WO1995021866A1 - Molecule immuno-interactive se liant au domaine extracellulaire du recepteur tie2/tek - Google Patents

Molecule immuno-interactive se liant au domaine extracellulaire du recepteur tie2/tek Download PDF

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Publication number
WO1995021866A1
WO1995021866A1 PCT/US1995/001743 US9501743W WO9521866A1 WO 1995021866 A1 WO1995021866 A1 WO 1995021866A1 US 9501743 W US9501743 W US 9501743W WO 9521866 A1 WO9521866 A1 WO 9521866A1
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WIPO (PCT)
Prior art keywords
antibody
molecule
animal
immunointeractive
tie2
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PCT/US1995/001743
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English (en)
Inventor
Andrew Stewart Runting
Andrew Frederick Wilks
Steven Alan Stacker
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Ludwig Institute For Cancer Research
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Publication date
Application filed by Ludwig Institute For Cancer Research filed Critical Ludwig Institute For Cancer Research
Priority to EP95910973A priority Critical patent/EP0812332A4/fr
Priority to JP7521374A priority patent/JPH10503081A/ja
Priority to AU18745/95A priority patent/AU689232B2/en
Publication of WO1995021866A1 publication Critical patent/WO1995021866A1/fr

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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
    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to immunointeractive molecules to an 5 animal growth factor receptor and, more particularly, to animal t ⁇ 2/Tek receptor.
  • the immunointeractive molecules provide the basis for a new range of therapeutic and diagnostic agents for use such as in the treatment, prophylaxis and diagnosis of an angiogenic-dependent phenotype.
  • Angiogenesis is the formation of new blood vessels from those which preexist 20 within the body (1). It is of fundamental importance for the development of the embryo and a number of roles in post-natal life (e.g. wound healing, tissue regeneration, cyclical growth of the corpus luteum and endometrium). Angiogenesis is also important in a number of pathological conditions including the growth of solid tumours (1). Recent studies have suggested that the acquisition of an angiogenic- 25 dependent phenotype is a key factor in the development of metastasis.
  • PCR polymerase chain reaction
  • NEGF has a mitagenic effect on endothelial cells but is also a potent mediator of vascular permeability. NEGF has in recent studies been shown to play a role in the hypoxia induced angiogenesis seen in a glioma model suggesting that NEGF may play a role in mediating angiogenesis in other tumour systems (6). Other studies have shown that anti-NEGF monoclonal antibodies have an anti-tumour effect in vivo (7).
  • the inventors developed a series of antibodies to the ⁇ YK (NEGFR2) receptor extracellular domain. These antibodies are useful in the development of a new range of therapeutic molecules such as agonists and antagonists to ⁇ YK-receptor interaction as well as a range of diagnostic agents capable of, for example, detecting normal, abnormal or mutant receptors, receptor expression on a cell surface and/or receptor-ligand interaction.
  • One aspect of the present invention contemplates an immunointeractive molecule capable of binding or otherwise associating with an animal rie2/Tek receptor extracellular domain.
  • the animal is a mammal such as a human or murine species.
  • the immunointeractive molecule is a polyclonal or monoclonal antibody.
  • Another aspect of the present invention is directed to a diagnostic agent comprising an immunointeractive molecule capable of binding or otherwise associating with an animal t e2/Tek receptor immunointeractive molecule is an antibody labelled with a reporter molecule.
  • SUBSTITUTE SHEET KSi 26 Yet another aspect of the present invention relates to a pharmaceutical composition comprising an immunointeractive molecule as contemplated above together with one or more pharmaceutically acceptable carriers and/or diluents.
  • Still yet another aspect of the present invention provides a method for treating an angiogenic-dependent phenotype or disease condition resulting therefrom in a mammal, said method comprising administering to said mammal an effective amount of an immunointeractive molecule capable of binding or otherwise associating with an animal tie2/Te receptor is particularly useful in the treatment of metastasis.
  • Figure 1 is a photographic representation of a silver stained 7.5 % SDS PAGE gel showing the analysis of purified tze2-FLAGTM construct eluted from the M2 affinity column. The elution of the bound protein is shown with the concentration of the peptide solution added shown at the top of the gel.
  • Figure 2 is a photographic representation demonstrating detection of 0.5 ⁇ g of tz ' e2-FLAGTM protein by Western blotting analysis using the monoclonal antibodies raised against tz ' e2-FLAG.
  • the positive control antibody (M2) is directed to the FLAGTM epitope.
  • the negative control antibody was directed to the extracellular domain of the receptor tyrosine kinase NYK.
  • the molecular weight standards are represented in kilodaltons.
  • Figure 3 is a graphical representation of an analysis of shared epitopes of the zvz ⁇ -tiel monoclonal antibodies by the use of real time interaction on a biosensor.
  • RU represents Response Units, which are proportional to the concentration of protein at the surface of the chip.
