sequences of the heavy chain variable region and/or of the light chain variable region may contain one or two mutations, in particular one or two conservative substitutions as disclosed above.

The antibody of the invention is a monoclonal antibody.

A “monoclonal antibody”, as used herein, means an antibody arising from a nearly homogeneous antibody population. More particularly, the subject antibodies of a population are identical except for a few possible naturally occurring mutations which can be found in minimal proportions. In other words, a monoclonal antibody consists of a homogeneous antibody arising from the growth of a single cell clone (for example a hybridoma, a eukaryotic host cell transfected with a DNA molecule coding for the homogeneous antibody, a prokaryotic host cell transfected with a DNA molecule coding for the homogeneous antibody, etc.) and is generally characterized by heavy chains of one and only one isotype and subtype, and light chains of only one type. In addition, in contrast with preparations of polyclonal antibodies, each monoclonal antibody is directed against a single epitope of an antigen.

To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from the immunized animal as described above and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art (e. g. the hybridoma technique originally developed by Kohler and Milstein (1975) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al_, 1983), the EBV-hybridoma technique to produce human monoclonal antibodies (Roder et al., 1986), and screening of combinatorial antibody libraries (Huse et al. 1989). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the target polypeptide(s) so that only monoclonal antibodies binding to said polypeptide(s) are isolated.

Humanized forms of antibodies of the invention are chimeric antibodies that contain minimal sequence derived from non- human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin (recipient antibody) are replaced by corresponding non-human residues of the donor antibody. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. In general, the humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin (donor antibody having the desired specificity, affinity, and capacity) and all or substantially all of the FRs are those of a human immunoglobulin sequence. In one embodiment, humanized antibodies comprise a humanized FR that exhibits at least 65% sequence identity with an acceptor (non-human) FR, e.g., murine FR. The humanized antibody also may comprise at least a portion of an immunoglobulin constant region (Fc), particularly a human immunoglobulin. Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source, which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization may be essentially performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. Other methods generally involve conferring donor CDR binding affinity onto an antibody acceptor variable region framework. One method involves simultaneously grafting and optimizing the binding affinity of a variable region binding fragment. Another method relates to optimizing the binding affinity of an antibody variable region. Humanization by CDR grafting is a proven, successful technique to take antibodies originating from murine, other xenogenic species or hybridomas and reduce the potential immunogenicity whilst retaining the binding and functional activity of the Parental antibody. Commonly starting from a chimeric antibody, the aim is to remove the foreign framework regions (FR) in the variable domains that can evoke an immune response (Bruggemann et al. 1987). The solution to the problem is to “graft” the complementarity determining regions (CDRs) of the Parental murine antibody onto a human Acceptor framework (Jones et al., 1986). However, CDR-grafting alone can lead to a significant reduction or complete loss of binding affinity, as a set of supporting framework residues in the Vernier zone are important for maintaining the conformation of the CDRs (Foote and Winter 1992). This problem can be solved by reintroducing murine residues into the human framework (Queen et al. , 1989); such substitutions are commonly called back-mutations.

As the most significant property of a therapeutic antibody is the activity, it is important that substitutions proposed during the humanization do not affect the affinity or stability of the antibody. A large amount of information has been collected in the last 20 years on humanisation and grafting of the CDRs (Jones et al., 1986, Foote and Winter 1992); the biophysical properties of antibodies (Ewert et al., 2003), the conformation of the CDR-loops (Chothia and lesk 1987; Al-Lazikani et al., 1997: North et al., 2011) and for the frameworks (Vargas-Madrazo and Paz-Garcia 2003; Honegger et al. 2009), which along with advance in protein modelling (Desmet et al. 2002; Almagro et al. 2011) makes it possible to accurately humanize antibodies with retained binding affinity and stability.

As an example, for each of the murine monoclonal antibodies, corresponding humanized V _H and V _L peptide sequences were determined by: identifying the CDR and framework regions in the murine sequences using the IMGT-ONTOLOGY database (Duroux et al. 2008 ; Giudicelli et al_ 1999) and IMGT® databases and tools (Ehrenmann et al., 2010;

Brochet et al., 2008) followed by identification of the amino acid sequence of the closest human framework region sequences in the IMGT/GENE-DB (Giudicelli et al., 2005), and grafting of the murine CDR sequences onto the human framework regions. More particularly, nucleotide and amino acid sequences of murine V _H and V _L domains were analyzed using IMGT/V-QUEST and IMGT/ DomainGapAlign to delimit the murine CDRs and framework regions, define CDR lengths and identify anchor amino acids. Anchor amino acids are residues at position 26, 39, 55, 66, 104 and 118 of IMGT “Collier de Perles” that support the CDR1-IMGT, CDR2-IMGT, CDR3-IMGT (Lefrancetal. 2007; Kaas et al., 2007). The closest human V (variable) and J (joining) genes to the murine sequences were identified and the most suitable genes chosen. Individual amino acids in the murine framework region were maintained if they were considered to possibly contribute to the specificity of the antibody by comparison with known 3D structures (Kaas et al. , 2007) using IMGT Collier de Perles on two layers. In a particular embodiment of the invention, the humanization procedure is performed as outlined below and based on Kabat nomenclature:

1) Parental mouse antibody domains and regions are identified;

2) Critical positions are identified: Antibody Fv’s have a number of critical positions that make up the VH/VL inter chain interface or are responsible for the discrete set of canonical structures that has been defined for 5 of the CDRs (Chothia and Lesk 1987; Martin and Thornton 1996; Al Laziniki et al. 1997): these positions should be considered in detail before substitutions are proposed for them. ) Based on the sequence analysis and the critical positions, optimal Acceptor human germline sequences are selected for each chain: based on the Parental mouse antibody sequence alignment to the human germlines, the closest matching entries are identified; the identification of the optimal human germlines as Acceptor is based on the ordered criteria listed below:

• Sequence identity across the whole V gene (framework + CDRs)

• Identical or compatible inter-chain interface residues

• Support loops with the Parental mouse CDRs canonical conformations;

4) A 3D structural model of the Parental mouse Fv regions is constructed;

5) Following a close inspection of the molecular model, an initial assessment of the possibility to substitute each position is made: positions are categorised as Neutral Contributing or Critical;

6) The CDR-grafting is performed by analyzing positions differing between the Parental mouse and Acceptor human sequences. All substitutions in Neutral positions were performed. For the design phase of a humanized antibody, the procedure is more accurately defined as germlining - replacing amino acids in the Parental mouse framework that differ from the chosen Acceptor with the corresponding human amino acid;

7) For the heavy chain, and as illustrated in the examples, four different human germlines, IGHV1 -46*01 and IGHV1-2*06, IGHV4-34*08 and IGHV’4-34*09 are used for the design of humanized versions. For the light chain, and as illustrated in the examples, two different human germlines IGKV2-30*02 and IGKV2-18*01 are used. Combinations of the different humanized VH and VL versions are produced, purified and tested for binding and biological activity;

8) The selection of the best humanized heavy and light chain combination between the different versions is performed by assessing the following criteria: a) The level of transient expression of the humanized versions produced in mammalian cells (HEK 293 or preferably CHO) as human lgG1/Kappa (as compared to the chimeric version). Using tissue culture supernatant from transfected cells before harvest for purification using ELISA or measurement by protein A using Octet label-free detection systems; b) The binding capacity (EC50 by ELISA or FACS; or preferably Kd by Biacore or Octet) as compared to the chimeric human lgG1/Kappa version (chimeric meaning the combination of the parental murine VH and VL fused to human constant regions); c) The biological activity of the humanized versions in a relevant in vitro cellular assay compared with that of the reference chimeric antibody; d) The cross-reactivity with relevant orthologue species (in vitro binding activity); e) A determination of the biophysical properties of the humanized versions as compared with chimeric:

• SEC-HPLC profile to determine the level of high molecular weight aggregates, · SDS-PAGE under non-reducing and reducing conditions,

• Analysis by differential scanning calorimetry (DSC) using Microcal™ VP capillary DSC system to determine the Tm of Fab, CH2 and CH3.

As disclosed in step 3) of the method of immunization, optimal Acceptor human germline sequences are selected for each chain.

Table 3 below summarizes examples of heavy and light variable chains from murine and selected human germlines for designing the humanized antibodies of the present invention. This table also discloses consensus sequence for each group and sub-group of sequences VH and VL

Table 4 below summarizes sequence identity between human germline and consensus sequences disclosed above

Another object of the present invention is a humanized monoclonal anti-vascular endothelial growth factor-C (VEGFC) antibody or a functional fragment thereof, comprising (i) the three heavy-chain CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 under Kabat (ii) the three light-chain CDRs of sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 under Kabat,

(iii) a heavy chain variable domain of sequence selected in the group consisting of consensus sequence SEQ ID NO: 144

QX1QLVQX2GAEVKKPGASVKVSCKASGYTFX3SYWMHWVRQAPGQGLEWX4GEINP SNGRTNYNEKFKS RX5TX6T V DX7 SX8STX9 YM ELSX10LRSX11 DTAVYYCASTSLFYYA

M N YWGQGTLVTVSS wherein X1: A or V X2: S or P X3: I or T X4: I or M

X5: V or A X6: L or M X7: K or R X8: I or T X9: A or V

X10: R or S X11: D or E consensus sequence SEQ ID NO: 145

QX1QLQX2X3GX4GLX5KPSX6TLSLTCAAYGYTFX7SYWMHWX8RQPPGKGLEWIG EIN PSNGRTNYNEKX9KSRVTX10SVDRSKNQASLKLSSVTAADTAVYYCASTSLFYYAMNY WGQGTLVTVSS Wherein X1: V orA X2: E or Q X3: P or S

X4: 1 or P X5: V or L X6: E or Q X7: S or I X8: I or V

X9: F or L X10: I or L, and consensus sequence SEQ ID NO: 147

MKHLWFFLLLVAAPRWVLSQAQLX1QX2GX3X4X5X6KPX7X8X9X10X11X12X13 CX14A X15GYTFISYWM H WVX16QX17PGX18GLEWIGEI N PSNGRTNYN EKX19KSKX20TLX21 VDRSX22X23X24AX25MX26LSSX27X28X29X30DX31AVYYCASTSLFYYAMNYWGQ G TX32VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPI EKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Wherein X1= R or V or Q X2= P or S X3= A or P X4= E or G X5= L or V

X6= V or K X7= G or S X8= A or Q X9= S or T X10= V or L

X11= KorS X12= L or V X13= SorT X14= KorA X15= S ou Y

X16= KorR X17= RorAorP X18= Q or K X19= ForL X20= A or V

X21= T or S X22= S or T or K X23= S or N X24= T or Q X25= Y or S

X26= Q or K X27= L or V X28= T or R X29= S or A X30= E or A

X31= SorT X32= S or L,

(iv) and a light chain variable domain of consensus sequence SEQ ID NO: 146 DX1VMTQX2PX3SLPVX4X5GX6PASISCRSSQSLVHSNGNTYLHWX7X8QX9PGQSPX1 0LLIYKVFNRLSGVPDRFSGSGSGX11DFTLKISX12VEAEDVGVYX13CSQNTHVPWTF G QGTKLEIK wherein X1: V or I X2: T or S X3: P or L X4: N or T X5: P or L X6: E or Q

X7: Y or F X8: L or Q X9: K or R X10: Q or R X11: T or S

X12: R or W X13: Y or F.

