WO2013139956A1

WO2013139956A1 - Antibodies abrogating cell binding to lactadherin

Info

Publication number: WO2013139956A1
Application number: PCT/EP2013/056057
Authority: WO
Inventors: Shirley LEYMAN; Hoa Thu NGO; Sofie NOTEBAERT; Richard Zwaal; Lorenzo TIBALDI; Clotilde THÉRY
Original assignee: Thrombogenics Nv; Institut Curie; Institut National De La Santé Et De La Recherche Médicale (Inserm)
Priority date: 2012-03-22
Filing date: 2013-03-22
Publication date: 2013-09-26

Abstract

The present invention relates to anti-lactadherin antibodies and their medical use.

Description

ANTIBODIES ABROGATING CELL BINDING TO LACTADHERIN FIELD OF THE INVENTION

BACKGROUND TO THE INVENTION

Lactadherin is a 46 kDa secreted glycoprotein initially identified as a major component in milk- fat globules (Stubbs 1990, Proc Natl Acad Sci USA 87, 8417-8421). It is also known as milk-fat globule-EGF factor 8 (MFGE8) and contains two distinct functional domains: an N-terminal epidermal growth factor (EGF)-like domain containing an Arg-Gly-Asp (RGD) cell adhesion sequence and C-terminally, two discoidin /F5/C8 domains (CI and C2) with homology to the C domains present in the blood coagulation factors V and VIII (Stubbs 1990, Proc Natl Acad Sci USA 87,8417-8421 ; Taylor 1997, DNA Cell Biol 16, 861-869; Andersen 2000, Biochemistry 39, 6200-6206; Hanayama 2002, Nature 9, 182-187). Lactadherin participates in a wide variety of cellular interactions: it acts as an antiviral protein for the protection to rotavirus infection (Newburg 1998, Lancet 351, 1 160-1164), facilitates fertilization by mediating sperm-egg binding (Ensslin and Shur 2003, Cell 114, 405-417), assists the phagocytosis of apoptotic cells (Hanayama 2002, Nature 9, 182-187; Hanayama 2004, Science 304, 1147-1150; Akakura 2004, Exp Cell Res 292, 403-416), maintains and repairs the intestinal epithelium (Bu 2007, J Clin Invest 117, 3673-3683), facilitates mammary gland branching morphogenesis (Ensslin and Shur 2007, Proc Natl Acad Sci USA 104, 2715-2720), and is involved in angiogenesis (Silvestre 2005, Nat Med 11 , 499-506). The RGD cell adhesion motif triggers different signaling pathways through binding to the cell surface integrin receptors α_νβ₃/5 (Y amaguchi 2008, J Leukoc Biol 83, 1300-1307; Miksa 2006, Shock 25, 586-593; Jinushi 2007, J Clin Invest 1 17, 1902-1913; Silvestre 2005, Nat Med 11 , 499-506; Motegi 201 1, Art Thr Vase Biol 9,2024-2034). With its CI discoidin domain lactadherin binds and targets collagen for cellular uptake and thus functions in diminishing the severity of pulmonary fibrosis (Atabai 2009, J Clin Invest 119, 3713-3722). The other two domains, EGF-like domain and C2 domain, are required for the phagocytic clearance of apoptotic cells. Apoptotic cells are bound via the C2 domain which consecutively triggers the phagocytic engulfment of these cells through the binding with the RGD-motif (in the EGF-like domain) to the 0C_V integrins, which are expressed on phagocytes (Hanayama 2002, Nature 9, 182-187; Hanayama 2004, Science 304, 1 147-1150; Leonardi-Essmann 2005, J Neur 160, 92-101). As a consequence of acting as an opsonin, lactadherin attenuates inflammation and immunity by stimulating the production of Treg cells (Jinushi 2007, J Clin Invest 1 17, 1902- 1913). Another activity involving ( νβ3/5 integrins implies lactadherin in blood vessel growth and maturation. Using a rabbit polyclonal anti-human lactadherin antibody and lactadherin-deficient animals, Silvestre and coworkers reported that lactadherin enhances vascular endothelial growth factor (VEGF)-dependent neovascularization and α_νβ₃/5 integrin-dependent endothelial cell survival (Silvestre 2005, Nat Med 1 1, 499-506). Progress of cancer is sustained by neo-angiogenesis required for nutrient and oxygen supply (Hanahan & Weinberg 2000, Cell 100, 57-70; Carmeliet & Jain 2000, Nature 407, 249-257). Therefore pathogenic neo-angiogenesis is selected as a therapeutic target pathway in order to suppress blood vessel formation in tumors. In the early eighties high expression of a 46 kDa component of human milk fat globule (HMFG), identified as lactadherin, was shown in human breast tumors (Ceriani et al. 1982, Proc Natl Acad Sci USA 79, 5420-5424; Larocca 1991, Can Res 51 , 4994-4998; Taylor 1997, DNA Cell Biol 16, 861 -869; Couto 1996, DNA Cell Biol 15, 281 -286). Upregulation of this glycoprotein in other cancer types, including triple negative breast-, ovarian-, pancreatic-, bladder- and prostate cancer and T cell leukemia was also reported (Sugano 2011 , Oncogene 30, 642-53; Yang 2011 , Cancer Res 71, 937-45). The pro-tumoral role of lactadherin was further elucidated in different xenograft mouse models (Neutzner 2007, Cancer Res 67, 6777-85; Jinushi 2008, Cancer Res 68, 8889-8898; Jinushi 2009, J Exp Med 206, 1317-26; Carrascosa 2011 Oncogene doi: 10.1038/onc.2011.356; Yang 201 1, Cancer Res 71 , 937-45; Motegi 2011 , Art Thr Vase Biol 9, 2024-34). Reduced tumor vessel permeability and decreased tumor burden was reported in a lactadherin-deficient Ripl-Tag2 pancreatic tumor mouse model (Silvestre 2005, Nat Med 1 1, 499-506; Neutzner 2007, Cancer Res 67, 6777-6785). In a murine melanoma mouse model Jinushi and coworkers demonstrated that lactadherin enhanced tumorigenicity and metastatic capacity (Jinushi 2008, Cancer Res 68, 8889-8898). Lactadherin impairs proliferation of HER2⁺ human breast cancer cells whereas it promotes survival to chemotherapy of triple negative breast cancer cells (Yang 201 1, Cancer Res 71 , 937-945). Furthermore, lactadherin is thought to stimulate tumor progression through coordinated signaling in both tumor and host cells within the tumor microenvironment. Jinushi and colleagues (using the same rabbit polyclonal anti- MFG-E8 antibody as described in Silvestre et al. 2005, Nat Med 11 , 499) explored the therapeutic potential of anti-lactadherin antibodies with/without combinatorial therapy in different settings (Jinushi 2009, J Exp Med 206, 1317-1326). In addition to high production of lactadherin in tumor-associated macrophages Jinushi and colleagues demonstrated that lactadherin renders cancer stem cells derived from murine and human tumor cells more resistant to chemotherapy-induced apoptosis and more tumorgenic (Jinushi 2008, Cancer Res 68, 8889- 8898; Jinushi 2011 , Proc Natl Acad Sci USA 108, 12425-12430). Recently, Motegi and coworkers demonstrated that pericytes are also important sources of lactadherin in vivo that regulate tumor-associated angiogenesis (Motegi 2011 , Art Thr Vase Biol 9, 2024-2034). Already in the eighties monoclonal antibodies were generated against different components of HMFG (Ceriani 1983, Som Cell Genet 9, 415-427; Peterson 1990, Hybridoma 9, 221 -235). Radiolabeled monoclonal antibodies against HMFG were tested for their effectiveness in destroying breast tumor cells and as such could control the tumor growth (Ceriani 1988, Cancer Res 48, 4664- 4672). Radioimmunotherapy with one of these, humanized Mc3, was shown to decrease growth of MX-I human breast tumors grafted in immunodeficient mice (Ceriani 1995, Cancer Res 55, 5852-5856).

SUMMARY OF THE INVENTION

The invention relates to isolated monoclonal antibodies binding to the antigen defined in SEQ ID NO:51 and being capable of inhibiting cell adhesion and/or migration and/or survival in the presence of human lactadherin.

Exemplary isolated antibodies binding to the antigen defined in SEQ ID NO:51 comprise antibodies comprising either one of the following combinations of complementarity determining region (CDR) amino acid sequences:

(i) heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 47 to 49, respectively; and light chain CDR 1 to 3 with amino acid sequences defined in SEQ

ID NO: 32, 37, and 38, respectively; (ii) heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 47 to 49, respectively; and light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 32 to 34, respectively;

(iii) heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 47 to 49, respectively; and light chain CDR 1 to 3 with amino acid sequences defined in SEQ

ID NO: 32, 35 and 36, respectively;

(iv) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively, or light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 29 to 31, respectively; and heavy chain CDR 1 and 2 with amino acid sequences defined in SEQ ID NO: 45 and 46, respectively, and heavy chain CDR 3 with amino acid sequence comprising SEQ ID NO: 50;

(v) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively, or light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 29 to 31, respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 45, 46, and 41 , respectively;

(vi) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively, or light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 29 to 31, respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 39 to 41, respectively;

(vii) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively, or light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 29 to 31 , respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 45, 46, and 44, respectively;

(viii) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively, or light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID

NO: 29 to 31, respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 42 to 44, respectively;

(ix) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 45, 46, and 41, respectively; (x) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 39 to 41 , respectively;

(xi) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 29 to 31 , respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 45, 46, and 44, respectively;

(xii) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 29 to 31 , respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 42 to 44, respectively; or

(xiii) an affinity matured antibody of any of (i) to (xii).

Any of the above antibodies can be a mammalian antibody, a non-rodent antibody such as a non- murine antibody, a human antibody or a humanized antibody. Further, any of the above antibodies can be a monovalent or multivalent antibody, and further be monospecific or multispecific provided that the binding specificity to said antigen (defined in SEQ ID NO:51) is maintained.

For example, the above-mentioned humanized antibody may be one comprising at least one of:

(i) a variable heavy chain as given in SEQ ID NO:22 or SEQ ID NO:25 carrying up to 12 mutations in the region outside the CDRs;

(ii) a variable light chain as given in SEQ ID NO:21 or SEQ ID NO:24 carrying up to 7 mutations in the region outside the CDRs;

(iii) a variable heavy chain as given in SEQ ID NO:22 carrying one or more of the following mutations: Gln5Val, Pro9Ala, Leul lVal, Argl9Lys, Asp41Pro, Lys43Gln, Thr44Gly, Ser76Thr, Gln82Glu, Thr87Arg, Ser91Thr, and/or Serl 16Thr;

(iv) a variable light chain as given in SEQ ID NO:21 carrying one or more of the following mutations: IlelOThr, Lysl 8Arg, Thr22Ser, Thr41Gln, Arg76Ser, Ala79Pro, and/or Ala82Phe;

(v) a variable heavy chain as given in SEQ ID NO:25 carrying one or more of the following mutations: GlylOThr, GlnBLys, SerHPro, Serl5Thr, Serl9Thr, Ser23Thr, Ser43Pro, Ala45Lys, Ser72Thr, Arg77Lys, Lys83Thr, and/or Serl20Leu or Serl20Thr;

(vi) a variable light chain as given in SEQ ID NO:24 carrying one or more of the following mutations: Leul5Val, Asp41Gly, Thr43Ala, Asn77Ser, Gln80Pro, Ala lOOGln, and/or Leu 106Ile.

More in particular, such humanized antibody may be one comprising a variable heavy chain as given in SEQ ID NO: 22 mutated according to (i) or (iii) and a variable light chain as given in SEQ ID NO:21 mutated according to (ii) or (iv); or may be one comprising a variable heavy chain as given in SEQ ID NO: 25 mutated according to (i) or (v) and a variable light chain as given in SEQ ID NO:24 mutated according to (ii) or (vi).

The invention also encompasses antigen-binding fragments of any of the above antibodies.

The invention further relates to isolated nucleic acids encoding any of the above antibodies or antigen-binding fragments.

Any of the above antibodies, antigen-binding fragments or nucleic acids encoding any of them is intended for use as a medicament. Such use can be a single use (monotherapy) or a use in combination with an additional therapeutic agent (combination therapy). To this end, at least one of the above antibodies, antigen-binding fragments or nucleic acids encoding any one of them may be formulated in a pharmaceutical composition which may further comprise a pharmaceutically acceptable carrier and, optionally, an additional therapeutic agent. Said medicament is intended for treatment of tumors including cancer, ophthalmologic disorders and disorders characterized by neo -vascularization.

Vectors comprising any of the above nucleic acids and host cells comprising such vector and/or expressing any of the above antibodies or antigen-binding fragments thereof are further part of the invention, as are method for producing any of the above antibodies or antigen-binding fragments thereof. The invention further relates to derivatives of any of the above-described antibodies or antigen binding fragments thereof.

The invention further relates to the use of any of the above-described antibodies or antigen binding fragments thereof, or of a nucleic acid encoding any thereof in the manufacture of a medicament.

FIGURE LEGENDS

FIGURE 1. The Biotin-Ahx-EISQEVRGDVFPSY-CONH₂ peptide (with Ahx = aminohexanoic acid, a 6-carbon spacer), as well as an irrelevant control peptide were obtained from Biomer Technology by custom synthesis. The peptide EISQEVRGDVFPSY is defined in SEQ ID NO:51. The Biotin-Ahx-EISQEVRGDVFPSY-CONH₂ peptide was coated on a streptavidin chip to a surface density of - 1100 RU, and the control peptide was coated on a reference flow cell at a surface density of ~ 830 RU. The different antibodies (250 nM) were then injected over both flow cells for 5 min at a flow rate of 5 μΐ/min. The surfaces were regenerated between each run by a 2-min injection of glycine -HCl pH 2.5. The data represent the sensorgrams obtained by subtracting the signal of the reference flow cell from the one obtained on the Biotin-Ahx- EISQEVRGDVFPSY-CONH₂ surface. The experiment was performed with the help of a Biacore 3000 instrument.

FIGURE 2. Illustration of the inhibitory activity of the anti-human lactadherin antibody 215A9 and the humanized positive control antibody Mc3 on Huvec adhesion to human lactadherin.

