RU2437677C2 - Inhibition of tumour metastases by antibodies against neuropiline-2 - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to field of medicine and pharmaceutics. As antagonist of neuropiline-2 applied is antibody against Nrp2B, or its antigen-binding fragment, which contains sequences of areas, which define complementarity, (CDR) of variable domains of light and/or heavy chain of antibody YW68.4.2 or antibody YW68.4.2.36, or version of claimed antibody or claimed antigen-binding fragment, where amino acid sequence Fab of YW68.4.2 of antibody fragment is shown in figure 10A and amino acid sequence Fab of YW68.4.2.36 of antibody fragment is shown in figure 10B.

EFFECT: group of inventions makes it possible to increase efficiency of prevention and treatment of tumour metastases.

21 cl, 13 dwg, 6 ex

Description

FIELD OF THE INVENTION

The present invention relates to antagonists of neuropilin-2 (Nrp2), in particular to antibodies against Nrp2, and to their use for the prevention and treatment of tumor metastases.

State of the art

It has now been established that angiogenesis is involved in the pathogenesis of a number of disorders. Such disorders are solid tumors and metastases, atherosclerosis, retrolental fibroplasia, hemangiomas, chronic inflammation, intraocular neovascular diseases, such as proliferating retinopathies, such as diabetic retinopathy, age-related macular degeneration (AMD), and other neovascular tissue-transmissible tissue glaucoma , rheumatoid arthritis and psoriasis. Folkman et al., J. Biol, Chem ., 267: 10931-10934 (1992); Klagsbrun et al., Annu. Rev. Physiol . 53: 217-239 (1991); and Garner A., "Vascular diseases", In: Pathobiology of Ocular Disease. A Dynamic Approach, Garner A., Klintworth GK, eds., 2nd Edition (Marcel Dekker, NY, 1994), pp. 1625-1710.

In the case of tumor growth, angiogenesis is obviously a key factor that plays an important role in the transition of hyperplasia to neoplasia and in providing the nutrients necessary for tumor growth and metastasis. Folkman et al., Nature 339: 58 (1989). Unlike normal cells, neovascularization is necessary for the growth of tumor cells and for their autonomous proliferation. Tumors are usually formed from one aberrant cell, which can proliferate only to a certain size, amounting to several cubic millimeters, which is determined by its distance from the capillary bed, and such a cell may remain “sleeping”, that is, it may not undergo further growth and dissemination for a long time period of time. Then, some tumor cells switch to an angiogenic phenotype, followed by activation of endothelial cells, which proliferate and mature, forming new capillary blood vessels. These newly formed blood vessels provide not only the constant growth of the primary tumor, but also the dissemination and recolonization of metastatic tumor cells. In accordance with this, there is a relationship between the density of microvessels in sections of the tumor and the survival of patients with breast cancer, as well as with tumors of some other types. Weidner et al., N. Engl. J. Med 324: 1-6 (1991); Horak et al., Lancet 340: 1120-1124 (1992); Macchiarini et al., Lancet 340: 145-146 (1992). The exact mechanism for regulating the switching of angiogenesis has not yet been well studied, however, it is clear that neovascularization of the tumor mass is due to the total balance of many stimulants and inhibitors of angiogenesis (Folkman Nat. Med . 1 (l): 27-31 (1995)).

Currently, it is believed that in the case of solid tumors, the vast majority, and according to experts, 90% of deaths are associated with metastases (Gupta and Massague, Cell 127, 679-695 (2006)). The complex process of metastasis formation consists of a number of separate stages, including the separation of tumor cells from the primary tumor, the intravization of tumor cells into lymphatic or blood vessels, and the extravasation and growth of tumor cells in the secondary regions. Analysis of regional lymph nodes in many types of tumors suggests that the lymphatic vasculature is an important channel for dissemination of human malignant tumors. In addition, in almost all carcinomas, the presence of tumor cells in the lymph nodes is the most important negative prognostic factor. Although it was previously thought that such metastases result from the passage of malignant cells through the lymphatic vessels that already exist near the tumors, recent experimental studies and clinical pathology reports (described in Achen et al., Br. J. Cancer 94 (2006) , 1355-1360 and Nathanson, Cancer 98, 413-423 (2003)) suggest that lymphangiogenesis can be induced by solid tumors and thereby stimulate the spread of these tumors. These and other recent studies suggest that targeted action on lymphatic vessels and lymphangiogenesis may serve as a therapeutic strategy to limit the development of cancer metastases, and such a strategy should provide a significant beneficial effect in a large number of patients.

VEGFC, which is a member of the family of vascular endothelial cell factors (VEGF), is one of the most well-studied mediators of lymphatic vascular development. Overexpression of VEGFC in tumor cells has been found to stimulate tumor lymphangiogenesis, leading to increased development of metastases in regional lymph nodes (Karpanen et al., Faseb. J. 20, 1462-1472 (2001); Mandriota et al. EMBO J. 20, 672 -682 (2001); Skobe et al., Nat. Med. 7, 192-198 (2001); Stacker et al., Nat. Rev. Cancer 2, 573-583 (2002); Stacker et al., Faseb. J. 16, 922-934 (2002)). VEGFC expression also correlates with tumor lymphangiogenesis and metastases in the lymph nodes for various human malignancies (as described in Achen et al., 2006, supra). In addition, it was found that blockade of VEGFC-mediated signal transmission leads to the suppression of tumor lymphangiogenesis and the development of metastases in lymph nodes in mice (Chen et al., Cancer Res 65, 9004-9011 (2005); He et al., J. Natl Cancer Inst 94, 8190825 (2002); Krishnan et al., Cancer Res 63, 713-722 (2003); Lin et al., Cancer Res 65, 6901-6909 (2005)).

VEGFC is known to bind to at least two receptors belonging to the cell surface receptor family, such as VEGF tyrosine kinase receptors and neuropilin receptors (Nrp).

VEGFC, one of the three VEGF receptors, can bind to VEGFR2 and VEGFR3, resulting in dimerization of this receptor (Shinkai et al., J Biol Chem 273, 31283-31288 (1998)), activation of kinase, and its autophosphorylation (Heldin, Cell 80, 213-223 (1995); Waltenberger et al., J. Biol Chem 269, 26988-26995 (1994)). The phosphorylated receptor induces the activation of many substrates, leading to angiogenesis and lymphangiogenesis (Ferrara et al., Nat. Med. 9, 669-676 (2003)).

The neuropilin family (Nrp) consists of two homologous proteins, neuropilin-1 (Nrp1) and neuropilin-2 (Nrp2). In addition to VEGF receptors, NEG2 also binds VEGFC, which was first identified as a class 3 semaphorin receptor and axon mediator (Favier et al., Blood 108, 1243-1250 (2006); Soker et al., J. Cell Biochem . 85, 357-368 (2002)). There is a lot of data indicating the involvement of Nrp2 in the development of the vascular and lymphatic systems. In homozygous Nrp2 mutants, a significant decrease in the number of small lymphatic vessels and capillaries in the prenatal period has been shown (Yuan et al., Development 129, 4797-4806 (2002)). In addition, serious abnormalities and defects leading to embryo death observed in mutant mice homozygous for Nrp1 are exacerbated by loss of Nrp2 function, leading to an early death (Takashima et al., Proc. Natl. Acad. Sci . USA 99, 3657-3662 (2002)). However, the role of Nrp2 in modulating biological processes in the blood and lymph vessels in adults and, in particular, its role in the development of metastases is still unclear.

Neuropilins (Nrp) have short intracellular domains; however, it is not known whether they have any enzymatic activity or signal transduction activity. It has been suggested that the function of Nrp is to enhance VEGFR signal transmission by increasing the binding level of the VEGF receptor to the ligand (Favier et al., 2006, see above; Soker et al., 2002, see above). In addition, sema3F, the Nrp2 semaphore ligand, has been found to modulate endothelial cell behavior in vitro o and in vivo (Bielenberg et al., J. Clin. Invest. 114, 1260-1271 (2004); Favier et al., Blood 1243-12503 (2006)). However, recent reports suggest a different assumption, namely that Nrp may function independently of VEGF receptors or that semaphorin acts as a modulator of endothelial cell (EC) migration (Murga et al., Blood 105, 1992-1999 (2005); Pan et al. , Cancer Cell 11, 53-67 (2007); Wang et al., J Biol Chem 278, 48848-48860 (2003)).

VEGF neutralizing antibodies inhibit the growth of various human tumor cell lines in nude mice (Kim et al., Nature 362: 841-844 (1993); Warren et al., J. Clin. Invest. 95: 1789-1797 ( 1995); Borgström et al., Cancer Res . 56: 4032-4039 (1996); Melnyk et al., Cancer Res . 56: 921-924 (1996)), and also inhibit intraocular angiogenesis in models with ischemic retinopathy (Adamis et al., Arch. Ophthalmol 114: 66-71 (1996)). Therefore, monoclonal antibodies against VEGF and other inhibitors of the action of VEGF are promising candidates for the treatment of tumors and various intraocular neovascular diseases. Such antibodies are described, for example, in EP 817648, published January 14, 1998; and in applications WO 98/45331 and WO 98/45332 published October 15, 1998. One of these anti-VEGF antibodies, namely bevacizumab, has been approved by the Food, Drug and Cosmetic Administration (FDA) for use in combination with chemotherapy for the treatment of metastatic colorectal cancer (CRC) and non-small cell lung cancer (NSCLC). A large number of clinical trials are currently underway on the use of bevacizumab for the treatment of various malignant tumors.

Other anti-VEGF antibodies and anti-Nrp1 antibodies are also known to those skilled in the art, as described, for example, in Liang et al., J. Mol. Biol. 366, 815-829 (2007); Pan et al., Cancer Cell 11, 53-67 (2007); and Liang et al., J. Biol. Chem. 281, 951-961 (2006).

Description of the invention

The present invention is based, at least in part, on experimental data obtained for a high affinity antibody blocking Nrp2 function. The results obtained for this antibody showed that Nrp2 plays a role in modulating the migration of lymphatic endothelial cells (LEC) and that its function goes beyond the previously attributed role of a VEGF receptor activation enhancer. In addition, the results demonstrated that blocking Nrp2 leads to inhibition of lymphangiogenesis and a significant reduction in metastases in the lymph nodes and distal organs.

In one aspect, the present invention relates to a method of inhibiting the migration of lymphatic vessel endothelial cells, comprising administering to the mammal, if necessary, an effective amount of a neuropilin-2 antagonist (Nrp2).

In another aspect, the present invention relates to a method for inhibiting tumor lymphangiogenesis, comprising administering to a mammal with a tumor an effective amount of a neuropilin-2 antagonist (Nrp2).

In another aspect, the present invention relates to a method for inhibiting tumor metastases, comprising administering to a mammal with a tumor an effective amount of a neuropilin-2 antagonist (Nrp2).

In all embodiments of the invention, the preferred mammal is a human, such as a patient with a malignant tumor, who may be diagnosed with metastases or who may be at risk of developing metastases.

In one embodiment of the invention, the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

In another embodiment, the cancer is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular cancer, gastric cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma , cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary carcinoma, kidney cancer or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma and various types of head and neck cancer, B-cell lymphoma, chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL);

hairy cell leukemia; chronic myeloid leukemia; lymphoproliferative disorder after transplantation (PTLD), pathological vascular proliferation associated with phacomatosis, edema associated with brain tumors, and Meigs syndrome.

In yet another embodiment of the invention, B-cell lymphoma is selected from the group consisting of low-grade / follicular non-Hodgkin's lymphoma (NHL); small cell lymphocytic (SL) NHL; NHL mid-stage differentiation / follicular NHL; diffuse NHL mid-stage differentiation; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small-cell undifferentiated NHL; generalized NHL; lymphoma of the cells of the cerebral cortex; AIDS related lymphoma; and Waldenstrom macroglobulinemia.

Nrp2 antagonists can be, but are not limited to, an anti-Nrp2 antibody, including anti-Nrp2B and Nrp2A antibodies, such as, for example, YW68.4.2, YW68.4.2.36, YW126.20 antibodies, and fragments and variants thereof, such as affinity -mature options.

In another aspect, the present invention relates to an anti-Nrp2B antibody comprising a variable region sequence of a heavy and / or light chain of an antibody selected from the group consisting of YW68.4.2, YW68.4.2.36, and a fragment or variant thereof.

In yet another aspect, the present invention relates to an anti-Nrp2A antibody comprising YW126.20 heavy and / or light chain variable region sequences, or a fragment or variant thereof.

The present invention also relates to a composition comprising an antibody according to any one of claims 33-38 in a mixture with a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a pharmaceutical composition for the prophylaxis or treatment of tumor metastases, comprising an effective amount of an Nrp2 antagonist in admixture with a pharmaceutically acceptable carrier.

In other aspects, the present invention relates to Nrp2 antagonists that can be used to prevent or treat metastases, and to the use of Nrp2 antagonists, such as anti-Nrp2 antibodies, to prevent or treat tumor metastases.

A brief description of the graphic material

Figure 1 . Characterization of monoclonal antibodies (mAb) against Nrp2 ^B (anti-Nrp2 ^B mAb). (A) Schematic representation of the localization of the Sema and VEGF binding regions on Nrp2 with respect to epitope regions for an anti-Nrp2 ^B antibody. (B) An ELISA assay showing the binding of an anti-Nrp2 ^B antibody to ECD hNrp2 (shaded squares) and B1-B2 hNrp2 domains (shaded circles), but not to ECD hNrp1 (unshaded squares) or to A1-A2 hNrp2 domains (unshaded circles) ) (C) Blocking the binding of VEGFC to Nrp2 by an anti-Nrp2 ^B antibody. increasing mAbs were pre-incubated in plates coated with human ECD Nrp2 (5 μg / ml) for 1-2 hours, and then pre-titrated biotinylated human VEGFC (1 nM) was added over 15 minutes. The percentage of bound VEGFC was determined using streptavidin-HRP (HRP) conjugates. (D) Blocking the binding of VEGF ₁₆₅ to Nrp2 by an anti-Nrp2 ^B antibody. (E) Blocking the binding of Sema3F to LEC. LECs were incubated with conditioned medium containing Sema3F bound to alkaline phosphatase (AP) (REF) in the presence or absence of an anti-Nrp2 ^B antibody. AP activity associated with AP Sema3F was detected colorimetrically with simultaneous manifestation. No binding to AP was observed (left panel). The anti-Nrp2 ^B antibody did not block the binding of Sema3F to LEC (middle panels). ECD Nrp2 was used as a positive control to block binding (right panel). Scale scale.

Figure 2. Antibody against Nrp2 ^B suppresses VEGFC-induced in vitro and in vivo function. (A) Image Examples of stained LECs migrating in response to 200 ng / ml VEGFC for 18 hours in the presence or absence of an anti-Nrp2 ^B antibody (50 μg / ml) or VEGFR3 ECD (50 μg / ml). (B) Quantification of LEC migration in response to 200 ng / ml VEGFC (n = 6 for each experimental condition). (C) Quantification of LEC migration in response to 10 ng / ml VEGF ₁₆₅ in the presence or absence of an anti-Nrp2 ^B antibody (50 μg / ml) or VEGFR3 ECD (50 μg / ml). N = 6 for each experimental condition. (D) A quantitative estimate of the number of pixels performed by analysis of the cornea micro-pocket and described in (E). * p <0.05. (E) Image Examples of a LYVE-1-stained cornea illustrating the action of 150 ng VEGFC (P) granules inside the cornea and systemic administration of an anti-Nrp2 ^B antibody (10 mg / kg, twice a week) or VEGFR3 ECD (25 mg / kg, two times a week). To facilitate visualization, LYVE-1 staining was performed, which gave pseudo-dye red, * p <0.05; the error value is presented as the standard error of the mean.

Figure 3. Treatment of Nrp2 ^B leads to a decrease in the activation of the VEGF receptor and to inhibition of the formation of the Nrp2 / VEGF receptor complex. (A) FACS analysis of Nrp2, VEGFR2 and VEGFR3 levels on the LEC surface after treatment with a control antibody (10 μg / ml; green line) or anti-Nrp2 ^B antibody (10 μg / ml) for 5 minutes (blue line) or for 20 hours (red line). (B) Quantification of LEC migration in response to 20 ng / ml HGF in the presence or absence of an anti-Nrp2 ^B antibody (50 μg / ml) or VEGFR3 ECD (50 μg / ml). N = 6 for each experimental condition. (C) The level of VEGFR2 phosphorylation in LEC detected by ELISA using antibodies that recognize total or tyrosine-phosphorylated VEGFR2. VEGFC (at indicated concentration) was added over 10 minutes in the presence or absence of an anti-Nrp2 ^B antibody (10 μg / ml) or VEGFR3 ECD (10 μg / ml) to induce VEGFR2 phosphorylation, n = 3 for each experimental condition. The level of VEGFR2 phosphorylation in cells treated with anti-Nrp2 ^B antibody (10 μg / ml) was significantly different from the level of VEGFC phosphorylation stimulated at 200 ng / ml, and consistently ranged from the level of phosphorylation induced by 175 ng / ml to the level of phosphorylation induced by 150 ng / ml VEGFC. (D) Quantification of LEC migration in response to VEGFC (at indicated concentration) in the presence or absence of an anti-Nrp2 ^B antibody (10 μg / ml) or VEGFR3 ECD (10 μg / ml). A significant reduction in migration was observed at 50 ng / ml VEGFC or when blocking under the influence of EEG VEGFR3. * p <0.05; the magnitude of the error is the standard error of the mean. Each experiment was repeated at least three times. (E) CO-IP.

Figure 4. Nrp2 is expressed in the lymphatic vessels of tumor mice. (AD) staining LYVE-1 (left column red) for labeling lymphatic vessels, staining Nrp2 (middle column green) and overlapping (A) in the small intestine and (B) in the lymph nodes of a healthy adult mouse. The Nrp2 signal does not coincide with the localization of LYVE-1-labeled lymphatic vessels in any organ. Nrp2 staining of inflammatory cells was rarely observed in the fibrostromal nucleus of intestinal villi and in the germinal centers of the lymph nodes. (C) In the lymph nodes of animals with tumors, the Nrp2 signal does not coincide with the localization of LYVE-1-positive lymphatic vessels lining the LN-sinuses. Additional staining of Nrp2 inflammatory cells was also observed. (D) Intensive Nrp2 staining has also been observed in the lymphatic vessels of 66cl4 tumors. On tumor cells, weak membrane staining could also be observed. On the second day, only the stained control did not detect any signal. The areas framed by the frame are shown as inserts with high magnification. Scale *** for AC and ** for D.

Figure 5. Antibody treatment with Nrp2 ^B resulted in a decrease in lung metastases in the 66c14 tumor model. (A) Below is a graph of the average tumor volume of 66c14 plotted in a study with a model of the indicated tumor. Twice a week ip, animals were dosed with a dose of 10 mg / kg anti-Nrp2 ^B antibody or control antibody after reaching an average tumor size of 100 mm ³ , and this dose was administered throughout the study. (B) Quantification was performed by visual observation of the number of nodes with metastases per lung in animals treated with control and anti-Nrp2 ^B antibody. (C) Examples of lung images for animals treated with control (left) and anti-Nrp2 ^B antibody (right). Before fixation, the lungs were inflated by perfusion of the right ventricle of the heart. To facilitate visualization, nodes were marked in white. (D, E) An example of a three-dimensional image of lungs scanned using micro-KT (computed tomography), illustrating nodes with metastases (red) in animals treated with control (D) and anti-Nrp2 ^B antibody (E). The positions of the longitudinal cut (upper insert) and the transverse cut (lower insert) are shown by black and red dashed lines, respectively. This analysis confirmed that most nodes are on the surface of the lungs. (F) Quantification of the number of nodes with lung metastases by micro-KT analysis of the lungs. (G) In vitro FACS analysis of Nrp2 levels on the surface of cultured 66c14 tumor cells. (H) staining of H&E lung nodes illustrating tumor cells with metastases. The magnitude of the error is presented as the standard error of the mean. Scale *** for C and ** for H.

Figure 6. Antibody treatment with Nrp2 ^B reduced the number of lung metastases in the C6 tumor model. (A) Below is a graph of the average C6 tumor volume plotted as a result of a study using a model with the indicated tumor. Twice a week ip, the animals were given a dose of an anti-Nrp2 ^B antibody (10 mg / kg), VEGFR3 ECD (25 mg / kg) or a control antibody (10 mg / kg) after reaching an average tumor size of 100 mm ³ , and this dose was administered on throughout the study. (B) Quantification was performed by visual observation of the number of nodes with lung metastases in the animals treated with control, VEGFR3 ECD and anti-Nrp2 ^B antibody. (C) Examples of images of lungs of animals treated with control (left), ECD VEGFR3 (middle) and anti-Nrp2 ^B antibody (right). Before fixation, the lungs were inflated by perfusion of the right ventricle of the heart. To facilitate visualization, nodes were marked in white. (D) An example of a three-dimensional image of lungs scanned using micro-KT (computed tomography) illustrating nodes with metastases (red) in animals treated with control (left) and anti-Nrp2 ^B antibody (right). The positions of the longitudinal cut (upper insert) and the transverse cut (lower insert) are shown by black and red dashed lines, respectively. This analysis confirmed that most nodes are on the surface of the lungs. (E) FACS analysis of Nrp2 levels on the surface of in vitro o cultured 66c14 tumor cells. (F) staining of H&E lung nodes showing tumor cells with metastases. The magnitude of the error is presented as the standard error of the mean. Scale *** for C and ** for F.