In a particular embodiment, the antibody of the invention comprises: (i) the three heavy-chain CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ

ID NO: 8 under Kabat

(ii) the three light-chain CDRs of sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 under Kabat,

(iii) a heavy chain variable domain (iii) comprising a consensus sequence selected in the groups consisting of SEQ ID NO: 14, SEQ ID NO: 47, SEQ ID NO: 81, SEQ ID

NO: 90 and SEQ ID NO: 147 and

(iv) a light chain variable domain (iv) comprising a consensus sequence SEQ ID NO: 129. In particular, its heavy chain variable domain (iii) comprises a sequence selected in the groups consisting of SEQ ID NO: 15 to 46, SEQ ID NO:48 to 79, SEQ ID NO: 82 to 89, SEQ ID NO: 91 to 98, SEQ ID NO: 150, and SEQ ID NO: 152 to 159 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with said sequences and its light chain variable domain (iv) comprises a sequence selected in the group consisting of SEQ ID NO: 130 to 133, and SEQ ID NO: 151 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with said sequences.

As a particular embodiment, its heavy chain variable domain (iii) comprises a sequence selected in the groups consisting of SEQ ID NO: 15 to 46, SEQ ID NO:48 to 79, SEQ ID NO: 82 to 89, SEQ ID NO: 91 to 98, SEQ ID NO: 150, and SEQ ID NO: 152 to 159 and its light chain variable domain (iv) comprises a sequence selected in the group consisting of SEQ ID NO: 130 to 133 and SEQ ID NO: 151. Each combination of one heavy chain variable domain sequence of each sub-group and one light chain variable domain sequence of the group of SEQ ID NO: 130 to 133 is encompassed by the present invention.

In another particular embodiment, the antibody of the invention comprises:

(i) the three heavy-chain CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 under Kabat

(ii) the three light-chain CDRs of sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 under Kabat,

(iii) a heavy chain variable domain (iii) comprising a consensus sequence selected in the groups consisting of SEQ ID NO: 14, SEQ ID NO: 47, SEQ ID NO: 81 , SEQ ID NO: 90 and SEQ ID NO: 147 and

(iv) a light chain variable domain (iv) comprising a consensus sequence SEQ ID NO: 135.

In particular, its heavy chain variable domain (iii) comprises a sequence selected in the groups consisting of SEQ ID NO: 15 to 46, SEQ ID NO:48 to 79, SEQ ID NO: 82 to 89, SEQ ID NO: 91 to 98 and SEQ ID NO: 150, and SEQ ID NO: 152 to 159 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with said sequences and its light chain variable domain (iv) comprises a sequence selected in the group consisting of SEQ ID NO: 136 to 143 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with said sequences.

As a particular embodiment, its heavy chain variable domain (iii) comprises a sequence selected in the groups consisting of SEQ ID NO: 15 to 46, SEQ ID NO:48 to 79, SEQ ID NO: 82 to 89, SEQ ID NO: 91 to 98 and SEQ ID NO: 150, and SEQ ID NO: 152 to 159 and its light chain variable domain (iv) comprises a sequence selected in the group consisting of SEQ ID NO: 136 to 143.

Each combination of one heavy chain variable domain sequence of each sub-group and one light chain variable domain sequence of the group of SEQ ID NO: 136 to 143 is encompassed by the present invention.

In another particular embodiment, the antibody of the invention comprises:

(i) the three heavy-chain CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 under Kabat

(ii) the three light-chain CDRs of sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 under Kabat, (iii) a heavy chain variable domain (iii) comprising a consensus sequence selected in the groups consisting of SEQ ID NO: 100, SEQ ID NO: 110, SEQ ID NO: 119 and SEQ ID NO: 147 and

(iv) a light chain variable domain (iv) comprising a consensus sequence SEQ ID NO: 129.

In particular, its heavy chain variable domain (iii) comprises a sequence selected in the groups consisting of SEQ ID NO: 101 to 108, SEQ ID NO: 111 to 118 and SEQ ID NO: 120 to 127 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with said sequences and its light chain variable domain (iv) comprises a sequence selected in the group consisting of SEQ I D NO: 130 to 133 and SEQ ID NO: 151, or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with said sequences.

As a particular embodiment, its heavy chain variable domain (iii) comprises a sequence selected in the groups consisting of SEQ ID NO: 101 to 108, SEQ ID NO: 111 to 118 and SEQ ID NO: 120 to 127 and its light chain variable domain (iv) comprises a sequence selected in the group consisting of SEQ ID NO:130 to 133 and SEQ ID NO: 151.

Each combination of one heavy chain variable domain sequence of each sub-group and one light chain variable domain sequence of the group of SEQ ID NO: 130 to 133 is encompassed by the present invention.

In another particular embodiment, the antibody of the invention comprises:

(i) the three heavy-chain CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 under Kabat

(ii) the three light-chain CDRs of sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 under Kabat,

(iii) a heavy chain variable domain (iii) comprising a consensus sequence selected in the groups consisting of SEQ ID NO: 100, SEQ ID NO: 110 and SEQ ID NO: 119 and

(iv) a light chain variable domain (iv) comprising a consensus sequence SEQ ID NO: 135.

In particular, its heavy chain variable domain (iii) comprises a sequence selected in the groups consisting of SEQ ID NO: 101 to 108, SEQ ID NO: 111 to 118 and SEQ ID NO: 120 to 127 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with said sequences and its light chain variable domain (iv) comprises a sequence selected in the group consisting of SEQ ID NO: 136 to 143 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with said sequences.

As a particular embodiment, its heavy chain variable domain (iii) comprises a sequence selected in the groups consisting of SEQ ID NO: 101 to 108, SEQ ID NO: 111 to 118 and SEQ ID NO: 120 to 127 and its light chain variable domain (iv) comprises a sequence selected in the group consisting of SEQ ID NO: 136 to 143.

Each combination of one heavy chain variable domain sequence of each sub-group and one light chain variable domain sequence of the group of SEQ ID NO: 136 to 143 is encompassed by the present invention.

In a particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO: 15 to 46 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO: 129, in particular a sequence of SEQ ID NO: 130 to SEQ ID NO: 133. In another particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO: 15 to 46 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO: 135, in particular a sequence of SEQ ID NO: 136 to SEQ ID NO: 143. In another particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:48 to 79 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO: 129, in particular a sequence of SEQ ID NO: 130 to SEQ ID NO: 133.

In another particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:48 to 79 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO: 135, in particular a sequence of SEQ ID NO: 136 to SEQ ID NO: 143.

In another particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:82 to 89 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO: 129, in particular a sequence of SEQ ID NO: 130 to SEQ ID NO: 133.

In another particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:82 to 89 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO:

135, in particular a sequence of SEQ ID NO: 136 to SEQ ID NO: 143. In another particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:91 to SEQ ID NO: 98 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO: 129, in particular a sequence of SEQ ID NO: 130 to SEQ ID NO: 133. In another particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:91 to SEQ ID NO: 98 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO: 135, in particular a sequence of SEQ ID NO: 136 to SEQ ID NO: 143. In another particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO: 101 to SEQ ID NO: 108 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO: 129, in particular a sequence of SEQ ID NO: 130 to SEQ ID NO: 133.

In another particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:101 to SEQ ID NO: 108 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO: 135, in particular a sequence of SEQ ID NO: 136 to SEQ ID NO: 143.

In another particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:111 to SEQ ID NO: 118 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO: 129, in particular a sequence of SEQ ID NO: 130 to SEQ ID NO: 133.

In another particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:111 to SEQ ID NO: 118 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO: 135, in particular a sequence of SEQ ID NO: 136 to SEQ ID NO: 143.

In another particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:120 to SEQ ID NO: 127 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO: 129, in particular a sequence of SEQ ID NO: 130 to SEQ ID NO: 133.

In another particular embodiment, the humanized antibody of the present invention comprises a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:120 to SEQ ID NO: 127 and a light chain variable region (VL) comprising a consensus sequence SEQ ID NO: 135, in particular a sequence of SEQ ID NO: 136 to SEQ ID NO: 143.

The present invention encompasses any humanized antibody comprising a heavy chain variable region (VH) comprising a sequence as disclosed above and a light chain variable region (VL) comprising a sequence as disclosed above, meaning that all combinations 2 to 2 are encompassed by the present invention.

In a particular embodiment, the table 5 discloses the amino acids sequences of each variable heavy chain (VH) and variable light chain (VL) for the murine chimeric antibody (SEQ ID NO: 148) and the humanized anti- VEGFC antibodies illustrated in the following examples.

