WO2004003211A1 - Anticorps bispecifiques qui se lient aux recepteurs de vegf - Google Patents

Anticorps bispecifiques qui se lient aux recepteurs de vegf Download PDF

Info

Publication number
WO2004003211A1
WO2004003211A1 PCT/US2002/041372 US0241372W WO2004003211A1 WO 2004003211 A1 WO2004003211 A1 WO 2004003211A1 US 0241372 W US0241372 W US 0241372W WO 2004003211 A1 WO2004003211 A1 WO 2004003211A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
ofthe
vegf receptor
kdr
Prior art date
Application number
PCT/US2002/041372
Other languages
English (en)
Inventor
Zhenping Zhu
Original Assignee
Imclone Systems Incorporated
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2002/020332 external-priority patent/WO2003002144A1/fr
Application filed by Imclone Systems Incorporated filed Critical Imclone Systems Incorporated
Priority to US10/520,026 priority Critical patent/US20090028859A1/en
Priority to AU2002368062A priority patent/AU2002368062A1/en
Publication of WO2004003211A1 publication Critical patent/WO2004003211A1/fr

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody

Definitions

  • the present invention is directed to production of bispecific antigen-binding proteins that bind specifically to the extracellular domains of two different VEGF receptors.
  • the bispecific antigen-binding proteins block activation ofthe VEGF receptors and are used to reduce or inhibit VEGF-induced cellular functions such as mitogenesis of vascular endothelial cells and migration of leukemia cells.
  • the antigen-binding proteins ofthe present invention have antigen-binding sites consisting of immunoglobulin heavy chain and light chain variable domains and may be monovalent or bivalent.
  • the antigen-binding proteins can further comprise immunoglobulin constant regions.
  • VEGF Vascular endothelial growth factors
  • P1GF placenta growth factor
  • VEGFR-l/Flt-1, VEGFR-2/KDR and VEGFR-3/Flt-4 have important roles in vasculogenesis, angiogenesis and growth of tumor cells.
  • VEGF Vascular endothelial growth factor
  • VEGF is a strong inducer of vascular permeability, stimulator of endothelial cell migration and proliferation, and is an important survival factor for newly formed blood vessels.
  • VEGF binds to and mediates its activity mainly through two tyrosine kinase receptors, VEGF receptor 1 (VEGFR-1), or fins-like tyrosine receptor 1 (Flt-1), and VEGF receptor 2 (VEGFR-2), or kinase insert domain-containing receptor (KDR; Flk-1 in mice).
  • VEGFR-1 VEGF receptor 1
  • Flt-1 fins-like tyrosine receptor 1
  • VEGFR-2 VEGF receptor 2
  • KDR kinase insert domain-containing receptor
  • Flt-1 and KDR have distinct functions in vascular development in embryos. Targeted deletion of genes encoding either receptor in mice is lethal to the embryo, demonstrating the physiological importance ofthe VEGF pathway in embryonic development. KDR-deficient mice have impaired blood island formation and lack mature endothelial cells, whereas Flt-1 null embryos fail to develop normal vasculature due to defective in the formation of vascular tubes, albeit with abundant endothelial cells. On the other hand, inactivation of Flt-1 signal transduction by truncation ofthe tyrosine kinase domain did not impair mouse embryonic angiogenesis and embryo development, suggesting that signaling through the Flt-1 receptor is not essential for vasculature development in the embryo.
  • Flt-1 and KDR The biological responses of Flt-1 and KDR to VEGF in the adult also appear to be different. It is generally believed that KDR is the main VEGF signal transducer that results in endothelial cell proliferation, migration, differentiation, tube formation, increase of vascular permeability, and maintenance of vascular integrity.
  • Flt-1 possesses a much weaker kinase activity, and is unable to generate a mitogenic response when stimulated by VEGF - although it binds to VEGF with an affinity that is approximately 10-fold higher than KDR.
  • Flt-1 has been implicated in VEGF and placenta growth factor (PlGF)-induced migration of monocytes/macrophage and production of tissue factor.
  • PlGF placenta growth factor
  • VEGF-B Apart from VEGF and PIGF, several other growth factors related to VEGF have been identified: VEGF-B, VEGF-C, VEGF-D, and VEGF-E.
  • VEGF-B like PIGF, binds to Flt-1.
  • VEGF-E is specific for KDR, while VEGF-C and VEGF-D can bind to KDR and another receptor, VEGFR-3 (Flt-4).
  • these ligands may form heterodimers that bind differentially to various receptor homo- or heterodimers and signal through different pathways.
  • Multispecific antibodies have been used in several small-scale clinical trials as cancer imaging and therapy agents, but broad clinical evaluation has been hampered by the lack of efficient production methods.
  • the design of such proteins thus far has been concerned primarily with providing multispecificity. In few cases has any attention been devoted to providing other useful functions associated with natural antibody molecules.
  • Bispecificity and/or bivalency has been accomplished by fusing two scFv molecules via flexible linkers, leucine zipper motifs, C H C L -heterodimerization, and by association of scFv molecules to form bivalent monospecific diabodies and related structures.
  • Multivalency has been achieved by the addition of multimerization sequences at the carboxy or amino terminus ofthe scFv or Fab fragments, by using for example, p53, streptavidin and helix-turn-helix motifs.
  • scFv fusion protein ofthe form (scFvl)-hinge-helix-turn-helix-(scFv2)
  • a tetravalent bispecific miniantibody is produced having two scFv binding sites for each of two target antigens.
  • Improved avidity may also been obtained by providing three functional antigen binding sites.
  • scFv molecules with shortened linkers connecting the V H and V L domains associate to for a triabody (Kortt et al, 1997, Protein Eng. 10:423-433).