  • the addition of lell antibody does not result in increased RU after the cessation of sample flow at 2450s.
  • FIG. 4 is a graphical representation showing analysis of the binding of the anti-tz ' e2-FLAGTM binding to native (a, b and c) and denatured (d, e and f) tiel- FLAGTM on the biosensor.
  • the response of the chip is shown in the figure with each graph representing the response in RU on the Y-axis versus time (X-axis).
  • An increase in RU corresponds to antibody binding to the rie2-FLAGTM derivatised chip.
  • t/e2-FLAGTM was denatured by passing a solution of 1.5% v/v 2-mercaptoethanol dissolved in 6 M guanidine-HCl and 1 M Tris-HCl pH 8.9.
  • Antibody lell demonstrates no binding to the denatured tz ' e2-FLAGTM immobilised on the biosensor chip ( Figure 4d).
  • Figure 5 is a graphical representation of response of the ft ' e2-EpoR expressing Ba/F3 cell line and a control Ba/F3 cell line to the lell anti-tie2 antibody.
  • 10 4 cells were incubated in 100 ⁇ l of growth medium lacking IL-3 with various concentrations of antibody, allowed to incubate for 4 days, then the number of proliferating cells ascertained by the use of a (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (12).
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
  • the result of the MTT assay is expressed in the difference in absorbances at 690 nm and 560 nm, which is proportional to the number of proliferating cells in the sample.
  • the control cells were not affected by the antibody, whereas the tz ' e2-EpoR expressing cells are stimulated to proliferate.
  • Figure 6 is a graphical representation showing detection of the tie! receptor expressed on Ba/F3 cells using the 4g8 antibody and FACS analysis.
  • the X-axis represents channel number and relates to the amount of fluorescence detected, while the Y axis shows the number of cells corresponding to that level of fluorescence.
  • the unshaded profile represents the fluorescence of untransfected Ba/F3 cells and the shaded region that of cells transfected with the tie! construct.
  • Binding of the 4g8 antibody was detected using a FITC-labelled anti-rat Ig antibody. Cells were analysed using a Becton Dickinson FACScan unit.
  • an immunointeractive molecule capable of binding or otherwise associating with an animal tie2/Tek receptor, and in particular, the extracellular domain of such a receptor.
  • the immunointeractive molecules are in the form of antibodies such as polyclonal or monoclonal antibodies although monoclonal antibodies are preferred.
  • the present invention also extends to immunologically interactive fragments, parts, derivatives, homologues or analogues of these antibodies.
  • Such antibodies may be in isolated or purified form meaning that a composition comprises at least 25 % , more preferably at least 35%, even more preferably at least 45-50%, still more preferably at least 60-75% and even still more preferably at least 95-100% of the antibodies as determined by weight, immunoreactivity or other convenient means.
  • the antibodies may be present in the form of isolated culture supernatant, tissue extract, serum, whole blood or ascites fluid.
  • the animal rie2/Tek receptor is of mammalian origin such as from a human, livestock animal (e.g. cow, horse, sheep, goat or donkey), laboratory test animal (e.g. rat, mouse or rabbit), companion animal (e.g. dog or cat) or captive wild animal (e.g. dingo, fox, wild boar or kangaroo).
  • the most preferred receptors are of human and laboratory test animal origin (e.g. mouse).
  • the antibodies are polyclonal antibodies, they may be generated in any convenient host including a human, livestock animal, companion animal or captive wild animal as hereinbefore described.
  • the antibodies may be prepared in any convenient hybridoma such as of murine (e.g. rat or mouse) origin.
  • the receptor used to generate the antibodies may be the whole receptor such as in purified, partially purified or isolated form including in the form of isolated membrane preparations.
  • the receptor may also be produced by recombinant procedures or synthetic procedures or a combination thereof.
  • a fragment of the receptor is used which, in an even more preferred embodiment, is fused to a suitable carrier or marker molecule such as FLAGTM protein or alkaline phosphatase CAP.
  • a suitable carrier or marker molecule such as FLAGTM protein or alkaline phosphatase CAP.
  • Another carrier is glutathione-S- transf erase (GST).
  • a molecule interactive with a non-full length tieHT k receptor fused to a carrier molecule The non-full length portion of the receptor acts an immunoreactive molecule.
  • the non-full length receptor comprises its extracellular domain.
  • the carrier molecule is FLAGTM or AP.
  • the resulting fusion molecule is then used to generate polyclonal or monoclonal antibodies which may undergo immunoadsorbent procedures to provide a composition of substantially, for example, extracellular domain-reactive receptor antibodies.
  • immunointeractive molecules is used herein in its broadest sense and includes antibodies, parts, fragments, derivatives, homologues or analogues thereof, peptide or non-peptide equivalents thereof and fusion molecules between two or more antibodies or between an antibody and another molecule.