Figure 7. Antibody treatment with Nrp2 ^B resulted in a decrease in the number of tumor lymphatic vessels. (A, B) Quantification of the density of the vascular system (A) detected by IHC PECAM-1 and the density of the lymphatic vessels (B) detected by IHC LYVE-1 in 66cl4 tumors treated with a control antibody or anti-Nrp2 ^B antibody. Vascular density was determined from 6 sample images obtained for each of 6 tumors per group and estimated by the average number of pixels using ImageJ. (C) Examples of images of PECAM-1 stained vessels (upper row) and LYVE-1 stained lymphatic vessels (middle and lower rows) in C6 tumors treated with a control antibody (left column), VEGFR3 ECD (middle column) or anti-Nrp2 ^B antibody (right column). The areas in the frame shown in the middle row are represented with a large increase in the lower row. To the right of these images is a quantitative assessment of the density of the vascular system (upper graph) and the density of lymphatic vessels (lower graph). (D) LYVE-1 stained tumors collected from animals treated with anti-Nrp2 ^B antibody (lower panels) on day 4 (collection 1) and on day 11 (collection 2) show lymphatic vessel destruction compared to lymphatic vessels collected from Control treated animals (upper panels). On the left are growth curves versus harvest time. The magnitude of the error is presented as the standard error of the mean. Scale scale ***.

Figure 8. Antibody treatment with Nrp2 ^B led to a decrease in the number of functional lymphatic vessels of the tumor and to a slowdown in the development of metastases in the primary lymph node. (A, B) After intradermal lymphangiography, polystyrene fluorescent microspheres (green) were observed exclusively in lymphatic vessels labeled with LYVE-1 IHC (red). The areas in the frame in Figure A are shown in Figure B with high magnification. (CD) Antibody treatment with Nrp2 ^B resulted in a decrease in Evans blue intensity in C6 (C) (P = 0.035) and 66cl4 (D) (P = 0.005) tumors, indicating a decrease in the number of functional lymphatic vessels in these treated tumors. (E) Percentage of animals with small lymph nodes (SLNs) containing β-gal-expressing C6 tumor cells at different times after tumor implantation in the ears of mice treated with control (black) and anti-Nrp2 ^B antibody (red). Antibody treatment with Nrp2 ^B slowed the entry of cells into SLN (p = 0.006). The number of animals per treatment condition in each time period was 7 (N = 7).

Figure 9. Expression of Nrp2 in various malignant human tumors. (AF) Affymetrix HG-U133A and B GeneChip® microarray data for assessing the level of Nrp2 expression in a healthy colon and adenocarcinoma of the rectum and colon (A), in healthy tissues of the head and neck, and in squamous cell carcinoma of the head and neck (B), in a healthy pancreas and pancreatic adenocarcinoma (C), in healthy skin and in malignant melanoma (D), in a healthy thyroid gland and in thyroid papillary carcinoma (E), and in a healthy mammary gland and in Her2-infiltrating duct adenocarcinoma (F). Each time period is presented for one patient.

Figure 10. Amino acid sequence of Fab fragments of anti-Nrp2 ^B antibody YW68.4.2 and YW68.4.2.36.

Figure 11. Amino acid sequence of a Fab fragment of an anti-Nrp2 ^A antibody YW126.20.

Figure 12. Alignment of the sequences of the variable domain of the light chain of the anti-Nrp2 ^A antibody YW126.20 with the human κ1 sequence.

Figure 13. Alignment of the sequences of the variable domain of the light chain of the antibody against Nrp2 ^A YW126.20 with the sequence III (hum III) of the person.

Additional figure 1. FACS analysis of receptor levels for VEGF of all types on the surface of in vitro o cultured LEC. FACS analysis of Nrp1, Nrp2, VEGFR1, VEGFR2 and VEGFR3 on cultured LECs.

Supplementary Figure 2. Anti-Nrp2 ^B antibody does not block VEGF- ₁₆₅ induced migration. (AB) Quantification of HUVEC (A) and LEC (B) cells migrating in response to 200 ng / ml VEGF for 18 hours in the presence or absence of an anti-Nrp1 ^B antibody, anti-Nrp2 ^B antibody (50 μg / ml) or both antibodies (50 μg / ml). * p <0.05. The magnitude of the error is presented as the standard error of the mean.

Additional figure 3. The effect of antibodies against Nrp2 ^B on VEGFC-mediated proliferation, vascular permeability and associated VEGFC intracellular signal transmission. (A) Quantification of LEC proliferation induced by 200 ng / ml VEGFC in the presence or absence of an anti-Nrp2 ^B antibody (50 μg / ml) or VEGFR3 ECD (50 μg / ml) as determined by BrdU incorporation (n = 6 per one condition of the experiment). (B) Analysis of vascular permeability of the skin of mice. Images were obtained for the skin of the same animal. Blue color indicates the leak of Evans blue from the vascular system in response to percutaneous delivery of VEGFC after systemic treatment with anti-Nrp2 ^B antibody (10 mg / kg) or VEGFR3 ECD (25 mg / kg). (C) Quantification of Evans Blue dye extracted from skin samples in a permeability assay. The indicated values are the average values obtained for 6 independent experiments. * p <0.05; the error value is presented as the standard error of the mean.

Supplementary Figure 4. Anti-Nrp2 ^B antibody does not block Sema3F-induced growth cone collapse. (A) Images of the growth cones of the hippocampus E17.5 stained with rhodamine conjugated with a phalloidin. In the control growth cones, large actin-rich structures were found at the end of each axon, which decreased after treatment with Sema3F. The anti-Nrp2 ^B antibody (50 μg / ml) does not block such a collapse. In contrast, ECD Nrp2 (10 μg / ml) blocks such a collapse. (B) Quality control for immunohistochemical analysis using antibodies against Nrp2. The image represents a phage clone that recognizes Nrp2 functioning in IHC on freshly frozen slices. The level of Nrp2 protein expression is similar to the level of Nrp2 expression observed using in situ hybridization (Chen et al., Neuron 19, 547-559 (1997)). This antibody was then used for immunohistological analysis (IHC) on freshly frozen sections of the tumor. Scale scale for main image = ** μm and for insertion images = ** μm.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The terms "neuropilin", "NRP" or "Nrp" are used interchangeably and mean neuropilin-1 (NRP1, Nrp1), neuropilin-2 (NRP2, Nrp2) and their isoforms and variants described in Rossignol et al. (2000) Genomics 70: 211-222. Neuropilins are non-tyrosine kinase receptors with a size of 120-130 kDa. There are many spliced variants and soluble isoforms of NRP-1 and NRP-2. The basic structure of neuropilins includes five domains: three extracellular domains (a1a2, b1b2 and c), a transmembrane domain and a cytoplasmic domain. The a1a2 domain is homologous to the complement components C1r and C1s (CUB), which usually contain four cysteine residues forming two disulfide bridges. The b1b2 domain is homologous to coagulation factors V and VIII. The central part of domain c is denoted by MAM, due to its homology with meprin, A5, and tyrosine phosphatase µ receptor proteins. Domains a1a2 and b1b2 are responsible for binding to the ligand, and domain c plays a crucial role in homodimerization or heterodimerization. Gu et al. (2002) J. Biol. Chem. 277; 18069-76; He and Tessier-Lavigne (1997) Cell 90: 739-51.

The term "neuropilin-mediated biological activity" means mainly physiological or pathological events in which neuropilin-1 and / or neuropilin-2 play an important role. Non-limiting examples of such activities are axon regulation during the development of the embryonic nervous system or neuron regeneration, angiogenesis (including vascular modeling), oncogenesis, and the formation of tumor metastases.

Used in the present description, the term "mediated neuropilin-2 biological activity" or "mediated Nrp2 biological activity" means mainly physiological or pathological events in which Nrp2 plays an important role, for example, such as increased activation of the VEGF receptor and, in particular, the ability to modulate the migration of endothelial cells (EC) of the lymphatic vessels, lymphangiogenesis in adults, in particular lymphangiogenesis and the formation of tumor metastases.

As used herein, the term “antibody” is used in its broadest sense, and in particular, the term encompasses monoclonal antibodies (including full-size monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, provided that they have the desired biological activity.

Used in the present description, the term "monoclonal antibody" means an antibody obtained from a population of mainly homogeneous antibodies, that is, individual antibodies that are part of this population and are identical, except for possible natural mutations that may be present in small amounts. Monoclonal antibodies are highly specific and target a single antigenic determinant. In addition, unlike standard preparations of (polyclonal) antibodies, which usually contain different antibodies that target different determinants (epitopes), each monoclonal antibody targets one determinant of the antigen. The term "monoclonal" indicates a type of antibody obtained essentially from a homogeneous population of antibodies, but this term does not imply that this antibody should be obtained by any specific method. For example, the monoclonal antibodies of the invention can be prepared using the hybridoma technology first described by Kohler et al. (1975) Nature 256: 495, or by recombinant DNA methods (see, for example, US Patent No. 4816567). "Monoclonal antibodies" can also be isolated from phage libraries of antibodies by methods described, for example, Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol . 222: 581-597.

The monoclonal antibodies provided herein in particular include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequences present in antibodies of a particular species or belonging to a particular class or subclass of antibodies, and the rest part of this chain (s) is identical or homologous to the corresponding sequences present in antibodies of another species or belonging to another class or subclass of antibodies; as well as fragments of these antibodies, provided that they have the desired biological activity (see US patent No. 4816567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. , USA, 81: 6851-6855).

“Humanized” forms of non-human (eg, murine) antibodies are chimeric antibodies that contain a minimal sequence of non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which the remnants of the reciprocal region of the recipient are replaced by the remnants of the hypervariable region of a non-human antibody (donor antibody), such as a mouse antibody, a rat antibody, a rabbit antibody, or a primate antibody, not being a person who has the desired specificity, affinity, or function. In some cases, residues of the Fv framework region (FR) of a human immunoglobulin can be replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues that are not present in the recipient antibody or in the donor antibody. Such modifications may be introduced to further improve antibody function. Typically, a humanized antibody can contain essentially all at least one, and usually two variable domains, in which all or essentially all of the hypervariable loops corresponding to the loops of non-human immunoglobulins, and all or essentially all of the FR regions are human immunoglobulin sequences . A humanized antibody may also, but not necessarily, contain at least a portion of the constant region of an immunoglobulin (Fc), typically a human immunoglobulin. For a more detailed description, see Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-329; and Presta (1992) Curr. Op. Struct. Biol . 2: 593-596.

The term “species-specific antibody” means an antibody that has a higher binding affinity for a mammalian antigen of the first species than for a homolog of a mammalian antigen of another species. Typically, a species-specific antibody “specifically binds” to a human antigen (ie, has a binding affinity (K _d ) of not more than about 1 × 10 ⁻⁷ M, preferably not more than about 1 × 10 ^-8 M, and most preferably not more than than about 1 × 10 ^-9 M), but has a binding affinity for a non-human mammalian antigen of a different species that is at least about 50 times, or at least about 500 times, or at least about 1000 times less antigen binding affinity person. The species-specific antibody may be any type of antibody as defined above, and a humanized or human antibody is preferred.

As used herein, the term “mutant antibody” or “antibody variant” means an amino acid sequence variant of a species-specific antibody in which one or more amino acid residues of the species-specific antibody are modified. Such mutants necessarily have a sequence that is less than 100% identical or similar to the sequence of a species-specific antibody. In a preferred embodiment, the mutant antibody has an amino acid sequence that is at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90% and most preferably at least 95% identical or similar to the amino acid sequence of the variable domain of the heavy or light chain of a species-specific antibody. Identity or similarity with this sequence is defined in the present description as the percentage of amino acid residues in the selected sequence that are identical (i.e., are the same) or similar to residues (i.e., amino acid residues belonging to the same group of amino acids classified by the general properties of the side chains, see below) species-specific antibodies, as determined by alignment of sequences with the introduction of gaps, if necessary to achieve maximum percent coagulant sequence identity. Moreover, none of the N-terminal modifications, C-terminal modifications or modifications such as internal additions, deletions or insertions in the sequence of antibodies located outside the variable domain should not be considered as a modification that affects the identity or similarity of the sequences.

An “isolated” antibody is an antibody that has been identified and separated from the components of its natural environment and / or isolated from such components. Impurity components of its natural environment are substances that can interfere with the diagnostic or therapeutic use of the indicated antibodies, and such substances can be enzymes, hormones and other protein or non-protein dissolved substances. In preferred embodiments of the invention, said antibody can be purified (1) by more than 95% by weight of the antibody, as can be determined by the Lauri method, and most preferably by more than 99% by weight of the antibody, (2) to a level sufficient to obtain N -terminal or internal amino acid sequence of at least 15 residues using a centrifuge cup sequencer, or (3) until homogeneity evaluated using SDS-PAGE under reducing or non-reducing conditions using Kumas and a blue dye, or preferably with silver. An isolated antibody is considered to be an in situ antibody in recombinant cells, since they lack at least one component of the natural environment of the antibody. However, as a rule, an isolated antibody can be obtained in at least one purification step.

As used herein, the term “variable domain of an antibody” means parts of the light and heavy chains of antibody molecules that include the amino acid sequences of the hypervariable regions (complementarity determining regions, CDRs; i.e., CDR1, CDR2 and CDR3) and framework regions (FR). V _H means the variable domain of the heavy chain. V _L means the variable domain of the light chain. In accordance with the methods used in the present invention, amino acid positions assigned to CDR and FR can be determined according to Kabat numbering (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1997 and 1991)) . The amino acid numbering of antibodies or antigen-binding fragments is also given according to Kabat.

Used in the present description, the term "hypervariable region" (CDR, that is, CDR1, CDR2 and CDR3) means the amino acid residues of the variable domain of the antibody, the presence of which is necessary for binding to the antigen. Each variable domain typically has three CDR regions designated as CDR1, CDR2, and CDR3. Each hypervariable region may contain amino acid residues of a “hypervariable region” defined by Kabat (that is, approximately residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain of the light chain and 31-35 ( H1), 50-65 (H2) and 95-102 (H3) in the variable domain of the heavy chain; Kabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ( 1991)), and / or residues of the "hypervariable loop" (that is, approximately residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain of the light chain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Chothia and Lesk (1987) J. Mol. Biol., 196: 901-917). In some cases, the hypervariable region may contain the amino acids of both regions of the CDRs defined by Kabat and the amino acids of the hypervariable loop. For example, the 4D5 antibody heavy chain CDRH1 contains amino acids 26-35.

"Frame regions" (hereinafter referred to as FR) are regions in which residues of the variable domains differ from residues of the CDR. Each variable domain usually has four FRs, designated as FR1, FR2, FR3, and FR4. If the CDRs are determined according to Kabat, the light chain FR residues have approximately the following positions: 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3) and 98-107 (LCFR4), and the heavy chain FR residues have approximately the following positions: 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3) and 103-113 (HCFR4). If the CDRs contain amino acid residues of the hypervariable loops, the light chain FR residues have approximately the following positions: 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3) and 97-107 (LCFR4), and the FR residues are heavy chains have approximately the following positions: 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3) and 102-113 (HCFR4). In some cases, if the CDR contains the amino acids of both the Kabat-determined CDR and the hypervariable loop, then the positions of the FR residues can be adjusted accordingly. For example, if CDRH1 contains amino acids H26-H35, then the heavy chain FR1 residues are present at positions 1-25, and the FR2 residues are present at positions 36-49.

Used in the present description, the term "set of codons" means a set of different sequences of nucleotide triplets encoding the desired amino acid variants. A set of oligonucleotides can be synthesized, for example, by solid-phase synthesis, including sequences that represent all possible combinations of nucleotide triplets provided by the specified set of codons, and which encode the desired group of amino acids. The standard form of codon designation is a known code, which was adopted by the International Biochemical Union (IUB) and described in this application. A set of codons is usually represented by 3 capital letters in italics, for example, NNK, NNS, XYZ, DVK , etc. For example, as used herein, the term “non-random set of codons” means a set of codons that encode specific amino acids, partially, and preferably completely, that satisfy the amino acid selection criteria described in this application. A method for synthesizing oligonucleotides with a certain degree of “degeneracy” in certain positions is well known to those skilled in the art, for example, the TRIM method (Knappek et al. (1999) J. Mol. Biol . 296: 57-86); Garrard & Henner (1993) Gene 128: 103). Such oligonucleotide sets having specific codon sets can be synthesized using commercially available nucleic acid synthesizers (available from, for example, Applied Biosystems, Foster City, CA), or they can be purchased (e.g. from Life Technologies, Rockville, MD) . Therefore, the synthesized set of oligonucleotides is a specific set of codons, which usually includes many oligonucleotides with different sequences, where these differences are determined by a specific set of codons throughout the sequence. The oligonucleotides used in accordance with the present invention have sequences that allow them to hybridize with the variable domain nucleic acid matrix, and may also, but not necessarily, contain restriction sites suitable, for example, for cloning.

An “Fv” fragment is an antibody fragment containing a full-sized antigen-recognizing site and an antigen-binding site. This region consists of a dimer of one variable domain of the heavy chain and one variable domain of the light chain, rigidly connected to each other by a bond, which by its nature can be covalent, for example, in scFv. This fragment has a configuration in which three CDRs in each variable domain interact with each other so that they form an antigen-binding site on the surface of the V _H -V _L dimer. In total, six CDRs or a subsequence of them impart antigen binding specificity to the antibody. However, even one variable domain (or half of the Fv, containing only three hypervariable regions specific for the antigen) has the ability to recognize the antigen and bind to it, albeit with less affinity than the entire binding site.

The "Fab" fragment contains the variable and constant domain of the light chain and the variable domain and the first constant domain (CH1) of the heavy chain. F (ab ') ₂ fragments of an antibody contain a pair of Fab fragments, which are usually covalently linked near their carboxy-ends by articulated cysteines located between them. Other chemical bonds connecting antibody fragments are also known to those skilled in the art.

An “single chain Fv fragment” or “scFv fragment” of an antibody contains the V _H and V _L domains of an antibody, where these domains are present in the same polypeptide chain. Typically, the Fv polypeptide also contains a polypeptide linker between the V _H and V _L domains, where the polypeptide provides the formation of scFv with the structure necessary for binding to the antigen. A description of scFv can be found in Pluckthun's publication The Pharmacology of Monoclonal Antibodies , vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” means small fragments of antibodies with two antigen-binding sites, where these fragments contain the variable domain of the heavy chain (V _H ) connected to the variable domain of the light chain (V _L ) in the same polypeptide chain (V _H -V _L ). If the linker is too short to form a pair between two domains on the same chain, then the domains are forced to mate with the complementary domains of the other chain and form two antigen-binding sites. Diabodies are described in more detail, for example, in EP 404097; WO 93/11161 and Hollinger et al. Proc. Natl. Acad. Sci., USA, 90: 6444-6448 (1993).

The term "linear antibodies" means antibodies described in the publication Zapata et al. (1995 Protein Eng , 8 (10): 1057-1062). Briefly, such antibodies contain a pair of tandem segments (V _H -C _H 1-V _H -C _H 1), which together with complementary light chain polypeptides form antigen-binding regions. Linear antibodies can be bispecific and monospecific.

As used herein, the term “library” means a plurality of antibody sequences or antibody fragments (eg, polypeptides of the invention) or nucleic acids encoding these sequences, wherein said sequences differ in combinations of different amino acids that are introduced into these sequences by the methods of the invention.

“Phage display” means a method by which polypeptide variants are presented as fusion proteins bound to at least a portion of a shell protein on the surface of a phage, such as filamentous phage particles. The phage display method is based on the ability to sort large libraries of randomized protein variants quickly and efficiently into sequences that bind to the target antigen with high affinity. The presentation of peptide and protein libraries on the phage is used to screen millions of polypeptides in order to detect polypeptides with specific binding properties. Polyvalent phage display methods are used to present small randomized peptides and small proteins by combining them with gene III or filamentous phage gene VIII. See Wells and Lowman (1992) Curr. Opin. Struct. Biol. 3: 355-362 and the references cited therein. In the monovalent phage display method, a protein or peptide library is attached to gene III or part thereof and expressed at low levels in the presence of wild-type protein encoded by gene III, so that phage particles present one copy of the fusion proteins or not present any of the fusion proteins . The avidity effects achieved in the case of using a monovalent phage are reduced in comparison with the effects achieved in the case of using a polyvalent phage, and therefore sorting is carried out on the basis of natural affinity for the ligand and using phagemid vectors to facilitate DNA manipulation. Lowman and Wells (1991) Methods: A companion to Methods in Enzymology 3: 205-0216.