  • IgG type bispecific antibodies which resemble IgG antibodies in that they possess a more or less complete IgG constant domain structure, has been achieved by chemical cross-linking of two different IgG molecules or by co-expression of two antibodies from the same cell.
  • One strategy developed to overcome unwanted pairings between two different sets of IgG heavy and light chains co-expressed in transfected cells is modification of he C H 3 domains of two heavy chains to reduce homodimerization between like antibody heavy chains.
  • CMC complement-mediated cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the present invention provides antibodies that have an antigen binding site specific for a first VEGF receptor and an antigen binding site specific for a second VEGF receptor.
  • the antibodies are at least bivalent and may be trivalent, tetravalent or multivalent.
  • the antibody is bispecific, having one antigen binding site specific for a first VEGF receptor and a second antigen binding site specific for a second VEGF receptor.
  • the antibody When bound to a VEGF receptor, the antibody effectively blocks interaction between the VEGF receptor and its ligand.
  • the antibody is effective to block dimerization ofthe VEGF receptor proteins. Compared to binding to a single VEGF receptor, dual binding can result in more potent inhibition of VEGF-stimulated cellular functions such as, for example, proliferation of endothelial cells and VEGF- and PlGF-induced migration of human leukemia cells.
  • Antigen-binding proteins are preferably specific for mammalian VEGF receptors or more preferably for human VEGF receptors.
  • VEGF receptors include human KDR, Flt-1 and Flt-4 and their mammalian homologs.
  • the antibody is specific for KDR and Flt-1.
  • an antibody can bind specifically to an extracellular domain of a VEGF receptor and neutralizing activation ofthe VEGF receptor, for example, by block ligand binding or receptor dimerization.
  • a bispecific antibody can bind specifically to a VEGF receptor and inhibit angiogenesis.
  • an antibody can bind specifically to an extracellular domain of a VEGF receptor and reduce tumor growth.
  • the invention further contemplates methods of producing bispecific antigen- binding proteins that are specific for two different VEGF receptors.
  • the antigen-binding proteins can be, for example, monovalent or bivalent.
  • diabodies are produced by coexpression and secretion of two protein chains in bacteria.
  • a first construct encodes the V H domain of a first antibody specific for the first VEGF receptor and the V L domain of a second antibody specific for the second VEGF receptor.
  • a second construct encodes the V L domain ofthe first antibody and the V H domain ofthe second antibody.
  • the two chains that are expressed associate as a heterodimer with one binding site for each VEGF receptor.
  • an Ig like antibody is produced wherein a first single chain Fv (scFv) specific for a first VEGF receptor is substituted for each ofthe V H domains and a second scFv specific for a second VEGF receptor is substituted for each ofthe V L domains.
  • the tetrameric antibody formed by association of two heavy and two light chains is bispecific and bivalent, and further comprises immunoglobulin constant regions.
  • the invention contemplates methods for neutralizing activation of a first VEGF receptor and a second VEGF receptor which comprise treating cells with a bispecific antibody ofthe invention. It is further contemplated to use the binding proteins in methods for inhibiting angiogenesis and reducing tumor growth.
  • Figure 1A is a schematic representation ofthe DNA constructs used for expression of scFv plCll, scFv 6.12 and the anti-KDR x anti-Fit- 1 bifunctional diabody comrising the pi CI 1 and Mab 6.12 antigen binding sites in E. coli.
  • Figure IB depicts expression and purification ofthe scFvs and the diabody.
  • the antibodies were expressed in E.coli, purified by affinity chromatography, and analyzed by SDS-PAGE. Lane 1, scFv plCll; lane 2, scFv 6.12; and lane 3, the bifunctional diabody. Molecular weights of markers are in kDa.
  • Figure 2 demonstrates the dual specificity ofthe anti-KDR x anti-Flt-1 bifunctional diabody.
  • Figure 2A shows simultaneous binding by the diabody to both KDR and Flt-1.
  • Figures 2B and 2C show specific binding ofthe antibodies to immobilized KDR (B) and Flt-1 (C).
  • Figure 3 shows inhibition of binding of KDR and Flt-1 to immobilized VEGF or PIGF by the anti-KDR x anti-Flt-1 bifunctional diabody.
  • Various concentrations of antibodies were incubated with a fixed concentration of KDR-AP (A) or Flt-l-Fc fusion proteins (B and C) in solution at RT for 1 h, after which the mixtures were transferred to 96-well plates coated with VEGF (A and B) or PIGF (C).
  • Figure 4 shows inhibition of PIGF and VEGF-induced migration of human leukemia cells by the anti-KDR x anti-Flt-1 bifunctional diabody.
  • Panel A and D PIGF (A) and VEGF (D) promote migration of HL60 and HEL cells in a dose-dependent manner.
  • Panels B, C, E and F Inhibition of PIGF (B and C), and VEGF (E and F) induced migration of human leukemia cells by the anti-KDR x anti-Flt-1 bifunctional diabody.
  • Figure 5 shows inhibition of VEGF-stimulated HUVEC mitogenesis by the anti-KDR x anti-Flt-1 bifunctional diabody.
  • the present invention provides bispecific antibodies that are capable of binding specifically to a first VEGF receptor and to a second VEGF receptor.
  • antibodies that bind to the extracellular domains of such receptors include the ligand-binding domain ofthe extracellular portion ofthe receptor, as well as extracellular portions that are involved in dimerization and overlapping epitopes.
  • the antibodies When bound to the extracellular domain of a VEGF receptor, the antibodies effectively block ligand binding and/or interfere with receptor dimerization. As a result of such binding, the antibodies neutralize activation ofthe VEGF receptor.
  • Neutralizing a receptor means diminishing and/or inactivating the intrinsic ability ofthe receptor to transduce a signal.
  • a reliable assay for VEGF receptor neutralization is inhibition of receptor phosphorylation.
