Anti-VEGF / NF (LVRF) monoclonal antibodies

Abstract

The present invention relates to monoclonal antibodies or fragments thereof that bind to a splice variant of vascular endothelial growth factor (VEGF) referred to as VEGF / NF (also known as "LVRF"), and the use of same as a drug, in particular for treating cancer.

Description

ANTI-VEGF-NF MONOCLONAL ANTIBODIES (LVRF) TECHNICAL FIELD

[0001] The present invention relates to monoclonal antibodies or fragments thereof which bind to a novel splice variant of vascular endothelial growth factor (VEGF), termed VEGF-NF, and their use as a medicament, particularly in the treatment of cancer. PREVIOUS ART

[0002] Angiogenesis, the development of new vessels by proliferation and branching from existing blood vessels, plays an important role in the growth (by providing oxygen and nutrients) and metastasis of malignant tumors. The main regulator of angiogenic processes is VEGF (Vascular Endothelial Growth Factor). VEGF is actually a generic term for a family of growth factors (VEGF-A, B, C, D and PIGF for Placental growth factor) that can bind to 3 types of receptor: VEGF-R1, R2 and R3. It is the interaction of VEGF-A with VEGF-R2 expressed on the surface of endothelial cells that represents the main target of anti-angiogenic treatments.

[0003] VEGF and the VEGF receptor (VEGFR) are prime targets for cancer treatment, particularly for solid tumors. Several anti-VEGF / VEGFR therapies have emerged, such as the monoclonal antibody bevacizumab, which was the first anti-angiogenic treatment marketed in 2005. Bevacizumab binds to VEGF-A and inhibits its binding to VEGF-R1 and VEGF-R2 receptors. Neutralizing the biological activity of VEGF-A causes tumor vessels to regress, normalizes remaining tumor vessels, and inhibits the formation of new tumor vessels, thereby inhibiting tumor growth.

[0004] Although inhibitors of VEGF-A signaling activation such as bevacizumab are used successfully in the clinic, some patients do not fully respond to these treatments.

[0005] The complexity of VEGF biology may partly explain the limitations of anti-VEGF therapies such as bevacizumab. Indeed, VEGF is regulated during all processes of its expression including transcription of its gene, splicing of its pre-mRNA, stabilization of its mRNA, destabilization of its mRNA and translation [1].

[0006] Since the discovery of VEGF in 1989, several splice variants have been identified. For example, alternative splicing of VEGF pre-mRNA generates mRNAs encoding pro-angiogenic isoforms known as VEGF^ (VEGF , VEGFI65, VEGFI89 and VEGF 206 , “xxx” corresponding to the number of amino acids minus the signal peptide of each isoform).

[0007] Recently, new VEGF splice variants designated VEGF-XXX / NF, hereinafter “VEGF-NF”, have been identified [2][3]. VEGF-NF is also referred to as “LVRF” for “Lymphatic and Vascular Resistance Factor”. Rabbit and mouse polyclonal antibodies binding to these variants have also been described [2][3].

[0008] However, no monoclonal antibodies capable of specifically binding to these variants and usable in therapy have been developed to date. There is therefore a need for monoclonal antibodies that are not only capable of specifically binding to VEGF-NF, but also exhibit biological activity that allows their use in therapy to be considered.

[0009] The inventors have not only developed monoclonal antibodies capable of specifically binding to VEGF-NF, but have also shown that these monoclonal antibodies have the ability to inhibit the signaling pathway mediated by VEGF receptors (VEGFR). These monoclonal antibodies therefore exhibit biological activity that allows their use as a drug to be considered, particularly for treating cancer. SUMMARY OF THE INVENTION

[0010] A first subject of the present invention relates to a monoclonal antibody or fragment thereof which binds to VEGF-NF (vascular endothelial growth factor-NF) of sequence SEQ ID NO: 40, said antibody or fragment thereof binds specifically to an epitope included in SEQ ID NO: 35 or to an epitope included in SEQ ID NO: 36.

[0011] In a first particular embodiment, the antibody or fragment thereof comprises: - three heavy chain variable region CDRs of sequences: SEQ ID NO: 1 for CDR1 VH, SEQ ID NO: 2 for CDR2 VH, and SEQ ID NO: 3 for CDR3 VH; and - three light chain variable region CDRs of sequences: SEQ ID NO: 4 for CDR1 VL, DVS for CDR2 VL, and SEQ ID NO: 5 for CDR3 VL; said antibody or fragment thereof specifically binds to an epitope included in SEQ ID NO: 35.

[0012] In a second particular embodiment, the antibody or fragment thereof may comprise: - three heavy chain variable region CDRs of sequences: SEQ ID NO: 6 for CDR1 VH, SEQ ID NO: 7 for CDR2 VH, and SEQ ID NO: 8 for CDR3 VH; and - three light chain variable region CDRs of sequences: SEQ ID NO: 9 for CDR1 VL, KVS for CDR2 VL, and SEQ ID NO: 10 for CDR3 VL; said antibody or fragment thereof specifically binds to an epitope included in SEQ ID NO: 36.

[0013] The antibody or fragment thereof binding to the antigen according to the invention may comprise: - a FRI H heavy chain hinge region selected from the group consisting of (i) SEQ ID NO: 11 or a sequence having at least 80% identity with SEQ ID NO: 11 and (ii) SEQ ID NO: 12 or a sequence having at least 80% identity with SEQ ID NO: 12; - a FR2 H heavy chain hinge region selected from the group consisting of (i) SEQ ID NO: 13 or a sequence having at least 80% identity with SEQ ID NO: 13 and (ii) SEQ ID NO: 14 or a sequence having at least 80% identity with SEQ ID NO: 14; - a FR3 H heavy chain hinge region selected from the group consisting of (i) SEQ ID NO: 15 or a sequence having at least 80% identity with SEQ ID NO: 15 and (ii) SEQ ID NO: 16 or a sequence having at least 80% identity with SEQ ID NO: 16; and - a FR4 H heavy chain hinge region selected from the group consisting of (i) SEQ ID NO: 17 or a sequence having at least 80% identity with SEQ ID NO: 17 and (ii) SEQ ID NO: 18 or a sequence having at least 80% identity with SEQ ID NO: 18.

[0014] The antibody or fragment thereof according to the invention may comprise: - a FRI L light chain hinge region selected from the group consisting of (i) SEQ ID NO: 19 or a sequence having at least 80% identity with SEQ ID NO: 19 and (ii) SEQ ID NO: 20 or a sequence having at least 80% identity with SEQ ID NO: 20; - an FR2 L light chain hinge region selected from the group consisting of (i) SEQ ID NO: 21 or a sequence having at least 80% identity with SEQ ID NO: 21 and (ii) SEQ ID NO: 22 or a sequence having at least 80% identity with SEQ ID NO: 22; - an FR3 L light chain hinge region selected from the group consisting of (i) SEQ ID NO: 23 or a sequence having at least 80% identity with SEQ ID NO: 23 and (ii) SEQ ID NO: 24 or a sequence having at least 80% identity with SEQ ID NO: 24; and - an FR4 L light chain hinge region selected from the group consisting of (i) SEQ ID NO: 25 or a sequence having at least 80% identity with SEQ ID NO: 25 and (ii) SEQ ID NO: 26 or a sequence having at least 80% identity with SEQ ID NO: 26.

[0015] The antibody or fragment thereof according to the first particular embodiment may comprise: - a FRI H heavy chain hinge region of sequence SEQ ID NO: 11 or a sequence having at least 80% identity with SEQ ID NO: 11; - a FR2 H heavy chain hinge region of sequence SEQ ID NO: 13 or a sequence having at least 80% identity with SEQ ID NO: 13; - a FR3 H heavy chain hinge region of sequence SEQ ID NO: 15 or a sequence having at least 80% identity with SEQ ID NO: 15; - a FR4 H heavy chain hinge region of sequence SEQ ID NO: 17 or a sequence having at least 80% identity with SEQ ID NO: 17; - a FRI L light chain hinge region of sequence SEQ ID NO: 19 or a sequence having at least 80% identity with SEQ ID NO: 19; - a FR2 L light chain hinge region of sequence SEQ ID NO: 21 or a sequence having at least 80% identity with SEQ ID NO: 21; - a FR3 L light chain hinge region of sequence SEQ ID NO: 23 or a sequence having at least 80% identity with SEQ ID NO: 23; and - a FR4 L light chain hinge region of sequence SEQ ID NO: 25 or a sequence having at least 80% identity with SEQ ID NO: 25.

[0016] Preferably, the antibody or fragment according to the first particular embodiment comprises: - a heavy chain variable region (VH) of sequence SEQ ID NO: 27 or a sequence having at least 80% identity with SEQ ID NO: 27; and - a light chain variable region (VL) of sequence SEQ ID NO: 29 or a sequence having at least 80% identity with SEQ ID NO: 29.

[0017] Preferably, the antibody or fragment according to the first particular embodiment comprises (i) a heavy chain of sequence SEQ ID NO: 46 or a sequence having at least 80% identity with SEQ ID NO: 46, and (ii) a light chain of sequence SEQ ID NO: 47 or a sequence having at least 80% identity with SEQ ID NO: 47.

[0018] The antibody or fragment according to the second particular embodiment may comprise: - a FRI H heavy chain hinge region of sequence SEQ ID NO: 12 or a sequence having at least 80% identity with SEQ ID NO: 12; - a FR2 H heavy chain hinge region of sequence SEQ ID NO: 14 or a sequence having at least 80% identity with SEQ ID NO: 14; - a FR3 H heavy chain hinge region of sequence SEQ ID NO: 16 or a sequence having at least 80% identity with SEQ ID NO: 16; and - a FR4 H heavy chain hinge region of sequence SEQ ID NO: 18 or a sequence having at least 80% identity with SEQ ID NO: 18. - a FRI L light chain hinge region of sequence SEQ ID NO: 20 or a sequence having at least 80% identity with SEQ ID NO: 20; - a FR2 L light chain hinge region of sequence SEQ ID NO: 22 or a sequence having at least 80% identity with SEQ ID NO: 22; - a FR3 L light chain hinge region of sequence SEQ ID NO: 24 or a sequence having at least 80% identity with SEQ ID NO: 24; and - a FR4 L light chain hinge region of sequence SEQ ID NO: 26 or a sequence having at least 80% identity with SEQ ID NO: 26.

[0019] Preferably, the antibody or fragment according to the second particular embodiment comprises: - a heavy chain variable region (VH) of sequence SEQ ID NO: 28 or a sequence having at least 80% identity with SEQ ID NO: 28; and - a light chain variable region (VL) of sequence SEQ ID NO: 30 or a sequence having at least 80% identity with SEQ ID NO: 30.

[0020] Preferably, the antibody or fragment according to the second particular embodiment comprises (i) a heavy chain of sequence SEQ ID NO: 44 or a sequence having at least 80% identity with SEQ ID NO: 44, and (ii) a light chain of sequence SEQ ID NO: 45 or a sequence having at least 80% identity with SEQ ID NO: 45.

[0021] In particular embodiments, the antibody or fragment thereof according to the invention binds to VEGF-NF of sequence SEQ ID NO: 40 with a dissociation constant (K D ) measured by SPR technology less than or equal to 100 nM.

[0022] Preferably, the antibody or fragment thereof according to the first particular embodiment binds to VEGF-NF of sequence SEQ ID NO: 40 with a dissociation constant (K D ) measured by SPR technology less than or equal to 25 nM, preferably less than or equal to 10 nM.

[0023] Preferably, the antibody or fragment thereof according to the second particular embodiment binds to VEGF-NF of sequence SEQ ID NO: 40 with a dissociation constant (K D ) measured by SPR technology less than or equal to 75 nM, preferably less than or equal to 50 nM.