  • the antibodies or other immunointeractive molecules may also be in recombinant or synthetic form. Accordingly, the present invention contemplates mutants and derivatives of the immunointeractive molecules, especially when such molecules are antibodies.
  • Mutants and derivatives of such antibodies include amino acid substitutions, deletions and/or additions. Furthermore, amino acids may be replaced by other amino acids having like properties, such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains, interactive and/or functional groups and so on. Glycosylation variants and hybrid antibodies are also contemplated by the present invention.
  • Amino acid substitutions are typically of single residues; insertions will usually be of the order of about 1-10 amino acid residues; and deletions will range from about 1-20 residues. Deletions or insertions preferably are made in adjacent pairs, i.e: a deletion of 2 residues or insertion of 2 residues.
  • amino acid variants referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known sequence are well known, for example through M13 mutagenesis. The manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are well known in the art.
  • KTr ⁇ E SHEET R LE 2 Other examples of recombinant or synthetic mutants and derivatives of the antibodies of the present invention include single or multiple substitutions, deletions and/or additions to any molecule associated with the ligand such as carbohydrates, lipids and/or proteins or polypeptides. Naturally occurring or altered glycosylated forms of the subject antibodies are particularly contemplated by the present invention.
  • Amino acid alterations to the subject polypeptide contemplated herein include insertions such as amino acid and/or carboxyl terminal fusions as well as intra- sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino or carboxyl terminal fusions, of the order of about 1 to 4 residues.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional variants are 'hose in which at least one residue in the sequence has been removed and a different r ⁇ . :due inserted in its place. Such substitutions may be made in accordance with Table 1.
  • analogues and derivatives also extend to any functional chemical equivalents of the antibodies characterised by their increased stability and/or efficacy in vivo or in vitro.
  • analogue and derivatives further extend to any amino acid derivative of the antibodies as described above.
  • Antibody analogues contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or derivatising the molecules and the use of crosslinkers and other methods which i ose conformational constraints on the antibodies.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5 '-phosphate followed by reduction with NaBH 4 .
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3- butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O- acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbomoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxy!ation with diethylpyrocarbonate .
  • Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t- butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6- methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • peptides could be conformationally constrained by, for example, incorporation of C ⁇ and N a - methylamino acids, introduction of double bonds between C ⁇ and C ⁇ atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the present invention extends to amino acid and/or chemical analogues of the subject antibodies having the identifying characteristics of being interactive with the extracellular domain of the t e2/Tek receptor.
  • an antibody includes the naturally occurring molecule, recombinant, synthetic and analogue forms thereof and to any mutants, derivatives and human and non-human homologues thereof including amino acid and glycosylation variants.
  • the immunointeractive molecules of the present invention may be used to develop a new range of therapeutic and diagnostic agents.
  • the antibodies or fragments or derivatives thereof may act as antagonists and be useful, for example, in the treatment of angiogenic-dependent phenotype and disease conditions resulting therefrom (e.g. metastasis). They may also be used for screening for agonists useful, for example, where angiogenesis is to be promoted. Normal, abnormal or mutant receptor structure or receptor expression can also be determined through immunoreactivity studies.
  • a method of detecting a rie2/Tek receptor on a cell in a biological sample comprising contacting said sample with an immunointeractive molecule capable of binding to the extracellular domain of said rie2/Tek receptor immobilised to a solid support for a
  • the immunointeractive molecule in rie2/Tek complex is detected by contacting the complex with an antibody against the immunointeractive molecule with the antibody being labelled with a reporter molecule.
  • the immunointeractive molecule itself is labelled with a reporter molecule.
  • the immunointeractive molecules are generally antibodies and these or antibodies directed against the immunointeractive molecules may be polyclonal or monoclonal antibodies and both are obtainable by immunisation of a suitable animal with the immunointeractive molecule (or tiel/Tek molecule) and either type is utilisable in the assay.
  • the methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of immunointeractive molecule or rie2/Tek preparation, or antigenic parts thereof, collecting serum from the animal, and isolating specific antibodies by any of the known immunoadsorbent techniques.
  • antibodies produced by this method are utilisable in virtually any type of assay, they are generally less favoured because of the potential heterogeneity of the product.
  • the use of monoclonal antibodies in the above immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitised against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol II , ed. by Schwartz, 1981; Kohler and Milstein, Nature 256: 495-499, " 975; European Journal of Immunology 6: 511 -519 , 1976) .
  • tie2/Tek receptor may be accomplished in a number of ways such as by Western blotting and ELISA procedures.
  • a wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4, 424,279 and 4,018,653. These, of course, include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
  • Sandwich assays are among the most useful and commonly used assays and are particularly useful in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an immunointeractive molecule is brought into contact with a biological sample comprising cells potentially carrying tz ' e2/Tek. After a suitable period of incubation, for a period of time sufficient to allow formation of an immunointeractive molecule - tie2/Tek complex, an antibody specific to the immunointeractive molecule, labelled with a reporter molecule capable of producing a detectable signal, is then added and incubated allowing sufficient time for the formation of a tertiary complex.