Table 5

In a particular embodiment, the humanized monoclonal anti-vascular endothelial growth factor-C (VEGFC) antibody of the present invention is selected in the group consisting of: a humanized anti- VEGFC antibody comprising a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:150 and a light chain variable region

(VL) comprising a sequence of SEQ ID NO: 151; a humanized anti- VEGFC antibody comprising a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:152 and a light chain variable region (VL) comprising a sequence of SEQ ID NO:151; a humanized anti- VEGFC antibody comprising a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:153 and a light chain variable region (VL) comprising a sequence of SEQ ID NO:151; a humanized anti- VEGFC antibody comprising a heavy chain variable region (VH) comprising a sequence of SEQ ID NO: 154 and a light chain variable region (VL) comprising a sequence of SEQ ID NO:151; a humanized anti- VEGFC antibody comprising a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:155 and a light chain variable region (VL) comprising a sequence of SEQ ID NO:151; a humanized anti- VEGFC antibody comprising a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:156 and a light chain variable region (VL) comprising a sequence of SEQ ID NO:151; a humanized anti- VEGFC antibody comprising a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:157 and a light chain variable region (VL) comprising a sequence of SEQ ID NO:151; a humanized anti- VEGFC antibody comprising a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:158 and a light chain variable region (VL) comprising a sequence of SEQ ID NO: 151; and a humanized anti- VEGFC antibody comprising a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:159 and a light chain variable region (VL) comprising a sequence of SEQ ID NO:151; preferably a humanized anti- VEGFC antibody comprising a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:150 and a light chain variable region (VL) comprising a sequence of SEQ ID NO:151; a humanized anti- VEGFC antibody comprising a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:152 and a light chain variable region (VL) comprising a sequence of SEQ ID NO:151; a humanized anti- VEGFC antibody comprising a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:153 and a light chain variable region (VL) comprising a sequence of SEQ ID NO: 151; and a humanized anti- VEGFC antibody comprising a heavy chain variable region (VH) comprising a sequence of SEQ ID NO:157 and a light chain variable region (VL) comprising a sequence of SEQ ID NO: 151. Antibodies may be isolated after production (e.g., from the blood or serum of the animals) or synthetized and further purified by well-known techniques. Antibodies specific for a protein can be selected or purified by affinity chromatography, ELISPOT or ELISA. For example, the proteolytically cleaved form of VEGFC can be covalently or non-covalently coupled to a solid support such as, for example, a chromatography column. The column can then be used to purify antibodies directed to the proteolytically cleaved form of VEGFC, from a sample containing antibodies directed against a large number of different epitopes, thereby generating a substantially purified antibody composition, i.e., one that is substantially free of contaminating antibodies. By a “substantially purified antibody composition” it is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those of the Strep-Tag II sequence, and preferably at most 20%, yet more preferably at most 10% and most preferably at most 5% (by dry weight) of the sample is contaminating antibodies. A “purified antibody composition” means that at least 99% of the antibodies in the composition are directed to the proteolytically cleaved form of VEGFC.

The humanized antibodies of the invention may be administered in their "naked" or unconjugated form or may have other agents conjugated to them. For examples the humanized antibodies may be in detectably labelled form. Antibodies can be detectably labelled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, and the like. Procedures for accomplishing such labelling are well known in the art.

In another embodiment, the humanized antibody of the invention is lyophilized. In such embodiment, the lyophilized antibody is admixed with a carrier or diluent such as those hereinabove described at the time of administration. In yet another embodiment, the humanized antibody of the invention is conjugated to a compound such as a polymer. In one embodiment, the polymer is a polyalkylene glycol. In one embodiment, the polyalkylene glycol is polyethylene glycol, or PEG.

For use in therapy, the humanized antibody(ies) will be suitably formulated together with pharmaceutically acceptable excipients. Suitable formulations for the preventive or therapeutical treatment can be administered parenterally, preferably intra-arterially, intraperitoneally, intravenously, subcutaneously, intramuscularly or via an aerosol, and they will contain an effective amount of the antibody(ies). Said amount will vary depending on the general conditions of the patient, the progression of the disease and other factors. Production of the said antibodies

The murine antibody 1E9 was previously obtained by immunizing mice, with peptides specific for human proteolytically cleaved form of VEGFC, particularly with the mature peptide of sequence

AHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNS EG LQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLDVYRQVHSIIRR (SEQ ID NO: 1) of the VEGFC human protein ((NCBI Reference Sequence NP_005420), following by screening methods to isolate antibody. Different screening methods to isolate antibody can be used including ELISA tests on said peptide.

In particular, the antibody of the invention is produced by the hybridoma.

The hybridoma has been obtained by immunization of mice with a peptide specific for human proteolytically cleaved form of VEGFC i.e. , the peptide of sequence AHYNTEILKSIDNEWRKTGCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEG LGCMNTSTSYLSKTLFEITVPLSGGPKPVTISFANHTSCRCMSKLDVYRGVHSIIRR (SEQ ID NO: 1) of the VEGFC human protein ((NCBI Reference Sequence NP_005420).

This strategy was developed to obtain specific hybridoma producing anti-VEGFC antibodies. The screening was based on the ability of the hybridoma to recognize the VEGFC antigen used in the immunization protocol by ELISA. The flow-chart of the experiment is described in Figure 2. Thirteen clones were selected for their ability to produce specific antibodies and the six best were amplified.

This hybridoma produces high yields of antibodies of the invention.

The humanized antibodies of the present invention may be produced accordingly by using the sequences of heavy and light variable chains as disclosed above in an expression vector under the control of a promoter which is effective in cells of non-human animal.

The humanized antibodies according to the invention have in particular the following advantages:

- They are unique described humanized antibodies directed to proteolytically cleaved form of VEGFC;

Compared to Bevacizumab (antibody directed to VEGF) which increases tumor growth in experimental models (Escudier et al., 2010), the antibodies of the invention inhibit tumors growth;

- The humanized antibodies of the invention target specifically the lymphatic pathway different of the classical angiogenic pathway. Hence by inhibiting lymphatic pathway, proliferation and/or migration of tumor cells (metastases) are prevented or inhibited by the antibodies of the invention.

The present invention also relates to a polynucleotide encoding a variable heavy chain (VH) for a humanized anti-VEGFC antibody according to the invention and a variable light chain (VL) for an anti- VEGFC antibody according to the invention, or both a variable heavy chain (VH) and a variable light chain (VL) for a humanized monoclonal anti- VEGFC antibody according to the invention. The invention also relates to a combination of two polynucleotides encoding a variable heavy chain (VH) and a variable light chain (VL) for a humanized monoclonal anti- VEGFC antibody according to the invention.

The term “polynucleotide” refers to a polymeric form of nucleotides, either deoxyribonucleotides or ribonucleotides, or analogues thereof.

The man skilled in the art is able to define the polynucleotide sequence corresponding to each amino acid sequence of heavy or light variable chain disclosed above.

In a particular embodiment, the table 6 discloses the nucleotididic sequences encoding variable heavy chain (V _H ) and variable light chain (VL) for the murine chimeric antibody and for the humanized anti- VEGFC antibodies illustrated in the following examples. Table 6

In a particular embodiment, the polynucleotide or combination of two polynucleotides encoding a variable heavy chain (VH) for a humanized anti-VEGFC antibody and a variable light chain (VL) for an anti- VEGFC antibody according to the invention, is selected in the group consisting of: a polynucleotide or combination of two polynucleotides comprising a sequence of SEQ ID NO: 162 encoding for a heavy chain variable region (VH) and a sequence of SEQ ID NO:163 encoding a light chain variable region (VL); a polynucleotide or combination of two polynucleotides comprising a sequence of SEQ ID NO:164 encoding for a heavy chain variable region (VH) and a sequence of SEQ ID NO:163 encoding a light chain variable region (VL); a polynucleotide or combination of two polynucleotides comprising a sequence of SEQ ID NO:165 encoding for a heavy chain variable region (VH) and a sequence of SEQ ID NO: 163 encoding a light chain variable region (VL); a polynucleotide or combination of two polynucleotides comprising a sequence of SEQ ID NO:166 encoding for a heavy chain variable region (VH) and a sequence of SEQ ID NO: 163 encoding a light chain variable region (VL); a polynucleotide or combination of two polynucleotides comprising a sequence of SEQ ID NO:167 encoding for a heavy chain variable region (VH) and a sequence of SEQ ID NO:163 encoding a light chain variable region (VL); a polynucleotide or combination of two polynucleotides comprising a sequence of SEQ ID NO:168 encoding for a heavy chain variable region (VH) and a sequence of SEQ ID NO:163 encoding a light chain variable region (VL); a polynucleotide or combination of two polynucleotides comprising a sequence of SEQ ID NO:169 encoding for a heavy chain variable region (VH) and a sequence of SEQ ID NO:163 encoding a light chain variable region (VL); a polynucleotide or combination of two polynucleotides comprising a sequence of SEQ ID NO: 170 encoding for a heavy chain variable region (VH) and a sequence of SEQ ID NO: 163 encoding a light chain variable region (VL); a polynucleotide or combination of two polynucleotides comprising a sequence of SEQ ID NO: 171 encoding for a heavy chain variable region (VH) and a sequence of SEQ ID NO:163 encoding a light chain variable region (VL); preferably a polynucleotide or combination of two polynucleotides comprising a sequence of SEQ ID NO:162 encoding for a heavy chain variable region (VH) and a sequence of SEQ ID NO:163 encoding a light chain variable region (VL); a polynucleotide or combination of two polynucleotides comprising a sequence of SEQ ID NO:164 encoding for a heavy chain variable region (VH) and a sequence of SEQ ID NO:163 encoding a light chain variable region (VL); a polynucleotide or combination of two polynucleotides comprising a sequence of SEQ ID NO:165 encoding for a heavy chain variable region (VH) and a sequence of SEQ ID NO: 163 encoding a light chain variable region (VL); and a polynucleotide or combination of two polynucleotides comprising a sequence of SEQ ID NO:169 encoding for a heavy chain variable region (VH) and a sequence of SEQ ID NO:163 encoding a light chain variable region (VL).

Another object of the invention is an expression vector comprising the polynucleotide or combination of two polynucleotides encoding a humanized antibody or a fragment thereof according to the invention.

The vectors can be viral vectors such as bacteriophages or non-viral such as plasmids.

