WO2013139956A1 - Cellule de suppression des anticorps liée à la lactadhérine - Google Patents

Cellule de suppression des anticorps liée à la lactadhérine Download PDF

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WO2013139956A1
WO2013139956A1 PCT/EP2013/056057 EP2013056057W WO2013139956A1 WO 2013139956 A1 WO2013139956 A1 WO 2013139956A1 EP 2013056057 W EP2013056057 W EP 2013056057W WO 2013139956 A1 WO2013139956 A1 WO 2013139956A1
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seq
antibody
amino acid
acid sequences
chain cdr
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Shirley LEYMAN
Hoa Thu NGO
Sofie NOTEBAERT
Richard Zwaal
Lorenzo TIBALDI
Clotilde THÉRY
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Thrombogenics Nv
Institut Curie
Institut National De La Santé Et De La Recherche Médicale (Inserm)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3015—Breast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to anti-lactadherin antibodies and their medical use.
  • 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).
  • GEF N-terminal epidermal growth factor
  • RGD Arg-
  • 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 ⁇ ⁇ ⁇ 3 /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).
  • 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).
  • lactadherin 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 ( ⁇ 3/5 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 ⁇ ⁇ ⁇ 3 /5 integrin-dependent endothelial cell survival (Silvestre 2005, Nat Med 1 1, 499-506).
  • VEGF vascular endothelial growth factor
  • 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.
  • HMFG human milk fat globule
  • lactadherin 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).
  • 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).
  • 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:
  • 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.
  • the above-mentioned humanized antibody may be one comprising at least one of:
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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.
  • 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.
  • the peptide EISQEVRGDVFPSY is defined in SEQ ID NO:51.
  • the Biotin-Ahx-EISQEVRGDVFPSY-CONH 2 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 ⁇ /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-CONH 2 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 5 ⁇ g/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 5 ⁇ g/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 5 ⁇ g/mL recombinant lactadherin in 0.1% fetal calf serum without antibodies. Mean of two experiments, with standard deviations are represented.
  • 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.
  • 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:
  • 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 invention also encompasses antigen-binding fragments of any of the above antibodies.
  • antibody 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.
  • VH variable domain
  • CL constant domain
  • Ig new antigen receptors IgNARs
  • CNAR constant domains
  • VNAR variable domain
  • 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).
  • Vab variable antigen binding domain of the heavy-chain immunoglobulin
  • V-NAR shk antibodies
  • Vab 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.
  • 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.
  • CDR complementarity determining region
  • 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).
  • 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 99/0
  • 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).
  • 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).
  • recombinant bispecific or bifunctional antibody formats have been developed (Kriangkum et al. 2001, Biomol Eng 18, 31- 40).
  • 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).
  • tandem scFv molecules 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).
  • 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.
  • knob-into-hole diabodies 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 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).
  • didiabody Li et al. 2004, J Biol Chem 279, 2856-2865.
  • 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).
  • 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).
  • 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 Fab 2 , mono- or tri-specific Fab 3 , 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.
  • 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.
  • 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).
  • "humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • 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.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the non-human immunoglobulin are replaced by corresponding human residues.
  • 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.
  • humanized versions of the murine antibodies of the invention include antibodies or fragments thereof comprising at least one of:
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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.
  • XaaNNXbb e.g., Gln5Val in SEQ ID NO:22
  • XaaNNXbb 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.
  • transgenic animals e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
  • 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;
  • 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).
  • SEQ ID NO:X refers to a biological sequence consisting of the sequence of amino acids or nucleotides given in the SEQ ID NO:X.
  • 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.
  • 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. At 211 , I 131 , I 125 , Y 90 , Re 186 ,
  • 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.
  • lactadherin- and tumor-antigen-bispecific antibodies may increase the efficiency of killing lactadherin-expressing cells.
  • 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.