  • any unreacted material is washed away, and the presence of the antibody bound to the immunointeractive molecule is determined by observation of a signal produced by the reporter molecule.
  • the results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten.
  • Variations on the forward assay include using an immunointeractive molecule labelled with a reporter molecule.
  • the immunointeractive molecule or cells may be immobilised onto a solid support. Suitable solid supports include glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, poly vinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing mLBP to the polymer.
  • Reporter molecule is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • SUBSTITUTE SHEET (R ⁇ iE 26
  • an enzyme is conjugated to the immunointeractive molecule or an antibody thereto generally by means of glutaraldehyde or periodate.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • the enzyme-labelled antibody is added to the immunointeractive molecule-receptor complex, allowed to bind, and then the excess reagent is washed away.
  • an enzyme labelled immunointeractive molecule is used.
  • a solution containing the appropriate substrate is then added to the tertiary complex.
  • the substrate will react with the enzyme linked to the antibody/immunointeractive molecule, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
  • Reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescent compounds may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome- labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome- labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • Immunofluorescence and El A techniques are both very well established in the art and are particularly useful for the present method.
  • other reporter molecules such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • the present invention also provides a pharmaceutical composition comprising an effective amount of an immunointeractive molecule capable of binding or otherwise associating with the extracellular domain of tie2/Tek receptor and one or more pharmaceutically acceptable carriers and/or diluents.
  • the active ingredients of a pharmaceutical composition comprising the immunointeractive molecules are contemplated to exhibit excellent therapeutic activity, for example, in the treatment of angiogenic-dependent phenotype and disease conditions resulting therefrom, such as metastasis, in an amount which depends on the particular case. For example, from about 0.5 ⁇ g to about 20 mg per kilogram of body weight per day may be administered. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (e.g. using slow release molecules).
  • the active ingredients which comprise the immunointeractive molecules may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients.
  • they will be coated by, or administered with, a material to prevent its inactivation.
  • the immunointeractive molecules may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes.
  • Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon.
  • Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
  • Liposomes include water-in-oil-in- water emulsions as well as conventional liposomes.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the active, compound may be orally administered, for example, with an inert diluent
  • compositions and preparations should contain at least 1 % by weight of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 10 ⁇ g and 2000 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and formulations.
  • pharmaceutically acceptable carrier and/or diluent includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/ml of carrier.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • the immunointeractive molecules used in a pharmaceutical composition are antibodies or mutants or derivatives thereof. Most preferably, the antibodies are monoclonal antibodies.
  • the present invention is further described by reference to the following non- limiting examples.
  • the murine myeloma P3X63Ag8.653(NS-l) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% v/v HI FBS, 5 mM L-glutamine, 50 ⁇ g/ml gentamycin, grown at 37° C in a humidified atmosphere of 10% v/v CO 2 .
  • Hybridomas were grown in supplemented DMEM plus 10 ⁇ g/ml of recombinant IL-6 (rIL-6) at all stages after removal from the hypoxanthine aminupterin thymidine (HAT) selection medium.
  • NIH-3T3 cells transfected with the AP-TAG- 1 constructs were selected in supplemented DMEM containing 700 ⁇ g/ml G418.
  • CHO cell lines were grown in Glasgow Minimal Essential Medium supplemented with 50 ⁇ g/ml gentamycin, non-essential amino acids, sodium pyruvate, glutamate and asparagine, nucleosides and 25 mM methionine sulphoxide (MSX).
  • COS cells were maintained in RPMI-1640 medium with supplements at 37 °C and 5% v/v C0 2 and transfected using the DEAE-dextran protocol.
  • EXAMPLE 2 Construction of the tze2-FLAGTM Plasmid
  • the full length clone of the mouse tie2 receptor described by Runting et al., (3) was subcloned into the mammalian expression vector pCDM8 (Invitrogen, San Diego, CA) using the BstXI restriction enzyme site.
  • Single stranded DNA was generated using the M13 origin of replication, and this DNA used as a template to make tie2 cDNA containing specific enzyme sites.
  • In frame BamHI sites were introduced prior to the transmembrane domain to allow ligation into the (5')HindIII-(3')BgIII site of the expression vector AP-TAG- 1.