  • Methods of determining receptor phosphorylation are well known in the art and include, for example, measurement of phosphotyrosine with monoclonal antibodies or radioactive labels.
  • a natural antibody molecule is composed of two identical heavy chains and two identical light chains. Each light chain is covalently linked to a heavy chain by an interchain disulfide bond. The two heavy chains are further linked to one another by multiple disulfide bonds.
  • Fig. 1 represents the structure of a typical IgG antibody. The individual chains fold into domains having similar sizes (110-125 amino acids) and structures, but different functions.
  • the light chain comprises one variable domain (Vf) and one constant domain (CJ.
  • the heavy chain comprises one variable domain (V JJ ) and, depending on the class or isotype of antibody, three or four constant domains (C H 1, C H 2, C H 3 and C H 4).
  • the isotypes are IgA, IgD, IgE, IgG, and IgM, with IgA and IgG further subdivided into subclasses or subtypes.
  • the portion of an antibody consisting of V L and V H domains is designated "Fv" and constitutes the antigen-binding site.
  • a single chain Fv (scFv) is an engineered protein containing a V L domain and a V H domain on one polypeptide chain, wherein the N terminus of one domain and the C terminus ofthe other domain are joined by a flexible linker.
  • Fab refers to the portion ofthe antibody consisting of V L , V H , C L and C H 1 domains.
  • variable domains show considerable amino acid sequence variablity from one antibody to the next, particularly at the location ofthe antigen binding site.
  • Three regions, called “hypervariable” or “complementarity-determining regions” (CDR's) are found in each of V L and V H .
  • Fc is the designation for the portion of an antibody which comprises paired heavy chain constant domains, i an IgG antibody, for example, the Fc comprises C H 2 and C H 3 domains.
  • the Fc of an IgA or an IgM antibody further comprises a C ⁇ domain.
  • the Fc is associated with Fc receptor binding, activation of complement-mediated cytotoxicity and antibody-dependent cellular-cytoxicity.
  • complex formation requires Fc constant domains.
  • antibody refers to a binding protein that comprises antibody V H and/or V L domains.
  • Antibody specificity refers to selective recognition ofthe antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. Bispecific antibodies (BsAbs) are antibodies which have two different antigen-binding specificities or sites. Where an antibody has more than one specificity, the recognized epitopes may be associated with a single antigen or with more than one antigen.
  • Antibodies ofthe present invention are specific for at least a first and a second VEGF receptor, which receptors include, but are not limited to, human KDR, Flt-1, Flt-4 and their non-human homologs.
  • Valency refers to the number of binding sites which an antibody has for a particular epitope.
  • a natural IgG antibody is monospecific and bivalent. Where an antibody has specificity for more than one epitope, valency is calculated for each epitope. For example, an antibody which has four binding sites and recognizes a single epitope is tetravalent. An antibody with four binding sites, two binding sites having one specificity and two binding sites having a second specificity, is considered bivalent.
  • V L and V H domains for use in the present invention can be obtained, e.g., from hybridomas or phage display libraries, or from antibodies previously identified as specific for a VEGF receptor.
  • Bispecific antibodies specific for two different receptors are exemplified, although antibodies with more than two binding sites can be engineered that are specific for more than two antigens.
  • an antibody ofthe invention binds to KDR and Flt-1.
  • an antibody ofthe invention binds to KDR and Flt-4.
  • scFv plCl 1 SEQ ID NOS: 27, 28
  • plCl 1 blocks VEGF-KDR interaction and inhibits VEGF-stimulated receptor phosphorylation and mitogenesis of human vascular endothelial cells (HUVEC).
  • VEC human vascular endothelial cells
  • Mab 6.12 is an example of an antibody that binds to soluble and cell surface- expressed Flt-1.
  • a hybridoma cell line producing Mab 6.12 has been deposited as ATCC number PTA-3344 under the provisions ofthe Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure and the regulations thereunder (Budapest Treaty).
  • antibodies to an individual growth factor such as VEGF would only neutralize specifically the angiogenic activity ofthe single ligand.
  • antagonistic antibodies to a VEGF receptor will not only block the angiogenic activity of VEGF, but also that of other growth factors exerting their angiogenic effects via the receptor.
  • an anti-KDR antibody will potentially block angiogenic activity of VEGF, VEGF-C, VEGF-D and VEGF-E, whereas an antibody to Flt-1 will inhibit the activity of VEGF, PIGF and VEGF-B.
  • antibodies ofthe invention are capable of binding to one or both monomers and blocking function.
  • KDR/Flt-1 heterodimers as well as KDR/KDR homodimers can be blocked by antibodies that are specific for KDR.
  • Antibodies specific for Flt-1 can block formation of KDR/Flt-1 heterodimers and Flt-l/Flt-1 homodimers.
  • Antibodies ofthe present invention have two or more binding sites and are at least bispecific. That is, the antibodies maybe bispecific even in cases where there are more than two binding sites.
  • Antibodies ofthe invention include, for example, multivalent single chain antibodies, diabodies and triabodies, as well as antibodies having the constant domain structure of naturally-occurring antibodies.
  • the antibodies can be wholly from a single species, or be chimerized or humanized.
  • some binding sites may be identical, so long as the protein has binding sites for two or more different antigens. That is, whereas a first binding site is specific for a first VEGF receptor, a second binding site is specific for a second, different VEGF receptor.
  • the antibodies are bispecific. In a more preferred embodiment, the antibodies are designed such that a population ofthe antibodies is homogeneous (i.e., each and every antibody in the population has a first binding site specific for a first VEGF receptor and a second binding site specific for a second VEGF receptor).
  • an antigen binding site of an antibody ofthe invention typically contains six complementarity determining regions (CDRs) which contribute in varying degrees to the affinity ofthe binding site for antigen.
