Neuropilin-1 peptide-based antibodies focused on novel epitopes associated with glycosaminoglycan-modified neuropilin-1 and methods of use thereof

Abstract

The present invention relates to the field of NRP-1 peptide vaccines that correspond to disease-associated epitopes and have the ability to elicit immune responses (e.g., generate anti-NRP-1 antibodies specific for GAG-modified NRP-1 that can block the DNA damage response (DDR)). In particular, NRP-1 peptide vaccines specific for epitopes present in the leptin-binding domain of NRP-1 and anti-NRP-1 antibodies derived therefrom (NRP-1 peptide antibodies) have the ability to enter the nucleus and inhibit the DNA damage response (DDR), possessing properties useful in the treatment of diseases involving the NRP-1 / OBR complex. The NRP-1 peptide vaccines and NRP-1 peptide antibodies are useful in diagnosis and therapy, particularly in diseases related to the DNA damage response and glycosaminoglycans.

Description

Detailed Description of the Invention

[0001] The present invention relates to the field of NRP-1 peptide vaccines that correspond to disease-associated epitopes and can elicit immune responses (e.g., the production of anti-NRP-1 antibodies specific for GAG-modified NRP-1 that can block the DNA damage response (DDR)). In particular, NRP-1 peptide vaccines and anti-NRP-1 antibodies derived therefrom (NRP-1 peptide antibodies) specific for epitopes located within the leptin-binding domain of NRP-1 have the ability to enter the cell nucleus and block the DNA damage response (DDR), and possess properties that make them attractive for the treatment of diseases involving the NRP-1 / OBR complex. The NRP-1 peptide vaccines and NRP-1 peptide antibodies are particularly attractive for the diagnosis and treatment of diseases related to the DNA damage response and glycosaminoglycans.

[0002] FIELD OF THE INVENTION Immunotherapy is one of the most powerful tools for the treatment of autoimmune diseases, infectious diseases, and cancer. Various approaches have been used in the development of immunotherapies, including monoclonal antibodies, cell therapy, cytokines, and vaccines.

[0003] In general, immunotherapeutic monoclonal antibodies are widely used, especially in cancer treatment. Compared with conventional antibodies, immunotherapy based on peptide antibodies or peptide vaccines has become a more useful approach for clinical diagnosis and targeted therapy. This is because they can act directly on either intracellular or extracellular domains, conserved regions, or specific conformations of peptides, or on post-translational modifications that are difficult to control when using proteins. Immunotherapies based on peptide vaccine strategies also aim to target cell-specific neoantigens or antigens expressed by cancer cells or infected cells to activate specific immune cells (e.g., lymphocytes (T cells and B cells), dendritic cells (DCs), natural killer cells (NKs), and macrophages). In the case of peptide-based vaccines, activation of the patient's immune system depends on the immunogenicity of the peptide, which is correlated with numerous factors arising from its composition, length, route of administration, and, more importantly, functional domains of the protein (e.g., ligand-binding domains, post-transcriptional modification domains, or cell-cell contact domains) (Lu RM et al., J Biomed Sci., 2020, 27:1; Zhang L et al., Theranostics, 2019, 9:7807-7825; Trier N et al., Int J Mol Sci., 2019, 20:6289).

[0004] In the present invention, NRP-1 peptide (Npep), identified by molecular docking as an NRP-1 peptide corresponding to the leptin-binding domain for NRP-1 / OBR complex signaling (WO2017050793), has shown great potential in the application of peptide-based vaccines due to its antigenic properties related to the functional domain of NRP-1 and its ability to induce neutralizing antibodies with high specificity against GAG-modified NRP-1, which may be involved in autoimmune diseases, viral infections, and cancer.

[0005] The antigenic potential of the NRP-1 peptide (Npep) of the present invention was demonstrated by inducing the production of anti-NRP-1 antibodies that neutralize leptin-induced NRP-1 / OBR signaling in a mouse model of peptide vaccination and by possessing immunotherapeutic properties in a mouse cancer model.

[0006] The cell surface glycoprotein neuropilin-1 (NRP-1), also known as CD304, is widely recognized as a promising target not only for cancer therapy targeting cancer cells, angiogenesis, or immune cells, but also for metabolic, inflammatory, and infectious diseases (Wilson AM et al., Sci Immunol., 2018, 3:4626; WO 2015 / 124588).

[0007] Neuropilin-1 (NRP-1) is a protein composed of (i) a large extracellular domain consisting of three major segments: two CUB domains (designated a1 / a2) and two coagulation factor V / VIII domains (designated FV / VIII or b1 / b2), followed by a MAM domain (designated c); and (ii) a short cytoplasmic tail (CP) lacking catalytic activity but containing a C-terminal SEA sequence that serves as a consensus binding motif for proteins containing PDZ (PSD-95, Dlg, ZO-1) domains, which promotes complex formation with signaling components (Gu C et al., J Biol Chem., 2002, 277:18069-76; Geretti E et al., Angiogenesis., 2008, 11:31-9; Prahst C et al., J Biol Chem 2008, 283:25110-25114).

[0008] As a signal transduction-defective protein, it has been implicated in angiogenesis (Soker S et al., Cell., 1998, 92:735-45), immune response (Tordjman R et al., Nat Immunol., 2002, 3:477-82; Lepelletier Y et al., Eur J Immunol., 2006, 36:1782-93; Lepelletier Y et al., Proc Natl Acad Sci U S A., 2007, 104:5545-50), neurogenesis (Kitsukawa T et al., Development., 1995, 121:4309-18; He Z and Tessier-Lavigne M., Cell., 1997, 90:739-51), and viral entry (Ghez D et al., J Virol., 2006, 104:5545-50). 80:6844-54; Cantuti-Castelvetri L et al., Science., 2020, 370:856-860; Wang HB et al., Nat Commun., 2015, 6:6240) and in hematopoietic regulation (Tordjman R et al., Blood., 1999, 94:2301-9 and Belaid-Choucair Z et al., Stem Cells. 2008, 26:1556-64). The multifunctionality of NRP-1 in the regulation of hematopoiesis (Tordjman R et al., Blood., 1999, 94:2301-9 and Belaid-Choucair Z et al., Stem Cells. 2008, 26:1556-64) is related to its interaction properties with ligands and receptors that possess specific binding domains, which induce specific signaling pathways corresponding to specific functions.

[0009] Several studies have described NRP-1 as a potential therapeutic target for cancer treatments that inhibit metastasis, drug resistance, and immune checkpoints, as well as for the treatment of viral infections (Chaudhary B et al., Cancer Immunol Immunother., 2014, 63:81-99; Chuckran CA et al., J Immunother Cancer., 2020, 8:e000967; Dumond A et al., Front Cell Dev Biol., 2020, 8:662). Drug candidates in clinical development targeting NRP-1 function are primarily antibodies that inhibit the binding of its two main ligands, semaphorins and VEGF: Astellas Pharma's ASP1948 / PTZ329 (Delgoffe GM et al., Nature., 2013, 501:252-6; Yano H et al., Immunology., 2019, 157:232-247; NCT03565445) and Roche's MNRP-1685A (Shumaker RC et al., Pharmacol., 2014, 73:1109-17; NCT00747734; NCT00954642). These two antibodies differ in their ability to bind exclusively to the a1 / a2 or b1 / b2 domains of NRP-1, respectively. These correspond, but not exclusively, to the semaphorin (Sema3A, Sema4A) and VEGF, VEGFB and PlGF2 binding domains to NRP-1.

[0010] Other strategies targeting NRP-1 (e.g., small molecules or peptide sequences) are also under development but do not have the properties of the present invention (Liu SD et al., Chin Med J (Engl)., 2020, 134:508-517).

[0011] Although many ligands and their corresponding receptors (SEMA3 / PLEXIN, VEGF / VEGFR, PlGF / VEGFR, HGF / cMET, TGFβ1 / TGFβRs, PDGF / PDGFR, FGF / FGFR2, Galectin, EGF / EGFR) share the same interaction domain with their common partner, NRP-1 (Dumond A et al., Front Cell Dev Biol., 2020, 8:662), the latter likely possess specificity that distinguishes them from each other, possessing peptide sequences specific to each ligand and receptor interaction with NRP-1.

[0012] Interestingly, the discovery of leptin and its corresponding receptor OBR (leptin receptor) supported this hypothesis by showing that although leptin and VEGF bind to the same domain (b1 domain), they can simultaneously bind to NRP-1, and therefore the interaction sequences must be different (WO2017050793; Polypeptide capable of inhibiting the binding between leptin and neuropilin-1).

[0013] It has been clearly shown that Sema3A competes with VEGF for binding to NRP-1 because it binds not only to the major a1 / a2-binding domain but also to the b1 domain. Meanwhile, Wei-Ching Liang et al. (WO2017050793) successfully generated antibodies that specifically bind to the a1-a2 NRP-1 domain or the b1-b2-NRP-1 domain (designated a-NRP1_SEM and a-NRP1_VE, respectively, as described below) (Liang WC et al., J Mol Biol. 2007 366:815-29). These antibodies were compared with the antibody of the present invention (designated a-NRP1_LEP, as described below) that specifically binds to a peptide sequence in the b1 domain that corresponds to leptin binding.

[0014] Although leptin and VEGF share the same b1-binding domain, their binding to NRP-1 is non-competitive, and it is speculated that Sema3a also has a distinct NRP-1 binding sequence compared to leptin.

[0015] In addition to the organization of the NRP-1 protein as a large extracellular domain composed of three major segments, alternative splicing of the primary NRP-1 transcript produces multiple NRP-1 protein isoforms that differ not only in morphology (soluble and membrane-bound), but also in size, ranging from 60 to 923 amino acids (Rossignol Met al., Genomics. 2000, 70:211-22; Cackowski FC et al., Genomics. 2004, 84:82-94; Gagnon ML et al., Proc Natl Acad Sci USA. 2000, 97:2573-8; Tao Q et al., Angiogenesis. 2003, 6:39-45), and post-translational modification properties. These post-translational modifications include glycosylation (Wu MH et al., Sci Rep., 2017, 7:11006), such as N-glycosylation (Huang X et al., Nat Commun., 2019, 10:3708) and O-glycosylation (Zacchi LF et al., Glycoconj J., 2016, 33:359-76), (Windwarder M et al., Glycoconj J. 2016, 33(3):387-97), and modifications with glycosaminoglycans (GAGs) such as chondroitin sulfate and heparan sulfate (Frankel P et al., EMBO Rep., 2008, 9:983-9; Shintani Y et al., EMBO J., 2006, 25:3045-55). In addition to Ser612, the major site of NRP1 CS-GAG modification, where point mutation to alanine induces complete loss of CS-GAG NRP1 modification, Frankel P et al. showed that point mutations to alanine at Ser115, Ser240, Ser283, Ser432, Ser439, Ser612, and Ser729 (mainly at Ser240 and Ser432) induce partial reduction in the level of NRP1-CS. These different CS-GAG modification sites may play important roles in NRP-1-related diseases, as reported in the detailed description of the present invention.

[0016] Existing antibodies target tumor progression by blocking VEGF binding to NRP-1, thereby blocking NRP-1 / VEGFR2 signaling in endothelial cells and inhibiting angiogenesis (Patnaik A et al., Cancer Chemother Pharmacol., 2014, 73:951-60; NCT00747734; NCT00954642), or by inhibiting semaphorin (SEM34 / SEMA4A) binding to NRP-1, thereby blocking the NRP-1 / plexin signaling pathway and inhibiting regulatory T lymphocytes (Tregs) to activate the immune response (Delgoffe GM, Nature., 2013, 501:252-6; Yano H et al., Immunology., 2019, 157:232-247; NCT03565445).

[0017] Although NRP-1 is an attractive therapeutic target in the treatment of cancer or infectious diseases, to date no antibody has received clinical approval or is part of a therapeutic armamentarium.

[0018] BRIEF DESCRIPTION OF THE INVENTION The present invention provides a novel class of NRP-1 antibodies that can enter the nuclei of cancer cells to induce DNA damage, centromere destabilization, and telomere shortening, and that can recognize GAG-modified NRP-1.

[0019] The first object of the present invention is to an Npep peptide with the sequence EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), or a peptide selected from N1, which has the sequence streptavidin-biotin-EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), N2, which has the sequence EGNKPVLFQGNTNPTDVVVAVFPK-biotin-streptavidin (SEQ ID NO: 54), or N3, which has the sequence EGNKPVLFQGNTNPTDVVC-KLH (SEQ ID NO: 55), The present invention relates to an anti-NRP-1 antibody or antibody fragment capable of recognizing

[0020] In a preferred embodiment, the anti-NRP-1 antibody or antibody fragment is capable of entering the nucleus of a cell.

[0021] The antibody of the present invention - a first immunization step: administering at least once to an immunocompetent non-human animal an immunogenic peptide sequence, for example an Npep selected from the group consisting of N1 having the sequence streptavidin-biotin-EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), N2 having the sequence EGNKPVLFQGNTNPTDVVVAVFPK-biotin-streptavidin (SEQ ID NO: 54), or N3 having the sequence EGNKPVLFQGNTNPTDVVC-KLH (SEQ ID NO: 55), - selecting animals with a desired phenotype, such as an obese phenotype; - recovering the antibody from said animal; - screening for antibodies capable of specifically recognizing the target protein and the corresponding peptide sequence, for example the Npep peptide as defined above, or a peptide selected from N1, N2 or N3, - selecting animals producing said antibodies capable of specifically recognizing said corresponding peptide sequence and said target protein; - a second immunization step: administering to the selected animals at least once the same immunogenic peptide as in the first immunization step; - recovering lymphocytes from the animal that has undergone the two-step immunization; - producing hybridomas from said lymphocytes; - a step of selecting hybridomas producing antibodies capable of specifically recognizing the target protein by cells expressing the protein, etc.; The compound is obtained by a method comprising:

[0022] The antibodies of the present invention may be obtained by using the Npep peptide as defined above, or a peptide selected from N1, N2 or N3.

[0023] In a preferred embodiment, the Npep is an N3 peptide.

[0024] In certain embodiments, the present invention relates to anti-NRP-1 antibodies or antibody fragments capable of specifically binding to NRP-1 isoforms containing GAG modifications at positions S612, and / or Ser115, and / or Ser283, and / or Ser240, and / or Ser432, and / or Ser439, and / or Ser729. In preferred embodiments, the recognized GAG modifications are at positions Ser240 and / or Ser432.

[0025] A second object of the present invention relates to an NRP-1 peptide corresponding to an epitope contained within the three-dimensional structure of post-transcriptionally modified NRP-1 isoforms, particularly within the Npep sequence. More specifically, the peptide is the N3 peptide, which is derived from the three-dimensional structure of glycosaminoglycan (GAG)-modified NRP-1 isoforms and has the sequence EGNKPVLFQGNTNPTDVVC-KLH (SEQ ID NO: 55).

[0026] The NRP-1 peptide corresponds to a functional domain of NRP-1 associated with NRP-1 / OBR complex signaling, particularly the leptin-binding domain. In certain embodiments, the NRP-1 peptide corresponds to the leptin-binding domain associated with NRP-1 / OBR complex signaling.

[0027] The peptides of the present invention, Npep peptide having the sequence EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), N1 peptide having the sequence streptavidin-biotin-EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), N2 peptide having the sequence EGNKPVLFQGNTNPTDVVVAVFPK-biotin-streptavidin (SEQ ID NO: 54), and N3 peptide having the sequence EGNKPVLFQGNTNPTDVVC-KLH (SEQ ID NO: 55), can be used as vaccines to induce immune responses and block the DNA damage response (DDR).

[0028] In the context of the present invention, the immune response involves at least an anti-NRP-1 antibody capable of targeting the leptin-binding domain on NRP-1, thereby blocking the signaling pathway of the NRP-1 / OBR complex, which is co-expressed in various types of cancer cells and cells of the tumor microenvironment (e.g., T lymphocytes, macrophages, cancer-associated fibroblasts) (Figure 1).

[0029] The present invention also relates to anti-NRP-1 antibodies generated by a vaccination strategy involving NRP-1 peptides, as defined above.

[0030] Another object of the invention relates to the use of an Npep peptide as defined above, or a peptide selected from N1, N2 or N3, for the production of an antibody according to the invention; and to a method for the production of an antibody according to the invention, based on a two-stage immunization with such a peptide and a selection step.

[0031] Anti-NRP-1 antibodies are characterized by their ability to inhibit the NRP-1 signaling pathway. The signaling pathway may correspond to any functional domain of NRP-1 involved in any signaling pathway and its corresponding ligand and receptor. In a preferred embodiment, this functional domain corresponds to the NRP-1 / OBR signaling pathway, more precisely, the functional domain corresponds to an epitope located on a functional domain of the NRP-1 protein isoform involved in leptin / OBR signaling, in particular the leptin recognition domain located on the b1 domain of the NRP-1 protein isoform.

[0032] In further embodiments, the anti-NRP-1 antibodies of the invention can specifically bind to GAG-modified NRP-1 isoforms on any epitope associated with any functional domain of the NRP-1 isoform associated with any signaling pathway and its corresponding ligand and receptor. The GAG ​​modification on the NRP-1 isoform can correspond to the presence of glycosylation associated with sulfate modification.

[0033] The glycosylation may correspond to O-glycosylation or N-glycosylation. The sulfate modification may correspond to the presence of chondroitin sulfate and / or heparan sulfate. As used herein, the antibody of the present invention is defined as "a-NRP1_LEP" in that it specifically inhibits leptin binding to NRP-1, as distinguished from the antibody "a-NRP1_VE" that exclusively inhibits VEGF binding to NRP-1, or the antibody "a-NRP1_SEM" that exclusively inhibits semaphorin binding to NRP-1.

[0034] Interestingly, neither a-NRP1_SEM nor a-NRP1_VE was able to bind to the peptide sequence used to generate a-NRP1_LEP, an antibody that inhibits leptin binding to NRP-1 (Table 2). In particular, the a-NRP1_LEP antibody is compared to TAB-264 (a-NRP1_VE), which exclusively inhibits VEGF binding to NRP-1, corresponding to Vesencumab (MNRP-1685A), an antibody clinically tested by Roche (Liang WC et al., J Mol Biol., 2007, 366:815-29; Weekes CD et al., Invest New Drugs., 2014, 32:653-60; Xin Y et al., Clin Cancer Res., 2012, 18:6040-8), and to YW64.3 (a-NRP1_SEM), which exclusively inhibits semaphorin binding to NRP-1, corresponding to Vesencumab (MNRP-1685A), an antibody clinically tested by Astellas (Liang WC et al., J Mol Biol., 2007, 366:815-29). ASP1948 (PTZ-329) (NCT03565445) is comparable to an antibody developed by Potenza (WO2018119171A1) in clinical trials (Delgoffe GM et al., Nature., 2013, 501:252-6; Chuckran CA et al., J Immunother Cancer., 2020, 8:e000967).

[0035] In contrast to a-NRP1_VE and a-NRP1_SEM, the antibody a-NRP1_LEP of the present invention exhibits high specificity for the GAG-modified NRP-1 expressed on the surface of various cancer cells (in particular, on circulating cancer stem-like cells (CTCs)) and associated with either chondroitin or heparan sulfate.

[0036] An exception was observed in the A459 lung cancer cell line, where a-NRP1_VE was able to recognize GAG-modified NRP-1, whereas a-NRP1_LEP, particularly the glycosylated form of NRP-1 (≦150 kDa), showed a poor ability to recognize said GAG-modified NRP-1 (>150 kDa), as confirmed in the CRISPR-Case9 NRP-1-A549 cell line (Figure 4A–C).

[0037] Thus, specificity can be inferred, related not only to the composition of GAGs (N-glycosaminoglycans and O-glycosaminoglycans), particularly chondroitin sulfate (CS) and heparan sulfate (HS), but also to their size, modifications by acetylation, sulfation, and / or epimerization (Morla S., Int J Mol Sci., 2019, 20(8):1963; Chen J et al., Front Cell Dev Biol., 2021, 30;9:760532), and the binding site for the core protein designated neuropilin-1. This specificity may also depend on the cell type and the responsiveness of the ligand and its corresponding receptor; VEGF / VGFR2 signaling has already been reported to associate with the GAG ​​binding site serine 612 in vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Shintani et al. report that although glycosylation increases VEGF binding in both cell types (ECs and SMCs), the different GAG composition of NRP-1 mediates opposite responsiveness to VEGF.