  • the mutant tie2 receptor containing the BamHI site located at the junction of the extracellular domain and the transmembrane domain was used to ligate a oligonucleotide linker sequence encoding the FLAGTM marker peptide, an in frame stop codon, Bglll compatible ends and an internal Clal site . (IBI; 5 '
  • Affinity Chromatographv tz ' e2-FLAGTM was purified from the expended tissue culture supernatant of tz ' e2-FLAGTM-CHO by affinity chromatography on a M2 (anti-FLAG) gel (IBI). Supernatant (100 ml) was passed over the M2 column then subsequently washed with
  • tz ' e2-FLAGTM Monoclonal Antibody Production Purified tz ' e2-FLAGTM (approximately 10 ⁇ g) was used to immunise female Wistar rats on day 54 (i.p.), 24 (i.p., and 3 (i.v. and i.p.) prior to fusion with the mouse myeloma P3X63Ag8.653 (NS-1). tz ' e2-FLAGTM was prepared for the first two i.p.
  • immunisations by combining with adjuvant containing trehalose dimycolate from Mycobacterium phlei, monophosphoryl lipid A from Salmonella minnesota R595, PBS/0.2% v/v Tween 80 and squalene as described in the manufactures instructions (RIBI Immunochem Research, Hamilton, MT).
  • the final immunisation was performed with purified fte2-FLAGTM diluted 1/1 with PBS. Rats were test bled on day 18 prior to fusion and the titre of anti-tz ' e2-FLAGTM antibodies determined by a solid phase El A and immunoprecipitation of tie2-AP.
  • Monoclonal antibodies to the tie2 extracellular domain were selected by screening the fusion on purified tz ' e2-FLAGTM and NYK extracellular domain-FLAGTM by an enzyme immunoassay. Briefly, 96 well PVC microtitre plates were coated with either rie2-FLAGTM or NYK-EX-FLAGTM at a level previously determined by reactivity
  • SUBSTITUTE SHEET RDU 26 with an anti-FLAGTM antibody (M2) to give an equivalent signal.
  • Hybridoma supernatants were added and incubated for 2 h at 4°C, followed by six washes with PBS/0.02% v/v Tween 20 (buffer). Incubation with a horse radish peroxidase conjugate anti-rat Ig followed for 1 h at 4°C. After washing, the assay was developed with an ABTS substrate system and the assay quantitated by reading absorbances at 405 mm in a multiwell plate reader (Flow Laboratories MCC/340, McLean, VA). Antibodies selected for further analysis were subcloned three times by limiting dilution. These antibodies were designated 3gl, 4g8, 6al2, lell and 3a6.
  • Rat monoclonal antibodies were purified using the technique of Darby et al. (13). Briefly, FCS was depleted of bovine IgG by serial passage over a protein G- Sepharose fast flow gel. The resulting bovine IgG depleted serum was used for the bulk culture of the rat hybridoma cell line. Cells were grown in roller bottles containing DMEM, 10% w/v IgG depleted FCS, 5 mM glutamine, 50 ⁇ g/ml gentamicin and 10 ⁇ g/ml recombinant IL-6 until expiration and the supernatant removed from the cellular debris. Rat IgG was subsequently purified by affinity chromatography on protein G-Sepharose and the yield assessed by OD 280 nm. Antibodies were dialysed versus PBS for use in assays. For coupling to
  • Immobilisation of tz ' e2-EX-FLAGTM and monoclonal antibodies were performed using standard NHS chemistry and using conditions recommended by the manufacturers instructions. Regeneration of the chip surface was carried out by passing 10 ⁇ l of
  • a construct was produced containing the tie2 extracellular domain fused in frame to the FLAGTM marker peptide. This was achieved by introducing a BamHI site at the junction of the extracellular domain and the transmembrane domain of the receptor by site-directed mutagenesis. An oligonucleotide linker sequence encoding the FLAGTM marker peptide, an in frame stop codon and having Bglll compatible ends was then ligated into this site and successfully ligated plasmids selected by a Clal site within the linker sequence. Expression analysis in COS cells demonstrated a protein of the expected size which could be specifically immunoprecipitated by the M2
  • the construct was subcloned into the pEE6 CHO cell expression vector for large scale production of the tz ' e2-FLAGTM protein.
  • the tz ' e2-FLAGTM protein was purified from CHO cell supernatants by affinity chromatography on M2 antibody gel and elution with the free FLAGTM peptide. Subsequent purification by ion-exchange chromatography removed the free peptide and other contaminants giving a homogenous species of Mr 95,000-100,000 by SDS-PAGE which is consistent with the predicted size of the extracellular domain plus glycosylation.
  • Rats were immunised with the rie2-FLAGTM protein and upon achieving an appropriate response their spleen cells were fused with the mouse myeloma NS-1 using standard somatic cell hybridisation techniques. Antibodies were selected on the basis of their reactivity to purified tz ' e2-FLAGTM compared to another fusion protein containing the extracellular domain of the NYK receptor ligated to the FLAGTM marker peptide.
  • cell lines expressing the t e2-FLAGTM and tie2-AP were biosynthetically labelled with 35 [S] Cys-Met and used for immunoprecipitation experiments.