FIGURE 3. Illustration of the inhibitory activity of the anti-human lactadherin antibodies 31 1A7, 346B6, 399A12 and 416H9 on Huvec adhesion to human lactadherin.

FIGURE 4. Illustration of the inhibitory activity of the anti -human lactadherin antibodies 31 1A7, 346B6, 399A12 and 416H9 on SKOV-3- and MDA-MB-231-cell adhesion to human lactadherin. Figure 4A: Dose-response effect of the antibodies, as compared to huMC3 in the adhesion assay. Mean of two experiments, with standard deviations are represented. Figure 4B: MDA-MB-231 adhesion assay on 5μg/mL lactadherin, and inhibition by l(^g/mL antibody, represented as slope values between 0 and 1 hour (left axes) or % of cell adhesion (right axis).

FIGURE 5. Illustration of the inhibitory activity of the anti-human lactadherin antibodies 311A7, 346B6, 399A12 and 416H9 on SKOV-3 cell migration. Dose-response effect of the antibodies, as compared to huMC3 in the migration assay. 100% migration corresponds to 5μg/mL lactadherin in the lower compartment of the xCelllgence CIM plate without antibodies. Mean of two experiments, with standard deviations are represented.

FIGURE 6. Illustration of the inhibitory activity of the anti-human lactadherin antibodies 31 1A7, 346B6, 399A12 and 416H9 on lactadherin-induced SKOV-3 cell survival. Dose- response effect of the antibodies, as compared to huMC3 in the survival assay. 100% survival corresponds to 5μg/mL recombinant lactadherin in 0.1% fetal calf serum without antibodies. Mean of two experiments, with standard deviations are represented.

DE TAILED DESCRIPTION OF THE INVENTION

Work on lactadherin as a therapeutic target led to the invention of a series of isolated monoclonal antibodies binding to the antigen defined in SEQ ID NO:51 and capable of inhibiting cell adhesion and/or migration and/or survival in the presence of human lactadherin.

(i) heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 47 to 49, respectively; and light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 32, 37, and 38, respectively;

(ii) heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 47 to 49, respectively; and light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 32 to 34, respectively;

(iii) heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 47 to 49, respectively; and light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 32, 35 and 36, respectively;

(vi) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively, or light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 29 to 31, respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 39 to 41, respectively; (vii) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively, or light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 29 to 31, respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 45, 46, and 44, respectively;

(viii) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively, or light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 29 to 31, respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 42 to 44, respectively;

(ix) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 45, 46, and 41, respectively;

(x) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 39 to 41 , respectively;

(xiii) an affinity matured antibody of any of (i) to (xii).

Any of the above antibodies can be a mammalian antibody, a human antibody or a humanized antibody. Further, any of the above antibodies can be a monovalent or multivalent antibody, and further be monospecific or multispecific provided that the binding specificity to said antigen (defined in SEQ ID NO:51) is maintained.

The term "antibody" as used herein refers to naturally occurring antibodies (immunoglobulins or IgGs). These comprise two heavy chains linked together by disulfide bonds and two light chains, one light chain being linked to each of the heavy chains by disulfide bonds. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains (three or four constant domains, CHI , CH2, CH3 and CH4, depending on the antibody class). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at its other end; the constant domains of the light chains each align with the first constant domains of the heavy chains, and the light chain variable domains each align with the variable domains of the heavy chains. This type of antibodies exist in camels, dromedaries and llamas along with an "unconventional" naturally occurring type of antibodies consisting of only two heavy chains, and thus being devoid of light chains. Other "unconventional" naturally occurring antibodies exist in in the serum of nurse sharks (Ginglymostomatidae) and wobbegong sharks (Orectolobidae). These latter antibodies are called Ig new antigen receptors (IgNARs). They are disulfi de -bonded homodimers consisting of five constant domains (CNAR) and one variable domain (VNAR). There is no light chain, and the individual variable domains are independent in solution and do not appear to associate across a hydrophobic interface (Greenberg et al. 1995, Nature 374, 168-173; Nuttall et al. 2001, Mol Immunol 38, 313-326; Diaz et al. 2002, Immunogenetics 54, 501-512; Nuttall et al. 2003, Eur J Biochem 270, 3543-3554). Due to the heavy chain structure characteristic of camelid and shark antibodies, these are sometimes termed "Heavy-Chain Mini-Antibodies" (mnHCAbs) or simply "Mini-Antibodies" (mnAbs) (Holliger & Hudson 2005, Nature Biotechnol 23, 1 126- 1 136). The complementary determining region 3 (CDR3) of camel antibodies and shark antibodies is usually longer (comprising about 16-21 amino acids, and about 16-27 amino acids, respectively) than the CDR3 of mouse VH region (comprising about 9 amino acids) (Muyldermans et al. 1994, Prot Eng 7, 1129-1135; Dooley & Flajnik 2005, Eur J Immunol 35, 936-945). Without the light chain, these heavy-chain antibodies bind to their antigens by one single domain, the variable antigen binding domain of the heavy-chain immunoglobulin, referred to as Vab (camelid antibodies) or V-NAR (shark antibodies). These smallest intact and independently functional antigen-binding fragment Vab are referred to as nano-antibody or nanobody (Muyldermans 2001, J Biotechnol 74, 277-302). Multivalent (etc. divalent, trivalent, tetravalent and pentavalent) Vab and/or V-NAR domains may be preferred in some instances due to their potentially higher cellular intake and retention and may be made by recombinant technology or by chemical means, such as described in WO 2010/033913. The variable domains of each pair of light and heavy chains are involved directly in binding the antibody to the antigen. The variable domains of naturally occurring light and heavy chains have the same general structure: four framework regions (FRs) connected by three complementarity determining regions (CDRs) (see e.g. Kabat et al. 1991 , Sequences of Proteins of Immunological Interest, 5^th Ed. Public Health Service, National Institutes of Health, Bethesda, MD). The CDRs in each chain are held in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site.

The term "complementarity determining region" or "CDR" as used herein in general refers to the hypervariable regions of light and heavy chains of classical 4-chain antibodies and as defined by the Kabat rules, more in particular the Kabat rules as available on http://www.bioinf.org.uk/abs/ and more particular in the "How to identify the CDRs by looking at a sequence" section.

Antibodies have been modified in order to increase their antigen-binding valency by several means including chemical homodimerization by introducing a thioether bond between 2 IgGs, e.g., Ghetie et al. 1997, Proc Natl Acad Sci USA 94, 7509-7514; WO 99/02567; Wolff et al. 1993, Cancer Res 53, 2560-2565), di- or polymerization via intermolecular disulfide bonding after engineering one or more cysteines into the heavy chain carboxyterminal ends (e.g. Shopes 1992, J Immunol 148, 2918-2922; W091/19515; Smith & Morrison 1994, BioTechnology 12, 683-688).

Bispecific or bifunctional antibodies have been produced using the quadroma technology based on the somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies with the desired specificities of the bispecific antibody (Milstein & Cuello 1983, Nature 305, 537-540). Alternatively, bispecific or bifunctional antibodies can be produced by chemical conjugation of two different mAbs or Ab fragments (Staerz et al. 1985, Nature 314, 628-631; Brennan et al. 1985, Science 229, 81-83). Further, recombinant bispecific or bifunctional antibody formats have been developed (Kriangkum et al. 2001, Biomol Eng 18, 31- 40). Amongst them tandem single-chain Fv molecules and diabodies starting from two single- chain Fv (scFv) fragments that recognize different antigens (see Economides et al. 2003, Nat Med 9, 47-52). In tandem scFv molecules (taFv), two scFv molecules with an additional peptide linker are simply connected together. Various linkers can be used to connect the two scFv fragments and linkers with a length of up to 63 residues (Nakanishi et al. 2001 , Annu Rev Immunol 19, 423-474). Using a very short Ala3 linker or long glycine/serine-rich linkers, expression of soluble tandem scFv molecules in bacteria has been achieved (Leung et al. 2000, J Immunol 164, 6495-6502; Ito et al.2003, J Immunol 170, 4802-4809; Kami et al. 2002, J Neuro immunol 125, 134-140) and a preferred linker thereto may be the one as described by Arndt & Krauss 2003, Methods Mol Biol 207, 305-321).

Diabodies are produced from scFv fragments by reducing the length of the linker connecting the VH and VL domain to approximately 5 residues (Peipp & Valerius 2002, Biochem Soc Trans 30, 507-51 1). This reduction of linker size facilitates dimerization of two polypeptide chains by crossover pairing of the VH and VL domains. Bispecific diabodies are produced by expressing, two polypeptide chains with, either the structure VHA-VLB and VHB-VLA (VH-VL configuration), or VLA-VHB and VLB-VHA (VL-VH configuration) within the same cell. Multivalent "abodies" also exist, such as triabodies and tetrabodies.

To avoid the problem of inactive homodimer formation, one approach to force generation of bispecific diabodies is the production of knob-into-hole diabodies (Holliger et al. 1993, Proc Natl Acad Sci USA 90, 6444-6448). By amino acid changes, a large knob is engineered in the VH domain and a complementary hole is engineered in the VL domain.

Single-chain diabodies (scDb) represent an alternative strategy to improve the formation of bispecific diabody-like molecules (Holliger & Winter 1997, Cancer Immunol Immunother 45, 128-130; Wu et al. 1996, Immunotechnology 2, 21-36). Bispecific single-chain diabodies are produced by connecting the two diabody-forming polypeptide chains with an additional middle linker with a length of approximately 15 amino acid residues. Consequently, all molecules with a molecular weight corresponding to monomeric single-chain diabodies (50-60 kDa) are bispecific.

Diabodies can be fused to Fc to generate a more Ig-like molecules, named didiabody (Lu et al. 2004, J Biol Chem 279, 2856-2865). In addition, multivalent antibody constructs comprising two Fab repeats in the heavy chain of an IgG and capable of binding four antigen molecules has been described (see WO 0177342A1 , and Miller et al. 2003, J Immunol 170, 4854-4861).

The term "antibody fragment" refers to any molecule comprising one or more fragments of an antibody (the parent antibody) such that it binds to the same antigen to which the parent antibody binds. Antibody fragments include Fv, Fab, Fab', Fab'-SH, single-chain antibody molecules (such as scFv), F(ab')₂, single variable VH domains, and single variable VL domains (Holliger & Hudson 2005, Nature Biotechnol 23, 1 126-1 136). The term further includes microantibodies, i.e. the minimum recognition unit of a parent antibody usually comprising just one CDR (Heap et al. 2005, J Gen Virol 86, 1791 -1800). Any of the fragments can be incorporated in a multivalent and/or multispecific larger molecule, e.g. mono- or bi-specific Fab₂, mono- or tri-specific Fab₃, bis-scFv (mono- or bispecific), diabodies (mono-or bi-specific), triabodies (e.g. trivalent monospecific), tetrabodies (e.g. tetravalent monospecific), minibodies and the like (Holliger & Hudson 2005, Nature Biotechnol 23, 1126-1 136). Any of the fragments can further be incorporated in e.g. V-NAR domains of shark antibodies, VhH domains of camelid antibodies, or nano(-anti)bodies. All these are included in the term "antibody fragment".

The term "monoclonal antibody" refers to a population of substantially homogeneous antibodies. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler & Milstein 1975, Nature 256, 495-497), or may be made by recombinant DNA methods (e.g. US 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, e.g., Clackson et al. 1991 , Nature 352, 624- 628 or Marks et al. 1991, J Mol Biol 222, 581 -597.

The term "chimeric antibody" refers to an antibody pieced together with portions derived from antibodies of the same species (e.g. antibodies of different classes) or different species, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (e.g. US 4,816,567; Morrison et al. 1984, Proc Natl Acad Sci USA 81 , 6851-6855). For instance, "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the non-human immunoglobulin are replaced by corresponding human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.

Examples of humanized versions of the murine antibodies of the invention include antibodies or fragments thereof comprising at least one of:

(iii) a variable heavy chain as given in SEQ ID NO: 22 carrying one or more of the following mutations: Gln5Val, Pro9Ala, Leul lVal, Argl9Lys, Asp41Pro, Lys43Gln, Thr44Gly, Ser76Thr, Gln82Glu, Thr87Arg, Ser91Thr, and/or Serl 16Thr;

(vi) a variable light chain as given in SEQ ID NO:24 carrying one or more of the following mutations: Leul5Val, Asp41Gly, Thr43Ala, Asn77Ser, Gln80Pro, AlalOOGln, and/or Leul06Ile. Herein, the generalize annotation "XaaNNXbb", e.g., Gln5Val in SEQ ID NO:22, refers to the mutation of an amino acid Xaa at position within the given sequence to an amino acid Xbb, e.g., the mutation of a glutamine at position 5 in SEQ ID NO:22 to a Valine. Such humanized antibodies or fragments thereof may for instance comprise any of the above- listed humanized versions of a variable heavy chain as given in SEQ ID NO:22 and any of the above-listed humanized versions of a variable light chain as given in SEQ ID NO:21. Such humanized antibodies or fragments thereof may for instance comprise any of the above-listed humanized versions of a variable heavy chain as given in SEQ ID NO:25 and any of the above - listed humanized versions of a variable light chain as given in SEQ ID NO:24.

A "human antibody" is an antibody produced by a human and/or that has been made using any of the techniques for making human antibodies. Human antibodies can be produced using various techniques known in the art such as by selection from a phage library expressing human antibodies (e.g.,Vaughan et al. 1996, Nature Biotechnol 14, 309-314 (1996); Sheets et al. 1998, Proc Natl Acad Sci USA 95, 6157-6162; Hoogenboom & Winter 1991, J Mol Biol 227, 381-388; Marks et al. 1991 , J Mol Biol 222, 581-597). Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed (e.g., US 5,545,807; US 5,545,806; US 5,569,825; US 5,625,126; US 5,633,425; US 5,661 ,016; Marks et al. 1992, BioTechnology 10, 779-783; Lonberg et al. 1994, Nature 368, 856-859; Morrison 1994, Nature 368, 812-813; Fishwild et al. 1996, Nature Biotechnol 14, 845-851; Lonberg & Huszar 1995, Internat Rev Immunol 13,65-93). Alternatively, the human antibody may be prepared via immortalization of human lymphocytes producing an antibody directed against a target antigen wherein such lymphocytes may be recovered from an individual or may have been immunized in vitro (e.g., Cole et al. 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77; Boerner et al. 1991, J Immunol 147, 86-95; US 5,750,373). The term "defined in/by SEQ ID NO:X" as used herein refers to a biological sequence consisting of the sequence of amino acids or nucleotides given in the SEQ ID NO:X. For instance, an antigen defined by SEQ ID NO:X consists of the amino acid sequence given in SEQ ID NO:X. A further example is an amino acid sequence comprising SEQ ID NO:X, which refers to an amino acid sequence longer than the amino acid sequence given in SEQ ID NO:X but entirely comprising the amino acid sequence given in SEQ ID NO:X (wherein the amino acid sequence given in SEQ ID NO:X can be located N-terminally or C-terminally in the longer amino acid sequence, or can be embedded in the longer amino acid sequence), or to an amino acid sequence consisting of the amino acid sequence given in SEQ ID NO:X.