[0038] By comparing the effects of corresponding neutralizing antibodies on different NRP-1 partners, different responses were observed. The neutralizing antibodies were a-NRP1_LEP, which inhibits leptin signaling, a-NRP1_VE, which inhibits VEGF signaling, and a-NRP1_SEM, which inhibits semaphorin signaling. As shown in Figure 4A-a, the antibody a-NRP1_LEP of the present invention can detect GAG-modified NRP-1 in CTC cells, whereas a-NRP1_VE could not. However, the prior art antibody a-NRP1_VE detects GAG-modified NRP-1 with higher specificity than the antibody a-NRP1_LEP of the present invention in the A549 cell line (Figure 4A-a).

[0039] Interestingly, in contrast to the prior art antibody a-NRP1_VE, we observed that the antibody a-NRP1_LEP of the present invention failed to detect doxycycline-induced unmodified NRP-1 and GAG-modified NRP1-FS, which correspond to the O-glycosylation site S612 (Figure 4A-b). Hendricks C et al. reported that S612 is associated with VEGF-induced angiogenesis, whereas different results were obtained in human breast cancer cell line MDA-MB231 and prostate cancer cell line PC3 when compared with the doxycycline-induced GAG-deficient form of S612, NRP1-Δ7 (Hendricks C et al., PLoS One, 2016, 11(10):e0165153).

[0040] However, the GAG-modified NRP-1 detected by our antibody (a-NRP1_LEP) is associated with either chondroitin sulfate or heparan sulfate (Figure 5), which likely corresponds to GAG modifications at serine S612, Ser240, and / or Ser432. However, the heavy size (>250 kDa) of the detected GAG forms suggests that they may have different GAG compositions and / or modification levels. NRP-1 has also been reported to be heavily glycosylated in the c / MAM domain (628-646), which is close to the known S612 glycosaminoglycan, and this may influence its pathophysiological properties (Windwarder M et al., Glycoconj J., 2016, 33(3):387-97).

[0041] Therefore, the specificity of glycosylation sites on NRP-1 and the impact of GAG composition on the function of GAG-modified NRP-1 should be considered in the development of targeted therapies. For example, in NRP-1 splice variants (deleting exons 4 and 5), loss of N-glycosylation at asparagine residues Asn150 or Asn261 results in a novel function involving Met and HGF, rather than GAG-modified NRP-1 at serine S612, which is involved in VEGF (Huang X et al., Nat Commun., 2019, 10(1):3708).

[0042] To propose a targeted NRP1 peptide vaccine with GAG-modified NRP-1 specific for each corresponding ligand, we aim to identify specificities that can be expanded to other known neuropilin-1 partners, such as TGFB, VEGF B, VEGFC, VEGFRs, TGFB RI / II / III, PlGF, PDGFs, PDGFRs, EGF, EGFR, FGF, FGFR, HGF, HGFR, SEMA4, and Plexin A1, as well as the recently identified partners PDL-1 / 2 and PD1 (Rossignol J et al., iScience., 2022, 25(6):104353).

[0043] In the case of viral infections, NRP-1 has been associated with HTLV-1, which is known to be involved in the entry of adult T-cell leukemia (ATL), SARS-CoV-2, and EBV viruses. In the case of NRP-1-dependent HTLV-1 and EBV entry, the mechanism of action involves the VEGFR and EGFR / RAS / ERK signaling pathways, respectively, and has links to glycosaminoglycans and the DNA damage response. Therefore, the identification of NRP-1 sequences associated with specific GAG-modified NRP-1s for each virus entry and DNA damage response, as reported for HTLV-1 (Boxus M et al., Retrovirology, 2012, 5;9:2), could be a highly innovative strategy for antiviral vaccines for cancer induction or other infectious diseases (Wang HB et al., Nat Commun., 2015, 11;6:6240; Jones KS et al., Viruses, 2011, 3(6):794-810; Gudowska-Sawczuk Met al., J Clin Med., 2021, 24;10(13):2772).

[0044] In vitro characterization of a-NRP1_LEP compared to a-NRP1_VE and a-NRP1_SEM in human and mouse cancer cell lines and in human stem cell-like cells derived from circulating tumor CTCs (Table 4) (Cayrefourcq L et al., Cancer Res., 2015, 75:892-901; Cayrefourcq L et al., Mol Cancer., 2021, 20:30; Soler A et al., Sci Rep., 2018, 8:15931) revealed unexpected properties of the antibody of the present invention. Indeed, we confirmed that the a-NRP1_LEP antibody penetrates into the nuclei of human and mouse cancer cell lines (breast, lung, colon, and prostate cancer cells) (Figures 6.1 and 6.2), as well as human peripheral blood mononuclear cells (PBMCs) from healthy donors (data not shown), demonstrating that the antibody can penetrate into the nuclei regardless of cell type specificity. This nuclear penetration is NRP-1 dependent, as demonstrated in wild-type and partial NRP-1 knockout A549 cell lines (Figures 7.1Aa and 7.1Ab). Interestingly, nuclear penetration of the a-NRP1_LEP antibody is mediated by the leptin-dependent NRP-1 / OBR complex, as observed by a dose-dependent increase in NRP-1 / OBR complex formation in a-NRP1_LEP-treated cells. In contrast, a-NRP1_VE has no effect (Figures 7.1Ba and 7.1Bb).

[0045] This observation is consistent with the leptin-induced entry of the a-NRP1_LEP antibody and NRP-1 protein into the cell nucleus (Figure 7.2), and is also consistent with previously reported data showing leptin-induced entry of the NRP-1 / OBR complex into the nucleus in a dose-dependent manner (WO / 2015 / 124588, Methods and pharmaceutical compositions for treating diseases mediated by the NRP-1 / OBR complex signaling pathway).

[0046] Regarding the effects of (i) leptin and its specific receptor OBR, (ii) glycosaminoglycans, and (iii) neuropilin-1 (NRP-1) on cancer cells, as reported in independent literature; regarding the high recognition of GAG-modified NRP-1 by the antibody a-NRP1_LEP of the present invention in CTCs compared to other cell lines and the prior art antibodies a-NRP1_VEGF and a-NRP1_SEM; and regarding the increased expression of NRP-1 and leptin receptor (OBR) genes in CTC415E, which is associated with drug resistance and treatment failure, compared to CTC41 collected before treatment. We decided to focus our research on the effect of nuclear entry of a-NRP1_LEP on cell fate in circulating cancer stem-like cells (CTC-41) from the same patient before treatment and in drug-resistant circulating cancer stem-like cells (CTC-415E) after chemotherapy and antiangiogenic treatment (Takakura N., Cancer Sci., 2012, 103:1177-81; Zheng Q et al., Endocr Relat Cancer., 2013, 20:797-808; Mishra AK et al., PLoS One., 2017, 12:e0178454; Tang YH et al., Breast Cancer Res., 2022, 24:8; Vitale D et al., FEBS J., 2019, 286:2870-2882).

[0047] Thus, our studies on the effects of a-NRP1_LEP on nuclear entry (Figures 6.1 and 6.2) and chromatin binding (Figure 10) in vitro and in vivo (Figure 11A and B) revealed genotoxic effects by inducing DNA damage, centromere destabilization, and telomere shortening in circulating tumor cells (CTCs) collected before and after treatment from patients resistant to chemotherapy and bevacizumab, both in vitro (Figure 8A-D) and in vivo (Figure 12A). However, these effects were not observed in human peripheral blood mononuclear cells (PBMCs) collected from healthy donors. This may be explained by the lack of excessive DNA damage response activity in healthy PBMCs (Figure 8E) compared with cancer cells. Additionally, the telomere-shortening effect of the a-NRP1_LEP antibody correlates with leptin, which has previously been reported as a regulator of hTERT gene expression and activity in cancers (e.g., breast cancer and hepatocellular carcinoma) (Stefanou N et al., BMC Cancer., 2010, 1186 / 1471-2407-10-442; Ren H et al., Biochem Biophys Res Commun., 2010, 394(1):59-63). Given that the a-NRP1_LEP antibody mimics leptin by binding to NRP-1 at the same binding domain (b1) involved in NRP-1 / OBR complex signaling and nuclear entry, we investigated whether leptin has a genotoxic effect on CTCs. As expected, no DNA damage, centromere destabilization, or telomere shortening was observed in CTCs treated with 10 nM leptin (Figure 8D).

[0048] Molecular characterization of CTCs by Dr. Catherine Alix-Panabieres and colleagues revealed the important role of genes regulating energy metabolism and DNA repair (Alix-Panabieres C et al., Clin Chem., 2017, 63:700-713). This correlates with the role of leptin in regulating energy metabolism (de Candia P et al., J Exp Med., 2021, 218:e20191593) and the potential neutralizing effect of the antibodies of the present invention on DNA repair. This is supported by the enrichment of proteins involved in DNA repair pathways, such as double-strand break (NHEJ:XRCC5 / XRCC6, HR:WRN) and single-strand break (BER:LIG3, NER:XPC) repair mechanisms, as revealed by mass spectrometry analysis of NRP-1 immunoprecipitated with a-NRP1_LEP antibody from CTC and A549 cell extracts compared to a-NRP1_VE (Table 5).

[0049] PARP1 protein is known to be involved in multiple DNA repair mechanisms and was found to be abundant in both cell types (CTC and A549) when a-NRP1_LEP targeted leptin-dependent NRP-1 / OBR complex signaling. In contrast, PARP1 protein did not increase when a-NRP1_VE targeted VEGF-induced NRP-1 / VEGFR signaling. This observation is consistent with previous data suggesting that NRP-1 is involved in resistance to PARPi (olaparib), and there is no evidence of a direct interaction between NRP-1 and PARPs (Vescarelli E et al., J Exp Clin Cancer Res., 2020, 2;39(1):3). This observation leads us to conclude that the mechanism of action of the leptin-dependent NRP-1 / OBR complex signaling pathway in DNA repair is completely distinct from that of the VEGF-induced NRP-1 / VEGFR complex signaling pathway. The VEGF / NRP-1 / VEGFR signaling pathway has been reported to regulate the expression of PARP1 mRNA and protein (Hoermann M et al., J Thromb Haemost., 2011, 9:1391-403; Mey L et al., Neurobiol Dis., 2013, 59:111-25). Meanwhile, leptin / NRP-1 / VEGFR directly interacts with the DNA repair machinery, as confirmed by DNA damage manifested by chromosome shattering, centromere destabilization, and telomere shortening after treatment of cells with the neutralizing antibody a-NRP1_LEP (Figures 8A-D). LC-MS / MS analysis using our antibody a-NRP1_LEP revealed some very interesting characteristics of cancer stem-like cells. In contrast to non-stem cell A549, CTCs possess key factors of different DNA repair mechanisms (NHEJ, HR, BER, NER, MMR, etc.) which may explain the resistance of cancer stem cells to multiple genotoxic therapies such as radiotherapy and chemotherapy observed in different cancer types involved in the DNA damage response (DDR) (Table 5) (Tayoun T et al., JCI Insight., 2022, 7(11)).The association between high GAG-NRP-1 morphology and global DNA repair mechanisms, as revealed by the antibody a-NRP1_LEP of the present invention in circulating cancer stem-like cells (CTCs), highlights the relationship between glycosaminoglycans and the DNA damage response (DDR), a mechanism reported to be associated with cancer stem cell therapy resistance and cancer recurrence (Vitale D et al., FEBS J., 2019, 286:2870-2882; Ahrens TD et al., Front Cell Dev Biol., 2020, 8:749).

[0050] In vivo analysis of the effects of the antibody a-NRP1_LEP of the present invention using the 4T1 breast cancer cell line in a syngeneic mouse model (BALB / c ByJ CRLF) showed that it significantly increased the expression of granzyme B (GrB) and perforin (Perf), as well as CD8 + GrB + and CD4 + Foxp3 - evidenced by cell-cell interactions between CD4 + Increased infiltration of T lymphocytes into tumors and activation of CD8+ T lymphocytes revealed immunomodulatory effects associated with reduced lung metastasis, as shown in Figures 13A-D.

[0051] The immunomodulatory effect of the antibody a-NRP1_LEP of the present invention is related to its genotoxic effect on cancer cells and may be explained by indirectly activating immune cells (indirect mechanism of action) or by acting directly on immune cells in the microenvironment that express the NRP-1 / OBR complex on their surface. Interestingly, a correlation between inhibition of the DNA damage response (DDR) and activation of the anti-cancer immune response has already been reported (Chabanon RM et al., Nat Rev Cancer., 2021, 21:701-717).

[0052] Different pathways have been described for the activation of innate and adaptive antitumor immune responses by DNA damage caused by DDR deficiency or DDR inhibitor therapy, including (i) promotion of de novo antigen generation, (ii) modulation of the immune synapse by upregulation of programmed death-ligand 1 (PD-L1), (iii) activation of cytoplasmic immunity via cGAS-STING signaling, and (iv) immunogenic cell death through exposure and release of multiple damage-associated molecular patterns (e.g., surface-exposed calreticulin), which promote the recruitment and activation of antigen-presenting cells (Chabanon RM et al., Nat Rev Cancer., 2021, 21(11):701-717; Fucikova J et al., Cell Death Dis., 2020, 11(11):1013; Galluzzi L et al., J Immunother Cancer., 2020, 8(1):e000337).

[0053] The reduction in lung metastasis may be the result of a DNA damage-dependent immune response, as evidenced by the detection of micronuclei in cells treated with a-NRP1_LEP. When a-NRP1_LEP was tested in immunodeficient mice using the 4T1 cancer cell line, it had no effect on reducing lung metastasis, indicating that the immune system is crucial for the efficacy of a-NRP1_LEP (the antibody of the present invention). These observations suggest that the leptin-induced NRP-1 / OBR complex is an excellent target for activating antitumor immune responses that are as important as those of PD-1, PD-L1, and anti-CTL4. While therapeutic anti-PD-1 antibodies such as Keytruda (pembrolizumab) and Opdivo (nivolumab) have a direct effect on reactivating exhausted cytotoxic CD8 lymphocytes and increasing their infiltration into tumors, the a-NRP1_LEP antibody inhibits the proliferation of T CD4 lymphocytes within tumors. + It acts to increase the number of cells, thereby suppressing exhausted cytotoxic CD8 + It has the effect of reactivating lymphocytes. CD4 + Foxp3 - and CD8 + GrB +The cells were detected by cell-cell contact (Figure 13A-D).

[0054] These observations suggest that these anti-NRP-1_LEP antibodies (i) are positioned as first-in-class antibodies compared to other developed antibodies, a-NRP1_VE and a-NRP1_SEM, or antibodies under development, (ii) have distinct effects on tumor cells and their environment, and (iii) act as immune system regulators dependent on suppression of the DNA damage response.

[0055] In conclusion, the NRP-1 peptide-based vaccine (Npep) and our antibody a-NRP1_LEP targeting GAG-modified NRP-1 and its partners (leptin and OBR) all show therapeutic potential in cancer and infectious diseases based on DNA damage response (DDR) and immune system interactions (Poplawski T., Curr Med Chem., 2019, 26:1423-1424).

[0056] Detailed Description of the Invention The first object of the present invention is to an Npep peptide with the sequence EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), or peptide N1, which has the sequence streptavidin-biotin-EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), N2, which has the sequence EGNKPVLFQGNTNPTDVVVAVFPK-biotin-streptavidin (SEQ ID NO: 54), or N3, which has the sequence EGNKPVLFQGNTNPTDVVC-KLH (SEQ ID NO: 55), The present invention relates to an anti-NRP-1 antibody or antibody fragment capable of recognizing

[0057] In a preferred embodiment, the anti-NRP-1 antibody or antibody fragment is capable of entering the nucleus of a cell.

[0058] The antibodies can enter the nuclei of immune cells, stimulating their activity and / or causing their proliferation. The antibodies can enter pathological cells (e.g., tumor cells or infected cells), causing DNA damage, centromere destabilization, and / or telomere shortening.

[0059] The antibody of the present invention - Phase 1 of immunization: immunogenic peptide sequence in an immunocompetent non-human animal; For example, the Npep peptide, which has the sequence EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), or a peptide selected from N1, which has the sequence streptavidin-biotin-EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), N2, which has the sequence EGNKPVLFQGNTNPTDVVVAVFPK-biotin-streptavidin (SEQ ID NO: 54), or N3, which has the sequence EGNKPVLFQGNTNPTDVVC-KLH (SEQ ID NO: 55), at least once administering - selecting animals with a desired phenotype, such as an obese phenotype; - recovering the antibody from said animal; - screening for antibodies capable of specifically recognizing the target protein and the corresponding peptide sequence, for example the Npep peptide as defined above, or a peptide selected from N1, N2 or N3, - selecting animals producing said antibodies capable of specifically recognizing said corresponding peptide sequence and said target protein; - a second immunization step: administering to the selected animals at least once the same immunogenic peptide as in the first immunization step; - recovering lymphocytes from the animal that has undergone the two-step immunization; - producing hybridomas from said lymphocytes; - a step of selecting hybridomas producing antibodies capable of specifically recognizing the target protein by cells expressing the protein, etc.; The method is prepared by a method comprising:

[0060] The antibodies of the present invention can be obtained by using Npep peptides, in particular peptides selected from the N1, N2 or N3 peptides defined above.

[0061] In a preferred embodiment, the Npep is the N3 peptide, which has the sequence EGNKPVLFQGNTNPTDVVC-KLH (SEQ ID NO: 55).

[0062] In certain embodiments, the present invention relates to anti-NRP-1 antibodies or antibody fragments capable of specifically binding to NRP-1 isoforms containing GAG modifications at positions S612, and / or Ser115, and / or Ser283, and / or Ser240, and / or Ser432, and / or Ser439, and / or Ser729. In preferred embodiments, the recognized GAG modifications are at positions Ser240 and / or Ser432.

[0063] It is noteworthy that the antibodies of the present invention do not recognize epitopes containing GAG modification at position Ser612, which are involved in the regulation of the VEGF-R2 pathway, as described by Shintani Yasunori et al. (The Embo journal Vol. 25, No. 13, 12 July 2006).

[0064] In a preferred embodiment, the anti-NRP-1 antibody or antibody fragment is capable of specifically binding to an NRP-1 protein isoform containing a GAG modification at positions Ser240 and / or Ser432.

[0065] Glycosaminoglycans (GAGs) are a class of biomolecules that can exist free in the extracellular matrix or covalently bind to proteins to form proteoglycans, which can be expressed on the cell surface or intracellular environment of mammalian cells. GAGs play important roles in a variety of physiological and pathological processes, including cancer, inflammation, infection, and many others (Morla S et al., Int J Mol Sci., 2019, 20:1963; Kamhi E et al., Biol Rev Camb Philos Soc., 2013, 88:928-43; Jinno A et al., Methods Mol Biol., 2015, 1229:567-85; Song Y et al., Adv Exp Med Biol., 2021, 1325:103-116).

[0066] In the experimental data provided herein, the anti-NRP-1 antibody or antibody fragment of the invention is referred to as a-NRP-1_LEP.

[0067] The anti-NRP-1 antibody a-NRP1_LEP, generated from the NRP-1 peptide-based vaccine (Npep), recognizes glycosylated NRP-1 (≦150 kDa) and, with even higher specificity, recognizes glycosaminoglycan (GAG)-modified NRP-1 (>150 kDa) on an epitope located on the b1 domain of the neuropilin-1 protein (described below). The GAG-modified form of NRP-1 corresponds to the presence of chondroitin sulfate and / or heparan sulfate.

[0068] As used herein, post-transcriptionally modified NRP-1 corresponds to glycosaminoglycan-modified NRP-1 (GAG-modified NRP-1) containing heparan sulfate (HS) and chondroitin sulfate (CS).

[0069] As used herein, HS, which corresponds to a linear polysaccharide, is composed of repeating GlcA-GlcNAc units and is modified by epimerization (C5-epimerase), N- and O-sulfation (NS, NS3S, 2S, 3S, and 6S), and desulfation (endosulfatase).

[0070] As used herein, CS, which consists of repeating disaccharide units, is composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) and undergoes significant structural modifications by O-sulfation (4S, 6S, and 4S6S) and epimerization.

[0071] The present invention provides NRP-1 peptides that have immunogenic properties, and these peptides can be used to generate antibodies.

[0072] In a particular embodiment, the present invention relates to anti-NRP-1 antibodies generated by a vaccination strategy comprising an NRP-1 peptide contained in the Npep sequence or a peptide selected from the N1, N2, or N3 peptides defined above.

[0073] The antibodies of the present invention exhibit functional properties that distinguish them from conventional anti-NRP-1 antibodies: in particular, they are capable of inhibiting the NRP-1 / OBR signaling pathway and, in particular, of binding to epitopes located on functional domains of the NRP-1 protein isoforms involved in leptin / OBR signaling.

[0074] In a preferred embodiment, this domain corresponding to the leptin recognition domain is located on the b1 domain of the NRP-1 protein isoform.