  • 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/.
  • adaptation of a nucleotide sequence to a given species i.e.
  • 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
  • Optologic 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 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” .
  • neo-vascularization 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.
  • combination therapy 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.
  • 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.
  • 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.
  • 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).
  • 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- ⁇ , Sirolimus (rapamycin) and endostatin.
  • VEGF vascular endothelial growth factor
  • anti-PlGF placental growth factor
  • agents such as macugen (pegaptanib sodium), trypthophanyl-tRNA synthetase (TrpRS), anecortave acetate, combrestatin A4 prodrug, AdPEDF (adenovector
  • 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).
  • anti-VEGF bevacizumab
  • ranibizumab anti-VEGF
  • aflibercept or VEGF Trap-Eye
  • rituximab and ibritumomab tiuxetan anti-CD20
  • trastuzumab anti-Her2
  • gemtuzumab ozogamicin anti-CD33
  • alemtuzumab anti-CD52
  • chemotherapeutic agents useful in the treatment of cancer include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXANTM); 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,
  • calicheamicin especially calicheamicin ⁇ and calicheamicin ⁇ (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,
  • paclitaxel TAXOL ® Bristol-Myers Squibb Oncology, Princeton, NJ
  • doxetaxel TAXOTERE®, Rhone-Poulenc Rorer, Antony, France
  • chlorambucil gemcitabine
  • 6-thioguanine mercaptopurine
  • platinum analogs such as cisplatin and carboplatin
  • vinblastine platinum
  • 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.
  • TAXOL Bristol-Myers Squibb On
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • 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.
  • anti-inflammatory agents examples include steroids (e.g. prednisolone, methylprednisolone, cortisone, hydrocortisone, prednisone, triamcinolone, dexamethasone) and non-steroidal anti- inflammatory agents (NSAIDs; e.g. acetaminophren, ibuprofen, aspirin).
  • steroids e.g. prednisolone, methylprednisolone, cortisone, hydrocortisone, prednisone, triamcinolone, dexamethasone
  • NSAIDs non-steroidal anti- inflammatory agents
  • antiviral agents examples include trifluridine, vidarabine, acyclovir, valacyclovir, famciclovir, and doxuridine.
  • antibacterial agents or antibiotics include ampicillin, penicillin, tetracycline, oxytetracycline, framycetin, gatifioxacin, gentamicin, tobramycin, bacitracin, neomycin and polymyxin.
  • anti-mycotic/fungistatic/antifungal agents examples include fluconazole, amphotericin, clotrimazole, econazole, itraconazole, miconazole, 5-fiuorocytosine, ketoconazole and natamycin.
  • anti-mitotic agents include mitomycin C and 5-fiuorouracyl.
  • antihistamines includes ketitofen fumarate and pheniramine maleate.
  • anesthetics include benzocaine, butamben, dibucaine, lidocaine, oxybuprocaine, pramoxine, proparacaine, proxymetacaine, tetracaine and amethocaine.
  • 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).
  • 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.
  • 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, lat
  • 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".
  • a pharmaceutically acceptable 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.
  • pharmaceutically acceptable compounds such as diluents, carriers and adjuvants
  • a “diluent”, or more in particular a “pharmaceutically acceptable diluent” includes diluents such as water, saline, physiological salt solutions, glycerol, ethanol, etc.
  • 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).
  • 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.
  • an adjuvant aluminium hydroxide (alum) is given, to which an active compound or ingredient of the invention can be adsorbed.
  • alum aluminium hydroxide
  • many other adjuvants are known in the art and can be used.
  • 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.
  • the pharmaceutically acceptable carrier may be a solid or a liquid or a gas which has been compressed to form a liquid, i.e.
  • 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.
  • 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.
  • 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.
  • 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.
  • the amount will vary from 0.01 to 1000 ⁇ g/dose, more particularly from 0.1 to 100 ⁇ g/dose.
  • the active substance may be administered at a dose between 1 ⁇ g/kg body weight and 10 mg kg body weight, or between 10 ⁇ g/kg body weight and 5 mg kg body weight, or between 100 ⁇ g 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 ⁇ g/kg/minute, between 1 and 50 ⁇ g/kg/minute, between 5 and 50 ⁇ g/kg/minute, or between 5 and 20 ⁇ g/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.
  • 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.
  • 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).
  • 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
  • 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:
  • 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).
  • huMC3 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.
  • Lactadherin knock-out mice were immunized with recombinant human and murine lactadherin (3 subcutaneous injections of human and murine lactadherin, 20 ⁇ g each, the first one in complete Freud adjuvant, the last two in incomplete Freud adjuvant).
  • the antigens were obtained from R&D Systems, UK.
  • mice received a final i.p. injection of 20 ⁇ g 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).
  • HAT hyperxanthine, aminopterine, thymidine
  • 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.