The invention further comprises methods for selecting antibodies with increased affinity to the antigen defined by SEQ ID NO:51, said methods including the steps of (i) subjecting any of the above described antibodies to a process of affinity maturation and (ii) selecting an antibody with increased affinity to the antigen defined by SEQ ID NO: 51.

An "affinity matured antibody" is the result of an affinity maturation process applied to a parent antibody. An affinity matured antibody usually differs from its parent antibody in one or more amino acid positions, including in one or more CDR regions, said differences resulting in an improved affinity of the affinity matured antibody for an antigen compared to the affinity of the parent antibody for the same antigen. Affinity maturation processes include those as described by e.g. Marks et al. 1992, BioTechnology 10, 779-783 (affinity maturation by VH and VL domain shuffling). Random mutagenesis of CDR and/or framework residues is described by e.g. Barbas et al. 1994, Proc Natl Acad Sci USA 91, 3809-3813; Schier et al. 1996, Gene 169: 147- 155; Yelton et al. 1995, J Immunol 155, 1994-2004; Jackson et al. 1995, J Immunol 154, 3310- 3319; Hawkins et al. 1992, J Mol Biol 226, 889-896. Derivatives of the antibodies of the invention, or of antigen-binding fragments of said antibodies, include, but are not limited to antibodies or fragments thereof labeled with an appropriate label, said label can for instance be of the enzymatic, colorimetric, chemiluminescent, fluorescent, or radioactive type. Derivatives of an antibody of the invention generally include all molecules resulting from conjugation of said antibody or fragment thereof with another compound. Such other compound may be, e.g., used to increase stability (e.g., half-life) and/or solubility of the antibody or antibody-fragment; an enzyme capable of converting a prodrug to its active form (e.g. for use in chemotherapy); or may itself have cytostatic and/or cytotoxic properties. Exemplary modifications include pegylation, introduction (by insertion or mutation) of a non-naturally occurring cysteine in the antibody or antibody-fragment backbone (to create a cross-linking site), glycosylation (synthetic or via recombinant means) and the like. A further example of derivation relates to linking of a cytotoxic agent (a substance that inhibits or prevents the function of cells and/or causes destruction of cells) such as radioactive isotopes (e.g. At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶,

Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), chemo therapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Other derivatives of the antibodies of the invention include bispecific antibodies. Such antibodies are specific for the lactadherin protein (as described above) on the one hand and specific for a second antigen on the other hand. The second antigen may, e.g., be any art-recognized tumor-specific antigen or tumor-associated antigen. Such lactadherin- and tumor-antigen-bispecific antibodies may increase the efficiency of killing lactadherin-expressing cells. Typically, the lactadherin-specificity of bispecific antibodies would be obtained by including one or more of the light- or heavy-chain variable domains or one or more of CDRs of the light- or heavy-chain variable domains of the antibodies of the invention.

The invention further relates to isolated nucleic acids encoding any of the above-described lactadherin-binding antibodies or antigen-binding fragments. Anyone somewhat familiar with the genetic code will be capable of translating any protein sequence into a nucleotide sequence. Moreover, tools to perform such "reverse translation" or "backtranslation" are widely available such as http://www.bioinformatics.org/sms2/rev_trans.html or http://arbl.cvmbs.colostate.edu/molkit/rtranslate/index.html or http://www.entelechon.com 2008/10/backtranslation-tool/. If required, adaptation of a nucleotide sequence to a given species, i.e. adaptation of the codon usage to optimize expression in a given species, nowadays also is common knowledge, and again freely accessible web-tools are available such as the codon usage database on http://www.kazusa.or.jp/codon/ or such as http://genomes.urv.es/OPTIMIZER/ (over 150 prokaryotic species). Any of the above antibodies, antigen-binding fragments or nucleic acids encoding any of them is intended for use as a medicament. Such use can be a single use (monotherapy) or a use in combination with an additional therapeutic agent (combination therapy). To this end, at least one of the above antibodies, antigen-binding fragments or nucleic acids encoding any one of them may be formulated in a pharmaceutical composition which may further comprise a pharmaceutically acceptable carrier and, optionally, an additional therapeutic agent.

Said medicament is intended for example for prevention, inhibition or treatment of benign, pre- malignant or malignant tumors, ophthalmologic disorders, and disorders characterized by neovascularization. "Cancer" refers to malignant neoplastic tumors and includes carcinomas (starting in the skin or in tissues that line or cover internal organs; includes skin-, lung-, colon-, pancreatic-, and ovarian cancers, and epithelial-, squamous- and basal cell carcinomas, melanomas, papillomas, and adenomas); sarcomas (starting in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue; includes bone- and soft tissue cancers, and osteosarcoma, synovialsarcoma, liposarcoma, angiosarcoma, rhabdosarcoma, and fibrosarcoma), leukemias (starting in blood- forming tissue such as the bone marrow and causes large numbers of abnormal blood cells to be produced and enter the blood; includes leukemia, lymphoblastic leukemias (ALL and CLL), myelogenous leukemias (AML and CML), T-cell leukemia, and hairy-cell leukemia), lymphomas and myelomas (starting in the cells of the immune system; including lymphoma, T-cell lymphomas, B-cell lymphomas, Hodgkin lymphomas, non-Hodgkin lymphoma, and lymphoproliferative lymphomas), central nervous system cancers (starting in the tissues of the brain and spinal cord; including brain and spinal cord tumors, gliomas, meningiomas, pituitary adenomas, vestibular schwannomas, primary CNS lymphomas, and primitive neuroectodermal tumors) and metastatic cancers (usually arising from a cell type listed above and now present in a tissue from which the cancer cells did not originally develop). A particular example of cancer is the triple-negative breast cancer which is not expressing the genes for estrogen receptor (ER), progesterone receptor (PR) or Her2/neu).

"Ophthalmologic disorders" include diabetic retinopathy (non-proliferative or proliferative), age- related macular edema, rubeosis iridis (growth of new abnormal blood vessels in the iris), choroidal neovascularization (CNV; growth of new blood vessels in the choroid layer of the eye), degenerative maculopathies, corneal neovascularization, neovascular glaucoma, retinopathy of prematurity, hyperplastic vitreous syndrome.

Disorders characterized by neo-vascularization, neo-angiogenesis or pathologic angiogenesis (all used interchangeably) refers to disorders or diseases characterized by the formation of new blood vessels (capillary ingrowth and endothelial proliferation) in unusual sites, a finding typical of so- called "angiogenic diseases" . An extensive list of disorders characterized by neo-vascularization is given in Table 1 of Carmeliet 2003 (Nature Medicine 9, 653-660) and includes, besides tumors and ophthalmologic disorders: infectious diseases, autoimmune disorders, vascular malformations, DiGeorge syndrome, HHT, cavernous hemangioma, atherosclerosis, transplant arteriopathy, obesity, psoriasis, warts, allergic dermatitis, scar keloids, pyogenic granulomas, blistering disease, pulmonary hypertension, asthma, nasal polyps, inflammatory bowel and periodontal disease, ascites, peritoneal adhesions, endometriosis, uterine bleeding, ovarian cysts, ovarian hyperstimulation, arthritis, synovitis, osteomyelitis, and osteophyte formation.

The term "combination therapy" as used herein refers to any type of combination. If technically feasible and clinically meaningful, two or more different active substances, one being an anti- lactadherin antibody or fragment thereof, or nucleic acid encoding any thereof, according to the invention, can be combined in a single medicament or formulation. Alternatively, the two or more different active substances are provided as individual medicaments (wherein the medicament not comprising an anti-lactadherin antibody or fragment thereof or nucleic acid encoding any thereof according to the invention can still be a combination of two or more other active substances) to be administered to the patient in a prescribed dosing regimen which can involve sequential and/or concurrent administration or administrations. It may well be that multiple administrations of the other active substance are required, one of which may be concurrent with the administration of the anti-lactadherin antibody or fragment thereof, or nucleic acid encoding any thereof, according to the invention. This may especially be the case if the pharmacokinetics of said other active substance is totally different from the pharmacokinetics of the anti-lactadherin antibody or fragment thereof, or nucleic acid encoding any thereof, according to the invention. Antibodies indeed have the advantage of having favorable pharmacokinetics in the sense that they have a relatively long half life. The sequential administration of two medicaments implies that the administration of the anti-lactadherin antibody or fragment thereof, or nucleic acid encoding any thereof, according to the invention is preceded or followed by at least one administration of the other active substance or medicament. The additional active substance or agent can be any agent recognized in the art as useful in the prevention, inhibition or treatment of the intended disease or disorder. In general, the additional active substance or agent can for instance be a chemical agent (e.g. targeting enzymes or proteins, DNA or RNA, including synthetic aptamers, siRNAs, antisense RNAs and the like) or chemotherapeutic agent (e.g. general cytostatic or cytotoxic agent), a biological agent (e.g. antibodies or fragments thereof or protein scaffold-based molecules), an anti-inflammatory agent, an antiviral agent, an antibacterial agent, an anti-angio genie agent, an anti -mitotic agent, an antihistamine, an anesthetic, an agent to induce mydriasis and an agent to induce cycloplegia. The additional active substance or agent may further be irradiation (e.g. in the treatment of some cancers) or laser therapy (e.g. in the treatment of some eye diseases).

Examples of anti-angiogenic agents include antibodies (or fragments thereof) such as anti-VEGF (vascular endothelial growth factor) or anti-PlGF (placental growth factor) antibodies and agents such as macugen (pegaptanib sodium), trypthophanyl-tRNA synthetase (TrpRS), anecortave acetate, combrestatin A4 prodrug, AdPEDF (adenovector capable of expressing pigment epithelium-derived factor), inhibitor of VEGF receptor-2, inhibitors of VEGF, P1GF or TGF-β, Sirolimus (rapamycin) and endostatin.

Examples of approved biological agents used in treatment of cancer or ophthalmologic disorders include bevacizumab (anti-VEGF), ranibizumab (anti-VEGF), aflibercept (or VEGF Trap-Eye), rituximab and ibritumomab tiuxetan (anti-CD20), trastuzumab (anti-Her2), gemtuzumab ozogamicin (anti-CD33), and alemtuzumab (anti-CD52).

Examples of chemotherapeutic agents useful in the treatment of cancer include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (e.g. bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1 065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin γ and calicheamicin θ (e.g., Nicolaou et al. 1994, Angew Chem Intl Ed Engl 33, 183-186); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifiuridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spiro germanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g. paclitaxel (TAXOL ® Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LYl 17018, onapristone, and toremifene (Fareston); and anti-androgens such as fiutamide, nilutamide, bicalutamide, leuprolide, and goserelin; ilnd pharmaceutically acceptable salts, acids or derivatives of any of the above.

Examples of anti-inflammatory agents include steroids (e.g. prednisolone, methylprednisolone, cortisone, hydrocortisone, prednisone, triamcinolone, dexamethasone) and non-steroidal anti- inflammatory agents (NSAIDs; e.g. acetaminophren, ibuprofen, aspirin).

Examples of antiviral agents include trifluridine, vidarabine, acyclovir, valacyclovir, famciclovir, and doxuridine. Examples of antibacterial agents or antibiotics include ampicillin, penicillin, tetracycline, oxytetracycline, framycetin, gatifioxacin, gentamicin, tobramycin, bacitracin, neomycin and polymyxin.

Examples of anti-mycotic/fungistatic/antifungal agents include fluconazole, amphotericin, clotrimazole, econazole, itraconazole, miconazole, 5-fiuorocytosine, ketoconazole and natamycin. Examples of anti-mitotic agents include mitomycin C and 5-fiuorouracyl.

Examples of antihistamines includes ketitofen fumarate and pheniramine maleate. Examples of anesthetics include benzocaine, butamben, dibucaine, lidocaine, oxybuprocaine, pramoxine, proparacaine, proxymetacaine, tetracaine and amethocaine.

For ophthalmological application or use, other adjunct agents or drugs can be used in conjunction with the anti-lactadherin antibody or fragment thereof according to the invention, or nucleic acid encoding any thereof, including agents inducing mydriasis/pupillary dilation (e.g. scopoloamine, atropine or tropicamide, and/or cycloplegia (paralysis of the eye focusing muscle). Lubricants may in the ophthalmological setting also be required, such include propylene glycerol, glycerin, carboxymethylcellulose, hydroxypropylmethylcellulose, soy lecithin, polyvinyl alcohol, white petrolatum, mineral oil, povidone, carbopol 980, polysorbate 80, and dextran 70.

In case of treating or inhibiting neovascular glaucoma, an agent for controlling the intra-ocular pressure may also be used in conjunction with the anti-lactadherin antibody or fragment thereof, or nucleic acid encoding any thereof, according to the invention. Such medicaments include adrenergic blocking agents (beta blockers or sympatholytic drugs such as betaxolol, carteolol, levobunolol, metipanolol and timolol), adrenergic stimulating agents (sympathomimetic drugs such as aproclonidine, epinephrine, hydroxy amphetamine, phenylephrine, naphazoline and tetrahydrozaline), carbonic anhydrase inhibitors (such as systemic acetozolamide, and topical brinzolamide and dorzolamide), miotics (cholinergic stimulating agents, parasympathomimetic drugs such as carbachol and pilocarpine), osmotic agents (such as glycerin and mannitol), prostaglandin and prostaglandin analogues (prostamides, bimatoprost, unoprostone isopropyl, travoprost, latanoprost, natural prostaglandin, prostaglandin F2a, and FP prostanoid receptor agonists). The anti-lactadherin antibody or fragment thereof according to the invention, or the nucleic acid encoding any of these, can be used for the manufacture of a medicament. Therefore, the active substance may need to be formulated into a "pharmaceutically acceptable formulation". Such formulation in general is a composition comprising a carrier, diluent or adjunvant compatible with the one or more active ingredients to be formulated, the whole formulation being compatible with the intended use in the intended tissue or organ, etc. Examples of pharmaceutically acceptable formulations as well as methods for making them can be found, e.g., in Remington's Pharmaceutical Sciences (e.g. 20^th Edition; Lippincott, Williams & Wilkins, 2000) or in any Pharmacopeia handbook (e.g. US-, European- or International Pharmacopeia).