[0024] In particular embodiments, the antibody or fragment thereof according to the invention also binds to one, two, three, four, five or six VEGF-NFs of sequence selected from the group consisting of SEQ ID NO: 37, 38, 39, 41, 42 and 43, preferably to the six VEGF-NFs of sequences SEQ IDs NO: 37, 38, 39, 41, 42 and 43.

[0025] In particular embodiments, the antibody or fragment thereof according to the invention inhibits the binding between VEGF-NF and one or more VEGF receptors (VEGFR) selected from VEGFR1, VEGFR2 and VEGFR3.

[0026] In particular embodiments, the antibody or fragment thereof according to the invention (i) inhibits the binding between VEGF-NF and one or more VEGF receptors (VEGFR). In particular, the antibody or fragment thereof according to the invention (i) inhibits the binding between VEGF-NF and one or more VEGFRs selected from VEGFR1, VEGFR2 and VEGFR3, and / or inhibits the binding between VEGF-NF and one or more neuroleptins (NRPs) selected from NRP1 and NRP2.

[0027] A second subject of the invention relates to a monoclonal antibody or antibody fragment which competes with the antibody or antibody fragment according to the first subject of the invention for binding to VEGF-NF of sequence SEQ ID NO: 40.

[0028] A third subject of the present invention relates to a composition comprising an antibody or a fragment thereof according to the invention.

[0029] A fourth subject of the present invention relates to a polynucleotide or combination of two polynucleotides encoding a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody or a fragment thereof according to the invention.

[0030] A fifth subject of the present invention relates to a polynucleotide or a combination of two polynucleotides encoding a heavy chain of an antibody or a fragment thereof according to the invention and a light chain of an antibody or a fragment thereof according to the invention.

[0031] A sixth subject of the present invention relates to an expression vector comprising a polynucleotide or a combination of two polynucleotides according to the invention.

[0032] A seventh subject of the present invention relates to a cell comprising a vector according to the invention.

[0033] An eighth subject of the present invention relates to an antibody or antibody fragment according to the invention or composition according to the invention, for use as a medicament.

[0034] A ninth subject of the present invention relates to an antibody or antibody fragment according to the invention or composition according to the invention, for use in the treatment of cancer in a patient. DETAILED DESCRIPTION OF THE INVENTION

[0035] Definitions

[0036] The term "antibody", also called "immunoglobulin", refers to a heterotetramer consisting of two heavy chains of approximately 50-70 kDa each (called the H chains for Heavy) and two light chains of approximately 25 kDa each (called the L chains for Light), linked together by intra- and inter-chain disulfide bridges. Each chain consists, in the N-terminal position, of a variable region or domain, called VL for the light chain, VH for the heavy chain, and in the C-terminal position, of a constant region, consisting of a single domain called CL for the light chain and three or four domains called CHI, CH2, CH3, CH4, for the heavy chain. Each variable domain generally includes 4 "hinge regions" (called FRI, FR2, FR3, FR4) and 3 regions directly responsible for binding to the antigen, called "CDR" (called CDR1, CDR2, CDR3).The antibody may be, for example, a mammalian antibody, such as a murine antibody, a chimeric antibody, a humanized antibody or a human antibody.

[0037] By "chimeric antibody" is meant an antibody whose sequences of the variable regions of the light chains and heavy chains belong to a different species than that of the sequences of the constant regions of the light chains and heavy chains. For the purposes of the invention, the sequences of the variable regions of the heavy and light chains are preferably of murine origin while the sequences of the constant regions of the heavy and light chains belong to a non-murine species. In this respect, for the constant regions, all species of non-murine mammals are likely to be used, and in particular humans, monkeys, suids, bovids, equines, felids, canids or even birds, this list not being exhaustive.Preferably, the chimeric antibodies according to the invention contain constant region sequences of the heavy and light chains of human origin and the variable region sequences of the heavy and light chains of murine origin.

[0038] By "humanized antibody" is meant an antibody in which all or part of the sequences of the regions involved in antigen recognition (Le., the hypervariable regions or CDR: Complementarity Determining Region) and sometimes certain amino acids of the FR regions (Framework regions) are of non-human origin while the sequences of the constant regions and variable regions not involved in antigen recognition are of human origin.

[0039] By "human antibody" is meant an antibody containing only human sequences, both for the variable and constant regions of the light chains and for the variable and constant regions of the heavy chains.

[0040] A “monoclonal antibody,” as used herein, has the same meaning as commonly understood by those skilled in the art. A monoclonal antibody refers to an antibody derived from a population of antibodies whose polypeptide sequence is homogeneous. In other words, a monoclonal antibody consists of a homogeneous antibody derived from the growth of a single cell clone (e.g., a hybridoma, a eukaryotic host cell transfected with a DNA molecule encoding the homogeneous antibody, a prokaryotic host cell transfected with DNA encoding the homogeneous antibody, etc.). To produce monoclonal antibodies, antibody-producing cells (lymphocytes) may be harvested from an animal immunized with an antigen and fused with myeloma cells by standard somatic cell fusion procedures, thereby immortalizing these cells and producing 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 for producing human monoclonal antibodies (Roder et al., 1986), and the screening of combinatorial antibody libraries (Huse et al., 1989).

[0041] Hybridoma cells can be screened immunochemically for the production of antibodies specifically reactive with the target polypeptide(s) so that only monoclonal antibodies binding to said polypeptide(s) are isolated.

[0042] Moreover, unlike polyclonal antibody preparations, each monoclonal antibody is directed against a single epitope of an antigen.

[0043] An "antigen-binding antibody fragment" is any part of an immunoglobulin obtained by digestion enzymatic, obtained by genetic engineering or obtained by bioproduction comprising at least one disulfide bridge and which binds to the antigen recognized by the whole antibody. This can be, for example, a Fab, Fab', F(ab')2, Fab'-SH fragment. Enzymatic digestion of immunoglobulins by papain generates two identical fragments, called Fab fragments (Fragment antigen binding), and an Fc fragment (Fragment crystallizable). Enzymatic digestion of immunoglobulins by pepsin generates an F(ab')2 fragment and an Fc fragment split into several peptides. F(ab')2 is formed from two Fab' fragments linked by interchain disulfide bridges. The Fab parts consist of the variable regions and the CH1 and CL domains. The Fab' fragment consists of the Fab region and a hinge region. Fab'-SH refers to a Fab' fragment in which the cysteine ​​residue of the hinge region carries a free thiol group.

[0044] The term "affinity" refers to the strength of all non-covalent interactions between a molecule, for example an antibody or antibody fragment, and the recognized antigen, for example an antigen such as G alpha protein. Affinity is usually represented by the dissociation constant (K D ). The dissociation constant (K D ) can be measured by well-known methods, for example SPR (Surface Plasmon Resonance). An implementation of the SPR method (BIACORE™) for measuring the affinity of an antibody or a fragment thereof binding to the antigen is described in Examples 3 and 4.

[0045] For the purposes of the present invention, "identity" is calculated by comparing two aligned sequences in a comparison window. The alignment of the sequences makes it possible to determine the number of positions (nucleotides or amino acids) in common for the two sequences in the comparison window. The number of positions in common is therefore divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percentage identity. The percentage identities referred to in the present invention are determined on the basis of a global alignment of sequences to be compared, i.e. on an alignment of sequences taken in their entirety over their entire length, using any algorithm well known to those skilled in the art, for example the algorithm of Needleman and Wunsch 1970.This sequence comparison can be performed using any software well known to those skilled in the art, for example using the Needle software 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 ebi.ac.uk website worldwide, under the name "Align". Other methods for determining the percentage of identity can also be used, for example those described in the following references: Computational Molecular Biology, Lesk AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and genome Projects, Smith DW, ed., Academic Press, New York. 1993; Computer Analysis of sequence Data, Part I, Griffin AM, and Griffin HG, eds., Humana Press. New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov M. and Devereux J., eds., M Stockton Press, New York, 1991; and Carillo H., and Lipman D„ SIAM J. Applied Math., 48: 1073 (1998). Methods for determining identity are designed to give the greatest match between the sequences tested.Additionally, methods for determining identity are codified in publicly available computer programs. Computer program methods for determining the identity between two sequences include, but are not limited to, GCG software (Devereux J. et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and PASTA (Altschul SF et al., J. Molec. Biol. 215:403-410 (1990)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul S. et al., NCBI NLM NUH Bethesda, MD 20894; Altschul S. et al., J. Mol Biol. 215:403-410 (1990)).

[0046] When a sequence of an antibody or antibody fragment according to the invention has an amino acid sequence which is not 100% identical to one of those described in the sequence listing (reference sequences) but which has at least 80% identity with such a reference sequence, it may have insertions, deletions or substitutions with respect to the reference sequence.

[0047] For the purposes of the invention, "at least 80% identity" means at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99%. Thus, in particular embodiments according to the invention, the term “at least 80% identity” corresponds to at least 81% identity, at least 82% identity, at least 83% identity. At least 84% identity. At least 85% identity. At least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99%

[0048] When a sequence has at least 80% identity with a reference sequence, it may have, for example, at least 1 insertion, at least 1 deletion and / or at least 1 substitution with respect to the reference sequence. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 insertions, substitutions and / or deletions with respect to the reference sequence. Thus, in particular embodiments according to the invention, the term “at least 80% identity” corresponds to at least 1 insertion, at least 1 deletion and / or at least 1 substitution with respect to the reference sequence. For example, the term “at least 80% identity” may correspond to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 insertions, substitutions and / or deletions with respect to the reference sequence.

[0049] For the purposes of the invention, a monoclonal antibody or antibody fragment according to the invention, one or more sequences of which have “at least 80% identity” with a reference sequence (see below) does not lose its capacity to bind specifically to an epitope included in SEQ ID NO: 35 or to an epitope included in SEQ ID NO: 36 of VEGF-NF of sequence SEQ ID NO: 40.

[0050] When it is a substitution, the substitution is preferably made by an “equivalent” amino acid, that is to say any amino acid whose structure is similar to that of the original amino acid and therefore unlikely to modify the biological activity of the antibody or antibody fragment. It may be at least one substitution, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, of an amino acid residue compared to the reference sequence. Said substitution of an amino acid residue is preferably a conservative substitution.

[0051] A "conservative substitution" is the replacement of one amino acid residue with another amino acid residue, having a side chain with similar properties. Amino acid families with side chains with similar properties are well known, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), polar and uncharged side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), and side chains apolar (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine).

[0052] Antibodies or antibody fragments having at least 80% identity therefore have certain amino acids that can be substituted by other amino acids in the constant regions and / or variable regions, without losing the antigen-binding capacity. It is preferable that this substitution is made within the DNA sequence that codes for the antibody or antibody fragment, i.e. that the substitution is conservative in nature. The person skilled in the art uses his general knowledge to determine the number of substitutions that can be made and their location in order to be able to preserve the function of the antibody or antibody fragment. In order to determine the capacity of one or more antibody or antibody fragment variants to bind specifically to an antigen, several suitable methods, well known to the person skilled in the art and described in the prior art, can be used.Antibodies or antibody fragments can therefore be tested by binding methods, such as ELISA, affinity chromatography, etc. Antibody or antibody fragment variants can be generated, for example, by the "phage display" method to generate a phage library. A large number of methods are known for generating a "phage display" library and targeting antibody or antibody fragment variants with the desired functional characteristics.

[0053] By "purified" and "isolated" is meant, when referring to a biological entity (e.g., an antibody, an antibody fragment, a cell), that said biological entity is present in the substantial absence of other biological entities of the same type. The term "purified" or "isolated" as used herein preferably means at least 75% by mass, more preferably at least 85% by mass, even more preferably at least 95% by mass, and most preferably at least 98% by mass or even at least 99% by mass of said biological entity, relative to all biological entities of the same type present.