  • CDRs complementarity determining regions
  • CDRHl, CDRH2 and CDRH3 three heavy chain variable domain CDRs
  • CDRL1, CDRL2 and CDRL3 three light chain variable domain CDRs
  • the extent of CDR and framework regions (FRs) is determined by comparison to a compiled database of amino acid sequences in which those regions have been defined according to variability among the sequences.
  • a binding protein specific for a VEGF receptor can consist of a single domain, i.e., a V H or a V L domain will bind specifically with high affinity.
  • Example of antibodies wherein binding affinity and specificity are contributed primarily by one or the other variable domain are known in the art. See, e.g., Jeffrey, P.D. et al., 1993, Proc. Nat.l. Acad. Sci. U S A 90:10310-10314, which discloses an anti-digoxin antibody which binds to digoxin primarily by the antibody heavy chain. Accordingly, single antibody domains can be identified that bind to VEGF receptors. Such single domain antibodies can be obtained, for example, from naturally occurring antibodies, or Fab or scFv phage display libraries.
  • Scaffolds for such single domain antibodies can be modified mouse or human variable domains. It is noted that single antibody domains can bind antigen in a variety of antigen binding modes. That is, the primary antibody-antigen interactions are not limited to amino acid residues corresponding to CDRs of V H -V L containing antibodies, and consideration can be given to binding interactions outside of CDR residues when optimizing the binding characteristics of such antibodies.
  • the antibodies ofthe present invention bind to VEGF receptors preferably with an affinity comparable to or greater than that ofthe natural ligand.
  • Affinity represented by the equilibrium constant for the association of an antigen with an immunoglobulin molecule (K), measures the binding strength between and antigenic determinant and an antigen binding site, irrespective ofthe number of binding sites.
  • K d the dissociation constant, is the reciprocal of K.
  • An antigenic determinant also known as an epitope, is the site on an antigen at which a given antibody binds.
  • Typical values of K d are 10 "5 M to 10 "11 M. Any K d greater than 10 "4 M is considered to be non-specific binding.
  • Avidity is a measure of the strength of binding between an immunoglobulin and its antigen. Unlike affinity, which measures the strength of binding at each binding site, avidity is determined by both the affinity and the number of antigen specific binding sites (valency) of an immunoglobulin molecule.
  • the antibodies ofthe invention may comprise only immunoglobulin variable domains, optionally linked by amino acid sequences of synthetic origin.
  • a typical diabody has two Fv domains and comprises two chains - the first chain incorporating the heavy chain variable domain of a first antibody linked to the light chain variable domain of a second antibody, and the second chain comprising the light chain variable domain ofthe first antibody linked to the heavy chain variable domain ofthe second antibody.
  • the domains are typically connected by a flexible polypeptide linker of about 5 to 10 amino acid residues, such as, for example, the 5 amino acid sequence Gly-Gly-Gly-Gly-Ser or the 10 amino acid sequence (Gly-Gly-Gly-Gly-Ser) 2 . Pairing of first and second chains is favored over pairing of like chains, and a substantially homogeneous population of diabodies is achieved.
  • antibodies ofthe invention further comprise immunoglobulin constant regions of one or more immunoglobulin classes.
  • Immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE isotypes and, in the case of IgG and IgA, their subtypes.
  • an antibody ofthe invention has a constant domain structure of an IgG type antibody, but has four antigen binding sites. This is accomplished by substituting a complete antigen binding sites (e.g., a single chain Fv) for each ofthe immunoglobulin variable domains.
  • the four antigen-binding sites preferably comprise two binding sites for each of two different binding specificities.
  • An antigen binding site for inclusion in an antibody having desired binding characteristics is obtained by a variety of methods.
  • the amino acid sequences ofthe V L and/or V H portions of a selected binding domain correspond to a naturally-occurring antibody or are chosen or modified to obtained desired immunogenic or binding characteristics.
  • V L and V H domains can be obtained directly from a monoclonal antibody which has the desired binding characteristics.
  • Anti-VEGFR-2 monoclonal antibodies include DC 101 (rat anti-mouse VEGFR-2; deposited as ATCC HB 11534), M25.18A1 (mouse anti-mouse VEGFR-2; deposited as ATCC HB 12152), and M73.24 (mouse anti-mouse VEGFR-2; deposited as ATCC HB 12153).
  • Anti-VEGFR-1 monoclonal antibodies include KM1730 (deposited as FERM BP-5697), KM1731 (deposited as FERM BP-5718), KM1732 (deposited as FERM BP-5698), KM1748 (deposited as FERM BP-5699), and KM1750 (deposited as FERM BP-5700), disclosed in WO 98/22616, WO 99/59636, Australian accepted application no. AU 1998 50666 B2, and Canadian application no. CA 2328893.
  • V L and V H domains can be from libraries of V gene sequences from a mammal of choice. Elements of such libraries express random combinations of V L and/or V H domains and are screened with any desired antigen to identify those elements which have desired binding characteristics. Particularly preferred is a human V gene library. Methods for such screening are known in the art.
  • V L and V H domains from a selected non- human source may be incorporated into chimeric antibodies. For example, for administration to a human, it may be desired to use a bispecific antibody with functional constant domains wherein the V L and V H domains have been selected from a non-human source. To maximize constant domain associated function or to reduce immunogenicity ofthe antibody, human constant regions are preferred.
  • a bispecific antibody can be made that is "humanized.”
  • Humanized variable domains are constructed in which amino acid sequences which comprise one or more complementarity determining regions (CDRs) of non-human origin are grafted to human framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs human framework regions
  • Variable domains have a high degree of structural homology, allowing easy identification of amino acid residues within variable domains which corresponding to CDRs and FRs. See, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest. 5th ed. National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD. Thus, amino acids which participate in antigen binding are easily identified.
