WO2016008498A1

WO2016008498A1 - Mfap4 binding antibody blocking the interaction between mfap4 and integrin receptors

Info

Publication number: WO2016008498A1
Application number: PCT/DK2015/050218
Authority: WO
Inventors: Grith Lykke SØRENSEN; Anders Schlosser; Uffe Holmskov
Original assignee: Syddansk Universitet
Priority date: 2014-07-17
Filing date: 2015-07-10
Publication date: 2016-01-21

Abstract

There is provided a novel antibody to prevent or to inhibit the proliferation and migration of vascular smooth muscles, vascular hyperplasia and remodeling, and pathological neovascularization of the eye. Furthermore, there is provided a novel antibody that effectively inhibits bronchial hyperplasia and inflammation in asthmatic allergy.

Description

MFAP4 BINDING ANTIBODY BLOCKING THE INTERACTION BETWEEN MFAP4 AND INTEGRIN RECEPTORS

FIELD OF THE INVENTION

The invention relates generally to medicine and the use of antibodies. The present invention specifically relates to a novel antibody, in particular monoclonal, that binds human Microfibrillar-associated protein 4 (MFAP4) and thereby inhibits MFAP4 interaction with integrin receptors.

BACKGROUND OF THE INVENTION

MFAP4 is a 36 kDa glycoprotein composed of a short N-terminal region that contains a potential integrin binding RGD motif followed by a fibrinogen related domain (FReD). The protein forms a homo-oligomeric structure under native conditions (1-

3). FReDs are found in a diverse group of human proteins involved in different functions such as coagulation, angiogenesis, tissue growth and remodeling, and innate immunity (4). MAGP-36/MFAP4 was first identified as a protein with tenascin resemblance in the amino acid composition and localized to ECM in arteries (2, 5-8). MAGP-36 was following demonstrated with direct interaction with ECM fibers including elastin, collagen, or calvasculin (2, 5-7, 9). The interaction between MAGP-36 and cellular integrin receptors was demonstrated using inhibition by RGD containing peptides of human aortic smooth muscle cells in attachment to immobilized MAGP-36 (6). All

RGD dependent integrins may potentially interact with this RGD site, however integrins α β3¾ are highly relevant for investigation of vascular remodeling. Integrins ανβ3/5, are known to induce VSMC responses both in vivo and in vitro (10, 11) and may be upregulated during restenosis (12-18). The integrin

is expressed in the media in normal arteries (19), yet highly upregulated very early after injury (20, ZHAO et al (1) discloses human microfibril-associated protein 4 (MFAP4). Zhao et al further discloses that the N-terminus of the protein bears an Arg-Gly-Asp (RGD) sequence that serves as the ligand motif for the cell surface receptor integrin. VASSILEV T.L. et al. (22) discloses antibodies against ligands to integrins, where the antibodies are against the RGD sequence, resulting in lack of ligand activation of the integrins.

KOKUBO T. et al. (11) discloses that the blockade of the integrin α_νβ3 by antagonists being either a blocking antibody to α_νβ3 or a a_vp₃-blocking RGD peptide reduced neointima by 70%. KOKUBO T. et al mentions vitronectin, fibronectin, osteopontin, fibrinogen and von Willebrand factor as ligands to α_νβ3.

Lorger et al. (61 ) dicloses that the activation status of the integrin α_νβ3 regulates angiogenese and cell growth of tumor metastasis in the brain. Thus, targeting and blockage of integrins like α_νβ3 provides the potential to detrimentally impact several important aspects of tumor biology, such as intracellular signaling, migration, angiogenese and the host tumor response (62). Meanwhile, these prior art documents do not identify a specific receptor for MFAP4 or antibodies directed to MFAP4 thereby inhibiting the known functions of the integrin receptor. Based on the prior art it could not be predicted whether MFAP4 would activate integrin or whether MFAP4-blocking antibodies would serve as agonists or an antagonists in MFAP4 integrin ligation or inhibition of neovascularization.

SUMMARY OF THE INVENTION

The subject of the present invention is to provide a medicament, in particular an antibody, for the prevention (the terms "prevention" or "prophylaxis" as used herein include the delaying of the onset of a disease or condition) and/or treatment of cardiovascular proliferative diseases. More specifically, it is the subject of the present invention to provide a medicament to prevent or to inhibit the proliferation or migration of endothelial cells or vascular smooth muscles implicated in vascular hyperplasia, remodeling or neovascularization. An additional subject of the present invention is to provide a medicament (preferably antibody) to prevent or to inhibit inflammatory infiltration and airway remodeling in allergic asthma.

Thus, the antibody of the present invention is used to block the interaction between MFAP4 and integrin receptors in order to treat, curatively or preventively, cardiovascular proliferative diseases.

In one aspect, the present invention provides an antibody, fragments hereof, or derivatives hereof, which specifically blocks the integrin interaction with human microfibrillar-associated protein 4 (MFAP4), wherein the antibody is characterized by:

• a light chain variable region comprising the amino acid sequence of SEQ ID NO 1 or sequences having at least 90% sequence identity, preferably at least 92%, 93%, 94% or at least 95% sequence identity, more preferably at least 96% or 97% sequence identity, and most preferably at least 98% or at least 99% sequence identity; and

• a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 2 or sequences having at least 90% sequence identity, preferably at least 92%, 93%, 94% or at least 95% sequence identity, more preferably at least 96% or 97% sequence identity, and most preferably at least 98% or at least 99% sequence identity.

In a preferred embodiment, the present invention provides the antibody, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO 1 or sequences having at least 95% sequence identity and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 2 or sequences having at least 95% sequence identity.

In another aspect of the present invention a pharmaceutical composition is provided.

The composition of the present invention may be a pharmaceutical composition, which comprises one or more physiologically acceptable carriers, excipients and/or diluents. The composition may, in addition, one or more stabilizing agents and/or one or more buffering agents.

In a further aspect, the antibody or the composition of the present invention are provided for use in the prevention or treatment of vascular proliferative diseases and/or related disorders in a mammal, preferably caused by hyperplasia or remodeling in blood vessels. The mammal is preferably a human.

In one embodiment, the antibody or composition of the present invention are used in the prevention or treatment of bronchiolar hyperplasia and eosinophilic inflammation in allergic asthma.

In another aspect, the antibody or composition of the present invention are used in the prevention or treatment of disorders characterized by pathological neovascularization in the eye, preferably, wherein the disorders characterized by pathological neovascularization in the eye is selected from the group consisting of age related macular degeneration (AMD), retinopathy, hhypertensive retinopathy and diabetic retinopathy (DR). Moreover, the present invention also provides for methods of prevention or treatment of vascular proliferative diseases and/or related disorders in a mammal .

Thus in a further aspect, the present invention provides a method of prevention or treatment of vascular proliferative diseases and/or related disorders in a mammal, said method comprising administering the antibody or the composition of the present invention to a mammal in need thereof.

DESCRIPTION OF THE FIGURES Figure 1 shows Immunohistochemical detection of MFAP4 in sections from a human vein with smooth muscle cell hyperplasia. MFAP4 and a-SMA were visualized by immunostaining, and elastin was visualized using Weigert's elastin stain. Upper panel, original magnification 25X. Lower panel, original magnification 1000X. Figure 2 shows identification of integrin receptors for MFAP4. (A) Silverstained elution profile for human placenta membrane proteins purified using an rMFAP4- coupled matrix. The double bands around 100 kDa labelled with and asterix represent potentially eluted integrins. (B) Immunodetection of integrins eluted in fractions 6-9 from (A) by Western Blotting using anti-integrin αν, βι , β3, or β₅ specific antibodies. (C) FACS analysis of fHAoSMCs using FITC-labelled anti-integrin αν, βι , β3, ββ or isotype control (iso) specific antibodies. (D) Adhesion assay assessing attachment of flourescently labelled fHAoSMCs onto various concentrations of immobilized BSA, laminin, rMFAP4, or fibronectin, respectively. (E) Adhesion assay assessing attachment of fHAoSMCs onto one fixed concentration of immobilized rMFAP4 in competition with integrin inhibitory RGD-containing peptide or DRG- containing control peptide, respectively. (F) Adhesion assay assessing attachment of fHAoSMCs onto one fixed concentration of immobilized rMFAP4 in competition with integrin-blocking antibodies specifically directed against integrins βι , α_ν, α β3, or α β_δ, respectively. Data points are means+SEM from experimental triplicates and representative for at least two independent experiments.

Figure 3 shows MFAP4 interaction with smooth muscle cells is inhibited by MFAP4 blocking antibodies and induced VSMC migration is inhibited by MFAP4 blocking anti-bodies in vitro. (A) fHAoSMCs were seeded in poly-D-lysine (PDL, negative control), or rMFAP4 coated micro-well plates with a central rounded area blocked by a cell stopper. The number of flourescently labelled cells was counted within this central area of the well (depicted) twenty hrs after removal of the cell stoppers and ± the anti-MFAP4 antibodies HG-HYB 7-5 (7-5), HG-HYB 7-14 (7-14), HG-HYB 7- 18 (7-18), or isotype control antibodies (iso) in order to quantitate cellular migration. (B) Cell counts from the migrations assay were performed using no coating (blank),

PDL-, rMFAP4-, or fibronection- coating and (C) using rMFAP4 coating in the presence of antibodies. The data are representative for 3 individual experiments, with observations made in quadruplicates. The data (B and C) are means + SEM obtained from two of these experiments. (D-E) The colourimetric MTT-assay was used to quantitate fHAoSMC proliferation. (D) fHAoSMCs were seeded in tissue culture wells with no coating (blank), rMFAP4-coating, or fibronectin-coating and + (black) or - (grey) PDGF-BB treatment 48 hrs after seeding. (E) fHAoSMCs were seeded in tissue culture wells with rMFAP4-coating ± PDGF-BB treatment after preincubation with anti-MFAP4 antibodies. Data are means+SEM measured in triplicates and representative for at least two different experiments.