[0054] The term “VEGF-NF+ cancer” or “VEGF-NF positive cancer” refers to a cancer expressing VEGF-NF. In particular, the term “VEGF-NF+ cancer” refers to any case of cancer in which the vessels supplying the tumor show expression of VEGF- NF. In particular embodiments, the VEGF-NF+ cancer is selected from cancers of the nervous system, gastrointestinal cancers (e.g., astrocytoma, ependymoma, glioblastoma, glioma, medulloblastoma, pancreatic cancer), liver cancers, gynecological cancers (e.g., endometrial cancer, cervical cancer, ovarian cancer, fallopian tube cancer), breast cancer, melanomas, cancers of the urinary system (e.g., bladder cancer, kidney cancer, in particular clear cell renal cell carcinoma (ccRCC)), cancers of the respiratory and ENT systems, head and neck cancer, bone cancers, hematological cancers (e.g., multiple myelomas).

[0055] The term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States or European Pharmacopoeia, or other generally recognized pharmacopoeia, for use in animals and humans. A "pharmaceutical composition" means a composition comprising a pharmaceutically acceptable carrier. For example, a pharmaceutically acceptable carrier may be a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such carriers may be sterile liquids, such as water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.Saline solutions and aqueous solutions of dextrose and glycerol may also be used as liquid vehicles, particularly for injectable solutions. Pharmaceutically acceptable excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like.Where the pharmaceutical composition is suitable for oral administration, the tablets or capsules may be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by well-known methods. known in the prior art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or may be presented as a dry product for constitution with water or another suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable vehicles such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl hydroxybenzoates or sorbic acid).The pharmaceutical compositions may also contain buffer salts, flavorings, colorings and sweeteners, as appropriate. The composition according to the invention is preferably a pharmaceutical composition.

[0056] The term “treat” or “treatment” encompasses any beneficial or desirable effect on a pathology or condition, and may even include a minimal reduction in one or more measurable markers of the pathology or condition. Treatment may, for example, involve either reducing or improving the symptoms of the pathology or condition, or delaying the progression of the disease or condition. The term “treatment” does not necessarily mean complete eradication or cure of the pathology or associated symptoms.

[0057] The term "patient" means a human or non-human mammal (such as a cat, dog, horse, or primate). Preferably, the patient is a human being, male or female.

[0058] Antibody or antibody fragment according to the invention

[0059] A first subject of the invention relates to a monoclonal antibody or fragment thereof which binds to VEGF-NF (vascular endothelial growth factor-NF) of sequence SEQ ID NO: 40, said antibody or fragment thereof binds specifically to an epitope included in SEQ ID NO: 35 or to an epitope included in SEQ ID NO: 36.

[0060] In a first particular embodiment, said antibody or fragment thereof comprises: - three heavy chain variable region CDRs of sequences: SEQ ID NO: 1 for CDR1 VH, SEQ ID NO: 2 for CDR2 VH, and SEQ ID NO: 3 for CDR3 VH; and - three light chain variable region CDRs of sequences: SEQ ID NO: 4 for CDR1 VL, DVS for CDR2 VL, and SEQ ID NO: 5 for CDR3 VL; said antibody or fragment thereof specifically binds to an epitope included in SEQ ID NO: 35.

[0061] The invention relates generally to an antibody or fragment thereof which binds to VEGF-NF (vascular endothelial growth factor-NF) of sequence SEQ ID NO: 40 comprising: - three heavy chain variable region CDRs of sequences: SEQ ID NO: 1 for CDR1 VH, SEQ ID NO: 2 for CDR2 VH, and SEQ ID NO: 3 for CDR3 VH; and - three light chain variable region CDRs of sequences: SEQ ID NO: 4 for CDR1 VL, DVS for CDR2 VL, and SEQ ID NO: 5 for CDR3 VL.

[0062] In a second particular embodiment, said antibody or fragment thereof comprises: - three heavy chain variable region CDRs of sequences: SEQ ID NO: 6 for CDR1 VH, SEQ ID NO: 7 for CDR2 VH, and SEQ ID NO: 8 for CDR3 VH; and - three light chain variable region CDRs of sequences: SEQ ID NO: 9 for CDR1 VL, KVS for CDR2 VL, and SEQ ID NO: 10 for CDR3 VL; said antibody or fragment thereof specifically binds to an epitope included in SEQ ID NO: 36.

[0063] The invention relates generally to an antibody or fragment thereof which binds to VEGF-NF (vascular endothelial growth factor-NF) of sequence SEQ ID NO: 40, comprising: - three heavy chain variable region CDRs of sequences: SEQ ID NO: 6 for CDR1 VH, SEQ ID NO: 7 for CDR2 VH, and SEQ ID NO: 8 for CDR3 VH; and - three light chain variable region CDRs of sequences: SEQ ID NO: 9 for CDR1 VL, KVS for CDR2 VL, and SEQ ID NO: 10 for CDR3 VL.

[0064] In particular embodiments, said antibody or fragment thereof comprises: - a FRI H heavy chain hinge region selected from the group consisting of (i) SEQ ID NO: 11 or a sequence having at least 80% identity with SEQ ID NO: 11 and (ii) SEQ ID NO: 12 or a sequence having at least 80% identity with SEQ ID NO: 12; - a FR2 H heavy chain hinge region selected from the group consisting of (i) SEQ ID NO: 13 or a sequence having at least 80% identity with SEQ ID NO: 13 and (ii) SEQ ID NO: 14 or a sequence having at least 80% identity with SEQ ID NO: 14; - a FR3 H heavy chain hinge region selected from the group consisting of (i) SEQ ID NO: 15 or a sequence having at least 80% identity with SEQ ID NO: 15 and (ii) SEQ ID NO: 16 or a sequence having at least 80% identity with SEQ ID NO: 16; and - a FR4 H heavy chain hinge region selected from the group consisting of (i) SEQ ID NO: 17 or a sequence having at least 80% identity with SEQ ID NO: 17 and (ii) SEQ ID NO: 18 or a sequence having at least 80% identity with SEQ ID NO: 18.

[0065] In particular embodiments, said antibody or fragment thereof comprises: - a FRI L light chain hinge region selected from the group consisting of (i) SEQ ID NO: 19 or a sequence having at least 80% identity with SEQ ID NO: 19 and (ii) SEQ ID NO: 20 or a sequence having at least 80% identity with SEQ ID NO: 20; - an FR2 L light chain hinge region selected from the group consisting of (i) SEQ ID NO: 21 or a sequence having at least 80% identity with SEQ ID NO: 21 and (ii) SEQ ID NO: 22 or a sequence having at least 80% identity with SEQ ID NO: 22; - an FR3 L light chain hinge region selected from the group consisting of (i) SEQ ID NO: 23 or a sequence having at least 80% identity with SEQ ID NO: 23 and (ii) SEQ ID NO: 24 or a sequence having at least 80% identity with SEQ ID NO: 24; and - an FR4 L light chain hinge region selected from the group consisting of (i) SEQ ID NO: 25 or a sequence having at least 80% identity with SEQ ID NO: 25 and (ii) SEQ ID NO: 26 or a sequence having at least 80% identity with SEQ ID NO: 26.

[0066] In particular embodiments, said antibody or fragment thereof comprises: - a heavy chain variable region (VH) selected from the group consisting of (i) SEQ ID NO: 27 or a sequence having at least 80% identity with SEQ ID NO: 27 and (ii) SEQ ID NO: 28 or a sequence having at least 80% identity with SEQ ID NO: 28; and - a light chain variable region (VL) selected from the group consisting of (i) SEQ ID NO: 29 or a sequence having at least 80% identity with SEQ ID NO: 29 and (ii) SEQ ID NO: 30 or a sequence having at least 80% identity with SEQ ID NO: 30.

[0067] In a first particularly preferred embodiment, said antibody or fragment thereof comprises: - a heavy chain variable region (VH) of sequence SEQ ID NO: 27 or a sequence having at least 80% identity with SEQ ID NO: 27; and - a light chain variable region (VL) of sequence SEQ ID NO: 29 or a sequence having at least 80% identity with SEQ ID NO: 29.

[0068] In a second particularly preferred embodiment, said antibody or fragment thereof comprises: - a heavy chain variable region (VH) of sequence SEQ ID NO: 28 or a sequence having at least 80% identity with SEQ ID NO: 28; and - a light chain variable region (VL) of sequence SEQ ID NO: 30 or a sequence having at least 80% identity with SEQ ID NO: 30.

[0069] In a preferred embodiment, the antibody or antibody fragment according to the invention comprises (i) a heavy chain of sequence SEQ ID NO: 46 or a sequence having at least 80% identity with SEQ ID NO: 46, and (ii) a light chain of sequence SEQ ID NO: 47 or a sequence having at least 80% identity with SEQ ID NO: 47. Preferably, the antibody or fragment thereof according to the invention corresponds to an antibody or antibody fragment whose sequences of the CDR, FR, VH and VL regions are those described in the sequence listing table for "P2-H1".

[0070] In another preferred embodiment, the antibody or antibody fragment according to the invention comprises (i) a heavy chain of sequence SEQ ID NO: 44 or a sequence having at least 80% identity with SEQ ID NO: 44, and (ii) a light chain of sequence SEQ ID NO: 45 or a sequence having at least 80% identity with SEQ ID NO: 45. Preferably, the antibody or fragment thereof according to the invention corresponds to an antibody or antibody fragment whose sequences of the CDR, FR, VH and VL regions are those described in the sequence listing table for "PIFS".

[0071] In particular embodiments, the antibody or antibody fragment according to the invention also binds to VEGF-NF of sequence SEQ ID NO: 37.

[0072] In particular embodiments, the antibody or antibody fragment according to the invention also binds to VEGF-NF of sequence SEQ ID NO: 38.

[0073] In particular embodiments, the antibody or antibody fragment according to the invention also binds to VEGF-NF of sequence SEQ ID NO: 39.

[0074] In particular embodiments, the antibody or antibody fragment according to the invention also binds to VEGF-NF of sequence SEQ ID NO: 41.

[0075] In particular embodiments, the antibody or antibody fragment according to the invention also binds to VEGF-NF of sequence SEQ ID NO: 42.

[0076] In particular embodiments, the antibody or antibody fragment according to the invention also binds to VEGF-NF of sequence SEQ ID NO: 43.

[0077] In particular embodiments, the antibody or antibody fragment according to the invention also binds to one, two, three, four, five or six VEGF-NFs of sequence selected from the group consisting of SEQ ID NO: 37, 38, 39, 41, 42 and 43.

[0078] In particular embodiments, the antibody or antibody fragment according to the invention also binds to the six VEGF-NFs of sequences SEQ ID Nos: 37, 38, 39, 41, 42 and 43. That is to say that the antibody or antibody fragment according to the invention binds to the seven VEGF-NFs of sequences SEQ ID Nos: 37-43.

[0079] The inventors have shown with an ELISA test (see Example 7) that the antibodies or antibody fragments according to the invention can inhibit the binding between VEGF-NF and VEGF receptors (VEGFR), such as VEGFR1, VEGFR2, VEGFR3, NRP1 and NRP2.

[0080] Thus, in particular embodiments, an antibody or antibody fragment according to the invention inhibits the binding between VEGF-NF and one or more VEGF receptors (VEGFR), such as VEGFR1, VEGFR2 and / or VEGFR3. In particular, an antibody or antibody fragment according to the invention inhibits the binding between VEGF-NF and one or more VEGF receptors selected from the group consisting of VEGFR1, VEGFR2, VEGFR3, NRP1 and NRP2. According to these embodiments, an antibody or antibody fragment according to the invention inhibits the binding between (i) one, two, three, four, five, six or seven splice variants of VEGF-NF of sequence selected from the group consisting of SEQ ID NO: 37, 38, 39, 40, 41, 42 and 43 and (ii) one or more VEGF receptors selected from the group consisting of VEGFR1, VEGFR2, VEGFR3, NRP1 and NRP2. For example, an antibody or antibody fragment according to the invention inhibits the binding between VEGF-NF of sequence SEQ ID NO: 40 and one or more VEGF receptors chosen from the group consisting of VEGFR1, VEGFR2, VEGFR3, NRP1 and NRP2.