  • methods have been developed to preserve or to enhance affinity for antigen of humanized binding domains comprising grafted CDRs.
  • One way is to include in the recipient variable domain the foreign framework residues which influence the conformation ofthe CDR regions.
  • a second way is to graft the foreign CDRs onto human variable domains with the closest homology to the foreign variable region.
  • CDRs are most easily grafted onto different FRs by first amplifying individual FR sequences using overlapping primers which include desired CDR sequences, and joining the resulting gene segments in subsequent amplification reactions. Grafting of a CDR onto a different variable domain can further involve the substitution of amino acid residues which are adjacent to the CDR in the amino acid sequence or packed against the CDR in the folded variable domain structure which affect the conformation ofthe CDR.
  • Humanized domains ofthe invention therefore include human antibodies which comprise one or more non-human CDRs as well as such domains in which additional substitutions or replacements have been made to preserve or enhance binding characteristics.
  • Antibodies ofthe invention also include antibodies which have been made less immunogenic by replacing surface-exposed residues to make the antibody appear as self to the immune system (Padlan, E.A. (1991) Mol. Immunol. 28, 489-498). Antibodies have been modified by this process with no loss of affinity (Roguska et al. (1994) Proc. Natl. Acad. Sci. USA 91, 969-973). Because the internal packing of amino acid residues in the vicinity ofthe antigen binding site remains unchanged, affinity is preserved. Substitution of surface-exposed residues according to the invention for the purpose of reduced immunogenicity does not mean substitution of CDR residues or adjacent residues which influence binding characteristics. [0045] The invention contemplates binding domains which are essentially human.
  • Human binding domains can be obtained from phage display libraries wherein human heavy or light chain variable domains or combinations thereof are displayed on the surface of filamentous phage (See, e.g., McCafferty et al. (1990) Nature 348, 552-554; Aujame et al. (1997) Human Antibodies 8, 155-168). Combinations of variable domains are typically displayed on filamentous phage in the form of Fabs or scFvs. The library is screened for phage bearing combinations of variable domains having desired antigen binding characteristics. Preferred variable domains and variable domain combinations display high affinity for a selected antigen and little cross-reactivity to other related antigens. By screening very large repertoires of antibody fragments, (see e.g. , Griffiths et al. (1994) EMBO J. 13, 3245-3260) a good diversity of high affinity Mabs are isolated, with many expected to have sub-nanomolar affinities for the desired antigen.
  • human binding domains can be obtained from transgenic animals into which unrearranged human Ig gene segments have been introduced and in which the endogenous mouse Ig genes have been inactivated (reviewed in Briiggemann and Taussig (1997) Curr. Opin. Biotechnol. 8, 455-458).
  • Preferred transgenic animals contain very large contiguous Ig gene fragments that are over 1 Mb in size (Mendez et al. (1997) Nature Genet. 15, 146-156) but human Mabs of moderate affinity can be raised from transgenic animals containing smaller gene loci (See, e.g., Wagner et al. (1994) Eur. J. Immunol. 42, 2672-2681; Green et al. (1994) Nature Genet. 7, 13-21).
  • V L and V H domains incorporated into antibodies ofthe invention can similarly be subject to in vitro mutation and screening procedures to obtain high affinity variants.
  • Binding domains of the invention include those for which binding characteristics have been improved by direct mutation or by methods of affinity maturation. Affinity and specificity may be modified or improved by mutating CDRs and screening for antigen binding sites having the desired characteristics (See, e.g., Yang et al. (1995) J. Mol. Bio. 254, 392-403).
  • CDRs are mutated in a variety of ways. One way is to randomize individual residues or combinations of residues so that in a population of otherwise identical antigen binding sites, all twenty amino acids, or a subset thereof, are found at particular positions. Alternatively, mutations are induced over a range of CDR residues by error prone PCR methods (See, e.g., Hawkins et al.
  • Phage display vectors containing heavy and light chain variable region genes are propagated in mutator strains of E. coli (See, e.g., Low et al. (1996) J. Mol. Bio. 250, 359-368). These methods of mutagenesis are illustrative ofthe many methods known to one of skill in the art.
  • variable domain ofthe antibodies ofthe present invention may be a complete immunoglobulin heavy or light chain variable domain, or it may be a functional equivalent or a mutant or derivative of a naturally occurring domain, or a synthetic domain constructed, for example, in vitro using a technique such as one described in WO 93/11236 (Medical Research Council et /./Griffiths et al). For instance, it is possible to join together domains corresponding to portions of antibody variable domains (i.e., variable domains which are missing at least one amino acid).
  • the important characterizing feature is the ability of each variable domain to associate with a complementary variable domain to form an antigen binding site.
  • the antibodies can be chemically or biosynthetically linked to anti-tumor agents or detectable signal-producing agents.
  • Anti- tumor agents linked to an antibody include any agents which destroy or damage a tumor to which the antibody has bound or in the environment ofthe cell to which the antibody has bound.
  • an anti-tumor agent is a toxic agent such as a chemotherapeutic agent or a radioisotope.
  • Suitable chemotherapeutic agents are known to those skilled in the art and include anthracyclines (e.g.
  • chemotherapeutic agents are conjugated to the antibody using conventional methods (See, e.g., Hermentin and Seiler (1988) Behringlnst. Mitt. 82, 197-215).
  • Detectable signal-producing agents are useful in vivo and in vitro for diagnostic purposes.
  • the signal producing agent produces a measurable signal which is detectible by external means, usually the measurement of electromagnetic radiation.
  • the signal producing agent is an enzyme or chromophore, or emits light by fluorescence, phosphorescence or chemiluminescence.
  • Chromophores include dyes which absorb light in the ultraviolet or visible region, and can be substrates or degradation products of enzyme catalyzed reactions.
  • the invention further contemplates antibodies to which target or reporter moieties are linked.