A “phagemid” is a plasmid vector having a origin of replication of a bacterial nature, for example ColEl, and a copy of the intergenic region of the bacteriophage. The phagemid can be used on any known bacteriophage, including filamentous bacteriophage and lambdoid bacteriophage. The plasmid also typically contains a selective marker of antibiotic resistance. DNA segments cloned into these vectors can be expanded as plasmids. If the cells containing these vectors have all the genes necessary for the production of phage particles, the plasmid replication mechanism is switched to “rolling ring” replication with the formation of copies of one strand of plasmid DNA and packed phage particles. The phagemid may form infectious or non-infectious phage particles. This term includes phagemids containing a phage coat protein gene or fragment thereof linked to a heterologous polypeptide gene in the form of a fusion gene that encodes a heterologous polypeptide present on the surface of a phage particle.

The term "phage vector" means a double-stranded replicative form of a bacteriophage containing a heterologous gene and capable of replication. The phage vector has a phage origin of replication, providing phage replication and the formation of phage particles. Said phage is preferably a filamentous bacteriophage, such as phage M13, fl, fd, Pf3 or a derivative thereof, or a lambdoid phage, such as phage lambda, 21, phi80, phi81, 82, 424, 434, etc. or their derivatives.

Used in the present description, the term "position accessible to the solvent" means the position of amino acid residues in the variable regions of the heavy and light chains of the original antibody or antigen-binding fragment, where the specified position is determined based on the structure, combination of structures and / or simulated structures of the antibody or antigen a binding fragment as available for solvent and / or for contact with a molecule, such as an antigen-specific antibody. Such positions are usually found in the CDR and outside the protein. The positions of the antibody or antigen binding fragment available for the solvent, as defined herein, can be determined using a number of algorithms known to those skilled in the art. The positions available for the solvent are preferably determined using the coordinates of a three-dimensional model of the antibody, preferably using a computer program such as the InsightII program (Accelrys, San Diego, CA). The positions available to the solvent can also be determined using algorithms known to those skilled in the art (e.g., Lee and Richards (1971) J. Mol. Biol . 55, 379 and Connolly (1983) J. Appl. Cryst . 16, 548). The positions available for the solvent can be determined using a computer program suitable for modeling proteins and searching for information about the 3-dimensional structure of antibodies. A computer program that can be used for this purpose is the SYBYL Biopolymer Module (Tripos Associates) program. Usually and preferably, when working with this algorithm (program), it is necessary for the user to enter a size parameter, and the “size” of the probe used in the calculations is set to approximately 1.4 angstroms or smaller radius. In addition, methods for determining regions and regions accessible to a solvent using a computer program developed for personal computers are described in Pacios (1994) Comput. Chem . 18 (4): 377-386.

An “angiogenic factor or agent” is a growth factor that stimulates the development of blood vessels, for example, stimulates angiogenesis, stimulates the growth of endothelial cells, increases the stability of blood vessels and / or vasculogenesis, etc. For example, angiogenic factors include, but are not limited to, VEGF and members of the VEGF, PIGF, PDGF family and the family of fibroblast growth factors (FGF), TIE ligands (angiopoietins), ethers, Del-1, fibroblast growth factors: acidic (aFGF) and basic (bFGF), follistatin, granulocyte colony stimulating factor (G-CSF), hepatocyte growth factor (HGF) / scattering factor (SF), interleukin-8 (IL-8), leptin, midkin, neuropilins, placental growth factor, platelet factor endothelial cell growth (PD-ECGF), platelet-derived growth factor, in particular PDGF-BB or PDGFR-beta, p eyotropin (PTN), progranulin, proliferin, transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), etc. Wound healing factors such as growth hormone, insulin-like growth factor-I (IGF-I), VIGF, epidermal growth factor (EGF), CTGF and its family members, and TGF-alpha, and TGF-beta are also such factors. . See, for example, Klagsbrun and D'Amore (1991) Annu. Rev. Physiol . 53: 217-39; Streit and Detmar (2003) Oncogene 22: 3172-3179; Ferrara & Alitalo (1999) Nature Medicine 5 (12): 1359-1364; Tonini et al. (2003) Oncogene 22: 6549-6556 (see, for example, table 1 for known angiogenic factors); and Sato (2003) Int. J. Clin. Oncol . 8: 200-206.

The term “anti-angiogenesis agent” or “angiogenesis inhibitor” means a low molecular weight substance, polynucleotide, polypeptide, isolated protein, recombinant protein, antibody or its conjugates, or fusion proteins that directly or indirectly inhibit angiogenesis, vasculogenesis, or unwanted vascular permeability. It should be noted that anti-angiogenesis agents are agents that bind to the angiogenic factor or its receptor and block the angiogenic activity of this factor or its receptor. For example, an anti-angiogenesis agent is an antibody or other antagonist of an angiogenic agent as defined above, for example an anti-VEGF-A antibody or an anti-VEGF-A receptor antibody (eg, KDR receptor or Fit-1 receptor), anti-PDGFR inhibitors such as Gleevec ™ (imatinib mesylate). Natural angiogenesis inhibitors such as angiostatin, endostatin, etc. are also anti-angiogenesis agents. See, for example, Klagsbrun and D'Amore (1991) Annu. Rev. Physiol . 53: 217-39; Streit and Detmar (2003) Oncogene 22: 3172-3179 (see, for example, table 3, for a list of antiangiogenic therapy regimens for malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5 (12): 1359-1364; Tonini et al. (2003) Oncogene 22: 6549-6556 (see, for example, table 2, for a list of known anti-angiogenic factors); and Sato (2003) Int. J. Clin. Oncol . 8: 200-206 (see, for example, table 1, which provides a list of antiangiogenic drugs used in clinical trials).

As used herein, the term “VEGF” or “VEGF-A” means human vascular endothelial cell growth factor consisting of 165 amino acids and related human vascular endothelial cell growth factors consisting of 121, 189 and 206 amino acids, as described in Leung et al. (1989) Science 246: 1306 and Houck et al. (1991) Mol. Endocrin, 5: 1806, together with their natural allelic and processed forms. The term "VEGF" also means VEGF of non-human animals, such as a mouse, rat or primate. Sometimes specific species of VEGF are indicated, for example, hVEGF for human VEGF, mVEGF for mouse VEGF, etc. The term "VEGF" is also used to denote truncated forms of a polypeptide containing amino acids 8-109 or 1-109 of human vascular endothelial cell growth factor consisting of 165 amino acids. In this regard, any form of VEGF can be identified in this application, for example, as "VEGF (8-109)", "VEGF (1-109)" or "VEGF ₁₆₅ ". The amino acid positions for the “truncated” native VEGF are numbered as indicated in the native VEGF sequence. For example, the position of amino acid 17 (methionine) in truncated native VEGF also corresponds to position 17 (methionine) in native VEGF. Truncated native VEGF compared to native VEGF has binding affinity for KDR and Fit-1 receptors, comparable to binding affinity for native VEGF.

An “anti-VEGF antibody” is an antibody that binds to VEGF with sufficient affinity and specificity. The anti-VEGF antibody of the invention can preferably be used as a therapeutic agent for the treatment and prevention of diseases or conditions that are associated with VEGF activity. An anti-VEGF antibody generally does not bind to other VEGF homologues, such as VEGF-B or VEGF-C, or to other growth factors, such as PIGF, PDGF or bFGF. The preferred anti-VEGF antibody is a monoclonal antibody that binds to the same epitope that the anti-VEGF antibody A4.6.1 produced by the ATCC HB 10709 hybridoma binds. A more preferred anti-VEGF antibody is a recombinant humanized anti-VEGF monoclonal antibody prepared as described in publications by Presta et al. (1997) Cancer Res . 57: 4593-4599, including, but not limited to, an antibody known as bevacizumab (BV; Avastin ™, avastin).

The Bevacizumab (BV) anti-VEGF antibody, also known as the “rhuMAb VEGF” or “Avastin®” antibody, is a recombinant humanized monoclonal anti-VEGF antibody prepared as described in Presta et al. (1997) Cancer Res . 57: 4593-4599. The specified antibody contains mutated framework regions of human IgG1 and antigen-binding hypervariable regions of monoclonal anti-hVEGF mouse antibodies A.4.6.1 and blocks the binding of human VEGF to receptors. Approximately 93% of the amino acid sequence of bevacizumab, including most of its framework regions, is human IgG1, and approximately 7% of the sequence is mouse antibody A4.6.1. Bevacizumab has a molecular weight of approximately 149,000 daltons and is glycosylated.

The terms “VEGFC” and “VEGF-C” are used interchangeably to mean a 419 amino acid human polypeptide (SwissProt: VEGFC_HUMAN P49767) and its non-human mammalian orthologs, first described in Joukov et al., EMBO J. 15, 290-98 (1996) and EMBO J. 15, 1751 (1996).

As used herein, the term “Nrp2 antagonist” means a molecule capable of neutralizing, blocking, inhibiting, reversing, decreasing or suppressing the ability of Nrp2 to modulate lymphatic vascular endothelial cell (EC) migration or lymphangiogenesis, in particular tumor lymphangiogenesis and the formation of tumor metastases.

The term "VEGF antagonist" means a molecule capable of neutralizing, blocking, inhibiting, abrogating, reducing or inhibiting the activity of VEGF, including, but not limited to, binding to one or more VEGF receptors. VEGF antagonists include, but are not limited to, anti-VEGF antibodies and antigen-binding fragments thereof, receptor molecules and derivatives that specifically bind to VEGF and thereby limit its binding to one or more receptors, anti-VEGF receptor antibodies and VEGF receptor antagonists, such as low molecular weight inhibitors of VEGFR tyrosine kinases. Used in the present description, the term "VEGF antagonist", in particular, includes molecules, namely antibodies, antibody fragments, other binding polypeptides, peptides and non-peptide small molecules that bind to neuropilin-1 and / or neuropilin-2 (Nrp -1 and / or Nrp-2) and have the ability to neutralize, block, inhibit, cancel, reduce or suppress the activity of VEGF, where these molecules are, but are not limited to, antibodies against Nrp1 and Nrp2 and antibodies cross-reacting with Nrp1 and Nrp2 provided that they possess capable of neutralizing, blocking, inhibiting, abrogating, reducing or inhibit the activity of VEGF. Thus, the term “VEGF activity” specifically includes neuropilin-mediated biological activity of VEGF (as defined above).

The term “semaphorin antagonists” means a molecule having the ability to neutralize, block, inhibit, reverse, reduce or prevent semaphorin activity, including, but not limited to, the ability to bind to one or more semaphorin receptors. Semaphorin antagonists include, but are not limited to, anti-semaphorin antibodies and their antigen-binding fragments; receptor molecules and their derivatives that specifically bind to semaphorin, limiting their binding to one or more receptors; antibodies against the semaphorin receptor; and antagonists of the semaphorin receptor, such as low molecular weight inhibitors of semaphorins. As used herein, the term “semaphorin antagonist” in particular means molecules, including antibodies, antibody fragments, other binding polypeptides, peptides, and non-peptide small molecules that bind to neuropilin-1 and / or neuropilin-2 (Nrp-1 and / or Nrp-2) and have the ability to neutralize, block, inhibit, cancel, reduce or prevent the activity of semaphorin, where these molecules are, but are not limited to, antibodies against Nrp1 and Nrp2 and antibodies cross-reacting with Nrp1 and Nrp2, when condition uu that they are capable of neutralizing, blocking, inhibiting, abrogating, reducing or preventing semaforinovuyu activity. Thus, the term “semaphorin activity”, in particular, includes neuropilin-mediated biological activity (as defined above) of class 3 semaphorins. Such biological activity is, for example, an action that inhibits the growth of neurites in the development of the embryonic nervous system and the regeneration of neurons.

The term "treatment" means both therapeutic and prophylactic treatment of a disease or a measure of its prevention. The violations to which the treatment is directed are already existing violations, as well as violations whose development must be prevented.

“Violation” is any condition whose symptoms can be alleviated as a result of treatment. An example is a disorder that a mammal suffers from or which requires preventive measures to eliminate pathological angiogenesis (excessive, inappropriate or uncontrolled angiogenesis) or pathological vascular permeability. This term means chronic and acute disorders or diseases, including pathological conditions that determine the predisposition of a mammal to this disorder. Non-limiting examples of the disorders described herein that are targeted for treatment are malignant and benign tumors; non-leukemia malignant tumors, and lymphoid malignant tumors, and in particular metastatic tumors (cancer).

The term “malignant tumor” means or refers to a physiological condition in mammals in which unregulated cell growth usually occurs. Examples of malignant tumors include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such malignancies include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma), peritoneal cancer, hepatocellular cancer, intestinal or stomach cancer (including gastrointestinal cancer), cancer pancreas, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colon and colon cancer, endometrial carcinoma or attacks, salivary carcinomas, kidney cancer or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma of the head and neck and various types of cancer, and B-cell lymphoma (including low-grade / non-Hodgkin follicular lymphoma (NHL); small cell lymphocytic (SL) NHL; mid-stage differentiation NHL / follicular NHL; diffuse mid-stage differentiation NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small-cell undifferentiated NHL; generalized NHL; lymphoma of the cells of the cerebral cortex; AIDS-related lymphoma and Waldenstrom macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia and lymphoproliferative disorder after transplantation (PTLD), as well as pathological vascular proliferation associated with phacomatosis, edema (such as edema associated with brain tumors) and Meigs syndrome.

The term "antitumor composition" means a composition that can be used to treat a malignant tumor and includes at least one active therapeutic agent, for example, "antitumor agent." Examples of therapeutic agents (antitumor agents) include, but are not limited to, chemotherapeutic agents, tumor growth inhibiting agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin drugs and other drugs used to treat malignant tumors, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor antagonist (EGFR) (e.g., tyrosine kinase inhibitor), HER1 / EGFR inhibitor (e.g., erlotinib (Tarceva ™)), platelet-derived growth factor inhibitors (e.g., glivec ™ (imatinib mesylate)), a COX-2 inhibitor (e.g. celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets, such as ErbB2 receptor, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF, as well as TRAIL / Apo2 and other biologically active and organic chemicals, etc. The present invention also includes combinations thereof.

Used in the present description, the term "cytotoxic agent" means a substance that inhibits or blocks the function of cells and / or causes the destruction of cells. This term includes radioactive isotopes (for example, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y and ¹⁸⁶ Re), chemotherapeutic agents and toxins, such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound used to treat a malignant tumor. Examples of chemotherapeutic agents are chemical compounds that can be used to treat a malignant tumor. Examples of chemotherapeutic agents are alkylating agents such as thiotepa and cytoxan® (cyclophosphamide); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboxwon, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylol melamine; acetogenins (in particular, bullatacin and bullatacinon); camptothecin (including the synthetic analogue of topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogues, such as adoselesin, carzelesin and biselezin); cryptophycin (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pankratistatin; sarcodiktiin; spongistatin; nitrogen mustard derivatives, such as chlorambucil, chlorofazine, holophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembikhine, phenesterol, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediin antibiotics (e.g. calicheamicin, in particular calicheamicin gamma II and calicheamicin omega II (see, for example, Agnew (1994) Chem. Intl. Ed. Engl. 33: 183-186); dinemycin, including dinemycin A ; bisphosphonates, such as clodronate; esperamycin, as well as neocarcinostatin chromophore and related chromoprotein enediin antibiotics-chromophores), aclacinomisins, actinomycin, autramycin, azaserin, bleomycin, caracinomycinomycinomycin, carcinomycinomycinomy carcinomycinomycinomycinomycin diazo-5-oxo-L-nor leucine, doxorubicin ADRIAMYCIN® (adriamycin) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mycitomycin, mycitomycin, mycitomycin, mycitomycin, mimetomycin, mimetomycin, mimetomycin, mimetomycin, mimetomycin, mimetomycin, mimetomycin, mimetomycin, mimetomycin, mimetomycin, mimetomycin, micitomycin, mimetomycin, micitomycin, mimetomycin, mimetomycin, mimetomycin, mimetomycin, micitomycin, mimetomycin, mimetomycin, mimetomycin, mimetomycin, mimetomycin, mimetomycin, mimetomycinum , potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU);

folic acid analogues such as denopterin, methotrexate, pteropterin, trimerexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, karmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, phloxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostan, testolactone; antiadrenergic agents such as aminoglutethimide, mitotan, trilostane; a folic acid substitute such as frolinic acid; Aceglaton; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisanthrene; edatraxate; defofamine; demecolcin; diaziquon; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pyrarubicin; losoxantrone; podophyllic acid; 2-ethyl hydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spiro germanium; tenuazonic acid; triaziquon; 2,2 ', 2 ”trichlorotriethylamine; trichotecenes (in particular, T-2 toxin, verracurin A, roridin A and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacitosin; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel TAXOL® (taxol) (Bristol-Myers Squibb Oncology, Princeton, NJ), a cremophor-free and albumin-based nanoparticle-based paclitaxel preparation, ABRAXANE ™ (abraxan) (American Pharmaceutical Partners, Schaumberg, Illinois), and Doxetaxel TAXOTERE® (Taxotere) (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine GEMZAR® (gemzar); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine NAVELBINE® (Navelbin); novantron; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar (camptosar), CPT-11) (including treatment with irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including a course of treatment with oxaliplatin (FOLFOX (folfox)); PKC-alpha, Raf, H-Ras, EGFR inhibitors (for example, erlotinib (Tarceva ™)) and VEGF-A, which reduces cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of all of the above compounds.

The definition of this term also includes anti-hormonal agents that regulate or inhibit the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen NOLVADEX® (nolvadex)), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifen, LY117018, onapriston and FARESTON (fareston) -toremifene; aromatase inhibitors that inhibit the aromatase enzyme and regulate the level of estrogen production in the adrenal cortex, such as, for example, 4 (5) -imidazoles, aminoglutethimide, megestrol acetate MEGASE®, exemestane AROMASIN® (aromazine), formestane, fadrozole, Vorozol RIVISOR® letrozole FEMARA® (femara) and anastrozole ARIMIDEX® (arimidex); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxacitabine (a nucleoside analog of 1,3-dioxolancytosine); antisense oligonucleotides, in particular oligonucleotides that inhibit gene expression by signaling involved in abnormal cell proliferation, such as, for example, PKC alpha, Raf and H-Ras; ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME® ribozyme (angiozyme)) and an HER2 expression inhibitor; vaccines such as a gene therapy vaccine, for example, ALLOVECTIN® vaccine (allovectin), LEUVECTIN® vaccine and VAXID® vaccine; rIL-2 PROLEUKIN® (proleukin); LURTOTECAN® topoisomerase 1 inhibitor (lurtotecan); rmRH ABARELIX® (abarelix); vinorelbine and esperamycins (see US patent No. 4675187) and their pharmaceutically acceptable salts, acids or derivatives of all of the above compounds.

As used herein, the term “prodrug” means a precursor or derivative of a pharmaceutically active substance that, compared to the parent drug, is less cytotoxic to tumor cells and is capable of enzymatically being activated or converted into a more active mature form. See, for example, Wilman (1986) “Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions , 14, pp. 375-382, 615th Meeting Belfast and Stella et al. (1985) “Prodrugs: A Chemical Approach to Targeted Drug Delivery”, Directed Drug Delivery , Borchardt et al. (ed.), pp. 247-267, Humana Press. Prodrugs of the invention are, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs; prodrugs containing optionally substituted phenoxyacetamide or prodrugs containing optionally substituted phenylacetamide; 5-fluorocytosine and other 5-fluoruridine prodrugs that can be converted into a more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized to produce a prodrug form for use in the present invention include, but are not limited to, the chemotherapeutic agents described above.

The term “low molecular weight compound” is defined herein as a compound with a molecular weight below about 500 daltons.

An “isolated” nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one impurity nucleic acid molecule with which it is usually associated in a natural source of nucleic acid encoding an antibody. An isolated nucleic acid molecule differs from another molecule in its shape or natural location. Therefore, the isolated nucleic acid molecules are different from the nucleic acid molecule present in natural cells. However, the isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that typically express an antibody, where, for example, the nucleic acid molecule has a chromosomal location that is different from its localization in natural cells.

The term "regulatory sequences" means DNA sequences necessary for the expression of a linked coding sequence in a particular host organism. Regulatory sequences suitable for prokaryotes are, for example, a promoter, optionally an operator sequence and a ribosome binding site. Eukaryotic cells are known to have promoters, polyadenylation signals, and enhancers.