[0081] The ability of an antibody or antibody fragment to inhibit the binding of VEGF-NF to one or more VEGFRs (e.g., VEGFR1, VEGFR2, VEGFR3, NRP1, or NRP2) can, for example, be tested by a competitive method. A “competitive method” involves testing an antibody (or antibody fragment) for its ability to block the binding of VEGF-NF to the VEGFR receptor. Many types of competitive methods can be used to determine whether an antibody or antibody fragment inhibits the binding of VEGF-NF to a VEGFR receptor (e.g., VEGFR1, VEGFR2, VEGFR3, NRP1, or NRP2), such as a competitive ELISA, a direct or indirect solid-phase radioimmunoassay (RIA), a direct or indirect solid-phase enzyme-linked immunosorbent assay (EIA), a surface plasmon resonance (SPR) technology (e.g.,via BIACORE™), by flow cytometry, by fluorescence polarization (e.g. between a fluorescent peptide and the antibody to be tested), etc. For example, the competitive ELISA method involves the use of a VEGFR bound to a solid surface or to cells, the unlabeled VEGF-NF and the labeled antibody or antibody fragment to be tested. Usually, VEGF-NF is present in a non-saturating concentration (relative to its dissociation constant K. D for VEGF-NF) and the signal is measured at increasing concentrations of the antibody or antibody fragment to be tested. When the concentration of the antibody or antibody fragment to be tested is in excess, it can block or inhibit (e.g., reduce) the specific binding of VEGF-NF to VEGFR by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97%, 98%, 99%, or even 100%.

[0082] Quite surprisingly, even if the antibodies or antibody fragments according to the invention are capable of inhibiting the binding between VEGF-NF and one or more VEGF receptors (e.g. VEGFR1, VEGFR2, VEGFR3, NRP1 or NRP2), the inventors have shown that they are not necessarily capable of blocking the signaling pathway mediated by VEGFR2 in the presence of VEGF-NF, which notably involves the phosphorylation of said VEGFR2. Thus, in particular embodiments, an antibody or antibody fragment according to the invention does not block the activation of the signaling pathway mediated by VEGFR2 in the presence of VEGF-NF, which notably involves the phosphorylation of said VEGFR2. In other words, an antibody or antibody fragment according to the invention does not block the phosphorylation of VEGFR2 in the presence of VEGF-NF.This property of the antibodies or antibody fragments according to the invention is all the more surprising since the inventors have shown that the anti-VEGF-NF polyclonal antibodies described in the literature (References [2] and [3]) are capable of blocking the signaling pathway mediated by VEGFR2 in the presence of VEGF-NF (see Example 8). This major difference suggests that the antibodies or antibody fragments according to the invention have properties and / or mechanisms of action different from the polyclonal antibodies described in references [2] and [3].

[0083] Without yet being able to explain the molecular mechanisms associated with the surprising properties of the antibodies and antibody fragments according to the invention, it is reasonably possible to deduce that they are associated with unique therapeutic effects compared to the anti-VEGF monoclonal antibodies described in the literature, such as bevacizumab,

[0084] The ability of an antibody or antibody fragment to inhibit the signaling pathway mediated by one or more VEGFRs can be tested in any model commonly used by those skilled in the art, for example the model described in reference [3] or in Example 8.

[0085] The antibody or antibody fragment according to the first object described above is called “reference antibody or antibody fragment” hereinafter concerning the second object according to the invention.

[0086] A second subject of the invention relates to a monoclonal antibody or antibody fragment (hereinafter "competitor antibody or antibody fragment") which competes with the antibody or antibody fragment according to the invention as described above (hereinafter "reference antibody or antibody fragment") for binding to VEGF-NF of sequence SEQ ID NO: 40.

[0087] The ability of an antibody or antibody fragment to compete with the reference antibody or antibody fragment for binding to VEGF-NF can be tested by a competitive method. A "competitive method" involves testing an antibody (or antibody fragment) for its ability to block the binding of a reference antibody or antibody fragment to an antigen or to compete with a reference antibody or antibody fragment for binding to the antigen. In other words, an antibody that competes with the reference antibody or antibody fragment binds to the same epitope as the reference antibody or antibody fragment or to an epitope that is sufficiently close to the epitope recognized by the reference antibody or antibody fragment to prevent binding of the reference antibody or antibody fragment due to steric hindrance.

[0088] Many types of competitive methods can be used to determine whether an antibody or antibody fragment competes with a reference antibody or antibody fragment, for example, by competitive ELISA, direct or indirect solid-phase radioimmunoassay (RIA), direct or indirect solid-phase enzyme-linked immunosorbent assay (EIA), surface plasmon resonance technology (e.g., BIACORE), flow cytometry, fluorescence polarization (e.g., between a fluorescent peptide and the test antibody), etc. For example, competitive ELISA involves the use of a purified antigen bound to a solid surface or cells, the test antibody that binds to the unlabeled antigen, and a labeled reference antibody or antibody fragment.Usually, the reference antibody or antibody fragment is present in a non-saturating concentration (relative to its dissociation constant K. D for VEGF-NF) and the signal is measured at increasing concentrations of the antibody or antibody fragment to be tested. When an antibody is present in excess, it may block or inhibit (e.g., reduce) the specific binding of a reference antibody or antibody fragment to an antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0089] The competing antibody or antibody fragment according to the invention generally has the same properties as the reference antibody with regard to (i) the capacity to inhibit the binding between VEGF-NF and one or more VEGF receptors and / or (ii) the capacity to block the VEGFR2 signaling pathway (see above). For example, an antibody or antibody fragment that competes with the P2-H1 antibody or the P1-F3 antibody is therefore capable of inhibiting the binding between VEGF-NF and one or more VEGF receptors selected from the group consisting of VEGFR1, VEGFR2, VEGFR3, NRP1 and NRP2.

[0090] In particular embodiments, the antibody or competing antibody fragment according to the invention also binds to VEGF-NF of sequence SEQ ID NO: 37.

[0091] In particular embodiments, the antibody or competing antibody fragment according to the invention also binds to VEGF-NF of sequence SEQ ID NO: 38.

[0092] In particular embodiments, the antibody or competing antibody fragment according to the invention also binds to VEGF-NF of sequence SEQ ID NO: 39.

[0093] In particular embodiments, the antibody or the competing antibody fragment according to the invention also binds to VEGF-NF of sequence SEQ ID NO: 41.

[0094] In particular embodiments, the antibody or the competing antibody fragment according to the invention also binds to VEGF-NF of sequence SEQ ID NO: 42.

[0095] In particular embodiments, the antibody or competing antibody fragment according to the invention also binds to VEGF-NF of sequence SEQ ID NO: 43.

[0096] In particular embodiments, the antibody or competing antibody fragment according to the invention also binds to one, two, three, four, five or six VEGF-NFs of sequence selected from the group consisting of SEQ ID NO: 37, 38, 39, 41, 42 and 43.

[0097] In particular embodiments, the competing antibody or antibody fragment according to the invention also binds to the six VEGF-NFs of sequences SEQ ID Nos: 37, 38, 39, 41, 42 and 43. That is to say that the competing antibody or antibody fragment according to the invention binds to the seven VEGF-NFs of sequences SEQ ID Nos: 37-43.

[0098] The reference antibodies or antibody fragments and the competing antibodies or antibody fragments as defined above are hereinafter jointly referred to as “antibodies or antibody fragments according to the invention”.

[0099] The antibody or antibody fragment according to the invention can bind to VEGF-NF in an isolated form and / or in its biological environment.

[0100] In particular embodiments, the antibody or antibody fragment according to the invention is of the IgG, IgM, IgA, IgD or IgE type, depending on the structure of its chain. heavy. Preferably, the antibody or antibody fragment according to the invention is of the IgG type, that is to say that its heavy chain is of the gamma (y) type. IgG antibodies are classified into four distinct subtypes, named IgG1, IgG2, IgG3 and IgG4, in order of abundance in the serum (IgG1 being the most abundant).

[0101] In particular embodiments, the antibody or antibody fragment according to the invention is of the IgG1, IgG2, IgG3 or IgG4 subtype. Preferably, the antibody or antibody fragment according to the invention is of the IgG1 subtype or the IgG2 subtype, preferably of the IgG1 subtype.

[0102] In particular embodiments, the antibody or antibody fragment according to the invention binds to VEGF-NF, in particular to VEGF-NF of sequence SEQ ID NO: 40, with a dissociation constant (K D ) measured by SPR technology (e.g. via BIACORE™) less than or equal to 100 nM, for example less than or equal to 90 nM, less than or equal to 80 nM, less than or equal to 70 nM, less than or equal to 60 nM, less than or equal to 50 nM, less than or equal to 40 nM, less than or equal to 30 nM, less than or equal to 10 nM. A dissociation constant of less than 100 nM is preferable for use in therapy. Advantageously, the antibody or antibody fragment according to the invention can bind to VEGF-NF with a dissociation constant (K D) measured by SPR technology (e.g. via BIACORE™) ranging from 0 to 100 nM (0 being excluded), ranging from 1 to 100 nM, ranging from 1 to 90 nM, ranging from 1 to 80 nM, ranging from 1 to 70 nM, ranging from 1 to 60 nM, ranging from 1 to 50 nM, ranging from 1 to 40 nM, ranging from 1 to 30 nM, ranging from 1 to 20 nM, ranging from 1 to 10 nM. A method for measuring K D of an antibody or an antibody fragment according to the invention by SPR technology (BIACORE™) is detailed in examples 2 and 3.

[0103] In preferred embodiments, the antibody or antibody fragment of the invention comprises a heavy chain variable region (VH) of sequence SEQ ID NO: 27 or a sequence having at least 80% identity with SEQ ID NO: 27 and a light chain variable region (VL) of sequence SEQ ID NO: 29 or a sequence having at least 80% identity with SEQ ID NO: 29, and binds to VEGF-NF of sequence SEQ ID NO: 40 with a dissociation constant (K D) measured by SPR technology (e.g. via BIACORE™) less than or equal to 100 nM, preferably less than 90 nM, for example less than 80 nM, less than 75 nM, less than 70 nM, less than 60 nM, or less than 50 nM, for example between 40 nM and 50 nM.

[0104] Preferably, the antibody or antibody fragment according to the invention comprises (i) a heavy chain of sequence SEQ ID NO: 46 or a sequence having at least 80% identity with SEQ ID NO: 46, and (ii) a light chain of sequence SEQ ID NO: 47 or a sequence having at least 80% identity with SEQ ID NO: 47, and binds to VEGF-NF of sequence SEQ ID NO: 40 with a dissociation constant (K D ) measured by SPR technology (e.g. via BIACORE™) less than or equal to 100 nM, preferably less than 90 nM, for example less than 80 nM, less than 75 nM, less than 70 nM, less than 60 nM, or less than 50 nM, for example between 40 nM and 50 nM.

[0105] In other preferred embodiments, the antibody or antibody fragment according to the invention comprises a heavy chain variable region (VH) of sequence SEQ ID NO: 28 or a sequence having at least 80% identity with SEQ ID NO: 28 and a light chain variable region (VL) of sequence SEQ ID NO: 30 or a sequence having at least 80% identity with SEQ ID NO: 30, and binds to VEGF-NF of sequence SEQ ID NO: 40 with a dissociation constant (K D ) measured by SPR technology (e.g. via BIACORE™) less than or equal to 25 nM, preferably less than 20 nM, for example less than 15 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, for example between 5 nM and 6 nM.