Figure 4 shows MFAP4 accelerates neointima formation and outward remodeling of the arterial wall. (A) The left common carotid artery was ligated at the bifurcation in both mfap4+/+ (+/+) and mfap4-/- (-/-) mice and the right common carotid artery was used as unligated control. At day 14 or day 28 after ligation the vessels were dissected, fixed and obtained sections were elastin stained. The shown sections are obtained 1 .5 mm distal to the bifurcation/ligation. (B) Morphometric analyses of cross sectional vessel areas were performed in unligated control carotid arteries and in the ligated carotid arteries 14 (n = 6 mice/group) or 28 (n = 3-6 mice/group) days after ligation. The ratios between the neointimal areas and the medial areas are depicted in the bottom panel. EEL = External elastic lamina. Black = mfap4-/- mice, white = mfap4+/+ mice. Data are means + SEM.

Figure 5 MFAP4 is increased in BAL and serum of allergic mice. (A) Pulmonary localization of MFAP4 in OVA- and HDM-treated mice. (B-G) MFAP4 levels are changed in asthma. MFAP4 concentrations were measured after OVA (B, D, F) and HDM (C, E, G) exposure in BAL (B, C), serum (D, E) and lung homogenate (F, G). Scale bar, 50 μηι. n=5-14. ^*p < 0.05, ^**p < 0.01 , ^***p < 0.001 , ^****p < 0.0001 .

Figure 6 MFAP4 deficiency attenuates eosinophilic inflammation. Total cell and eosinophil numbers in BAL were lowered in MFAP4 KO mice after OVA (A) or HDM

(B) exposure. (C-D) Representative pictures of H&E-stained lungs from OVA-treated

(C) and HDM-treated (D) mice. Parenchymal inflammation was dampened in OVA- treated (E) but not HDM-treated (F) MFAP4-deficient animals. Eosinophil counts in BAL correlated positively with MFAP4 concentration in serum of WT animals from OVA (G) and HDM (H) models. Scale bar, 100 μπτι. n=6-8 (A, E), 10-19 (B, F). ^*p <

0.05, ^**p < 0.01 , ^***p < 0.001 , ns = not significant.

Figure 7 MFAP4 influences production of eotaxins. Levels of eotaxin-1 (CCL-1 1 ) and eotaxin-2 (CCL-24) were measured in BAL (A-B) and lung homogenate (C-D) of OVA-treated mice. Lung levels of both chemokines correlated positively with MFAP4 levels in serum of WT mice (E-F). n=10-15 (A-B), 5-9 (C-D). ^*p < 0.05, ^**p < 0.01 , ns = not significant.

Figure 8 MFAP4 promotes ASM remodeling. Representative pictures. Scale bar, 50 μΠΊ.

Figure 9 MFAP4 deficiency reduces airway hyper responsiveness (AHR). Airway reactivity to increased doses of methacholine (MCh), measured as increase in resistance (A), central airway resistance (B) and tissue damping (C), was lowered in treated MFAP4 KO mice. ^*p < 0.05, ^**p < 0.01 , ^****p < 0.0001 , ns = not significant. Figure 10 MFAP4 is upregulated in asthmatic BSMCs. (A) MFAP4 mRNA expression is increased in asthmatic cells regardless of seeding on MFAP4. (B-C) Western blot analysis reveals increased MFAP4 protein levels in asthmatic BSMCs. Data are means + SEM (A, C) or representative (B) of 3 independent experiments. ^*p < 0.05, ^**p < 0.01 .

Figure 1 1 MFAP4 promotes BSMC attachment through integrin νβ5. MFAP4 increases BSMC adhesion in a dose-dependent manner (A). MFAP4-dependent adhesion can be inhibited by RGD blocking peptide (B) but not DGR control peptide (C). BSMC adhesion to MFAP4 is dependent on integrin ανβ5 (D). Western blot analysis shows FAK phosphorylation after seeding cells on MFAP4 (E). Data are means + SEM (A-D) or representative (E) of at least 3 independent experiments. ^*p < 0.05, ^**p < 0.01.

Figure 12 MFAP4 promotes BSMC proliferation. MFAP4 enhances BSMC proliferation (A), which can be inhibited by anti-MFAP4 antibodies (B). Data are means + SEM of at least 3 independent experiments. ^*p < 0.05, ^**p < 0.01 .

Figure 13 MFAP4 blocking antibody HG-HYB 7-1 has similar epitope recognition as HG-HYB 7-14. Combinations of labeled and unlabeled antibodies were added to MFAP4 coated microwells using serial dilution of the unlabeled antibody as illustrated on the abscissa. The combinations are described in the legend with the biotinylated antibody and the unlabeled antibody was diluted in a 2-fold serie.

Figure 14 Antibody validation was conducted to ensure reactivity and specificity of the aMFAP4 (HG-HYB 7-1 ) antibody. Immunofluorescent staining of kidney tissue with 1 μg ml aMFAP4 (green) and DAPI (blue) (A), and negative control staining with ^g/ml IgG (green) and DAPI (blue) (B) Scale bars: 25μηι Immunohistochemical staining of MFAP4 in kidney tissue (C) (Wulf-Johansson et al., 2013 PLoS One)

Scale bar: 100μπι

Figure 15 Fundus fluorescein angiography at day 7 shows a decrease in lesion size and lesion density in eyes treated with aMFAP4 compared to IgG and aVEGF-A treated eyes. Representative images of lesions from eyes treated with IgG, aVEGF-A and aMFAP4 at 1 μg and 5μg (A). Treatment with 5μg aMFAP4 reduced average lesion size (B) and average integrated density of lesion (C) compared to IgG and aVEGF- A treated eyes.

Scale bars: 100μηι, n=lesions (mice)

Figure 16 Fundus fluorescein angiography at day 14 shows a decrease in lesion size and lesion density in eyes treated with aMFAP4 or aVEGF-A compared to IgG treated eyes.

Representative images of lesions from eyes treated with IgG, aVEGF-A and aMFAP4 at 1 μg and 5μg (A). aMFAP4 at 1 μg and 5μg is able to reduce both average lesion size (B) and average integrated density of lesion (C) compared to IgG controls.

Scale bars: 100μηι, n=lesions(mice)

Figure 17 IB4 staining of flatmounted choroids shows a decreased average density of burn in 5 g aMFAP4 treated eyes, but no significant difference in burn area.

Representative images of lesions from eyes treated with IgG, aVEGF-A and aMFAP4 at ^ μg and 5 g (A). Treatment with aVEGF-A, or aMFAP4 at either concentration produces no significant difference in average burn area (B). A significant decrease is seen in average integrated density of lesions from 5 g aMFAP4 treated eyes compared to IgG control (C).

Scale bars: 100μηι, n=lesions(mice)

Figure 18 CD45 staining of macrophages shows a decrease in macrophage infiltration with aMFAP4 or aVEGF-A treatment.

Representative images of lesions from eyes treated with IgG, aVEGF-A and aMFAP4 at 1 g and 5 g (A). Treatment with aVEGF-A or 5μg aMFAP4 results in a significant decrease in macrophage infiltration compared to IgG control (B).

Scale bars: δθμπι, n=lesions(mice)

Figure 19 Asthmatic BSMC proliferation. Asthmatic primary bronchial smooth muscle cell (BSMC) proliferation as measured by absorption (OD590) were measured against treatment with varying HG-HYB 7-1 concentrations (0-20 μςΛπΙ).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

The antibodies and antigen binding domains of the invention bind selectively to MFAP4 that is they bind preferentially to MFAP4 with a greater binding affinity than to other antigens. The antibodies may bind selectively to human MFAP4, but also bind detectably to non-human MFAP4, such as murine MFAP4. Alternatively, the antibodies may bind exclusively to human MFAP4, with no detectable binding to non-human MFAP4.

The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, wherein each monoclonal antibody will typically recognize a single epitope on the antigen. The term "monoclonal" is not limited to any particular method for making the antibody. For example, monoclonal antibodies of the invention may be made by the hybridoma method as described in Kohler et al. Nature 256, 495 (1975) or may be isolated from phage libraries using the techniques as described herein, for example.

The term "antigen binding domain" or "antigen binding region" or "fragment or derivative thereof" refers to that portion of the selective binding agent (such as an antibody molecule) which contains the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen.

Preferably, the antigen binding region will be of human origin. In other embodiments, the antigen binding region can be derived from other animal species, in particular domestic animal and rodents such as rabbit, rat or hamster.

The terms "effective amount" and "therapeutically effective amount" when used in relation to an antibody or antigen binding domain, fragment or derivative thereof, immunoreactive with a MFAP4 peptide, refer to an amount of a selective binding agent that is useful or necessary to support an observable change in the level of one or more biological activities of MFAP4, wherein said change may be either an increase or decrease in the level of MFAP4 activity.