A nucleic acid is "functionally linked" if it is in functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader sequence is considered to be functionally linked to the DNA of the polypeptide if it is expressed as a pre-protein that is involved in the secretion of the polypeptide; either a promoter or enhancer is considered to be functionally linked to the coding sequence if they affect the transcription of the sequence; or the ribosome binding site is considered to be functionally linked to the coding sequence, if its location helps facilitate translation. Generally, the term “operably linked” means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, they are contiguous and are in the same reading frame. However, enhancers should not be contiguous. Binding is accomplished by ligation at suitable restriction sites. If such sites are absent, then synthetic oligonucleotide adapters or linkers are used in accordance with generally accepted practice.

Used in the present description, the terms "cell", "cell line" and "cell culture" are used interchangeably, and all these terms also include their offspring. Thus, the terms “transformants” and “transformed cells” include the primary cell under consideration and its cultures without taking into account the number of passages. It should also be noted that all offspring cannot be absolutely identical in terms of DNA content, which is due to specially introduced or spontaneous mutations. This term includes mutated offspring that have the same functions or biological activity that screening has shown is found in the original transformed cells. If any other terms are used below, they will be understood from the context of the description of the invention.

MODES FOR CARRYING OUT THE INVENTION

In one aspect, the present invention is based on experimental data indicating that blocking Nrp2 function leads to inhibition of tumor metastases.

The main event in the multi-stage process of the formation of metastases is the exit of tumor cells from the primary tumor mass. In solid tumors, the lymphatic system often provides a pathway for cell separation. It is known that VEGF is the main modulator of lymphangiogenesis and metastases in many tumor models, and inhibition of VEGF of all types is considered as a promising strategy for inhibiting the development of metastases. Prior to the invention, Nrp2, a VEGFC coreceptor, was not considered a target for inhibiting tumor metastases, which is probably due to the absence of damage to the lymphatic system in adult Nrp2 mutant mice.

The studies underlying the present invention and described in the examples below confirmed the important role of Nrp2 in lymphangiogenesis and tumor metastases, partly only due to modulation of the transmission of the VEGFR3 signal. In addition, the data presented in the examples demonstrated the presence of functional lymphatic vessels in tumors and showed that treatment with an anti-Nrp2 ^B antibody leads to a decrease in the number of these functional lymphatic vessels.

Selective VEGFC functions regulated by Nrp2 partially according to a mechanism independent of VEGF receptor activation

It is now known that the induction of cell migration and proliferation is accomplished by the two central VEGFC cellular functions described in the literature (Joukov et al., Embo J. 16, 3898-3911 (1997)). Thus, it was unexpectedly discovered by the present inventors that blocking Nrp2 by an anti-Nrp2 ^B antibody results in blocking the migration but not proliferation of LEC (Figures 2, 3). Such selectivity has recently been reported in Nrp2 siRNA inhibition experiments, but this fact has been explained by the technical flaws of the experiment (Favier et al., Blood 108, 1243-1250 (2006)). The data presented in the present description showed that the functional selectivity of Nrp2 is also observed in vivo , where treatment with an anti-Nrp2 ^B antibody resulted in a decrease in the level of VEGFC-regulated lymphangiogenesis rather than vascular permeability (Figures 2, 3). Such observations suggested that inhibition by the anti-Nrp2 ^B antibody is not only a result of impaired VEGFC signal transmission. However, it was found that blocking Nrp2 leads to a moderate decrease in the level of phosphorylation of the VEGF receptor (Figure 3), which confirms the presence of a mechanism in which one of the roles of Nrp2 is to enhance the function of the VEGF receptor. This increases the likelihood that different levels of VEGF receptor activation may be required to carry out various VEGF-induced physiological events. Thus, a decrease in the level of receptor activation may be sufficient to suppress migration, but not for proliferation or vascular permeability.

To identify this fact, the dependence of the dose of VEGFC on the response to phosphorylation of the VEGF receptor was compared with the dependence of the dose on the response to migration of LEC (figure 3). Doses of VEGFC, which lead to a certain level of receptor phosphorylation, equivalent to the level observed when treated with anti-Nrp2 ^B antibody, did not reduce or inhibit migration. This indicated that a decrease in the level of activation of the receptor alone could not explain the effects of blocking function under the action of antibodies against Nrp2 ^B.

In this regard, other mechanisms were investigated under the influence of which Nrp2 blocking can selectively affect migration, such as modulation of adhesion or mobility. Antibody treatment with Nrp2 ^B did not have any effect on LEC-mediated adhesion or migration induced by VEGF ₁₆₅ (FIG. 2), HGF (FIG. 3) or FGF-2, which indicated that such treatment did not substantially or any effect on migration through impaired mobility. In addition, it has been suggested that sema3F, another Nrp2 ligand, can modulate LEC or EC migration by acting as a chemorepellant (Bielenberg et al., J. Clin. Invest 114, 1260-1271 (2004); Favier et al., Blood 108, 1243-1250 (2006)). However, the anti-Nrp2 ^B antibody did not inhibit or enhance the binding of sema3F to Nrp2 (Figure 1) or the functional effect of sema3F on susceptible neurons (Supplementary Figure 4). Thus, it is unlikely that a decrease in the level of VEGFC-induced migration by the anti-Nrp2 ^B antibody can be explained by modulation of the sema3F function.

The effect of the anti-Nrp2 ^B antibody on the formation of the Nrp2 / VEGF receptor complex was also evaluated. Unlike Nrp1, Nrp2 forms a complex with VEGFR2 and VEGFR3 in the absence of a ligand (Favier et al., 2006, see above; Karpanen et al., Faseb . J 20, 1462-1472 (2006)). It is important to note that the anti-Nrp2 ^B antibody significantly inhibits the formation of these complexes. In addition, this observation, in addition to the fact that Nrp2 plays an important role not only in enhancing the function of the VEGF receptor, allows us to create a model in which Nrp2 will provide additional functionality in the specific modulation of migration, possibly by communicating with the VEGF receptor complex additional function.

Nrp2 plays an important role in modulating adult lymphangiogenesis

Analysis of Nrp2 in KO mice demonstrates that Nrp2 is a modulator of developing lymphangiogenesis, probably due to its role as a VEGFC co-receptor (Yuan et al., 2002, supra). However, in these mutant mice, functional lymphatic vessels form after their birth, which indicates that in this case either a disorder occurs that is not inhibition but in slowing the growth of lymphatic vessels, or functional compensation occurs due to another molecular mediator. Therefore, the role of Nrp2 in the preservation of mature lymphatic vessels and in the modulation of lymphangiogenesis in adults is still unclear. The expression analysis (Figure 4) did not confirm the role of Nrp2 in the preservation of lymphatic vessels. It is interesting to note that Nrp2 is largely expressed in the lymphatic vessels that are present in tumors and in the lymph nodes (LN) adjacent to the tumors, suggesting that Nrp2 may play a role in the activation or growth of lymphatic vessels. In vitro observations show that an anti-Nrp2 ^B antibody is an effective tool for evaluating the role of Nrp2 in these processes. Therefore, the anti-Nrp2 ^B antibody was tested in vivo in the analysis of the cornea micro-pocket (Figure 2). The anti-Nrp2 ^B antibody effectively blocked VEGFC-induced lymphangiogenesis, and it was unexpectedly found that such blocking is commensurate with the blocking of ECD of VEGFR3-induced lymphangiogenesis. It is interesting to note that the anti-Nrp2 ^B antibody has a selective in vivo inhibitory function and also does not affect VEGFC-induced vascular permeability. These data are consistent with in vitro observations indicating that Nrp2 specifically modulates migration, that is, a process that plays an important role in lymphangiogenesis, but is unlikely to play any role in vascular permeability. Finally, in healthy adult animals treated with an anti-Nrp2 ^B antibody, no changes in the lymphatic vessels of the small intestine were observed, indicating that Nrp2 does not play a role in preserving mature lymphatic vessels.

Inhibition of Nrp2 leads to a decrease in the number of functional lymphatic vessels in the tumor and to a decrease in the number of metastases, which is probably due to the inhibition of tumor cells leaving the main tumor mass along the lymphatic pathways

Inhibition of all types of VEGFC, most often using VEGFR3 ECD, is one of the most common strategies for reducing the number of metastases (Chen et al., 2005, see above; He et al., 2002, see above; Krishnan et al., 2003 , see above; Lin et al., 2005, see above). VEGFC can help reduce the number of metastases by initiating lymphangiogenesis and thereby increase the surface area of tumor cells when contacted with LEC by modulating the adhesive properties of LEC or expressing cytokines or by increasing vascular permeability (Alitalo and Carmeliet, Cancer Cell 1, 219-227 (2002)) . Because the anti-Nrp2 ^B antibody modulates selective VEGFC-mediated functions, including inhibition of VEGFC-induced lymphangiogenesis in adults, studies have been conducted on the effect of Nrp2 blocking on metastases. To minimize unnecessary concomitant variables and to unambiguously evaluate the effect of the anti-Nrp2 ^B antibody on metastases, the authors of the present invention created models where Nrp2 blocking did not affect the growth of the primary tumor, and then all animals in this study were analyzed at regular intervals (Withers and Lee, Semin Radiat Oncol . 16, 111-119 (2006)).

In both models with 66c14 and C6 tumors, treatment with an anti-Nrp2 ^B antibody resulted in a significant decrease in the number of metastatic nodes in the lungs upon visual observation (Figures 5, 6). This is confirmed by more sensitive and quantitative micro-CT methods (Li et al., Technol. Cancer Res. Treat . 5, 147-155 (2006)). Comparison of anti-Nrp2 ^B antibody and VEGFR3 ECD treatment data in 66c14 tumors was not possible due to a decrease in the size of the primary tumor after VEGFR3 treatment. However, this analysis was performed on C6 tumors because their growth was not affected by the ECD VEGFR3 treatment. As with the analysis carried out in the cornea micro pocket, treatment with an anti-Nrp2 ^B antibody resulted in blocking of metastases, commensurate with the blocking observed with VEGFR3 ECD.

The histological analysis performed by the authors showed the absence of any effect on the primary tumor cells. Thus, the authors evaluated two potential pathways for the development of metastases that are accessible to tumor cells, namely metastases in blood and lymph vessels (Figure 7). The introduction of antibodies against Nrp2 ^B did not affect the structure or density of blood vessels. Based on in vitro data and in vivo data obtained by analysis in the corneal micro pocket, it was hypothesized that blocking Nrp2 should lead to a decrease in the number of tumor lymph vessels. The introduction of antibodies against Nrp2 ^B led to a sharp decrease in the density of the lymphatic vessels, but in this case, the degree of such a decrease was equivalent to the degree of decrease observed during EEG VEGFR3 treatment. However, these two injections differ in their effect on the morphology of the resulting lymphatic vessels. ECD VEGFR3 administration resulted in the formation of a rare network of lymphatic vessels consisting of apparently-infected lymphocytes. On the other hand, administration of an anti-Nrp2 ^B antibody led to the development of short vessels and pockets from individual healthy-looking lymphatic cells. In addition, these differences were confirmed by a model in which Nrp2 not only acts as an enhancer of activation of the VEGF receptor, but also reports unique functional properties mediating the biological effect of VEGFC. These results also showed that the tested experimental paradigms are that the anti-Nrp2 ^B antibody acts as an inhibitor of lymphangiogenesis (Figure 7). However, it cannot be ruled out that an anti-Nrp2 ^B antibody can also destroy more developed lymphatic vessels in tumors.

The authors of the present invention also conducted a study to determine whether the intratumoral lymphatic vessels are functional and, therefore, whether they contribute to the formation of metastases. To identify rare functional intratumoral lymphatic vessels, lymphangiography was performed (Figure 8). The method used is not an adequately analytical method for determining the number of functional intratumoral lymphatic vessels. However, they obviously represent a small fraction of the total number of lymphatic vessels (Padera et al., Mol. Imaging 1, 9-15 (2002)). Since it is possible that the authors were able to reduce the total density of lymphatic vessels with a small number of functional vessels (which may have different sensitivity to anti-Nrp2 ^B antibodies), the effect of Nrp2 blocking on the formation of functional lymphatic vessels was evaluated. The anti-Nrp2 ^B antibody suppressed the formation of functional vessels, indicating a more direct correlation of the effect on tumor lymphatic vessels with the observed reduction in the number of metastases.

Finally, to confirm the effects of a decrease in the number of functional lymphatic vessels, the effect of the anti-Nrp2 ^B antibody on metastases in SLN was evaluated. SLNs are the first tissue where tumor cells enter after they migrate from the tumor through the lymphatic vessels. Thus, they represent tissue where one of the earliest stages of the formation of metastases in the peripheral organs occurs (Stracke and Liotta, In vivo 6, 309-316 (1992)). As predicted, treatment with an anti-Nrp2 ^B antibody slows down the formation of metastases in SLN, which is consistent with the belief that fewer cells outflow from the primary tumor mass. This is consistent with evidence that VEGFC increases metastases by inducing lymphatic hyperplasia and increasing the delivery of cancer cells to the lymph nodes (Hoshida et al., Cancer Research 66, 8065-8075 (2006)). Thus, there is a wealth of data indicating a mechanism by which Nrp2 blocking leads to a decrease in the number of functional tumor lymphatic vessels and thereby to preventing the involvement of tumor cells in the metastatic process as a result of the release of these cells from the primary tumor mass.

Nrp2 as a target for the formation of metastases

Numerous clinical and pathological studies have shown that the expression of VEGFC and VEGFR3 is directly related to the formation of metastases in the lymph nodes and distal organs in various human malignant tumors (for a detailed review, see Stacker et al., Nat. Rev. Cancer , 2002, see above; Stacker et al., Faseb J , 2002, see above, and He et al., 2004, see above). However, information related to the expression of Nrp2 and its relation to the development of metastases is very limited. Indeed, only the relationship between the expression of Nrp2 and the development of malignant tumors, in particular pancreatic cancer, was established (Cohen et al., Biochem Biophys Res Commun 284, 395-403 (2001); Fukahi et al., Clin Cancer Res 10, 581- 590 (2004)) and lung cancer (Kawakami et al., Cancer 95, 2196-2201 (2002); Lantuejoul et al., J Pathol 200, 336-347 (2003)). Similarly, it was found that the expression of Nrp2, compared with expression in the corresponding control tissues, is observed at a higher level not only in the cells of the pancreas, but also in the cells of the colon adenocarcinoma, squamous cell carcinoma of the head and neck, melanoma, papillary thyroid carcinoma glands and infiltrating adenocarcinomas of the ducts of the mammary gland (figure 9). More important is the fact that when these tumors were divided into subgroups of metastatic and non-metastatic tumors, a statistically higher level of Nrp2 expression was observed in most tumors of this type belonging to the metastatic group. It is interesting to note that all tumors of this type showed intratumoral lymphangiogenesis, which, in addition, is directly associated with metastases in the lymph nodes (Achen et al., 2006, see above; Achen and Stacker, Int. J. Cancer 119, 1755-1760 ( 2006)). This indicates that the results obtained in the experiments under discussion suggest that such studies can be continued with the participation of people with tumors of various types.

In conclusion, it should be noted that the data discussed in the present description and presented in the following examples indicate that Nrp2 plays a role in modulating VEGFC-induced cell migration, and suggest that Nrp2 can act in many ways, including amplification activation of the VEGF receptor, and by mechanisms that are independent of VEGF receptor signaling. In addition, blocking Nrp2 function using an anti-Nrp2 ^B antibody leads to a sharp decrease in VEGFC-induced lymphangiogenesis in adult mice. Such treatment also leads to a reduction in the number of metastases, probably due to the suppression of the development of functional lymphatic vessels. These data, along with analyzes for the expression of Nrp2 in a number of human tumors, suggest that Nrp2 is a true target for modulating metastases.

Obtaining antibodies against Nrp2

The present invention contemplates the preparation and use of antibodies against Nrp2. Examples of methods for producing antibodies are described in more detail in the sections below.

Antibodies against NRP2 are selected using the mammalian NRP2 antigen. The preferred antigen is human NRP2 (hNRP2). However, other mammalian NRP2s such as mouse NRP2 (mNRP2) can also be used as a target antigen. Mammalian NRP2 antigens of various species can be isolated from natural sources. In other embodiments, the antigen is produced by recombinant methods or using other synthetic methods known to those skilled in the art.

The selected antibody usually has a sufficiently high binding affinity for the NRP2 antigen. For example, an antibody can bind to hNRP2 with a K _d value of not more than about 5 nM, preferably not more than about 2 nM, and more preferably not more than about 500 pM. The affinity of antibodies can be determined by analysis, for example, based on surface plasmon resonance (such as the BIAcore analysis described in the examples); enzyme-linked immunosorbent assay (ELISA); and competitive binding assays (e.g., RIA).

In addition, the antibody can be analyzed using other tests for biological activity, for example, to assess the effectiveness of this antibody as a therapeutic agent. Such assays are known to those skilled in the art, and their choice depends on the target antigen and the purpose of the antibody. Examples are HUVEC inhibition assays (described in the examples below); tumor cell growth inhibition assays (described, for example, in WO 89/06692); antibody-dependent cell cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (US Pat. No. 5,500,362); and tests for agonistic activity or hematopoiesis (see WO 95/27062).

To screen for antibodies that bind to a particular epitope on an antigen of interest, a routine cross-blocking assay can be performed, such as that described in Antibodies, A Laboratory Manual , Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, in order to determine whether an antibody binds to an epitope of interest, epitope mapping can be performed, for example, as described in Champe et al. (1995) J. Biol. Chem . 270: 1388-1394.

Obtaining antibodies against NRP2 from synthesized phage antibody libraries

In a preferred embodiment, antibodies against NRP2 are selected using a unique phage display method. Such a method involves the preparation of synthesized phage antibody libraries on a single framework matrix, the creation of a sufficient variety of variable domains, the presentation of polypeptides having diverse variable domains, the selection of candidate antibodies with high affinity for the NRP target antigen, and the selection of selected antibodies.

A detailed description of phage display methods can be found, for example, in WO 03/102157, published December 11, 2003.

In one aspect of the invention, antibody libraries can be obtained by mutations introduced at positions accessible to the solvent and / or at positions with a high diversity of the at least one variable domain CDR of the antibody. Some or all of the CDRs may be mutated using the methods described herein. In some embodiments of the invention, it may be preferable to obtain a variety of antibody libraries by mutations introduced at the positions of CDRH1, CDRH2 and CDRH3 to form one library, or by mutations introduced at the positions of CDRL3 and CDRH3 to form one library, or by mutations introduced at the positions of CDRL3 and CDRH1, CDRH2 and CDRH3 to form one library.

A library of variable antibody domains can be obtained, for example, by introducing mutations at positions accessible to the solvent and / or at positions with a high diversity of CDRH1, CDRH2 and CDRH3. Another library can be obtained by introducing mutations in CDRL1, CDRL2 and CDRL3. Such libraries can also be used in combination with each other to obtain binding agents with the desired affinity. For example, after one or more rounds of selection of the heavy chain library for binding to the target antigen, the light chain library can be replaced by a series of heavy chain binding agents to conduct additional selection rounds to increase the affinity of the binding agents.

Preferably, the library is created by replacing the starting amino acids with amino acid variants in the CDRH3 variable region of the heavy chain sequence. The resulting library may contain many antibody sequences, where sequence diversity is achieved mainly in the region of the CDRH3 sequence of the heavy chain.

In one aspect of the invention, a library is created based on a sequence of a humanized 4D5 antibody or a frame amino acid sequence of a humanized 4D5 antibody. Preferably, the library is created by replacing at least 95-100 amino acid residues of the heavy chain with amino acids encoded by the DVK codon set, where the specified set of DVK codons is used to encode a series of amino acid variants for each of these positions. An example of a set of oligonucleotides that can be used to create such substitutions is the sequence ( DVK ) ₇ . In some embodiments of the invention, the library is created by replacing at least 95-100 amino acid residues with amino acids encoded by both codon sets DVK and NNK . An example of a set of oligonucleotides that can be used to create such substitutions is the sequence ( DVK ) ₆ ( NNK ). In another embodiment of the invention, the library is created by replacing at least 95-100 amino acid residues with amino acids encoded by both sets of codons DVK and NNK . An example of a set of oligonucleotides that can be used to create such substitutions is the sequence ( DVK ) ₅ ( NNK ). Another example of a set of oligonucleotides that can be used to create such substitutions is the sequence ( NNK ) ₆ . Other examples of suitable oligonucleotide sequences may be determined by those skilled in the art in accordance with the criteria described herein.

In another embodiment of the invention, various CDRH3 constructs are used to isolate binding agents with high affinity and to isolate binding agents for a number of epitopes. CDRH3 obtained in this library has a length of 11 to 13 amino acids, although CDRH3 with a different length can be obtained. The diversity of H3 can be increased using the set of codons NNK, DVK and NVK , and it can also be limited at the N- and / or C-ends.