[0106] Preferably, the antibody or antibody fragment according to the invention comprises (i) a heavy chain of sequence SEQ ID NO: 44 or a sequence having at least 80% identity with SEQ ID NO: 44, and (ii) a light chain of sequence SEQ ID NO: 45 or a sequence having at least 80% identity with SEQ ID NO: 45, and binds to VEGF-NF of sequence SEQ ID NO: 40 with a dissociation constant (K D ) measured by SPR technology (e.g. via BIACORE™) less than or equal to 25 nM, preferably less than 20 nM, for example less than 15 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, for example between 5 nM and 6 nM.

[0107] In a particular embodiment, the antibody or antibody fragment according to the invention is isolated or purified. The antibodies or antibody fragment according to the invention may be isolated after production by a cell, for example a cell according to the invention, or synthesized and further purified by well-known techniques. The antibody or antibody fragment according to the invention may be purified by well-known techniques from a culture medium, in particular by affinity chromatography.

[0108] Composition of antibody or antibody fragment

[0109] Another subject of the invention relates to a composition comprising an antibody or an antibody fragment according to the invention. The composition is preferably a pharmaceutical composition, i.e. a composition comprising one or more pharmaceutically acceptable excipients.

[0110] Other objects

[0111] Another subject of the present invention relates to a polynucleotide or combination of two polynucleotides encoding a heavy chain variable region (VH) of an antibody or a fragment thereof and a light chain variable region (VL) of an antibody or a fragment thereof according to the invention.

[0112] In a particular embodiment, the invention relates to a polynucleotide or a combination of two polynucleotides comprising a sequence SEQ ID NO: 31 coding for a heavy chain variable region (VH) and a sequence SEQ ID NO: 33 coding for a light chain variable region (VL). In this particular embodiment, said polynucleotide or combination of two polynucleotides codes for a heavy chain variable region (VH) of sequence SEQ ID NO: 27 and for a light chain variable region (VL) of sequence SEQ ID NO: 29.

[0113] In another particular embodiment, the invention relates to a polynucleotide or a combination of two polynucleotides comprising a sequence SEQ ID NO: 32 encoding a heavy chain variable region (VH) and a sequence SEQ ID NO: 34 encoding a light chain variable region (VL). In this other particular embodiment, said polynucleotide or combination of two polynucleotides encodes a heavy chain variable region (VH) of sequence SEQ ID NO: 28 and a light chain variable region (VL) of sequence SEQ ID NO: 30.

[0114] Another subject of the present invention relates to a polynucleotide or a combination of two polynucleotides encoding a heavy chain of an antibody or a fragment thereof according to the invention and a light chain of an antibody or a fragment thereof according to the invention.

[0115] In a particular embodiment, the invention relates to a polynucleotide or a combination of two polynucleotides comprising a sequence SEQ ID NO: 48 coding for a heavy chain and a sequence SEQ ID NO: 49 coding for a chain Light Tl. In this particular embodiment, said polynucleotide or combination of two polynucleotides codes for a heavy chain of sequence SEQ ID NO: 44 and for a light chain of sequence SEQ ID NO: 45.

[0116] In another particular embodiment, the invention relates to a polynucleotide or a combination of two polynucleotides comprising a sequence SEQ ID NO: 50 coding for a heavy chain and a sequence SEQ ID NO: 51 coding for a light chain. In this particular embodiment, said polynucleotide or combination of two polynucleotides codes for a heavy chain of sequence SEQ ID NO: 46 and for a light chain of sequence SEQ ID NO: 47.

[0117] Another subject of the present invention relates to an expression vector comprising a polynucleotide or a combination of two polynucleotides according to the invention. Any type of vector suitable for the production of antibodies can be used within the scope of the invention. The vectors can be viral vectors such as bacteriophages or non-viral vectors such as plasmids. The vector can comprise nucleic sequences which make it possible to produce the antibody according to the invention, for example promoter sequences, regulatory sequences, etc. The preparation of a suitable expression vector does not present any particular difficulty for the person skilled in the art.

[0118] Another subject of the invention relates to a cell, for example an isolated cell, comprising an expression vector according to the invention. The cell according to the invention can be obtained by methods widely described in the literature, for example by preparing a cell clone comprising an expression vector according to the invention and capable of producing the antibody or antibody fragment according to the invention. The invention is not limited to a particular cell type. Any cell capable of producing antibodies can be used within the scope of the invention. These may be eukaryotic cells, such as mammalian cells, for example human cells, or murine cells or prokaryotic cells, for example bacteria or yeasts.

[0119] Therapeutic use

[0120] The invention also relates to an antibody or a fragment thereof according to the invention or a composition according to the invention, for use as a medicament.

[0121] The invention also relates to an antibody or fragment thereof according to the invention or a composition according to the invention, for use in the treatment of cancer in a patient, for example for use in the treatment of a VEGF-NF+ cancer.

[0122] In a particular embodiment, the cancer is chosen from cancers of the nervous system, gastrointestinal cancers (e.g. astrocytoma, ependymoma, glioblastoma, glioma, medulloblastoma, pancreatic cancer), liver cancers, gynecological cancers (e.g. endometrial cancer, cervical cancer, ovarian cancer, fallopian tube cancer), breast cancer, melanomas, cancers of the urinary system (e.g. bladder cancer, kidney cancer, in particular clear cell renal cell carcinoma (ccRCC)), cancers of the respiratory and ENT systems, head and neck cancer, bone cancers, hematological cancers (e.g. multiple myelomas).

[0123] In a particular embodiment, the cancer is refractory to treatment with bevacizumab, i.e., a cancer that does not respond to treatment with bevacizumab. The inventors have in fact noticed that patients treated with bevacizumab, who have become refractory to this treatment, overexpress VEGF-NF, which makes treatment with the antibody of the invention or the fragment thereof particularly interesting.

[0124] In a particular embodiment, the patient is refractory to bevacizumab treatment, i.e., a patient who does not respond to bevacizumab treatment. For example, the patient has been previously treated with bevacizumab and has become refractory to bevacizumab treatment.

[0125] The Applicant has notably observed an increase in the expression of VEGF NF in patients treated with bevacizumab [3]. Similarly, in a bevacizumab-resistant mouse model, mice treated with bevacizumab show an increase in VEGF-NF in the blood [3].

[0126] The antibody or antibody fragment of the invention is preferably formulated for parenteral administration, for example, intravascular (intravenous or intra-arterial), intraperitoneal, or intramuscular administration. The term "parenterally administered," as used herein, refers to modes of administration other than enteral and topical administration, generally by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intra- arterial, intrathapsal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, by injection, transtracheal infusion, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal. Intravenous administration is preferred for the purposes of the present invention, for example by intravenous infusion.

[0127] The dose of the antibody or antibody fragment of the invention administered to a subject in need thereof will vary depending on several factors, including, without limitation, the route of administration, the type and severity of the condition being treated, the condition of the patient, the body size of the patient, the age of the patient, etc. One skilled in the art can readily determine, based on his or her knowledge in this field, the required dosage range based on these and other factors. The appropriate dose can also be determined with animal models or with clinical trials. For example, typical doses of antibodies or antibody fragments according to the invention administered intravenously may be from 1 mg / kg to 50 mg / kg, for example from 2 mg / kg to 40 mg / kg, from 3 mg / kg to 30 mg / kg, from 3 mg / kg to 25 mg / kg, from 3 mg / kg to 20 mg / kg, from 3 mg / kg to 15 mg / kg, from 5 mg / kg to 15 mg / kg, for example equal to 5 mg / kg, 10 mg / kg, 15 mg / kg.Administration can be done in one dose or, more commonly, in several doses. The administration schedule may include an initial loading dose followed by maintenance doses, for example, weekly, every two weeks, every three weeks, every month, or more frequently. The duration of treatment may vary depending on the condition being treated and the individual.

[0128] The antibody or antibody fragment according to the invention can be used as monotherapy or in combination with drugs whose therapeutic interest is recognized in the pathology considered. By "combination" is meant simultaneous (concurrent) or consecutive administration in any order.

[0129] A drug whose therapeutic interest is recognized in the pathology considered may, for example, be an anti-angiogenic compound. As used herein, the term "anti-angiogenic compound" designates any compound that inhibits the development of a pathological vascular network. In particular, the anti-angiogenic compound may inhibit pro-angiogenic factors such as VEGF, receptors for pro-angiogenic factors such as VEGF receptors and CXCL / ELR+ chemokine receptors. In particular, the anti-angiogenic compound is chosen from the group consisting of: (i) anti-VEGFA antibodies, such as bevacizumab, aflibercept, (ii) inhibitors of receptors involved in angiogenesis, in particular VEGFR1, 2, 3, CSFR, PDGFR, such as sunitinib, sorafenib, axitinib, cabozantinib, pazopanib, lenvatinib, regorafenib, (iii) m-TOR inhibitors, such as everolimus, temsirolimus, and (iv) mixtures thereof.

[0130] A drug whose therapeutic interest is recognized in the pathology considered can also be chosen from intercalating agents / DNA crosslinking agents (such as oxaliplatin, mitoxantrone), DNA synthesis inhibitors (such as cytosine bD-arabinofuranoside, 5-fluorouracil), DNA-RNA transcription regulators (doxorubicin, actinomycin D), microtubule inhibitors (paclitaxel, nocodazole).

[0131] Another drug whose therapeutic interest is recognized in the pathology considered can also be an antibody used in anti-cancer immunotherapy such as an anti-PDl or anti-PD-Ll.

[0132] The antibody or antibody fragment according to the invention may also be used in combination with radiotherapy, such as conventional radiotherapy or vectorized internal radiotherapy.

[0133] The invention will be further illustrated by the following figures and examples. However, these examples and figures should in no way be construed as limiting the scope of the invention. Brief description of the figures

[0135] Figure 1 represents the SPR signal (Biacore™) of the anti-VEGF-NF antibody P1-F3 against VEGF-222 / NF, allowing the determination of the dissociation constant (K D ).

[0136] Figure 2 represents the SPR signal (Biacore™) of the anti-VEGF-NF antibody P2-H1 against VEGF-222 / NF, allowing the determination of the dissociation constant (K D ).

[0137] Figure 3 represents the measurements of the recognition of peptides PI: SGFREPDLSPGKTD (SEQ ID NO: 36), peptide P2: DVTSRGGEPGRRKE (SEQ ID NO: 35), or of the total protein VEGF-222 / NF by the different antibodies in ELISA. ** p<0.01, **** p<0.0001 VS CTRL.

[0138] Figure 4 shows the measurements of the recognition of the total VEGF-222 / NF protein by the different antibodies in sandwich ELISA. **** p<0.0001 vs CTRL.

[0139] Figure 5 shows the viability measurements of 786-0 cells after treatment with F3, H1 antibodies or bevacizumab. ** p<0.01 vs CTRL.

[0140] Figure 6 represents the effect of P1-F3 and P2-H1 antibodies on VEGF-NF binding to the NRP1 receptor. Values ​​in mean + / - SD, n=7 for all conditions, **** p<0.0001 versus VEGF-NF alone.

[0141] Figure 7 represents the effect of P1-F3 and P2-H1 antibodies on VEGF-NF binding to the NRP2 receptor. Values ​​in mean + / - SD, n=7 for all conditions, **** p<0.0001 versus VEGF-NF alone.

[0142] Figure 8 represents the effect of P1-F3 and P2-H1 antibodies on VEGF-NF binding to the VEGFR1 receptor. Values ​​in mean + / - SD, n=7 for all conditions, **** p<0.0001 versus VEGF-NF alone.