The present invention also concerns a non-human host cell transformed with a polynucleotide ora combination of two polynucleotides suitable forexpressing a humanized anti-VEGFC according to the invention.

The invention also relates to a host cell comprising the polynucleotide or a combination of two polynucleotides encoding a humanized antibody or a functional fragment thereof according to the invention ora vector comprising the polynucleotide or a combination of two polynucleotides.

Another object of the invention is a method of producing a humanized anti-VEGFC antibody according to the invention comprising: culturing the non-human host cell of the invention transformed with a polynucleotide according to the invention or a combination of two polynucleotides suitable for expressing an anti-VEGFC according to the invention, in particular non-human fibroblasts, under suitable conditions and recovering the humanized anti-VEGFC antibody from the culture medium or from the said cultured cells.

Combination

Another object of the invention is a combination of a humanized monoclonal antibody anti- VEGFC according to the invention and an anti-angiogenic compound.

By "combination", it refers to simultaneous (concurrent) or consecutive administration in any order. The combined administration includes co- administration, using separate formulations or a single pharmaceutical formulation or composition, and consecutive administration in any order. Thanks to the present invention, the subject receiving the combined treatment will be completely treated (cured), i.e., the subject will survive to the cancer [beneficial impact on the "overall survival" (OS)], or will have a much longer disease- free survival (DFS) or metastasis free survival chance than a subject who does not receive said combined treatment.

As used herein, the term “anti-angiogenic compound” refers to any compound that inhibits the development of a pathological vascular network. In particular, the anti-angiogenic compound can inhibit receptors of pro-angiogenic factors like VEGF receptors and receptors of CXCL/ELR+ chemokines (e.g., CXCR1 and CXCR2).

In particular, the anti-angiogenic compound is selected from the group consisting of:

- Antibodies anti-VEGFA, like bevacizumab, aflibercept (particularly for the treatment and/or prevention of cancers, in particular colorectal cancer);

Inhibitors of receptors involved in angiogenesis including VEGFR1, 2, 3, CSFR, PDGFR, like sunitinib, sorafenib, axitinib, cabozantinib, pazopanib, lenvatinib, regorafenib;

Inhibitors of m-TOR, like everolimus, temsirolimus; - And mixtures thereof.

In a particular embodiment, the anti-angiogenic compound is selected from the group consisting of antibodies anti-VEGF distinct from the anti-VEGFC humanized antibody of the invention, inhibitors of receptors involve in angiogenesis including VEGFR1, 2, 3, CSFR, PDGFR, inhibitors of m-TOR, preferably an anti-VEGF antibody distinct from the anti- VEGFC antibody of the invention. In a particular embodiment, the anti-angiogenic compound is an anti-VEGF antibody distinct from the humanized anti-VEGFC antibody of the invention.

In a particular embodiment, the anti-VEGF antibody distinct from the humanized anti- VEGFC antibody of the invention is selected from the group consisting of bevacizumab and aflibercept.

In a particular embodiment, the combination may further comprise another anti-angiogenic compound selected from inhibitors of receptors involved in angiogenesis including VEGFR1 , 2, 3, CSFR, PDGFR, in particular selected from the group consisting of sunitinib, sorafenib, axitinib, carbozantinib, pazopanib, lenvatinib, and regorafenib.

The combination may also comprise at least another compound of interest (e.g. an anti tumor agent, an anti-inflammatory compound). As used herein, the term “anti-tumor agent” refers to any compound that prevents tumor growth or promotes tumor shrinking. Anti-tumor agents can be anti-angiogenic compounds, DNA intercalators/ Cross-linkers (like oxaliplatin, mitoxantrone), DNA synthesis inhibitors (like cytosine b-D-arabinofuranoside, 5-Fluorouracil), DNA-RNA Transcription Regulators (doxorubicin, actinomycin D), Microtubule Inhibitors (paclitaxel, nocodazole).

As used herein, the term “anti-inflammatory compound” refers to any compound that reduces inflammation. In particular, anti-inflammatory compound can be corticoid and non steroid anti-inflammatory agent ibuprofen derivative. So, the invention also relates to a combination product, which comprises:

- a humanized anti-VEGFC antibody or a functional fragment thereof according to the invention;

- an anti-angiogenic compound; and

- optionally another compound selected from anti-tumor compound and anti- inflammatory compound for simultaneous, separate or sequential use as a medicament.

Pharmaceutical composition Another object of the invention is a pharmaceutical composition comprising a humanized anti-VEGFC antibody according to the invention ora combination according to the invention and an excipient, carrier, and/or diluent. Such humanized antibody or combination can be present in the pharmaceutical composition or medicament according to the invention in a therapeutically effective amount (active and non-toxic amount). A therapeutically effective amount refers to that amount of compound which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the amount therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The amount ratio of toxic to therapeutic effects is the therapeutic index and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. For example, the antibody according to the invention, can be administered to a patient, in particular intravenously, in an amount, within the range from 0.1 pg/kg to 100 mg/kg, particularly from 1 mg/kg to 50 mg/kg of body weight of said patient at least every month, particularly at least every three weeks, more particularly at least every two weeks. In the context of the invention, the humanized anti-VEGFC antibody, or functional fragment thereof, is administered to the subject in a therapeutically effective amount [amount effective to reduce the number of cancer cells, kill cancer cells, reduce the tumor volume/size, or inhibit tumor growth, in particular when the anti-VEGFC humanized antibody is used in combination with the conventional treatment] or prophylactically effective amount [amount effective to reduce or suppress lymphatic vessels formation or development and/or reduce or prevent the occurrence of new metastasis(es) or the development of existing metastasis(es)], typically at a concentration range from about 2 to 20 mg/kg of body weight, preferably from about 5 to 15 mg/kg of body weight, for example 6, 7, 8, 9, 10, 11, 12, 13 or 14 mg/kg of body weight, preferably 10 mg/kg of body weight.

The pharmaceutical composition according to the invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, rectal means or ocular.

In addition to the active ingredients, the pharmaceutical composition of the invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. In particular, the pharmaceutical composition according to the invention is formulated in a pharmaceutical acceptable carrier. Pharmaceutical acceptable carriers are well known by one skilled in the art. Further details on techniques for formulation and administration may be found in the latest edition of Remington’s Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.). In particular, the pharmaceutical acceptable carrier comprises or is an isotonic solution.

A further object herein described is a kit or composition, comprising (i) a conventional treatment, and (ii) a humanized anti-VEGFC antibody, or functional fragment thereof as herein described.

A "conventional treatment of cancer" may be selected from a chemotherapy, a radiotherapy, an hormonotherapy, an immunotherapy, a specific kinase inhibitor-based therapy, an antiangiogenic compound based-therapy, an antibody-based therapy, in particular a monoclonal antibody-based therapy, and surgery.

In particular, the conventional treatment comprises an anti-angiogenic compound.

Such anti-angiogenic compounds are disclosed above.

Uses

The present invention also relates to a humanized monoclonal antibody as defined in the invention, or a combination of the invention or a pharmaceutical composition of the invention, for use in therapy or diagnosis, in particular in the treatment of cancers or disorders characterized by undesirable lymphatic endothelial cell migration and/or proliferation, or in an in vitro method for diagnosing or predicting the risk to relapse and/or to develop a tumor metastasis in a subject treated by its conventional treatment comprising an anti-angiogenic compound

Therapeutic use

The present invention also relates to a humanized antibody as defined in the invention, or a combination of the invention or a pharmaceutical composition of the invention, for use as a medicament.

In the context of the present invention, the patient or subject is a mammal. The mammal may be a primate, particularly a human being (also herein identified as "human"). In a preferred embodiment, the mammal is a human being, whatever its age or sex.

In a particular embodiment, the subject has a tumor or cancer, typically a malignant tumor, in particular a metastatic (malignant) tumor, or a pathological characterized by exacerbated lymphangiogenesis and/or VEGFC-dependent fibrosis. In the context of the present invention, the cancer or "malignant tumor" may be any kind of cancer or neoplasia, typically metastatic cancer or neoplasia. The cancer or tumor can be selected from a carcinoma, a sarcoma, and a. The cancer is preferably selected from a renal or kidney cancer, preferably a renal cancer carcinoma (RCC), in particular a clear cell renal cancer carcinoma (ccRCC); a breast cancer; ovarian cancers, lung cancers, head and neck cancer, pancreatic cancers and colon cancers. In a particular embodiment, the cancer is a renal cancer carcinoma (RCC), in particular a clear cell renal cancer carcinoma (ccRCC).

In a particular embodiment, the said antibody or the said combination or the said pharmaceutical composition are used in the treatment of cancers or disorders characterized by undesirable lymphatic endothelial cell migration and/or proliferation, in particular renal cancer carcinoma (RCC), breast cancer, head and neck cancers, , retinopathies, arthritis or rheumatoid arthritis, preferably renal cancer carcinoma (RCC).

The terms "treat", "treating" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the aim is to prevent or ameliorate cancer or prevent or slow down cancer progression or metastases appearance, development or multiplication. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

The terms "preventing", "prevention", "preventative" or "prophylactic" refers to keeping from occurring, or to hinder, defend from, or protect from the occurrence of a condition, disease, disorder, or phenotype, including an abnormality or symptom. A subject in need of prevention may be prone to develop the condition.

By ‘disorders characterized by undesirable lymphatic endothelial cell migration and/or proliferation’, it means in particular disorders selected from the group consisting of: cancers with abnormal angiogenesis, in particular solid tumors with abnormal angiogenesis, more particularly renal cancers including clear cell renal cell carcinoma, breast cancers, head and neck cancers, ovarian cancers, lung cancers, pancreatic cancers and colon cancers; ophthalmological diseases with abnormal angiogenesis, in particular age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa and uveitis; rheumatoid arthritis;

Particularly, said pathological angiogenesis disease is a cancer, and preferably a clear cell renal cell carcinoma. In a particular and preferred embodiment, the said humanized antibody or the said combination or the said pharmaceutical composition are used in a subject who is relapsed from or refractory to its conventional treatment, and/or who is developing or is at risk of developing tumor metastasis.