In the context of the present invention, the term "sequence identity" or "homologue" indicates a quantitative measure of the degree of homology between two amino acid sequences or between two nucleic acid sequences. If the two sequences to be compared are not of equal length, they must be aligned to give the best possible fit, allowing the insertion of gaps or, alternatively, truncation at the ends of the polypeptide sequences or nucleotide sequences. The sequence identity can be

(N,_ef-Ndif)l00

calculated as ^NrEf , wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Ndif=2 and Nref =8). A gap is counted as non-identity of the specific residue(s), i.e. the DNA sequence AGTGTC will have a sequence identity of 75% with the DNA sequence AGTCAGTC (Ndif=2 and Nref=8).

With respect to all embodiments of the invention relating to amino acid sequences or nucleotide sequences, the percentage of sequence identity between one or more sequences may also be based on alignments using the clustalW software

(http:/www.ebi. ac.uk/clustalW/index. html) with default settings. For nucleotide sequence alignments these settings are: Alignment=3Dfull, Gap Open 10.00, Gap Ext. 0.20, Gap separation Dist. 4, DNA weight matrix: identity (IUB). For amino acid sequence alignments the settings are as follows: Alignment=3Dfull, Gap Open 1 0.00, Gap Ext. 0.20, Gap separation Dist. 4, Protein weight matrix: Gonnet.

Alternatively, nucleotide sequences may be analysed using programme DNASIS Max and the comparison of the sequences may be done at http :/^'/www.paral ig ri.org/. This service is based on the two comparison algorithms called Smith -Waterman (SW) and ParAlign. The first algorithm was published by Smith and Waterman (1981 ) and is a well-established method that finds the optimal local alignment of two sequences. The other algorithm, ParAlign, is a heuristic method for sequence alignment; details on the method are published in Rognes (2001 ). Default settings for score matrix and Gap penalties as well as E-values were used. Aspects and embodiments of the invention

Thus, the antibody of the present invention is used to block the interaction between MFAP4 and integrin receptors in order to treat, curatively or preventively, cardiovascular proliferative diseases, such as vein graft intimal hyperplasia, restenosis after endovascular interventions, cardiac transplant arteriopathy, pulmonary hypertension and additional obstructive diseases atherosclerosis and restenosis after percutaneus coronary intervention operations, or disorders characterized by pathological vessel growth such as age related macular degeneration or diabetic retinopathy.

In another aspect of the present invention the antibody is used to block the interaction between MFAP4 and integrin receptors in order to treat, curatively or preventively, allergic asthma.

Specifically the present invention provides an antibody, which specifically blocks MFAP4 interaction with integrins. Preferably, the antibody is selected from isolated polyclonal antiserum, or a preparation of purified polyclonal antibodies. The antibody of the present invention has the following amino-acid sequence in the light chain variable region or homologues thereof:

SEQ ID NO 1 (HG-HYB 7-1 )

MESQTQVLMFLLLWVSGACADIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSN NQKNYLAWYQQKSGQSPKLLIYWASTRESGVPDRFVGSGSGTDFTLTISSVKAE DLAVYYCQQYYTSTWTFGGGTKLEIKRAKRADAAPTVSIFPPSSEQLTSGGASVV CFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYE RHNSYTCEATHKTSTSPIVKSFNRNEC

The antibody of the present invention has the following amino-acid sequence in the heavy chain variable region or homologues thereof:

SEQ ID NO 2 (HG-HYB 7-1 )

MGWSYIILFFVATATGVHSQVQLQQPGADLVKPGTSVKLSCKASGFTFTSYWMH WVKQRPGQGLEWIGVIHPNSGNTKYNEKFRSEATLTVDKSSNTAYIQLSSLTSED SAVYYCAREMWNYGNSWYFDVWGTGTTVTVSSAKTTPPSVYPLAPGSAAQTNS MVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTW PSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITL TPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMH QDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSL TCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEA GNTFTCSVLHEGLHNHHTEKSLSHSPGK

Accordingly and more specifically the present invention provides an antibody, preferably monoclonal, fragments hereof, or derivatives hereof, which specifically blocks the interaction between MFAP4 and integrins.

Antibodies and antigen binding domains, and fragments, variants and derivatives thereof, of the invention will retain the ability to bind selectively to MFAP4, preferably to human MFAP4.

In one aspect, the present invention provides an antibody, fragments hereof, or derivatives hereof, which specifically blocks the integrin interaction with human microfibrillar-associated protein 4 (MFAP4), wherein the antibody is characterized by: · a light chain variable region comprising the amino acid sequence of SEQ

ID NO 1 or sequences having at least 90% sequence identity, preferably at least 92%, 93%, 94% or at least 95% sequence identity, more preferably at least 96% or 97% sequence identity, and most preferably at least 98% or at least 99% sequence identity; and

· a heavy chain variable region comprising the amino acid sequence of

SEQ ID NO 2 or sequences having at least 90% sequence identity, preferably at least 92%, 93%, 94% or at least 95% sequence identity, more preferably at least 96% or 97% sequence identity, and most preferably at least 98% or at least 99% sequence identity. In a preferred embodiment, the present invention provides the antibody, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO 1 or sequences having at least 95% sequence identity and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 2 or sequences having at least 95% sequence identity.

In another embodiment, the antibody of the present invention is monoclonal. In a further embodiment, the antibody of the present invention may be coupled to a detectable label or a substance having toxic or therapeutic activity.

In another aspect of the present invention a pharmaceutical composition is provided. The composition of the present invention may be a pharmaceutical composition, which comprises one or more physiologically acceptable carriers, excipients and/or diluents. The composition may, in addition, one or more stabilizing agents and/or one or more buffering agents.

Moreover, in one embodiment, compositions of the present invention may comprise at least one stabilizing agent, such as a surfactant, in particular a surfactant selected from polysorbate and polyoxypropylene-polyethylene esters (Pluronic^®). The surfactant may also be selected from polysorbate 20 and polysorbate 80.

In one embodiment, the antibody or composition for use according to the present invention, in the prevention or treatment of vascular proliferative diseases and/or related disorders caused by pathological neovascularization. In another embodiment, the antibody or composition of the present invention are usedin the prevention or treatment of bronchiolar hyperplasia and eosinophilic inflammation in allergic asthma. In another aspect, the antibody or composition of the present invention are used in the prevention or treatment of disorders characterized by pathological neovascularization in the eye, preferably, wherein the disorders characterized by pathological neovascularization in the eye is selected from the group consisting of age related macular degeneration (AMD), retinopathy, hypertensive retinopathy and diabetic retinopathy (DR).

In a further aspect of the present invention, the antibody or composition of the present invention are used in the prevention or treatment of cancer and other malignancies. In one embodiment, the cancer or malignancy is selected from glioblastoma, head, neck and lung cancer.

Moreover, the present invention also provides for methods of prevention or treatment of vascular proliferative diseases and/or related disorders in a mammal . Thus in a further aspect, the present invention provides a method of prevention or treatment of vascular proliferative diseases and/or related disorders in a mammal, said method comprising administering the antibody or the composition of the present invention to a mammal in need thereof. The mammal may preferably be a human. The method according to the present invention comprises administration intravenously or subcutaneously of the compounds or compositions of the present invention.

In one embodiment of the present invention the method the vascular proliferative diseases and/or related disorders are caused by pathological neovascularization .

In another embodiment of the present invention, the method comprises prevention or treatment of bronchiolar hyperplasia and eosinophilic inflammation in allergic asthma. In another embodiment of the present invention, the method comprises prevention or treatment of disorders characterized by pathological neovascularization in the eye. The said disorders characterized by pathological neovascularization in the eye may be selected from the group consisting of age related macular degeneration (AMD), retinopathy, hhypertensive retinopathy and diabetic retinopathy (DR).

The antibody or composition of the present invention may preferably be administered intravenously, ocularly or subcutaneously. MFAP4 crosslinks VSMCs to ECM fibrils and induces cellular migration and proliferation through integrin ανβ^β_δ ligation

In order to elucidate the presence of MFAP4 in human vascular tissue with pathological remodeling processes sections from a human vein with intimal hyperplasia were obtained from a patient with lower extremity PAD that underwent surgical reconstitution following bypass surgery induced restenosis. The neointima appeared with inhomogeneous staining for MFAP4. The most intense MFAP4 staining was localized closest to the outer periphery of the vessel. A similar staining pattern was found for both a-smooth muscle actin (a-SMA) and elastin. MFAP4 staining appeared to colocalize with the elastic fibers, whereas the a-SMA staining was intracellular (Figure 1 ). Immunostaning detected dispersed integrin α_νβ3 staining throughout the neointimal area with highest intensity in capillary endothelium. Few intervening CD45-positive inflammatory cells were also observed.