[0143] Figure 9 represents the effect of P1-F3 and P2-H1 antibodies on VEGF-NF binding to the VEGFR2 receptor. Values ​​in mean + / - SD, n=7 for all conditions, **** p<0.0001 versus VEGF-NF alone.

[0144] Figure 10 represents the effect of P1-F3 and P2-H1 antibodies on VEGF-NF binding to the VEGFR3 receptor. Values ​​in mean + / - SD, n=7 for all conditions, **** p<0.0001 versus VEGF-NF alone.

[0145] Figure 11 is a Western Blot representing the effect of P1-F3 and P2-H1 antibodies on VEGFR2 activation in the presence of VEGF-NF.

[0146] Figure 12 represents the effect of P1-F3 and P2-H1 antibodies on mouse weight in an in vivo 786-0 cell tumor growth model.

[0147] Figure 13 represents the effect of P1-F3 and P2-H1 antibodies on tumor growth of 786-0 cells in vivo in mice.

[0148] Figure 14 represents the effect of P1-H3 and P2-H1 antibodies on tumor weight at the end of the in vivo tumor growth experiment of 786-0 cells in mice. EXAMPLES

[0150] Example 1: Identification of antibodies according to the invention

[0151] Anti-VEGF-NF antibodies were identified by the Phage Display method (polyclonal). For this, tubes were coated with the peptides C-SGFREPDLSP (SEQ ID NO: 36 to which a cysteine ​​was added at the N-terminus to allow conjugation with the carriers) and DVTSRGGEPGRRKE-C (SEQ ID NO: 35 to which a cysteine ​​was added at the C-terminus to allow conjugation with the carriers), previously conjugated with carriers (ovalbumin (OVA), KLH, or BSA). After washing steps, the peptides were incubated with a proprietary phage library previously depleted against the carriers only. Phages binding the peptides were eluted and then amplified. The most relevant phages were selected by ELISA. For this, plates were coated with the aforementioned peptides and incubated with the eluted phages.After washing, an HRP-coupled anti-phage antibody was added to each well and TMB was added to reveal the ELISA. The reaction was stopped using HCl. The plate was then read at 450 nm. Quantity _ Round 1 _ Round 2 _ Round 3 _ Round 4 _ of phages Ag 1 CN 2 Ag 1 CN 2 Ag 1 CN 2 Ag 1 CN 2 (pfu / well) lx lO 12 0.450 0.201 1.972 0.988 2.624 0.930 3.753 0.902 3.3xlO n 0.133 0.074 1.656 0.997 2.111 0.968 3.529 0.891 1.1x10“ 0.063 0.031 1.492 0.558 1.991 0.799 3.326 0.768 3.6xlO 10 0.029 0.023 0.914 0.424 1.546 0.523 3.343 0.619 l.2xlO 10 0.019 0.011 0.572 0.314 1.339 0.377 3.184 0.354 4x l0 9 0.014 0.011 0.397 0.212 0.903 0.263 2.525 0.139 l.3xl0 9 0.011 0.010 0.134 0.110 0.555 0.123 1.894 0.054 0 0.009 0.009 0.009 0.009 0.010 0.009 0.010 0.016

[0152] Table 1: ELISA results. 1 Wells coated with a mixture of the two peptides conjugated to BSA + OVA + KLH; Negative control (NC) = Wells coated with carriers alone: ​​BSA + OVA + KLH

[0153] The data confirmed significant enrichment over the cycles, as indicated by good specific binding to peptides (in bold). The background was moderate and due to the difficulty of eliminating nonspecific binding against carriers, a known phenomenon. The result of the fourth series showing the best enrichment, it was selected for a first ELISA (monoclonal).

[0154] At this stage, 192 phage clones from the fourth enrichment round were tested by monoclonal ELISA for their ability to bind peptides conjugated with the above-mentioned carriers and their ability not to bind carriers alone. Among the 192 phage clones, 132 were selected (data not shown). Among these 132 phage clones, 22 unique sequences were identified after sequencing.

[0155] The 22 unique phage clones identified above were tested a second time by ELISA using a higher phage concentration. This confirmation step was essential to ensure the specificity of each of the unique clones. The test was performed on the peptide mixture and on the full-length protein to select the phage clones of interest. (Table 2).

[0156]

[0157] Table 2: Absorbance results (ELISA). Negative control: wells coated with a mixture of the three carriers alone (BSA + OVA + KLH); Negative control = Wells coated with the wash buffer only

[0158] The results show that all phage clones presented in Table 2 bind specifically to peptides. Among the 22 peptide-binding clones, only 5 clones also bind to the full-length VEGF-NF protein (P1-H2, P1-A3, P1-F3, P2-H1, and P2-C2). The amino acid sequences of the P1-F3 and P2-H1 antibodies are described in the "Sequence Listing" table.

[0159] Example 2: Determination of dissociation constants (K D ) of the anti-VEGF-NF antibody P1-F3 (BIACORE)

[0160] The dissociation constant (K D) of the anti-VEGF-NF antibody P1-F3 against VEGF-222 / NF (SEQ ID NO: 40) was measured by surface plasmon resonance (SPR) technology. VEGF-222 / NF (10 pg / ml, contained in 20 mM NaAc buffer pH 4.5) was immobilized on a biosensor (CM5 sensor) using EDC / NHS maleimide coupling until the response reached 143 resonance units (RU). The response measures changes in refractive index and is related to mass variations near the sensor surface. The response is therefore proportional to the number of antibody molecules interacting with the antigen. The binding of the analyte (anti-VEGF-NF P1-F3 antibody contained in a 0.01 M Hepes pH 7.4 buffer, 0.15 M NaCl, 3 mM EDTA, 0.01% surfactant P20) was measured during its injection by a microfluidic system.The antibody at a defined concentration was passed over the CM5 sensor and the signal was recorded over time, showing the progression of the interaction and association / dissociation cycle (Figure 1). After successively testing different concentrations, the kinetic parameters and affinity were calculated using the BIA evaluation software (Table 3).

[0161] Table 3: Kinetic parameters and affinity value of the anti-VEGF-NF antibody P1-F3 against VEGF-222 / NF.

[0162] The P1-F3 antibody has a dissociation constant (K D ) in the nanomolar range, indicating a high affinity towards VEGF-222 / NF. The sensorgrams of the P1-F3 antibody demonstrated a clear concentration / response correlation with smooth and consistent curves indicating good interactions.

[0163] Example 3: Determination of dissociation constants (K D) of the anti-VEGF-222 / NF P2-H1 antibody (BIACORE)

[0164] The dissociation constant of the anti-VEGF-NF antibody P2-H1 against VEGF-222 / NF (SEQ ID NO: 40) was measured by surface plasmon resonance (SPR) technology. VEGF-222 / NF (10 pg / ml, contained in 20 mM NaAc buffer pH 4.5) was immobilized on a biosensor (CM5 sensor) using EDC / NHS maleimide coupling until the response reached 143 resonance units (RU). The response measures changes in refractive index and is related to mass variations near the sensor surface. The response is therefore proportional to the number of antibody molecules interacting with the antigen. The binding of the analyte (anti-VEGF-NF P2-H1 antibody contained in a 0.01 M Hepes pH7.4 buffer, 0.15 M NaCl, 3 mM EDTA, 0.01% surfactant P20) was measured during its injection by a microfluidic system.The antibody at a defined concentration was passed over the CM5 sensor and the signal was recorded over time, showing the progression of the interaction and association / dissociation cycle (Figure 2). After successively testing different concentrations, the kinetic parameters and affinity were calculated using the BIA evaluation software (Table 4).

[0165] Table 4: Kinetic parameters and affinity value of the anti-VEGF-NF antibody P2-H1 against VEGF-222 / NF

[0166] The P2-H1 antibody has a dissociation constant (K D ) in the nanomolar range, indicating high affinity for VEGF-222 / NF. Sensorgrams of the P2-H1 antibody demonstrated a clear concentration / response correlation with smooth and consistent curves indicating good interactions.

[0167] Example 4: Peptide and Total Protein Binding Assays (ELISA)

[0168] 1. Materials and Methods

[0169] Experiments were performed on 96-well plates coated with human peptides: negative control peptide: KLH, P1 peptide: SGFREPDLSPGKTD (SEQ ID NO: 36), P2 peptide: DVTSRGGEPGRRKE (SEQ ID NO: 35), or total purified protein: VEGF-222 / NF (SEQ ID NO: 40). For this, the peptides or total purified protein were incubated in the wells of the plates at 4 °C, overnight, in PBS at a rate of 100 ng per well. The wells were washed three times with PBS-0.05% Tween-20 and blocked with PBS-1% BSA for 2 hours at room temperature. The wells were rinsed three times with PBS-0.05% Tween.

[0170] Antibodies A3 (i.e. P1-A3), C2 (i.e. P2-C2), F3 (i.e. P1-F3), H1 (i.e. P2-H1), H2 (i.e. P1-H2) were diluted in PBS - 0.1% BSA - 0.05% Tween-20 at 1 pg / ml and incubated for 2 h at room temperature. The wells were washed three times with PBS-Tween-20 0.05%, and anti-human HRP (Thermo Fischer, Goat anti-Human IgG Fc Secondary Antibody, HRP, reference A18817) was added at a dilution of 1 / 2000 in PBS - 0.1% BSA - 0.05% Tween-20 for 30 min at room temperature. After three washes, the ELISA was revealed after addition of TMB. Optical density (OD) was read at 450 nm after stopping the reaction using a 10% maleic acid / <1% hydrochloric acid solution (Biolegend, Stop Solution for TMB Substrate, Cat. No. 4230001).

[0171] 2. Results

[0172] F3 and H1 antibodies recognize peptide 1 and peptide 2, respectively, unlike A3, C2, and H2 antibodies, which do not recognize any of peptides 1 and 2. Evaluation of these antibodies on total VEGF222-NF protein demonstrated that F3 and H1 antibodies outperform A3, C2, and H2 antibodies (Figure 3). The CTRL corresponds to the anti-human HRP secondary antibody alone, which allows us to know the background noise associated with the use of this antibody.

[0173] Example 5: Total Protein Binding Assays (Sandwich ELISA - Using the VEGFA ELISA kit from Peprotech)

[0174] 1. Materials and methods

[0175] Experiments were performed in wells of 96-well plates coated overnight at room temperature with monoclonal antibodies A3, C2, F3, H1 or H2 at a concentration of 1.5 pg / ml in PBS. Each well was saturated with PBS-BSA 2% for 2 h at room temperature. VEGF-222 / NF (SEQ ID NO: 40) was incubated at 10 ng per well in PBS - 0.05% Tween-20 - 0.1% BSA for 2h at room temperature. The wells were rinsed three times with PBS - 0.05% Tween-20. The detection antibody of the kit is a biotin-coupled antibody that recognizes all forms of human VEGF (VEGF-A and VEGF-NF) was added (1 μg / ml, in PBS - 0.1% BSA - 0.05% Tween-20) for 2h at room temperature. After three washes, an avidin-HRP antibody was incubated (1 / 5000 in PBS - 0.1% BSA - 0.05% Tween-20) for 30 min at room temperature. After three washes, the ELISA was revealed after addition of TMB. The OD was read at 450 nm after stopping the reaction using a 10% maleic acid / <1% hydrochloric acid solution (Biolegend, Stop Solution for TMB Substrate, Cat. No. 4230001).

[0176] 2. Results

[0177] The F3 and H1 antibodies were the only two to recognize the total and native form of VEGF-222 / NF (Figure 4). These results obtained on the native form confirm those obtained via the direct ELISA method. The CTRL corresponds to antibodies directed against rabbit antibodies.

[0178] Example 6: In vitro tests of antibodies on cancer cells

[0179] 1. Materials and methods

[0180] 1.1. Cell culture

[0181] The 786-0 cell line (ATCC CRL-1932), a human renal adenocarcinoma cell, was cultured in RPMI-1640 medium supplemented with 10% serum.