A "diluent, carrier or adjuvant", is any suitable excipient, diluent, carrier and/or adjuvant which, by itself, does not induce the production of antibodies harmful to the individual receiving the composition. Typically, pharmaceutically acceptable compounds (such as diluents, carriers and adjuvants) can be found in, e.g., a Pharmacopeia handbook (e.g. US-, European- or International Pharmacopeia). A "diluent", or more in particular a "pharmaceutically acceptable diluent", includes diluents such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives may be included in such diluents. A (pharmaceutically acceptable) carrier or adjuvant may enhance the response elicited by an antibody or fragment thereof according to the invention, e.g., by providing a continuous release of the antibody or fragment thereof, or nucleic acid encoding any thereof, according to the invention over a prolonged period of time (slow-release formulations). The term "adjuvant" usually refers to a pharmacological or immunological agent that modifies (preferably increases) the effect of other agents (e.g., drugs, vaccines) while having few if any direct effects when given by themselves. As one example of an adjuvant aluminium hydroxide (alum) is given, to which an active compound or ingredient of the invention can be adsorbed. Further, many other adjuvants are known in the art and can be used. The term "pharmaceutically acceptable carrier" means any material or substance with which the active ingredient is formulated in order to facilitate its application or dissemination to the locus to be treated, for instance by dissolving, dispersing or diffusing the said composition, and/or to facilitate its storage, transport or handling without impairing its effectiveness. They may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents (for example sorbic acid or chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like, provided these are consistent with pharmaceutical practice, i.e. carriers and additives which do not create permanent damage to the subject receiving the carrier. Additional ingredients may be included in order to control the duration of action of the active ingredient in the composition. The pharmaceutically acceptable carrier may be a solid or a liquid or a gas which has been compressed to form a liquid, i.e. the compositions of this invention can suitably be used as concentrates, emulsions, solutions, granulates, dusts, sprays, aerosols, suspensions, ointments, creams, tablets, pellets or powders. Suitable pharmaceutical carriers for use in said pharmaceutical compositions and their formulation are well known to those skilled in the art, and there is no particular restriction to their selection within the present invention. The pharmaceutical compositions of the present invention may be prepared in any known manner, for instance by homogeneously mixing, coating and/or grinding the active ingredients, in a one-step or multi-step procedure, with the selected carrier material and, where appropriate, the other additives such as surface-active agents. They may also be prepared by micronisation, for instance in view to obtain them in the form of microspheres usually having a diameter of about 1 to 10 μηι, namely for the manufacture of microcapsules for controlled or sustained release of the active ingredients. The medicament according to the invention may be prepared as an injectable, either as a liquid solution or suspension. Injection may be subcutaneous, intramuscular, intravenous, intra-arterial, intraperitoneal, intrathecal, intradermal, intraepidermal. In the case of local administration to the eye, intravitreal injection, injection into the anterior chamber or subconjunctival injection may for instance be performed. The composition may also be prepared to make it suitable for other types of administration such as implantation, suppositories, oral ingestion, enteric application, inhalation, aerosolization, eye drops, nasal spray or drops, or administration through medical devices such as stents. Solid forms, suitable for dissolving in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or encapsulated in liposomes for enhancing its effect. The preparation may be administered to a subject as a bolus dose or by continuous infusion. The preparation may also be administered continuously via an osmotic minipump. For the purpose of treating, preventing or inhibiting an intended disease or disorder, and in methods for treating, preventing or inhibiting an intended disease or disorder, an effective amount of the anti-lactadherin antibody or fragment thereof according to the invention, or the nucleic acid encoding any of these, is administered to a subject in need thereof. An "effective amount" of an active substance in a composition is the amount of said substance required and sufficient to elicit an adequate response in preventing or treating or reducing the intended or targeted medical indication. It will be clear to the skilled artisan that such response may require successive (in time) administrations with the composition as part of an administration scheme or -schedule. The effective amount may vary depending on the health and physical condition of the individual to be treated, the age of the individual to be treated (e.g. dosing for infants may be lower than for adults) the taxonomic group of the individual to be treated (e.g. human, non-human primate, primate, etc.), the capacity of the individual's system to respond effectively, the degree of the desired response, the formulation of the active substance, the treating doctor's assessment and other relevant factors. The effective amount further may vary depending on whether it is used in monotherapy or in combination therapy. It is expected that the effective amount of the active substance of the invention (anti-lactadherin antibody or fragment thereof or derivative thereof) will fall in a relatively broad range that can be determined through routine trials. Usually, the amount will vary from 0.01 to 1000 μg/dose, more particularly from 0.1 to 100 μg/dose. Alternatively, the active substance may be administered at a dose between 1 μg/kg body weight and 10 mg kg body weight, or between 10 μg/kg body weight and 5 mg kg body weight, or between 100 μg kg body weight and 2 mg/kg body weight. Dosage treatment may be a single dose schedule or a multiple dose schedule. If the active substance is administered continuously, administered doses may be between 1 and 100 μg/kg/minute, between 1 and 50 μg/kg/minute, between 5 and 50 μg/kg/minute, or between 5 and 20 μg/kg/minute.

Preventive/prophylactic administration of (an effective amount of) the anti-lactadherin antibody or fragment thereof according to the invention, or the nucleic acid encoding any of these, may be useful in e.g. the ophthalmological setting. Indeed, in case of an ophthalmological disease or disorder that responds to treatment with the anti-lactadherin antibody or fragment thereof according to the invention, or the nucleic acid encoding any of these, a subject may suffer from such disease or disorder in a single eye. It is known in the art, however, that in such cases the companion other eye of the same subject is susceptible to develop the same disease or disorder. In such instances, the unaffected eye could be prophylactically treated with the anti-lactadherin antibody or fragment thereof according to the invention, or the nucleic acid encoding any of these.

"Treatment" refers to any rate of reduction or retardation of the progress of the disease or disorder compared to the progress or expected progress of the disease or disorder when left untreated. More desirable, the treatment results in no/zero progress of the disease or disorder (i.e. "inhibition") or even in any rate of regression of the already developed disease or disorder.

The invention further relates to an antibody or fragment thereof, or derivative of any thereof, according to the invention for use as diagnostic tool. One exemplary diagnostic method in which the antibodies (or fragments or derivatives of any thereof) according to the invention can be used is the detection of lactadherin protein in isolated cells (isolated such as by, e.g., biopsy) suspected to be tumor cells, or in biological fluids (such as e.g. blood, ascites). The presence of lactadherin in the isolated cells or fluids, especially if overexpressed, can subsequently be used as criterion for applying the anti- lactadherin antibodies in a therapy for eradicating the tumor cells. The isolated nucleic acids encoding the anti-lactadherin antibody or fragment thereof according to the invention may be comprised in a recombinant vector, in particular an expression vector.

A further aspect of the invention relates to isolated cell lines or recombinant host cells expressing an anti-lactadherin antibody or fragment thereof according to the invention. Said host cell can be any cell capable of expressing said antibody or fragment thereof. Suitable host cells include, but are not limited to, cultured mammalian (such as HEK293) or insect cells, cultured plant cells or transgenic plants, yeasts such a Saccharomyces, Schizosaccharomyces, Pichia, Hansenula, Torulopsis, and bacterial cells. Expression of the antibody of the invention or functionally equivalent fragment thereof may be transient or constitutive. In particular, the host cell is a hybridoma cell line such as the ones described above. Another aspect of the invention covers hybridoma cell lines expressing an antibody of the invention, in particular the hybridoma cell lines with any of the biological deposit accession numbers LMBP 9488CB (anti-lactadherin antibody 215A9), LMBP 9489CB (anti-lactadherin antibody 31 1A7), LMBP 9490CB (anti-lactadherin antibody 346B6), LMBP 9491CB (anti- lactadherin antibody 399A12), or LMBP 9492CB (anti-lactadherin antibody 416H9). These deposits were done under the conditions of the Budapest Treaty at the Belgian Co-ordinated Collections of Micro-organisms/Laboratory of Molecular Biology-Plasmid collection (BCCM/LMBP), Technologiepark 927, 9052 Gent (Zwijnaarde), Belgium on 15 March 2012.

Methods of producing the above -described lactadherin-binding antibodies, or antigen-binding fragments thereof, form an integral aspect of the invention. In particular, such methods can comprise the steps of:

(i) obtaining a crude preparation of said antibody or antibody fragment by means of recombinant expression of said antibody or antibody fragment, or by means of chemical synthesis of said antibody or antibody fragment;

(ii) purifying said antibody or antibody fragment from the crude preparation obtained in

(i).

Alternatively, an antigen-binding fragment of the antibodies of the invention binding to lactadherin can be obtained or produced by a method comprising the steps of:

(i) obtaining a crude preparation of an antibody comprising said fragment by means of recombinant expression of said antibody or by means of chemical synthesis of the antibody ;

(ii) purifying said antibody from the crude preparation obtained in (i).

(iii) isolating the antigen-binding fragment from the antibody purified in (ii).

EXAMPLES

The Examples included hereafter demonstrate the invention and are not construed to be limiting the scope of the invention in any way. EXAMPLE 1. Cloning and production of huMc3

The amino acid sequence of the huMc3 heavy and light chain variable domains is described in Couto et al, Cancer Res (1995) 55:1717-1722. DNA sequences encoding the described amino acid sequences were designed in silico, whereby codons were chosen in accordance with the average codon usage in over 90,000 human coding sequences as described in the codon usage database (http://www.kazusa.or.jp/codon/). The designed DNA sequences flanked by restriction sites were synthesized de novo by Integrated DNA Technologies (Coralville, Iowa) and cloned in a pBudCE4.1 (Life Technologies, Carlsbad, CA)-based mammalian expression vector with two expression cassettes. One expression cassette contained sequences for a signal peptide (GenBank accession number XI 7263) and the human kappa light chain constant region (GenBank accession number J00241) while the other cassette contained sequences for a signal peptide (GenBank accession number X70197) and the human IgGl heavy chain constant region (GenBank accession number J00228). The resulting, sequence-verified plasmid was grown in TOP 10 bacteria and prepared using the Qiagen Plasmid Maxi Kit (Qiagen, Venlo, The Netherlands). The huMc3 -encoding construct was subsequently transferred to the in -house developed expression vector pTG330 (huMc3/pTG330).

The production of huMC3 was carried out by transient transfection of the HEK-293 cells with huMc3/pTG330. Following the standard protocol recommended in the 293 FreeStyle Expression System from Invitrogen (cat n°: K900001), 5 L of huMC3 -containing supernatant at 20-25 mg/L was obtained. The huMC3 antibody was then purified on recombinant Protein A and dialyzed against a 10 mM Histidine-HCl buffer (pH 5.5) containing 6mg/ml Mannitol, 0.01% Tween-20.

EXAMPLE 2. Generation of murine monoclonal anti-lactadherin antibodies

Lactadherin knock-out mice were immunized with recombinant human and murine lactadherin (3 subcutaneous injections of human and murine lactadherin, 20 μg each, the first one in complete Freud adjuvant, the last two in incomplete Freud adjuvant). The antigens were obtained from R&D Systems, UK.

Blood samples were collected from the tail of the mice, and the sera were tested for the presence of anti-human and anti-murine lactadherin antibodies by ELISA. Briefly, 96-well ELISA plates were coated overnight with 1 μg/ml of human or murine lactadherin in PBS (100 μΐ/well, 4°C). The plates were blocked with 1 % BSA for 1 h at room temperature, the plasma samples (100 μΐ/well) were added to the plates and the antibodies were allowed to bind for 1 h at room temperature. Bound antibodies were detected with an HRP-labeled, goat anti-murine IgG (Sigma) (100 μΐ/well, 1 h at room temperature) followed by reaction with OPD. The mice then received an additional i.p. injection of 20 μg of both antigens in saline buffer.

At least 6 weeks after the third subcutaneous injection, the mice received a final i.p. injection of 20 μg of both antigens in saline buffer, and the fusion was performed. The mice were sacrificed and spleen cells were fused with SP2/0 myeloma cells according to the procedure of Galfre & Milstein (Methods Enzymol. 73, 3-46, 1981). After selection in HAT (hypoxanthine, aminopterine, thymidine) medium, positives clones were selected by screening the culture supematants by ELISA as described above. Positive clones were expanded, and the antibodies were purified by Protein A chromatography to allow further characterization. ELISA confirmed the binding of the purified antibodies to human lactadherin.