  • Five monoclonal antibodies were shown to specifically immunoprecipitate the tz ' e2-FLAGTM and tie2-AP protein indicating they recognise the native tz ' e2 extracellular domain.
  • the role of the anti-t e2 antibodies as an agonist in endothelial cell growth and development would rely on their binding to a growth factor receptor and causing its dimerization and subsequent activation.
  • the ti ⁇ l receptor is though to be such a receptor, so the action of an antibody which crosslinks tie2 may be agonistic.
  • an artificial system which would detect the dimerization of the extracellular domain was established.
  • a chimeric molecule consisting of the extracellular domain of tie2 receptor fused to the intracellular and transmembrane domain of the Erythropoietin receptor (EpoR) was constructed and transfected into a factor dependent cell line (Ba/F3).
  • the control cell line is stimulated to proliferate by the antibody - all cells die within 48 hours.
  • the ?ze2-EpoR cell line shows a dose dependent response to the levels of purified 1 lei beginning at a concentration of 0.1 ⁇ g/ml.
  • the 4g8 antibody also stimulates the tze2-EpoR transfected Ba/F3 cells to a lesser degree, whereas the 3gl antibody does not stimulate these cells at all (data not shown).
  • Antibody 4g8 Can Detect the tie2-2 Receptor on the Surface of Cells by FACS
  • FACS Fluorescent Activated Cell Sorting
  • a control cell line gives low fluorescence ( Figure 6, unshaded), whereas the transfected cell line shows a higher fluorescence, consistent with the presence of a large number of receptors on the surface ( Figure 6, shaded).
  • the 4g8 antibody has also been used detect expression of native tie2 on an endothelial cell line of mouse origin, as well as chimeric tie2 molecules transfected into COS and Ba/F3 cells, respectively.
  • TELECOMMUNICATION INFORMATION (A) TELEPHONE: (212) 688-9200 (B) TELEFAX: (212) 838-3884 (2) INFORMATION FOR SEQ ID NO: 1:

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Abstract

La présente invention concerne des molécules présentant une immuno-interactivité avec le récepteur du facteur de croissance animal, et en particulier, le récepteur animal tie2/Tek. Ces molécules immuno-interactives constituent la base de nouveaux agents thérapeutiques et diagnostiques, utiles pour le traitement, la prophylaxie et le diagnostic du phénotype angiogéno-dépendant.
PCT/US1995/001743 1994-02-10 1995-02-09 Molecule immuno-interactive se liant au domaine extracellulaire du recepteur tie2/tek WO1995021866A1 (fr)

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EP95910973A EP0812332A4 (fr) 1994-02-10 1995-02-09 Molecule immuno-interactive se liant au domaine extracellulaire du recepteur tie2/tek
JP7521374A JPH10503081A (ja) 1994-02-10 1995-02-09 Ｔｉｅ２／ｔｅｋレセプタ細胞外ドメインに結合する免疫相互作用性分子
AU18745/95A AU689232B2 (en) 1994-02-10 1995-02-09 An immunointeractive molecule which binds the (TIE)2/TEK receptor extracellular domain

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AUPM3794A AUPM379494A0 (en) 1994-02-10 1994-02-10 Immunointeractive molecules - ii

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Cited By (26)

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US5851797A (en) * 1996-06-19 1998-12-22 Regeneron Pharmaceuticals, Inc. Tie ligand-3, methods of making and uses thereof
WO1999043801A1 (fr) * 1998-02-26 1999-09-02 Cancer Research Campaign Technology Limited Vaccins anti-angiogeniques: substances et methodes afferentes
US6030831A (en) * 1997-09-19 2000-02-29 Genetech, Inc. Tie ligand homologues
WO2000018437A1 (fr) * 1998-09-28 2000-04-06 Smithkline Beecham Corporation Anticorps antagonistes de tie2
US6057435A (en) * 1997-09-19 2000-05-02 Genentech, Inc. Tie ligand homologues
EP1046715A1 (fr) * 1999-04-23 2000-10-25 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Interaction de la protéine vasculaire endothelial tyrosine phosphatase et le récepteur Tie-2 d'angiopoietine
WO2000075323A1 (fr) * 1999-06-07 2000-12-14 Immunex Corporation Antagonistes tek