[0182] 1.2. MTT Test

[0183] An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to measure cell viability after treatment with F3, H1, or bevacizumab antibodies. A 5X MTT solution (2.5 mg / mL) was prepared in phosphate-buffered saline (PBS). To determine the effect of antibodies on cell proliferation, 5,000 786-0 cells were seeded into wells of 96-well plates the day before treatment in RPMI + 10% serum. The next day, cells were rinsed three times with RPMI - 1% serum. F3, H1, or bevacizumab antibodies were pre-incubated in RPMI - 1% serum at 37°C / 5% CO2 at a concentration of 10 pg / mL. For each experiment, cells were treated on days 1 and 3. The effect on cell proliferation was determined after 7 days. The control condition was treated with a non-specific anti-rabbit secondary antibody. Then, 200 μL of 1X MTT solution was added and incubated at 37°C for 3 h. The formazan crystals were dissolved at room temperature for 30 min after replacing them with 100 μL of dimethyl sulfoxide (DMSO). Fluorescence was measured at excitation and emission wavelengths of 570 and 690 nm using a microplate reader. The results were expressed as % survival relative to untreated cells (CTRL).

[0184] 2. Results

[0185] Treatment with F3 and H1 antibodies resulted in a significant decrease in 786-0 cell survival (Figure 5). The results obtained with F3 and H1 antibodies appear even better than the results obtained with bevacizumab, which targets all forms of VEGF.

[0186] Example 7: Test for inhibition of binding between VEGF-NF and NRP1, NRP2, VEGFR1, VEGFR2 and VEGFR3 receptors

[0187] The receptors used are: Human recombinant Neuropilin-1 (3870-Nl, R&D Systems), Human recombinant Neuropilin-2 chimeric Fc (2215-N2, R&D Systems), Human recombinant VEGFR3 / Flt-4 chimeric Fc (349-F4, R&D Systems), Human recombinant VEGFR2 / KDR chimeric Fc (357-KD / CF, R&D Systems), Human recombinant VEGFRl / Flt-1 chimeric Fc (321-FL / CF, R&D Systems).

[0188] The antibodies tested are: P1-F3 (F3), P2-H1 (Hl) and bevacizumab (BVZ).

[0189] On ELISA plates, different receptors (NRP1, NRP2, VEGFR1, VEGFR2 or VEGFR3) were added to wells at a concentration of 100 ng / well in PBS. The plate was incubated overnight at 4°C.

[0190] The next day, the wells were rinsed three times with PBS containing 0.05% Tween. Then, 100 μL of VEGF-NF (SEQ NO: 40 produced in HEK) at a concentration of 100 ng / mL in PBS was added to each well in the presence or absence of 10 μg / mL of the tested antibody, and left to incubate for 2 hours at room temperature. After this incubation, the wells were rinsed three times with PBS / 0.05% Tween.

[0191] A polyclonal rabbit anti-VEGF-NF antibody (references [2] and [3]) diluted in PBS was added, and the plates were then incubated for 2 hours at room temperature. After three additional washes with PBS / 0.05% Tween, a secondary anti-rabbit IgG antibody coupled to horseradish peroxidase (HRP, Anti-rabbit IgG, HRP-linked Antibody #7074, Cell Signaling Technology) diluted in PBS was added to the wells and incubated for 30 minutes at room temperature, followed by three rinses with PBS / 0.05% Tween.

[0192] For visualization, 100 μL of TMB (3,3',5,5'-Tetramethylbenzidine, T0440, Sigma-Aldrich) was added to each well. The reaction was stopped with 100 μL of STOP solution (#7002, Cell Signaling Technology) as soon as a blue color appeared. The absorbance of the samples was measured at a wavelength of 450 nm to assess the binding of VEGF-NF to the receptors.

[0193] The results are presented in Figures 6 to 12.

[0194] Figure 6 shows that both F3 and H1 antibodies significantly inhibited VEGF-NF binding to NRP1. Conversely, BVZ had little effect on inhibiting VEGF-NF binding to NRP1. The inhibition was particularly marked for the F3 antibody.

[0195] Figure 7 shows that both F3 and H1 antibodies significantly inhibited VEGF-NF binding to NRP2. Conversely, BVZ had little effect on inhibiting VEGF-NF binding to NRP2. The inhibition was particularly marked for the F3 antibody.

[0196] Figure 8 shows that F3 and H1 antibodies significantly inhibited VEGF-NF binding to VEGFR1. Conversely, BVZ only slightly inhibited VEGF-NF binding to VEGFR1. The inhibition was particularly marked for the F3 antibody.

[0197] Figure 9 shows that F3 and H1 antibodies significantly inhibited VEGF-NF binding to VEGFR2. Conversely, BVZ did not inhibit VEGF-NF binding to VEGFR2. The inhibition was particularly marked for the F3 antibody.

[0198] Figure 10 shows that F3 and H1 antibodies significantly inhibited VEGF-NF binding to VEGFR3. Conversely, BVZ did not inhibit VEGF-NF binding to VEGFR3. The inhibition was particularly marked for the F3 antibody.

[0199] Example 8: Test for inhibition of VEGFR2 activation in the presence of VEGF-NF

[0200] Human endothelial cells (TIME) were seeded in 6-well plates at 250,000 cells per well.

[0201] The next day, confluent cells were washed twice with phosphate-buffered saline (PBS). Then, endothelial basal medium (EBM, without serum or growth factors, Promocell) was added for 2 hours to deprive the cells of any exogenous factors.

[0202] Cells were then stimulated for 30 minutes with 100 ng / mL of VEGF (human recombinant VEGF165, R&D Systems, Minneapolis, MN, USA) or 100 ng / mL of VEGF-NF (SEQ NO: 40 produced in HEK), in the presence or absence of antibodies.

[0203] The antibodies tested are: rabbit polyclonal antibody anti-VEGF-NF #2.2 & #1.1 (References [2] and [3]), P1-F3 (F3), P2-H1 (Hl) and bevacizumab. The antibody concentrations tested are: 10 pg / mL for all antibodies and 20 pg / mL for P1-F3 (F3), P2-H1 (Hl) and bevacizumab.

[0204] All treatment conditions were prepared in EBM medium and preincubated for 30 minutes at 37°C before application to cells. After treatment, cells were rinsed with cold PBS and then lysed with 1.5X Laemmli buffer containing 2% SDS, 10% glycerol, 60 mM Tris-HCl (pH 6.8), and Hait™ Phosphatase Inhibitor Cocktail (Thermo Fisher). DNA was fragmented by sonication. Lysates, supplemented with 0.002% bromophenol blue and 100 mM DTT, were heated to 96°C, separated by SDS-PAGE, and then transferred to PVDF membranes (Millipore).

[0205] Membranes were incubated with the following antibodies: pVEGFR2 (Tyrll75, CST, 2478S) and VEGFR2 (CST, 2479S), p44 / 42 MAPK (pERKl / 2, CST, 9102S), ERK1 / 2 (Sigma Aldrich, M8159). Reactive bands were visualized using Immobilon™ HRP Western Chemiluminescent Substrate (Merck Millipore®).

[0206] The results are shown in Figure 11.

[0207] Figure 11 shows that F3 and H1 antibodies did not prevent VEGFR2 phosphorylation in the presence of VEGF-NF. Conversely, bevacizumab blocked VEGFR2 phosphorylation in the presence of VEGF-NF, as did the polyclonal antibodies described in references [1] and [2].

[0208] These results show that F3 and H1 antibodies do not block the VEGFR2-mediated signaling pathway in the presence of VEGF-NF, unlike the other antibodies tested (polyclonal and bevacizumab). This confirms the unique properties of F3 and H1 antibodies.

[0209] Example 9: In vivo efficacy of F3 and Hl antibodies on mice grafted with 786-0 tumors

[0210] The efficacy of P2-H1 and P1-F3 antibodies was evaluated compared to axitinib, a standard conventional treatment for metastatic CRC (clear cell renal cell carcinoma). 786-0 cells (1x10 7cells) were implanted subcutaneously into the flank of female Nu / Nu mice. When the tumors began to grow exponentially, the mice were divided into the different groups to ensure a homogeneous distribution of the initial tumor volume. The animals were treated for 4 weeks and then remained for an additional week without treatment. The experimental groups were as follows: Isotype control (n=5, 7.5mg / kg, IP, 2 times per week), Axitinib (n=8, 10mg / kg, PO, 2 times per week), PIFS (n=8, 7.5mg / kg, IP, 2 times per week), P2-H1 (n=8, 7.5mg / kg, IP, 2 times per week). Tumor volume and weight of the mice were determined 3 times per week. At the end of the study, the animals were sacrificed, the tumor tissues were resected and weighed. The percentage of tumor growth inhibition (%TGI) was used to evaluate the anti-tumor efficacy and was calculated using the formula:

[0211] %TGI=[l-(tumor volume variation in the treated group / tumor volume variation in the control group)] x 100.

[0212] The results are presented in Figures 12 to 15.

[0213] The results show that, in this model, P2-H1 and P1-F3 antibodies have superior efficacy to axitinib (Figure 13 and 14) without weight loss in mice (Figure 12). In addition, the P2-H1 antibody was found to be more effective than P1-F3 in this experimental model.

[0214]

[0215] The results show that P1-F3 and P2-H1 antibodies were significantly more effective than the reference treatment with Axitinib.

[0216] The efficacy of the polyclonal antibody of references [2] and [3] and bevacizumab was also evaluated according to the protocol detailed above, with the difference of the following parameters: • n=6 for the control group and n=5 for the treated groups • Doses administered: 5mg / kg • Measurements were taken after 4 weeks of treatment

[0217] The %TGIs obtained were: 63.54% for the polyclonal and 17.16% for the bevacizumab.

[0218] The efficacy of the P1-F3 antibody is slightly lower than the polyclonal antibody, despite the fact that the immune coverage of the polyclonal antibody is significantly higher than that of the monoclonal antibody. The efficacy of the P2-H1 antibody is higher than that of the polyclonal antibody, which was not expected. Unsurprisingly, bevacizumab has very little activity compared to the other products tested.

[0219] Example 10: Specificity of P2-H1 and P1-F3 antibodies against VEGF-NF

[0220] The dissociation constant (K D) of the anti-VEGF-NF antibody P1-F3, the anti-VEG-NF antibody P2-H1 and the anti-VEGFA antibody Bevacizumab against the antigens VEGF-222 / NF (SEQ ID NO: 40), VEGF-A and VEGF-C was measured by octet technique. The antibodies were first loaded onto the sensors coated with anti-Fc, then after a reference step in kinetics buffer (K D ), they were then immersed in wells containing varying concentrations of antigen. Dissociation was measured by immersing the sensors in wells containing KB. Association and dissociation signals were recorded, and sensograms were were fitted using the appropriate 1:1 linkage model of the DataAnalysisHT VIO software.

[0221] The results are presented in the table below.

[0222] The results show that all antibodies evaluated bound to VEGF-222 / NF and none to VEGF-C. Bevacizumab bound to both VEGF222 / NF and VEGF-A. P1-F3 bound to VEGF222 / NF with an avidity of 9.3 nM. P1-F3 did not bind to VEGF-A (signal too weak, kinetic constants could not be calculated). P2-H1 bound strongly to VEGF-222 / NF with a calculated avidity of 6.7 nM. P2-H1 did not bind to VEGF-A (signal too weak, kinetic constants could not be calculated). SEQUENCE LISTING REFERENCES [1] Apte et al. VEGF in Signaling and Disease: Beyond Discovery and Development. Cell, 2019, 176, 1248-1264. 10.1016 / j.cell.2019.01.021 [2] W02022090414A1 [3] Christopher Montemagno, Jérôme Durivault, Cécile Gastaldi, Maeva Dufies, Valérie Vial, et al.. A group of novel VEGF splice variants as alternative therapeutic targets in renal cell carcinoma. Molecular Oncology, 2023, 17 (7), pp.1379-1401. 10.1002 / 1878- 0261.13401

Claims

CLAIMS 1. A monoclonal antibody or fragment thereof that binds to VEGF-NF (vascular endothelial growth factor-NF) of sequence SEQ ID NO: 40, said antibody or fragment thereof specifically binds to an epitope included in SEQ ID NO: 35 or to an epitope included in SEQ ID NO:

36.