In a particular embodiment of the present invention, the subject is typically a subject undergoing a treatment of cancer, in particular a conventional treatment of cancer (for example chemotherapy and/or radiotherapy). The term "conventionally" means that the therapy is applied or, if not routinely applied, is appropriate and at least recommended by health authorities. The "conventional" treatment is selected by the cancerologist depending on the specific cancer to be prevented or treated.

In the context of the present invention, a "conventional treatment of cancer" may be selected from a chemotherapy, a radiotherapy, a hormonotherapy, an immunotherapy, a specific kinase inhibitor-based therapy, an antiangiogenic compound based-therapy, an antibody- based therapy, in particular a monoclonal antibody-based therapy, and surgery.

The invention also relates to a method of treatment of diseases characterized by undesirable lymphatic endothelial cell migration and/or proliferation, said method comprises the step of administering to a patient in need thereof a therapeutically or prophylactic amount of at least one humanized anti-VEGFC antibody or a functional fragment thereof according to the invention, a combination according to the invention or a pharmaceutical composition according to the invention, as disclosed above.

Diagnosis and/or prognosis uses and dedicated kits

• Risk to relapse and/or to develop a tumor metastasis

Another object of the invention is an in vitro method for diagnosing or predicting the risk to relapse and/or to develop a tumor metastasis in a subject treated by its conventional treatment comprising an anti-angiogenic compound, characterized in that said method comprises a step of contacting a biological sample of said subject with a humanized anti- VEGFC antibody of the invention, it being possible for said antibody to be, if necessary, labelled.

In a particular embodiment, the subject is affected by a cancer or a disease related to undesirable lymphatic cell proliferation and/or migration and is treated by a conventional treatment. Such conventional treatments are disclosed above. In a more particular embodiment, the conventional treatment comprises an anti-angiogenic treatment, in particular an anti-VEGF antibody distinct from a humanized anti-VEGFC antibody according to the invention.

In particular, the method comprises a step of determining the expression and/or the level of expression of at least one human proteolytically cleaved form of VEGFC in a biological sample, by detecting and/or quantifying the binding of said antibody according to the invention with the mature form of VEGFC that specifically binds to VEGF-R3 expressed on lymphatic endothelial cells. So, the invention also relates to an in vitro method for diagnosing or predicting the risk to relapse and/or to develop a tumor metastasis in a subject treated by its conventional treatment comprising an anti-angiogenic compound, said method comprising: i) determining the expression and/or the level of expression of at least one human proteolytically cleaved form of VEGFC in a biological sample of said subject using at least one humanized anti-VEGFC antibody and/or a functional fragment thereof according to the invention; ii) comparing said level of expression determined at step i) to a control level and determining if said subject has a risk to relapse and/or to develop a tumor metastasis.

For example, step i) can be performed by an ELISA assay, wherein the humanized anti- VEGFC antibody or a functional fragment thereof of the invention is immobilized on a microtiter plate, said plate being thereafter incubated with at least one labelled secondary antibody directed proteolytically cleaved form of VEGFC, which recognize the antibody and/or a functional fragment thereof according to the invention, in appropriate conditions well-known in the art.

In a preferred embodiment, the method of the invention comprises the step ii) of comparing said level of expression determined at step i) to a control level and determining if said level of expression determined at step i) is significantly higher than said control level; said significantly higher level of expression indicates that the subject is has a risk to relapse and/or to develop a tumor metastasis; wherein said control level is the expression level of said proteolytically cleaved form of VEGFC determined in at least a biological sample from an healthy subject, or from a subject who is not affected with a disease related to undesirable lymphatic cell proliferation and/or migration or a cancer.

The term "biological sample" encompasses a variety of sample types obtained from an organism that may be used in a diagnostic or prognostic assay. The term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen, or tissue cultures or cells derived there from and the progeny thereof. Additionally, the term may encompass circulating tumors or other cells. The term specifically encompasses a clinical sample, and further includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, biological fluids including aqueous humour and vitreous for eyes samples, and tissue samples. The term also encompasses samples that have been manipulated in any way after procurement, such as treatment with reagents, solubilisation, or enrichment for certain components.

Advantageously, the biological sample can be selected from the group comprising a bodily fluid, a fraction thereof, tissue extract and, cell extract. Particularly the biological sample can be selected from the group comprising plasma sample and tumor extract.

The method for detecting in a sample the proteolytically cleaved form of VEGFC that specifically binds to VEGF-R3 according to the invention can be based on various techniques, well known by one skilled in the art, including, but not limited to:

- a western blot assay (the proteolytically cleaved form of VEGFC present in a cell lysate or in a solution being immobilized on a membrane, the said membrane being thereafter incubated with the antibody of the invention, preferably labelled, in appropriate conditions well-known in the art),

- an ELISA assay (the proteolytically cleaved form of VEGFC being immobilized on a microtiter plate, the said plate being thereafter incubated with the antibody of the invention, preferably labelled, in appropriate conditions well-known in the art),

- an immunohistochemistry assay (the recombinant antibody, preferably labelled, being used to stain a sample containing fixed cells or tissues expressing the VEGFC),

- a flow cytometry assay (the recombinant antibody, preferably labelled, being used to stain a sample containing fixed or living cells expressing the VEGFC, in appropriate conditions well-known in the art).

In one embodiment of the method for detecting in a sample a proteolytically cleaved form of VEGFC according to the invention, the antibody of the invention is coated on a solid support. These detection techniques are well-described in Sambrook, Fritsch and Maniatis -

“Molecular Cloning - A Laboratory Manual” Second Edition Cold Spring Harbor Laboratory, 1989. Any other detection techniques requiring the use of an antibody are herein encompassed. The presence and eventually the amount of said cells expressing proteolytically cleaved form of VEGFC in said sample can be determined thanks to these techniques. Some of these techniques require labelling the antibody of the invention with a detectable marker, preferably a fluorescent or a luminescent marker, as disclosed above. Such in vitro method permits to identify and/or select subjects (patients) that may benefit of a combined therapy comprising a humanized anti-VEGFC antibody of the invention and anti-angiogenic compound.

• Diagnostic and/or prognostic method of a disease related to undesirable lymphatic cell proliferation and/or migration

The invention also relates to in vitro or ex vitro diagnostic and/or prognostic method of a disease related to undesirable lymphatic cell proliferation and/or migration, in particular of clear cell renal cell carcinoma, in a subject, said method comprising: i) determining the expression and/or the level of expression of at least one human proteolytically cleaved form of VEGFC in a biological sample of said subject using at least one humanized anti-VEGFC antibody and/or a functional fragment thereof according to the invention; ii) comparing said level of expression determined at step i) to a control level and determining if said subject is affected with a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly with a clear cell renal cell carcinoma.

In particular, step i) can be performed by further using at least one labelled secondary antibody directed to proteolytically cleaved form of VEGFC that recognizes the humanized anti-VEGFC antibody and/or a functional fragment thereof of the invention.

For example, step i) can be performed by an ELISA assay, wherein the humanized anti-VEGFC antibody or a functional fragment thereof of the invention is immobilized on a microtiter plate, said plate being thereafter incubated with at least one labelled secondary antibody directed proteolytically cleaved form of VEGFC, which recognize the humanized antibody and/or a functional fragment thereof according to the invention, in appropriate conditions well-known in the art.

In a preferred embodiment, the method of the invention comprises the step ii) of comparing said level of expression determined at step i) to a control level and determining if said level of expression determined at step i) is significantly higher than said control level; said significantly higher level of expression indicates that the subject is affected with a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly with clear cell renal cell carcinoma; wherein said control level is the expression level of said proteolytically cleaved form of VEGFC determined in at least a biological sample from an healthy subject, or from a subject who is not affected with a disease related to undesirable lymphatic cell proliferation and/or migration, particularly with clear cell renal cell carcinoma. • In vitro method to determine a bad outcome of a disease related to undesirable lymphatic cell proliferation and/or migration

The invention also relates to in vitro or ex vitro method to determine a bad outcome of a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma, in a subject, said method comprising: a) determining the expression and/or the level of expression of at least one human proteolytically cleaved form of VEGFC in a biological sample of said subject using at least one antibody and/or a functional fragment thereof according to the invention. The method of the invention can further comprise the step b) of comparing said level of expression determined at step a) to a control level and determining if said subject is or not undergoing a bad outcome of a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of a clear cell renal cell carcinoma.

Suitable “control level” include the expression level of said proteolytically cleaved form of VEGFC determined in at least one reference sample and a reference threshold value.

A “reference sample” is a biological sample from a subject with a known state of disease related to undesirable lymphatic cell proliferation and/or migration, more particularly a known clear cell clear cell renal cell carcinoma state, or from a healthy subject, or from a subject who is not affected with a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly with clear cell renal cell carcinoma.

Preferably, the control level is the mean expression levels of said proteolytically cleaved form of VEGFC determined in several reference samples, from several subjects.

Preferably, the reference sample is the same type of biological sample (i. e. a biological sample of corresponding physiological nature) than said biological sample of step i) or a).

In a preferred embodiment, the method of the invention comprises the step b) of comparing said level of expression determined at step a) to a control level and determining if said level of expression determined at step a) is significantly higher than said control level; said significantly higher level of expression indicates a bad outcome of disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma in said subject; wherein said control level is the expression level of said proteolytically cleaved form of VEGFC determined in at least a biological sample from a healthy subject, or from an subject who is not affected with a disease related to undesirable lymphatic cell proliferation and/or migration,, particularly with a pathological angiogenesis disease, more particularly with clear cell renal cell carcinoma.

Suitable “control level” include the expression level of said proteolytically cleaved form of VEGFC determined in the same type of biological sample of a healthy subject, of a subject who is not affected with a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly with clear cell renal cell carcinoma.

Preferably, the control level is the mean expression levels of said proteolytically cleaved form of VEGFC determined in the same type of biological sample of several healthy subjects, of several subjects who are not affected with a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly with clear cell renal cell carcinoma.

Said significant higher level of expression of said proteolytically cleaved form of VEGFC can corresponds to an increase of expression of at least more than 10 %, more than 15 %, more than 20 %, more than 25 %, more than 30 %, preferably more than 35 % of said control level.