  • Target moieties are first members of binding pairs.
  • Anti-tumor agents for example, are conjugated to second members of such pairs and are thereby directed to the site where the antibody is bound.
  • a common example of such a binding pair is avidin and biotin.
  • biotin is conjugated to an antibody ofthe invention, and thereby provides a target for an anti-tumor agent or other moiety which is conjugated to avidin or sfreptavidin.
  • biotin or another such moiety is linked to an antibody of the invention and used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or sfreptavidin.
  • Suitable radioisotopes for use as anti-tumor agents are also known to those skilled in the art. For example, 131 I or 211 At is used. These isotopes are attached to the antibody using conventional techniques (See, e.g., Pedley et al. (1993) Br. J. Cancer 68, 69-73). Alternatively, the anti-tumor agent which is attached to the antibody is an enzyme which activates a prodrug. In this way, a prodrug is administered which remains in its inactive form until it reaches the tumor site where it is converted to its cytotoxin form once the antibody complex is administered.
  • the antibody-enzyme conjugate is administered to the patient and allowed to localize in the region ofthe tissue to be treated.
  • the prodrug is then administered to the patient so that conversion to the cytotoxic drug occurs in the region ofthe tissue to be treated.
  • the anti-tumor agent conjugated to the antibody is a cytokine such as interleukin-2 (LL-2), interleukin-4 (LL-4) or tumor necrosis factor alpha (TNF- ⁇ ).
  • the antibody targets the cytokine to the tumor so that the cytokine mediates damage to or destruction ofthe tumor without affecting other tissues.
  • the cytokine is fused to the antibody at the DNA level using conventional recombinant DNA techniques.
  • proteins ofthe invention can be fused to additional amino acid residues such as a peptide tag to facilitate isolation or purification, or a signal sequence to promote secretion or membrane transport in any particular host in which the protein is expressed.
  • additional amino acid residues such as a peptide tag to facilitate isolation or purification, or a signal sequence to promote secretion or membrane transport in any particular host in which the protein is expressed.
  • V L and V H gene combinations encoding binding sites specific for a particular antigen are isolated from cDNA of B cell hybridomas.
  • random combinations of V L and V H genes are obtained from genomic DNA and the products then screened for binding to an antigen of interest.
  • the polymerase chain reaction (PCR) is employed for cloning, using primers which are compatible with restriction sites in the cloning vector. See, e.g, Dreher, MX. et al. (1991) J. Immunol. Methods 139:197-205; Ward, E.S. (1993) Adv. Pharmacol. 24:1-20; Chowdhury, P.S. and Pastan, I. (1999) N «t. Biotechnol. 17:568-572.
  • V genes encoding those domains are assembled into a bacterial expression vector.
  • a vector can be used which has sequences encoding a bacterial secretion signal sequence and a peptide linker and which has convenient restriction sites for insertion of V L and V H genes.
  • PCR primers specific to the sequences encoding those domains are used.
  • mixtures of primers are used which amplify multiple sequences.
  • Preferred diabodies ofthe invention are made by expressing 1) a first polypeptide comprising a heavy chain variable domain corresponding to a first specificity connected to a light chain variable domain of a second specificity; and 2) a second polypeptide comprising a light chain variable domain corresponding to the first specificity connected to the heavy chain variable domain of to the second specificity.
  • Diabodies are commonly produced in E. coli using D A constructs which comprise bacterial secretion signal sequences at the start of each polypeptide chain.
  • binding proteins ofthe invention expression in other host cells may be desired.
  • binding proteins comprising constant domains are often more efficiently expressed in eukaryotic cells, including yeast, insect, vertebrate and mammalian cells. It will be necessary to use such cells where it is desired that the expressed product be glycosylated.
  • the D ⁇ A fragments coding for the first and second polypeptides can be cloned, e.g., into HCMV vectors designed to express human light chains of human heavy chains in mammalian cells. (See, e.g., Bendig, et al, U.S. Patent 5,840,299; Maeda, et al. (1991) Hum. Antibod.
  • Hybridomas 2, 124-134 Such vectors contain the human cytomegalo virus (HCMV) promoter and enhancer for high level transcription ofthe light chain and heavy chain constructs, hi a preferred embodiment, the light chain expression vector is pKNIOO (gift of Dr. S. Tarran Jones, MRC Collaborative Center, London, England), which encodes a human kappa light chain, and the heavy chain expression vector is pGlD105 (gift of Dr. S. Tarran Jones), which encodes a human gamma- 1 heavy chain. Both vectors contain HCMV promoters and enhancers, replication origins and selectable markers functional in mammalian cells and E. coli.
  • HCMV human cytomegalo virus
  • a selectable marker is a gene which encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium.
  • Typical selectable markers encode proteins that (a) confer resistance to antibiotics or other toxins, e.g. ampicillin, neomycin, methofrexate, or tetracycline, (b) complement auxofrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g. the gene encoding D-alanine racemase for Bacilli.
  • a particularly useful selectable marker confers resistance to methofrexate.
  • cells transformed with the DHFR selection gene are first identified by culturing all ofthe transformants in a culture medium that contains methofrexate (Mtx), a competitive antagonist of DHFR.
  • Mtx methofrexate
  • An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 11, 4216.
  • the transformed cells are then exposed to increased levels of methofrexate. This leads to the synthesis of multiple copies ofthe DHFR gene, and, concomitantly, multiple copies of other DNA comprising the expression vectors, such as the DNA encoding the antibody or antibody fragment.
  • mutant myeloma cells that are deficient for thymidine kinase are unable to use exogenously supplied thymidine when aminopterin is used to block DNA synthesis.
  • Useful vectors for transfection carry an intact TK gene which allows growth in media supplemented with thymidine.
  • a suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7. Stinchcomb et al, 1979 Nature, 282, 39; Kingsman et al, 1979, Gene 7, 141.