EP1117692A1 (fr) * 1998-09-28 2001-07-25 SmithKline Beecham Corporation Anticorps agonistes de tie2
US6348350B1 (en) 1997-09-19 2002-02-19 Genentech, Inc. Ligand homologues
US6350450B1 (en) 1997-09-19 2002-02-26 Genentech, Inc. TIE ligand homologue antibody
US6368853B1 (en) 1997-09-19 2002-04-09 Genentech, Inc. Tie ligand homologues
US6521424B2 (en) 1999-06-07 2003-02-18 Immunex Corporation Recombinant expression of Tek antagonists
WO2003034990A2 (fr) * 2001-10-25 2003-05-01 Regeneron Pharmaceuticals, Inc. Angiopoietines et methodes d'utilisation
US7138370B2 (en) 2001-10-11 2006-11-21 Amgen Inc. Specific binding agents of human angiopoietin-2
US7205275B2 (en) 2001-10-11 2007-04-17 Amgen Inc. Methods of treatment using specific binding agents of human angiopoietin-2
US7485297B2 (en) 2003-08-12 2009-02-03 Dyax Corp. Method of inhibition of vascular development using an antibody
US7521053B2 (en) 2001-10-11 2009-04-21 Amgen Inc. Angiopoietin-2 specific binding agents
US7658924B2 (en) 2001-10-11 2010-02-09 Amgen Inc. Angiopoietin-2 specific binding agents
WO2012162561A2 (fr) 2011-05-24 2012-11-29 Zyngenia, Inc. Complexes plurispécifiques multivalents et monovalents, et leurs utilisations
EP2671891A2 (fr) 2008-06-27 2013-12-11 Amgen Inc. Inhibition d'ang-2 pour traiter la sclérose en plaques
US9017670B2 (en) 2011-08-19 2015-04-28 Regeneron Pharmaceuticals, Inc. Anti-Tie2 antibodies and uses thereof
US9902782B2 (en) 2014-07-15 2018-02-27 Astellas Pharma Inc. Anti-human tie-2 antibody
EP3424530A1 (fr) 2013-03-15 2019-01-09 Zyngenia, Inc. Complexes multispécifiques monovalents et multivalents et leurs utilisations
US10316105B2 (en) 2011-08-19 2019-06-11 Regeneron Pharmaceuticals, Inc. Anti-TIE2 antibodies and uses thereof
US10336820B2 (en) 2008-02-20 2019-07-02 Amgen Inc. Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof
EP4061852A4 (fr) * 2019-11-21 2024-03-13 Unity Biotechnology Anticorps dirigés contre tie-2 et méthodes d'utilisation

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US4683295A (en) * 1984-05-24 1987-07-28 Scripps Clinic And Research Foundation Method for the preparation of anti-receptor antibodies
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PROC. NATL. ACAD. SCI. U.S.A., Volume 90, issued October 1993, SATO et al., "Tie-1 and Tie-2 Define Another Class of Putative Receptor Tyrosine Kinase Genes Expressed in Early Embryonic Vascular System", pages 9355-9358. *
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Cited By (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5851797A (en) * 1996-06-19 1998-12-22 Regeneron Pharmaceuticals, Inc. Tie ligand-3, methods of making and uses thereof
US6521234B1 (en) 1997-09-19 2003-02-18 Genentech, Inc. NL3 TIE ligand homologue
US6551822B1 (en) 1997-09-19 2003-04-22 Genentech, Inc. NL4 tie ligand homologue
US6586397B1 (en) 1997-09-19 2003-07-01 Genentech, Inc. Tie ligand homologues
US6057435A (en) * 1997-09-19 2000-05-02 Genentech, Inc. Tie ligand homologues
US6492331B1 (en) 1997-09-19 2002-12-10 Genentech, Inc. NL8 tie ligands homologues
US6426218B1 (en) 1997-09-19 2002-07-30 Genentech, Inc. NL3 TIE ligand homologue nucleic acids
US6030831A (en) * 1997-09-19 2000-02-29 Genetech, Inc. Tie ligand homologues
US6420542B1 (en) 1997-09-19 2002-07-16 Paul J. Godowski Tie ligands
US6074873A (en) * 1997-09-19 2000-06-13 Genentech, Inc. Nucleic acids encoding NL-3
US6348350B1 (en) 1997-09-19 2002-02-19 Genentech, Inc. Ligand homologues
US6348351B1 (en) 1997-09-19 2002-02-19 Genentech, Inc. Tie receptor tyrosine kinase ligand homologues
US6350450B1 (en) 1997-09-19 2002-02-26 Genentech, Inc. TIE ligand homologue antibody
US6455496B1 (en) 1997-09-19 2002-09-24 Genentech, Inc. NL2 TIE ligand homologue polypeptide
US6368853B1 (en) 1997-09-19 2002-04-09 Genentech, Inc. Tie ligand homologues
US6372491B1 (en) 1997-09-19 2002-04-16 Genentech, Inc. Tie ligands
WO1999043801A1 (fr) * 1998-02-26 1999-09-02 Cancer Research Campaign Technology Limited Vaccins anti-angiogeniques: substances et methodes afferentes