[0075] Furthermore, the antibodies of the present invention can specifically bind to GAG-modified NRP-1 protein isoforms at epitopes located on the leptin recognition domain of the NRP-1 protein isoforms; the GAG ​​modification on the NRP-1 protein isoforms can correspond to the presence of glycosylation associated with sulfate modifications. The glycosylation can correspond to O-glycosylation or N-glycosylation, and the sulfate modifications can correspond to the presence of chondroitin sulfate and / or heparan sulfate and their modifications.

[0076] In a preferred embodiment, the NRP-1 peptide-based vaccine generates antibodies that recognize non-GAG-glycosylated and GAG-modified NRP-1 protein isoforms that have O-glycosylation or N-glycosylation associated with heparan sulfate and chondroitin sulfate (Ahrens TD et al., Front Cell Dev Biol., 2020, 8:749).

[0077] The inventors have demonstrated that the antibodies of the present invention can recognize a form of GAG-modified NRP-1 that is different from that recognized by conventional antibodies.

[0078] The NRP-1 peptide-based vaccine (the NRP-1 peptide of the present invention) can be used to treat glycosaminoglycan-related diseases, including cancer, inflammation, and infectious diseases.

[0079] The inventors demonstrated that the epitope of the interaction domain on RP-1 for the antibodies of the present invention is distinct from the epitope of the a-NRP1_VE antibody against NRP-1, which shares the same NRP-1 b1 domain but does not share the binding sequence (Figure 3). They demonstrated by ELISA that murine, chimeric, and humanized variants of the a-NRP1_LEP antibody specifically bound to a synthetic peptide corresponding to the leptin-binding sequence on NRP-1, whereas unrelated human immunoglobulins and conventional antibodies (a-NRP1_VE and a-NRP1_SEM) did not bind (Table 2). Thus, the antibodies of the present invention recognize a previously unreported specific epitope located within the b1 domain of NRP-1.

[0080] In addition to said epitope specificity, the antibodies of the invention show specificity for GAG-modified NRP-1 forms, as observed in CTCs and the A549 cell line.

[0081] The anti-NRP-1 antibody of the present invention, a-NRP1_LEP, has the ability to bind with high specificity to the glycosaminoglycan (GAG)-modified form of NRP-1 protein, particularly in the case of "stem cell-like" cell lines (CTCs); in contrast, anti-NRP-1 antibodies targeting the binding domains of other ligands, such as the well-known a-NRP1_VE and a-NRP1_SEM, do not bind to GAG-modified NRP-1 in this type of cell.

[0082] Furthermore, the antibodies of the present invention bind very weakly to non-GAG-modified NRP-1.

[0083] In certain embodiments, the antibodies of the invention are capable of entering the nucleus of cancer cells and causing DNA damage (chromosome shattering) and / or centromere destabilization and / or telomere shortening.

[0084] In another embodiment, the genotoxic effect of a-NRP1_LEP appears to be specific to pathological cells, since no significant DNA damage was observed in vitro in human hepatocyte and lymphoblastoid cell lines and in human peripheral blood mononuclear cells (PBMCs) from healthy donors, and in vivo in bone marrow cells isolated from a-NRP1_LEP-treated SCID mice.

[0085] The present invention also provides specific antibodies defined by their sequences.

[0086] In a preferred embodiment, the present invention relates to an antibody derived from the mouse a-NRP1_LEP n°1 (also called 21B10 mouse antibody) and its humanized variants, which comprise the sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 defined below: CDR-H1 is represented by the sequence SX1X2MH (SEQ ID NO: 1), where: X1=F or Y X2=G, S or Y - CDR-H2 has the sequence X3ISX4X5X6X7X8X9X 10 YAX 11 X 12 X 13 X 14 G (SEQ ID NO: 2), where: X3=Y, V or I, X4=S, Y or P, X5=G, S or D, X6=S or G, X7=S or G, X8=T or S, X9=I, K or T, X 10 = H, Y or S, X 11 =D or Q, X 12 = S, K or T, X13 = V or F, X 14 = K or Q, CDR-H3 is represented by the sequence RHYGSSRYWYFDV (SEQ ID NO: 3), - CDR-L1 is the sequence X 15 ASQX 16 X 17 X 18 SX 19 LX 20 (SEQ ID NO: 4), where: X 15 = K or R, X 16 = D or S, X 17 = I or V, X 18 = K or S, X 19 = Y or W, X 20 = S or A, - CDR-L2 is the sequence X 21 AX 22 SX 23 X 24 X 25 (SEQ ID NO: 5), where: X 21 = Y or D, X 22 = T or S, X 23 = L or R, X 24 = A or E, X 25 = G, S or T, - CDR-L3 is the sequence X 26 QYX 27 X 28 SX 29 YT (SEQ ID NO: 6), where: X 26 = L or Q, X 27 = G or S, X 28 = E or S, X 29 =P or S.

[0087] In a preferred embodiment, the antibody of the invention is selected from the following antibodies: An antibody or antibody fragment comprising the sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 as defined below: CDR-H1: SFGMH (SEQ ID NO: 7), CDR-H2: YISSGSSTIHYADTVKG (SEQ ID NO: 8), or YISSGSSTIYYADTVKG (SEQ ID NO: 35), CDR-H3: RHYGSSRYWYFDV (SEQ ID NO: 9), or RHYGRSRYWYFDV (SEQ ID NO: 36), CDR-L1: KASQDIKSYLS (SEQ ID NO: 10), CDR-L2: YATSLAG (SEQ ID NO: 11) or YATSLAD (SEQ ID NO: 38), CDR-L3: LQYGESPYT (SEQ ID NO: 12).

[0088] Even better: Antibody a-NRP1_LEP n°1 of a fragment thereof comprising the following CDRs: CDR-H1: SFGMH (SEQ ID NO: 7), CDR-H2: YISSGSSTIHYADTVKG (SEQ ID NO: 8), CDR-H3: RHYGSSRYWYFDV (SEQ ID NO: 9), CDR-L1: KASQDIKSYLS (SEQ ID NO: 10), CDR-L2: YATSLAG (SEQ ID NO: 11), CDR-L3: LQYGESPYT (SEQ ID NO: 12).

[0089] In other embodiments, an antibody of the invention: a heavy chain having a sequence selected from SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, and a light chain having a sequence selected from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27; Includes:

[0090] In a preferred embodiment, the antibody of the invention: - variant 4 of a-NRP1_LEP n°1, combining a heavy chain of SEQ ID NO: 13 or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NO: 13, and a light chain of SEQ ID NO: 26 or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NO: 26; - variant 6 of a-NRP1_LEP n°1, combining a heavy chain of SEQ ID NO: 14 or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NO: 14, and a light chain of SEQ ID NO: 23 or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NO: 23; - variant 8 of a-NRP1_LEP n°1, combining a heavy chain of SEQ ID NO: 14 or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NO: 14, and a light chain of SEQ ID NO: 26 or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NO: 26; Corresponds to.

[0091] In another embodiment, the present invention relates to an antibody derived from murine a-NRP1_LEP n°2 (also called 27H10) and its humanized variants, which comprise the sequences of CDR-H4, CDR-H5, CDR-H6, ​​CDR-L4, CDR-L5 and CDR-L6 as defined below: - CDR-H4 has the sequence SX 30 X 31 MX 32 (SEQ ID NO: 28), where: X 30 =F or Y X 31 = G, S, Y or A X 32 =H or S - CDR-H5 has the sequence X333 ISX3 34 X 35 X 36 STX 37 YYADX 38 VKG (SEQ ID NO: 29), where: X 33 = Y or V, X 34 = S or Y, X 35 = S or G, X 36 = S or G, X 37 = I or K, X 38 = T or S, CDR-H6 is represented by the sequence RHYGRSRYWYFDV (SEQ ID NO: 30), - CDR-L4 is the sequence X 39 ASQX 40 X 41 X 42 SYLX 43 (SEQ ID NO: 31), where: X 39 = K or R, X 40 = D or S, X 41 = I or V, X 42 = K or S, X 43 = S or A, -CDR-L5 is the sequence X 44 AX 45 SX 46 X 47 X 48 (SEQ ID NO: 32), where: X 44 = Y or G, X 45 = T or S, X 46 = L or R, X 47 = A or E, X 48 = D, S or T, -CDR-L6 is the sequence X49 QYGESX 50 YT (SEQ ID NO: 33), where: X 49 = L or Q, X 50 =P or S.

[0092] In a preferred embodiment, the antibody of the invention is selected from the following antibodies: Antibody a-NRP1_LEP n°2 comprising the above CDRs: CDR-H1: SFGMH (SEQ ID NO: 34, identical to SEQ ID NO: 7), CDR-H2: YISSGSSTIYYADTVKG (SEQ ID NO: 35), CDR-H3: RHYGRSRYWYFDV (SEQ ID NO: 36), CDR-L1: KASQDIKSYLS (SEQ ID NO: 37, identical to SEQ ID NO: 10), CDR-L2: YATSLAD (SEQ ID NO: 38), CDR-L3: LQYGESPYT (SEQ ID NO: 39, identical to SEQ ID NO: 12).

[0093] In other embodiments, an antibody of the invention: a heavy chain having a sequence selected from SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, or a sequence having at least 80% sequence identity to the non-CDR regions of SEQ ID NOs: 40 to 47; and a light chain having a sequence selected from SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, or a sequence having at least 80% sequence identity to the non-CDR regions of SEQ ID NOs: 48 to 53; Includes:

[0094] As used herein, "antibody" refers to an intact antibody or an antibody fragment.

[0095] "Antibody fragment" refers to a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', Fd, dAb, dsFv, scFv, sc(Fv)2, CDRs, diabodies formed from antibody fragments, and multispecific antibodies.

[0096] The term "Fab" refers to an antibody monovalent fragment having a molecular weight of approximately 50,000, antigen-binding activity, and consisting of VL, VH, CL, and CH1 domains.

[0097] The term "VH" refers to the variable region of an antibody immunoglobulin heavy chain, including the heavy chain of an Fv, scFv, dsFv, Fab, Fab' or F(ab)' fragment.

[0098] The term "VL" refers to the variable region of an immunoglobulin light chain of an antibody, including the light chain of an Fv, scFv, dsFv, Fab, Fab' or F(ab)' fragment.

[0099] The Fv fragment is the N-terminal portion of the Fab fragment and consists of the variable regions of one light chain and one heavy chain.

[0100] The term "F(ab)'" refers to a bivalent antibody fragment having a molecular weight of approximately 100,000 and antigen-binding activity, which comprises two Fab fragments linked by disulfide bridges at the hinge region.

[0101] The term "Fab'" refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, which can be obtained by cleaving the disulfide bond in the hinge region of the F(ab')2.

[0102] The term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains.

[0103] The term "dAb" refers to a single variable domain antibody, i.e., an antibody fragment consisting of a VH or VL domain.

[0104] Single-chain Fv ("scFv") polypeptides are covalently linked VH:VL heterodimers and are typically expressed from gene fusions comprising genes encoding VH and VL, joined by a peptide-encoding linker.

[0105] The term "dsFv" refers to a VH:VL heterodimer stabilized by a disulfide bond. Divalent and multivalent antibody fragments form spontaneously by association of monovalent scFvs or can be generated by coupling monovalent scFvs with a peptide linker, such as a bivalent sc(Fv)2.

[0106] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a VH domain connected to a VL domain (VH-VL) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain to form two antigen-binding sites.

[0107] The term "chimeric antibody" refers to an antibody in which the constant region or a portion thereof has been modified, substituted or exchanged so that the variable region is linked to a constant region of a different species or belongs to another antibody class or subclass. The term "chimeric antibody" also refers to an antibody in which the variable region or a portion thereof has been modified, substituted or exchanged so that the constant region is linked to a variable region of a different species or belongs to another antibody class or subclass.

[0108] Furthermore, the antibody format of the present invention can be selected from different antibody subclasses depending on the distribution of target cells and the biological activity resulting from binding affinity. In cancer treatment, preferred subclasses are generally IgG1, IgG2, or IgG4. In a preferred embodiment, the format is IgG4.

[0109] This specific recognition of the glycosaminoglycan-modified neuropilin-1 (GAG-modified NRP-1) form correlates with the original property of this antibody, which specifically binds to a peptide sequence corresponding to leptin binding to NRP-1, as previously described. The MNRP-1685A anti-NRP-1 antibody inhibits VEGF binding to NRP-1 (a-NRP1_VE), and the YW64.3 anti-NRP-1 antibody blocks Sema3A binding to NRP-1 (a-NRP1_SEM).

[0110] The anti-NRP-1 antibodies of the present invention have the ability to specifically inhibit the interaction between the NRP-1 receptor and its ligand, leptin, and act on the NRP-1 / OBR signaling pathway, which is deeply involved in cancer and infectious diseases.

[0111] The original properties of this new class of anti-NRP-1 antibodies are described in the experimental section, and the data strongly suggest that such antibodies are promising tools for cancer therapy.

[0112] The antibodies of the invention may be characterized by their ability to enter the nuclei of cells expressing the NRP-1 / OBR complex and induce DNA damage and / or centromere destabilization and / or telomere shortening.

[0113] The antibodies or antibody fragments of the invention can also elicit a therapeutic immune response.

[0114] In certain embodiments, the antibody of the invention is an antibody drug conjugate (ADC) or a radionuclide-conjugated antibody, or may be part of a chimeric antigen receptor.

[0115] A second object of the invention relates to Npep peptides, in particular peptide N3, of sequence EGNKPVLFQGNTNPTDVVC-KLH (SEQ ID NO: 55).

[0116] The NRP-1 peptides of the present invention correspond to consensus sequences associated with post-transcriptionally modified splice variants of different isoforms of NRP-1.

[0117] In a preferred embodiment, the NRP-1 peptide of the invention is part of the three-dimensional structure of glycosaminoglycan-modified NRP-1.

[0118] In a preferred embodiment, the NRP-1 peptide corresponds to a functional domain of NRP-1 associated with the specific binding domain of its known ligand and the corresponding signaling pathway.

[0119] The NRP-1 peptide of the present invention further corresponds to a functional domain of NRP-1 associated with NRP-1 / OBR complex signaling, particularly the leptin-binding domain. This peptide has the ability to induce an immune response, particularly by activating adaptive and innate immune system cells. Thus, this peptide is immunogenic and capable of inducing a cytotoxic immune response and the production of anti-NRP-1 antibodies. A third object of the present invention is the use of the Npep peptide, or a peptide selected from the N1, N2 or N3 peptides defined above, in vaccination strategies in the treatment of diseases involving glycosaminoglycans and the DNA damage response (DDR).

[0120] As used herein, a "DDR-associated disease" may be cancer, inflammatory disease, or infectious disease.

[0121] As defined above, this NRP-1 peptide corresponds to an epitope contained within the three-dimensional structure of post-transcriptionally modified NRP-1, and is capable of activating cells of the adaptive and innate immune systems, which means inducing the production of anti-NRP-1 antibodies and inducing a cytotoxic immune response.

[0122] The Npep peptides, and in particular the N3 peptide, can be used to generate antibodies as defined above.

[0123] The fourth object of the present invention is to - Phase 1 of immunization: immunogenic peptide sequence in an immunocompetent non-human animal; For example, the Npep peptide, which has the sequence EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), or a peptide selected from N1, which has the sequence streptavidin-biotin-EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), N2, which has the sequence EGNKPVLFQGNTNPTDVVVAVFPK-biotin-streptavidin (SEQ ID NO: 54), or N3, which has the sequence EGNKPVLFQGNTNPTDVVC-KLH (SEQ ID NO: 55), at least once administering - selecting animals with a desired phenotype, such as an obese phenotype; - recovering the antibody from said animal; - screening for antibodies capable of specifically recognizing the target protein and the corresponding peptide sequence, for example the Npep peptide as defined above, or a peptide selected from N1, N2 or N3, - selecting animals producing said antibodies capable of specifically recognizing said corresponding peptide sequence and said target protein; - a second immunization step: administering to the selected animals at least once the same immunogenic peptide as in the first immunization step; - recovering lymphocytes from the animal that has undergone the two-step immunization; - producing hybridomas from said lymphocytes; - a step of selecting hybridomas producing antibodies capable of specifically recognizing the target protein by cells expressing the protein, etc.; The present invention relates to a method for producing an antibody as defined above, comprising:

[0124] In human cells, endogenous sources of DNA during normal and abnormal metabolic activity (chronic inflammation), as well as environmental factors such as exposure to carcinogens (X-rays, ultraviolet (UV) light, various genotoxic chemicals) and viral infections, can cause DNA damage, i.e., mutations in the chemical structure of DNA, resulting in up to 1 million molecular lesions per cell per day (Tubbs A et al., Cell. 2017, 168(4):644-656; Chatterjee N et al., Environ Mol Mutagen. 2017, 58(5):235-26 3; Ryan EL et al., Biomolecules, 2016, 6(1):2; Kay J et al., DNA Repair (Amst)., 2019, 8 3:102673).

[0125] Many of these lesions cause different structural damage to DNA molecules, such as apurinic / apyrimidinic (AP) sites (abasic sites), adducts, single-strand breaks (SSBs), double-strand breaks (DSBs), DNA-protein cross-links, and insertion / deletion mismatches, which can lead to cellular stress and damage that is involved in many diseases (Martin LJ., J Neuropathol Exp Neurol., 2008, 67(5):377-87).

[0126] The human genome encodes information for maintaining genetic stability and protecting its own integrity. The DNA damage response (DDR) represents a series of processes that detect (damage-specific sensor proteins), signal, and correct (DNA repair proteins) DNA damage (Chatterjee N et al., Environ Mol Mutagen. 2017, 58(5):235-263; Martin LJ., J Neuropathol Exp Neurol., 2008, 67(5):377-87; Wood RD et al., Mutat Res., 2005, 577(1-2):275-83).

[0127] Multiple DNA repair and damage tolerance pathways involve over 450 DDR proteins that contribute to damage removal and tolerance, thereby enabling cell survival. These enzymes are often characterized by the type of DNA damage they repair, such as base excision repair (BER) enzymes, nucleotide excision repair (NER) enzymes, mismatch repair (MMR) enzymes, DNA helicases, and DNA polymerases (Giglia-Mari G et al., Cold Spring Harb Perspect Biol., 2011, 3(1):a000745; Wood RD et al., Mutat Res., 2005, 577(1-2):275-83).

[0128] Unfortunately, errors can occur. Malfunction, defects, or inactivation of certain DNA repair enzymes can lead to the accumulation of DNA damage. Cells can then enter one of three possible states: (i) irreversible dormancy known as senescence; (ii) cell suicide, also known as apoptosis or programmed cell death; and (iii) uncontrolled cell division, which can lead to tumorigenesis (Tubbs A et al., Cell. 2017, 168(4):644-656).

[0129] Standard cancer treatments, such as chemotherapy, which typically involves direct DNA-damaging agents that cause mutations or genomic instability (Van den Boogaard WMC et al., Cancers (Basel)., 2022, 14(3):627), or radiation therapy, in which ROS react with bases and deoxyribose, causing significant oxidation reactions, both destabilize the DNA double helix and cause DNA damage (Juan CA et al., Int J Mol Sci., 2021, 22(9):4642).

[0130] The present invention particularly relates to "DNA repair targeted therapies" corresponding to a-NRP1_LEP antibodies, which cause DNA damage repair deficiency and affect the DNA damage response (DDR) in different cancer cell types.

[0131] In another embodiment, the antibodies of the invention inhibit the proliferation of CD4 T cells in tumors by inducing the production of cytolytic molecules (granzyme B and perforin). + Foxp3 - Increased T cell counts and CD8 + Promoting T cell reactivation can reduce cancer cell metastasis and mobilize anti-tumor immune responses.

[0132] The anti-NRP-1 antibodies of the present invention were developed using KLH and biotin-streptavidin conjugate vaccine strategies, comprising an immunogenic sequence derived from the leptin-interacting domain of NRP-1 as described in WO2017 / 050793; the immunogenic sequence is N1: sequence streptavidin-biotin-EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), N2: the sequence EGNKPVLFQGNTNPTDVVVAVFPK-biotin-streptavidin (SEQ ID NO: 54), and N3: sequence EGNKPVLFQGNTNPTDVVC-KLH (SEQ ID NO: 55) The antibodies of the present invention specifically recognize the NRP-1 peptides (Npep) on glycosylated NRP-1 and GAG-modified NRP-1.