Such a reference threshold value may vary depending on the type of tested biological sample and the method used for determining the level of expression of said proteolytically cleaved form of VEGFC. However, for particular experimental conditions (same type of tested biological sample, same method for determining the level of expression of said at least one chemokine), said threshold value may be determined based on a reference pool of patients comprising both a population of patients with stable disease related to undesirable lymphatic cell proliferation and/or migration, more particularly stable clear cell renal cell carcinoma (alive patients) and a population of patients undergoing a bad outcome of disease related to undesirable lymphatic cell proliferation and/or migration, in particular clear cell renal cell carcinoma (deceased patients). By measuring the levels of expression of said proteolytically cleaved form of VEGFC of these patients, a reference threshold value T can be determined by the following features: all or most reference patients with stable disease related to undesirable lymphatic cell proliferation and/or migration, more particularly stable clear cell renal cell carcinoma (alive patients) have at least proteolytically cleaved form of VEGFC expression level values inferior to T; and all or most reference patients undergoing a bad outcome of disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma (deceased patients) have at least proteolytically cleaved form of VEGFC expression level values superior to T.

In this case, if said level of expression determined at step i) is significantly higher than said threshold value T, it indicates a bad outcome of disease related undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma in said subject.

The term "determination" as used herein may mean both qualitatively detecting and quantifying. The term "expression" generally refers to the process by which a polynucleotide sequence undergoes successful transcription and translation such that detectable levels of the amino acid sequence or protein are expressed. In certain context herein, expression refers to the production of mRNA. In other contexts, expression refers to the production of protein or fragments thereof. The functional fragments may be produced via enzymatic cleavage or biological processes characteristic of normal or diseased conditions.

Any of a variety of known methods may be used for detection of said proteolytically cleaved form of VEGFC in said biological sample of said individual, including, but not limited to, immunoassay, using antibody or a functional fragment thereof according to the invention, e.g., by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and the like.

• Kit for carrying diagnostic and/or prognostic methods

The invention also relates to a kit for carrying out the in vitro method for diagnosing or predicting the risk to relapse and/or to develop a tumor metastasis in a subject treated by its conventional treatment comprising an anti-angiogenic compound, said kit comprises at least one humanized anti-VEGFC antibody and/or a functional fragment thereof according to the invention.

In a particular embodiment, the invention also relates to a kit for carrying out the in vitro diagnostic and/or prognostic method of a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma, in a subject, said kit comprises at least one humanized anti-VEGFC antibody and/or a functional fragment thereof according to the invention.

The said kits may provide additional components that are useful in methods of the invention, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, means for detection, control samples, standards, instructions, and interpretive information for determining if a subject is affected with a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma.

The kit can also comprise: at least one reagent for detecting said humanized anti-VEGFC antibody or a functional fragment thereof according to the invention.

• Personalized treatment

The invention also relates to a method comprising: G) diagnosing or predicting the risk to relapse and/or to develop a tumor metastasis in a subject treated by its conventional treatment comprising an anti-angiogenic compound, in particular according to the method of the invention disclosed above; and ii”) treating subject having the risk to relapse and/or to develop a tumor metastasis in a subject treated by its conventional treatment comprising an anti-angiogenic compound, with a pharmaceutical composition or combination of the invention as disclosed above.

In particular, said method comprises:

G) diagnosing or predicting the risk to relapse and/or to develop a tumor metastasis in a subject treated by its said conventional treatment, comprising the determination of the expression and/or the level of expression of at least one human proteolytically cleaved form of VEGFC in a biological sample of said subject, and ii”) treating a subject having the risk to relapse and/or to develop a tumor metastasis in a subject treated by its conventional treatment comprising an anti-angiogenic compound, with a pharmaceutical composition or combination of the invention, comprising the administration of at least an humanized anti-VEGFC antibody or a functional fragment thereof according to the invention; or a combination of said humanized anti-VEGFC antibody with at least an anti-angiogenic compound and optionally another compound selected from anti-tumor compound and anti-inflammatory compound, for simultaneous, separate or sequential administration.

The invention also relates to a method comprising:

I”) diagnosing a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma, in a subject, in particular according to the in vitro diagnostic method of the invention; and ii”) treating subject affected with a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma, in a subject, with a pharmaceutical composition or combination of the invention as disclosed above.

In particular, said method comprises: i") diagnosing a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma, in a subject, comprising the determination of the expression and/or the level of expression of at least one human proteolytically cleaved form of VEGFC in a biological sample of said subject; and ii”) treating a subject affected with a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma, with a pharmaceutical composition or combination of the invention, comprising the administration of at least a humanized anti-VEGFC antibody or a functional fragment thereof according to the invention; or a combination of said humanized anti-VEGFC antibody with at least an anti-angiogenic compound and optionally another compound selected from anti-tumor compound and anti-inflammatory compound, for simultaneous, separate or sequential administration.

The invention also relates to a method comprising:

G”) determining a bad outcome of a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma in a subject, in particular according to the method of the invention; li’”) treating a subject who is undergoing a bad outcome of a disease related to disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma, with a pharmaceutical composition or combination of the invention, comprising the administration of at least a humanized anti-VEGFC antibody or a functional fragment thereof according to the invention; or a combination of said humanized anti-VEGFC antibody with at least an anti-angiogenic compound and optionally another compound selected from anti-tumor compound and anti-inflammatory compound, for simultaneous, separate or sequential administration.

In particular, said method comprises:

G”) determining a bad outcome of a disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma in a subject, comprising the determination of the expression and/or the level of expression of at least one human proteolytically cleaved form of VEGFC in a biological sample of said subject; and li’”) treating a subject who is undergoing a bad outcome of a disease related to disease related to undesirable lymphatic cell proliferation and/or migration, more particularly of clear cell renal cell carcinoma, with a pharmaceutical composition or combination of the invention, comprising the administration of at least a humanized antibody or a functional fragment thereof according to the invention; or a combination of said humanized antibody with at least an anti-angiogenic compound and optionally another compound selected from anti-tumor compound and anti-inflammatory compound, for simultaneous, separate or sequential administration. BRIEF DESCRIPTION OF THE FIGURES Figure 1 : The VEGFC sequence

Figure 2: Flow chart of the selection of anti-VEGFC hybridomas.

Figure 3: Isotyping of the different antibodies

Figure 4: Effect of antibodies 1 E9 on the phosphorylation of VEGFR3 and VEGFR2 in endothelial cells: ELISA assay forVEGFR3 (a) or immunobloting for VEGFR2 activation (b). Figure 5: Effect of antibodies 1E9 on the viability of vascular endothelial cells HuVEC (Figure 5a) and on the migration of lymphatic endothelial cells (LEC) (Figure 5b).

Figure 6: Effect of antibodies 1 E9 on the proliferation of kidney and breast tumor cells expressing VEGFC and its receptor/co-receptor through NRP2 signaling: effect on proliferation of kidney and breast tumor cells - Figure 6a, and proliferation curves of 786-0 and 786-0_K0_NRP2-Figure 6b.

Figure 7: The nucleotide and the protein sequences of the variable regions of the light and heavy chains of the chimeric antibodies: nucleic sequence of the variable region heavy chain of the 1 E9 antibodies (SEQ ID NO: 2); nucleic sequence of the variable region light chain of the 1E9 antibodies (SEQ ID NO: 3); amino acid sequence of the heavy chain variable region (VH) of the 1E9 antibodies (SEQ ID NO: 4) and amino acid sequence of the light chain variable region (VL) of the 1 E9 antibodies (SEQ ID NO:5).

Figure 8: Effect of antibodies 1 E9 alone or in combination with Bevacizumab (BVZ) on experimental RCC: effect on the growth (Figure 8a) and on the tumor weight (Figure 8b). Figure 9: Effect of bevacizumab on the expression of VEGFC on 786-0 and A498 cells. Figure 10: Purification profile of the 10 antibodies. Non-reduced SDS-PAGE with Coomassie blue staining. IN. Input sample. FT. Flow through. W. Wash step. E. Eluted fractions. MW. Molecular weight marker (kDa).

Figure 11 : Anti-VEGFC antibodies inhibit the metabolism of breast and kidney cancer cells. (A) breast (MDA-MB231) and (B) Kidney (786-0) cancer cells’ metabolism was assessed by XTT assays after 48 h of incubation with 10 pg/mL of purified antibodies in DM EM medium containing 1% fetal bovine serum. The results are presented as the means ± SEM * p< 0.05, ** p < 0.01 , ***p<0.001 , **** pO.0001.

Figure 12: consensus of heavy sequence of the humanized antibodies of 1E9.

The present invention will be explained with examples in the following text, but the technical scope of the present invention is not limited to these examples. EXAMPLES

Example 1 : Preparation of anti-VEGFC monoclonal antibodies

CHO expressing the VH and VL chains (cloning of the corresponding gene in pFUSE vectors (InVivoGen) are cultivated in F12 medium with 5% low IgG serum. Cells were cultivated until they reached confluency. Then medium was replaced by a production medium containing 1.25% low IgG serum. The conditioned medium is recovered 20 days after for antibody purification. Cell supernatant is loaded on a HiTrapiM Protein G HP- column (flow 0.2 to 1 ml/minute for a 5 ml column). Fixed antibodies were desorbed in the elution buffer (1 M Tris-HCI pH 2.7) and immediately neutralized in neutralizing buffer (1 M Tris pH 9), 100 mI per ml of fraction).

Cloning of VEGFC in pGEX-6P-3

The immunogen comprising the sequence coding for the mature peptide (SEQ ID NO:1) of the VEGFC that specifically stimulates VEGFR3, was subcloned in the pGEX6P3 vector. The Figure 1 presents the sequence of VEGF-C with the mature peptide in bold characters.

Production and purification of a fusion protein GST-VEGFC

This plasmid was transformed in BL21 bacteria for the production of a GST-VEGFC fusion protein.

Mice immunization, cell fusion and hybridoma screening

Mice were injected several times with the immunogen comprising the sequence coding for the mature peptide (SEQ ID NO:1) of the VEGFC.