EP1117692A1 (fr) * 1998-09-28 2001-07-25 SmithKline Beecham Corporation Anticorps agonistes de tie2
US6365154B1 (en) 1998-09-28 2002-04-02 Smithkline Beecham Corporation Tie2 agonist antibodies
EP1117692A4 (fr) * 1998-09-28 2002-11-13 Smithkline Beecham Corp Anticorps agonistes de tie2
WO2000018437A1 (fr) * 1998-09-28 2000-04-06 Smithkline Beecham Corporation Anticorps antagonistes de tie2
WO2000065085A1 (fr) * 1999-04-23 2000-11-02 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Interaction de proteine-tyrosine phosphatase vasculaire-endotheliale avec le recepteur angiopoietine tie-2
EP1046715A1 (fr) * 1999-04-23 2000-10-25 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Interaction de la protéine vasculaire endothelial tyrosine phosphatase et le récepteur Tie-2 d'angiopoietine
WO2000075323A1 (fr) * 1999-06-07 2000-12-14 Immunex Corporation Antagonistes tek
US7067475B2 (en) 1999-06-07 2006-06-27 Immunex Corporation Tek antagonists
US6413932B1 (en) 1999-06-07 2002-07-02 Immunex Corporation Tek antagonists comprising soluble tek extracellular binding domain
US6521424B2 (en) 1999-06-07 2003-02-18 Immunex Corporation Recombinant expression of Tek antagonists
US7205275B2 (en) 2001-10-11 2007-04-17 Amgen Inc. Methods of treatment using specific binding agents of human angiopoietin-2
US9200040B2 (en) 2001-10-11 2015-12-01 Amgen Inc. Specific binding agents of human angiopoietin-2
US7138370B2 (en) 2001-10-11 2006-11-21 Amgen Inc. Specific binding agents of human angiopoietin-2
US7723499B2 (en) 2001-10-11 2010-05-25 Amgen Inc. Specific binding agents of human angiopoietin-2
US7790674B2 (en) 2001-10-11 2010-09-07 Amgen Inc. Methods of treatment using specific binding agents of human angiopoietin-2
US7521053B2 (en) 2001-10-11 2009-04-21 Amgen Inc. Angiopoietin-2 specific binding agents
US7658924B2 (en) 2001-10-11 2010-02-09 Amgen Inc. Angiopoietin-2 specific binding agents
US7666832B2 (en) 2001-10-11 2010-02-23 Amgen Inc. Methods of treatment using specific binding agents of human angiopoietin-2
US7666839B2 (en) 2001-10-11 2010-02-23 Amgen Inc. Methods of treatment using specific binding agents of human angiopoietin-2
US7666831B2 (en) 2001-10-11 2010-02-23 Amgen Inc. Methods of treatment using specific binding agents of human angiopoietin-2
WO2003034990A2 (fr) * 2001-10-25 2003-05-01 Regeneron Pharmaceuticals, Inc. Angiopoietines et methodes d'utilisation
WO2003034990A3 (fr) * 2001-10-25 2003-12-04 Regeneron Pharma Angiopoietines et methodes d'utilisation
US7485297B2 (en) 2003-08-12 2009-02-03 Dyax Corp. Method of inhibition of vascular development using an antibody
US10336820B2 (en) 2008-02-20 2019-07-02 Amgen Inc. Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof
EP2671891A2 (fr) 2008-06-27 2013-12-11 Amgen Inc. Inhibition d'ang-2 pour traiter la sclérose en plaques
WO2012162561A2 (fr) 2011-05-24 2012-11-29 Zyngenia, Inc. Complexes plurispécifiques multivalents et monovalents, et leurs utilisations
US10316105B2 (en) 2011-08-19 2019-06-11 Regeneron Pharmaceuticals, Inc. Anti-TIE2 antibodies and uses thereof
US10023641B2 (en) 2011-08-19 2018-07-17 Regeneron Pharmaceuticals, Inc. Anti-TIE2 antibodies and uses thereof
US9546218B2 (en) 2011-08-19 2017-01-17 Regeneron Pharmaceuticals, Inc. Anti-Tie2 antibodies and uses thereof
US9017670B2 (en) 2011-08-19 2015-04-28 Regeneron Pharmaceuticals, Inc. Anti-Tie2 antibodies and uses thereof
US10442864B2 (en) 2011-08-19 2019-10-15 Regeneron Pharmaceuticals, Inc. Anti-Tie2 antibodies and uses thereof
US10752702B2 (en) 2011-08-19 2020-08-25 Regeneron Pharmaceuticals, Inc. Anti-TIE2 antibodies and uses thereof
EP3424530A1 (fr) 2013-03-15 2019-01-09 Zyngenia, Inc. Complexes multispécifiques monovalents et multivalents et leurs utilisations
US9902782B2 (en) 2014-07-15 2018-02-27 Astellas Pharma Inc. Anti-human tie-2 antibody
EP4061852A4 (fr) * 2019-11-21 2024-03-13 Unity Biotechnology Anticorps dirigés contre tie-2 et méthodes d'utilisation

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CA2182681A1 (fr) 1995-08-17

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