[0133] Thus, in a particular embodiment, the present invention relates to an immunization method using a peptide comprising the sequence of SEQ ID NO: 54 conjugated to biotin-streptavidin and the sequence of SEQ ID NO: 55 conjugated to KLH.The present invention aims to generate anti-NRP-1 antibodies capable of inhibiting the NRP-1 / OBR signaling pathway, characterized by specifically binding to an epitope located in the leptin recognition domain on glycosylated, non-GAG-modified NRP-1 proteins (≦150 kDa) and GAG-modified NRP-1 proteins (>150 kDa).

[0134] Because the NRP-1 / OBR complex is expressed in tumor cells and cells in the microenvironment, antibodies that can inhibit the NRP-1 / OBR signaling pathway are particularly suitable for the treatment of cancers, whether or not associated with infections.

[0135] As used herein, "tumor cells" refers to cancer cells, cancer stem cells, circulating cancer cells derived from liquid and solid tumors.

[0136] As used herein, "tumor cells" refers to cancer cells, cancer stem cells, metastatic tumors, and circulating cancer cells derived from primary tumors.

[0137] As used herein, "cells of the microenvironment" means immune cells and stromal cells.

[0138] As used herein, "immune cells" refers to tumor-associated macrophages (TAMs), natural killer (NK) cells, neutrophils, mast cells, dendritic cells (DCs), CD4 + and CD8 + It refers to T cells and B cells.

[0139] As used herein, "stromal cells" refers to CAFs (cancer-associated fibroblasts) and CAF precursors (stationary fibroblasts and mesenchymal stromal cells).

[0140] Therefore, a fifth object of the present invention relates to the use of an antibody or antibody fragment as defined above for the treatment of a disease associated with glycosaminoglycans and DNA damage response, selected from cancer, inflammatory diseases and infectious diseases. In a preferred embodiment, the target disease is cancer.

[0141] In particular, these antibodies may be used in the treatment of solid tumors such as colon, lung, breast, brain, skin, and prostate cancer, but may also be useful in the treatment of hematological cancers, as well as other diseases in which inhibition of the NRP-1 / OBR signaling pathway is beneficial.

[0142] In a particular embodiment, the present invention relates to the use of an anti-NRP-1 antibody as defined above, which is characterized by its ability to enter the nucleus of pathological cells expressing the NRP-1 / OBR complex and to induce DNA damage (chromosome shattering) and / or centromere destabilization and / or telomere shortening.

[0143] Indeed, the inventors have shown that the properties of the antibodies of the invention depend on their ability to enter the nucleus in vitro and in vivo, where they induce DNA damage (chromosome shattering) and / or centromere destabilization and / or telomere shortening (Figures 6-12).

[0144] As used herein, "inducing DNA damage" means that the antibody has the ability to inhibit DNA repair. As used herein, "inhibiting DNA repair" refers to the inhibition of homologous recombination (HR), canonical non-homologous end-joining (c-NHEJ) recombination, alternative non-homologous end-joining (alt-NHEJ) recombination, nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), ribonucleotide excision repair (RER), and ADP-ribose-mediated chromatin regulation.

[0145] In certain embodiments, the invention relates to the use of antibodies capable of raising an immune response in vivo.

[0146] In certain embodiments, the present invention relates to the use of antibodies that are capable of increasing the infiltration of immune cells in the tumor microenvironment.

[0147] The inventors have shown that the antibodies of the present invention can induce anti-tumor responses by increasing the infiltration of immune cells within the tumor micro-environment (Figure 13).

[0148] As used herein, "tumor microenvironment" refers to the environment of a primary tumor and metastasis, including the surrounding blood vessels, immune cells, fibroblasts, signaling molecules, and extracellular matrix. The tumor and its surrounding microenvironment are closely related and constantly interacting.

[0149] As used herein, "immune cells" refers to non-cytotoxic and cytotoxic cells, including tumor-associated macrophages (TAMs), natural killer (NK) cells, neutrophils, mast cells, dendritic cells (DCs), CD4 + and CD8 +These include T cells and B cells. In particular, immune cells encompass cells that express NRP-1 / OBR and are capable of modulating anti-tumor immune responses in vitro and in vivo.

[0150] As used herein, "cytotoxic cells" means cells that express granzyme B and perforin.

[0151] Furthermore, the antibodies of the present invention are capable of inhibiting CD4 + By increasing T cells, it becomes possible to inhibit metastasis and regulate immune cells.

[0152] As used herein, "CD4 + By "T lymphocyte cells" is meant CD4 T granzyme B negative cells, CD4 T perforin negative cells, cytotoxic CD4 T granzyme B positive cells, and cytotoxic CD4 T perforin positive cells.

[0153] As used herein, "CD8 + By "T cells" is meant CD8 T granzyme B negative cells, CD8 T perforin negative cells, cytotoxic CD8 T granzyme B positive cells, and cytotoxic CD8 T perforin positive cells.

[0154] In certain embodiments of the invention, the anti-NRP-1 antibodies are used in combination with chemotherapy or radiation therapy.

[0155] In other specific embodiments of the invention, the anti-NRP-1 antibody is used in combination with at least one agent selected from a chemotherapeutic agent, and / or an anti-checkpoint inhibitor such as anti-PD1, anti-PDL1, anti-CTLA4, and / or an anti-angiogenesis inhibitor such as anti-VEGF, and / or a casein kinase 2 inhibitor (anti-CK2).

[0156] In certain embodiments of the invention, the anti-NRP-1 antibody is used as a free monoclonal antibody, as an antibody-drug conjugate (ADC), or as an antibody associated with CAR cells (anti-NRP-1-CAR cells), which may be CAR-T cells, NK cells, or other immune cells, or radionuclide-conjugated antibodies.

[0157] The antibodies of the invention are administered to a patient by a suitable route, which may be selected from oral, intramuscular, subcutaneous, intravenous, intraperitoneal, or local intratumoral injection. The composition is formulated to be compatible with the intended route of administration.

[0158] DESCRIPTION OF THE DRAWINGS Figure 1: Expression of the NRP-1 / OBR complex in cancer cells and immune cells in various human cancers.

[0159] Tissue sections from different human cancers are shown. (A) Nuclear staining of the NRP-1 / OBR complex (thick arrow: most tumor cells), cytoplasmic staining (thin arrow: some tumor cells), and staining of stromal cells (*: fibroblasts) in breast cancer tissue; (B) NRP-1 / OBR complex-positive tumor cells, lymphocytes (thin arrow), and macrophages (thick arrow) in lung cancer tissue; (C) NRP-1 / OBR complex in many positive stromal lymphocytes and tumor cells (T: tumor cells) in lung metastasis; (D) Nuclear staining of the NRP-1 / OBR complex (thick arrow: most tumor cells), cytoplasmic staining (thin arrow: some tumor cells), and staining of stromal cells (*: macrophages) in colon cancer tissue; (E) NRP-1 / OBR in positive tumor cells and positive fibroblasts (black arrow) in metastasis from colon cancer.

[0160] Figure 2: Representative histograms of body weight monitoring of mice immunized with three different immunogenic peptides generated from the NRP-1 peptide Npep (N1, N2, N3).

[0161] Figure 3: Schematic representation of the structure of NRP-1, its natural ligands, and different classes of mAbs.

[0162] (A) Overview of the different domains of NRP-1. The natural ligands are shown in italics, and gray arrows indicate the respective binding domains. Different classes of inhibitory antibodies are shown in black, and the boxes indicate the classes of antibodies of the present invention that inhibit leptin binding to the NRP-1 bl domain. (B) Crystal structure of NRP-1685A Fab. Only CDR-H3 and CDR-L1 bind to a 10-amino acid epitope on the bl domain (gray open circle, PDB: 2QQN). The peptide Npep (dark circle) used in the present invention to generate a new class of mAbs (a-NRP1_LEP) is also shown.

[0163] Figure 4: The a-NRP1_LEP antibody of the invention specifically recognizes the NRP-1 protein.

[0164] (Aa) Representative results of Western blot analysis showing the detection of NRP-1 (forms ≦150 kDa and forms exhibiting higher molecular weights >150 kDa) after immunoprecipitation using protein lysates from circulating tumor cells (CTC41.5E) and A549 lung cancer cell lines with the prior art antibody a-NRP1_VE, human a-NRP1_LEP, the chimeric a-NRP1_LEP antibody of the present invention, and their respective isotope controls (hIgG4 and hIgG1).

[0165] (Ab) Representative results of Western blotting to detect unmodified and GAG-modified NRP-1 after immunoprecipitation of lysates from prostate PC3 cells overexpressing doxycycline-inducible NRP-1 with a-NRP1_LEP or the conventional antibody a-NRP1_VE and their respective isotype controls (hIgG4 and hIgG1).

[0166] (B) Representative Western blot analysis showing detection of NRP-1 (<150 kDa and higher molecular weight forms >150 kDa) after immunoprecipitation with the prior art antibodies a-NRP1_VE and a-NRP1_SEM or the mouse and chimeric a-NRP1_LEP antibodies of the present invention using protein lysates from the human MDA-MB-231 breast cancer cell line. MDA-MB-231 cells were treated with 10 nM recombinant human leptin for 3 hours before cell lysis, demonstrating the specificity of the a-NRP1_LEP antibody of the present invention for the leptin-dependent NRP-1 / OBR complex.

[0167] (C) Representative Western blot analysis showing the detection of NRP-1 (forms ≦150 kDa and forms with higher molecular weights >150 kDa) after immunoprecipitation of NRP-1 with the prior art antibody a-NRP1_SEM or the chimeric a-NRP1_LEP antibody of the present invention using protein lysates from the human COLO205 colon cancer cell line. COLO205 cells were treated with 10 nM recombinant human leptin for 3 hours before cell lysis, demonstrating the specificity of the a-NRP1_LEP antibody of the present invention for the leptin-dependent NRP-1 / OBR complex.

[0168] (D) Representative results of Western blot analysis showing the detection of NRP-1 (high molecular weight form >150 kDa) after immunoprecipitation against OBR using protein lysates from human CTC41.4 cells.

[0169] a-NRP1_LEP: an antibody of the present invention that targets the binding domain of leptin to NRP-1; a-NRP1_SEM: a prior art antibody targeting the binding domain of semaphorin to NRP-1; a-NRP1_VE: a prior art antibody targeting the binding domain of VEGF to NRP-1; Chimeric: a form of the murine antibody a-NRP1_LEP of the invention in which the constant domains of a human immunoglobulin have been grafted onto the variable domains of a murine antibody; CTC: circulating tumor cells; GAG: glycosaminoglycan; hIgG: human immunoglobulin isotype G; IP: immunoprecipitation; KO: knockout; mIgG: mouse immunoglobulin isotype G; Mouse: antibodies produced in mice; NRP-1: neuropilin-1; OBR: leptin receptor; rh: recombinant human protein; Variant: a form of the humanized antibody a-NRP1_LEP of the invention; WB: Western blot.

[0170] FIG. 5: The antibody a-NRP1_LEP of the present invention binds more specifically to GAG-modified NRP-1 containing chondroitin and heparin sulfate motifs within its epitope compared to prior art antibodies.

[0171] (A) Representative flow cytometry histograms showing the expression of GAG-modified NRP-1 by MDA-MB-231 cells treated or untreated with chondroitinase using the antibodies of the present invention a-NRP1_LEP (chimeric a-NRP1_LEP and variant 4 a-NRP1_LEP) compared to prior art antibodies (a-NRP1_VE and a-NRP1_SEM). The black curve represents untreated cells, and the gray curve represents chondroitinase-treated cells.

[0172] (B) Representative Western blot analysis showing the detection of NRP-1 (<150 kDa and higher molecular weight forms >150 kDa) after immunoprecipitation of NRP-1 with antibodies from human MDA-MB-231 cells. The immunoprecipitated NRP-1 was either untreated or treated with chondroitinase or heparinase to remove chondroitin sulfate or heparin sulfate glycosaminoglycans, respectively.

[0173] (C) Representative Western blot analysis of protein lysates from human CTC41 cells showing NRP-1 (<150 kDa and higher molecular weight forms >150 kDa) after immunoprecipitation with the humanized variant of the a-NRP1_LEP antibody of the present invention. The immunoprecipitated NRP-1 was either untreated or treated with PNGase to remove N-linked oligosaccharides.

[0174] a-NRP1_LEP: an antibody of the present invention that targets the binding domain of leptin to NRP-1; a-NRP1_SEM: a prior art antibody targeting the binding domain of semaphorin to NRP-1; a-NRP1_VE: a prior art antibody targeting the binding domain of VEGF to NRP-1; Chimeric: a form of the murine antibody a-NRP1_LEP of the invention in which the constant domains of a human immunoglobulin have been grafted onto the variable domains of a murine antibody; chondro: chondroitinase CTC: circulating tumor cells; GAG: glycosaminoglycan; hepa: heparinase hIgG: human immunoglobulin isotype G; IP: immunoprecipitation; KO: knockout; NRP-1: neuropilin-1; Variant: a form of the humanized antibody a-NRP1_LEP of the invention; WB: Western blot.

[0175] FIG. 6: Detection of NRP-1 in the cell nuclei of different cancer cell types by the anti-NRP-1 antibody of the present invention, a-NRP1_LEP.

[0176] [6.1(Aa)] Representative fluorescent staining of CTC41.5E cells pretreated for 72 hours with the mouse or humanized form of the antibody a-NRP1_LEP of the present invention. Detection was performed using FITC-labeled secondary antibodies (FITC-anti-mouse IgG and FITC-anti-human IgG), as indicated by the light gray lines. Nuclei were then stained with DAPI (nuclei, dark gray) to detect the intracellular localization of the antibody.

[0177] [6.1 and 6.2] Representative results of Western blot analysis showing mouse or human IgG in the cytoplasm and / or nucleus of circulating tumor cells CTC41.5E (6.1Ab, Ac, and 6.2E) and CTC41 (6.2D), as well as the human prostate cancer cell line PC3 (6.2B) and the mouse breast cancer cell line 4T1 (6.2C) after immunoprecipitation with protein A and protein G Sepharose. Cells were pretreated for 5 hours with either the prior art antibodies a-NRP1_VE and a-NRP1_SEM or the mouse (1B3, 21B10, and 27H10), chimeric, and humanized variants of the applicant's antibody a-NRP1_LEP. Specific nuclear localization of HDAC2 protein is used as an indicator of subcellular fractionation efficiency.

[0178] a-NRP1_LEP: an antibody of the present invention that targets the binding domain of leptin to NRP-1; a-NRP1_SEM: a prior art antibody targeting the binding domain of semaphorin to NRP-1; a-NRP1_VE: a prior art antibody targeting the binding domain of VEGF to NRP-1; Chimeric: a form of the murine antibody a-NRP1_LEP of the invention in which the constant domains of the murine immunoglobulin have been replaced by human constant domains; C: cytoplasm; CTC: circulating tumor cells; DAPI: 4',6-diamidino-phenylindole (4',6-diamidino-2-phenylindole); GAG: glycosaminoglycan; HDAC2: histone deacetylase 2; hIgG: human immunoglobulin isotype G; IP: immunoprecipitation; mIgG: mouse immunoglobulin isotype G; Mouse: antibodies produced in mice; N: Nucleus; NRP-1: neuropilin-1; Variant: A form of the humanized antibody a-NRP1_LEP of the invention.

[0179] FIG. 7.1: NRP-1-dependent nuclear entry of the antibody of the invention a-NRP1_LEP via the NRP-1 / OBR complex in tumor cells.

[0180] (Aa) Representative results of Western blot analysis of NRP-1 expression in whole cell extracts of wild-type and NRP-1-KO A549 cell lines using primary rabbit polyclonal antibody anti-NRP-1.

[0181] (Ab) Representative results of Western blot analysis of humanized a-NRP1_LEP antibody in the cytoplasm and / or nucleus of wild-type and NRP-1-KO A549 cell lines after immunoprecipitation with Protein A and Protein G Sepharose. Cells were pretreated for 5 hours with the humanized variant of the applicant-developed antibody a-NRP1_LEP or its control isotype, hIGg4. Specific nuclear localization of HDAC2 protein is used as an indicator of subcellular fractionation efficiency.

[0182] (Ba) Representative flow cytometry histograms showing the detection of NRP-1 / OBR complexes by flow PLA-APC analysis performed with a commercially available rabbit monoclonal anti-human NRP-1 antibody and a mouse monoclonal anti-OBR antibody. The dark gray curve represents MDA-MB-231 cells (pretreated) treated with the mouse a-NRP1_LEP antibody of the present invention at 10, 20, and 40 ng / ml for 2 days, while the light gray curve represents cells preincubated with the corresponding mIgG2b control isotype.

[0183] (Bb) Representative flow cytometry histograms showing APC intensities obtained after PLA experiments performed with commercially available NRP-1 and OBR antibodies. The dark gray curve represents MDA-MB-231 cells (pretreated) treated with the murine a-NRP1_LEP antibody of the present invention and its control isotype at 20 ng / ml for 2 days (positive PLA signal), while the light gray curve represents cells preincubated with the prior art a-NRP1_VE antibody and its irrelevant control isotype at the same concentrations (negative PLA signal).

[0184] a-NRP1_LEP: an antibody of the present invention that targets the binding domain of leptin to NRP-1; a-NRP1_VE: a prior art antibody targeting the binding domain of VEGF to NRP-1; C: cytoplasm; HDAC2: histone deacetylase 2; hIgG: human immunoglobulin isotype G; KO: knockout; mIgG: mouse immunoglobulin isotype G; Mouse: antibodies produced in mice; N: Nucleus; NRP-1: neuropilin-1; OBR: leptin receptor; PLA: Proximity ligation assay Variant: a form of the humanized antibody a-NRP1_LEP of the invention; WT: wild type.

[0185] Figure 7.2: Leptin-dependent induction of NRP-1 and a-NRP1_LEP antibody translocation into the nucleus of the CTC41.5E cell line.

[0186] Representative results of Western blot analysis showing detection of a-NRP1_LEP or NRP-1 in the cytoplasm and / or nucleus of the CTC41.5E cell line after immunoprecipitation with protein A and protein G sepharose. Cells were serum-starved overnight and then treated with variant 4α-NRP1_LEP (10 μg / ml) in the presence or absence of leptin (10 nM) for 3 h at 37°C. Specific nuclear localization of HDAC2 protein was used as an indicator of subcellular fractionation efficiency.

[0187] a-NRP1_LEP: an antibody of the present invention that targets the binding domain of leptin to NRP-1; C: cytoplasm; HDAC2: histone deacetylase 2; Human IgG: human immunoglobulin isotype G; MW: molecular weight; N: Nucleus; NRP-1: neuropilin-1; Prot.A / G Sepharose: 1:1 mixture of Protein A and Protein G Sepharose 4 Fast Flow beads; Variant: A form of the humanized antibody a-NRP1_LEP of the invention.

[0188] FIG. 8: The antibody a-NRP-1-LEP of the invention induces DNA damage (chromosome shattering) and / or centromere destabilization and / or telomere shortening in circulating tumor cells.

[0189] (Aa) Representative cytogenetic detection of DNA damage in CTC41 and CTC41.5E cells pretreated for 72 hours with a control isotype or a humanized variant of the inventive antibody a-NRP1_LEP, and the prior art antibodies a-NRP1_VE and a-NRP1_SEM, using telomere and centromere staining followed by M-FISH technology. Karyotypes of CTC41.5E cells obtained by M-FISH reveal normal chromosomes (mainly in untreated cells) and shattered chromosomes (mainly in cells treated with the inventive antibodies).

[0190] (Ab) Representative fluorescence images of CTC41 and CTC41.5E cells at metaphase. After pretreatment as described above (Fig. 8A-a), cells were analyzed for the detection of telomeres (black dots) and centromeres (light gray signals) following DAPI staining (dark gray signals). Arrows indicate acentric fragments.

[0191] (B) Quantification of the percentage of cells (Figure 8A) scored according to the number of chromosome fragments: (i) ≥10 (dark gray), (ii) <10 (light gray), or (iii) no fragments (black).

[0192] (C) Representative phase-contrast images of CTC41.5E cells stained with DAPI and anti-IgG-FITC after pretreatment as described above (Figure 8A-a and Ab). Arrows indicate micronuclei.

[0193] (Cb) Quantification of scored micronuclei in CTC41 and CTC41.5E cells after cytokinesis-block micronucleus assay in association with telomere and centromere staining.

[0194] (D) Representative cytogenetic detection of DNA damage, as indicated by chromosome shattering / multiple acentric fragments (Da), micronuclei (Db), and telomere / centromere intensity quantification (Dc and Dd), in CTC41.5E cells pretreated for 3 and 24 hours with a control isotype or a humanized variant of the antibody a-NRP1_LEP of the present invention and leptin.