The screening of specific hybridoma producing anti-VEGFC antibodies was based on the ability of the hybridoma to recognize the VEGFC antigen used in the immunization protocol by ELISA. The flow-chart of the experiment is described in Figure 2.

Serum from each mouse was tested by ELISA against the VEGFC antigen harvested from the mice with the strongest immune response.

Supernatants of hybridomas were screened by ELISA for the ability to bind the VEGFC antigen. Several rounds of screening were performed to ensure that only hybridomas stably producing antibodies recognizing the VEGFC antigen were selected.

Thirteen clones were selected for their ability to produce specific antibodies and the six best were amplified. Isotyping of the different antibodies

Isotyping of the six clones was performed using specific commercially available kits, and the results were presented in Figure 3. Efficacy of the different antibodies

Cell lines and culture conditions

HuVECs (Human umbilical vein endothelial cells), LECs (Lymphatic endothelial cells), 786- O, A498 and MDA-MB231 were purchased from the American Tissue Culture Collection (ATCC). 786-0_#NRP2 knock-out (KO) cells were generated in the laboratory as previously described in Dumond, A (2021). Cells were cultured as indicated by ATCC and as already described in Signoretti, S et al. (2018) and Grepin, R et al. (2014).

The different antibodies (six clones) were tested for their ability to inhibit the phosphorylation of VEGFR3 but not of VEGFR2 in endothelial cells (Figure 4), the viability of endothelial cells (HuVEC) expressing VEGFR2 (Figure 5a), the migration of lymphatic endothelial cells (LECs) expressing VEGFR3 (Figure 5b) and the proliferation of tumor cells (Figure 6) considering that aggressive cancer cells, including RCC cells, express VEGFC and their receptors (VEGFR2/3) or their co-receptors the neuropilins especially neuropilin 2. The effects of the antibodies 1 E9 that were the most potent were described in the said figures. Of note VEGFR2 and VEGFR3 can be stimulated by VEGFC.

Effect of antibodies 1E9 on the phosphorylation of VEGFR3 and of VEGFR2 in endothelial cells For 786-0 and 786-0_#NRP2-K0, cells were incubated with an irrelevant or 1 E9 antibodies (10 pg/mL) for 96h. Cell proliferation was monitored during 96h by cell counting. Results are expressed as % of day 0.

An ELISA assay for VEGFR3 (Figure 4a) or immunobloting for VEGFR2 activation (Figure 4b) were carried out. Statistical significance and P values were determined with the two tailed t test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Immunoblotting

HuVECs and LECs cells were starved for 2h and then treated for 20 min with VEGFC (100 ng/mL) in presence or not of 1E9 antibodies (10 pg/mL). Cells were lysed in Laemmli buffer containing 2% SDS, 10% Glycerol, 60mM Tris-HCI, 1X Halt™ phosphatase inhibitor cocktail (Thermo Fischer). DNA was fragmented by sonification. Lysates supplemented with 0.002% bromophenol blue and 100mM DTT were heated to 96°C, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore). Membranes were probed with the following antibobies (pVEGFR2 (Tyr1175), CST, 2478S; VEGFR2, CST, 2479S and b-actin (D6A8) CST, 8457.

ELISA assay

The production of VEGFC following bevacizumab exposure was determined in vitro in 786- O and A498 cells by ELISA using R&D systems ELISA kit. Cells were plated in 6-well plates and treated with bevacizumab (10 pg/mL) for 48h. Supernatants were collected and ELISA performed according to the manufacturer recommendations. Results are expressed as pg/mL/millions of cells. The activation of VEGF receptor 3 (VEGFR3) was performed by ELISA using Human phospho-VEGFR3 DuoSet IC ELISA, R&D systems). HuVECs and LECs cells were starved for 2h and then treated for 20 min with VEGFC (100 ng/mL) in presence or not of 1 E9 antibodies (10 pg/mL). Results are expressed as pg of phospho- VEGFR3/ pg of proteins

The results presented in Figure 4 showed that 1 E9 antibodies decreased the VEGFC- dependent phosphorylation of VEGFR3 but not VEGFR2.

Effect of antibodies 1E9 on the viability of vascular endothelia cells.

Human vascular endothelial cells (HUVEC) viability was assessed as already described using the ADAM technology (Giuliano et al. 2015). Cells were incubated during the indicated times in a medium specific for endothelial cells (Promocell) without growth factor (-ser), in the presence of 2% fetal bovine serum (-2%), in the presence of 50 ng/ml of the non- maturated form of VEGFC that can stimulate VEGFR2 and VEGFR3 (VC 50), in the presence of 10 mg/ml of 1E9 antibodies (1E9) and 50 ng/ml of VEGFC plus 10mg/ml of 1E9 antibodies (Vc 50 n 1 E9). **p<0.01.

Briefly, human endothelial cells (HuVEC) kept in low concentration of serum (0.1%) were stimulated or not with serum (2%) or VEGFC (50 ng/ml) in the presence or not of 1E9 antibodies. 1 E9 antibodies decreased the basal and VEGFC-stimulated viability of HuVEC cells (p< 0.01).

The results presented in Figure 5a showed that antibodies 1E9 inhibit the viability of vascular endothelial cells expressing VEGFC.

Effect of antibodies 1E9 on the migration of lymphatic endothelial cells (LEC).

A confluent LEC monolayer was scratched with a yellow tip. The wound closure was assessed after 10 hours in absence of growth factors (CT), in the presence of 50 ng/ml of VEGFC (VEGFC), in control condition plus 5 pg/ml of 1 E9 (1 E9), and in the presence of 5 or 10 pg/ml 1 E9 plus 50 ng/ml of VEGFC (1 E9 5 or 10 VC). ** p < 0.01. The results presented in Figure 5b showed that 1E9 antibodies inhibit the migration of lymphatic endothelial cells. Therefore, 1E9 antibodies inhibited the VEGFC-dependent migration of lymphatic endothelial cells. Effect of antibodies 1E9 on the proliferation of kidney and breast tumor cells expressing VEGFC and its receptor/co-receptor.

Several papers showed that tumor cells aberrantly overexpress VEGF and VEGFC and their receptors (VEGFR1, VEGFR2) creating autocrine proliferation loops. However, ccRCC cells do not exert these autocrine loops via VEGFR2 or VEGFR3 but depend on their respective co-receptors Neuropilin 1 and Neuropilin 2. Triple negative breast cancer cells overexpress VEGF and VEGFC but do not express VEGFR. Their proliferation greatly depends on a VEGF/VEGFC/NRP1/NRP2 autocrine proliferation loops. Considering the potent effects of the 1E9 antibodies on the VEGFC-dependent signaling, we hypothesized that they should inhibit the proliferation of tumor cells presenting such autocrine loops. Therefore, we tested the 1E9 antibodies on ccRCC (A498 and 786-0, Neuropilin 1 and 2 positive) and breast (MDA-MB231, VEGFR2 and Neuropilin 1 positive) tumor cells.

Kidney (A498, 786-0) and breast (MDA-MB231) cancer cells proliferation was assessed by MTT assays after 48h of incubation with 10pg/ml of purified 1E9 antibodies in DM EM medium containing 2% fetal bovine serum. The three independent cell lines express high amounts of VEGFC (around 1 ng/ml/10 ⁶ cells). Kidney cells only express the VEGFC co receptor neuropilin 2 and the MDA-MB231 only express the VEGFC receptor VEGFR3. *** p < 0.001.

For the proliferation curves of 786-0 and 786-0_K0_NRP2 (Figure 6b), cells were incubated with 1 E9 antibodies and counted at the indicated times. ** p < 0.01 , *** p < 0.001. The results presented in Figure 6 showed that antibodies 1E9 inhibit the proliferation of kidney and breast tumor cells expressing VEGFC and its receptor/co-receptor. At 10 pg/mL, 1 E9 antibodies decreased by 40% the proliferation of cancer cells (Figure 6a). These effects on 786-0 cell proliferation were found to be dependent on NRP2 signaling (Figure 6b). Therefore, in addition to its impact on angiogenesis/lymphangiogenesis (inhibition of survival/proliferation/migration of vascular and lymphatic endothelial cells), 1E9 antibodies have a strong impact on tumor cell proliferation.

Example 2: Preparation of chimeric antibodies

Two monoclonal antibodies produced by 1 E9 hybridoma were cloned and sequenced using techniques known to those skilled in the art. The variable regions of these 1E9 antibodies were sequenced (heavy and light chains). The CDR of these two antibodies were fused to human lgG1 light and heavy chains to obtain chimeric antibodies that can be used in the clinic. Expression vectors were transfected in CHO cells to obtain stable clones expressing the chimeric antibodies. The chimeric antibodies recognized with a high affinity the human VEGFC. In particular, the variable region of the light and heavy chain of these antibodies were sequenced and cloned in the pFUSE2ss-CLIg-hk and pFUSEss-CHIg-hG1 vectors (In vivoGen San Diego, CA 92121 - USA) respectively. The murine parts of the monoclonal antibodies were fused to the constant light and heavy chain of human lgG1 antibodies.

The vectors were transfected in CHO cells sequentially. CHO clones thank can grow in suspension and that produced the VEGFC antibodies were obtained. The yield of production is about 10mg of chimeric antibodies per liter of medium without serum. Equivalent results for anti-proliferation and anti-migration abilities were obtained with the murine monoclonal antibodies and the chimeric antibodies. The nucleotide and the protein sequences of the variable regions of the light and heavy chains are the following (Figure 7).

SEQ ID NO: 2: nucleic sequence of the variable region heavy chain SEQ ID NO: 3: nucleic sequence of the variable region light chain

SEQ ID NO: 4: amino acid sequence of the variable region heavy chain SEQ ID NO: 5: amino acid sequence of the variable region light chain

Example 3: Efficacy of anti-VEGFC antibodies on experimental models of ccRCC

Effect of antibodies 1E9 on the growth of experimental RCC.

Chimeric antibodies were used to performed in vivo experiments. For these experiments, the efficacy of anti-VEGFC antibodies alone or combined with the anti-VEGF antibodies used in the clinic (Bevacizumab/Avastin) was tested on the growth of experimental RCC in nude mice (Figure 8).