To identify relevant MFAP4 binding integrins, human placenta membrane proteins were affinity purified on immobilized rMFAP4. Proteins, which might correspond to integrins according to molecular weight, were eluted from the column and integrin monomers α_ν, βι , β3, and β₅ were detected specifically in the collected fractions (Figure 2A and B). Similar results were obtained using fluorescence-activated cell sorting (FACS) analysis of fHAoSMC (Figure 2C). A cellular adhesion assay following demonstrated that calcein AM f luorogenic dye labeled f HAoSMCs attached to rMFAP4, fibronectin, and laminin and failed to adhere to bovine serum albumin (BSA) (Figure 2D). The synthetic peptide GRGDSP completely inhibited cellular attachment of to rMFAP4, whereas the control peptide S DGRG showed no significant inhibition (Figure 2E). Anti-integrin a and anti-integrin ανβ3 antibodies completely blocked the cellular adhesion to rMFAP4, while anti-integrin ανβδ antibodies showed a small but significant reduction in adhesion. In contrast, anti- integrin βι had no effect on the cellular adhesion to rMFAP4 (Figure 2F). The integrins were detectable through all tested cell culture conditions. Yet, integrin av and integrin β₅ were coordinately expressed with MFAP4 while integrin β₃ expression was diminished when fHAoSMCs differentiated from the proliferating to the contractile phenotype.

Monoclonal anti-MFAP4 antibodies block VSMC interaction with MFAP4

Monoclonal anti-MFAP4 antibodies were raised in mfap4-/- mice because the mouse MFAP4 homologue has very high sequence similarity to certain regions within the human protein. ELISA based assays demonstrated that produced antibodies with reactivity against MFAP4; anti-MFAP4 HG-Hyb 7-14 and 7-18 antibodies clearly bind double (AGA) and triple (AAA) RGD mutated rMFAP4 in the Chinese hamster ovary (CHO) cell culture supernatant. In contrast, the rMFAP4 detection signals were reduced for the point-mutated proteins when the HG-Hyb 7-5 was used as capture antibody suggesting that HG-Hyb 7-5 binds to an epitope covering the RGD sequence in rMFAP4. HG-HYB 7-5 and HG-HYB 7-14 both prohibited the cellular adhesion to rMFAP4. This latter observation suggests that HG-HYB 7-14 may bind at close proximity to the RGD site. It was further observed that focal adhesions and cellular stress fibers (vinculin and F-actin, respectively) formed within 20 hrs of exposure to either f ibronectin or rMFAP4, but not with poly-D-lysin. Inhibition of focal adhesion and stress fiber formation was observed when fHAoSMCs were incubated with the blocking antibodies. Cellular migration and proliferation of VSMCs are induced by MFAP4 and inhibited by MFAP4 blocking antibodies

The fHAoSMC migration was increased almost 2-fold towards immobilized rMFAP4 (Figure 3A and B). Incubation with HG-HYB 7-5 or 7-14 antibodies reduced the numbers of migrating cells significantly while no effect was seen when using the non-blocking HG-HYB 7-18 antibody or the isotype control (Figure 3A and C).

The effect of rMFAP4 on proliferating fHAoSMC was further assessed using a Thiazoyl blue tetra-zolium bromide (MTT)-assay. The cells were allowed to proliferate for 48 hrs, either in the presence or absence of 5 ng/mL platelet-derived growth factor-BB (PDGF-BB) and the proliferation was significantly induced when cells were seeded onto either rMFAP4 or fibronectin (Figure 3D). Microplates coated with rMFAP4 were following blocked with anti-MFAP4 antibodies before seeded with fHAoSMC. HG-HYB 7-5 and 7-14 both lead to a significant reduction of cellular proliferation to a non-PDGF-BB treated level (Figure 3E) in parallel with integrin ανββ blocking antibodies (data not shown).

Decreased vessel diameter and neointima formation in mfap4-/- mice after carotid artery ligation is associated with reduced VSMC proliferation and infiltration with CD45 positive cells

A ligated carotid artery will undergo initial outward remodeling, followed by vessel shrinkage and neointima formation, resulting in narrowing of the lumen (23). The remodeling responses 14 days and 28 days were compared after left carotid arterial ligation in mfap4+/+ and mfap4-/- littermates of the C57BL/6N strain in order to examine whether the lack of MFAP4 affected the arterial response to ligation. Transverse sections were obtained 0.5, 1.0, and 1 .5 mm proximal to the ligature/bifurcation and at corresponding locations in the contralateral vessel and stained with Verhoeff-van Gieson elastin staining (Figure 4A). Neointimal growth appeared delayed in the mfap4-/- mice, with very limited or no formation after 14 days, but with neointimal areas comparable to mfap4+/+ after 28 days (Figure 4B). Furthermore, the external elastic lamina (EEL) of the ligated mfap4-/- vessel was significantly decreased compared to the mfap4+/+ vessel (Figure 4B). Thus, at day 28 the lumen in mfap4-/- vessels was significantly decreased. No apparent differences in the vessel diameter or in the lumen diameter were observed in the contralateral control arteries.

Circulating MFAP4 levels are increased by allergic asthma

We investigated the role of MFAP4 in allergic airway disease in two distinct models of asthma. Initially, we used OVA-induced sensitization and challenge model. Then, to focus particularly on airway remodeling, we performed prolonged, chronic HDM exposure model. To determine whether MFAP4 levels are influenced by asthma development, we measured lung MFAP4 expression as well as soluble MFAP4 concentration in relevant body fluids. Histological staining revealed MFAP4 localization within the basement membrane, in close proximity to airway epithelial cells and smooth muscle cells (Fig. 5A). MFAP4 mRNA expression did not change after OVA or HDM treatment (data not shown). However, upon allergen challenge soluble MFAP4 concentrations significantly increased in serum of WT mice (Fig. 5B- C). When we investigated local MFAP4 production, we found that MFAP4 levels were raised in BAL of HDM-treated WT mice (Fig. 5D-E), with a corresponding decrease of MFAP4 content in lung homogenate (Fig. 5F-G).

MFAP4 deficiency attenuates allergy-induced airway eosinophilia and production of eotaxins

We then investigated if MFAP4 deficiency affected allergic airway inflammation. In both models, MFAP4 KO mice showed significantly decreased total cell and eosinophil numbers in BAL compared to WT littermates (Fig. 6A-B). The same pattern was observed in the lung of OVA-treated mice, where WT animals exhibited more severe parenchymal infiltration than their MFAP4-deficient counterparts (Fig. 6C, E). However, we detected only a slight trend towards reduced inflammation in the parenchyma in HDM-challenged MFAP4 KO mice (Fig. 6D, F). Moreover, we found a significant positive correlation between BAL eosinophil number and MFAP4 serum concentration in WT animals (Fig. 6G-H).

Due to differences in eosinophil count, we investigated potential mediators responsible for eosinophil attraction. We concentrated on two canonical chemokines involved in eosinophil recruitment, CCL1 1 (eotaxin-1 ) and CCL24 (eotaxin-2). We found that OVA-treated MFAP4 KO mice had lowered amounts of both CCL1 1 and

CCL24 in BAL as well as in lung homogenates (Fig. 7A-D). Furthermore, the levels of lung CCL1 1 and CCL24 correlated with serum MFAP4 concentrations (Fig. 7E- F).

MFAP4-deficient mice are partially protected from airway smooth muscle deposition

To investigate the influence of MFAP4 on airway remodeling, we performed morphometric analysis on a-SMA-stained lung tissue sections (Fig. 8A-B). The increase in peribronchial smooth muscle layer thickness was absent or significantly reduced in MFAP4 KO mice (Fig. 8E-F). MFAP4 deficiency is protective against airway hyper-reactivity (AHR)

To specify the role of MFAP4 in AHR, we measured lung function parameters after MCh challenge. Baseline resistance values were similar in all study groups. However, OVA-treated MFAP4 KO mice demonstrated significantly lowered MCh- induced increase in pulmonary resistance than their WT littermates (Fig. 9A). The same tendency was observed in chronically treated animals (not shown). We then analyzed data obtained from the so-called constant phase model, which divides resistance into central airway resistance of the conducting airways and tissue damping, a measure of resistance of lung parenchyma. We found that mainly changes in the parenchymal resistance are responsible for the overall attenuation of AHR in MFAP4-deficient mice (Fig. 9B-C). MFAP4 is upregulated in asthmatic BSMCs

To validate our in vivo findings and further define the mechanism by which MFAP4 contributes to asthmatic airway disease, we studied the effects of MFAP4 on healthy or asthmatic human BSMCs in vitro. BSMCs turned out to be a potent source of MFAP4; in addition, we observed that asthmatic BSMCs produce increased levels of MFAP4 relative to BSMCs derived from the non-asthmatic donor, both on mRNA and protein level (Fig. 10). MFAP4 production was not affected by exogenous MFAP4 (Fig. 10A).

MFAP4 binds to BSMCs via ανβ5

We analyzed expression of various integrin receptors on BSMC surface. We detected high levels of integrins β1 , aV and α\/β5, and considerably lower levels of α\/β3, whereas β6 and βδ subunits were undetected (not shown). We then investigated the capacity and integrin dependence of MFAP4 to mediate BSMC adhesion. MFAP4 promoted adhesion of BSMCs in a dose-dependent manner, similarly in both healthy and asthmatic cells (Fig. 1 1 A) and to the level of positive control fibronectin (data not shown). MFAP4-dependent adhesion could be inhibited with soluble RGD peptide but not with control DGR peptide, suggesting that MFAP4 binds RGD-dependent integrins on BSMC surface (Fig. 1 1 B-C). To define which integrin receptor serves as a BSMC ligand for MFAP4, we performed adhesion assay with integrin-blocking antibodies. Anti-aV and anti-aVp5 antibodies prohibited BSMC adhesion, showing that α\/β5 is a main MFAP4-binding partner on BSMCs

(Fig. 1 1 D). However, we did not detect changes in either subunit in cells grown on MFAP4 compared to control cells seeded on PDL (data not shown). MFAP4 promotes BSMC proliferation through PI3Kand ERK1/2

BSMC proliferation is a known phenomenon in the airways of asthmatics and contributes to airway obstruction and AHR. We explored the influence of MFAP4 on proliferative capacity of BSMCs. MFAP4 promoted PDGF-induced BSMC proliferation in a specific manner that could be inhibited using anti-MFAP4 antibodies

(Fig. 12A-B). Asthmatic BSMCs raised stronger response to PDGF than healthy BSMCs.