[0195] (Da) Quantification of the percentage of cells (Figure 8A) scored as having (i) 10 or more chromosome fragments (dark gray), (ii) less than 10 chromosome fragments (light gray), or (iii) no fragments (black).

[0196] (Db) Quantification of scored micronuclei in CTC41.5E cells after cytokinesis-block micronucleus assay in association with telomere and centromere staining.

[0197] (Dc and Dd) Quantification and analysis of telomere (Dc) and centromere (Dd) intensities.

[0198] (E) Detection of DNA damage using a cytokinesis-block micronucleus assay, followed by quantification and analysis of telomere and centromere staining in lymphocytes treated with a-NRP1_LEP variant 4, a-NRP1_VE, and their respective controls, hIgG4 and hIgG1. Cells were treated for 72 hours, and cytochalasin B was added 24 hours before cell harvest. Chromosomal aberrations analysis using telomere and centromere staining in lymphocytes treated with a-NRP1_LEP variant 4, a-NRP1_VE, and their respective controls, hIgG4 and hIgG1. Cells were treated for 72 hours, and colcemid was added 2 hours before cell harvest.

[0199] a-NRP1_LEP: an antibody of the present invention that targets the binding domain of leptin to NRP-1; CTC: circulating tumor cells; hIgG: human immunoglobulin isotype G; MN: micronucleus; NRP-1: neuropilin-1; Variant: A form of the humanized antibody a-NRP1_LEP of the invention.

[0200] Figure 9: The interactome of NRP-1 with various DNA repair proteins revealed by the antibody a-NRP1_LEP of the present invention.

[0201] (A) STRING: Functional protein association network. The interaction network of NRP-1 with various DNA repair proteins immunoprecipitated from whole-cell extracts of CTC41.5E and A549WT cell lines using the antibody a-NRP1_LEP of the present invention and detected by LC-MS / MS. All gene names listed in Figure 9 are clearly defined in Table 5.

[0202] (B) Representative results of Western blot analysis showing the detection of a-NRP1_LEP and NRP-1 in the cytoplasm and / or nucleus of the CTC41.5E cell line after co-immunoprecipitation with free protein A and protein G Sepharose. Cells were previously treated with variant 4 a-NRP1_LEP (10 μg / ml) for 30 min, 1 h, 24 h, or 48 h at 37°C. Specific nuclear localization of HDAC2 protein is used as an indicator of subcellular fractionation efficiency.

[0203] a-NRP1_LEP: an antibody of the present invention that targets the binding domain of leptin to NRP-1; C: cytoplasm; HDAC2: histone deacetylase 2; Human IgG: human immunoglobulin isotype G; MW: molecular weight; N: Nucleus; NRP-1: neuropilin-1; PARP: poly(ADP-ribose) polymerase; Prot.A / G Sepharose: 1:1 mixture of Protein A and Protein G Sepharose 4 Fast Flow beads; Variant: A form of the humanized antibody a-NRP1_LEP of the invention.

[0204] Figure 10: The anti-NRP-1 antibody of the invention, a-NRP1_LEP, binds to chromatin in CTC cells.

[0205] Representative results of Western blot analysis showing humanized variants of the a-NRP1_LEP antibody (variants 4, 6, and 8) bound to chromatin of circulating tumor cells CTC41.5E after immunoprecipitation with protein A and protein G Sepharose. Cells were pretreated for 24 hours with either the humanized variants of the a-NRP1_LEP antibody or its isotype control, human IgG4.

[0206] a-NRP1_LEP: an antibody of the present invention that targets the binding domain of leptin to NRP-1; CTC: circulating tumor cells; CBF: chromatin bound fraction; CF: cytoplasmic fraction; hIgG: human immunoglobulin isotype G; IP: immunoprecipitation; MF: membrane fraction; NRP-1: neuropilin-1; NSF: nuclear soluble fraction KO: knockout; Variant: a form of the humanized antibody a-NRP1_LEP of the invention; WB: Western blot.

[0207] FIG. 11: Detection of the anti-NRP-1 antibody of the invention, a-NRP1_LEP, in the nuclei of CTC cells xenografted subcutaneously in SCID mice.

[0208] (A) Representative Western blot analysis showing humanized a-NRP1_LEP in the nuclei of circulating tumor cells from mouse tumors after immunoprecipitation with protein A and protein G Sepharose. Mice were pretreated with either humanized variant 4 of the a-NRP1_LEP antibody or its isotype control, human IgG4 (hIgG4), via tail vein injection once a week for a total of five times.

[0209] (B) Representative immunofluorescence staining of tumor sections from subcutaneously xenografted CTC41.5E cells in SCID mice treated with either humanized a-NRP1_LEP antibody or its isotype control, human IgG4 (hIgG4). FITC-labeled antibody (FITC-anti-human IgG) was used as the secondary antibody, as indicated by the light gray line. Subcellular localization of the antibody was then detected by DAPI staining (nuclei, dark gray).

[0210] a-NRP1_LEP: an antibody of the present invention that targets the binding domain of leptin to NRP-1; C: cytoplasm; CTC: circulating tumor cells; DAPI: 4',6-diamidino-2-phenylindole; FITC: Fluorescein isothiocyanate; HDAC2: histone deacetylase 2; hIgG: human immunoglobulin isotype G; IP: immunoprecipitation; N: Nucleus; NRP-1: neuropilin-1; Prot.A / G Sepharose: 1:1 mixture of Protein A and Protein G Sepharose 4 Fast Flow beads; SCID: Severe combined immunodeficiency; Variant: A form of the humanized antibody a-NRP1_LEP of the invention.

[0211] FIG. 12: The antibody of the invention, a-NRP-1-LEP, induces telomere shortening in CTC cells xenografted subcutaneously in SCID mice.

[0212] (A) Representative results of telomere and centromere staining in 5 μm tumor frozen sections taken from CTC cells subcutaneously xenografted into SCID mice.

[0213] (B) Representative results of telomere staining in mouse bone marrow cells isolated from SCID mice subcutaneously xenografted with CTC cells.

[0214] Mice were pretreated with either the humanized variant 4 of the a-NRP1_LEP antibody or its isotype control, human IgG4 (hIgG4), via tail vein injection once a week for a total of five times.

[0215] a-NRP1_LEP: an antibody of the present invention that targets the binding domain of leptin to NRP-1; CTC: circulating tumor cells; SCID: Severe combined immunodeficiency; Variant: A form of the humanized antibody a-NRP1_LEP of the invention.

[0216] FIG. 13: In vivo efficacy of the murine a-NRP1_LEP antibody of the invention on lung metastases from primary tumors of the 4T1 mouse breast cancer cell line grafted orthotopically in syngeneic BALB / c mice.

[0217] (A) Representative lung histology (HES staining).

[0218] (B) CD4 in lung metastases + Foxp3 - cells / mm 2 Histogram showing quantification of. Values ​​are means ± SEM for the PBS group (6 mice), the mIgG2b group (9 mice), and the mouse a-NRP1_LEP group (11 mice).

[0219] (C)CD4 + Foxp3 - Correlation between cell density and metastasis size in the lung.

[0220] (D) CD8 expression in lung metastases of mice treated with PBS or isotype control IgG2b compared to the antibody of the present invention a-NRP1_LEP. + GRB + , and CD4 + Foxp3 - Representative areas of staining are shown. Zoomed areas of murine a-NRP1_LEP-treated mice show activated CD8 T cells, as indicated by cell-cell interactions (black arrows). + GRB + , and CD4 + Foxp3 - shows the clusters.

[0221] a-NRP1_LEP: an antibody of the present invention that targets the binding domain of leptin to NRP-1; CD: cluster of differentiation; Foxp3: Forkhead box P3; HES: hematoxylin and eosin stain; mIgG: mouse immunoglobulin isotype G; Mouse: antibodies produced in mice; PBS: phosphate-buffered saline; R2: Coefficient of determination. *: p≦0.05, **p≦0.01 (one-way ANOVA).

[0222] [Experimental part] I-Materials and Methods The a-NRP1_LEP antibody of the present invention was produced by CRO Biotem (https: / / www.biotem.fr / ), and TAB-264 (a-NRP1_VE) and YW64.3 (a-NRP1_SEM) were purchased from Creative Biolabs (https: / / www.creative-biolabs.com / ).

[0223] (Generation of mouse a-NRP1_LEP antibody) OF1 female mice (Charles River) were immunized subcutaneously and intraperitoneally with three different immunogenic peptides generated from the NRP-1 protein Npep (EGNKPVLFQGNTNPTDVVVAVFPK) (SEQ ID NO: 54): N1: sequence streptavidin-EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), N2: sequence EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54)-streptavidin, and N3: sequence EGNKPVLFQGNTNPTDVVC-KLH (SEQ ID NO: 55). Prior to streptavidin binding, N1 and N2 were first biotinylated at their N- and C-termini, respectively. The N3 peptide sequence of NRP-1 was first modified at the C-terminus, substituting cytosine for the last six amino acids to enable conjugation with keyhole limpet hemocyanin (KLH). Mice were injected with either a high dose (50 μg) or a low dose (20 μg) of each immunogenic peptide in Freund's Complete Adjuvant. On days 22 and 42, mice were intraperitoneally boosted with the immunogenic peptide in Freund's Incomplete Adjuvant. Mice were weighed weekly to monitor the phenotypic effects of immunization as an indicator of neutralizing antibody production, which may be related to leptin function in energy balance and the impact of its dysfunction on obesity. On day 52, the production of anti-NRP-1 antibodies (a-NRP1_LEP) in immunized mice was analyzed by ELISA as described below. Anti-NRP-1 (a-NRP1_LEP) neutralizing antibodies for leptin signaling were selected based on two main criteria: (i) the obese phenotype, and (ii) the ability to recognize human and mouse NRP-1, as verified by a positive ELISA signal against recombinant NRP-1 corresponding to the extracellular domain, and by flow cytometry analysis of binding to human and mouse cell surfaces expressing native membrane NRP-1 protein.After the fourth intraperitoneal immunization, or without any immunization, a subset of mice meeting the above criteria were boosted intravenously with a high (10 μg) or low (5 μg) dose of antigen (N3 peptide) without adjuvant. Selected mice were sacrificed, and hybridomas were generated using the classical PEG-based method at a myeloma:lymphocyte ratio of 1:5. Hybridomas were then aliquoted into 96-well plates, and cell culture supernatants were screened for reactivity and isotyping by ELISA.

[0224] The ELISA method used to screen for anti-NRP-1 (α-NRP1_LEP) antibodies in either mouse serum or hybridoma cell culture supernatant was based on antigen binding detection. The antigen binding detection was performed against the following: recombinant human NRP-1 (3870-N1-025, Bio-Techne), recombinant mouse NRP-1 (5994-N1-050, Bio-Techne), and NRP-1 peptides (containing the target peptide sequence of NRP-1), Npep, and unconjugated N3 (without KLH). Only clones that showed positive reactions in three tests (Table 1) were selected and compared with the prior art antibody. In contrast, the prior art antibody reacted only with mouse and human NRP-1 but not with the NRP-1 peptides (Npep and unconjugated N3). Culture medium, non-immune serum from unimmunized OF1 mice, and NRP-1-negative cancer cells were used as negative controls.

[0225] For further research and engineering steps, murine antibodies 21B10 and 27H10 were selected and designated a-NRP1_LEP n°1 and a-NRP1_LEP n°2, respectively.

[0226] (Chimerization and humanization of a-NRP1_LEP antibody) The selected a-NRP1_LEP monoclonal antibody was produced as a recombinant chimeric antibody using standard procedures. High-quality RNA was extracted from the hybridoma, and cDNA was prepared using high-fidelity reverse transcriptase (SuperScript™ IV, Invitrogen). Specific cDNA encoding the antibody variable region was amplified by high-fidelity PCR using Q5® High-Fidelity DNA Polymerase (NEB) and degenerated primers (a primer kit designed and improved by Biotem). The resulting PCR products were directly sequenced. The sequences encoding the heavy chain variable domain (VH) and light chain variable domain (VL) were then synthesized and optimized for codon expression in mammalian cells. The corresponding synthetic genes were cloned into Biotem's proprietary vector system. The vector systems contained either the human constant region of an IgG1, IgG1 silenced, or IgG4 heavy chain, or the human constant region of a kappa light chain. The vectors were verified by sequence analysis and then amplified to prepare low-endotoxin plasmid DNA, which was again verified by sequence analysis.

[0227] Chimeric antibodies were produced by transient expression in CHO DG44 cells for 15 days. The supernatants were collected and antibody titers were measured by dual-layer interferometry using a protein A biosensor on the BLITZ system (Pall ForteBio) and estimated based on a human IgG1 standard.

[0228] The supernatant was then purified by protein A affinity chromatography (Purolite Praesto Jetted A50). After dialysis in PBS, pH 7.4, the total protein concentration was determined by spectrophotometry at 280 nm (Eppendorf Biospectrometer).

[0229] A murine antibody was humanized by grafting three CDRs (using the Kabat and IMGT nomenclature) from the light chain variable region (VL) into a selected human germline VL that closely resembles the murine antibody VL. Similarly, the three CDRs of the heavy chain variable region (VH) were grafted into a selected human germline VL that closely resembles the murine antibody VL. Furthermore, some amino acid residues in the framework regions (FR) of the selected human germline VL were reverted to the corresponding murine amino acid residues (back-mutation). Based on information collected about the structure of immunoglobulin variable regions and using the guidance of molecular models of mAbs (VH and VL), several residues in the FR were identified as potentially important for maintaining the conformation of the CDRs or as playing a role in the interface between the heavy and light chain variable regions. The VL and VH are two domains that interact without forming covalent bonds. Residues involved in this interaction must also be maintained. Otherwise, the paratope may be altered, potentially altering the affinity of the antibody. Therefore, these residues were retained in humanized version V1 ("low risk") or mutated with corresponding human germline amino acid residues in humanized version V2. Using molecular modeling, some CDR residues were also replaced with corresponding human germline residues in humanized version V2. Here, the combination of structural modeling and pure sequence analysis enabled identification of truly paratope-oriented and non-paratope residues in the CDR regions. Additionally, the structural model allowed for guiding backmutation selection based on the selected germline backbone used, thereby accelerating the humanization process. Humanized version V1, provided for both VH and VL, is a conservative version designed to avoid (or significantly minimize) paratope alterations. These versions were expected to exhibit binding / potency activities similar to those of the chimeric antibody used as the reference molecule.The humanized version V2 was designed to have at least 85% sequence identity with the closest human germline sequences in both VH and VL (Germinality index as described by Pelat et al. [J Mol Biol 2008]). Three human germline sequences were selected for the design of the CDR-grafted versions of mouse VH (mouse a-NRP1_LEP n°1 and mouse a-NRP1_LEP n°2). The selection was based on high sequence identity across the V genes or because they are germline sequences widely used in human antibody production (according to the IMGT / GeneFrequency database). These germline sequences have other interesting features and offer the possibility of CDR-grafting in different molecular environments. For the design of the CDR-grafted versions of mouse VLκ (a-NRP1_LEP n°1 and a-NRP1_LEP n°2), two human germline sequences were selected because their homology was sufficiently high. The first step in the humanization process was to select the optimal heavy and light chain pair between the humanized VH and VL versions: (i) Humanization was performed using three different human germline sequences, each with two versions (V1 and V2), to obtain a total of six different humanized VHs. (ii) Humanization was performed using two different human germline sequences, each with two versions (V1 and V2), to obtain a total of two different humanized VLs. Finally, a total of 6 (HC) × 4 (LC) = 24 monoclonal antibodies were transiently produced and purified in HEK cells. The monoclonal antibodies were then tested for their ability to bind to NRP-1, as well as for other properties such as production efficiency, aggregation tendency, and functional activity.

[0230] (Preparation of protein lysates from cancer cells) After two washes with ice-cold PBS, plated or suspended cells were harvested on ice in 20 mM Tris pH 7, 0.1 M NaCl, 10 mM NaPO, 1% Triton X-100, 1% protease and phosphatase inhibitor cocktail (1861281, Thermo Scientific), incubated on ice for 30 min with vortexing for 20 s every 15 min, and then centrifuged at 4400 rpm for 10 min at 4°C. The protein lysates were harvested for protein concentration quantification (BCA kit, Thermo Scientific) prior to immunoprecipitation, followed by Western blot analysis.

[0231] In some experiments, human breast cancer cell line MDA-MB-231, human colon cell line COLO205, and patient-derived circulating tumor cells CTC41.5E were serum-starved overnight and then treated with 10 nM recombinant human leptin (398-LP, R&D Systems) for 3 hours at 37°C before preparation of protein lysates.

[0232] One hundred micrograms of protein lysate from MDA-MB-231, COLO205, and CTC41.4 (circulating tumor cells isolated from patients with metastatic colon cancer; cell source: Laboratory of Rare Human Circulating Cells (LCCRH), Saint-Eloi Hospital, University Medical Centre, Montpellier, France), and wild-type and NRP-1-KO A549 (human lung cancer cell line) cells was applied to Protein A and Protein G Sepharose 4 Fast Flow beads (17-6002-35, GE). The cells were incubated overnight at 4°C with a 1:1 mixture of α-NRP1 and α-NRP1_VE antibodies (Bio-Rad Laboratories, Inc., San Diego, CA) and 2 μg of each of the following antibodies: the antibody a-NRP1_LEP (mouse, chimeric, and humanized variants) of the present invention; and the conventional antibodies a-NRP1_VE and a-NRP1_SEM, or their respective isotype controls. To investigate the NRP-1 / OBR interaction in CTC41.4 cells, 100 μg of protein lysate was immunoprecipitated as described above using an anti-OBR antibody (MAB867, R&D Systems). The next day, the immunoprecipitated samples were washed three times with PBS, resuspended in 10% β-mercaptoethanol 4X Laemmli Sample Buffer (1610747, Bio-Rad Laboratories, San Diego, CA) and heated at 95°C for 5 minutes. They were then either stored at -20°C or subjected to Western blot analysis.

[0233] To identify the nature of the glycosaminoglycans within GAG-modified NRP-1, immunoprecipitated samples were treated with either chondroitinase (C2905, Sigma) (25 mU / ml), heparinase I and III (H3917, Sigma) (1.25 mU / ml), or PNGase F (P0708s, New England BioLabs) according to the manufacturer's instructions before Western blot analysis.

[0234] (Western blot analysis) The immunoprecipitated proteins or total protein lysates were separated by electrophoresis through 4–20% acrylamide gels (Product No. 4568095, BioRad) and transferred to PVDF membranes using a Trans-Blot Turbo Transfer System (Product No. 1704272, BioRad). After 45 min of incubation with blocking solution (3% BSA), the membranes were incubated overnight at 4°C with the primary rabbit polyclonal antibody anti-NRP-1 (in 1% BSA) (a gift from Alex L. Kolodkin, The Johns Hopkins University School of Medicine, Baltimore, USA). The following day, the membranes were washed three times for 15 min each and then incubated with the secondary antibody anti-rabbit immunoglobulin HRP-conjugated to 1 / 1000 (Product No. 18-8816-33, Rockland). Following addition of ECL substrate (1705060, Biorad), chemiluminescent signals were acquired using a ChemiDoc Imaging System (Bio-Rad).

[0235] (Single stain for flow cytometry) Adherent cells were detached using a non-enzymatic cell dissociation solution (C5914, Sigma). After washing the cells (adherent or suspension cells) with PBS containing 2% FBS, they were incubated with the present invention's a-NRP1_LEP anti-NRP-1 non-binding antibody (mouse, chimeric, and humanized variants) or conventional non-binding antibodies (a-NRP1_VE and a-NRP1_SEM). A specific secondary antibody was added to detect non-binding antibodies. After the final wash, fluorescence intensity, cell size, and granularity were measured using an LSR Fortessa flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software. Commercially available PE-Vio770-conjugated anti-NRP-1 was used as a positive control.

[0236] (Proximity Ligation Assay (PLA)) To verify the recognition of native cellular NRP-1 protein by a-NRP1_LEP, an antibody proximity ligation assay was performed. MDA-MB-231 cells were detached using a non-enzymatic cell detachment solution (C5914, Sigma). 1.5 million cells per condition were washed, fixed, permeabilized, and blocked using a transcription factor buffer set (562574, BD Biosciences). The cells were then incubated for 1 hour at 37°C with a mixture of a commercially available anti-NRP-1 antibody (NBP2-67539, Novus) that specifically binds to the C-terminal domain of NRP-1 and our antibody (mouse a-NRP1_LEP). The different PLA steps (probe incubation, ligation, amplification, and detection) were performed using the Duolink® Flow PLA Detection Kit (DUO94004, Sigma) according to the manufacturer's instructions. The PLA positive signal (FarRed) was detected by an LSR Fortessa flow cytometer (BD Biosciences). As a negative control for the PLA, cells were incubated with a commercially available anti-NRP-1 antibody alone.