EXAMPLE 3. Cloning of cD As encoding the VH and VL chains of murine monoclonal anti-lactadherin antibodies

RNA was isolated from 5 -10 * 10⁶ hybridoma cells using the RNeasy Mini Kit from Qiagen (Venlo, The Netherlands) and cDNA was prepared using the QuantiTect Reverse Transcription Kit from Qiagen (Venlo, The Netherlands). PCR was performed with primers specific for the leader sequence and the constant region, respectively. Thus, the variable regions were contained in the PCR products but did not include the primer sequences. The primer combinations depicted in Tables 1 and 2 were used for each of the hybridomas. Table 1. Heavy Chain primers

R = A or G; W = A or T; K = G or T; H = A or C or T; S = C or G; Y = C or T; M = A or C; V = A or C or G

To amplify the V_H and V_L chains a touchdown PCR protocol was used with Accuprime Pfx Supermix (Life Technologies, Carlsbad, CA) in 25 μΐ reactions. After 30 seconds at 95°C, 3 cycles were run of 30 seconds 95°C, 30 seconds 64°C and 1 minute at 68°C, followed by 3 cycles of 30 seconds 95°C, 30 seconds 61°C and 1 minute at 68°C, 3 cycles of 30 seconds 95°C, 30 seconds 58°C and 1 minute at 68°C, 3 cycles of 30 seconds 95°C, 30 seconds 55°C and 1 minute at 68°C, 25 cycles of 30 seconds 95°C, 30 seconds 52°C and 1 minute at 68°C, to finish with 5 minutes at 72°C. All reactions were done at least twice. For the heavy chains, reactions were also repeated with a regular PCR (30 seconds at 95°C, 35 cycles of 30 seconds 95°C, 30 seconds 56°C and 1 minute at 68°C, 5 minutes at 72°C). The obtained PCR products were isolated from gel and inserted into pCR4Blunt-TOPO (Life Technologies, Carlsbad, CA) and sequenced with the Ml 3 Forward (-20) primer (GTAAAACGACGGCCAG; SEQ ID NO:9) and the M13 reverse primer (C AGG AAAC AGCT AT G AC ; SEQ ID NO: 10) using the BigDye Terminator V3.1 Cycle Sequencing Kit (Life Technologies, San Diego, CA).

Antibody sequences were isolated from 5 hybridomas. The hybridoma 215A9 resulted from one mouse, the hybridomas 399A12 and 416H9 resulted from a second mouse, and the hybridomas 31 1A7 and 346B6 resulted from a third mouse. The light and heavy chain variable region sequences isolated from the hybridomas 399A12 and 416H9 both are identical. The heavy chain variable region sequences isolated from the hybridomas 31 1A7 and 346B6, originating from the same mouse, are identical. Their light chain variable region sequences are not identical but highly similar. None of the determined sequences is identical to the huMc3 sequences. Light chain variable region, heavy chain variable region and complemenarity determining region (CDR) sequences of the different antibodies are given hereafter.

Light and Heavy Chain DNA sequences

Hybridoma 215 A9 - light chain

CAGGCTGTTGTGACTCAGGAATCTGCGCTCACCACATCACCCGGTGAAACCGTAACA CTC ACTTGTCGCTC AAGC ACTGGT GCTGTGACCACTAACAATTAT GCCAACTGGGTC CAGGAGAAACCGGATCATCTGTTTACTGGGCTGATAGGCGGGACCAACAATCGAAC TCCTGGCGTTCCAGCCAGGTTCTCAGGGTCCCTGATTGGAGACAAGGCTGCCCTCAC CATCACAGGCGCACAGACTGAGGATGAGGCGATCTACTTCTGCGCCCTGTGGTACA GCAATCTTTGGGTGTTTGGAGGTGGCACCAAACTGACTGTGCTCGGA (SEQ ID NO: l l)

Hybridoma 215 A9 - heavy chain

CAGGTTTCTCTGAAAGAGTCTGGCCCTGGGATATTGCAGTCCTCCCAGACCCTCAGT CTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTGAGTTGGA TTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGAATGATG ACAAGCGCTATAACCCATCCCTGAAGAGTCGACTCACAATCTCCAAGGATACCTCTA GAGACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACATACT TCTGTGCTCGAAGACCTAGGTTCTACCGTGATTACGACGAGGGGATTGACTTCTGGG GCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 12)

Hybridoma 31 1A7 - light chain

CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTC ACCATAACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTGGTTCCAGCAGAAG CCAGGCACTTCTCCCAAACTCTGGATTTATAGTACATCCAACCTGGCTTCTGGAGTC CCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGA ATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGAGTAGTTACCCACG GACGTTCGGTGGAGGCACCAAGCTGGAAATCAAGCGG (SEQ ID NO: 13)

Hybridoma 31 1 A7 - heavy chain

GAGTTCCAGCTGCAGCAGTCTGGACCTGAACTGGTGAAGCCTGGCGCTTCAGTGAG GATATCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACAACATGAACTGGGTGAA GCAGAGCGATGGAAAGACCCTTGAGTGGATTGGAGTTATTAATCCTGACTATGGTAC TACTAACAACAATCAGAGGTTCAGGGGCAAGGCCACATTAACTCTAGACCTATCTTC CAGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTA CTGTGCAAGAGGGGGAGATTACGGCGCGGGGTATGCTTTGGATTACTGGGGTCAAG GAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 14) Hybridoma 346B6 - light chain

CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTC ACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAA GCCAGGATCCTCCCCCAGACTCCTGATTTATGACACATCCAACCTGGCTTCTGGAGT CCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCCG AATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTTACCCACC CACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG (SEQ ID NO:15)

Hybridoma 346B6 -heavy chain

GAGTTCCAGCTGCAGCAGTCTGGACCTGAACTGGTGAAGCCTGGCGCTTCAGTGAG GATTTCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACAACATGAACTGGGTGAA GCAGAGCGATGGAAAGACCCTTGAGTGGATTGGAGTTATTAATCCTGACTATGGTAC TACTAACAACAATCAGAGGTTCAGGGGCAAGGCCACATTAACTCTAGACCTATCTTC CAGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTA CTGTGCAAGAGGGGGGGATTACGGCGCGGGGTATGCTTTGGATTACTGGGGTCAAG GAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 16) Hybridoma 399A12 - light chain = Hybridoma 416H9 - light chain

GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTC ACCATCACTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAG AAACCAGATGGAACTATTAAACTCCTGATCTACTACGCATCAAGATTACACTCAGGA GTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGC AACCTGGAACAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCG CTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG (SEQ ID NO: 17)

Hybridoma 399A12 - heavy chain = Hybridoma 416H9 -heavy chain

CAGGTCACTCTGAAAGAGTCTGGCCCTGGGGTCTTGCAGTCCTCCCAGACCCTCAGT CTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTTTGGGTGTGATCTGGA TTCGTCAGCCTTCAGGAGCGGGTCTGGAGTGGCTGGCACAAATTTACTGGGATGATA ACAAGCGCTATAACCCATCCCTGAAGAGCCGCCTCACAATCTCCAAGGATACCTCCA GAAACCAGGTATTCCTCAAGATCACCCCTGTGGACACTGCAGATACTGCCACATACT ACTGTGCTCGAAGGACCTTCTATGATTACGACGAAGGTTTCTATGCTATGGACTACT GGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:l 8)

Light Chain and Heavy Chain amino acid sequences (variable regions)

Hybridoma 215 A9 - light chain (Lambda)

QAWTQESALTTSPGETVTLTCRSSTGAVTTNNYANWVQEKPDHLFTGLIGGTNNRTPG VPARFSGSLIGDKAALT GAQTEDEAIYFCALWYSNLWVFGGGTKLTVLG (SEQ ID NO: 19)

Hybridoma 215 A9 - heavy chain

QVSLKESGPGILQSSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWNDDKR YNPSLKSRLTISKDTSRDQVFLKTTSVDTADTATYFCARRPRFYRDYDEGIDFWGQGTTL TVSS (SEQ ID NO:20) Hybridoma 311 A7- light chain (Kappa)

QIVLTQSPAIMSASPGEKVTITCSASSSVSYMYWFQQKPGTSPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPRTFGGGTKLEIKR (SEQ ID NO:21) Hybridoma 311 A7- heavy chain = Hybridoma 346B6 - heavy chain

EFQLQQSGPELVKPGASVRISCKASGYSFTDY MNWVKQSDGKTLEWIGVINPDYGTT N NQRFRGKATLTLDLSSSTAYMQLNSLTSEDSAVYYCARGGDYGAGYALDYWGQGT SVTVSS (SEQ ID NO:22)

Hybridoma 346B6 - light chain (Kappa)

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVR FSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGAGTKLELKR (SEQ ID NO:23) Hybridoma 346B6 - heavy chain = Hybridoma 31 1A7- heavy chain

Hybridoma 399A12- light chain (Kappa) = Hybridoma 416H9 - light chain (Kappa)

DIQMTQTTSSLSASLGDRVTITCRASQDISNYLNWYQQKPDGTIKLLIYYASRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPLTFGAGTKLELKR (SEQ ID NO:24) Hybridoma 399A12 - heavy chain = Hybridoma 416H9 - heavy chain

QVTLKESGPGVLQSSQTLSLTCSFSGFSLSTSGLGVIWIRQPSGAGLEWLAQIYWDDNKR Y PSLKSRLTISKDTSRNQVFLKTTPVDTADTATYYCARRTFYDYDEGFYAMDYWGQG TSVTVSS (SEQ ID NO:25)

Complementarity determining regions (CDRs) in the above-described monoclonal antibodies are depicted in Tables 3 and 4. Table 3. Light chain Complementarity determining regions (CDRs)

Based on the similarity between CDR2 and CDR3 sequences of hybridomas 31 1A7 and 346B6 (both having identical light chain CDRl sequence and heavy chain CDRl , CDR2 and CDR3 sequences), a generalized CDR2 sequence XTSNLAS (SEQ ID NO:37 with X being Ser or Asp) and CDR3 sequence QQXSSYPXT (SEQ ID NO: 38, with X at position 3 being Arg or Trp, and X at position 8 being Arg or Pro), respectively, can be designed.

Table 4. Heavy chain Complementarity determining regions (CDRs)

Based on the similarity between CDRl and CDR2 sequences of hybridomas 215A9 and 399A12, a generalized CDRl sequence TSGXGVX (SEQ ID NO:45 with X at position 4 being Met or Leu, and X at position 7 being Ser or He) and CDR2 sequence XIYWXDXKRYNPSLKS (SEQ ID NO:46, with X at position 1 being His or Ala, and X at positions 5 and 7 being Asn or Asp), respectively, can be designed. In addition, the CDR3 of both hybridomas comprise a common sequence FYXDYDEG (SEQ ID NO:50, with X being absent or being Arg). The different hybridoma cell lines expressing these antibodies were deposited under the conditions of the Budapest Treaty at the Belgian Co-ordinated Collections of Microorganisms/Laboratory of Molecular Biology-Plasmid collection (BCCM/LMBP), Technologiepark 927, 9052 Gent (Zwijnaarde), Belgium on 15 March 2012, and the following biological deposit accession numbers were assigned: LMBP 9488CB (anti-lactadherin antibody 215A9), LMBP 9489CB (anti-lactadherin antibody 311A7), LMBP 9490CB (anti-lactadherin antibody 346B6), LMBP 9491CB (anti-lactadherin antibody 399A12), or LMBP 9492CB (anti- lactadherin antibody 416H9). EXAMPLE 4. Binding of murine monoclonal anti-lactadherin to RGD peptide

The question of whether the 215A9, 346B6, 311 A7, 399A12 or 416H9 antibodies share the same epitope than huMc3 was addressed using the Biacore technology. A huMc3 antibody (see Example 1) surface was prepared by coating the antibody on a CM5 surface. Human lactadherin was then captured to the surface via the coated antibody, and the different antibodies to be tested (namely 215A9, 346B6, 31 1A7, 399A12 or 416H9) were flowed over the surface. The huMc3 antibody itself and the AB3 antibody, a murine monoclonal antibody generated within ThromboGenics and which is known to bind to an epitope distinct from the one of huMc3, were used as controls. In this experimental setup, the lactadherin being captured to the surface via the huMc3 antibody, only antibodies binding to an epitope that doesn't overlap with the one of huMc3 will show binding. None of the tested antibodies showed binding, indicating that they all bind to an epitope that overlaps with the epitope recognized by huMc3. By contrast, as expected, clear binding was observed for the positive control antibody AB3.

In a further experiment, the Biacore technology was used to determine whether the 215A9, 346B6, 311A7, 399A12 or 416H9 antibodies bind to a peptide of sequence EISQEVRGDVFPSY (SEQ ID NO:51) which contains the RGD motif.A biotinylated version of the peptide was coated to a streptavidin chip surface, and the different antibodies to be tested where injected over the chip surface. Clear binding to the peptide was observed with 215A9, 346B6, 311 A7, 399A12 and 416H9, but not with huMc3. These data are given in Figure 1. This was analyzed in further detail by ELISA wherein the binding of various antibodies to 14- mer peptides was assessed. The peptides included either have (i) the RGD domain in the center (SEQ ID NO:51), (ii) a mutant variant of the RGD domain in the center (1 or 2 amino acids mutated into alanine; SEQ ID NOs: 74-80), (iii) part of the RGD domain at the C-terminus (SEQ ID NOs: 81 -82) or at the N-terminus (SEQ ID NOs: 84-85), or (iv) are either fully N-terminal or C-terminal of the RGD sequence (SEQ ID NO: 83 and 86, respectively), thus not including the RGD motif itself. Antibodies included in the ELISA assay were huMc3, a rabbit polyclonal antibody preparation raised by C. Thery against the peptide with SEQ ID NO:51 (Silvestre et al. 2005, Nature Medicine 1 1, 499-506; referred to as "rabbit polyclonal" in Table 5), a rabbit polyclonal antibody preparation raised against amino acids 56-190 of murine MFG-E8 (immunogen containing the RGD motif and defined by SEQ ID NO:87 (TGLVCNETERGPCSPNPCYNDAKCLVTLDTQRGDIFTEYICQCPVGYSGIHCETETNYY NLDGEYMFTTAVPNTAVPTPAPTPDLSNNLASRCSTQLGMEGGAIADSQISASSVYMGF MGLQRWGPELARLYRTG); commercially available at Santa Cruz, catalog no. sc-33546), and the monoclonal antibodies 215A9, 346B6, 311A7, 399A12 or 416H9 as herein described. The results are given in Table 5. For the ELISA, the peptides where synthesized in an N-terminally biotinylated form and coated onto a neutravidin plate (Thermo Scientific, Cat. 15127) at a concentration of 10 μg/ml. The antibodies were incubated in each well at a concentration of 1 μg/ml and, following washing, were then detected in a standard ELISA format using an appropriate, HRP-labeled secondary antibody. Results are expressed as the difference in the absorbance read at 450 nm minus the absorbance read at 540 nm. Blank 2 was obtained in the absence of peptide using only the secondary antibody, while Blank 1 was obtained in the absence of peptide using both the primary and the secondary antibodies. Positive binding was arbitrarily defined for absorbance values above 0.15 (values highlighted in yellow), except for the sc-33546 antibody because of the higher background.