Variety can also be created in CDRH1 and CDRH2. The creation of varieties of CDR-H1 and H2 is achieved in accordance with a strategy aimed at the repertoire of the described natural mimetic antibodies, while the modification is aimed at obtaining a variety that is more consistent with natural diversity than the diversity of previous designs.

To obtain CDRH3 diversity, many libraries with different H3 lengths can be separately constructed, which can then be combined to select agents that bind to target antigens. Many libraries can be combined and sorted by selection methods on a solid carrier and by methods of sorting in solution, as described previously and as described below in this application. Many sorting strategies can be used. So, for example, one option involves selecting a target associated with a solid, followed by label sorting, which may be present on a fused polypeptide (e.g., an anti-gD tag), and subsequent other sorting by target associated with a solid. Alternatively, libraries can be first sorted by target bound to a solid surface, and then eluted binding agents can be sorted by binding in solution with decreasing concentrations of the target antigen. Using combinations of different sorting methods allows one to minimize the selection of only highly expressed sequences and to select a number of different clones with high affinity.

High affinity binding agents for the NRP2 target antigen can be isolated from libraries. The limited diversity of the H1 / H2 region reduces the degeneracy of the code by about 10 ⁴ -10 ⁵ times, and a higher diversity of the H3 region requires a higher affinity of the binding agent. To use libraries with different types of CDRH3 diversity (for example, using DVK or NVT codons), it is necessary to isolate binding agents that can bind to different epitopes of the target antigen.

After isolation of binding agents from the pooled libraries described above, it was found that affinity can be further increased by limiting diversity in the light chain. In this embodiment, light chain diversity can be created as follows: in CDRL1: the amino acid at position 28 is encoded by the RDT codon; the amino acid at position 29 is encoded by the RKT codon; the amino acid at position 30 is encoded by the RVW codon; the amino acid at position 31 is encoded by the ANW codon; the amino acid at position 32 is encoded by the THT codon; optionally, the amino acid at position 33 is encoded by the CTG codon; in CDRL2: the amino acid at position 50 is encoded by the KBG codon; the amino acid at position 53 is encoded by the AVC codon; and optionally, the amino acid at position 55 is encoded by a GMA codon; in CDRL3: the amino acid at position 91 is encoded by TMT or SRT codons, or both; the amino acid at position 92 is encoded by the DMC codon; the amino acid at position 93 is encoded by the RVT codon; the amino acid at position 94 is encoded by the NHT codon; and the amino acid at position 96 is encoded by TWT or YKG codons, or both.

In another embodiment, a library or libraries with a variety of CDRH1, CDRH2, and CDRH3 regions are prepared. In this embodiment of the invention, the diversity of CDRH3 is achieved using H3 regions of different lengths and mainly using codon sets XYZ and NNK or NNS . Libraries can be obtained using individual oligonucleotides and their combinations, or oligonucleotides can be combined to form a subsequence of libraries. In this embodiment of the invention, libraries can be sorted by target associated with a solid. Clones isolated from many sorting procedures can be screened for specificity and affinity using ELISA assays. To determine the specificity of the clones can be screened for the desired target antigens, as well as other antigens that are not targets. Agents that bind to the NRP1 target antigen can then be screened for affinity in an ELISA assay for competitive binding in solution or in a point assay for competitive binding. High affinity binding agents can be isolated from the library using XYZ codon sets prepared as described above. These binding agents can be easily obtained in cell culture in the form of antibodies or antigen-binding fragments in high yield.

In some embodiments of the invention, it may be desirable to obtain libraries with a wide variety of CDRH3 region lengths. For example, it may be desirable to obtain libraries with CDRH3 regions having a length of about 7 to 19 amino acids.

High affinity binding agents isolated from the libraries of these variants of the invention can be easily obtained in a high yield bacterial and eukaryotic cell culture. These vectors can be designed so that sequences such as gD tags and the sequence of the viral envelope protein component can be easily removed and / or added to constant region sequences to produce full length antibodies or antigen-binding fragments thereof in high yield.

A library with mutations in CDRH3 can be combined with a library containing modified versions of other CDRs, for example CDRL1, CDRL2, CDRL3, CDRH1 and / or CDRH2. For example, in one embodiment of the invention, the CDRH3 library is combined with a CDRL3 library based on the sequence of a humanized 4D5 antibody with amino acid variants at positions 28, 29, 30, 31 and / or 32 using predefined codon sets. In another embodiment of the invention, a library with mutations in CDRH3 may be combined with a library containing the heavy chain variable domains of the CDRH1 and / or CDRH2 variants. In one embodiment of the invention, a CDRH1 library is created using the sequence of a humanized 4D5 antibody with amino acid variants at positions 28, 30, 31, 32 and 33. A CDRH2 library can be created using a sequence of a humanized 4D5 antibody with amino acid variants at positions 50, 52, 53 , 54, 56, and 58 and using predefined sets of codons.

Anti-NRP2 Antibody Mutants

An anti-NRP2 antibody obtained from phage libraries can be further modified to produce mutant antibodies, which, in comparison with the parent antibody, have improved physical, chemical or biological properties. If the assay used is a biological activity assay, then in the selected assay, the mutant antibody should preferably have a biological activity that is at least about 10 times, preferably at least about 20 times, more preferably at least about 50 times, and sometimes at least about 100 or 200 times the biological activity of the parent antibody in this assay. Thus, for example, a mutant anti-NRP1 antibody preferably has an NRP binding affinity that is at least about 10 times, preferably at least about 20 times, more preferably at least about 50 times, and sometimes at least about 100 or 200 times the binding affinity of the parent anti-NRP antibody.

To obtain a mutant antibody, one or more amino acid modifications (e.g., substitutions) are introduced into one or more hypervariable regions of the parent antibody. Alternatively or additionally, one or more modifications (for example, substitutions) of framework region residues can be introduced into the parent antibody, which will lead to an increase in the binding affinity of the mutant antibody to a mammalian antigen of another species. Examples of modifiable framework region residues are those that directly and non-covalently bind to the antigen (Amit et al. (1986) Science 233: 747-753); interact with CDR / affect the conformation of CDR (Chothia et al. (1987) J. Mol. Biol. 196: 901-917); and / or participate in the formation of the boundary region V _L -V _H (EP 239400 B1). In some embodiments of the invention, the modification of one or more such framework region residues results in an increase in the binding affinity of the antibody to a mammalian antigen of another species. For example, in this embodiment of the invention, about one to five framework residues can be modified. Sometimes it may be sufficient to obtain a mutant antibody with a yield suitable for use in preclinical trials even if none of the residues of the hypervariable region is modified. However, usually a mutant antibody contains additional modification (s) in the hypervariable region.

Remains of the hypervariable region that are modified can be replaced arbitrarily, especially if the parent antibody has an initial binding affinity at which such randomly obtained antibody mutants can be easily screened.

One technique suitable for producing such mutant antibodies is a technique called “alanine scanning mutagenesis” (Cunningham and Wells (1989) Science 244: 1081-1085). In this case, one or more residues of the hypervariable region is replaced with alanine (s) or polyalanine (s) residue (s) in order to influence the interaction of amino acids with a mammalian antigen of another species. This (s) residue (s) of the hypervariable region, having (s) functional sensitivity to substitutions, is then clarified by introducing additional or other mutations in or near substitution regions. Thus, although the site for introducing changes in the amino acid sequence is predetermined, the nature of the per se mutation need not be predetermined. Such ala-mutants obtained by this method are screened for biological activity, as described in this application.

Typically, the replacement procedure should begin with the introduction of conservative substitutions, such as the substitutions presented in the table below, entitled “Preferred Substitutions”. If such substitutions result in a change in biological activity (for example, binding affinity), then more substantial substitutions, referred to in the table below as “characteristic substitutions”, or substitutions further described below indicating the classes of amino acids, can be introduced, and the resulting products can be screened.

Preferred Substitutions: Original balance Characteristic replacements Preferred Substitutions Ala (A) Val; Leu Ile Val Arg (R) Lys; Gln; Asn Lys Asn (n) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (s) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu Val; Met; Ala; Phe; norleucine Leu Leu (L) norleucine; Ile Val; Met; Ala; Phe Ile Lys (C) Arg; Gln; Asn Arg Met (M) Leu Phe; Ile Leu Phe (f) Leu Val; Ile Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (w) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile Leu Met; Phe; Ala; norleucine Leu

More significant changes in the biological properties of the antibody can be achieved by selecting substitutions that have a significant effect on the conservation of (a) the structure of the polypeptide backbone in the region of substitution, for example, a folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the desired site, or (c) side chain volume. Natural residues are divided into the following groups according to the general properties of the side chains:

(1) hydrophobic residues: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic residues: Cys, Ser, Thr; Asn; Gln;

(3) acidic residues: Asp, Glu;

(4) basic residues: His, Lys, Arg;

(5) residues that affect chain orientation: Gly, Pro; and

(6) aromatic residues: Trp, Tyr, Phe.

Non-conservative substitutions lead to replacements of a member of one of these classes with a member of another class.

In another embodiment of the invention, sites selected for modification are subjected to affinity maturation by phage display method (see above).

Nucleic acid molecules encoding mutant amino acid sequences are prepared by various methods known to those skilled in the art. Such methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cluster mutagenesis of an already obtained mutant or non-mutant variant of the parent antibody. A preferred method for producing mutants is site directed mutagenesis (see, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488).

In some embodiments of the invention, the mutant antibody has only one residue substitution in the hypervariable region. In other embodiments of the invention, two or more residues of the hypervariable region of the parent antibody have been replaced, for example, from about two to ten residues.

Typically, a mutant antibody with improved biological properties has an amino acid sequence that is at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90% and most preferably at least 95% identical or similar to the amino acid sequence of the variable domain of the heavy or light chain of the parent antibody. The identity or similarity of these sequences is defined in the present description as the percentage of amino acid residues in the candidate sequence that are identical (i.e., the same residues) or similar (i.e., amino acid residues belonging to the same group based on their common properties side chains) residues of the parent antibody after alignment of sequences and the introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity. None of the N-terminal, C-terminal or internal modifications, such as extensions, deletions or insertions introduced into the sequence of antibodies outside the variable domain, should not adversely affect the identity or similarity of the sequences.

After obtaining a mutant antibody, the biological activity of the obtained molecule is determined in comparison with the parent antibody. As indicated above, this can be done by determining the binding affinity and / or other biological activities of the antibody. In a preferred embodiment, a panel of mutant antibodies is prepared and screened for binding affinity to an antigen, such as NRP1 or a fragment thereof. One or more mutant antibodies selected as a result of the initial screening is, but not necessarily, analyzed for one or more additional biological activities in order to determine if such mutant antibody (s) can (gut) with an increased binding affinity to be used (s), for example, in preclinical studies.

The mutated antibody (s) thus selected can be further modified, often depending on the purpose of the antibody. Such modifications may include an additional modification of the amino acid sequence, fusion with the heterologous (s) polypeptide (s) and / or covalent modifications, such as the modifications described below. Typical amino acid sequence modifications are described above. So, for example, any cysteine residue that is not involved in maintaining the appropriate conformation of the mutant antibody can also be replaced, usually with serine, to increase the oxidation stability of the molecule and prevent undesired cross-linking. Conversely, cysteine bond (s) can be introduced into said antibody to increase its stability (in particular, if said antibody is an antibody fragment, such as an Fv fragment). Another type of amino acid mutant has an altered glycosylation pattern. This character can be achieved by deletion of one or more carbohydrate groups present in the antibody and / or by adding one or more glycosylation sites not present in the antibody. Glycosylation of antibodies is usually either N-linked or O-linked. N-linked glycosylation means the attachment of a carbohydrate group to the side chain of an asparagine residue. The tripeptide sequences “asparagine-X-serine” and “asparagine-X-threonine”, where X is any amino acid except proline, are recognition sequences for the enzymatic attachment of a carbohydrate group to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in the polypeptide contributes to the creation of a potential glycosylation site. O-linked glycosylation means the addition of one of the sugars, such as N-acetylgalactosamine, galactose or xylose, to a hydroxy amino acid, mainly to serine or threonine, although they may also be 5-hydroxyproline or 5-hydroxylysine. The attachment of glycosylation sites to an antibody is usually carried out by modifying the amino acid sequence such that the amino acid sequence will contain one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Such a modification can also be carried out by adding one or more serine or threonine residues to the sequence or replacing them in the sequence of the original antibody (for O-linked glycosylation sites).

Vectors, host cells and recombinant methods

Antibodies against Nrp2 according to the invention can be obtained recombinantly using readily available methods and materials.

For recombinant production of an anti-NRP2 antibody, a nucleic acid encoding such an antibody is isolated and inserted into a replicated vector for subsequent cloning (DNA amplification) or expression. DNA encoding an antibody can be easily isolated or synthesized by standard methods (for example, using oligonucleotide probes capable of specifically binding to DNA encoding the heavy and light chains of an antibody). Many vectors are publicly available. Vector components are typically, but are not limited to, one or more of the following components, such as a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

(i) Component signal sequence

An antibody of the invention can be recombinantly produced not only by a direct method, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of a mature protein or polypeptide. The selected heterologous signal sequence is preferably a sequence that is recognized and processed (i.e., cleaved by signal peptidase) in the host cell. If prokaryotic host cells do not recognize and process the native signal sequence of an antibody, then this signal sequence can be replaced by a prokaryotic signal sequence selected, for example, from the group consisting of leader sequences of alkaline phosphatase, penicillinase, lpp or thermostable enterotoxin II. For yeast secretion, the native signal sequence may be replaced, for example, with a yeast invertase leader sequence, an α-factor leader sequence (including Saccharomyces and Kluyveromyces α-factor leader sequences) or an acid phosphatase leader sequence, a C. albicans glucoamylase leader sequence, or a signal sequence, described in WO 90/13646. For expression in mammalian cells, mammalian signal sequences are used, as well as viral secretory leader sequences, for example, the herpes simplex virus gD signal sequence.

DNA for such a precursor region is ligated to the DNA encoding the antibody, while maintaining the reading frame.

(ii) The replication origin component

Expression and cloning vectors contain a nucleic acid sequence that facilitates the replication of this vector in one or more selected host cells. Typically, in cloning vectors, such a sequence is a sequence that allows a given vector to replicate independently of the host chromosome DNA and that contains a replication origin or autonomously replicating sequence. Such sequences for various bacteria, yeast and viruses are well known. The replication origin of plasmid pBR322 is suitable for most gram-negative bacteria, namely, the replication origin of a 2 μm plasmid is suitable for yeast, and various replication origin of viruses (SV40, polyomavirus, adenovirus, VSV or BPV) can be used to clone vectors in cells mammals. Typically, the component of the origin of replication is not necessary for vectors expressed in mammalian cells (the origin of SV40 replication is usually only used when it contains an early promoter).

(iii) Component Selective Gene

The expression and cloning vectors may contain a selective gene, also called a selective marker. Typically, selective genes encode proteins that (a) show resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate or tetracycline, (b) complement deficiency due to auxotrophy, or (c) provide important nutrients that are not delivered from complex media, for example, a gene encoding Bacillus D-alanine racemase.

One example of a screening scheme is the use of a drug that stops the growth of a host cell. Cells that have been successfully transformed with a heterologous gene produce a protein that reports drug resistance and thereby contributes to the survival of these cells in a selective medium. For such dominant selection, for example, drugs such as neomycin, mycophenolic acid and hygromycin can be used.

Other examples of suitable selective markers for mammalian cells are markers that allow identification of cells capable of incorporating an antibody nucleic acid encoding antibodies such as DHFR, thymidine kinase, metallothionein-I and II genes, and preferably genes encoding primate metallothionein, adenosine deaminase, ornithinide etc.

Thus, for example, cells transformed with the DHFR selective gene are first identified by culturing all transformants in culture medium containing methotrexate (Mtx), a competitive DHFR antagonist. When using wild-type DHFR, a suitable host cell is the Chinese Hamster Ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (in particular wild-type hosts containing endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, wild-type DHFR protein and another selective marker, such as aminoglycoside-3'-phosphotransferase (ARN) can be selected by culturing cells in a medium containing a selection agent carried out using a selective marker, such as an aminoglycoside antibiotic, for example kanamycin, neomycin or G418. See U.S. Patent No. 4,965,199.

A suitable selective gene for use in yeast is the trp 1 gene present in the yeast plasmid YRp7 (Stinchcomb et al. (1979) Nature 282: 39). The trp1 gene is a selective marker for a mutant strain of yeast that is not able to grow in the presence of tryptophan, for example, a strain deposited in ATCC No. 44076 or PEP4-1. Jones (1977) Genetics , 85:12. The presence of a mutation in the trp1 gene in the genome of the yeast host cell allows efficient detection of transformation by cultivation in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20622 or 38626) were complemented with known plasmids carrying the Leu2 gene.

In addition, vectors of the circular 1.6-μm plasmid pKD1 can be used to transform Kluyveromyces yeast. Alternatively, an expression system for large-scale production of recombinant calf chymosin has been described for K.lactis . Van den Berg (1990) Bio / Technology , 8: 135. Stable multi-copy expression vectors for the secretion of mature recombinant human serum albumin by industrial Kluyveromyces strains have also been described. Fleer et al. (1991) Bio / Technology , 9: 968-975.

(iv) Component promoter

Expression and cloning vectors usually contain a promoter recognized by the host organism and functionally linked to the nucleic acid encoding the antibody. The promoters suitable for use in prokaryotic hosts are the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan ( trp ) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are also suitable. The promoters used in bacterial systems also contain a Shine-Dalgarno (SD) sequence functionally linked to the DNA encoding the antibody.

Promoter sequences suitable for eukaryotes are known to those skilled in the art. In fact, all eukaryotic genes have an AT-rich region, located approximately in the region located 25-30 nucleotides higher from the site of transcription initiation. Another such sequence, located approximately in the region located 70-80 nucleotides higher from the transcription initiation site of many genes, is the CNCAAT region, where N can be any nucleotide. At the 3'-end of most eukaryotic genes is the AATAAA sequence, which can serve as a signal to attach the poly A tail to the 3'-end of the coding sequence. All of these sequences can be appropriately inserted into eukaryotic expression vectors.

Examples of promoter sequences suitable for use in yeast host cells are promoters of 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose phosphate fructosease 3, glucose triosophosphatisomerase, phosphoglucose isomerase and glucokinase.

Other yeast promoters, which are inducible promoters that have the additional advantage of being able to regulate transcription under certain cultivation conditions, are the promoter regions of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, destructive enzymes associated with the metabolism of nitrogen, metallothionein, glyceraldehyde-3 -phosphate dehydrogenase and enzymes responsible for the utilization of maltose and galactose. Vectors and promoters suitable for expression in yeast are also described in EP 73657. Along with yeast promoters, yeast enhancers are also predominantly used.

Transcription of antibodies from vectors in mammalian host cells is regulated, for example, by promoters derived from genomes of viruses such as polyoma virus, poultry smallpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and most preferably monkey virus 40 (SV40); heterologous mammalian promoters, for example an actin promoter or an immunoglobulin promoter; and promoters of heat shock proteins, provided that such promoters are compatible with host cell systems.

The early and late promoters of the SV40 virus are usually obtained as an SV40 restriction fragment, which also contains the origin of the SV40 virus replication. The early early promoter of human cytomegalovirus is usually obtained in the form of a restriction HindIII fragment E. A DNA expression system in mammalian host cells derived from bovine papillomavirus used as a vector is described in US Pat. No. 4,419,446. A modification of such a system is described in US Pat. No. 4,601,978. Expression of human β-interferon cDNA in mouse cells under the control of the herpes simplex virus thymidine kinase promoter is also described in Reyers et al. (1982) Nature 297: 598-601. Alternatively, a Rous sarcoma virus having a long terminal repeat may be used as a promoter.

(v) Component enhancer element

Transcription of the DNA encoding the antibody of the invention in higher eukaryotes is often enhanced when an enhancer sequence is inserted into a vector. Currently, many enhancer sequences of mammalian genes are known (globin, elastase, albumin, α-fetoprotein and insulin genes). However, an enhancer from a eukaryotic cell virus is commonly used. Examples are the SV40 enhancer located in the late region of the replication start point (100-270 bp), the cytomegalovirus early promoter enhancer, the polyomavirus enhancer located in the late region of the replication start point, and adnovirus enhancers. See also Yaniv (1982) Nature 297: 17-18, which describes enhancer elements for activating eukaryotic promoters. An enhancer can be spliced into a vector at the 5'- or 3'-position with respect to the sequence encoding the antibody, but preferably it is located at the 5'-position from the promoter.

(vi) Component transcription termination site

Expression vectors used in eukaryotic host cells (in yeast, fungi, insects, plants, animals, humans, or in nucleus-containing cells of other multicellular organisms) also contain the sequences needed to terminate transcription and to stabilize mRNA. Such sequences are usually located at the 5'-end, and sometimes the 3'-end of the untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. One suitable component of transcription termination is the bovine growth hormone polyadenylation region. See the description of expression vectors in WO 94/11026 and in this application.