To further define signaling pathways involved in MFAP4-dependent BSMC proliferation, we cultured cells with presence of specific pharmacologic inhibitors of PI3K and MEK (MEK is an immediate upstream kinase crucial for ERK1 /2 activation). We found that both inhibitors abolished MFAP4-mediated proliferation (Fig. 12C).

MFAP4 blocking antibody HG-HYB 7-1 has same epitope recognition as HG- HYB 7-14 In the above described experiments, MFAP4 blocking effects were investigated using HG-HYB 7-5 and HG-HYB 7-14. More antibodies have been produced and identified as MFAP4-blocking antibodies in cellular adhesion assays. One antibody HG-HYB 7-1 had similar epitope recognition as HG-HYB 7-14 (Figure 13). This clone was selected for patenting as the HG-HYB 7-14 antibodies precipitated during storage, whereas HG-HYB 7-1 did not.

DATA INTERPRETATION

One mechanistic role for MFAP4 is in integrin α_ν 35 activation of VSMC adhesion, migration, and proliferation. In line with this, MFAP4 deficiency delayed neointima formation after flow-cessation induced vascular injury. Yet, the lack of MFAP4 additionally reduced arterial outward remodeling and consequentially resulted in overall accelerated lumen reduction. Other roles for MFAP4 are as positive modulator of airway inflammation and airway remodeling through α_ν δ activation.

\n vitro data generated in this study identifies VSMCs as sites of synthesis for MFAP4. The localization of human MFAP4 to VSMCs combined with the observation that MFAP4 binds the ECM fibrils supported a role for MFAP4 in maintaining homeostatic functions in the vessel wall as known for other matricellular proteins and/or integrin a_vp₃ ligands like osteopontin and vitronectin (24-26). The presence of integrin receptors for MFAP4 in the VSMCs was following characterized. The utilized fetal cell line had a relatively high expression of integrin ανβ3 and therefore may represent partly dedifferentiated cells as commonly observed in ligated or otherwise injuried arteries. An alternatively tested adult

HAoSMC line predominantly expressed integrin α_νβ5 and interacted with MFAP4 through this receptor (data not shown). The almost complete disruption of cellular adhesion onto immobilized MFAP4 with blocking antibodies strongly indicates that integrin α_νβ3 is the dominating MFAP4 interaction partner in the present investigations.

The relatively high level of MFAP4 in the diseased as well as in the normal artery separates the expressional regulation of MFAP4 from the common transient high expression of matricellular proteins and well-known integrin α_νβ3/5 ligands and suggests that MFAP4 mediated cellular effects primarily are regulated by other means than expression.

In order to study the effects of MFAP4 on VSMC biology in vivo the mouse mfap4 gene was inactivated. Histological examinations of tissues including arteries, skin and lung from unchallenged mfap4-/- mice showed a normal gross appearance up till at least 3 months of age. The mean blood pressure did not differ between wild- type, and homozygous mfap4-/- mice when measured through catheterization. Blood pressure responses to phenylephrine infusions were normal in homozygous mfap4-/- mice, indicating that the mfap4 gene deficiency did not alter the intrinsic pharmacological properties of smooth muscle cells in mice. Thus, no relevant cardiovascular phenotype was found in the unchallenged mfap4-/- mice. These observations supported that MFAP4 is not essential for survival or normal cardiovascular development like for many other matricellular proteins including the integrin α_νβ3 ligand osteopontin (27).

Mfap4-/- mice underwent ligation of the left carotid artery in order to stop the blood flow and thereby causing the vessel to shrink in the luminal area due to the neointima formation and additional arterial remodeling (23). During the next 14 days, the EEL of the ligated vessels in mfap4-/- mice was reduced when compared to the unligated control vessels, without prominent acquisition of intimal mass. Intravascular ultrasound has previously confirmed the presence of both outward and constricting remodeling after angioplasty suggesting that an increase in the total E EL confined area is adaptive, whereas a decrease in the EEL area contributes to restenosis with occlusion of the lumen (28). As the mfap4-/- mice did not appear with prominent outward arterial remodeling, neither 14 nor 28 days after ligation, the delayed neointima formation ultimately resulted in a narrowing of the lumen 28 days after ligation.

As known for integrin

antibodies (11) and other integrin ανββ antagonists (29) the MFAP4 blocking antibodies may be anticipated to target vasculoproliferative processes including VSMC driven restenosis and neovascularization. One putative advantage with MFAP4 blocking antibodies could be the selective inhibition of cellular integrins engaged in complexes with MFAP4, and the possible reduction of side effects from the integrin inhibition. Moreover, prophylactic anti-MFAP4 treatment could be initiated prior to an expected vascular damage due to the constitutive presence of MFAP4 in the vessels. However, such treatment may require relatively high amounts of antibody, unless applied topically. Moreover, although concern has existed regarding the safety of long -term systemic administration of integrin α_νβ3 antagonists, including inhibition of wound healing and promotion of paradoxical cancerous activity (30) recent evidence has lessened this concern. Integrin α_νβ3 antagonism appears with an acceptable level of adverse effects (31-33), and sustained systemic exposure with integrin α_νβ3- or

antibodies did not inhibit wound healing in monkeys and humans (34, 35).

In summary of vascular effects, the results of this study show that MFAP4 plays a surprisingly multifacetted role in the vascular stenotic responses by promoting protective outward vessel remodeling but also the cellular growth and migration leading to hyperplasia. MFAP4 is constitutively expressed and thus has the potential to serve as prophylactic therapeutical target for inhibition of VSMC growth and migration. In the present study of allergic asthma, we evaluated the role of MFAP4 in OVA- and HDM-mediated allergic asthma models. We show that OVA or HDM exposure results in elevated serum MFAP4. We demonstrate that challenged MFAP4-deficient mice show reduced eosinophilia, airway smooth muscle deposition and AHR compared to WT mice, whereas IgE responses and most Th2-related cytokines remain unchanged. We also show that MFAP4 is upregulated in asthmatic BSMCs, and that it promotes BSMC integrin-dependent adhesion, proliferation and CCL1 1 release. Collectively, we suggest MFAP4 as an important contributor to allergic asthma. MFAP4 levels were increased in serum in OVA-treated mice and both serum and

BAL in HDM-treated mice but decreased in lung homogenates. This may suggest that MFAP4 is released to the circulation due to increased matrix turnover and remodeling accompanying disease progression. Our in vitro data indicate that increased local MFAP4 production can also contribute to elevated circulating MFAP4. We show that MFAP4 is upregulated in asthmatic BSMCs, but other cell types such as vascular smooth muscle cells may also constitute a potential source of MFAP4.

Correlations between serum MFAP4 and eosinophil numbers and eosinophil chemoattractants suggest that MFAP4 plays an important role in allergic airway disease. To date, there are no reports about MFAP4 expression in asthmatic patients. It would be interesting to assess if MFAP4 may be a good predictor of eosinophilic asthma in humans, as was shown for other ECM proteins such as periostin (36).

Pronounced eosinophilic inflammation is present in the majority of asthmatic cases (37). Eotaxins are potent chemokines playing a crucial role in eosinophil accumulation in asthmatic airways (37-39). Disturbing eotaxins or their receptor CCR3 impaired lung eosinophil recruitment after antigen challenge, which was accompanied in most, but not all, studies by a decrease in goblet cell metaplasia and AHR (40-43). Our results indicate that MFAP4 contributes to antigen -induced eosinophilia through BSMC-derived eotaxin-1. BSMCs were shown to upregulate

CCL1 1 and other eosinophil-related chemoattractants after stimulation with ECM

(43).

AHR is responsible for decrease in lung function and clinical symptoms seen in asthma (44). ASM proliferation is known to increase in asthma (45) and is one of the most important causes of airway narrowing and AHR (46, 47). We found that BSMC proliferation is directly influenced by MFAP4, and that lack of MFAP4 partially normalizes peribronchial ASM thickness and consequently AHR. Moreover, MFAP4 may also contribute to increased BSMC deposition through eosinophil-dependent hyperplasia (48).

We assessed the contributions of central airways and lung parenchyma to the overall AHR increase. The main effect was mediated through changes in tissue damping, a parameter reflecting parenchymal resistance, although we detected a significant decrease also in central airway resistance in MFAP4-deficient mice. As the increase in tissue damping is a manifestation of small airway constriction (49), MFAP4-related effect on AHR is most probably caused by inflammation and remodeling in the parenchyma.