The first and second series of experiments demonstrate that the five herein described monoclonal anti-lactadherin antibodies, although binding in the vicinity of the binding site of huMc3, do not bind to the same lactadherin epitope as does huMc3. The second series of experiments (Table 5) first of all indicates that a polyclonal antiserum raised against a RGD-containing peptide derived from murine lactadherin ("sc-33546") does, relatively to the blanks, not at all bind to any of the human lactadherin peptides as listed in Table 5. Monoclonal antibodies 346B6 and 31 1A7 are requiring the presence of an intact RGD-motif, whereas monoclonal antibodies 215A9, 399A12 and 416H9 require the presence of an intact RGD-motif or a variant RGD-motif wherein the arginine and/or glycine are mutated to alanine (at the cost of losing much of the binding); mutation of the aspartate residue in the RGD-motif, however, fully destroys all binding of these antibodies. Further, the results indicate that half of the binding of the antibodies in the polyclonal antiserum raised against SEQ ID NO:51 ("rabbit polyclonal") can be attributed to the epitopes present N-terminal of the RGD-motif and thus not containing the RGD-motif (compare SEQ ID NO:51 and SEQ ID NO:83; and see SEQ ID NOs: 79 and 80). The remainder of the binding seems to require the presence of arginine or aspartate in the RGD motif (see SEQ ID NOs: 74- 78, 82 and 85). Overall, however, binding of this polyclonal antibodies is shifted towards the N- terminus of SEQ ID NO:51 , and nearly all of the binding is targeting (i) the 7 N-terminal amino acids of SEQ ID NO:51 (compare SEQ ID NO:51 and SEQ ID NO:82) not requiring the full RGD motif (R residue suffices) plus some binding to (ii) the 6 C-terminal amino acids of SEQ ID NO:51 , again not requiring the full RGD motif (D residue suffices) . This is different from the herein disclosed monoclonal antibodies which do not at all show binding outside the RGD motif, require a full RGD motif for binding, and, for some of the antibodies, allow limited variation in the RGD motif as described above. Table 5. Binding of indicated antibodies to listed peptides. Highlighted in the peptides are (part of) the RGD motif or mutated RGD motif. Highlighted in the ELISA results are those values higher than 0.15 (except for sc-33546 because of the higher blank 1 value).

Antibody tested Hu rabbit sc- 215A9 346B6 311A7 399A12 416H9

MC3 polyclonal 33546

SEQ ID NO: Peptide

51 EISQEVRGDVFPSY 0,04 0.28 1 , 10 1 ,00 1 ,47 1 ,62 1 ,76

74 EISQEVAGDVFPSY 0,04 1 ,75 0.24 0,52 0,02 0,02 0,24 0,25

75 EISQEVRADVFPSY 0,03 1 ,70 0.22 ; 0,74 0,01 0,01 0,69 ί 0,70

76 EISQEVRGAVFPSY 0,06 1 ,35 0.24 0,10 0,02 0,01 0,03 0,02

77 EISQEVAADVFPSY 0,05 1 ,40 { 0.24 0,23 0,01 0,01 0,20 0, 19

78 EISQEVRAAVFPSY 0,06 1 _j2 ^*"J 0.28 0,02 0,02 0,02 0,02 0,02

79 EISQEVAGAVFPSY 0,04 1 ,12 0.28 0,03 0,07 0,05 0,03 0,05 80 E I SQEVAAAVFPSY 0,04 0,84 0.31 0,02 0,01 0,02 0,02 0,02

81 NGGLCEE I SQEVRG 0,09 1 ,98 0.27 0,03 0,02 0,01 0,03 0,02

82 HNGGLCEE I SQEVR 0,08 1 ,87 0.24 0,03 0,02 0,02 0,02 0,03

83 CHNGGLCEE I SQEV 0,09 0,98 0.24 0,03 0,02 0,02 0,02 0,03

84 GDVFPSYTCTCLKG 0,08 0,17 0.29 0,04 0,02 0,02 0,03 0,03

85 DVFPSYTCTCLKGY 0,09 0,29 0.41 0,04 0,03 0,02 0,04 0,03

86 VFPSYTCTCLKGYA 0,07 0,14 0.34 0,05 0,03 0,02 0,03 0,03

Blank 1 (no peptide; 0,03 0,04 0.26 0,06 0,07 0,03 0,06 0,03 primary + secondary

Ab)

Blank 2 (no peptide; 0,02 0,02 0.05 0,02 0,02 0,02 0,02 0,01 secondary Ab)

EXAMPLE 5. Inhibitory activity of the anti-human lactadherin antibodies on cell adhesion to human lactadherin

Principle of the cell adhesion assay

To evaluate the inhibitory activity of anti-human lactadherin antibodies on cell adhesion to human lactadherin, the xCELLigence system (jointly developed by Roche Applied Science and ACEA Biosciences Inc.) was used. It consists of a RTCA (Real-Time Cell Analyzer) instrument that allows real-time monitoring of cellular events. The instrument measures electrical impedance across micro -electrodes integrated on the bottom of tissue culture microplates (E- plates). The presence of cells on top of the electrodes leads to an increase in impedance since the cells act as insulators. The more cells are attached to the electrodes, the larger the increase in electrical impedance. In addition, the impedance depends on the quality of the cell interaction with the electrodes. Increased cell attachment or spreading leads to higher cell index (CI) values (RTCA software, Roche), which displays the electrical impedance. The slope of CFs variation over time, calculated by the RTCA software, quantifies the efficiency of cell binding to the wells. Overview of the HUVEC cell adhesion assay

For the cell adhesion assay, primary pre-screened human umbilical vein endothelial cells (Huvec; S200-05n from Cell Applications Inc.) were used. Frozen cells were stored in liquid nitrogen. Upon thawing, cells were resuspended in pre -warmed endothelial cell growth medium (21 1K-500 from Cell Applications Inc.) and cultured in a 5% C0₂ incubator at 37°C. When 70% confluence was reached, cells were passaged using 0.05% trypsin-EDTA (25300-054 from Life Technologies™). A seeding density of 4000 to 5000 cells/cm² was applied during routine culture. Cells used for the cell adhesion assay were subcultured maximally 5 times.

To start the cell adhesion assay, an E-plate (05232368001 from Roche Applied Science) was coated with human lactadherin (2767-MF from R&D Systems) at a concentration of 5μg/ml and a volume of 50 μΐ per well and put overnight at 4°C. Following two washes with 100 μΐ PBS (phosphate-buffered saline; 14190-136 from Life Technologies™) per well, 50 μΐ of PBS + 1 % BSA (10735086001 from Roche Applied Science) was added and the plate was incubated at 37°C for 2 hours. After another wash, 50 μΐ of endothelial cell basal medium (1 13-500 from Cell Applications Inc.) was added per well and the plate was incubated at 37°C for 1 hour. With the xCELLigence system, the background CI was measured first. Then, per well 50 μΐ of anti- lactadherin antibody was added and another 50 μΐ of 20.000 Huvec, both in basal medium. Following 30 minutes incubation at room temperature, the plate was put back in the RTCA instrument in a 5% C0₂ incubator at 37°C. Electrical impedance was measured every 15 seconds during 3 hours. As a result curves with CI values for each condition over time were available. With the software of the xCELLigence system, the slope of each curve over a 1.5 hour time period was calculated. This slope or steepness of each curve is indicative for the degree of Huvec attachment to coated human lactadherin. With GraphPad Prism^® software, a non-linear regression analysis was performed using the obtained slope values in order to generate antibody dose-response curves with variable slope. IC50 values (i.e. half maximal inhibitory concentration) were also calculated. All conditions were tested in duplicate. As a positive control, huMc3 (see Example 1) was employed. Mouse monoclonal IgGl (anti-KLH antibody; ab34607 from Abeam) and IgG2b (anti-GST antibody; sc-57753 from Santa Cruz Biotechnology) antibodies were used as isotype controls.

Inhibitory activity of the anti-human lactadherin antibodies in the HUVEC cell adhesion assay The 5 anti-human lactadherin antibodies 215A9, 311A7, 346B6, 399A12 and 416H9 were first tested at 2 different concentrations in the cell adhesion assay: 10 and 50 μg/ml. At these concentrations, all antibodies were able to completely block the attachment of Huvec to coated human lactadherin. The positive control antibody huMc3 also fully inhibited Huvec adhesion to human lactadherin at these quantities. The mouse monoclonal IgGl and IgG2b isotype control antibodies did not show any inhibition of cell adhesion to human lactadherin (data not shown). The anti-human lactadherin antibodies were also tested at more and lower concentrations: 10, 5, 1 , 0.5, 0.1, 0.05 and 0.01 μg/ml in order to determine their IC50 values. The percentage of inhibition of Huvec attachment to human lactadherin was similar among all antibodies, including the humanized positive control antibody Mc3: while the highest antibody doses led to complete inhibition, the antibodies failed to block Huvec adhesion to human lactadherin at the lowest concentrations (see Figures 2 and 3). In accordance, the IC50 values of all antibodies were in the low nanomolar range (Table 6), which is indicative for them being potent inhibitors of cell adhesion to lactadherin.

Table 6. IC50 values for the 5 anti-human lactadherin antibodies and antibody huMc3 in the HUVEC cell adhesion assay.

Cell adhesion assay with SKOV-3 ovarian cancer cells and MDA-MB-231 breast cancer cells Two cancer cell lines (SKOV-3, MDA-MB-231) were cultured respectively in RPMI or DMEM Glutamax medium (Gibco) supplemented with Penicilline, Streptomycine and 10% Fetal Calf Serum (FCS). SKOV-3 were purchased from ATCC, MDA-MB-231 was provided by Dr V. Soumelis (Institut Curie). Both cell lines express ανβ3 and ανβ3 integrins, as observed by flow cytometry using anti -human ανβ3 (MAB1976Z) and ανβ5 (MAB1961Z) antibodies (Millipore) (not shown).

The adhesion assay was performed essentially as described for HUVECs, using the xCELLigence system (Roche). E-Plate wells were coated overnight at 4°C with 5 μg/mL recombinant human lactadherin (R&D systems) diluted in PBS. After two washes with PBS, wells were satured for 1 hour with 1% BSA in PBS. After one last wash in PBS, 50 μΐ. serum-free RPMI or DMEM was added per well and E-Plates were set in the xCELLIgence device in a 37°C and 5% C0₂ incubator for 1 hour, to equilibrate wells and electrodes. Background of impedance was measured after this step (= tO). E-Plates were taken out of the xCELLIgence device, 50 μΕ/well of antibodies (diluted at 3x final concentration in RPMI) were added, followed by 2.10⁴ SKOV-3 or MDA-MB-231 cells in 50 μΐ, serum-free medium well. E-Plates were transferred back onto the xCELLigence instrument, which was left again to equilibrate for 20 min before beginning of impedance recording. Impedance was then measured every 15 seconds for the first 4 h, followed by every 5 minutes for the next 44 hours. Data were plotted using the RTCA software as Cell Index (CI) value over time, and the slope of CI between the first time of recording after cell seeding and the time required for impedance to reach a first plateau (90 minutes for SKOV-3 or 60 min for MDA- MB-231) was calculated.

Inhibitory activity of the anti-human lactadherin antibodies in the SKOV-3 ovarian cancer cells and MDA-MB-231 breast cancer cells adhesion assay

The herein disclosed monoclonal antibodies displaying specific binding to human lactadherin in vitro, and no binding to other proteins with common structural domains (i.e. vitronectin and Factor VIII) were tested in the SKOV-3 adhesion assay. The different antibodies displayed different inhibitory activities in this assay. To determine the efficacy of these antibodies, decreasing amounts from 50 to 0.5 μg/mL were used in the adhesion assay (Figure 4A). At 5 μg/mL, all clones, like hMc3, still inhibited more than 70% of cell adhesion, except 346B6 (only 25%). Two clones still displayed inhibitory activity when used at the lowest concentration (416H9 and 311A7), whereas hMc3 inhibited cell adhesion by less than 10% at this concentration. Furthermore, the anti-lactadherin antibodies behaved similarly on MDA-MB-231 as they did on SKOV-3 cells (Figure 4B).

EXAMPLE 6. Inhibitory activity of the anti-human lactadherin antibodies on cell migration For the migration assay, the xCELLigence system was used with CIM tissue culture microplates (Roche), i.e. Boyden chamber-type tissue culture wells, with a lower and an upper compartments separated by a 8 μηι pore -containing membrane, with the impedance-measuring electrodes at its lower side. The bottom chamber of CIM -Plate wells were loaded with 160 μL· medium-0.1 % FCS containing lactadherin and/or antibodies at the final concentration, the transwell insert was positioned and 50μΙ νε11 of medium-0.1% FCS was seeded in the upper chamber. CIM-Plates were transferred onto the xCELLigence instrument and left to equilibrate for 1 hr, at which time background impedance was measured (= tO). Plates were taken out and 50 of medium-0.1% FCS containing 2.10⁴ SKOV-3 cells were loaded in the upper chamber of each well. Plates were placed back in the xCELLigence device, which was set to measure impedance every 5 minutes for the next 72 hours. Data were analyzed as above, and the slope of CI between 0 and 18 h was chosen as the shortest and most discriminant time point to evidence the differences in the different conditions. When tested in a dose-response setting in the migration assay (Figure 5), all antibodies abolished migration when used at the highest concentration (20 μg/mL), but only three clones (416H9, 399A12, 31 1 A7), still decreased migration by more than 80% when used at 5 μg/mL.

EXAMPLE 7. Inhibitory activity of the anti-human lactadherin antibodies on lactadherin- induced cell survival

To measure survival in low-serum conditions, 2.10⁴ SKOV-3 cells were seeded in tissue culture 96-well plates, in 50 μL medium-0.1% FCS/well. Lactadherin and antibody dilutions were added as 2x concentrated solutions in RPMI-0.1 % in 50 μΕΛνεΙΙ each. Cells were kept in culture at 37°C and 5% C02 for the next 96 h. Cell viability was determined by adding \0 μL· of Cell titer blue reagent (Promega) and incubating plates for another 2 h at 37°C, before reading absorbance at 616 nm (emission) after excitation at 544 nm.