(vii) Selection and transformation of host cells

Host cells suitable for cloning or expression of DNA in the vectors described herein are prokaryotic, yeast, or higher eukaryotic cells described above. Prokaryotes suitable for this purpose are Eubacteria, such as Gram-negative or Gram-positive microorganisms such as Enterobacteriaceae, such as Escherichia, such as E.coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, for example Serratia marcescans and Shigella as well as Bacilli , such as B. subtilis and B.licheniformis (e.g. B.licheniformis 41P, described in patent DD 266710, published April 12, 1989), Pseudomonas , such as P.aeruginosa , and Streptomyces . The preferred cloning host of E. coli is E. coli 294 (ATCC 31446), although other strains such as E. coli B, E. coli X1776 (ATCC 31537) and E. coli W3110 (ATCC 27325) can be used. These examples are provided for illustration only and do not limit the scope of the invention.

In addition to prokaryotes, eukaryotic microorganisms such as hyphomycetes or yeast are suitable hosts for cloning or expression of vectors encoding the antibody. Of the lower eukaryotic host microorganisms, Saccharomyces cerevisiae or standard baker's yeast are most commonly used. However, in this case, other commonly used and available microorganisms of a different kind, of other species and strains, such as Schizosaccharomyces pombe ; Kluyveromyces hosts, such as, for example, K.lactis , K.fragilis (ATCC 12424), K.bulgaricus (ATCC 16045), K.wickeramii (ATCC 24178), K.waltii (ATCC 56500), K.drosophilarum (ATCC 36906 ), K.thermotolerans and K.marxianus; yarrowia (EP 402226); Pichia pastoris (EP 183070); Candida Trichoderma reesia (EP 244234); Neurospora crassa ; Schwanniomyces such as Schwanniomyces occidentalis ; and hyphomycetes, such as, for example, Neurospora , Penicillium, Tolypocladium and Aspergillus , such as A.nidulans and A.niger .

Host cells suitable for expression of a glycosylated antibody are cells of multicellular organisms. Examples of invertebrate cells are plant and insect cells. Were identified by various baculoviral strains and variants and corresponding permissive insect host cells such as Spodoptera frugiperda (caterpillar cutworm), Aedes aegypti (zheltolihoradochny mosquito), Aedes albopictus (belopyatnisty mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori ( silkworm). Currently, there are a number of viral strains available that can be used for transfection, for example, Autographa californica NPV variant L-1 and Bombyx mori NPV strain Bm-5, and such viruses can be used in the present invention, in particular for transfection of Spodoptera cells frugiperda . Cells of plants of cotton, corn, potato, soy, petunia, tomatoes and tobacco can also be used as host cells.

However, vertebrate cells are of the greatest interest, and the reproduction of vertebrate cells in culture (tissue culture) is already becoming a routine. Examples of commonly used mammalian host cell lines are monkey kidney cell line CV1 transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al. (1977) J. Gen. Virol . 36:59); hamster cub kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al. (1980) Proc. Natl. Acad. Sci. USA 77: 4216); Mouse Sertoli cells (TM4, Mather (1980) Biol. Reprod. 23: 243-251); monkey kidney cells (CVCC ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); Buffalo laboratory rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL 51); TR1 cells (Mather et al. (1982) Annals NY Acad. Sci . 383: 44-68); MRC 5 cells; FS4 cells; human hepatoma cell line (Hep G2).

Host cells are transformed with the above expression or cloning vectors to produce antibodies and cultured in suitable culture media, modified, if necessary, to induce promoters, select transformants, or amplify genes encoding the desired sequences.

(viii) Cultivation of host cells

The host cells used to produce the antibodies of the invention can be cultured in various media. Media suitable for culturing host cells are commercially available media such as Hams F10 (Sigma), Minimum Support Medium ((MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle Medium ((DMEM ), Sigma) In addition, any medium described in Ham et al. (1979) Meth. Enz. 58:44, Barnes et al. (1980) Anal. Can be used as a culture medium for culturing host cells . Biochem 102:.. 255, U.S. Pat №№ 4,767,704, 4,657,866, 4,927,762, 4,560,655 or 5,122,469, in WO 90/03430, WO 87/00195 or U.S. Patent Re № 30985. any of these media may be added, If necessary, hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride and calcium and magnesium phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine) antibiotics (such as the drug gentamicin ™), micronutrients (defined as inorganic compounds usually present in final concentrations in micromolar doses), and glucose or an equivalent energy source. Any other necessary additives at appropriate concentrations known to those skilled in the art may also be included. Culturing conditions, such as temperature, pH, and the like, are similar to those previously used to express selected host cells, and are known to one of ordinary skill in the art.

(ix) Purification of antibodies

Using recombinant DNA techniques, an antibody can be obtained inside cells or in the periplasmic space, or it can be directly secreted into the medium. If at the first stage the antibody is obtained inside the cells, then the cell debris, or host cells, or their lysed fragments, are removed, for example, by centrifugation or ultrafiltration. In a publication by Carter et al. (1992) Bio / Technology 10: 163-167 describes a technique for isolating antibodies that are secreted into the periplasmic space of E. coli . Briefly, the cell composition is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatants from such expression systems are typically concentrated first using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device. A protease inhibitor, such as PMSF, can be introduced at any of the above steps to inhibit proteolysis, and antibiotics can be introduced to prevent the growth of randomly introduced impurity microorganisms.

An antibody composition obtained from cells can be purified, for example, by hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, with affinity chromatography being the preferred purification method. Whether protein A is suitable as an affinity ligand depends on the type and isotype of the Fc region of any immunoglobulin present in the antibodies. Protein A can be used to purify antibodies containing heavy chains of the human immunoglobulin γ1, γ2 or γ4 (Lindmark et al. (1983) J. Immunol. Meth . 62: 1-13). For all isotypes of mice and for γ3 of human immunoglobulin, protein G is recommended (Guss et al. (1986) EMBO J. 5: 1567-1575). Agarose is most commonly used as the matrix to which the affinity ligand binds, but other matrices can be used. Mechanically stable matrices, such as glass with adjustable pore size or poly (styrene divinyl) benzene, provide a higher flow rate and shorter processing time than can be achieved using agarose. If the antibody contains a C _H 3 domain, then Bakerbond ABX ™ resin (JT Baker, Phillipsburg, NJ) can be used for purification. Other methods of protein purification, such as fractionation on an ion-exchange column, precipitation with ethanol, reverse phase HPLC, chromatography on silica, chromatography on heparin-sepharose ™, chromatography on anion or cation exchange resin ( for example, on a column with polyaspartic acid), chromatofocusing, SDS-PAGE and precipitation with ammonium sulfate.

After any pre-treatment step (s), the mixture containing the antibody of interest and impurities can be subjected to hydrophobic chromatography at low pH using an elution buffer at a pH of about 2.5-4.5, preferably at low salt concentrations ( for example, about 0-0.25 M).

Pharmaceutical Compositions

Therapeutic compositions containing the antibody are prepared in convenient storage forms by mixing the antibody of the desired purity with optional physiologically acceptable carriers, excipients or stabilizers ( Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) to produce lyophilized preparations or aqueous solutions. Acceptable carriers, excipients or stabilizers at the dosages and concentrations used should be non-toxic to the recipients, and these are buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-phenol;) low molecular weight polypeptides (having about less than 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counterions, such as sodium; metal complexes (e.g. Zn protein complexes); and / or nonionic surfactants such as Tween ™, Pluronic ™ or polyethylene glycol (PEG).

Presented in the present description, the composition may also contain several active compounds necessary for a particular indication, preferably having additional activity and not adversely affect each other. For example, additional administration of an immunosuppressant, antitumor agent, anti-angiogenesis agent, antineoplastic agent, cytotoxic agent and / or chemotherapeutic agent may be desirable. Such molecules may be present in combination in amounts effective to be used for the intended purpose.

The active ingredients can also be introduced into a microcapsule obtained, for example, by coacervation methods or by interfacial polymerization, for example, a hydroxymethyl cellulose or gelatin microcapsule and a polymethyl methacrylate microcapsule, respectively, into systems for the delivery of colloidal drugs (for example, liposomes, albumin microsulfides nanoparticles and nanocapsules) or in macroemulsions. Such methods are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Compositions used for in vivo administration must be sterile. This can be easily achieved by filtration through sterile filtering membranes.

Sustained release preparations may be prepared. Suitable examples of sustained release formulations are semipermeable matrices of solid hydrophobic polymers containing an antibody, wherein said matrices are in the form of finished products, such as films or microcapsules. Examples of sustained release matrices are polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or polyvinyl alcohol), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, an indecomposable ethylene-vinyl acetate copolymer, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT ™ (injection microspheres consisting of a copolymer of lactic acid-glycolic acid and leuprolide acetate) and poly-D - (-) - 3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate copolymer and lactic acid-glycolic acid copolymer are capable of releasing molecules for more than 100 days, and some hydrogels release proteins in shorter periods of time. If the encapsulated antibodies remain in the body for a long period of time, they can denature or aggregate under the influence of moisture at 37 ° C, which leads to a loss of biological activity and possible changes in immunogenicity. To stabilize, depending on the specific mechanism, rational strategies can be developed. So, for example, if it was discovered that the aggregation mechanism is the formation of an intermolecular bond SS through thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acid solutions, regulating the moisture content using appropriate additives and obtaining specific compositions based on a polymer matrix .

Therapeutic use

It is believed that the antibodies of the invention can be used to treat a mammal. In one embodiment of the invention, the antibody is administered to a non-human mammal in order to obtain, for example, preclinical data. Examples of non-human mammals that can receive treatment are non-human primates, dogs, cats, rodents, and other mammals involved in preclinical studies. Such mammals can be animals with an established disease model obtained by treatment with an antibody, or such animals can be used to study the toxicity of an antibody of interest. In each of these embodiments of the invention, dose-increasing studies can be performed on mammals. If the indicated antibody is an anti-NRP2 antibody, then it can be administered, for example, to a rodent host with a solid tumor model.

Additionally or alternatively, the antibody is used to treat a person, for example, a patient suffering from a disease or disorder whose symptoms can be reduced after administration of the indicated antibody.

The present invention includes the prevention and treatment of tumor lymphangiogenesis, the prevention and treatment of metastatic tumor and anti-angiogenic cancer therapy, a new cancer treatment strategy aimed at inhibiting the development of tumor blood vessels that require nutrients to support their growth. The present invention, in particular, includes the inhibition of neoplastic tumor growth in the area of its primary formation, as well as the prevention and / or treatment of tumor metastases in the field of secondary tumor formation and, therefore, exposure to tumors with other therapeutic agents. Examples of cancerous tumors described herein that are treated (including the prophylaxis of these tumors) include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such malignancies include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma), peritoneal cancer, hepatocellular cancer, intestinal or stomach cancer (including gastrointestinal cancer), cancer pancreas, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colon and colon cancer, endometrial carcinoma or attacks, salivary carcinoma, kidney cancer or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma and various types of head and neck cancer, and B-cell lymphoma (including low-grade / non-Hodgkin follicular lymphoma (NHL); small cell lymphocytic (SL) NHL; mid-stage differentiation NHL / follicular NHL; diffuse mid-stage differentiation NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small-cell undifferentiated NHL; generalized NHL; lymphoma of the cells of the cerebral cortex; AIDS-related lymphoma and Waldenstrom macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia and lymphoproliferative disorder after transplantation (PTLD), as well as pathological vascular proliferation associated with phacomatosis, edema (such as edema associated with brain tumors) and Meigs syndrome. More specifically, malignant tumors that can be treated with the antibodies of the invention are breast cancer, colorectal cancer, colon cancer, non-small cell lung cancer, non-Hodgkin lymphoma (NHL), renal cell cancer, prostate cancer, cancer liver, pancreatic cancer, soft tissue sarcoma, Kaposi’s sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma and multiple myeloma.

The present invention provides that the antibodies of the invention, when used to treat various diseases, can be combined with other therapeutic agents suitable for treating the same or similar diseases. The antibodies of the invention, when used to treat cancer, can be used in combination with standard cancer therapies such as surgery, radiation, chemotherapy, or combinations thereof.

In some aspects of the invention, other therapeutic agents used in the combination therapy of cancer with the antibody of the invention are other anti-angiogenesis agents. Many anti-angiogenesis agents have been identified and are well known in the art, including those listed in Carmeliet and Jain (2000).

In one aspect, an antibody of the invention is used in combination with a VEGF antagonist or a VEGF receptor antagonist, such as anti-VEGF antibodies, VEGF variants, fragments of soluble VEGF receptors, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, VEGFRNA inhibitors and any combination of them. Alternatively or additionally, two or more anti-NRP1 antibodies may be co-administered to a patient. In a more preferred embodiment, an anti-NRP1 ^A or NRP ^B antibody of the invention is used in combination with an anti-VEGF antibody to produce additive or synergistic effects. Preferred anti-VEGF antibodies are antibodies that bind to the same epitope that the anti-hVEGF A4.6 antibody binds to. A more preferred anti-VEGF antibody is bevacizumab or ranibizumab.

In some other aspects of the invention, other therapeutic agents used to combine the treatment of a tumor with the antibody of the invention are an antagonist of other factors that are involved in tumor growth, such as EGFR, ErbB2 (also known as Her2), ErbB3, ErbB4 or TNF. Preferably, the anti-NRP1 antibody of the invention can be used in combination with low molecular weight tyrosine kinase receptor inhibitors (RTKIs) that target one or more tyrosine kinase receptors, such as VEGF receptors, FGF receptors, EGF receptors, and PDGF receptors. Many therapeutic low molecular weight RTKIs are known to those skilled in the art, including, but not limited to, vatalanib (PTK787), erlotinib (Tarceva, TARCEVA®), OSI-7904, ZD6474 (zaktima, ZACTIMA®), ZD6126 (ANG453), ZDini39, s, SUTENT®), Semaxanib (SU5416), AMG706, AG013736, Imatinib (Gleevec, GLEEVEC®), MLN-518, CEP-701, PKC-412, Lapatinib (GSK572016), VELCADE® (Velcade), AZD2171, Sorafenib (Nexav NEXAVAR®), XL880 and CHIR-265.

An anti-Nrp antibody of the invention, taken alone or in combination with another therapeutic agent (such as an anti-VEGF antibody), can also be used in combination with one or more chemotherapeutic agents. Various chemotherapeutic agents can be used in the combination treatment methods of the invention. Examples and a non-limiting list of chemotherapeutic agents considered are given in the present description in the "Definitions" section.

When administering an anti-Nrp antibody together with a second therapeutic agent, a second therapeutic agent can be administered first, and then an anti-Nrp antibody. However, it is also contemplated that these agents are administered simultaneously or the anti-Nrp antibody is administered first. Suitable doses of the second therapeutic agent are the doses used in the present invention, and such doses can be reduced due to the combined (synergistic) effect of said agent and anti-Nrp antibody.

The appropriate dose of antibody used to prevent or treat a disease depends on the type of disease being treated, the severity and stage of development of the disease, regardless of whether the antibody is administered for prophylactic or therapeutic purposes, the type of treatment given before, the patient’s history and response the patient for this antibody, as well as from the appointment of the attending physician. The antibody can be administered to the patient once or after certain periods of time during treatment.

Depending on the type and severity of the disease, approximately 1 μg / kg-50 mg / kg (e.g. 0.1-20 mg / kg) of the antibody is a possible starting dose for administration to a patient, either by, for example, one or more separate administrations or by continuous infusion. A typical daily dose may be from about 1 μg / kg to 100 mg / kg or more, depending on the above factors. When re-administered for several days or longer, depending on the condition of the patient, treatment may be continued until the desired alleviation of the symptoms of the disease is achieved. However, other dosage regimens may be used. In a preferred aspect of the invention, the antibody of the invention is administered every two to three weeks at a dose of about 5 mg / kg to 15 mg / kg. More preferably, such a dosage regimen is used in combination with chemotherapy as first-line therapy for the treatment of metastatic colorectal cancer. In some aspects of the invention, chemotherapy includes the traditional periodic administration of high doses. In some other aspects of the invention, chemotherapeutic agents are administered in lower doses and more frequently, which allows for continuous therapy (“metronome chemotherapy”). An advantage of the therapy of the invention is that it can be easily monitored using standard techniques and analyzes.

The antibody composition is prepared, dosed and administered in accordance with standardized international requirements for the quality of the treatment process. Factors considered in this regard are the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the etiology of the particular disorder, the delivery site of the drug, route of administration, route of administration, and other factors known to practicing physicians. A “therapeutically effective amount” of antibody administered is administered based on an assessment of these factors and is the minimum amount necessary to prevent, ameliorate symptoms, or treat a disease or disorder. This antibody does not have to be obtained together with one or more agents currently used to prevent or treat the disorder in question. An effective amount of such other agents depends on the amount of antibody present in the composition, on the type of disorder or treatment, and other factors discussed above. Typically, such an amount is administered at the same doses and the same routes of administration as described above, or at doses ranging from about 1 to 99% of the above doses. Typically, improving or treating a disease or disorder includes alleviating one or more symptoms or pathologies associated with the disease or disorder. In the case of cancer, a therapeutically effective amount of the drug can be administered alone or in combination with the following therapy, aimed at: reducing the number of cancer cells; tumor size reduction; inhibition (i.e., reduction to some extent and / or cessation) of cancer cell infiltration into peripheral organs; inhibition of tumor metastases; inhibition of tumor growth to some extent; and / or weakening to some extent one or more symptoms associated with cancer. A drug, depending on the extent to which it is able to prevent the growth of cancer cells and / or destroy existing cancer cells, can have a cytostatic and / or cytotoxic effect. In some embodiments of the invention, the composition of the invention can be used to prevent the development or relapse of a disease or disorder in an individual or mammal.

The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention. The contents of all cited patents and scientific publications in their entirety are incorporated into this description by reference.

EXAMPLES

Example 1

Obtaining and characterization of antibodies against Nrp2 ^B

The anti-Nrp2 ^B antibody was isolated from synthesized phage libraries of human antibodies, as described previously (Lee et al., J Mol. Biol. 340, 1073-1093 (2004)). Briefly, synthesized antibody libraries for phage display were constructed on a single human framework region by artificially introducing diversity into solvent accessible positions of the heavy chain hypervariable regions (CDRs). To improve the properties of the library, libraries of monovalent and divalent antigen-binding fragments (Fab) were constructed, which were analyzed for a variety of different CDR-H3 by changing the amino acid composition and length of the CDR. Then this library was expanded by increasing the variability of the lengths of CDR-H3 and the use of specially designed codons that mimic the amino acid composition of the natural sequences of CDR-H3. Using such libraries with fully synthesized CDRs presented on the same framework, high affinity antibodies were prepared. A more detailed description of strategies and methods for producing synthesized single-matrix antibody libraries can be found, for example, in WO 03/102157, published December 11, 2003, the entire contents of which are incorporated herein by reference.

The selection procedures for anti-Nrp clones consist of various combinations of sorting known to those skilled in the art and carried out on the basis of binding on a solid support and in solution. When sorting on a solid support, the phage library of antibodies was panned using the target antigen applied to a 96-well NUNC Maxisorp immunoplate at a concentration of 5 μg / ml. In a solution-based sorting method, a phage library was incubated with a decreasing concentration of biotinylated antigen in the solution, which was then captured by neutravidin coated on a 96-well Maxisorp plate (2-5 μg / ml). Decreasing concentrations make it possible to create more stringent conditions when panning is carried out in order to search for more closely binding agents.

By combining solid-state and solution sorting methods, one V _H library clone (YW68.4) and another V _H V _L library clone (YW126.20) were identified as NRP-2 binding agents. A number of in vitro assays were performed to evaluate the properties and activities of the selected new anti-NRP antibodies, including affinity binding assays (such as BIAcore) and blocking assays (such as semaphorin-induced growth cone collapse assays and HUVEC assays).

CDRs of "untrained" clones were designed to increase their affinity and stability, and then antibodies against Nrp2 YW68.4.2 and YW68.4.2.36 were obtained. CHO cells expressing the mNrp2 (a1a2b1b2) -His, hNrp2 (a1a2b1b2) -Fc fusion protein, and insect cells expressing hNrp2 (b1b2) were used to screen and characterize the antibodies. The V _L and V _H regions of the anti-Nrp2 ^B phage antibody (originally designated YW68.4.2.36) were cloned into the mammalian expression vector, respectively. Human antibodies against Nrp2 ^B IgG1 or mIgG2a were expressed in mammalian CHO cells and purified on protein A affinity column.