There is limited knowledge on expression of integrin receptors on BSMCs. In several studies, a5, av and β1 subunits were found to be universally expressed, together with some expression of α1 -α8 and low levels of β3 (50). In agreement with existing literature, our data show that BSMCs express high levels of β1 and av and low levels of β3 integrin. We also found high expression of ανβ5 heterodimer, and show that it serves as a recognition ligand for MFAP4 on BSMCs. Integrin ανβ5 was previously found to activate TGF-β and promote airway remodeling (51). Furthermore, a recent genome-wide study identified ITGB5 as the gene with the strongest association to AHR severity in asthmatic patients (52). It suggests that interaction between ανβ5 and its ligands, contributes to the asthmatic airway disease. However, the underlying mechanism remains unknown, as in vitro studies investigating BSMC responses to integrin ligands focused on β1 and νβ3 integrins.

We detected changes in FAK activation shortly after plating cells onto MFAP4, suggesting that FAK is directly activated by MFAP4-binding integrins. Indeed, FAK was previously shown to modulate cell behavior after ligation of ανβ3 and ανβ5 by periostin (14). To our knowledge, there is only one study addressing the role of FAK in integrin signaling specifically in ASM, showing that FAK promotes collagen I- induced bovine ASM proliferation together with following activation of PI3K and ERK (53). Both these pathways regulate growth factor-induced cell growth (54, 55).

In summary of allergic asthma effects, our data indicate that MFAP4 contributes to allergic asthma by promoting airway eosinophilia, AHR and lung remodeling through regulation of ASM proliferation and chemokine secretion. Targeting MFAP4 may be thus suggested as a novel approach for the treatment of asthmatic patients.

HG-HYB 7-1 was found to have similar epitope recognition and function as the antibody HG-HYB 7-14 and was therefore a candidate treatment for inhibition of MFAP4 interaction with integrins in human disease. aMFAP4 (HG-HYB 7-1) Treatment Reduces Lesion Density and Macrophage Infiltration Treatment with aMFAP4 (HG-HYB 7-1 ), particularly at 5Mg, was able to consistently reduce lesion size and density and limit infiltration of macrophages into the burn area. While this study highlights the effectiveness of targeting this protein in the treatment of AMD, it is not clear through which pathways this therapy is influencing the development of this disease. It may be that reduction in active MFAP4 levels through aMFAP4 treatment reduces activation of the complement system in a similar way to ficolins, which leads to the reduction of macrophage recruitment, which therefore reduces production of pro-angiogenic cytokines, ultimately reducing CNV.

The invention will now be illustrated by way of the following examples:

Examples Production and purification of wild-type rMFAP4 and RGD mutants

Wild-type rMFAP4 and different genetically modified versions of the protein was performed as previously described (2).

Production of anti-MFAP4 monoclonal antibodies

C57BL/6/N MFAP4 deficient mice were immunized for the production of monoclonal antibodies (HG HYB 7-5, 7-14, and 7-18) against rMFAP4.

Immunohistochemical analysis

Freeze sections from a human vein with intimal hyperplasia were obtained from the Vascular Research Unit, Viborg Hospital. Formalin fixed normal human tissue was obtained from the tissue bank at the Department of Pathology, Odense University Hospital (Odense, Denmark). The local ethical committee in Odense approved the use of the human tissue sections (Ref. No. VF20050070). Mouse tissue was obtained from mfap4-/- or mfap4+/+ mice. Utilized antibodies included; anti-MFAP4 (HG-HYB 7-14), fluorescein isothiocyanate (FITC)-anti-MFAP4 (HG-HYB 7-14), anti-a-SMA (Dako #M0851 ), FITC-anti-a-SMA (Sigma, clone 1 A4), anti-integrin α_νβ₃ (Santa Cruz #SC-7312), anti-human CD45 (Roche #760-4279), anti-mouse CD45 (BD pharmingen, clone 30-F1 1 ), anti-Ki-67 (Dako, clone MIB-1 ), anti-caspase-3 (Cell Signaling #9664), and anti-FITC antibody (P5100, Dako).

Ligand binding studies

Insoluble type I collagen from bovine Achilles tendon and insoluble elastin from bovine aorta were supplied by Sigma (St. Louis, MO, USA) and Elastin Products Company, Inc. (Owensville, MO, USA), respectively. Five milligram of collagen or elastin was hydrated overnight in 10 mM tris buffered saline (TBS) 0.05% (w/w) TWEEN 20, and 5 mM CaCI₂ (TBS/Tw-Ca²⁺) or 10 mM Ethylenediaminetetraacetic acid (EDTA) (TBS/Tw-EDTA) at 4^<€ and mixed with rMFAP4 in TBS/Tw-Ca²⁺ or

TBS/Tw-EDTA. After incubation at room temperature for 1 h, the water phase was recovered by centrifugation and analyzed by ELISA.

Detection of MFAP4 by ELISA

Sandwich ELISA assays were performed in 96-well Maxisorb Microplates (Nunc) essentially as described in Molleken et al. 2009 (56).

Statistical Methods

Statistical significance between groups in in vitro and in vivo experiments was assessed by one-way or (paired or unpaired) two-way ANOVA with Bonferroni adjusted t-tests when relevant. Data were analyzed using Graph Pad Prism 5. P < 0.05 was considered statistically significant.

Human aorta smooth muscle cell (HAoSMC) cultures

Cells were grown at 37°C in 5% C0₂ humidified incubator (Hera cell, Heraeus). fHAoSMC's or adult cells (Cell application, inc.) derived from normal human tunica intima and media of either fetal or adult aorta, were cultured in a smooth muscle cell growth medium (Cell application, inc), or when allowed to differentiate in a smooth muscle cell differentiating medium (Cell application, inc.). Cells were used in passages 3-7.

Human bronchial smooth muscle cell (BSMC) cultures

Primary human BSMCs derived from healthy or asthmatic donors (39 and 27 years respectively, both non-smoking Caucasian males) were obtained from Lonza. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin, 50 μg/ml streptomycin and 2mM L-glutamine (Gibco). Cells between passages 5-10 were used in all experiments.

Immunofluorescence microscopy

Fixed and permeabilized cells were stained for 1 h at room temperature using 10 μς/ηιΙ FITC-anti-MFAP4 in phosphate buffered saline (PBS)/BSA containing 0.2% saponin (w/w). Cell adhesion assay

Black 96-well Maxisorp FluoroNunc™ microtiter plates (Nunc) were basically coated as above. In blocking experiments well were further incubated with 20 μg/mL of MFAP4 blocking antibodies or cells were pre-incubated with either 25-100 μg/mL synthetic GRGDS or SDGRG peptides (Sigma-Aldrich) or 10 μg/mL anti-integrin antibodies; anti-integrin a , monoclonal mouse anti-human antibody clone L230

(Alexis Biochemicals); anti-integrin βι , monoclonal mouse anti-human antibody clone P4C10 (Millipore); anti-integrin αν/βδ, monoclonal mouse anti-human antibody clone P1 F6 (Santa Cruz Biotechnologies); anti-integrin α_ν/ 3, monoclonal mouse anti-human antibody clone LM609 (Millipore); monoclonal mouse anti-fibrinogen C domain-containing protein 1 (anti-FIBCD1 ) antibody clone 12-5 (control antibody produced in-house (57)). A Vybrant™ cell adhesion assay kit (Molecular Probes, Invitrogen) was used.

Cell migration assay

The migration assay was performed using the Oris™ Migration Assembly Kit

(Platypus Technologies Madison, Wl) with coating as above. Cells were serum- starved before the addition of 0.5% (w/w) fetal calf serum and 5 ng/ml PDGF-BB allowing cell migration. Some well were incubated with anti-MFAP4 antibody clones. Migrated cells were detected using 4',6-diamidino-2-phenylindole (DAPI) solution (Invitrogen). In some experiments, cells were seeded in serum-free DMEM for 4 h to allow attachment. Subsequently, anti-integrin antibodies or inhibitors of PI3K and MEK (LY294002 and PD98059, respectively, both from Cell Signaling Technology) were added 1 h prior to PDGF stimulation. Cell proliferation assay Cells were serum starved before seeding onto immobilized rMFAP4 or fibronectin. Blocking experiments were performed by incubating the protein coated wells with 20 μς/ιτιΙ. anti-MFAP4 antibodies, or by preincubating suspended cells with anti-integrin antibody in the presence of 0.3% (w/w) fetal calf serum ± 5 ng/mL recombinant human PDGF-BB. The number of viable cells was following determined using an

MTT-assay.

SDS-PAGE and Western Blotting

SDS-PAGE and Western Blotting were performed using standard methods. Primary antibodies included; anti-av (CD51 ), monoclonal mouse IgG clone 21 (BD

Biosciences); anti-βι, monoclonal mouse IgG clone BV7 (abeam); anti- ₃, polyclonal goat IgG clone C-20 (Santa Cruz Biotechnology); anti-p₅, polyclonal rabbit IgG clone H-96 (Santa Cruz Biotechnology); anti-MFAP4, monoclonal HG-HYB 7-5; Anti- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), monoclonal mouse IgG clone 6C5 (Santa Cruz Biotechnology); anti-a-SMA, monoclonal mouse IgG clone

1 A4 (Sigma-Aldrich). Secondary antibodies included: horseraddish peroxidase (HRP)-labelled donkey anti-goat immunoglobulin (Santa Cruz Biotechnology), goat anti-rabbit immunoglobulin HRP-labelled (Dako), and rabbit anti-mouse immunoglobulin HRP-labelled (Dako).