[0237] To analyze the effect of the a-NRP1_LEP antibody on the NRP-1 / OBR complex formation, the MDA-MB-231 cells were cultured in human serum (H4522, Sigma) for 2 days. The cells were then treated with the murine a-NRP1_LEP antibody of the present invention or its mIgG2b isotype control at concentrations of 10, 20, or 40 μg / ml, or with the prior art antibody a-NRP1_VE and its hIgG1 isotype control at a concentration of 20 μg / ml, for an additional 2 days. After washing, fixation, permeabilization, and blocking, the cells were incubated with 10 μg / ml of a commercially available anti-NRP-1 antibody (NBP2-67539, Novus) and an anti-OBR antibody (AF389, R&D Systems) at 37°C for 1 hour. As a negative control for the PLA, cells were incubated with the commercially available anti-NRP-1 antibody alone. The PLA positive signal (FarRed) was detected by an LSR Fortessa flow cytometer (BD Biosciences).

[0238] (Detection of chondroitin sulfate-modified NRP-1 by flow cytometry) Chondroitinase (C2905, Sigma) was reconstituted in 0.01% BSA aqueous solution. The MDA-MB-231 cells were treated with chondroitinase (1 U / ml) for 2 hours at 37°C. The cells were detached using a non-enzymatic cell dissociation solution (C5914, Sigma). 300,000 cells per condition were washed with 2% FBS-containing PBS and then incubated with 40 μg / ml of the different antibodies of the present invention or their respective isotype controls for 30 minutes at 4°C. After washing, 10 μg / ml of Alexa488-conjugated anti-human immunoglobulin antibody (20022-1, Biotium) was added for 30 minutes at 4°C. After the final wash, fluorescence intensity, cell size, and granularity were measured using an LSR Fortessa flow cytometer (BD Biosciences).

[0239] (Subcellular fractionation: separation of cytoplasmic, membrane, nuclear soluble, chromatin-bound, and cytoskeletal proteins) The wild-type (ab259777, Abcam) and NRP-1-KO A549 cell lines (ab269507, Abcam), the mouse breast cancer cell line 4T1 (purchased from ATCC), the human prostate cancer cell line PC3 (gift from Dr. Christophe Deroanne, Laboratory of Connective Tissues Biology, University of Liege, Belgium), and circulating tumor cells CTC41 / 41.5E (purchased from Dr. Catherine Panabieres, Laboratory of Rare Human Circulating Cells, Saint-Eloi Hospital, University Medical Centre, Montpellier, France) were cultured in human serum and treated with 10 μg / ml of the following antibodies at 37°C for 5 minutes: mouse (a-NRP1_LEP The antibodies tested were: 1B3, 21B10, and 27H10), three different humanized variants of the antibody a-NRP1_LEP of the present invention (variants 4, 6, and 8), the prior art antibodies a-NRP1_VE and a-NRP1_SEM, and their respective irrelevant isotype controls: mIgG2b (mouse a-NRP1_LEP), hIgG4 (humanized a-NRP1_LEP), and hIgG1 (prior art antibody). Adherent cells were collected by scraping. Both adherent and suspended cells were collected by centrifugation at 500 g for 5 minutes and washed three times by suspending the cell pellet in ice-cold PBS.

[0240] Cytoplasmic and nuclear protein fractions were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagent (78833) or Subcellular Protein Fractionation Kit for Cultured Cells (78840, Thermo Scientific) according to the manufacturer's instructions.

[0241] To confirm the purity of the extracts (contamination between nuclear and cytoplasmic fractions), 30 μg of each protein fraction was separated by Western blot, and the membranes were incubated overnight at 4°C with anti-HDAC2 antibody (05-814, Millipore) specific for nuclear HDAC2 protein.

[0242] To confirm the intracellular localization of the antibodies, a mixture of Protein A and Protein G Sepharose 4 Fast Flow beads (1:1) was added to both the cytoplasmic and nuclear protein fractions. The samples were incubated on an orbital rotator mixer at room temperature for 2 hours. The immunoprecipitated samples were washed and then subjected to Western blot analysis using anti-human IgG-HRP1 / 2000 (A18805, ThermoFisher) or anti-mouse IgG-HRP1 / 8000 (1030-05, SouthernBiotech) antibodies.

[0243] Leptin- and a-NRP1-dependent translocation of a-NRP1_LEP into the nucleus of CTC41.5E cell line. Circulating tumor cells CTC41.5E were serum-starved overnight and then treated with α-NRP1_LEP antibody (variant 4) (10 μg / ml) in the presence or absence of leptin (10 nM) for 3 h at 37°C. Cytoplasmic and nuclear fractions were prepared as described above.

[0244] NRP-1 and a-NRP1_LEP antibody complexes were immunoprecipitated by adding a mixture of Protein A and Protein G Sepharose 4 Fast Flow beads (1:1) to both the cytoplasmic and nuclear protein fractions. Samples were incubated on an orbital rotor mixer at room temperature for 2 hours. After washing, the immunoprecipitated samples were subjected to Western blot analysis using either anti-human IgG-HRP (A18805, ThermoFisher) to detect the a-NRP1_LEP antibody or primary rabbit polyclonal antibody anti-NRP-1, followed by anti-rabbit HRP-conjugated antibody.

[0245] (Immunofluorescence microscopy detection of nuclear a-NRP1_LEP) CTC41.5E cells were incubated with mouse α-NRP1_LEP, humanized variants of the α-NRP1_LEP, the antibodies of the present invention, and their respective control isotypes (10 μg / ml) for 3 days. The cells were cytospun onto slides at 1000 g for 5 minutes and fixed with ice-cold acetone for 5 minutes. After washing and blocking, the cells were incubated with anti-human IgG-FITC (20022-1, Biotium) or anti-mouse IgG-FITC (A-11029, ThermoFisher) antibodies (light gray signal) at 1 / 1000 for 1 hour at 37°C. The slides were counterstained with DAPI (dark gray signal) and mounted in PPD. Immunofluorescence (Figure 6.1Aa) was acquired using a ZEISS Plan-Apochromat 63x / 1.40 oil and a CoolCube 1 Digital High-Resolution CCD Camera, using the automated acquisition module Autocapt software (MetaSystems, version 3.9.1).

[0246] (Detection of DNA damaging effects with a-NRP1_LEP antibody using M-FISH technique after telomere and centromere staining) This approach was used to reliably and accurately assess the genotoxic effects of the anti-NRP-1 antibody of the present invention (a-NRP1_LEP) compared to prior art antibodies (a-NRP1_VE and a-NRP1_SEM) in CTC cells.

[0247] For chromosome preparation and mitotic index quantification, circulating tumor cells (CTC41 and CTC41.5E) were cultured in human serum and treated with the corresponding anti-NRP-1 antibodies and their respective isotype controls (mouse IgG2b, human IgG4, and IgG1) as described above. After 72 hours of treatment, colcemid (100 ng / ml) was added 2 hours before cell harvest, and metaphase cells were prepared according to a standard methanol / acetic acid (3 / 1, v / v) procedure. Treated cells were stored at -20°C until use.

[0248] Briefly, telomeres and centromeres were hybridized with a Cy-3-labeled PNA probe specific for telomeric TTAGGG and an FITC-labeled probe specific for centromeric sequences (Cell Environment, Evry, France) as previously described (M'kacher et al., 2014; M'kacher et al., 2015). Briefly, slides were washed with 1X PBS and fixed with 4% formaldehyde at room temperature. After rinsing three times with PBS (3 × 5 min), slides were treated with pepsin (0.5 mg / ml) at 37°C for 5 min. After rinsing three times with PBS (3 × 5 min), the slides were dehydrated sequentially with 50%, 70%, and 100% ethanol and air-dried. The telomere and centromere probes were added to the slides, denatured on a hot plate at 80°C for 3 minutes, and incubated at room temperature for 1 hour in the dark. The slides were then rinsed twice with 70% formamide / 10 mM Tris pH 7.2 for 15 minutes each (2 × 15 minutes), followed by rinsing in 50 mM Tris pH 7.2 / 150 mM NaCl / 0.05% Tween-20 (3 × 5 minutes). After a final rinse with PBS, the slides were counterstained with DAPI and mounted in PPD. After telomere quantification and automated photography of metaphases with telomere and centromere staining, the same slides were washed with 2× SCC at 70°C for 30 minutes, rinsed with 0.1× SSC at room temperature for 1 minute, and then denatured with NaOH. After washing with 0.1× SCC and 2× SSC for 1 minute, the slides were dehydrated sequentially with 70%, 95%, and 100% ethanol and air-dried. M-FISH probes (M-FISH 24XCyte, Metasystems, Altlussheim, Germany) were denatured at 75°C for 5 minutes. The probes were added to the slides and incubated at 37°C for 2 days. The slides were then rinsed with 0.4× SSC at 72°C for 2 minutes, followed by rinsing with 2× SSC / 0.005% Tween-20.The slides were counterstained with DAPI and mounted in PPD (Kaddour et al. 2017; M'kacher et al. 2020).

[0249] Chromosome aberrations were assessed. A total of 100 metaphases were scored for each condition. Metaphase images were acquired using a ZEISS Plan-Apochromat 63x / 1.40 oil and a CoolCube 1 Digital High-Resolution CCD Camera with the Autocapt software (MetaSystems, version 3.9.1) automated acquisition module. Exposure and gain settings were kept constant between images. Analysis was performed using Isis software (MetaSystems, version 3.9.1) as previously described.

[0250] (Micronucleus assay) Circulating tumor cells (CTC41 and CTC41.5E) were cultured in RPMI 1640 Medium GlutaMA™ (Product No. 61870036, Gibco) supplemented with human EGF Premium (Product No. 130-097-749, Miltenyi), human FGF-2 Premium (Product No. 130-093-564, Miltenyi), insulin-trans-sel-A (Product No. 51300044, Gibco), and 10% inactivated human serum (Sigma-Aldrich). Cells were treated with 10 μg / ml of humanized variants of the inventive antibody a-NRP1_LEP (Variant 4, Variant 6, and Variant 8) and the prior art antibodies a-NRP1_VE and a-NRP1_SEM at 37°C for 3 days. Human IgG4 and IgG1 were used as negative controls. Cytochalasin B (from Drechslera dematioidea, Sigma) solution, prepared in dimethyl sulfoxide at a concentration of 6 μg / mL, was added 24 hours before treatment termination according to standard procedures.

[0251] Automated scoring of micronuclei (MN) was performed using MNScore software (version 3.8.101, MetaSystems, Althaussen, Germany) with a Metafer 4 image analyzer (MetaSystems, Althaussen, Germany) equipped with a Zeiss Axioplan 2 imager to detect MN. False-positive MN in binucleated cells were manually identified and excluded.

[0252] (Liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) analysis) 100 μg of protein lysates from circulating tumor cells CTC41.5E and human lung cancer cells A549 were incubated overnight at 4° C. with a 1:1 mixture of Protein A and Protein G Sepharose 4 Fast Flow beads (17-6002-35, GE Healthcare) and 2 μg of the humanized antibody a-NRP1_LEP (variant 4) of the present invention or the prior art antibody a-NRP1_VE and their respective isotype controls (human IgG4 and IgG1). Proteins immunoprecipitated by the above antibodies or their corresponding isotype controls were detected by LC-MS / MS.

[0253] Reagents: MS-grade acetonitrile (ACN), MS-grade HO, and MS-grade formic acid (FA) were from ThermoFisher Scientific (Waltham, MA, USA). Sequencing-grade trypsin, Lys-C, and Mass Spec Grade were from Promega (Madison, WI, USA). Dithiothreitol (DTT) and iodoacetamide (IAA) were from Sigma-Aldrich.

[0254] Sample preparation before LC-MS / MS analysis: Beads were subjected to double enzymatic digestion in 20 μl of 25 mM NH4HCO3 buffer containing 2 μg of trypsin and a Lys-C mixture (1:1) per sample overnight at 37° C. The resulting peptides were loaded onto an Evosep (Odense, Denmark) and desalted according to the manufacturer's protocol.

[0255] LC-MS / MS Acquisition: Samples were analyzed using an Evosep one system (Evosep, Odense, Denmark) coupled to a timsTOF Pro 2 mass spectrometer (Bruker Daltonics, Bremen, Germany) using the manufacturer's developed 30SPD method. Briefly, the method was based on a 44-minute gradient and 48-minute total cycle time using a C18 analytical column (0.15 x 150 mm, 1.9 μm beads, ref. EV-1106), equilibrated at room temperature, and operated at a flow rate of 500 nl / min. H2O / 0.1% FA was used as solvent B, and ACN / 0.1% FA was used as solvent B.

[0256] The timsTOF Pro 2 was operated in PASEF mode (Meier F; Beck S; Grassl N; Lubeck M; Park MA; Raether O; Mann M. Parallel Accumulation-Serial Fragmentation (PASEF): Multiplying Sequencing Speed ​​and Sensitivity by Synchronized Scans in a Trapped Ion Mobility Device. J. Proteome Res. 2015, 14 (12), 5378-5387) with a cycle time of 1.3 seconds. Mass spectra for MS and MS / MS scans were recorded between 100 and 1700 m / z. Ion mobilities were 0.75–1.25 V·s / cm with a ramp time of 180 ms.2 The time was set to 0. Data-dependent acquisition was performed using 10 PASEF MS / MS scans per cycle at a duty cycle of approximately 100%. Low m / z and singly charged ions were excluded from PASEF precursor selection by applying filters in m / z and ion mobility space. Dynamic exclusion was enabled and set for 0.8 min. A target value of 16,000 was specified with an intensity threshold of 1,000. Collision energy was ramped in steps as a function of ion mobility.

[0257] Data analysis: MS raw data files were processed using PEAKS OnlineX (build 1.5, Bioinformatics Solutions Inc.). Data were searched against the Human Uniprot release 2021_03 database (20,387 total entries), which consists only of reviewed sequences. Parent mass tolerance was set at 20 ppm, along with a fragment mass tolerance of 0.05 Da. Specific tryptic cleavages were selected, and up to two missed cleavages were allowed. For identification, the following post-translational modifications were included: acetylation (protein N-terminus) as variables and half of a disulfide bridge, oxidation (M), deamidation (NQ), or carbamidomethylation (C) as fixed. Identifications were filtered based on a 1% FDR (False Discovery Rate) threshold at both the peptide and protein group levels.

[0258] (In vivo validation of the ability of the humanized anti-NRP-1 antibody a-NRP1_LEP of the present invention: ability to enter the nucleus of subcutaneously xenografted CTC cells in SCID mice and ability to induce DNA damage) Eight SCID mice (Jackson Mouse SN 1803 F individual mice, CBySmn.Cg-Prkdcscid / J, Ho, F, 7 wo) were injected subcutaneously into the right flank with CTC41.5E cells in 100 μl of medium / Matrigel (Corning # 356255) (1:1). The mice were continuously monitored for tumor growth. Total tumor volume reached 300 mm 3 At the time of reaching 100 mg / kg, the mice were injected weekly with 200 μg of either humanized a-NRP1_LEP variant 4 or its isotype control (human IgG4) via the tail vein. At the end of the experiment, tumor samples (primary tumors) and organs (e.g., lungs and liver) were collected for histological and biochemical analysis. The harvested tumors were either (i) dissociated using a Tumor Dissociation Kit (Miltenyi #130-096-730), and the resulting tumor cells were lysed using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo #78833), followed by immunoprecipitation using a 1:1 mixture of Protein A and Protein G Sepharose 4 Fast Flow beads alone, followed by Western blot analysis to detect the localization of humanized a-NRP1_LEP variant 4; or (ii) placed in plastic molds, covered with a thick layer of Cryomatrix™ cryo-embedding medium (Thermo Scientific), and stored at -80°C until use. For immunofluorescence analysis, 5 μm sections of the samples were cut at -20°C using a CryoStar NX70 Cryostat (Thermo Scientific), and the frozen tissue sections were mounted on slides and stored at -20°C until use.

[0259] Telomeres and centromeres were stained using a Cy-3-labeled PNA probe specific for telomeric TTAGGG and an FITC-labeled probe specific for centromeric sequences (Cell Environment, Evry, France). Briefly, slides with 5 μm tumor sections were fixed with acetic acid / methanol (1 volume:3 volumes) for 20 minutes. The slides were dried overnight at room temperature.

[0260] After washing with PBS, tumor sections were fixed with 4% formaldehyde at room temperature. After rinsing three times with PBS, they were permeabilized with pepsin (0.5 mg / ml) at 37°C for 5 minutes. After washing three more times with PBS, tumor sections were dehydrated sequentially with 50%, 70%, and 100% ethanol and air-dried. The telomere and centromere probes were added to the tumor sections, denatured on a hot plate at 80°C for 3 minutes, and then incubated in the dark at room temperature for 1 hour. The slides were then rinsed three times with 70% formamide / 10 mM Tris pH 7.2 for 15 minutes each, and finally rinsed in 50 mM Tris pH 7.2 / 150 mM NaCl pH 7.5 / 0.05% Tween-20 (3 x 5 minutes). After a final wash with PBS, the tumor sections were counterstained with DAPI and mounted in PPD at the appropriate pH.

[0261] In vivo analysis of the effects of mouse a-NRP1_LEP antibody on metastasis and the immune system using the highly metastatic breast cancer cell line 4T1 in a compatible syngeneic mouse model. All experimental procedures were approved by the French Animal Experiments Ethics Committee n°026 in 2020, in accordance with French and European legislation. Five- to six-week-old female BALB / c mice were purchased from Charles River Laboratories (L'Arbresle, France). Mice were housed under SPF conditions, with a maximum of five mice per cage, and were allowed one week to adapt to the new environment. Subsequently, mouse breast cancer cells 4T1 (50,000 cells / 20 μl of PBS) were orthotopically implanted into the third mammary gland (lower left side of the mouse). Tumor volume and body weight were measured three times weekly. The average tumor volume was 150 mm. 3 At the time of the appearance of lung metastases, mice were randomly assigned to experimental groups. They were then intravenously injected three times (once weekly) with PBS, mIgG2b isotype control (InVivoPlus mouse IgG2b isotype control, unknown-specificity - Catalog #BP0086), and mouse a-NRP1_LEP antibody at 2.5 mg / kg (equivalent to 50 μg for a mouse with an average body weight of 20 g). Mice were euthanized 10 days after the last injection, and organs were harvested and fixed for histological analysis.

[0262] (Histology of lung metastases and immune system infiltration) Histological analysis was performed at the Gustave Roussy Laboratory's ISO-certified AMMICa PETRA platform (ISO 9001:2015). Lungs were fixed with 4% paraformaldehyde solution (F8775, Sigma), dehydrated, and embedded in paraffin. Samples were cut using a microtome to obtain 4-μm-thick sections. All samples were then stained with hematoxylin / eosin / saffron (HES). Immunohistochemical staining of lung sections was performed using an automated Bond RX (Leica) system. All samples and slides were anonymized to ensure that technicians were blinded to the treatment of the mice.

[0263] To detect CD4 / FOXP3 double staining (Cell Signaling #25229, clone D7D2Z, 1:50 / #12653 clone D608R, 1:200), paraffin sections were unmasked by heating in ER2 (EDTA buffer pH 9) at 100°C for 20 minutes, and then incubated with the two primary antibodies sequentially for 1 hour at room temperature. The antibodies were detected using the Bond Polymer Refine Red Detection kit (DS9390, Leica Biosystems) and the Bond Polymer Refine Detection kit (DS9800, Leica Biosystems), respectively, and the HIGHDEF® black IHC chromogen (HRP) kit (ADI-950-171-0030, Enzo Lifesciences). In the case of a positive signal, red staining (Fast Red) indicates CD4 on the membrane, and black staining appears for FOXP3 in the nucleus.

[0264] For CD4 / CD8 / GrB or CD4 / CD8 / Perf triple marker detection, slides were incubated in ER2 buffer (pH 9.0) at 100°C for 20 min for antigen retrieval. Subsequently, the slides were sequentially incubated with the antibodies CD4 (Cell Signaling #25229, clone D7D2Z, 1:50), CD8 (Cell Signaling #98941, clone D4W2Z, 1:400), granzyme B (Cell Signaling #44153, clone 5EV2L, 1:100), or perforin (Cell Signaling #31647S, clone E3W4I, 1:100) for 30 min at room temperature, followed by detection with polymer anti-rabbit HRP OPAL (Akoya, #NEL830001KT). The signals were revealed by OPAL 480 (Akoya, #FP1500001KT) for CD4, OPAL 570 (Akoya, #FP1488001KT) for CD8, and OPAL 690 (Akoya, #FP1497001KT) for granzyme B or perforin. Finally, the sections were counterstained with DAPI (Akoya) and the slides were mounted for immunofluorescence scanning using an Akoya scanner and image analyzer, Vectra POLARIS.