Amino acid sequences of antibodies against Nrp2 ^B YW68.4.2 and YW68.4.2.36

shown in Figure 10. The amino acid sequence of the anti-Nrp2 ^B antibody YW126.20 is shown in Figure 11. The sequence alignment of the anti-Nrp2 ^A antibody YW126.20 variable domain with the human κ1 sequence is shown in Figure 12. The sequence of the anti-Nrp2 antibody heavy chain variable domain sequence ^A YW126.20 with a human III sequence (hum III) is shown in FIG. 13.

In the following experiments, an anti-Nrp2 ^B antibody YW68.4.2.36 was used.

which is further referred to as "anti-Nrp2 ^B ". This antibody targets the coagulation factor V / VII (b1-b2) Nrp2 domains (Figure 1A), as these domains are necessary for binding of VEGFC to neuropilins (Karpanen et al., Faseb. J. 20, 1462-1472 (2006)) . In addition, this antibody binds with similar affinity to murine and human Nrp2, but does not bind to Nrp1 (Figure 1B). It has been confirmed that anti-Nrp2 ^B binds exclusively to the b1-b2 domains and does not bind to the CUB (a1-a2) domains of human Nrp2, which are mainly responsible for binding to semaphorin (Chen et al., Neuron 21, 1283- 1290 (1998); Giger et al., Neuron 21, 1079-1092 (1998)).

To determine the binding affinities of antibodies against Nrp2 ^B IgG1, surface plasmon resonance (SRP) measurements were performed on BIAcore ™ -3000 equipment. To obtain approximately 200 response units (RU, EO), first of all, human anti-Nrp2 ^B IgG was immobilized on CM5 biosensor chips coated with rabbit anti-human IgG antibody. To measure kinetics, double serial dilutions of mouse or human Nrp2 (a1a2b1b2) (0.5 nM-250 nM) were separately injected into PBT buffer (PBS with 0.05% (v / v) Tween 20) at 25 ° C at flow rates 30 μl / min. Association rates (k _on ) and dissociation rates (k _off ) were calculated using a simple 1: 1 Langmuir binding model (BIAcore Evaluation Software version 3.2).

The equilibrium dissociation constant (K _D ) was calculated as the ratio k _off / k _on .

Anti-Nrp2 ^B binds to mouse Nrp with a K _{d of} 4.9 nM and human Nrp2 with a K _{d of} 5.3 nM, as estimated by surface plasmon resonance measurement.

The ability of anti-Nrp2 ^{B to} block the binding of VEGFC to Nrp2 was tested in ELISA format and in cell binding assays.

To conduct ELISA assays for binding specificity, three-fold serial dilutions of anti-Nrp2 ^B IgG (0.002 nM-500 nM) in PBST buffer (in PBT buffer with 0.5% (w / v) BSA) were incubated with 1 μg / ml antigen applied to 96-well Maxisorp plates for at least 1 hour, and then the plates were washed with PBT buffer. Bound antibodies were detected using an HRP (HRP) -conjugated anti-human antibody diluted 1: 2500 in PBST buffer, and then developed using a TMB substrate for approximately 5 minutes, quenched by the addition of 1M H ₃ PO ₄ and read on a spectrophotometer at 450 nm.

To evaluate the blocking of Nrp2 binding to VEGF, three-fold serial dilutions of anti-Nrp2 ^B IgG were first incubated on a Maxisorp 96-well plate sensitized with NRP2-Fc (5 μg / ml), in PBST buffer for 2 hours, and then biotinylated VEGF ₁₆₅ or VEGFC (full size) for 15 minutes. The level of binding of biotinylated VEGF to Nrp2 was determined using streptavidin-HRP (HRP) conjugates.

To assess the level of cell binding, biotinylated VEGF ₁₆₅ or VEGFC was coupled to LEC as previously described (Jia et al., J. Biol. Chem. 281, 13493-13502 (2006)), and then binding was determined using streptavidin-alkaline conjugates phosphatase. " Binding to Sema3F was carried out as described previously (Chen et al., 1998, see above).

Anti-Nrp2 ^B substantially blocked the binding of VEGFC to Nrp2 (Figure 1C) and to HEK-293 cells transfected with full-length Nrp2. Since Nrp2 can also bind to VEGF (Gluzman-Poltorak et al., J Biol Chem 275, 29922 (2000)), probably involving the same domains, the ability of anti-Nrp2 ^{B to} block VEGF ₁₆₅ binding to Nrp2 was also analyzed. . Anti-Nrp2 ^B also substantially blocks the binding of VEGF ₁₆₅ to Nrp2 (Figure 1D) with a similar IC ₅₀ (0.1 nM). However, anti-Nrp2 ^B did not have the ability to block the binding of Sema3F to LEC (Figure 1E), which largely express Nrp2 (Supplementary Figure 1). The data obtained are consistent with previous observations, which indicate that the a1-a2 domains are mainly responsible for binding to semaphorin, and the b1-b2 domains are responsible for binding to VEGF (Figure 1A).

Example 2

Antibody against Nrp2 ^B blocks selective VEGFC-mediated functions in vitro o

Materials and methods

Cell culture

HMVEC-dLyAd - endothelial cells of lymphatic microvessels (LEC) of human skin and HUVEC were purchased from Cambrex and cultured in EGM-2 medium (Cambrex). C6 LacZ cells were purchased from ATCC. 66C14 was kindly provided by Dr. Fred R. Miller. Tumor cells were cultured in DMEM (Gibco), to which 10% FBS was added. All cells were kept at 37 ° C in 5% CO ₂ in an incubator with 95% humidity.

Cell Proliferation Assay

A 96-well plate with a black transparent bottom (VWR) was sensitized with 5 μg / ml fibronectin (Invitrogen) at 37 ° C for 2 hours. LECs were collected and resuspended in assay medium (0.1% BSA, EGM-2) containing 3000 cells / 100 μl, and then added to the wells. Cells were incubated at 37 ° C for 16 hours. Then, a solution for BrdU labeling (ELISA assay kit for cell proliferation; Roche) was added and the cells were incubated for another 24 hours at 37 ° C. The incorporation of BrdU was determined using chemiluminescent immunoassay (6 wells per experiment).

results

The role of Nrp2 in VEGFC-mediated migration and proliferation was evaluated. The main cellular activities induced by VEGFC are known (Joukov et al., Embo J. 16, 3898-3911 (1997)). It has been previously shown that LECs are highly susceptible to VEGFC (Makinen et al., Embo J. 20, 4762-4773 (2001); Veikkola et al., Faseb 17, 2006-2013 (2003); Whitehurst et al. Lymphat Res Biol 4, 119-142 (2006)). Using a multi-well system, human LECs were introduced into the top of the chamber, and VEGFC was added to the bottom of the chamber to stimulate migration. Then, LECs that migrated to the bottom were fixed, stained (Figure 2A) and quantified (Figure 2B). The VEGFR3 extracellular domain protein (ECD) containing the first three (ligand binding) VEGFR3 Ig domains was used as a positive control to block VEGFC-mediated migration in this and subsequent experiments (Makinen et al., Nat Med 7, 199-205 (2001)). To determine whether Nrp2 is required for LEC migration, anti-Nrp2 ^B mAbs were added to cells located in the upper part of the chamber, immediately before the addition of VEGFC. Anti-Nrp2 ^B significantly reduced the level of VEGFC-mediated migration of LEC (Figure 2A, 2B; p = 0.004). The level of inhibition was less than the level of inhibition observed with VEGFR3 ECD, which completely inhibited VEGFC-mediated LEC migration (Figure 2A, 2B; p <0.001 compared to control; p = 0.002 compared to anti-Nrp2 ^B ). Similar results were obtained using other VEGFC-susceptible primary cell lines, HUVEC.

Since anti-Nrp2 ^B also blocked the binding of VEGF ₁₆₅ to Nrp2, the role of Nrp2 in modulating VEGF _165- mediated migration was evaluated. VEGF ₁₆₅ significantly induced LEC migration as previously described (Hirakawa et al., Am J Pathol 162, 575-586 (203); Hong et al., Faseb J 18, 1111-1113 (2004); Makinen et al. , Embo J. 20, 4762-4773 (2001); Veikkola et al., Faseb J 17, 2006-2013 (2003)). The cross-reactivity of the anti-VEGF antibody (B20.4.1) in many species was used as a positive control to block such VEGF-induced migration (Liang et al., J Biol Chem 281, 951-961 (2006)). It is interesting to note that anti-Nrp2 ^B did not have any effect on VEGF _165- mediated migration (Figure 2C), which may be due to the presence of Nrp1 (Supplementary Figure 1). Blocking the function of Nrp1 under the action of anti-Nrp1 mAb, anti-Nrp1 ^B (Pan et al., Cancer Cell 11, 53-67 (2007)) leads to a sharp decrease in the level of VEGF-mediated migration, which confirms this hypothesis (additional figure 2) . The addition of anti-Nrp1 ^B and anti-Nrp2 ^B did not lead to any additional inhibition of migration, in contrast to the inhibition observed in the presence of anti-Nrp1 ^B alone (additional figure 2), which indicates that Nrp2 does not play any role in VEGF _165- mediated migration.

Then, the effect of anti-Nrp2 ^B on VEGFC-induced LEC proliferation was analyzed. It is noteworthy that anti-Nrp2 ^B does not affect LEC proliferation, while VEGFR3 ECD significantly blocks LEC proliferation (additional figure 3), which is consistent with previously obtained data indicating that Nrp2 siRNA does not inhibit VEGFC-induced proliferation of endothelial cells (Favier et al., Blood 108, 1243-1250 (2006)). Thus, it is obvious that Nrp2 plays an important role in VEGFC-induced migration, but not proliferation.

The ability of anti-Nrp2 ^{B to} modulate semaphorin function was then tested. The authors of the present invention analyzed the collapse of the growth cone of hippocampal neurons, which previously showed that Nrp2 is necessary for Sema3F-mediated trimming of actin-rich structures (Pozas et al., 2001). The addition of anti-Nrp2 ^B did not have any effect on the semaphorein-induced collapse, and the addition of either the recombinant Nrp2 A1A2 domain or ECD Nrp2 completely inhibited this collapse (additional figure 4). The obtained result was consistent with the results obtained in the preliminary studies carried out by the authors, which showed that anti-Nrp2 ^B does not bind to the semaphorin-binding region and does not block the binding of Sema3F to Nrp2. Thus, anti-Nrp2 ^B blocks specific aspects of Nrp2 function by inhibiting VEGFC responses but not VEGF or Sema3F-mediated cell responses.

Example 3

Antibody against Nrp2 ^B blocks VEGFC-mediated lymphangiogenesis in vivo

Materials and methods

Analysis carried out in the micro pocket of the cornea of mice

Adult CD-1 mice (Charles-River) were anesthetized and a 2 × 3 mm pocket was created by microdissection at a distance of 1 mm from the center of the cornea in the epithelium (Polverini et al., Methods Enzymol 198, 440-450 (1991)). Agents tested for lymphangiogenic activity were immobilized on inert hydron granules (2 × 2 mm). Then these granules were implanted into the base of the pocket. The animals were injected intraperitoneally (i.r.) with a control antibody (10 mg / kg), anti-Nrp2 ^B (10 mg / kg) or VEGFR3 ECD (25 mg / kg) twice a week for 2 weeks. Then the animals were euthanized and corneas were dissected. Lymph nodes were visualized by complete histological analysis (IHC) on a slide using an anti-LYVE-1 antibody (R&D Systems 1: 500). Corneas were photographed and quantified by LYVE-1-positive limb lymphatic vessels.

Mouse skin permeability analysis

The backs and sides of adult female C57BL6J mice were shaved and divided into 4 zones in the area of the nipples. Then mice i.v. 150 μl of 0.5% Evans blue solution were injected. 1 hour after Evans blue injection, mice were injected percutaneously and randomly in any one of the four zones with 20 μl of PBS containing BSA or hVEGF (7.5 μg / ml) in the presence or absence of an antibody (0.5 mg / ml). After 1 hour, the animals were sacrificed, and then the skin was excised and photographed. Skin samples for each injection zone were cut and incubated in a formamide solution at 55 ° C for 48 hours to extract the blue dye. Then, the optical density of the solution was measured on a spectrometer at 600 nm.

results

Given the significant reduction in LEC migration by blocking Nrp2 in vitro , it was checked whether Nrp2 is required to modulate VEGFC function in vivo . The authors of the present invention investigated two well-characterized VEGFC-mediated in vivo activity against adult lymphangiogenesis and vascular permeability (Cao et al., Circ Res 94, 664-670 (2004); Joukov et al., J Biol Chem 273, 6599 -6602 (1998); Kubo et al., Proc Natl Acad Sci USA 99, 8868-8873 (2002); Saaristo et al., Faseb J 16, 1041-1049 (2002)). To study lymphangiogenesis, the analysis was performed in the micro pocket of the mouse cornea (Kubo et al., 2002, see above). In this assay, VEGFC granules were introduced into the non-vascular part of the cornea of an adult mouse. After 14 days, in response to VEGFC, proliferation of the dense plexus of the lymphatic vessels into the cornea from the limb was observed (Figure 2E; 12,000 pixels ² for VEGFC treatment compared to 2284 pixels ² for control). These vessels were labeled with LYVE-1 by the immunohistochemical method (IHC) and then quantified (Figure 2D). Systemic administration of ECD VEGFR3 almost completely blocked such VEGFC-induced lymphangiogenesis (2671 pixels ² ; p <0.001). Anti-Nrp2 ^B also effectively blocked the lymphangiogenic response in the cornea (3281 pixels ² ; p <0.001). This blocking was similar to the blocking observed using VEGFR3 ECD (Figure 2E, 2D; p = 0.67).

Numerous assays have been performed to evaluate vascular permeability (Brkovic and Sirois, J. Cell Biochem. 100, 727-37 (2007); Eriksson et al., Circulation 107, 532-1538 (2003)). This analysis was performed using transdermal injection of VEGFC to induce vascular permeability and intravascular injection of Evans blue dye, used as an indicator for detecting and quantifying skin vessel permeability (additional figure 3). It is noteworthy that treatment with an anti-Nrp2 ^B antibody did not affect VEGFC-induced permeability, in contrast to the blocking observed with VEGFR3 ECD treatment (p = 0.038). These results showed that, in accordance with the data observed in vitro , Nrp2 obviously plays an important role in selective VEGFC-mediated functions in vivo .

Example 4

Antibody against Nrp2 ^B  modulates VEGFC function by inhibiting the formation of the Nrp2 / VEGF receptor complex

Materials and methods

Cell Migration Analysis

Cell migration assays were performed using a modified Boyden chamber containing an integrated Falcon 24-well system with a pore size of 8 μM (BD Biosciences). The tablets were sensitized with 5 μg / ml fibronectin (Invitrogen) for 2 hours at 37 ° C. Cells were added to the upper chamber in 100 μl of assay medium (0.1% BSA, EGM-2) in the presence / absence of antibodies. A chemoattractant in 500 μl of assay medium was added to the lower chamber and the cells were incubated at 37 ° C for 16 hours. Cells on the upper membrane were removed with a sponge swab, and cells on the lower surface were fixed in 70% ethanol and stained with green Sytox stain (Molecular Probes). Then, an image of the entire lower surface of the well was obtained and the number of migrated cells was counted (6 wells per experimental condition).

FACS analysis

Confluent LECs were incubated with control antibodies against Nrp2 ^B (10 μg / ml) for 5 minutes, 2 hours or 20 hours at 37 ° C. Cells were collected using an enzyme-free cell dissociation buffer (Gibco) and incubated with biotinylated antibody at a ratio of 1: 100 in FACS buffer (PBS, 2% FBS, 2 mM EDTA, 0.1% sodium azide) containing 5% normal mouse serum, 2% normal rat serum, and 10% 10 μg / ml human IgG. Antibodies were biotinylated using a FluoReporter mini-kit containing biotin-xx protein for labeling (Molecular Probes). The cells were then washed with FACS buffer and stained with streptavidin-PE (PE) (BD Biosciences). The data obtained were analyzed using the FacsCalibur system (BD Biosciences).

Cell adhesion assay

Subconfluent LECs were preincubated in 100 μl of 199 medium containing control or anti-Nrp2 ^B antibodies (10 μg / ml) for 30 minutes at 37 ° C, and then they were plated on 96-well flat bottom NUNC Maxisorp plates (eBioscience) coated with 1 μg / ml fibronectin (Roche) at a density of 10,000 cells per well. The plates were centrifuged for 1 minute at 140g to synchronize cell contact with the substrate and incubated at 37 ° C for 30 minutes. Then the tablets were washed 3 times with PBS and frozen at -80 ° C. Cell density was determined using the CyQuant kit (Molecular Probes).

VEGF receptor signaling assays

HUVEC confluent cells were stimulated for 10 minutes with 200 ng / ml VEGFC in the presence or absence of a control antibody or an anti-Nrp2 ^B antibody. Cells were lysed and assayed for a variety of mediators, which are known to play a role in VEGF receptor signaling. VEGFR3 activation was evaluated by ELISA assays for total VEGFR2 and phospho-VEGFR2 (ELISA kit, DuoSet IC ELISA kit, R&D). VEGFR3 activation was evaluated using a kinase receptor activation assay (KIRA) using the VEGFR3-293 cell line described in the literature (Sadick et al., J Pharm. Biomed Anal 19, 883-891 (1999)). Briefly, stable 293 cell lines expressing full-length Flag-tagged human hVEGFR3 were analyzed for phosphorylation of the receptor after stimulation. 5 × 10 ⁴ cells were cultured on minimal medium overnight (DMEM with 0.1% BSA) and then stimulated with 40 ng / ml VEGFA (Genentech South San Francisco, CA) or 200 ng / ml VEGFC (Genentech South San Francisco, CA) within 8 minutes. Cells were lysed in PBS containing 1% triton and sodium orthovanadate. ELISA plates were coated with immobilized Flag antibody (Sigma St Louis, MO). The plates were then sensitized (PBS + 1 μg / ml antibody) overnight and blocked (PBS + 0.5% BSA) for 1 hour. After 3 washes (PBS + 0.05% Tween 20), lysates were added for 2 hours, washed three times, and then 4G10 antibody was added to detect phosphorylation (Upstate Lake Placed, NY) for 2 hours. Detection was performed using HRP (HRP) conjugated antibody (Amersham Piscataway, NJ) and TMB substrate. Plates were read at 450 nm. Then evaluated for total AKT, phospho-AKT, total Erk1 / 2, phospho-Erk1 / 2, total Src, phospho-Src, total p38 MAPK and phospho-p38 MAPK using sandwich ELISA kits from BioSource.

results

The fact that anti-Nrp2 ^B suppresses VEGFC function is consistent with evidence that this antibody blocks the binding of VEGFC to Nrp2. In addition, the fact that this antibody blocks only selective functions both in vitro and in vivo was extremely unexpected. One possible explanation for why impaired LEC migration and lymphangiogenesis, rather than LEC proliferation or vascular permeability, is that anti-Nrp2 ^B can substantially inhibit migration, possibly due to impaired LEC adhesion

to the extracellular matrix. To verify this fact, the effect of anti-Nrp2 ^B on LEC migration induced by various motogens was evaluated. Anti-Nrp2 ^B had no effect on migration induced by VEGF (Figure 2C), HGF (Figure 3B), or FGF. Therefore, anti-Nrp2 ^B did not substantially interfere with LEC migration. In addition, anti-Nrp2 ^B did not have any effect on the adhesion of LEC to fibronectin or collagen, that is, to two extracellular matrix substrates.

A second possible explanation is that anti-Nrp2 ^B mAb can cause internalization of Nrp2 (Jaramillo et al., Exp. Cell Res. 312, 2778-2790 (2006)). Since Nrp2 forms a complex with VEGFR3 even in the absence of a ligand (Favier et al., 2006, see above; Karkkainen and Alitalo, Semin Cell Dev Biol 13, 9-18 (2002)), this leads to selective co-internalization of VEGFR3, which affects on specific VEGFC-mediated functions. This possibility is also supported by evidence indicating that VEGFC-induced vascular permeability is mediated by VEGFR2 rather than VEGFR3 (Joukov et al., 1998, supra). To test this possibility, LECs were incubated with anti-Nrp2 ^B at 37 ° C for 5 minutes, 2 hours or 20 hours, and then FACS analysis was performed using antibodies against Nrp2, VEGFR2 and VEGFR3 to determine the level of receptors on the cell surface. There was no difference between the results of these treatments, suggesting that anti-Nrp2 ^B did not cause significant internalization of Nrp2, VEGFR2 or VEGFR3 (Figure 3A).