Flow cytometry analysis

Pelleted FHAoSMCs were resuspended with relevant primary anti-integrin antibodies described under "cell adhesion assay" or isotype matched anti-chicken ovalbumin (The State Serum Institute, Copenhagen) and polyclonal anti-mouse FITC-conjugated goat F(ab')₂ (Dako) as secondary antibody. Cells were analyzed using a Becton Dickinson (BD) Flow Cytometry FACScan™ (BD Biosciences) and BD Cell quest™ Software (BD Biosciences).

Carotid artery ligation model

All mouse experimens were performed under a license obtained from The National

Animal Experiments Inspectorate who also approved the study (ref.no. 2012-15- 2934-00095). The arterial ligation model was essentially performed as described in Kumar and Lindner 1997 (23). Allergic asthma models Female 8 to 12-week-old mfap4-/- (knockout, KO) mice and littermate mfap4+/+ (wild type, WT) mice, bred into the BALB/c background for more than 10 generations, were used. The animals had access to pelleted chow and water ad libitum. The National Animal Ethics Committee approved all animal experiments (permit 2012-15-2934-00354).

Mice were sensitized intraperitoneally with 200 μg ovalbumin (OVA; grade VI, Sigma-Aldrich) and 2 mg alum (Thermo Scientific) in 200 μΙ phosphate-buffered saline (PBS) on days 0 and 7. On days 14-16, mice were challenged intranasally with 20 μ9 OVA in 50 μΙ PBS under light isoflurane anaesthesia. Alum-sensitized, PBS-challenged mice were used as controls.

Alternatively, mice were challenged intranasally with 25 μg house dust mite extract (HDM; Greer, endotoxin content 51 .5 EU/mg) 5 days/week for seven weeks as described previously (58). PBS-treated mice were used as controls.

Measurement of lung mechanics and AHR 24 h after the last challenge mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), tracheostomized and connected to computer-controlled small animal ventilator (Flexivent, SCIREQ). Mechanical ventilation was set at 150 breaths/min with a tidal volume of 10 ml/kg and a positive end -expiratory pressure of 3 cm H₂0. Lung function parameters were measured in the steady state and after exposure to increasing doses of nebulized methacholine (MCh; Sigma-Aldrich). For each parameter, a coefficient of determination of 0.90 was the lower limit for accepting a measurement.

Bronchoalveolar lavage (BAL)

Immediately after AHR measurements mice were sacrificed by cardiac puncture. Lungs were washed four times with 0.5 ml ice-cold PBS. BAL fluids were centrifuged at 1 ,000 g for 10 min at 4°C, and supernatants were stored in -80°C until further analysis. Cells were washed in red blood cell lysis buffer (Sigma), resuspended in PBS, counted, cytospun and stained with Hemacolor (Merck Millipore). For differential count, 200 cells/sample were counted based on morphological criteria. Preparation of lung homogenates Frozen lungs were homogenized in 1 ml PBS with protease inhibitors (Sigma). Homogenates were centrifuged for 10,000 g at 4°C. The supernatants were stored in -80°C until further analysis.

Measurement of specific IgE Specific IgE was measured by ELISA. Briefly, wells were coated with 5 μg/ml OVA or HDM overnight at 4°C. Wells were blocked with PBS/0,05% Tween/1 % BSA for 1 h. Serum samples diluted 1 :20 in blocking buffer were then incubated for 2 h, after which wells were washed and incubated with anti-lgE-HRP antibody (Southern Biotech). Results are shown as relative absorbance units (OD450). Lung histology and immunohistochemistry

Formalin-fixed, paraffin-embedded lung tissues were cut in 4 μιη-thick slides and stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), Trichrome or Picrosirius Red.

Morphometric analysis of lung tissue Lung inflammation was graded on H&E-stained slides by point counting using CAST software. Briefly, 36-point grid was laid onto the field of vision. In each of 25 randomly selected fields, points hitting the inflamed area as well as all points hitting lung parenchyma were counted. The degree of inflammation was quantified as the percentage of counted points hitting the area of interest. Subepithelial fibrosis was quantified by color threshold analysis as Trichrome- positive area and normalized to the length of basement membrane. Goblet cell hyperplasia was assessed by counting PAS-positive cells. Smooth muscle cell remodeling was quantified by measuring the thickness of the smooth muscle cell layer around airways. At least 5 same-sized bronchioles were counted in each slide. All analyses were performed using ImageJ software (59).

Cytokine quantifications in mouse lungs

The levels of IL-4, IL-5, IL-13, CCL1 1 , CCL24 and TGF-p1 in BAL or lung homogenates were measured using commercial ELISA kits (BioLegend, eBioscience, R&D) according to the manufacturer's instructions. The minimum detection limits were: 1 pg/ml for IL-4, 4 pg/ml for IL-5 and IL-13, 3 pg/ml for CCL1 1 and CCL24, and 15,4 pg/ml for TGF-βΙ .

Hydroxyproline assay

Levels of hydroxyproline in lung tissues were measured using Hydroxyproline Colorimetric Assay Kit (BioVision) according to manufacturer's instructions.

Detection of soluble MFAP4

MFAP4 levels were measured by AlphaLISA Technique as described previously (60) and shown as U/ml. 1 U/ml corresponds to 38 ng/ml in human serum. Competitive analysis of monoclonal antibodies against MFAP4

Microtiter plates were coated with recombinant MFAP4 (250 ng/ml). Combinations of labeled and unlabeled antibodies were added using serial dilution of the unlabeled antibody as illustrated on the abscissa. The combinations are described in the legend with the biotinylated antibody (500 ng/ml) and the unlabeled antibody from 4000 ng/ml and diluted 2-fold.

Validation of aMFAP4 (HG-HYB 7-1) antibody aMFAP4 antibody used throughout this study was HG-HYB 7-1 of the present invention. Antibody validation was conducted to ensure reactivity and specificity of the aMFAP4 antibody. 18μηη post fixed kidney sections embedded in OCT were blocked and stained for

MFAP4 (Ι ςΛπΙ). Secondary Alexafluor 488 rabbit anti-mouse (Life Technologies) was used to detect MFAP4 staining, and coverslips were mounted with Fluoroshield with DAPI (Sigma).

Western blots were also conducted on both lung and choroidal tissues, however the aMFAP4 antibody used here was unable to detect MFAP4 reliably (data not shown).

Animals

Female C57/BL6 J mice were used for this study, and were treated in accordance with ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, at the University of Nottingham Biological Services Unit, under a UK Home Office licence.

Animals were anaesthetised with an intraperitoneal (IP) injection of 50mg/kg ketamine, and recovered with 0.5mg/kg IP Sedastart. Pupils were dilated with topical applications of 5% phenylephrine hydrochloride and 0.8% tropicamide, and eyes were coated with GenTeal Gel (Novartis) to prevent dehydration.

Lasering and Drug Administration

Lesions were produced using a Meridian Merilas 532a Green Laser Photocoagulator to penetrate Bruch's membrane at 4 points per eye, in both eyes. Presence of a bubble was used to determine the successful rupture in each burn. Laser settings were maintained at 450mW for 130ms for each burn.

Eyes were injected with either ^g aMFAP4, 5μg aMFAP4, ^g mouse IgG (DAKO), ^g aVEGF-A (Biolegend), or uninjected. Antibodies were diluted to Ο.δμς/μΙ (2.5μς/μΙ for 5μg aMFAP4) in sterile PBS and 2μΙ administered with a 36 gauge Hamilton needle (World Precision Instruments) with fine forceps used to stabilise the eye (World Precision Instruments).

Fundus Fluorescein Angiography (FFA)

To visualise the vasculature and burn development in vivo, 200μΙ of 10mg/ml sodium fluorescein (Sigma) in saline was injected IP and allowed to circulate before imaging with a Phoenix Micron IV Retinal Imaging Microscope. Development of cataracts in some eyes meant that some burns had to be excluded from FFA imaging.

Immunofluorescence

After dissection, choroids were blocked in flatmounting serum (5% Goat Serum, 3% Triton X 100, 1% BSA) and stained with lsolectin-B4 (IB4) (Sigma Aldrich, biotin conjugated) 5 g/ml and CD45 (Abeam) 5μg/ml overnight at 4°C. Streptavidin conjugated Alexafluor 488 2μg/ml and donkey anti-rabbit Alexafluor 555 4 g/ml were used to detect IB4 and CD45 staining respectively, and coverslips were mounted with Fluoroshield with DAPI. Images were obtained using a Leica TCS SPE confocal microscope, and all settings were maintained between images. All image analysis was conducted in ImageJ, and statistics and graphs produced in GraphPad Prism 6.

Area and density were calculated by drawing arou nd the lesion with the freehand select tool. Burn area in μηη² was measured directly by ImageJ, and integrated density was used to measure fluorescence, which was used as a proxy for the density of vasculature in the highlighted area.