[0265] (Slide scanning) HES slides were scanned at 20x magnification using a Hamamatsu Nanozoomer 2.0-HT C9600-13, and CD4 and FoxP3 double-stained slides were scanned at 20x magnification using an Olympus VS 120.

[0266] (Stain detection and quantification) All slides were processed using QuPath software [Bankhead, P. et al. QuPath: Open source software for digital pathology image analysis. Scientific Reports (2017) https: / / doi.org / 10.1038 / s41598-017-17204-5]. The quantification process involved four steps: drawing regions of interest, cell detection, cell classification, and, optionally, cell measurement (e.g., calculating the distance to tumor compartments). Automatic selection of regions of interest (whole tissue or tumor area) was performed by a classifier (trained by machine learning to recognize these compartments), which was then manually refined to exclude artifacts and misclassifications.

[0267] For CD4 / FoxP3 staining, cell detection was performed using the Cell detection function, and phenotypic analysis was performed using two sequential classifiers specialized for recognizing red membrane staining and black nuclear staining, respectively. Distance to the tumor edge was calculated using the Distance to annotation 2D function. The size of lung metastases was determined manually by a senior pathologist using Hamamatsu digital pathology NDP.view2 U12388-01 viewing software.

[0268] For CD4 / CD8 / GrB or CD4 / CD8 / Perf staining, cell detection was performed using the Cell detection function, and phenotypic analysis was performed using three consecutive classifiers specialized in recognizing Opal480 staining (CD4), Opal570 staining (CD8), and Opal690 staining (GrB or Perf).

[0269] The results are shown as the density of stained cells (per mm of tumor tissue) for each phenotype. 2 The numbers are shown as the number of stained cells of each phenotype per 1000 cells / well.

[0270] (statistical analysis) Statistical analysis was performed using GraphPad Prism 6 software. Differences between groups were tested for significance by one-way ANOVA. A p value of ≤ 0.05 was considered statistically significant.

[0271] [II-Results] Example 1: NRP-1 peptide vaccine strategy induces an immune response as demonstrated by the production of neutralizing antibodies capable of recognizing the NRP-1 protein. Generally, the development of antibodies using peptide sequences that mimic the epitope of an antigen is not used because the antibodies generated do not necessarily recognize the native antigen. Furthermore, when developing monoclonal antibodies (MAbs) using proteins, it is difficult to generate antibodies against specific sites, such as modifications or specific domains.

[0272] Interestingly, the prior art anti-NRP-1 antibodies (a-NRP1_VE and a-NRP1_SEM) were developed using phage display technology. On the other hand, the antibody of the present invention, a-NRP1_LEP, was generated using an NRP-1 peptide-based vaccine conjugated to a carrier protein for mouse immunization, thus demonstrating the therapeutic potential of NRP-1 peptides (Npep) as cancer and infectious disease vaccines. Among the vaccination strategies previously described in the Materials and Methods section, only the N3 sequence conjugated to KLH was able to induce antibody production with the requisite properties compared with the synthetic N1 and N2 sequences conjugated to biotin-streptavidin. Thus, N3 peptide vaccination generated distinct antibody clones, which were classified into two series: (i) 1B3, 2C2, 2C5, 8E11, PB4, and PD7 antibodies, which were developed before the mice developed the obese phenotype, and (ii) 21B10, 21H6, 23H8, and 27H10 antibodies, which were developed after the fourth intraperitoneal immunization followed by an intravenous boost with the antigen (N3 peptide) from mice with the obese phenotype. All a-NRP1_LEP clones generated were able to recognize the Npep sequence and the recombinant NRP-1 protein, but only antibodies generated after the mice developed the obese phenotype were able to enter the cell nuclei. Details are shown in Example 4 (Figure 6.1Ac).

[0273] Example 2: a-NRP1_LEP antibody neutralizes NRP-1 / OBR complex signaling NRP-1 functions as a coreceptor for leptin, as a complex of NRP-1 and the leptin receptor (OBR) was detected by immunohistochemistry in tissue microarrays of different cancer tissues (Figure 1). The formation of this complex is leptin-dependent (Methods and Pharmaceutical Compositions for the Treatment of Diseases Mediated by the NRP-1 / OBR Complex Signaling Pathway, WO 2015 / 124588). Consequently, to develop specific neutralizing antibodies that inhibit leptin binding to NRP-1 and prevent NRP-1 / OBR signaling, mice immunized with a peptide sequence of NRP-1 corresponding to leptin binding exhibited an obese phenotype as proof of concept (Figure 2). The anti-NRP-1 (a-NRP1_LEP) neutralizing antibody targeting leptin signaling was selected based on two main criteria: (i) the obese phenotype, and (ii) the ability to recognize human and mouse NRP-1 (verified by ELISA-positive signals on recombinant proteins, as shown in Figure 2 and Table 1), all of which contribute to its ability to bind to native NRP-1 protein on the surface of human and mouse cells.

[0274] [Table 1]

[0275] Table 1: Extraction results of a screening ELISA aimed at selecting hybridomas showing interesting reactions to human and mouse recombinant NRP-1 and to the synthetic peptide Npep.

[0276] For further research and engineering steps, mouse a-NRP1_LEP antibodies 21B10 and 27H10 were selected and named a-NRP1_LEP n°1 and a-NRP1_LEP n°2, respectively.

[0277] Example 3: a-NRP1_LEP antibody that recognizes a specific epitope present on the b1 domain of NRP-1 and has higher specificity for GAG-modified NRP-1 compared to prior art antibodies The chimeric and humanized a-NRP1_LEP antibodies of the present invention have been shown to have epitope specificity corresponding to the leptin-binding site in the b1 domain of NRP-1 (Figure 3A). This epitope specificity is demonstrated by the fact that the prior art antibodies a-NRP1_VE and a-NRP1_SEM, which specifically target the VEGF and SEMA binding sites on NRP-1, respectively, do not recognize this peptide sequence (Table 2). In contrast, all of the above-described antibodies were able to bind to recombinant human and mouse NRP-1 and to cells expressing native NRP-1 protein on their surfaces. The epitopes of a-NRP1_LEP and a-NRP1_VE are shown in Figure 3B.

[0278] [Table 2]

[0279] Table 2: Specificity of chimeric and humanized variants of the a-NRP1_LEP antibody confirmed by ELISA for binding (10 μg / ml) to immobilized human and mouse recombinant NRP-1 (1 μg) and the synthetic peptide Npep (2 μg) in comparison with irrelevant human immunoglobulins and prior art antibodies (a-NRP1_VE and a-NRP1_SEM).

[0280] The specificity of the antibody a-NRP1_LEP of the present invention for NRP-1 protein was demonstrated in A549WT and NRP-1-KO cells using the prior art antibody a-NRP1_VE as a positive control for NRP-1 protein detection. As shown in Figure 4A, neither the antibody a-NRP1_LEP of the present invention nor the prior art antibody a-NRP1_VE detected NRP-1 protein in the A549NRP-1-KO cell line. Interestingly, the specificity of both the antibody a-NRP1_LEP and a-NRP1_VE for GAG-modified NRP-1 (>150 kDa) was confirmed. The antibody a-NRP1_LEP of the present invention can bind to GAG-modified NRP-1 (>150 kDa) with high specificity, as demonstrated in CTC41.5E cells (Figure 4A) and multiple cell types (human breast cancer cell line MDA-MB231, human colon cancer cell line COLO205, and mouse breast cancer cell line 4T1). On the other hand, prior art antibodies only weakly recognize GAG-modified NRP-1 (>150 kDa), as shown in Figure 4A.

[0281] The exclusive and unique ability of the a-NRP1_LEP antibody to bind with high specificity to GAG-modified NRP-1 protein may depend on the cell type and its state. Thus, highly GAG-modified forms of NRP-1 are (i) The stress resistance observed in serum-starved MDA-MB-231 breast cancer cell line (MDA-MB-231), which exclusively expresses the GAG-modified form (>150 kDa), compared with unstressed cells (Figure 4B), which express both non-GAG-modified NRP-1 (≤150 kDa) and highly GAG-modified NRP-1 (>150 kDa). (ii) Therapy escape mechanisms observed in circulating tumor cells, considered to be “stem-like” cells, isolated from a patient with treatment-resistant metastatic colon cancer CTC41.5E; may be useful.

[0282] These cells appear to express a specific, highly GAG-modified form of NRP-1 that is sensitively detectable by our a-NRP1_LEP antibody, as shown in Figure 4A, in contrast to other anti-NRP-1 ligands (e.g., the known a-NRP1_VE antibody). Furthermore, our antibody binds weakly to non-GAG-modified NRP-1 in both immortalized cells (A549) and patient-derived cancer cells (CTCs) (Figure 4).

[0283] Association of GAG-modified NRP-1 with the a-NRP1_LEP target, i.e., the leptin-dependent NRP-1 / OBR complex, has been demonstrated in human breast cancer (MDA-MB-231), human colon cancer (COLO205), and mouse breast cancer (4T1) cell lines (Fig. 4B and C). NRP-1 immunoprecipitation performed in leptin-treated cells after overnight serum starvation demonstrated that the a-NRP1_LEP antibody could bind to the high-molecular-weight form of GAG-modified NRP-1 (>150 kDa) in a leptin-dependent manner, confirming that the antibody of the present invention is specific for the leptin-binding site on the NRP-1 protein, unlike the prior art antibodies a-NRP1_VE and a-NRP1_SEM, which target the VEGF- and semaphorin-binding sites on NRP-1, respectively (Fig. 4C). Under leptin stimulation, two historical antibodies, a-NRP1_VE and a-NRP1_SEM, from the prior art, primarily bind to glycosylated forms of NRP-1 (≦150 kDa) and, to a lesser extent, to high molecular weight forms of NRP-1 (>250 kDa) (Figure 4C). Furthermore, when cells were incubated with leptin for 3 hours, the signal became even higher, indicating that this treatment induces increased NRP-1 / OBR protein complex formation at the cell membrane (Figure 4C).

[0284] Taken together, these data suggest that the a-NRP1_LEP antibody of the present invention, in contrast to the prior art a-NRP1_VE and a-NRP1_SEM antibodies, binds primarily to the post-translationally modified, high molecular weight form of NRP-1 (>250 kDa) (GAG-modified NRP-1) associated with the NRP-1 / OBR complex, as confirmed by co-immunoprecipitation of CTCs from patients (Fig. 4D).

[0285] Because the a-NRP1_LEP antibody binds to high molecular weight forms of NRP-1, we decided to investigate the nature of this post-translational modification (Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling. Yamamoto, Tomi Fukushima, Kazuyuki Sugahara, Masafumi Kitakaze, Masatsugu Hori. EMBO J 2006 Jul 12;25(13):3045-55 and Chondroitin sulphate-modified neuropilin 1 is expressed in human tumor cells and modulates 3D invasion in the U87MG human glioblastoma cell line through a p130Cas-mediated pathway. Paul Frankel, Caroline Pellet-Many, Pauliina Lehtolainen, Giovanna M. D'Abaco, Michelle L Tickner, Lili Cheng, Ian C Zachary. EMBO Rep 2008 Oct;9(10):983-9). We focused on chondroitin sulfate and heparan sulfate from four different classes of GAGs. Flow cytometry analysis of MDA-MB-231 cells treated with chondroitinase for 2 hours showed a decrease in the percentage of cells staining with a-NRP1_LEP (mouse, chimeric, and humanized variants) compared with the prior art antibodies a-NRP1_VE and a-NRP1_SEM.This was indicated by a shift in the fluorescence intensity histogram (Fig. 5A, gray histogram), signifying decreased binding of a-NRP1_LEP to GAG-modified NRP-1 on the cell surface (Table 3).

[0286] [Table 3]

[0287] Table 3: Percentage of a-NRP1_LEP antibodies of the invention binding to native NRP-1 on MDA-MB-231 cells under chondroitinase treatment compared to prior art antibodies (a-NRP1_SEM and a-NRP1_LEP) and the corresponding isotype controls.

[0288] These data are validated by Western blotting performed after chondroitinase treatment of NRP-1 immunoprecipitated with a-NRP1_LEP, which revealed a decrease in the GAG-modified form of NRP-1 compared with NRP-1 immunoprecipitated with a-NRP1_VE and a-NRP1_SEM (Fig. 5B ).

[0289] As expected, a-NRP1_VE and a-NRP1_SEM (especially a-NRP1_VE) bind primarily to glycosylated forms of NRP-1 (≦150 kDa) and, to a lesser extent, to higher molecular weight forms of NRP-1 (>250 kDa) (Figure 5B). This result correlates with flow cytometry analysis, which showed no significant reduction in binding to the cell surface of MDA-MB-231 cells. In contrast to flow cytometry analysis, treatment of immunoprecipitated cell lysates with chondroitinase or heparinase indicates that a-NRP1_VE recognizes the lower molecular weight chondroitin sulfate form (Figure 5B). On the other hand, as already shown above, the chimeric and variant a-NRP1_LEP antibodies bind to the higher molecular weight GAG-modified NRP-1 rather than the glycosylated forms (≦150 kDa) of NRP-1 (Figure 5B). Interestingly, a-NRP1_LEP appears to recognize both chondroitin and heparan sulfate GAGs of NRP-1 (Fig. 5B).

[0290] These data demonstrated that the antibodies of the present invention recognize a new epitope that is distinct from those recognized by prior art antibodies.

[0291] Further confirmation of the recognition of GAG-modified NRP-1 by the a-NRP1_LEP antibody was assessed by PNGase treatment of immunoprecipitated samples obtained with the humanized antibody of the present invention from cell lysates of CTCs obtained from patients. Interestingly, a loss of the highest molecular weight form of GAG-modified NRP-1 (>250 kDa) and an enrichment of glycosylated forms of NRP-1 (<150 kDa) were observed (Figure 5C).

[0292] Example 4: a-NRP1_LEP antibody capable of penetrating into the nuclei of multiple types of cancer cells Evaluation of the properties of a-NRP1_LEP (cell surface binding, intracellular entry, and intracellular localization within CTCs) using FITC-labeled secondary anti-human IgG and anti-mouse IgG antibodies revealed the unexpected nuclear localization of our a-NRP1_LEP antibody, a unique property that has not been reported for any anti-NRP-1 antibody in basic research or drug development (Figure 6.1Aa).

[0293] Validation of the nuclear entry of the a-NRP1_LEP antibody was demonstrated by its detection in the nuclear fractions of several a-NRP1_LEP-treated cell types after immunoprecipitation with Protein A and Protein G Sepharose 4 Fast Flow beads and Western blot analysis (Figures 6.1 and 6.2). This unexpected ability of the inventive antibody to enter the nucleus was not observed in any of the cell types (human prostate cell line PC3, mouse breast cancer cell line 4T1, and human circulating tumor cells CTCs obtained from patients) treated with prior art antibodies (a-NRP1_VE and a-NRP1_SEM) (Figure 6.2). The validity of the previously employed cell fractionation technique was confirmed using the specific nuclear marker HDAC2 protein.

[0294] Example 5: NRP-1-dependent nuclear entry of the antibody of the present invention, a-NRP1_LEP, in tumor cells via the NRP-1 / OBR complex. The NRP-1-dependent nuclear entry of the a-NRP1_LEP antibody was confirmed by the significantly higher detection rate of the a-NRP1_LEP antibody in wild-type A549 lung cancer cell lines compared to NRP-1 knockout cell lines (Figure 7.1Aa and Ab).

[0295] The NRP-1 / OBR complex-dependent entry of a-NRP1_LEP antibody is demonstrated by the dose-dependent formation of NRP-1 / OBR complexes in MDA-MB-231 cells treated with a-NRP1_LEP (Fig. 7.1Ba and Bb). The significant increase in NRP-1 / OBR complex formation detected by proximity ligation assay was specific to a-NRP1_LEP; this effect was not observed with the isotype controls, mouse IgG2b and a-NRP1_VE (Fig. 7.1Ba and Bb).

[0296] Example 6: Leptin-dependent induction of co-translocation of NRP-1 and a-NRP1_LEP antibody into the nucleus of the CTC41.5E cell line Leptin-dependent NRP-1 / OBR complex formation and nuclear translocation was reported in a previous patent (WO 2015 / 124588) and builds on the recent discovery of NRP-1-dependent nuclear translocation of our antibody a-NRP1_LEP (Example 4). We investigated the simultaneous nuclear localization of the a-NRP1_LEP antibody and GAG-modified NRP-1 protein. Western blots performed on nuclear fractions of CTCs serum-starved overnight and stimulated with leptin for 3 hours after immunoprecipitation using Protein A and Protein G Sepharose 4 Fast Flow beads showed simultaneous detection of the a-NRP1_LEP antibody and GAG-modified NRP-1 protein in a leptin-dependent manner (Figure 7.2).

[0297] Example 7: Antibody a-NRP-1-LEP of the invention induces DNA damage (chromosome shattering) and / or centromere destabilization and / or telomere shortening in circulating tumor cells. The effect of the inventive antibody a-NRP1_LEP on nuclear entry was investigated in circulating tumor cells (CTCs) treated with variants a-NRP1_LEP (variants no. 4, 6, and 8), their isotype control hIgG4, or the prior art antibody a-NRP1_VE. These cells were selected due to (i) the high expression of GAG-modified NRP-1 protein in CTCs from patients before treatment (CTC41) and after second-line treatment and relapse, (ii) the high association of this NRP-1 form with treatment resistance (e.g., to chemotherapy and antiangiogenic drugs), and (iii) the binding of the a-NRP1_LEP antibody to native NRP-1 protein on the CTC cell surface (Table 4).

[0298] [Table 4]

[0299] Table 4: Percentage of circulating tumor cells (CTCs) bound by the anti-NRP-1 antibody of the invention (a-NRP1_LEP).

[0300] Cytogenetic analysis revealed that the antibodies of the present invention have genotoxicity, inducing DNA damage. This DNA damage is manifested by chromosome shattering (Figure 8A), multiple acentric fragments (Figure 8B), and micronuclei (Figure 8C). This genotoxic effect was barely observed with the prior art antibodies a-NRP1_VE and a-NRP1_SEM, correlating with their inability to enter the cell nucleus. Furthermore, this genotoxic effect was not observed with leptin, the ligand that binds to NRP-1, the target of the a-NRP1_LEP antibody of the present invention (Figure 8D).

[0301] Quantification of the genotoxic effect of the inventive antibody a-NRP1_LEP, as indicated by (i) the number of acentric fragments, was statistically significant in both CTC41 and CTC41.5E cell lines when compared with the isotype control hIgG4 (p<0.001, Fisher's test). In contrast, the number of acentric fragments induced by the prior art antibodies a-NRP1_VE and a-NRP1_SEM was less significant in the CTC41 cell line (p<0.05, Fisher's test) and not significant in the CTC41.5E cell line (p>0.05, Fisher's test) when compared with the isotype control hIgG1 (Figure 8B). Furthermore, quantification of the genotoxic effect of the inventive antibody a-NRP1_LEP, as indicated by (ii) the number of micronuclei, was statistically significant in both CTC41 (p<0.05, Fisher's test) and CTC41.5E (p<0.001, Fisher's test) cell lines when compared with the isotype control hIgG4. In contrast, the number of micronuclei induced by the prior art antibodies a-NRP1_VE and a-NRP1_SEM was less significant in the CTC41.5E cell line (p<0.05) and not significant in the CTC41 cell line (p>0.05) when compared with the isotype control hIgG1 (Figure 8C).

[0302] Quantification of telomere (Fig. 8D-c) and centromere (Fig. 8D-d) intensity was significantly reduced after treatment of CTCs with the antibody a-NRP1_LEP of the present invention compared to the isotype control hIgG4 (p<0.001, chi-squared test) and untreated cells (p<0.001, chi-squared test), correlating with the ability of the a-NRP-1_LEP antibody to induce telomere shortening and centromere destabilization in pathological cells.