Since it has been suggested that Nrp2 enhances signal transmission by the VEGF receptor (Favier et al., 2006, see above), the effect of anti-Nrp2 ^B on the activation of VEGFR2 and VEGFR3 has been investigated, where VEGFC stimulation leads to dimerization and autophosphorylation of the receptor. According to previously obtained in vitro and in vivo data, it can be argued that VEGFR3 ECD completely blocks the VEGFC-mediated phosphorylation of VEGFR2 (Figure 3C; p <0.001) and VEGFR3. Antibody treatment with Nrp2 ^B reduced activation of VEGFR2 (Figure 3C; p <0.001) and VEGFR3, but to a much lesser extent than the treatment of VEGFR3 ECD (p <0.001). This observation increases the likelihood that the selective inhibitory activity of anti-Nrp2 ^B may be the result of various VEGF receptor activation conditions necessary for migration and proliferation, with higher levels of receptor activation required for migration than for proliferation. To verify this assumption, a dose-response relationship was evaluated for phosphorylation of VEGFR2 associated with VEGFC stimulation (Figure 3C). It is absolutely obvious that the decrease in VEGFR2 phosphorylation caused by treatment with an anti-Nrp2 ^B antibody was equivalent to VEGFR2 phosphorylation achieved by stimulation of 175 ng / ml or 150 ng / ml VEGFC. Then, a dose-response analysis was performed for migration associated with VEGFC stimulation (Figure 3D). It was noted that LEC stimulated with 175 ng / ml or 150 ng / ml VEGFC migrated to the same extent as cells stimulated with 200 ng / ml VEGFC. Indeed, a significant decrease in the level of migration was not observed until VEGFC levels were reduced to 50 ng / ml. In this regard, the authors of the present invention argue that the decrease in the level of phosphorylation of VEGFR2 induced by anti-Nrp2 ^B was in itself insufficient to reduce the level of migration.

The authors of the present invention conducted an additional assessment of the effect of anti-Nrp2 ^B on subsequent events of signal transmission mediated by VEGF receptors. Antibody treatment with Nrp2 ^B or stimulation of 175 ng / ml or 150 ng / ml VEGFC did not significantly reduce the level of Erk1 / 2, Akt, or p38 MAPK activation, which modulates VEGFR2-mediated proliferation, permeability, and motility, respectively, i.e., reduces similar to that observed for Nrp1 (Pan et al., 2007). This indicates that Nrp2 can regulate LEC migration and lymphangiogenesis by a mechanism different from the mechanism for enhancing activation of the VEGF receptor or subsequent signal transmission by this receptor.

Finally, we have tested the effect of anti-Nrp2 ^B on the formation of the Nrp2 / VEGF receptor complex. As previously reported, Nrp2 can be co-immunoprecipitated with VEGFR2 and VEGFR3 in the presence or absence of VEGFC (Favier et al., 2006, see above; Karpanen et al., 2006, see above) (Figure 3E). This interaction was sharply attenuated by anti-Nrp2 ^B (Figure 3E). The obtained result suggests that the Nrp2 / VEGF receptor complex plays an important role in the implementation of specific VEGFC-mediated functions. In addition, the role of Nrp2 is not only solely in enhancing signal transmission by the VEGF receptor in response to ligand stimulation.

Example 5

Nrp2 is expressed in the lymph vessels associated with the tumor

Materials and methods

Immunohistochemical analysis

Tissue sections 18 μm thick were cut and placed on glass slides. These sections were incubated overnight with the “first” antibody (anti-NRP2 ^B ) (1: 500, control staining was performed in the mouse spinal cord E12.5, where expression was well characterized), with anti-LYVE-1 antibody (anti-R & D , 1: 200), anti-PECAM-1 antibody (Benton Dickinson, 1: 500), anti-PROX-1 antibody (Chemicon, 1: 1000) or Ki67 (Neovision, 1: 100) at 4 ° C. Then the samples were stained with "second" antibodies Alexa 488 or Alexa 568 (1: 200; Molecular Probes) for 4 hours at room temperature. Staining with the “second” antibody only was used as a control. TUNNEL staining was performed using a commercially available kit (Roche). Images were captured on a Zeiss Axiophot fluorescence microscope. The areas of blood and lymph vessels were determined from 6 representative images for each of the 6 tumors per group, which were estimated by the average number of pixels using ImageJ.

results

To determine whether Nrp2 plays any role in the biology of lymphatic vessels in adult animals, the expression of Nrp2 in lymphatic vessels in adult animals was evaluated. For the vascular system, Nrp2 expression in veins and lymphatic vessels has been described previously (Herzog et al., Mech Dev 109, 115-119 (2001); Moyon et al., Development 128, 3359-3370 (2001); Yuan et al., Development 129, 4797-4806 (2002)). As described above, LECs in culture express Nrp2 at a high level (additional figure 1). However, the authors of the present invention failed to detect Nrp2 using IHC in the LYVE-I-positive lymphatic vessels of the colon of healthy adult mice (Figure 4A; staining of the positive control, see additional figure 4). The colon was evaluated because it is rich in plexuses of the lymphatic vessels with a fairly stereotyped picture. The authors of the present invention also failed to detect the expression of Nrp2 using IHC in the lymphatic vessels of the lymph nodes (Figure 4B) and in the skin of healthy adult mice. These results were confirmed by in situ hybridization of Nrp2 (ISH). In contrast, a high level of Nrp2 expression was observed in LYVE-1-positive lymphatic vessels of the lymph nodes located next to orthotopically or heterotopically transplanted tumors (Figure 4C). This level was observed for a number of tumor lines, including the orthotopically transplanted mouse mammary adenocarcinoma line, 66cl4 (Aslakson and Miller, Cancer Res 52, 1399-1405 (1992)), and the heterotopically transplanted rat glioblastoma line, C6 (data not shown). Nrp2 was also observed in the near-tumor and intratumoral lymphatic vessels (Figure 4D) for a number of tumor lines, including 66c14, C6 and PC3 (human prostate carcinoma line). This expression was confirmed by ISH analysis performed to subpopulate tumors of various types.

Example 6

Antibody against Nrp2 ^B  reduces lung metastases in many tumor models

Materials and methods

All animal studies were carried out in accordance with the guidelines for the care and use of laboratory animals published by the National Institute of Health (NIH Publication 85-23, revised 1985). All animal research protocols have been approved by the Institutional Committee for Animal Care and Use (IACUC).

Tumor Models

In the case of 66C14 cells, these cells were harvested by trypsinization, washed and resuspended in PBS at a concentration of 2 × 10 ⁵ cells in 10 μl of PBS. Mice were anesthetized using 75 mg / kg ketamine and 7.5 mg / kg xylazine and an incision was made under the right forelimb. Cells at a concentration of 2 × 10 ⁵ cells in 10 μl of PBS were injected directly into the accessible 4th mammary adipose body of 6-8-week-old female balb-C mice. In the case of using C6 cells, 2 × 10 ⁶ tumor cells in 100 μl of PBS were injected subcutaneously in the right flank of 6-8-week-old female balb-C nude mice. At both stages of the study, tumor growth was monitored 3 times a week. When the tumor reached an average size of 80-120 mm ³ , the mice were divided according to the average size of the tumor into almost identical groups and treatment was started. In each study, such treatment was considered as day 1. Animals were treated with a control antibody (10 mg / kg), anti-Nrp2 ^B (10 mg / kg) or VEGFR3 ECD (25 mg / kg) i.r. twice a week until studies are completed. To ensure reproducibility, all studies were repeated 3 times.

At the end of the studies, the animals were anesthetized and then they were injected with 4% PFA. Tumors were collected, kept under cryoprotection conditions and frozen in OCT (Tissue-Tek). The lungs were inflated by right ventricular perfusion with 10 ml PBS,

and then 4% PFA, and metastatic lesions were visualized before micro-CT analysis.

SLN metastases were evaluated by heterotopic implantation of C6 tumor cells in the ear. Briefly, a group of animals was pretreated with a control antibody (10 mg / kg) or anti-Nrp2 ^B (10 mg / kg) one day before tumor cell implantation and twice a week after such implantation. 1 × 10 ⁵ C6 lacZ cells were subcutaneously injected into the ear of 70 female balb / c nude mice. Mice were euthanized on days 3, 6, 9, 13, and 15, and then the “sentinel” lymph nodes were identified by subcutaneous lymphangiography and excised. The lymph nodes were homogenized and the lysate was analyzed for β-galactosidase activity (Pierce).

Intradermal lymphangiography was performed on control and tumor mice as follows. Evans Blue dye (2 μl, 3% wt.), Containing 1% 20 nm polystyrene fluorescent microbeads (Molecular Probes), was injected transdermally into the top of the tumor. The animals were left for 2 hours to recover, and then killed. Tumors were carefully excised so that they did not include perituminal tissue. Tumors were either fixed and then subjected to histochemical analysis or incubated in formamide for the extraction of Evans blue and quantified by measuring the optical density of OD ₆₀₀ on a spectrophotometer.

Micro-KT analysis of the lungs

The lungs were immersed in 10% NBF for 24 hours and then immersed for 24 hours in a 20% solution of iodine-containing contrast agent for X-ray computed tomography, Isovue370 (Bracco Diagnostics Inc, Princeton, NJ), diluted in PBS. The lungs were then immersed and perfused through a tracheotomy tube with 20 ml of soybean oil (Sigma-Aldrich, St. Louis, MO) at a rate of 0.25 ml / min. Soybean oil was used to remove excess contrast media and provide a background medium for images.

The lungs of mice were visualized ex vivo using a VivaCT X-ray microcomputer tomography (micro CT) system (SCANCO Medical, Basserdorf, Switzerland). A sagittal overview image comparable to a standard flat X-ray image was obtained in order to determine the start and end points for axial compilation of a series of micro-CT images of sections. The position and number of axial images were selected taking into account the full coverage of the lungs. The lungs were immersed in soybean oil, used as a background medium. Micro-CT images were created using an X-ray tube operating in the following mode: energy level of 45 kV, current of 160 μA, and integration time of 450 milliseconds. Axial images were obtained at an isotropic resolution of 21 μm. Evaluation of lung tumors (number and volume) was carried out using a semi-automated image analysis algorithm, which includes the stage of examination using a specially designed reader. Tumors of the lungs looked like a hyperintensive solid mass in contrast to the porous sieve-like structure of normal lungs. This is due to the absorption of iodine-containing contrast agent by solid structures (walls of the bronchi and alveoli, tumors, trachea and mediastinum) present in the lungs. Excess contrast medium was removed from the air-filled space at the oil perfusion stage. Potential tumor mass was extracted by a series of image processing steps. An image analysis program was developed by the laboratory. This program was written in the C ++ programming language, and it used the functions of the library functions of the program for image analysis Analyze (AnalyzeDirect Inc., Lenexa, KS, USA). The following parameters were used in this algorithm: intensity threshold, morphological filtration, and formation of tumor regions for extraction of all potential tumor masses. The intensity threshold (1480 Hounsfield units) was determined by analyzing histograms of 5 randomly selected lungs used to develop the algorithm, and the optimal threshold was chosen so that it included elements of the volumetric image of the tumor and excluded any background signal. Morphological parameters (erosion, dilatation) and the formation of tumor regions were applicable for joining volumetric image elements of hyperintensive areas and for removing any volumetric image elements with the same density observed in thin walls of bronchioles and alveoli. The stage of formation of tumor regions requires a minimum volume of 2300 related elements of the volumetric image (more than 0.0231 mm ³ ), taken as an object (mass). The identified objects were then evaluated on a specially designed reader using the Analyze 3D visualization software. Individual objects were taken or discarded as possible tumors by their appearance and localization in the lungs. Objects were discarded if they were outside the lungs (for example, in the mediastinum, in the debris of other tissues) or looked like blood vessels. For each lung, the number of tumors, the volume of an individual tumor, and the total volume of tumors were counted. Such an analytical method was confirmed by a well-developed model of tumor metastases. Eleven animals with orthotopic transplantation of tumor cells of mammary adenocarcinoma 4T1 were examined for lung metastases using this micro-CT method followed by serial histological analysis of the lungs. The volume of lung tumors was highly correlated (r = 0.9, p = 0.0002) with a histological assessment of tumor size (pixel count; additional figure 5).

results

One of the main methods for inhibiting metastases is the inhibition of all types of VEGFC (Chen et al., Cancer Res 65, 9004-9011 (2005); He et al., J Natl Cancer Inst 94, 819-825 (2002); Krishnan et al. , Cancer Res 63, 713-722 (2003); Lin et al., Cancer Res 65, 6901-6909 (2005)). To determine whether blocking Nrp2 function can also modulate the development of metastases, we evaluated the effect of anti-Nrp2 ^B antibody treatment on the formation of lung metastases in two different tumor models - model 66c14, model of breast cancer, and model of glioblastoma C6. 66cl4 is a mouse mammary adenocarcinoma lineage derived from a spontaneously developing breast tumor in Balb / c mice (Aslakson and Miller, Cancer Res 52, 1399-1405 (1992)). These cells express VEGFC and form metastases in the lungs under the influence of the lymphatic system (Aslakson and Miller, 1992, supra). Orthotopic transplantation of these cells in Balb / c mice resulted in reproducible development of tumors and metastases in the lungs. Antibody treatment with Nrp2 ^B did not affect the growth rate of the primary tumor (Figure 5A). Since VEGFR3 ECD did not cause a sharp decrease in the growth rate of the primary tumor, it was excluded from any subsequent metastasis analysis. Groups of animals (N = 6 in each group) with similar tumor sizes in both treatment groups were simultaneously euthanized, and then the lungs were dissected and inflated to facilitate analysis of metastatic nodes. Anti-Nrp2 ^B caused a sharp decrease in the average number of visually detectable metastatic nodes per lung compared to the number of metastatic nodes in animals treated with control IgG (Figure 5B, C), averaging 3.5-0.8 (P = 0, 03). To confirm this result and use these methods to evaluate metastases in the lung parenchyma, which was not available for visual assessment, the authors of the present invention conducted micro-CT analysis (Li et al., Technol Cancer Res Treat 5, 147-155 (2006 )) lung after autopsy. This analysis confirmed that in animals treated with anti-Nrp2 ^B , compared with control animals, there was a decrease in the number of lung metastases (Figure 5D-F). However, micro-CT analysis was more sensitive than visual analysis, and revealed a greater absolute number of metastatic nodes in both groups. Micro-CT analysis allowed the inventors to determine the total metastatic load in the lungs. Antibody treatment with Nrp2 ^B also led to a reduction in the total metastasis volume (0.74 cm ³ ) compared with the control treatment (1.78 cm ³ ). In addition, this analysis confirmed that the vast majority of lesions were present on the surface of the lungs in both control animals and animals treated with anti-Nrp2 ^B antibody. Therefore, anti-Nrp2 ^B did not cause a decrease in the number of metastases due to the movement of nodes from the surface to the lung parenchyma.

FACS analysis carried out as described in example 4 showed that Nrp2, but not VEGFR2 or VEGFR3, was expressed on 66cl4 tumor cells. This increases the likelihood that treatment with an anti-Nrp2 ^B antibody has a direct effect on the behavior of tumor cells, leading to the formation of metastases. This fact is unlikely, given the absence of the effect of such treatment with an anti-Nrp2 ^B antibody on the growth of a primary tumor. In addition, anti-Nrp2 ^B did not have any effect on the proliferation, apoptosis or migration of tumor cells in vitro o. However, given the likelihood that the decrease in the number of metastases is due to the effect of anti-Nrp2 ^B on tumor cells, the authors of the present invention evaluated the effect of anti-Nrp2 ^B on the rat C6 glioblastoma model. These cells did not express Nrp2 to their surface to any significant degree (Figure 6E), but expressed VEGFC and probably formed lung metastases through the lymphatic system (Bernstein and Woodard, 1995). In addition, these cells were obtained so that they express β-galactosidase, which can be used as a marker that facilitates the detection of tumor cells.

Subcutaneous transplantation of these cells to “naked” mice led to the continuous development of tumors and metastases in the lungs. Antibody treatment with Nrp2 ^B did not affect the growth rate of these primary tumors (Figure 6A). In addition, VEGFR3 ECD did not produce a sharp decrease in the growth rate of the primary tumor in this tumor model. This allows comparisons of the antimetastatic effects of VEGFR3 ECD and anti-Nrp2. And in this case, a group of animals (N = 10) with similar tumor sizes in all treatment groups were euthanized, lungs were dissected and inflated to facilitate analysis of metastatic nodes. Antibody treatment with Nrp2 ^B and VEGFR3 ECD caused a decrease in the average number of visually detectable metastatic nodes per lung (Figure 6B, C). The decrease observed using anti-Nrp2 ^B was comparable to the decrease observed using VEGFR3 ECD. Micro-CT analysis of the lungs confirmed these data (Figure 6D), and also confirmed that the vast majority of metastases are localized on the surface of the lungs in all treatment groups. As shown by histological analysis of both tumor models, the nodes were metastatic lesions (Figures 5H and 6G). In addition, general autopsy did not reveal any nodes on the surface of other organs in any model with a tumor.

Although the above description of the present invention is illustrated with reference to specific options for its implementation, however, the present invention is not limited to these options. Indeed, various modifications of the present invention, in addition to those indicated and described in this application, will be obvious to a person skilled in the art based on the above description and are included in the scope of the attached claims.

All publications cited in the present description, and the works cited in these publications, accurately and in their entirety are incorporated into this description by reference.

Claims (21)

1. An anti-Nrp2B antibody or antigen binding fragment thereof which comprises sequences of complementarity determining regions (CDRs) of the light and / or heavy chain variable domains of the YW68.4.2 antibody or YW68.4.2.36 antibody or a variant of said antibody or said antigen binding fragment, where the amino acid the Fab sequence of the YW68.4.2 antibody fragment is shown in Figure 10A and the amino acid sequence of the Fab fragment of the YW68.4.2.36 antibody is shown in Figure 10B. 2. The anti-Nrp2B antibody or antigen binding fragment thereof according to claim 1, which comprises CDR sequences of the light and heavy chain variable domains of the YW68.4.2 antibody or YW68.4.2.36 antibody or a variant of said antibody or said antigen binding fragment. 3. The anti-Nrp2B antibody or its antigen binding fragment according to claim 1, which contains the sequence of the variable regions of the light and / or heavy chain of the antibody YW68.4.2 or antibody YW68.4.2.36 or a variant of the indicated antibody or the specified antigen binding fragment having one or more changes in frame regions where the amino acid sequence of the Fab fragment of the YW68.4.2 antibody is shown in Figure 10A and the amino acid sequence Fab of the YW68.4.2.36 antibody fragment is shown in Figure 10B. 4. The anti-Nrp2B antibody or antigen binding fragment of claim 3, which comprises sequences of the light and heavy chain variable regions of the YW68.4.2 antibody or YW68.4.2.36 antibody or a variant of said antibody or said antigen binding fragment having one or more changes in frame areas where the amino acid sequence of the Fab fragment of the YW68.4.2 antibody is shown in Figure 10A and the amino acid sequence of the Fab fragment of the YW68.4.2.36 antibody is shown in Figure 10B. 5. The anti-Nrp2B antibody according to claim 1, which contains YW68.4.2 or YW68.4.2.36 or an antigen-binding fragment of the antibody YW68.4.2 or YW68.4.2.36. 6. The anti-Nrp2B antibody of claim 1, which comprises YW68.4.2.36, or an antigen binding fragment thereof. 7. The anti-Nrp2B antibody of claim 1, which is YW68.4.2.36 or an antigen binding fragment thereof. 8. An anti-Nrp2B antibody, or an antigen binding fragment thereof, or a variant according to any one of claims 1 to 7, which is bispecific. 9. The anti-Nrp2B antibody, or antigen binding fragment, or the variant of claim 8, which further binds to VEGF or to the VEGF receptor. 10. Antibody against Nrp2B, or antigen binding fragment, or a variant according to any one of claims 1 to 7, where the specified fragment is selected from the group consisting of fragments Fab, F (ab ') ₂ and scFv. 11. Antibody against Nrp2B, or antigen-binding fragment, or a variant according to any one of claims 1 to 7, where the specified variant is an affinity-matured variant. 12. The anti-Nrp2B antibody according to any one of claims 1 to 7, wherein said antibody, antigen binding fragment, or variant is chimeric or humanized. 13. A composition comprising an antibody, antigen binding fragment, or a variant according to any one of claims 1 to 7 in a mixture with a carrier. 14. A pharmaceutical composition for the prophylaxis or treatment of tumor metastases, containing an effective amount of an anti-Nrp2B antibody, antigen binding fragment or variant according to any one of claims 1 to 7 in a mixture with a pharmaceutically acceptable carrier. 15. A method of inhibiting the migration of lymphatic endothelial cells, comprising administering to a mammal in need an effective amount of an anti-Nrp2B antibody, an antigen binding fragment, or a variant according to any one of claims 1 to 7. 16. The method of claim 15, wherein said mammal is a human. 17. The method according to clause 16, where the specified person is diagnosed with cancer. 18. The method according to 17, where the specified person is diagnosed with tumor metastases or there is a risk of tumor metastases. 19. The method according to p, where the specified metastasis is in the lymphatic system or in a distant organ. 20. The method according to 17, which further includes the introduction of antibodies against VEGF. 21. The method of claim 21, wherein said anti-VEGF antibody is bevacizumab.

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