  • 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
  • Antibody sequences were isolated from 5 hybridomas.
  • the hybridoma 215A9 resulted from one mouse
  • the hybridomas 399A12 and 416H9 resulted from a second mouse
  • 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.
  • Hybridoma 399A12 - light chain Hybridoma 416H9 - light chain
  • Hybridoma 399A12 - heavy chain Hybridoma 416H9 -heavy chain
  • Hybridoma 215 A9 - light chain (Lambda)
  • Hybridoma 311 A7- heavy chain Hybridoma 346B6 - heavy chain
  • Hybridoma 346B6 - light chain (Kappa)
  • Hybridoma 346B6 - heavy chain Hybridoma 31 1A7- heavy chain
  • Hybridoma 399A12- light chain Hybridoma 416H9 - light chain (Kappa)
  • Hybridoma 399A12 - heavy chain Hybridoma 416H9 - heavy chain
  • CDRs Complementarity determining regions in the above-described monoclonal antibodies are depicted in Tables 3 and 4. Table 3. Light chain Complementarity determining regions (CDRs)
  • 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
  • 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
  • CDRl and CDR2 sequences of hybridomas 215A9 and 399A12 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.
  • the CDR3 of both hybridomas comprise a common sequence FYXDYDEG (SEQ ID NO:50, with X being absent or being Arg).
  • 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.
  • 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.
  • clear binding was observed for the positive control antibody AB3.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • RTCA Real-Time Cell Analyzer
  • an E-plate (05232368001 from Roche Applied Science) was coated with human lactadherin (2767-MF from R&D Systems) at a concentration of 5 ⁇ g/ml and a volume of 50 ⁇ per well and put overnight at 4°C. Following two washes with 100 ⁇ PBS (phosphate-buffered saline; 14190-136 from Life TechnologiesTM) per well, 50 ⁇ of PBS + 1 % BSA (10735086001 from Roche Applied Science) was added and the plate was incubated at 37°C for 2 hours.
  • PBS phosphate-buffered saline
  • 14190-136 from Life TechnologiesTM
  • 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 ⁇ of anti- lactadherin antibody was added and another 50 ⁇ 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% C0 2 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.
  • 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 ⁇ g/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).
  • 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.
  • SKOV-3 ovarian cancer cells
  • MDA-MB-231 breast cancer cells
  • SKOV-3, MDA-MB-231 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 ⁇ 3 and ⁇ 3 integrins, as observed by flow cytometry using anti -human ⁇ 3 (MAB1976Z) and ⁇ 5 (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 ⁇ g/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 ⁇ . serum-free RPMI or DMEM was added per well and E-Plates were set in the xCELLIgence device in a 37°C and 5% C0 2 incubator for 1 hour, to equilibrate wells and electrodes.
  • R&D systems recombinant human lactadherin
  • E-Plates were taken out of the xCELLIgence device, 50 ⁇ /well of antibodies (diluted at 3x final concentration in RPMI) were added, followed by 2.10 4 SKOV-3 or MDA-MB-231 cells in 50 ⁇ , 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.
  • CI Cell Index
  • 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.
  • decreasing amounts from 50 to 0.5 ⁇ g/mL were used in the adhesion assay ( Figure 4A). At 5 ⁇ g/mL, all clones, like hMc3, still inhibited more than 70% of cell adhesion, except 346B6 (only 25%).
  • EXAMPLE 6 Inhibitory activity of the anti-human lactadherin antibodies on cell migration
  • 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 ⁇ pore -containing membrane, with the impedance-measuring electrodes at its lower side.
  • the bottom chamber of CIM -Plate wells were loaded with 160 ⁇ L ⁇ medium-0.1 % FCS containing lactadherin and/or antibodies at the final concentration, the transwell insert was positioned and 50 ⁇ ⁇ 11 of medium-0.1% FCS was seeded in the upper chamber.
  • EXAMPLE 7 Inhibitory activity of the anti-human lactadherin antibodies on lactadherin-induced cell survival
  • SKOV-3 cells were seeded in tissue culture 96-well plates, in 50 ⁇ L medium-0.1% FCS/well. Lactadherin and antibody dilutions were added as 2x concentrated solutions in RPMI-0.1 % in 50 ⁇ each. Cells were kept in culture at 37°C and 5% C02 for the next 96 h. Cell viability was determined by adding ⁇ 0 ⁇ L ⁇ 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.
  • Cell titer blue reagent Promega
  • lactadherin 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 anti-tumor activity of the anti-lactadherin antibodies is evaluated at several dose-levels (initially at 4 and 10 mg kg).
  • the anti-lactadherin antibodies are administered intraperitoneally, twice weekly, until the tumors reach a volume of 1000 mm 3 in the control group.
  • the control group is treated with PBS alone.
  • Tumor volumes are calculated using the formula: volume
  • % TGI tumor growth inhibition
  • 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).
  • MTD maximum tolerated dose
  • 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.
  • PR Partial Regressions
  • CR Complete Regressions
  • 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 mm 3 . 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mouse antibodies 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.
  • 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.
  • 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 .
  • Table 9 Overview of humanized antibody constructs indicating the number of mutations heavy and light chains.
  • 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

Abstract

La présente invention concerne des anticorps anti-lactadhérine et leur utilisation médicale.
PCT/EP2013/056057 2012-03-22 2013-03-22 Cellule de suppression des anticorps liée à la lactadhérine WO2013139956A1 (fr)

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