2. Antibody or fragment thereof according to claim 1, comprising: - three heavy chain variable region CDRs of sequences: SEQ ID NO: 1 for CDR1 VH, SEQ ID NO: 2 for CDR2 VH, and SEQ ID NO: 3 for CDR3 VH; and - three light chain variable region CDRs of sequences: SEQ ID NO: 4 for CDR1 VL, DVS for CDR2 VL, and SEQ ID NO: 5 for CDR3 VL; said antibody or fragment thereof specifically binds to an epitope included in SEQ ID NO:

35.

3. Antibody or fragment thereof according to claim 1, comprising: - three heavy chain variable region CDRs of sequences: SEQ ID NO: 6 for CDR1 VH, SEQ ID NO: 7 for CDR2 VH, and SEQ ID NO: 8 for CDR3 VH; and - three light chain variable region CDRs of sequences: SEQ ID NO: 9 for CDR1 VL, KVS for CDR2 VL, and SEQ ID NO: 10 for CDR3 VL; said antibody or fragment thereof specifically binds to an epitope included in SEQ ID NO:

36.

4. An antibody or fragment thereof according to any one of the preceding claims, comprising: - a FRI H heavy chain hinge region selected from the group consisting of (i) SEQ ID NO: 11 or a sequence having at least 80% identity with SEQ ID NO: 11 and (ii) SEQ ID NO: 12 or a sequence having at least 80% identity with SEQ ID NO: 12; - a FR2 H heavy chain hinge region selected from the group consisting of (i) SEQ ID NO: 13 or a sequence having at least 80% identity with SEQ ID NO: 13 and (ii) SEQ ID NO: 14 or a sequence having at least 80% identity with SEQ ID NO: 14; - a FR3 H heavy chain hinge region selected from the group consisting of (i) SEQ ID NO: 15 or a sequence having at least 80% identity with SEQ ID NO: 15 and (ii) SEQ ID NO: 16 or a sequence having at least 80% identity with SEQ ID NO: 16; and - a FR4 H heavy chain hinge region selected from the group consisting of (i) SEQ ID NO: 17 or a sequence having at least 80% identity with SEQ ID NO: 17 and (ii) SEQ ID NO: 18 or a sequence having at least 80% identity with SEQ ID NO:

18.

5. An antibody or fragment thereof according to any one of the preceding claims, comprising: - a FRI L light chain hinge region selected from the group consisting of (i) SEQ ID NO: 19 or a sequence having at least 80% identity with SEQ ID NO: 19 and (ii) SEQ ID NO: 20 or a sequence having at least 80% identity with SEQ ID NO: 20; - an FR2 L light chain hinge region selected from the group consisting of (i) SEQ ID NO: 21 or a sequence having at least 80% identity with SEQ ID NO: 21 and (ii) SEQ ID NO: 22 or a sequence having at least 80% identity with SEQ ID NO: 22; - an FR3 L light chain hinge region selected from the group consisting of (i) SEQ ID NO: 23 or a sequence having at least 80% identity with SEQ ID NO: 23 and (ii) SEQ ID NO: 24 or a sequence having at least 80% identity with SEQ ID NO: 24; and - an FR4 L light chain hinge region selected from the group consisting of (i) SEQ ID NO: 25 or a sequence having at least 80% identity with SEQ ID NO: 25 and (ii) SEQ ID NO: 26 or a sequence having at least 80% identity with SEQ ID NO:

26.

6. An antibody or fragment thereof according to any one of claims 1, 2, 4 or 5, comprising: - a FRI H heavy chain hinge region of sequence SEQ ID NO: 11 or a sequence having at least 80% identity with SEQ ID NO: 11; - a FR2 H heavy chain hinge region of sequence SEQ ID NO: 13 or a sequence having at least 80% identity with SEQ ID NO: 13; - a FR3 H heavy chain hinge region of sequence SEQ ID NO: 15 or a sequence having at least 80% identity with SEQ ID NO: 15; - a FR4 H heavy chain hinge region of sequence SEQ ID NO: 17 or a sequence having at least 80% identity with SEQ ID NO: 17; - a FRI L light chain hinge region of sequence SEQ ID NO: 19 or a sequence having at least 80% identity with SEQ ID NO: 19; - a FR2 L light chain hinge region of sequence SEQ ID NO: 21 or a sequence having at least 80% identity with SEQ ID NO: 21; - a FR3 L light chain hinge region of sequence SEQ ID NO: 23 or a sequence having at least 80% identity with SEQ ID NO: 23; and - a FR4 L light chain hinge region of sequence SEQ ID NO: 25 or a sequence having at least 80% identity with SEQ ID NO:

25.

7. An antibody or fragment thereof according to any one of claims 1, 3, 4 or 5, comprising: - a FRI H heavy chain hinge region of sequence SEQ ID NO: 12 or a sequence having at least 80% identity with SEQ ID NO: 12; - a FR2 H heavy chain hinge region of sequence SEQ ID NO: 14 or a sequence having at least 80% identity with SEQ ID NO: 14; - a FR3 H heavy chain hinge region of sequence SEQ ID NO: 16 or a sequence having at least 80% identity with SEQ ID NO: 16; and - a FR4 H heavy chain hinge region of sequence SEQ ID NO: 18 or a sequence having at least 80% identity with SEQ ID NO:

18. - a FRI L light chain hinge region of sequence SEQ ID NO: 20 or a sequence having at least 80% identity with SEQ ID NO: 20; - a FR2 L light chain hinge region of sequence SEQ ID NO: 22 or a sequence having at least 80% identity with SEQ ID NO: 22; - a FR3 L light chain hinge region of sequence SEQ ID NO: 24 or a sequence having at least 80% identity with SEQ ID NO: 24; and - a FR4 L light chain hinge region of sequence SEQ ID NO: 26 or a sequence having at least 80% identity with SEQ ID NO:

26.

8. An antibody or fragment thereof according to any one of claims 1, 2, 4, 5 or 6, comprising: - a heavy chain variable region (VH) of sequence SEQ ID NO: 27 or a sequence having at least 80% identity with SEQ ID NO: 27; and - a light chain variable region (VL) of sequence SEQ ID NO: 29 or a sequence having at least 80% identity with SEQ ID NO:

29.

9. An antibody or fragment thereof according to any one of claims 1, 3, 4, 5 or 7, comprising: - a heavy chain variable region (VH) of sequence SEQ ID NO: 28 or a sequence having at least 80% identity with SEQ ID NO: 28; and - a light chain variable region (VL) of sequence SEQ ID NO: 30 or a sequence having at least 80% identity with SEQ ID NO:

30.

10. An antibody or fragment thereof according to any one of claims 1, 2, 4, 5, 6 or 8, comprising (i) a heavy chain of sequence SEQ ID NO: 46 or a sequence having at least 80% identity with SEQ ID NO: 46, and (ii) a light chain of sequence SEQ ID NO: 47 or a sequence having at least 80% identity with SEQ ID NO:

47.

11. An antibody or fragment thereof according to any one of claims 1, 3, 4, 5, 7 or 9, comprising (i) a heavy chain of sequence SEQ ID NO: 44 or a sequence having at least 80% identity with SEQ ID NO: 44, and (ii) a light chain of sequence SEQ ID NO: 45 or a sequence having at least 80% identity with SEQ ID NO:

45.

12. An antibody or fragment thereof according to any one of the preceding claims, said antibody or fragment thereof binds to VEGF-NF of sequence SEQ ID NO: 40 with a dissociation constant (K D) measured by SPR technology less than or equal to 100 nM.

13. An antibody or fragment thereof according to any one of claims 6, 8 or 10, said antibody or fragment thereof binds to VEGF-NF of sequence SEQ ID NO: 40 with a dissociation constant (K D ) measured by SPR technology less than or equal to 25 nM, preferably less than or equal to 10 nM.

14. An antibody or fragment thereof according to any one of claims 7, 9 or 11, said antibody or fragment thereof binds to VEGF-NF of sequence SEQ ID NO: 40 with a dissociation constant (K D ) measured by SPR technology less than or equal to 75 nM, preferably less than or equal to 50 nM.

15. An antibody or fragment thereof according to any one of the preceding claims, said antibody or fragment thereof also binds to one, two, three, four, five or six VEGF-NFs of sequence selected from the group consisting of SEQ ID NO: 37, 38, 39, 41, 42 and 43, preferably to the six VEGF-NFs of sequences SEQ IDs NO: 37, 38, 39, 41, 42 and 43.

16. An antibody or fragment thereof according to any preceding claim, said antibody or fragment thereof inhibits binding between VEGF-NF and one or more VEGF receptors (VEGFR).

17. A monoclonal antibody or antibody fragment which competes with the antibody or antibody fragment according to any one of the preceding claims for binding to VEGF-NF of sequence SEQ ID NO:

40.

18. A pharmaceutical composition comprising an antibody or fragment thereof according to any one of the preceding claims.

19. A polynucleotide or combination of two polynucleotides encoding a heavy chain variable region (VH) of an antibody or fragment thereof according to any one of claims 1 to 17 and a light chain variable region (VL) of an antibody or fragment thereof according to any one of claims 1 to 17.

20. Polynucleotide or combination of two polynucleotides according to claim 19, comprising a sequence SEQ ID NO: 31 coding for a chain variable region heavy chain (VH) and a sequence SEQ ID NO: 33 coding for a light chain variable region (VL).

21. Polynucleotide or combination of two polynucleotides according to claim 19, comprising a sequence SEQ ID NO: 32 coding for a heavy chain variable region (VH) and a sequence SEQ ID NO: 34 coding for a light chain variable region (VL).

22. A polynucleotide or combination of two polynucleotides encoding a heavy chain of an antibody or fragment thereof according to any one of claims 1 to 17 and a light chain of an antibody or fragment thereof according to any one of claims 1 to 17.

23. Polynucleotide or combination of two polynucleotides according to claim 22, comprising a sequence SEQ ID NO: 48 coding for a heavy chain (VH) and a sequence SEQ ID NO: 49 coding for a light chain (VL).

24. Polynucleotide or combination of two polynucleotides according to claim 19, comprising a sequence SEQ ID NO: 50 coding for a heavy chain (VH) and a sequence SEQ ID NO: 51 coding for a light chain (VL).

25. Expression vector comprising a polynucleotide or a combination of two polynucleotides according to any one of claims 19 to 24.

26. Cell comprising an expression vector according to claim 25.

27. An antibody or fragment thereof according to any one of claims 1 to 17 or a composition according to claim 18, for use as a medicament.

28. An antibody or fragment thereof according to any one of claims 1 to 17 or a composition according to claim 18, for use in the treatment of cancer in a patient, for example for use in the treatment of a VEGF-NF+ cancer.

29. An antibody or fragment thereof or composition for use according to claim 28, said cancer is selected from cancers of the nervous system, gastrointestinal cancers, liver cancers, gynecological cancers, breast cancers, melanomas, urinary system cancers, respiratory and ENT cancers, head and neck cancers, bone cancers, hematological cancers.

30. An antibody or fragment thereof or composition for use according to any one of claims 28 or 29, said patient is refractory to treatment with bevacizumab.

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