The antibodies were tested in the survival assay in the presence of 5 μg/mL lactadherin (Figure 6), i.e. a dose of lactadherin allowing reproducible increase of SKOV-3 survival in the conditions described above. 346B6, the least efficient clone in the other two assays, did not decrease survival at any concentration, two others induced at most 30% inhibition when used at the highest concentration (2154A9 and 311A7), whereas the last two (416H9 and 399A12) were as efficient as hMc3, and decreased cell number to less than 50% of the lactadherin-alone treated cells. The experiments described in examples 6 and 7 thus show that, in addition to inhibiting binding of cells to lactadherin, the new anti-lactadherin antibodies also display novel inhibitory activities on functional outcomes of cell exposure to lactadherin: survival in starvation conditions and migration towards lactadherin.

EXAMPLE 8. Anti-tumor activity of the anti-human lactadherin antibodies

The anti-tumor activity of the anti-lactadherin antibodies is evaluated at several dose-levels (initially at 4 and 10 mg kg). The anti-tumor activity is determined against human xenograft mammary tumors implanted subcutaneously in female BALB/c nude mice (n= 10 mice per group, treated and control). Treatment starts when tumor size reaches a volume of 150 mm . The anti-lactadherin antibodies are administered intraperitoneally, twice weekly, until the tumors reach a volume of 1000 mm³ in the control group. The control group is treated with PBS alone.

For the evaluation of the anti-tumor activity, animals are weighed and tumors are measured 2-3 times weekly by means of a caliper. Tumor volumes are calculated using the formula: volume

(mm 3 ) = [length (mm) x width (mm 2 )]/2. Anti-tumor activity is determined according to the % TGI (tumor growth inhibition) criteria, which is determined by dividing the tumor volumes from treated groups by the control groups and multiplied by 100. Alternatively, or additionally, tumors can be removed from the animals and %TGI can be determined based on tumor weight. Data is analyzed using the statistical tools known to the person skilled in the art.

Treatment with anti-lactadherin antibodies is well tolerated at all dose-levels. The growth of tumors is significantly slowed by the anti- lactadherin antibody treatment compared to the PBS- control group. EXAMPLE 9. Anti-tumor activity of the anti-human lactadherin antibodies in combination with chemotherapy

The improved efficacy of a combination may be established by determination of the therapeutic synergy. A combination shows therapeutic synergy if it is therapeutically superior to each of the constituents used alone at its maximum tolerated dose (MTD). To demonstrate the efficacy of a combination, it may be necessary to compare the maximum tolerated dose of the combination with the maximum tolerated dose of each of the separate constituents in the study in question.

This efficacy may be quantified by determining the % TGI (tumor growth inhibition), which is calculated by dividing the tumor volumes from treated groups by the control groups and multiplied by 100. Toxicity is declared at dosages inducing >15% body weight loss or >10 % drug death. Another criteria to be used is the response rate: Partial Regressions (PR) correspond to regression > 50 % initial tumor burden, and Complete Regressions (CR) to regression below the limit of palpation. Data is analyzed using the statistical tools known to the person skilled in the art.

The efficacy of the combinations in cancer may be determined experimentally in the following manner: Immunodeficient animals are subcutaneously grafted with mammary or ovarian cancer cell lines. Treatment starts when tumor size reaches a volume of 150 mm³. The anti-lactadherin antibody is administered intraperitoneally, twice weekly at 3-5 dose-levels, until the tumors reach a volume of 1000 mm in the control group. Chemotherapy with doxorubicin, cisplatin or taxanes, at 3-dose-levels, starts simultaneously with the anti-lactadherin antibody using their best treatment schedules previously determined in a pilot study. The different animal groups are weighed 3 or 4 times a week until the maximum weight loss is reached, and the groups are then weighed at least once a week until the end of the trial. The tumors are measured 2 or 3 times a week until the tumor reaches approximately 2 g, or until the animal dies if this occurs before the tumor reaches 2 g. The animals are autopsied when sacrificed.

For a study of the combinations on leukemias or lymphomas the animals are grafted with a particular number of cells, and the antitumour activity is determined by the increase in the survival time of the treated mice relative to the controls. The product is considered to be active if the increase in survival time is greater than 27%, and is considered to be very active if it is greater than 75%.

The results obtained with treatment of chemotherapy and anti-lactadherin antibody alone, and the combinations being used at their optimum dose are analyzed using the statistical tools known to the person skilled in the art.

Treatment with anti-lactadherin antibody is well tolerated at all dose-levels. The combination of anti-lactadherin with the standard of care chemotheraputics at their MTD is also well-tolerated. The growth of tumors is significantly slowed by the anti-lactadherin antibody treatment compared to the PBS-control group. The combination is synergistic, with a greater activity and statistical significance than the one observed for the best single agent.

EXAMPLE 10. Cloning of murine anti-lactadherin antibodies

The huMc3 antibody (see Example 1) was made as a IgGl heavy chain with a kappa light chain. Restriction sites have been engineered to facilitate further cloning of other antibody sequences into the huMc3 mammalian expression vector. For the heavy chain, a BsrGI site, a Nhel site and a Xhol were incorporated just upstream the variable region, just downstream the variable region and just downstream the constant region, respectively. For the light chain, a Xbal site, a BsiWI site and a BamHI were incorporated just upstream the variable region, just downstream the variable region and just downstream the constant region, respectively.

To generate vectors expressing the murine antibodies 311A7, 346B6, 416H9 and 215A9, a two- step strategy was used. First, the human constant heavy and light chain regions were replaced by their murine IgGl and kappa counterparts using Nhel - Xhol and BsiWI - BamHI, respectively. Then, the human heavy and light chain variable regions were replaced by their murine IgGl and kappa counterparts using BsrGI - Nhel and Xbal - BsiWI, respectively.

EXAMPLE 11. Cloning of chimeric anti-lactadherin antibodies

Chimeric versions of the murine antibodies 31 1A7, 346B6, 416H9 and 215A9 were made comprising of variable regions originating from the murine antibodies linked to the constant regions originating from human antibodies. Murine 31 1A7, 346B6 and 416H9 contain an IgGl heavy chain and a kappa light chain. The huMc3 antibody (see Example 1) was also made as a IgGl heavy chain with a kappa light chain. For making the chimeric antibody constructs, variable region fragments of huMc3 were simply exchanged with those from 311A7, 346B6 and 416H9 using BsrGI - Nhel for the heavy chain and Xbal - BsiWI for the light chain, respectively.

Murine 215 A9 contains an IgG 1 heavy chain and a lambda light chain. The chimeric heavy chain was made as described above for 31 1A7, 346B6 and 416H9. As the 215A9 light chain was of the lambda isotype while that of huMc3 was of the kappa isotype, a full length chimeric gene containing the mouse 215A9 variable light chain and the human constant lambda light chain was synthesized de novo by Integrated DNA Technologies (Coralville, Iowa) and exchanged for the full length huMc3 light chain.

EXAMPLE 12. Humanization of murine antibody sequences

When injected in humans, mouse antibodies can induce a human anti-mouse antibody response, which could hamper the therapeutic efficacy. To limit this response, the mouse antibodies are made to look more like endogenous human sequences in a process called humanization. Mouse and human antibody sequences are compared, and where appropriate, mouse amino acid residues are substituted by human amino acid residues. Substitutions that have a negative effect on properties such as affinity and stability are to be avoided, and humanized variants of the mouse antibody need to be evaluated for conservation of affinity.

Computer modeling-assisted humanization was selected as method for humanizing the murine antibodies of the current invention. It will be clear to the skilled person that other humanization methods may result in humanized murine antibodies different from those described hereafter. The herein described humanized murine antibodies therefore are not be considered as limiting the number of possible humanized antibody species.

In a first step of the chosen humanization method, the structures of the mouse antibodies 311 A7 and 416H9 were modeled using the program MOE (Chemical Computing Group Inc, Canada). This modeling included the steps of selecting an antibody template sequence from the PDB (Protein Data Bank, www.rcsb.org/). For 31 1A7, structure 2W9D was selected as template, while for 416H9, structure 1N5Y was selected as template. In a next step, human monoclonal sequences with a high homology with the target sequences were searched with a blastp search in the database of human proteins ( h t p : / b 1 a s t . n c b i . n 1 m . n i h . go v ) with omission of antibodies probably of non-human origin and already humanized mouse antibodies. Side-chains different between the mouse and the human sequence were considered for humanization if 5 A or more away from the CDRs and if having a relative accessibility >25%. By visual inspection on a graphical stereo equipped screen all these residues were checked for mutation to the human variant. Residues not disturbing the overall three dimensional conformation were considered for change into the human counterpart residue.

Thus, for both 311A7 and 416H9, 12 residues in the heavy chain variable region and 7 residues in the light chain variable region were identified as candidates for humanizing these murine antibodies. These residues are summarized in Table 7 (heavy chains) and Table 8 (light chains).

Table 7. Heavy chain residues of murine antibodies 311A7 and 416H9 that are candidates for humanization

Table 8. Heavy chain residues of murine antibodies 311A7 and 416H9 that are candidates for humanization 31 1A7 VL 416H9 VL

Residue Mouse % Human Residue Mouse % Human

number variant access variant number variant access variant

L10 He 51.0 Thr L15 Leu 42.5 Val

L18 Lys 59.1 Arg L41 Asp 68.3 Gly

L22 Thr 29.0 Ser L43 Thr 32.8 Ala

L41 Thr 43.7 Gin L77 Asn 31.5 Ser

L76 Arg 54.6 Ser L80 Gin 46.7 Pro

L79 Ala 50.7 Pro L100 Ala 58.3 Gin

L82 Ala 28.3 Phe L106 Leu 44.1 He

As way of example, and for both 31 1A7 and 416H9, different expression constructs were designed harboring an increasing number of mutations, hence creating a set of different humanized antibodies for each. The heavy and light chain variable regions were codon optimized and synthesized de novo by GeneArt and cloned into the same mammalian expression vector described in Example 1. An overview of the different constructs with the number of mutations is given in Table 9.

Table 9. Overview of humanized antibody constructs indicating the number of mutations heavy and light chains.

The resulting amino acid and nucleic acid sequences are listed hereafter, with the mutations being indicated in bold underlined characters in the amino acid sequences.

>h31 1A7.H1 - amino acid sequence

EFQLVQSGAEVVKPGASVKISCKASGYSFTDYNMNWVKQSPGQTLEWIGVINPDYGTT NNNQRFRGKATLTLDLSTSTAYMQLNSLTSEDTAVYYCARGGDYGAGYALDYWGQG TSVTVSS (SEQ ID NO:52) > h31 1A7.H2 - amino acid sequence

EFQLVQSGAEVVKPGASVKISCKASGYSFTDY MNWVKQSPGQTLEWIGVINPDYGTT N NQRFRGKATLTLDLSTSTAYMELNSLTSEDTAVYYCARGGDYGAGYALDYWGQGT TVTVSS (SEQ ID NO:53)

> h31 1A7.H3 - amino acid sequence

EFOLVOSGAEVVKPGASVKISCKASGYSFTDY MNWVKQSPGOGLEWIGVINPDYGTT N NQRFRGKATLTLDLSTSTAYMELNSLRSEDTAVYYCARGGDYGAGYALDYWGQG TTVTVSS (SEQ ID NO:54)

>h31 1A7.L1 - amino acid sequence

QIVLTQSPATMSASPGERVTrrCSASSSVSYMYWFQQKPGTSPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTISSMEPEDAATYYCQQRSSYPRTFGGGTKLEIK (SEQ ID NO:55)

> h311 A7.L2 - amino acid sequence

QIVLTQSPATMSASPGERVTISCSASSSVSYMYWFQQKPGTSPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTISSMEPEDFATYYCQQRSSYPRTFGGGTKLEIK (SEQ ID NO:56)

> h311 A7.L3 - amino acid sequence

QIVLTQSPATMSASPGERVTISCSASSSVSYMYWFQQKPG^SPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTISSMEPEDFATYYCQQRSSYPRTFGGGTKLEIK (SEQ ID NO:57)

>h31 1A7.H1 - nucleic acid sequence

GAATTTCAGCTGGTGCAGTCTGGCGCCGAGGTCGTGAAACCTGGCGCCTCCGTGAAG

ATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCGACTACAACATGAACTGGGTCAA

GCAGAGCCCTGGCCAGACCCTGGAATGGATCGGCGTGATCAACCCCGACTACGGCA

CCACCAACAACAACCAGCGGTTCCGGGGCAAGGCCACCCTGACACTGGATCTGAGC

ACCAGCACCGCCTACATGCAGCTGAACAGCCTGACCAGCGAGGACACCGCCGTGTA

CTACTGTGCCAGAGGCGGCGATTACGGCGCTGGCTACGCCCTGGATTATTGGGGCCA

GGGCACAAGCGTGACCGTGTCCTCT (SEQ ID NO:58) > h31 1A7.H2 - nucleic acid sequence

GAATTTCAGCTGGTGCAGTCTGGCGCCGAGGTCGTGAAACCTGGCGCCTCCGTGAAG ATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCGACTACAACATGAACTGGGTCAA GCAGAGCCCTGGCCAGACCCTGGAATGGATCGGCGTGATCAACCCCGACTACGGCA CCACCAACAACAACCAGCGGTTCCGGGGCAAGGCCACCCTGACACTGGATCTGAGC ACCAGCACCGCCTACATGGAACTGAACAGCCTGACCAGCGAGGACACCGCCGTGTA CTACTGTGCCAGAGGCGGCGATTACGGCGCTGGCTACGCCCTGGATTATTGGGGCCA GGGCACCACCGTGACCGTGTCTAGT (SEQ ID NO:59)

> h31 1A7.H3 - nucleic acid sequence

GAATTTCAGCTGGTGCAGTCTGGCGCCGAGGTCGTGAAACCTGGCGCCTCCGTGAAG ATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCGACTACAACATGAACTGGGTCAA GCAGAGCCCAGGCCAGGGCCTGGAATGGATCGGCGTGATCAACCCCGACTACGGCA CCACCAACAACAACCAGCGGTTCCGGGGCAAGGCCACCCTGACACTGGATCTGAGC ACCAGCACCGCCTACATGGAACTGAACAGCCTGCGGAGCGAGGACACCGCCGTGTA CTATTGTGCCAGAGGCGGCGACTATGGCGCCGGATACGCCCTGGATTATTGGGGCCA GGGCACCACCGTGACCGTGTCTAGT (SEQ ID NO:60)