Significant differences were indicated on graphs as asterisks, where:

P≤ 0.05

P≤0.01

P≤ 0.001

Any burns which had merged or animals in which the contralateral eye had burns measuring greater than 2 standard deviations from the mean were excluded from analysis. aMFAP4 (HG-HYB 7-1 ) Antibody Validation

Initial experiments were conducted to ensure reactivity and specificity of the aMFAP4 (HG-HYB 7-1 ) antibody. Immunofluorescent staining of kidney sections was used to visualise localisation of MFAP4 protein. MFAP4 staining was localised to the lumen of tubules (Fig 14A), and compared to immunohistochemical staining from other groups (Fig 14C). Rabbit anti-mouse IgG was used as a negative control (Fig 14B).

Western blots were also conducted on lung and choroidal tissues, however the antibody was not able to distinguish MFAP4 protein from IgG (not shown). This was established by running samples which had been precleared of IgG with the same samples before preclearing. Any bands seen in unprecleared samples were not detected after preclearing, indicating that bands detected were IgG. aMFAP4 (HG-HYB 7-1) Reduces Average Lesion Size and Density by Day 7 Injection with sodium fluorescein highlighted vasculature within the eye, and showed burns and regions of leakage. At day 7, there was no significant difference between the IgG negative control and the aVEGF-A positive control, in either average lesion size (Fig 15B) or average integrated density of lesion (Fig 15C). Treatment with 5Mg aMFAP4 significantly reduced average lesion size and average integrated density compared to IgG negative controls (p<0.01 ) and aVEGF-A positive controls (P<0.05). There was also a reduction in average lesion size and average lesion density in mice treated with 1 μg aMFAP4 (HG-HYB 7-1 ), but this was not statistically significant. aMFAP4 (HG-HYB 7-1) and aVEGF-A Reduce Average Lesion Size and Density by Day 14

At day 14, further fluorescein angiograms were taken before culling and tissue collection. As seen at day 7, 5μg aMFAP4 (HG-HYB 7-1 ) was able to significantly reduce average lesion size and average integrated density compared to IgG controls (p<0.001 ) (Fig 16). By day 14, a significant decrease in average lesion size (p<0.05) and average lesion area (p<0.01 ) is also apparent in ^g aMFAP4 treated eyes compared to IgG controls, which was not seen at day 7. A significant decrease in average lesion density (p<0.05) is also seen in aVEGF-A treated positive controls over IgG negative controls, although a corresponding significant decrease in average lesion area is not observed. aMFAP4 (HG-HYB 7-1) and aVEGF-A Reduces Lesion Density but Not Lesion Size When Assessed by IB4

After assessment of burns via fluorescein angiograms, eyes were collected, fixed, dissected and stained. Staining of vasculature with IB4 showed no significant differences in average burn area between any of the treatment groups (Fig 17B).

There was, however a significant decrease in average integrated density in 5 g aMFAP4 (HG-HYB 7-1 ) treated eyes compared to IgG controls (Fig 17C), as previously indicated by fluorescein angiography, ^g aMFAP4 (HG-HYB 7-1 ) treatment appeared to have little effect on either average burn area (Fig 17B) or average integrated density (Fig 17C) when compared to IgG controls. Treatment with aVEGF-A does appear to reduce average integrated density, although this is not statistically significant. aMFAP4 (HG-HYB 7-1) and aVEGF-A Reduce Infiltration of Macrophages Choroids were also stained with CD45 to detect infiltration of macrophages into burn area (Fig 18B). 5μg aMFAP4 (HG-HYB 7-1 ) treatment and positive control aVEGF- A both significantly reduced infiltration of macrophages (p<0.01 and p<0.05 respectively). There was also a decrease in average integrated density in the 1 μς aMFAP4 treated group, although this was not statistically significant.

Asthmatic BSMC proliferation

A 96-well plate was coated with 10 ug/ml MFAP4 overnight at 4 degrees. The next day the plate was washed with PBS and blocked with 10 mg/ml BSA for 1 h at room temperature, after which the wells were washed again and incubated with HG-HYB

7-1 anti-MFAP4 antibody (concentration range 5-20 ug/ml) for 1 h at room temperature. After the final wash wells were left at room temperature to dry.

Asthmatic primary bronchial smooth muscle cells (BSMCs) were serum-starved in serum-free DMEM for 24 h. The next day cells were detached with trypsin, collected, counted and seeded (10,000 cells/well) in serum-free DMEM. After 48 h, MTT reagent was added to the final concentration of 1 mg/ml. After another 4 h, medium was removed and MTT crystals were dissolved in 4 mM hydrochloric acid in isopropanol. The plate was incubated at room temperature with shaking for 15 min, after which the absorbance was read at wavelength 590 nm versus 620 nm (Fig 19).

ITEMS

The invention may be disclosed according to the following items: 1 . An antibody, fragments hereof, or derivatives hereof, which specifically blocks the integrin interaction with human microfibrillar-associated protein 4 (MFAP4), wherein the antibody is characterized by:

• a light chain variable region comprising the amino acid sequence of SEQ ID NO 1 or sequences which are at least 90% homologous, preferably at least 95% homologous, and more preferably at least 98%, or at least

99% homologous thereto; and

• a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 2 or sequences which are at least 90% homologous, preferably at least 95% homologous, and more preferably at least 98%, or at least 99% homologous thereto.

2. Antibody according to item 1 , wherein the antibody is monoclonal.

3. Antibody of item 1 or 2 coupled to a detectable label or a substance having toxic or therapeutic activity.

4. Antibody of any one of items 1 -3 for use in the prevention or treatment of vascular proliferative diseases and/or related disorders.

5. Antibody for use according to item 4, wherein the vascular proliferative diseases and/or related disorders are caused by hyperplasia or remodeling in blood vessels. 6. Antibody of any one of items 1 -3 for use in the prevention or treatment of bronchiolar hyperplasia and eosinophilic inflammation in allergic asthma.

7. Antibody of any one of items 1 -3 for use in the prevention or treatment of disorders characterized by pathological neovascularization in the eye, such as AMD and DR.

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Claims

1 . An antibody, fragments hereof, or derivatives hereof, which specifically blocks the integrin interaction with human microfibrillar-associated protein 4 (MFAP4), wherein the antibody is characterized by:

• a light chain variable region comprising the amino acid sequence of SEQ ID NO 1 or sequences having at least 90% sequence identity; and

• a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 2 or sequences having at least 90% sequence identity.

2. The antibody according to claim 1 , wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO 1 or sequences having at least 95% sequence identity and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 2 or sequences having at least 95% sequence identity.

3. The antibody according to claim 1 or 2, wherein the antibody is monoclonal.

4. The antibody according to any one of claims 1 -3 coupled to a detectable label or a substance having toxic or therapeutic activity.

5. A composition comprising the antibody according to any one of claims 1 -4 and one or more physiologically acceptable carriers, excipients and/or diluents.

6. The composition of claim 5, said composition comprising one or more stabilizing agents and/or one or more buffering agents.

7. The composition for use according to claim 6, wherein at least one stabilizing agent is a surfactant.

8. The antibody of any one of claims 1 -4 or the composition according to any one of claims 5-7 for use in the prevention or treatment of vascular proliferative diseases and/or related disorders in a mammal.

9. The antibody or composition for use according to claim 8, wherein the vascular proliferative diseases and/or related disorders are caused by hyperplasia or remodeling in blood vessels.

1 0. The antibody or composition for use according to claim 8, wherein the vascular proliferative diseases and/or related disorders are caused by pathological neovascularization.

1 1 . The antibody or composition for use according to claim 8, wherein vascular proliferative diseases and/or related disorders are bronchiolar hyperplasia or eosinophilic inflammation in allergic asthma.

1 2. The antibody or composition for use according to claim 8 wherein the vascular proliferative diseases and/or related disorders is characterized by pathological neovascularization in the eye.

1 3. The antibody or composition for use according to claim 1 2, wherein the disorders characterized by pathological neovascularization in the eye is selected from the group consisting of age related macular degeneration (AMD), retinopathy, hypertensive retinopathy and diabetic retinopathy (DR).

14. The antibody or composition for use according to claim 8 wherein the vascular proliferative diseases and/or related disorders are cancers or other malignancies.

1 5. The antibody or composition for use according to claim 14, wherein the cancer or malignancy is selected from the group consisting of glioblastoma, head, neck and lung cancer.

1 6. The antibody or composition for use according to claim 8-15, wherein the mammal is a human.

17. A method of prevention or treatment of vascular proliferative diseases and/or related disorders in a mammal, said method comprising administering the antibody of any one of claims 1 -4, or the composition as defined in any of claims 5-7.

18. The method of claim 17 for prevention or treatment of the vascular proliferative diseases and/or related disorders caused by pathological neovascularization.

19. The method of claim 17 for prevention or treatment of bronchiolar hyperplasia and eosinophilic inflammation in allergic asthma.

20. The method of claim 17 for prevention or treatment of disorders characterized by pathological neovascularization in the eye.

21 . The method of claim 20, wherein the disorders characterized by pathological neovascularization in the eye is selected from the group consisting of age related macular degeneration (AMD), retinopathy, hypertensive retinopathy and diabetic retinopathy (DR).

22. The method according to claim 17, wherein the vascular proliferative diseases and/or related disorders are cancers or other malignancies.

23. The method according to claim 22, wherein the cancer or malignancy is selected the group consisting of from glioblastoma, head, neck and lung cancer.

24. The method according to any one of claims 17-23, wherein said mammal is a human.

25. The method according to any one of claims 17-24, wherein said antibody or composition is administered intravenously, ocularly or subcutaneously.