3x10 ⁶ 786-0 RCC cells were subcutaneously injected in the flank of nude mice. Tumor growth was evaluated with a caliper as already described [5] 28 days after tumor cell injection, the average tumor size in each group was determined and used as the reference tumor size before treatments (100 %). Treatments started at 28 days for mice bearing tumors ranging between 70 and 100 mm ³ in size (5 mg/kg twice a week during the indicated times). Tumor size in mm ³ was then evaluated for the indicated time points and reported to the reference size at 28 days for each tumor (10 per group). The fold increase for each tumor was reported in the figure. High and low thresholds were indicated by black (plain dotted) lines respectively. Statistics are indicated; *, p<0.05; ***, p<0.001. Effect of antibodies 1E9 alone or in combination with Bevacizumab (BVZ) on tumor weight Tumor weight at the end of the experiments was significantly lower for tumors of mice treated with the 1E9 antibodies alone or combined with anti-VEGF antibodies (Figure 8b). So, as we previously described, bevacizumab (BVZ) had a trend to accelerate tumor growth (Figure 8a) although it did not modify tumor weight at sacrifice (Figure 8b) as compared to control tumors from mice receiving irrelevant antibodies. 1E9 antibodies (1E9) decreased the growth of 786-0 xenografts (Figure 8a) and their weight (Figure 8b). The combination of 1E9 antibodies with bevacizumab (1E9 + BVZ) inhibited more efficiently tumor growth (Figure 8a). Tumor weight was significantly decreased as compared to the control condition and as compared to 1E9 antibodies alone (Figure 8b). ***, p<0.001. These results suggest that 1 E9 antibodies had a therapeutic efficacy at least on experimental models but that they also revert the detrimental effects of bevacizumab alone that we previously described.

Furthermore, Figure 9 shows that the treatment of 786-0 and A498 cells in vitro with bevacizumab for 48 h significantly increased the expression of VEGFC. *, p<0.05.

The additive effect of anti-VEGFC plus anti-VEGF antibodies was consistent with previous results showing that resistance to anti-VEGF antibodies or to tyrosine kinase inhibitors of the VEGF/VEGFR signaling was dependent on VEGFC and the subsequent development of lymphatic vessels[5, 6] .

So, the anti-VEGFC antibodies of the invention inhibit the growth of experimental kidney tumors in nude mice. Whereas anti-VEGF antibodies have no effect or even favor experimental tumor growth in this model, the anti-VEGF plus anti-VEGFC combination have an additive effect on the inhibition of tumor growth. Therefore, these antibodies may constitute a new therapeutic strategy for metastatic kidney tumors.

Although tyrosine kinase inhibitors TKI or anti-VEGF reduced transiently the size of distant metastasis, they promote at the same time the development of an alternative vessel network participating in further metastatic dissemination. It was also observed that one common denominator of the response of tumor cells to several types of treatments was the induction of VEGFC; i) platinum salts for lung cancer cells, oral squamous cell carcinoma, melanoma, MOB ii) taxanes for breast or prostate cancers; iii) radiotherapy for oral squamous cell carcinoma (OSCC) (Lupu-Plesu et al. , 2017). Hence, a bundle of evidence links tumor resistance to a VEGFC-dependent lymphangiogenesis and further metastatic dissemination. Although the experiments illustrated above are centered on RCC, anti- VEGFC antibodies of the invention combined with the reference treatment of a specific tumor, may be proposed in the first line or at relapse. In addition, bevacizumab was also approved for the treatment of breast cancers, but it lost its FDA approval for insufficient benefits. We observed that several cell lines representative of triple negative breast cancers including MDA-MB231 overexpress VEGF and VEGFC. Hence, combining anti-VEGF and anti-VEGFC appears relevant for the treatment of could triple negative breast cancer for which the VEGFC/VEGFR3/NRP2 signaling is detrimental.

Example 4: In silico humanization from 1E9 murine antibody

From the heavy and light chain sequences of 1E9 mouse antibody (SEQ ID NO:4 and SEQ ID NO: 5) with CDR defined by Kabat method (SEQ ID NO: 6 to SEQ ID NO: 11), in silico methods were used to define potential humanized antibodies sequences.

The humanization procedure was performed as outlined below:

1) Parental mouse antibody domains and regions were identified

2) Critical positions were identified: antibody Fv’s have a number of critical positions that make up the VH/VL inter chain interface or are responsible for the discrete set of canonical structures that has been defined for 5 of the CDRs (Chothia and Lesk 1987; Martin and Thornton 1996; Al Laziniki et al. 1997): these positions should be considered in detail before substitutions are proposed for them.

3) Based on the sequence analysis and the critical positions, optimal Acceptor human germline sequences were selected for each chain: based on the Parental antibody sequence alignment to the human germlines, the closest matching entries were identified. The identification of the optimal human germlines as Acceptor was based on the ordered criteria listed below:

• Sequence identity across the whole V gene (framework + CDRs)

• Identical or compatible inter-chain interface residues

• Support loops with the Parental CDRs canonical conformations

For the light chain, human germline IGKV2-30*02 and human germline IGKV2- 18*01 were selected.

For the heavy chain, four human germlines were selected: human germline IGHV1-46*01, human germline IGHV1-2*06, human germline IGHV4-34*08 and human germline IGHV4-34*09.

4) A 3D structural model of the Parental mouse Fv regions was constructed

5) Following a close inspection of the molecular model an initial assessment of the possibility to substitute each position was made: positions were categorized as Neutral Contributing or Critical. 6) The CDR-grafting was performed by analyzing positions differing between the Parental and Acceptor sequences. All substitutions in Neutral positions were performed. For the design phase of a humanized antibody, the procedure is more accurately defined as germlining - replacing amino acids in the Parental framework that differ from the chosen Acceptor with the corresponding human amino acid.

7) For the heavy chain four different human germlines, IGHV1-46*01 and IGHV1- 2*06, IGHV4-34*08 and IGHV’4-34*09 were used for the design of humanized versions. For the light chain two different human germlines IGKV2-30*02 and IGKV2-18*01 were used for the design of humanized versions. Combinations of the different humanized VH and VL versions are produced, purified and tested for binding and biological activity.

8) The selection of the best humanized heavy and light chain combination between the different versions is performed by assessing the following criteria: a) The level of transient expression of the humanized versions produced in mammalian cells (HEK 293 or preferably CHO) as human lgG1/Kappa (as compared to the chimeric version). Using tissue culture supernatant from transfected cells before harvest for purification using ELISA or measurement by protein A using Octet label-free detection systems. b) The binding capacity (EC50 by ELISA or FACS; or preferably Kd by Biacore or Octet) as compared to the chimeric human lgG1/Kappa version (chimeric meaning the combination of the parental murine VH and VL fused to human constant regions). c) The biological activity of the humanized versions in a relevant in vitro cellular assay compared with that of the reference chimeric antibody. d) The cross-reactivity with relevant orthologue species (in vitro binding activity). e) A determination of the biophysical properties of the humanized versions as compared with chimeric:

• SEC-HPLC profile to determine the level of high molecular weight aggregates,

• SDS-PAGE under non-reducing and reducing conditions,

• Analysis by differential scanning calorimetry (DSC) using Microcal™ VP capillary DSC system to determine the Tm of Fab, CH2 and CH3. The table 3 disclosed above summarizes all the heavy and light variable sequences designed according to this in silico humanization method. Example 5: Preparation of the 1E9 murine chimeric antibody (named antibody 1) and 9 “humanized” variants (named antibodies 2 to 10) of the 1E9 and evaluation of their efficiency

We previously evaluated the activity of 1 E9 by in vitro tests. More specifically we previously showed that 1 E9 inhibited the proliferation of kidney and breast tumor cells.

Therefore, we used these tests to determine among the different “humanized” variants of 1 E9 obtained by changing the residues involved in the six complementarity-determining regions but also by modifying framework residues to change from human to murine.

5-1 Construction of expression vectors

Gene Synthesis of optimized sequences has been performed for expression in CHO cells (see above). The sequences of heavy chains have been subcloned in the pTXs1-h1 expression vector(humanlgG1 backbone). The sequences of light chains have been subcloned in the pTXs1-hk (human kappa backbone).

5-2 Expression & Purification Test

An endotoxin-free DNA preparation was done for the vector constructions. Plasmids were then transiently co-transfected in Xten CHO cells (30ml_-culture in animal product-free medium). Culture medium was collected when viability dropped <50% (14 days post transfection), and recombinant antibodies were then purified on a Protein A/G resin using a standard method: i) Equilibration binding and wash with PBS ph7.5; ii) Elution with 20mM citric acid pH2.7; iii) Neutralization with 1M Tris HCI pH 9; iv) Pool of fraction of interest and buffer exchange with PBS pH 7.5 plus sterilization on 0.22 mm filtration; vi) Final sample QC qualitative SDS PAGE and quantification by Bradford. The results are presented in Figure 10.

5-3 Effect of the different humanized antibodies on the proliferation of kidney and breast tumor cells expressing VEGFC and its receptor/co-receptor.

The proliferation of kidney (786-0) and breast (MDA-MB231) cancer cells has been evaluated by MTT assays after 48h of incubation with 10pg/ml of the different purified antibodies in DM EM medium containing 1% fetal bovine serum. The two independent cell lines express important amounts of VEGFC (superior or equal to 1 ng/ml/10 ⁶ cells).

The results presented in Figure 11 showed that antibodies inhibit to different extent the proliferation of kidney and breast tumor cells expressing VEGFC and its receptor/co receptor. According to these results, the classification of the different antibodies from the less to the most efficient is the following: 10 = 9 = 7 = 6 = 5 < 8 < 4 < 3 < 2 = 1 (Control 1 E9). According to the relationship between the activity and the mutation introduced in the amino acid sequence we established the consensus of heavy sequence as represented on Figure 12. Amino acids in white color on a dark background are different from the mouse antibody. Antibodies with these “humanized” mutations keep their biological activity. Underlined amino acids are present in antibodies with or without activity.

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