>h31 1A7.L1 - nucleic acid sequence

CAGATCGTGCTGACCCAGAGCCCCGCCACAATGAGCGCTTCTCCAGGCGAGCGCGT GACCATCACATGTAGCGCCAGCAGCAGCGTGTCCTACATGTACTGGTTCCAGCAGA AGCCCGGCACCAGCCCCAAGCTGTGGATCTACAGCACCAGCAACCTGGCCAGCGGC GTGCCAGCCAGATTTTCTGGCAGCGGCTCTGGCACCAGCTACAGCCTGACCATCAGC AGCATGGAACCCGAGGACGCCGCCACCTACTACTGCCAGCAGAGAAGCAGCTACCC CCGGACCTTTGGCGGAGGCACCAAGCTGGAAATCAAG (SEQ ID NO:61)

> h311 A7.L2 - nucleic acid sequence

CAGATCGTGCTGACCCAGAGCCCCGCCACAATGAGCGCTTCTCCAGGCGAGAGAGT GACCATCAGCTGCAGCGCCAGCAGCAGCGTGTCCTACATGTACTGGTTCCAGCAGA AGCCCGGCACCAGCCCCAAGCTGTGGATCTACAGCACCAGCAACCTGGCCAGCGGC GTGCCAGCCAGATTTTCTGGCAGCGGCTCTGGCACCAGCTACAGCCTGACCATCTCC AGCATGGAACCCGAGGACTTCGCCACCTACTACTGCCAGCAGCGGAGCAGCTACCC CAGAACCTTTGGCGGAGGCACCAAGCTGGAAATCAAG (SEQ ID NO:62)

> h311 A7.L3 - nucleic acid sequence

CAGATCGTGCTGACCCAGAGCCCCGCCACAATGAGCGCTTCTCCAGGCGAGAGAGT GACCATCAGCTGCAGCGCCAGCAGCAGCGTGTCCTACATGTACTGGTTCCAGCAGA AGCCCGGCCAGTCCCCCAAGCTGTGGATCTACAGCACCAGCAACCTGGCCAGCGGC GTGCCAGCCAGATTTTCTGGCAGCGGCTCTGGCACCAGCTACAGCCTGACCATCTCC AGCATGGAACCCGAGGACTTCGCCACCTACTACTGCCAGCAGCGGAGCAGCTACCC CAGAACCTTTGGCGGAGGCACCAAGCTGGAAATCAAG (SEQ ID NO:63)

>h416H9.Hl - amino acid sequence

QVTLKESGPGVLQPTQTLTLTCSFSGFSLSTSGLGVIWIRQPSGAGLEWLAQIYWDDNK RY PSLKSRLTITKDTSKNQVFLTITPVDTADTATYYCARRTFYDYDEGFYAMDYWGQ GTTVTVSS (SEQ ID NO:64)

>h416H9.H2 - amino acid sequence

QVTLKESGPTVLKPTQTLTLTCSFSGFSLSTSGLGVIWIRQPSGAGLEWLAQIYWDDNK RY PSLKSRLTITKDTSKNQVFLTITPVDTADTATYYCARRTFYDYDEGFYAMDYWGQ GTTVTVSS (SEQ ID NO:65)

>h416H9.H3 - amino acid sequence

QVTLKESGPTVLKPTQTLTLTCSFSGFSLSTSGLGVIWIRQPPGKGLEWLAQIYWDDNK RY PSLKSRLTITKDTSKNQVFLTITPVDTADTATYYCARRTFYDYDEGFYAMDYWGQ GTTVTVSS (SEQ ID NO:66)

>h416H9.Ll - amino acid sequence

DIQMTQTTSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGTIKLLIYYASRLHSGVPS RFSGSGSGTDYSLTISSLEPEDIATYFCQQGNTLPLTFGAGTKLEIK (SEQ ID NO:67)

>h416H9.L2 - amino acid sequence DIQMTQTTSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAIKLLIYYASRLHSGVPS RFSGSGSGTDYSLTISSLEPEDIATYFCQQGNTLPLTFGQGTKLEIK (SEQ ID NO:68)

>h416H9.Hl - nucleic acid sequence

TGTACAGTGTCAAGTGACCCTGAAAGAAAGCGGCCCTGGCGTGCTGCAGCCTACCC AGAC ACT GACCCTGACCT GC AGCTTCAGCGGCTTC AGCCTGAGC ACATCTGGCCTGG GCGTGATCTGGATCAGACAGCCTTCTGGCGCCGGACTGGAATGGCTGGCCCAGATCT ACTGGGACGACAACAAGCGGTACAACCCCAGCCTGAAGTCCCGGCTGACCATCACC AAGGACACCAGCAAGAACCAGGTGTTCCTGACAATCACCCCCGTGGACACCGCCGA TACCGCCACCTACTACTGTGCCAGACGGACCTTCTACGACTACGACGAGGGCTTCTA CGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCATCTGCTAGC (SEQ ID NO:69)

>h416H9.H2 - nucleic acid sequence

TGTACAGTGTCAAGTGACCCTGAAAGAAAGCGGCCCCACCGTGCTGAAGCCCACCC AGAC ACT GACCCTGACCT GCAGCTTCAGCGGCTTCAGCCTGAGC ACATCTGGCCTGG GCGTGATCTGGATCAGACAGCCTTCTGGCGCCGGACTGGAATGGCTGGCCCAGATCT ACTGGGACGACAACAAGCGGTACAACCCCAGCCTGAAGTCCCGGCTGACCATCACC AAGGACACCAGCAAGAACCAGGTGTTCCTGACAATCACCCCCGTGGACACCGCCGA TACCGCCACCTACTACTGTGCCAGACGGACCTTCTACGACTACGACGAGGGCTTCTA CGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCATCTGCTAGC (SEQ ID NO:70)

>h416H9.H3 - nucleic acid sequence

TGTACAGTGTCAAGTGACCCTGAAAGAAAGCGGCCCCACCGTGCTGAAGCCCACCC AGAC ACT GACCCTGACCT GCAGCTTCAGCGGCTTCAGCCTGAGC ACATCTGGCCTGG GCGTGATCTGGATCAGACAGCCTCCTGGCAAGGGCCTGGAATGGCTGGCCCAGATC TACTGGGACGACAACAAGCGGTACAACCCCAGCCTGAAGTCCCGGCTGACCATCAC CAAGGACACCAGCAAGAACCAGGTGTTCCTGACAATCACCCCCGTGGACACCGCCG ATACCGCCACCTACTACTGCGCCAGACGGACCTTCTACGACTACGACGAGGGCTTCT ACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCATCTGCTAGC (SEQ ID NO:71)

>h416H9.Ll - nucleic acid sequence

TCTAGATGCGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCGTGGG CGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACATCAGCAACTACCTGAACT GGTATCAGCAGAAGCCTGGCGGCACCATCAAGCTGCTGATCTACTACGCCAGCAGA CTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTACAG CCTGACAATCAGCTCCCTGGAACCCGAGGATATCGCTACCTACTTCTGTCAGCAAGG CAACACCCTGCCCCT GACCTTTGGAGCCGGCACCAAGCT GGAAATCAAGCGT ACG

(SEQ ID NO:72)

>h416H9.L2 - nucleic acid sequence

TCTAGATGCGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCGTGGG CGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACATCAGCAACTACCTGAACT GGTATCAGCAGAAGCCTGGCGGAGCCATCAAGCTGCTGATCTACTACGCCAGCAGA CTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTACAG CCTGACAATCAGCTCCCTGGAACCCGAGGATATCGCTACCTACTTCTGTCAGCAAGG CAACACCCTGCCCCT GACCTTTGGCCAGGGCACCAAGCT GGAAATCAAGCGT ACG

(SEQ ID NO:73)

EXAMPLE 13. Inhibitory activity of humanized and chimeric anti-human lactadherin antibodies on cell adhesion to human lactadherin

The humanized and chimeric 31 1A7 antibodies as described in Examples 1 1 and 12 were compared with the original murine 311A7 antibody in terms of their capacity of inhibiting adhesion of cells to human lactadherin (assay as outlined in Example 5). The results are given in Table 10 and indicate that all of the humanized and chimeric 311A7 antibodies are as potent as the original murine 31 1A7 antibody. Table 10. Inhibitory activity of humanized and chimeric 311A7 antibodies on cell adhesion to human lactadherin

Antibody IC50 (nM)

311A7.1 (see Table 9) 8.67

311A7.2 (see Table 9) 9.08

311A7.3 (see Table 9) 7.34

chimeric 31 1A7 (see Example 11) 7.36

murine 311A7 7.36

huMc-3 (see Example 1) 6.48

Claims

1. An isolated monoclonal antibody binding to the antigen defined in SEQ ID NO:51 and inhibiting cell adhesion to human lactadherin.

2. An isolated monoclonal antibody binding to the antigen defined in SEQ ID NO:51 further characterized in that it comprises one of the following combinations of complementarity determining region (CDR) amino acid sequences:

(v) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively, or light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 29 to 31 , respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 45, 46, and 41 , respectively;

(vi) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively, or light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 29 to 31, respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 39 to 41, respectively; (vii) light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 26 to 28, respectively, or light chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 29 to 31 , respectively; and heavy chain CDR 1 to 3 with amino acid sequences defined in SEQ ID NO: 45, 46, and 44, respectively;

(xiii) an affinity matured antibody of any of (i) to (xii).

The antibody according to claim 1 or 2 which is a mammalian antibody, a human antibody or a humanized antibody.

The antibody according to any one of claims 1 to 3 which is a monovalent or multivalent antibody further being monospecific or multispecific provided that the binding specificity to said antigen is maintained.

An antigen -binding fragment of the antibody according to any one of claims 1 to 4.

6. An isolated nucleic acid encoding the antibody according to claims 1 to 4 or encoding the antigen-binding fragment according to claim 5.

7. The antibody according to claims 1 to 4, the antigen-binding fragment according to claim 5, or the nucleic acid according to claim 6 for use as a medicament.

8. The antibody according to claims 1 to 4, the antigen-binding fragment according to claim 5, or the nucleic acid according to claim 6 for use as a medicament in combination with an additional therapeutic agent.

9. A pharmaceutical composition comprising at least one of the antibody according to claims 1 to 4, the antigen-binding fragment according to claim 5, or the nucleic acid according to claim 6 and further comprising at least one of a pharmaceutically acceptable diluents, carrier or adjuvant.

10. The pharmaceutical composition according to claim 8 further comprising an additional therapeutic agent.

1 1. The antibody according to claims 1 to 4, the antigen-binding fragment according to claim 5, or the nucleic acid according to claim 6 for treating or inhibiting a benign, pre-malignant or malignant tumor.

12. The antibody according to claims 1 to 4, the antigen-binding fragment according to claim 5, or the nucleic acid according to claim 6 for preventing, treating or inhibiting an ophthalmologic disorder.

13. The antibody according to claims 1 to 4, the antigen-binding fragment according to claim 5, or the nucleic acid according to claim 6 for preventing, treating or inhibiting a disorder characterized by neovascularization.

14. The antibody, the antigen-binding fragment, or nucleotide sequence according to claim 13 which is part of a combination therapy with an additional therapeutic agent.

15. An isolated host cell comprising the isolated nucleic acid according to claim 6.

16. An isolated host cell expressing the antibody according to claims 1 to 4 or the antigen- binding fragment according to claim 5.

17. A method of producing the antibody according to claims 1 to 4 or the antigen-binding fragment according to claim 5, said method comprising the steps of:

(i).

18. A method of producing the antibody according to claims 1 to 4 or the antigen-binding fragment according to claim 5, said method comprising the steps of:

(i) obtaining a crude preparation of an antibody comprising said fragment by means of recombinant expression of said antibody or by means of chemical synthesis of the antibody;

(ii) purifying said antibody from the crude preparation obtained in (i);

19. A derivative of the antibody according to claims 1 to 4 or of the antigen-binding fragment according to claim 5.

20. Use of the antibody according to claims 1 to 4, the antigen-binding fragment according to claim 5, or the nucleic acid according to claim 6 in the manufacture of a medicament.

21. The humanized antibody according to claim 3, or a fragment thereof, comprising at least one of:

(vii) a variable heavy chain as given in SEQ ID NO:22 or SEQ ID NO:25 carrying up to 12 mutations in the region outside the CDRs; (viii) a variable light chain as given in SEQ ID N0:21 or SEQ ID NO:24 carrying up to 7 mutations in the region outside the CDRs;

(ix) a variable heavy chain as given in SEQ ID NO:22 carrying one or more of the following mutations: Gln5Val, Pro9Ala, Leul lVal, Argl9Lys, Asp41Pro, Lys43Gln, Thr44Gly, Ser76Thr, Gln82Glu, Thr87Arg, Ser91Thr, and/or Serl 16Thr;

(x) a variable light chain as given in SEQ ID NO:21 carrying one or more of the following mutations: IlelOThr, Lysl 8Arg, Thr22Ser, Thr41Gln, Arg76Ser, Ala79Pro, and/or Ala82Phe;

(xi) a variable heavy chain as given in SEQ ID NO:25 carrying one or more of the following mutations: GlylOThr, GlnBLys, SerHPro, Serl5Thr, Serl9Thr, Ser23Thr, Ser43Pro, Ala45Lys, Ser72Thr, Arg77Lys, Lys83Thr, and/or Serl20Leu or Serl20Thr;

(xii) a variable light chain as given in SEQ ID NO:24 carrying one or more of the following mutations: Leul5Val, Asp41Gly, Thr43Ala, Asn77Ser, Gln80Pro, AlalOOGln, and/or Leul06Ile.

22. The humanized antibody or fragment thereof according to claim 21 , or a fragment thereof, comprising a variable heavy chain as given in SEQ ID NO:22 mutated according to (i) or (iii) and a variable light chain as given in SEQ ID NO:21 mutated according to (ii) or (iv).

23. The humanized antibody or fragment thereof according to claim 21, or a fragment thereof, comprising a variable heavy chain as given in SEQ ID NO: 25 mutated according to (i) or (v) and a variable light chain as given in SEQ ID NO:24 mutated according to (ii) or (vi).