[0303] Although leptin and the antibody of the present invention share the same property (binding to NRP-1 and inducing its entry into the cell nucleus), no genotoxic effect was observed after treating CTCs with 10 nM leptin. As shown in Figure 8D, the (i) increase in the number of acentric fragments (Figure 8D-a), (ii) increase in the number of micronuclei (Figure 8D-b), (iii) decrease in the intensity of telomeres (Figure 8D-c), and (iv) decrease in the intensity of centromeres (Figure 8D-d) induced in CTCs treated with a-NRP-1_LEP for 24 hours were statistically significant compared to leptin (p<0.001, Fisher's test (i); p<0.001, chi-square test (ii); p<0.001, chi-square tests (iii) and (iv)). To better understand this genotoxic effect of the inventive antibody a-NRP1_LEP, we tested its ability to interact with proteins involved in DNA repair mechanisms (described in detail in Example 8) or to remodel chromatin. Subcellular fractionation of a-NRP1_LEP-treated cells revealed that the inventive antibody a-NRP1_LEP binds to chromatin after entering the nucleus of CTC cells (Figure 10).

[0304] Example 8: Interaction between NRP-1 and proteins involved in DNA repair mechanisms revealed by the antibody a-NRP1_LEP of the present invention The involvement of GAG-modified NRP-1 in DNA damage through co-nuclear entry with the genotoxic antibody a-NRP1_LEP of the present invention was demonstrated by co-immunoprecipitation with DNA repair proteins using the a-NRP1_LEP antibody, as revealed by mass spectrometry. Table 5 shows the enrichment of proteins involved in different DNA repair pathways detected in samples immunoprecipitated from CTC41.5E and A459 lung cancer cell lysates using the a-NRP1_LEP variant 4 antibody (V4: light gray) and a prior art antibody (a-NRP1_VE: selected for its minimal toxic effects) (TAB: dark gray). The DNA repair-related proteins shown in Table 5 were selected based on the number of enriched unique peptides and acceptable (significant) peak area values ​​(a-NRP1_LEP and a-NRP1_VE antibodies are shown in bold).

[0305] The functional protein association network of various DNA repair proteins and unknown NRP-1 interaction networks immunoprecipitated by the a-NRP1_LEP antibody of the present invention and detected by LC-MS / MS, in comparison with the prior art antibody a-NRP1_VE, is shown in Figure 9A. These enriched proteins appear to have experimentally determined or predicted interactions, such as gene neighborhood, gene fusion, gene co-occurrence / co-expression, or protein homology.

[0306] Verification of the interaction of NRP-1 with proteins involved in DNA repair machinery was revealed by Western blot analysis, which showed the detection of a-NRP1_LEP and PARP in the cytoplasm and / or nucleus of the CTC41.5E cell line treated with the variant a-NRP1_LEP antibody (variant 4) after coimmunoprecipitation using only protein A and protein G Sepharose (Fig. 9B ).

[0307] [Table 5]

[0308] Table 5: List of immunoprecipitated proteins associated with the a-NRP1_LEP antibody of the invention and detected by LC-MS / MS. DSB: double-strand break, SSB: single-strand break, NHEJ: non-homologous end joining, HR: homologous recombination, BER: base excision repair, NER: nucleotide excision repair, RER: ribonucleotide excision repair, MMR: mismatch repair, V4: variant 4 a-NRP1_LEP, TAB: a-NRP1_VE, PRKDC: DNA-dependent protein kinase catalytic subunit (DNA-PK), XRCC6: X-ray repair cross-complementing protein 6, XRCC5: X-ray repair cross-complementing protein 5, PARP1: poly[ADP-ribose] polymerase 1, LIG3: DNA ligase 3, TOP1: DNA topoisomerase 1, RPA1: replication protein A 70 kDa DNA binding subunit, SUPT16H: FACT complex subunit SPT16, PDS5B: sister chromatid cohesion protein PDS5 homolog B, MACROH2A1: core histone macro-H2A.1, MSH2: DNA mismatch repair protein Msh2, TOP2A: DNA topoisomerase 2-α, POLR1A: DNA-dependent RNA polymerase I subunit RPA1, MSH6: DNA mismatch repair protein Msh6, WRN: Warner syndrome ATP-dependent helicase, XPC: XP-C cells Complementary DNA repair proteins: H2AZ2: histone H2A.V, H2AZ1: histone H2A.Z, SMARCA5: SWI / SNF-related matrix-binding actin-dependent chromatin regulator subfamily A member 5, NMNAT1: nicotinamide / nicotinic acid mononucleotide adenylyltransferase 1, H3C1: histone H3.1, TOP2B: DNA topoisomerase 2-β, RAD23B: UV excision repair protein RAD23 homolog B, PARP2: poly[ADP-ribose] polymerase 2.

[0309] Example 9: In vivo validation of the ability of the anti-NRP-1 antibody a-NRP1_LEP of the present invention to enter the nuclei of CTC cells xenografted subcutaneously into SCID mice and to induce DNA damage and / or centromere instability and / or telomere shortening. To investigate the ability of the a-NRP1_LEP antibody of the present invention to diffuse through the tumor vasculature, and in particular to reach the nucleus of cancer cells and induce DNA damage as demonstrated in vitro, CTC41.5E cells were xenografted subcutaneously into the right flank of female SCID mice. Tumors with an average volume of 300 mm were cultured. 3 At the time of 10 days, mice were injected weekly with 200 μg of humanized a-NRP1_LEP variant 4 antibody or its isotype control (human IgG4) via the tail vein. At the end of the experiment, tumor samples (primary tumors) and organs (lungs, liver, etc.) were collected for histological and biochemical analysis.

[0310] The presence of the a-NRP1_LEP antibody in the nuclei of cancer cells was detected by Western blotting after immunoprecipitation of the antibody with protein A and protein G Sepharose (Fig. 11A) or by immunofluorescence in frozen tissue sections (Fig. 11B).

[0311] Stained sections were then analyzed for telomere and centromere detection, followed by DAPI staining. Interestingly, in contrast to IgG4 isotype control-treated mice, we observed a decrease in the intensity of human centromere and telomere signals in xenografted CTC tumors derived from SCID mice treated with a-NRP1_LEP antibody. This decrease was associated with telomere shortening, DNA degradation accompanied by the presence of micronuclei, and morphological modifications in subcutaneously xenografted CTC cells in SCID mice treated with a-NRP1_LEP antibody (Figure 12A). As demonstrated in vitro in human PBMCs, the genotoxic effect of a-NRP1_LEP antibody was specific to pathological cells (human xenografted CTCs); no significant telomere shortening was observed in healthy mouse bone marrow cells (Figure 12A-B).

[0312] Example 10: In vivo effects of a-NRP1_LEP antibody on metastasis and anti-tumor immune response To investigate the impact and therapeutic efficacy of the a-NRP1_LEP antibody of the present invention on metastasis and anti-tumor immune responses in vivo, the highly metastatic murine breast cancer cell line 4T1, known for its lung tropism, was orthotopically implanted into female BALB / c mice. The dose of the a-NRP1_LEP antibody or its isotype control, 2.5 mg / kg (equivalent to 50 μg for a mouse with an average body weight of 20 g), was chosen to be lower than the doses used for the previously published prior art antibodies a-NRP1_VE (5 mg / kg) and a-NRP1_SEM (500 μg / dose). The mean tumor volume was 150 mm 3At the time of reaching 100 mg / kg / day, mice were intravenously injected with PBS, mIgG2b isotype control, and mouse α-NRP1_LEP antibody three times (once a week). At the end of the experiment, tumor samples (primary tumors) and organs (lungs, liver, etc.) were harvested, fixed, and then subjected to HES staining. CD4 (a glycoprotein, i.e., a co-receptor for the T cell receptor (TCR)) and FOXP3 markers were detected by immunohistochemistry. In the lung HES slides (Figure 13A), mice treated with mouse α-NRP1_LEP antibody clearly showed reduced lung metastases compared to both PBS- and mIgG2b-treated mice. This reduction in metastases was due to the CD4 expression in infiltrating lung metastases. + FOXP3 - The density of CD4+ was inversely correlated with the density of CD4+ in the lungs of mice treated with mouse a-NRP1_LEP antibody compared with both control groups (Fig. 13B and C). + FOXP3 - This suggests a strong recruitment of T cells. + FOXP3 - There was a statistically significant negative correlation between density and lung metastasis size (R 2 =0.63), the highest CD4 + FOXP3 - Mice characterized by a-NRP1_LEP levels had the smallest metastasis size (Fig. 10D). This suggests that a subpopulation of CD4 T cells may have cytotoxic activity. + FOXP3 -Infiltration shows very promising results (Revisiting the role of CD4 T cells in cancer immunotherapy - new insights into old paradigms. Rong En Tay, Emma K Richardson, Han Chong Toh. Cancer Gene Ther 2021 Feb;28(1-2):5-17). In primary mammary tumors, the a-NRP1_LEP antibody shows a tendency to increase CD4 infiltration. The control isotype used in this study was established from Merwin plasma cell tumor-II introduced into BALB / c mice in the 1970s, and its specificity is unknown. Therefore, we cannot exclude the possibility that the mouse immune system is modulated in response to this control antibody. Regarding CD8 positive T cells, a tendency to increase was observed in the lungs of a-NRP1_LEP-treated mice compared to control mice. However, the most interesting observation was the CD4 + and CD8 + / GrB + This is cell-cell contact between CD8 + In the activation of CD4 + These data suggest that the murine a-NRP1_LEP antibody of the present invention may be involved in the activation of the immune system in the lung, particularly the cytotoxic CD8 + These findings suggest that IL-16 has the ability to recruit CD4 T cells to activate and kill metastatic cells. [Brief explanation of the drawings]

[0313] [Figure 1] Expression of the NRP-1 / OBR complex in cancer cells and immune cells in a variety of human cancers. [Figure 2] Representative histograms of body weight monitoring of mice immunized with three different immunogenic peptides generated from the NRP-1 peptide Npep (N1, N2, N3). [Figure 3]Schematic diagram of the structure of NRP-1, its natural ligand, and different classes of mAbs. [Figure 4] The a-NRP1_LEP antibody of the present invention specifically recognizes the NRP-1 protein. [Figure 5] The antibody a-NRP1_LEP of the present invention binds more specifically to GAG-modified NRP-1 containing chondroitin and heparin sulfate motifs within its epitope than prior art antibodies. [Figure 6] Detection of NRP-1 in the nuclei of different cancer cell types by the anti-NRP-1 antibody of the present invention, a-NRP1_LEP. [Figure 7] 1 shows that the antibody of the present invention, a-NRP1_LEP, enters the nucleus in tumor cells in an NRP-1-dependent manner via the NRP-1 / OBR complex. [Figure 8] The antibody a-NRP-1-LEP of the invention induces DNA damage (chromosome shattering) and / or centromere destabilization and / or telomere shortening in circulating tumor cells. [Figure 9] This shows the interaction network between NRP-1 and various DNA repair proteins revealed by the antibody a-NRP1_LEP of the present invention. [Figure 10] The anti-NRP-1 antibody of the present invention, a-NRP1_LEP, binds to the chromatin of CTC cells. [Figure 11] 1 shows the detection of the anti-NRP-1 antibody of the present invention, a-NRP1_LEP, in the nuclei of CTC cells xenografted subcutaneously into SCID mice. [Figure 12] The antibody a-NRP-1-LEP of the present invention induces telomere shortening in CTC cells xenografted subcutaneously in SCID mice. [Figure 13] 1 shows the in vivo effect of the murine a-NRP1_LEP antibody of the present invention on lung metastases derived from primary tumors of the 4T1 mouse breast cancer cell line orthotopically implanted in syngeneic BALB / c mice.

Claims

1. an Npep peptide with the sequence EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), or a peptide selected from N1 having the sequence streptavidin-biotin-EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), N2 having the sequence EGNKPVLFQGNTNPTDVVVAVFPK-biotin-streptavidin (SEQ ID NO: 54), or N3 having the sequence EGNKPVLFQGNTNPTDVVC-KLH (SEQ ID NO: 55), An anti-NRP-1 antibody or antibody fragment capable of recognizing

2. 2. The anti-NRP-1 antibody or antibody fragment of claim 1, which is capable of entering the nucleus of a cell.

3. 20. An anti-NRP-1 antibody or antibody fragment according to claim 1 or 2, obtained by the use according to claim 19 or the method according to claim 20.

4. The anti-NRP-1 antibody or antibody fragment of any one of claims 1 to 3, which is capable of specifically binding to an NRP-1 isoform containing a GAG modification.

5. The anti-NRP-1 antibody or antibody fragment of any one of claims 1 to 4, which is capable of specifically binding to an NRP-1 isoform containing a GAG modification at positions S612, and / or Ser115, and / or Ser283, and / or Ser240, and / or Ser432, and / or Ser439, and / or Ser729.

6. The anti-NRP-1 antibody or antibody fragment of claim 5, which is capable of specifically binding to NRP-1 isoforms containing GAG modifications at positions Ser240 and / or Ser432.

7. The antibody or antibody fragment of any one of claims 1 to 6, comprising the sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 as defined below: - CDR-H1 has the sequence SX １ X ２ MH (SEQ ID NO: 1), where: X １ = F or Y X ２ = G, S or Y - CDR-H2 has the sequence X ３ ISX ４ X ５ X ６ X ７ X ８ X ９ X １０ YAX １１ X １２ X １３ X １４ G (SEQ ID NO: 2), where: X ３ = Y, V or I, X ４ = S, Y or P, X ５ = G, S or D, X ６ = S or G, X ７ = S or G, X ８ = T or S, X ９ = I, K or T, X １０ = H, Y or S, X １１ = D or Q, X １２ = S, K or T, X １３ = V or F, X １４ = K or Q, CDR-H3 is represented by the sequence RHYGSSRYWYFDV (SEQ ID NO: 3), - CDR-L1 has the sequence X １５ ASQX １６ X １７ X １８ SX １９ LX ２０ (SEQ ID NO: 4), where: X １５ = K or R, X １６ = D or S, X １７ = I or V, X １８ = K or S, X １９ = Y or W, X ２０ = S or A, - CDR-L2 has the sequence X ２１ AX ２２ SX ２３ X ２４ X ２５ (SEQ ID NO: 5), where: X ２１ = Y or D, X ２２ = T or S, X ２３ = L or R, X ２４ = A or E, X ２５ = G, S or T, - CDR-L3 has the sequence X ２６ QYX ２７ X ２８ SX ２９ YT (SEQ ID NO: 6), where: X ２６ = L or Q, X ２７ = G or S, X ２８ = E or S, X ２９ =P or S.

8. 8. The antibody or antibody fragment of claim 7, comprising the sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 as defined below: CDR-H1: SFGMH (SEQ ID NO: 7), CDR-H2: YISSGSSTIHYADTVKG (SEQ ID NO: 8), or YISSGSSTIYYADTVKG (SEQ ID NO: 35), CDR-H3: RHYGSSRYWYFDV (SEQ ID NO: 9), or RHYGRSRYWYFDV (SEQ ID NO: 36), CDR-L1: KASQDIKSYLS (SEQ ID NO: 10), CDR-L2: YATSLAG (SEQ ID NO: 11), or YATSLAD (SEQ ID NO: 38), CDR-L3: LQYGESPYT (SEQ ID NO: 12).

9. 8. The antibody or antibody fragment of claim 7, comprising the following CDRs: CDR-H1: SFGMH (SEQ ID NO: 7), CDR-H2: YISSGSSTIHYADTVKG (SEQ ID NO: 8), CDR-H3: RHYGSSRYWYFDV (SEQ ID NO: 9), CDR-L1: KASQDIKSYLS (SEQ ID NO: 10), CDR-L2: YATSLAG (SEQ ID NO: 11), CDR-L3: LQYGESPYT (SEQ ID NO: 12).

10. 8. The antibody or antibody fragment of claim 7, comprising the following CDRs: CDR-H1: SFGMH (SEQ ID NO: 7), CDR-H2: YISSGSSTIYYADTVKG (SEQ ID NO: 35), CDR-H3: RHYGRSRYWYFDV (SEQ ID NO: 36), CDR-L1: KASQDIKSYLS (SEQ ID NO: 10), CDR-L2: YATSLAD (SEQ ID NO: 38), CDR-L3: LQYGESPYT (SEQ ID NO: 12).

11. a heavy chain having SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NOs: 40-47, and a light chain which is SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NOs: 48-53; The antibody or antibody fragment of any one of claims 1 to 7, comprising:

12. a heavy chain having SEQ ID NO: 13 or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NO: 13, and a light chain having SEQ ID NO: 26 or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NO: 26; a heavy chain having SEQ ID NO: 14 or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NO: 14, and a light chain having SEQ ID NO: 23 or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NO: 23; a heavy chain having SEQ ID NO: 14 or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NO: 14, and a light chain having SEQ ID NO: 26 or a sequence having at least 80% sequence identity with the non-CDR regions of SEQ ID NO: 26; The antibody or antibody fragment of any one of claims 1 to 7, comprising:

13. The antibody or antibody fragment of any one of claims 1 to 12, wherein the antibody is capable of eliciting a therapeutic immune response.

14. 14. The antibody or antibody fragment of any one of claims 1 to 13, wherein the antibody is capable of entering the nucleus of cells expressing the NRP-1 / OBR complex and capable of causing DNA damage and / or centromere destabilization and / or telomere shortening in pathological cells.

15. The antibody or antibody fragment of any one of claims 1 to 14, wherein the antibody is an antibody drug conjugate (ADC) or a radionuclide-conjugated antibody.

16. The antibody or antibody fragment of any one of claims 1 to 15, wherein the antibody is part of a chimeric antigen receptor.

17. A peptide with the sequence EGNKPVLFQGNTNPTDVVC-KLH (SEQ ID NO: 55).

18. 16. A peptide having the sequence EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), or the sequence streptavidin-biotin-EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), or the sequence EGNKPVLFQGNTNPTDVVVAVFPK-biotin-streptavidin (SEQ ID NO: 54), or the peptide according to claim 15, for use in vaccination strategies in the treatment of diseases involving glycosaminoglycans and the DNA damage response (DDR), selected from cancer, inflammatory diseases and infectious diseases.

19. Use of a peptide with the sequence EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), or a peptide with the sequence streptavidin-biotin-EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), or a peptide with the sequence EGNKPVLFQGNTNPTDVVVAVFPK-biotin-streptavidin (SEQ ID NO: 54), or a peptide according to claim 17, for the production of an antibody according to any one of claims 1 to 16.

20. - a first immunization step: administering an immunogenic peptide sequence at least once to an immunocompetent non-human animal; - selecting animals with a desired phenotype, such as an obese phenotype; - recovering the antibodies from said animal; - screening for antibodies that can specifically recognize the target protein and the corresponding peptide sequence; - selecting animals producing said antibodies capable of specifically recognizing said corresponding peptide sequence and said target protein, - a second immunization step: administering to the selected animals at least once the same immunogenic peptide as in the first immunization step; - recovering lymphocytes from the animals that have undergone the two-stage immunization; - producing hybridomas from said lymphocytes, - selecting hybridomas producing antibodies capable of specifically recognizing the target protein by cells expressing the protein; A method for producing the antibody according to any one of claims 1 to 16, comprising:

21. the immunogenic peptide is selected from peptide Npep having the sequence EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), or peptide N1 having the sequence streptavidin-biotin-EGNKPVLFQGNTNPTDVVVAVFPK (SEQ ID NO: 54), or peptide N2 having the sequence EGNKPVLFQGNTNPTDVVVAVFPK-biotin-streptavidin (SEQ ID NO: 54), or peptide N3 according to claim 17, The target protein is a GAG-modified NRP-1 protein, and the desired phenotype is an obesity phenotype; 21. The method of claim 20.

22. The antibody or antibody fragment of any one of claims 1 to 16 for use in the treatment of a disease involving glycosaminoglycans and the DNA damage response (DDR), selected from cancer, inflammatory diseases and infectious diseases.

23. 23. The antibody or antibody fragment of claim 22, for the use defined in claim 22, wherein the disease associated with glycosaminoglycans and DNA damage response is cancer.

24. 23. The antibody or antibody fragment of claim 22 for the use defined in claim 22, wherein the antibody is administered in combination with an agent selected from an anti-checkpoint inhibitor, and / or an anti-angiogenesis inhibitor, and / or a casein kinase 2 inhibitor (anti-CK2).

25. 25. The antibody or antibody fragment of any one of claims 22 to 24 for the use as defined in any one of claims 22 to 24 in combination with an anti-PD1, anti-PDL1, anti-CTLA4 agent, and / or anti-VEGF, and / or anti-CK2.

26. 26. The antibody or antibody fragment of any one of claims 22 to 25 for use as defined in any one of claims 22 to 25 in combination with a therapy that induces a DNA damage response, including chemotherapy and radiotherapy.

27. The antibody or antibody fragment of any one of claims 22 to 26 for use as defined in any one of claims 22 to 26 for increasing the infiltration of immune cells in the tumor microenvironment.

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