  • the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones (1977) Genetics 85, 12. The presence ofthe trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.
  • Preferred host cells for transformation of vectors and expression of antibodies ofthe present invention are bacterial cells, yeast cells and mammalian cells, e.g., COS-7 cells, Chinese hamster ovary (CHO) cells, and cell lines of lymphoid origin such as lymphoma, myeloma, or hybridoma cells.
  • the transformed host cells are cultured by methods known in the art in a liquid medium containing assimilable sources of carbon, e.g. carbohydrates such as glucose or lactose, nitrogen, e.g. amino acids, peptides, proteins or their degradation products such as peptones, ammonium salts or the like, and inorganic salts, e.g. sulfates, phosphates and/or carbonates of sodium, potassium, magnesium and calcium.
  • the medium furthermore contains, for example, growth-promoting substances, such as trace elements, for example iron, zinc, manganese and the like.
  • Antibodies ofthe instant invention have dual specificity and capable of binding to two different antigens simultaneously.
  • the different antigens can be located on different cells or on the same cell.
  • Cross linking of antigen can be shown in vitro, for example by providing a solid surface to which a first antigen has been bound, adding a bispecific antibodies specific for the first antigen and a second antigen for which the binding protein is also specific and detecting the presence of bound second antigen.
  • Antibodies ofthe invention can of block the interaction between two receptors and their respective ligands.
  • a diabody specific for KDR and Flt-1 inhibits VEGF induced cell migration as well as PIGF induced cell migration.
  • combination of two receptor binding specificities either as a mixture of single chains antibodies (scFvs) or in a bispecific diabody, is more efficacious in inhibiting cell migration that the individual parent antibodies.
  • bispecific antibodies can be more potent inhibitors of cellular function.
  • VEGF-stimulated cellular functions such as, for example, proliferation of endothelial cells and VEGF- and PlGF-induced migration of human leukemia cells can be more efficiently inhibited by bispecific antibodies, even where affinity for one or both ofthe two target antigens is reduced, hi one embodiment ofthe invention, a diabody was made that was specific for KDR and Flt-1.
  • scFv corresponding to either ofthe target antigens was unable to completely inhibit VEGF- or PlGF-induced cell migration, even at the highest scFv concentrations tested, h contrast, a diabody specific for both ofthe target antigens completely abolished cell migration, even though the affinity ofthe diabody for Flt-1 was reduced compared to the corresponding scFv.
  • the antibodies ofthe present invention are useful for treating diseases in humans and other mammals.
  • the antibodies are used for the same purposes and in the same manner as heretofore known for natural and engineered antibodies.
  • the present antibodies thus can be used in vivo and in vitro for investigative, diagnostic or treatment methods which are well known in the art.
  • the present antibodies can be administered for therapeutic treatments to a patient suffering from a tumor in an amount sufficient to prevent or reduce the progression of the tumor, e.g, the growth, invasiveness, metastases and/or recurrence ofthe tumor.
  • An amount adequate to accomplish this is defined as a therapeutically effective dose. Amounts effective for this use will depend upon the severity ofthe disease and the general state ofthe patient's own immune system. Dosing schedules will also vary with the disease state and status ofthe patient, and will typically range from a single bolus dosage or continuous infusion to multiple administrations per day (e.g., every 4-6 hours), or as indicated by the treating physician and the patient's condition.
  • Antibodies ofthe invention can be administered in a single dosages as high as 40 mg/kg body-weight or higher. More preferably, the antibodies are administered in dosages that range from 0.2 mg/kg to 20 mg/kg body-weight. It should be noted, however, that the present invention is not limited to any particular dose.
  • the present invention can be used to treat any suitable tumor, including, for example, tumors ofthe breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles, cervix or liver.
  • Tumors ofthe present invention preferably have aberrant expression or signaling of VEGFR.
  • Enhanced signaling by VEGFR has been observed in many different human cancers. High levels of VEGFR-2 are expressed by endothelial cells that infiltrate gliomas (Plate, K. et al., (1992) Nature 359:845-848).
  • VEGFR-2 levels are specifically upregulated by VEGF produced by human glioblastomas (Plate, K. et al. (1993) Cancer Res. 53:5822-5827).
  • the finding of high levels of VEGFR-2 expression in glioblastoma associated endothelial cells (GAEC) indicates that receptor activity is probably induced during tumor formation since VEGFR-2 transcripts are barely detectable in normal brain endothelial cells. This upregulation is confined to the vascular endothelial cells in close proximity to the tumor.
  • the antibodies ofthe invention are also to be used in combined treatment methods.
  • the bispecific antibodies can be administered with an anti-neoplastic agent such as a chemotherapeutic agent or a radioisotope.
  • chemotherapeutic agents include anthracyclines (e.g. daunomycin and doxorubicin), paclitaxel, irinotecan (CPT-11), topotecan, methofrexate, vindesine, neocarzinostatin, cisplatin, chlorambucil, cytosine arabinoside, 5-fluorouridine, melphalan, ricin, calicheamicin, and combinations thereof.
  • anthracyclines e.g. daunomycin and doxorubicin
  • paclitaxel paclitaxel
  • irinotecan CPT-11
  • topotecan methofrexate
  • vindesine neocarzinostatin
  • cisplatin chlor
  • bispecific antibody and an anti-neoplastic agent are admininstered to a patient in amounts effective to inhibit angiogenesis and reduce tumor growth.
  • the antibodies are also to be administered in combination with other treatment regimes.
  • bispecific antigen binding proteins ofthe invention can be administered with radiation, either external (external beam radiation therapy) or internal (brachytherapy).
  • antibodies of the invention where used in the human body for the purpose of diagnosis or treatment, will be administered in the form of a composition additionally comprising a pharmaceutically-acceptable carrier.
  • suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness ofthe binding proteins.
  • the compositions of this invention may be in a variety of forms.
  • solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions, dispersions or suspensions, liposomes, suppositories, injectable and infusible solutions.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • the preferred compositions are in the form of injectable or infusible solutions.
  • compositions of this invention are similar to those generally used for passive immunization of humans with antibodies as are known to those of skill in the art, and include but are not limited to intraveneous, intraperitoneal, subsutaneous, and intramuscular administration. Further, it is understood that combination treatments may involve administration of antibodies and, e.g., chemotherapeutic agents, by different methods.
  • EXAMPLE 1 Materials and Methods Cell lines.
  • a hybridoma cell line (ATC No. PTA-334) producing the anti-Flt-1 antibody, Mab6.12 (IgGl, K), was established at nClone Systems Incorporated (New York, NY) from a mouse immunized with a recombinant form ofthe receptor.
  • Primary-cultured human umbilical vein endothelial cells (HUVEC) were obtained from Dr. S. Rafii at Cornell Medical Center, New York, and maintained in EBM-2 medium (Clonetics, Walkersville, MD) at 37°C, 5% CO 2 .
  • the leukemia cell lines, HL60 and HEL were maintained in RPMI containing 10% of fetal calf serum and grown at 37°C with 5% CO 2 . Proteins and antibodies.
  • soluble fusion protein KDR-alkaline phosphatase (AP) was expressed in stably transfected NLH 3T3 and purified from cell culture supernatant by affinity chromatography using immobilized monoclonal antibody to AP as described by Lu, D., et al, 2000, J. Biol Chem., 275:14321-14330.
  • VEGF165 protein was expressed in baculovirus and purified following the procedures described. Id. PIGF and Flt-1 -Fc fusion proteins were purchased from R&D Systems (Minneapolis, MN). Preparation ofscFv specific for Flt-1.
  • V H and V L genes of Mab 6.12 were cloned by RT-PCR from mRNA isolated from the hybridoma cells, following the procedures of Bendig et al. (1996) hi: Antibody Engineering: A Practical Approach, McCafferty, J., Hoogenboom, H.R., Chiswell, D.J., eds., Oxford University Press, Incorporated; pl47-168. Eleven 5' primers, specifically designed to hybridize to the 5' ends of mouse antibody light chain leader sequences, and one 3' primer that hybridizes to the 5' end of mouse K light chain constant region, were used to clone the V L gene.
  • PCR fragments encoding the V L and the V H genes of MAB 6.12 were used to assemble scFv 6.12, using overlapping PCR.
  • this scFv the C-terminal of Mab 6.12 V H is linked to the N-terminal of Mab 6.12 V L via a 15 amino acid linker,
  • CDRH3, CDRLl, CDRL2, and CDRL2 are presented by SEQ ID NOS:35, 36, 37, 38, 39, and
  • a single chain antibody directed against KDR, scFv p 1 C 11 was isolated from a phage display library constructed from the splenocytes of a mouse immunized with KDR (Zhu, Z. et al, 1998, Cancer Res. 58:3209-3214).
  • Female BALB/C mice were given two intraperitoneal (i.p.) injections of 10 ⁇ g KDR-AP in 200 ⁇ l of REBI Adjuvant System followed by one i.p. injection without REBI adjuvant over a period of two months. The mice were also given a subcutaneous (s.c.) injection of 10 ⁇ g KDR-AP in 200 ⁇ l of REBI at the time ofthe first immunization.
  • mice were boosted i.p. with 20 ⁇ g of KDR-AP three days before euthanasia.
  • mRNA was purified from total RNA extracted from splenocytes.
  • cDNAs corresponding to expressed V L and V H genes were separately amplified.
  • the amplified products were inserted into a vector designed to accept each gene separately or linked to nucleotides encoding a secretion signal sequence and polypeptide linker (e.g., by PCR amplification) and the fused product inserted into a desired vector. See, e.g., Zhu et al, 1998.
  • the scFv-gene HI constructs were ligated into the pCANTAB 5E vector.
  • Transformed TGI cells were plated onto 2YTAG plates (17 g/1 tryptone, 10 g/1 yeast extract, 5 g/1 NaCl, 20 g/1 glucose, 100 ⁇ g/ml ampicillin, 15 g/1 Bacto-agar) and incubated. The colonies were scraped into 10 ml of 2YT medium (17 g/1 tryptone, 10 g/1 yeast extract, 5 g/1 NaCl), mixed with 5 ml 50% glycerol and stored at -70°C as the library stock.
  • the library stock was grown to log phase, rescued with M13K07 helper phage and amplified overnight in 2YTAK medium (2YT containing 100 ⁇ g/ml of ampicillin and 50 ⁇ g/ml of kanamycin) at 30°C.
  • the phage preparation was precipitated in 4% PEG/0.5M NaCl, resuspended in 3% fat-free milk/PBS containing 500 ⁇ g/ml of alkaline phosphatase (AP) and incubated at 37°C for 1 h to block phage-scFv having specificity for AP scFv and to block other nonspecific binding.
  • 2YTAK medium 2YT containing 100 ⁇ g/ml of ampicillin and 50 ⁇ g/ml of kanamycin
  • KDR-AP (10 ⁇ g/ml) coated Maxisorp Star tubes were first blocked with 3% milk/PBS at 37°C for 1 h, and then incubated with the phage preparation at room temperature for 1 h.
  • the tubes were washed 10 times with PBST (PBS containing 0.1% Tween 20), followed by 10 times with PBS.
  • the bound phage were eluted at room temperature for 10 min. with 1 ml of a freshly prepared solution of 100 mM triethylamine.
  • the eluted phage were incubated with 10 ml of mid-log phase TGI cells at 37°C for 30 min. stationary and 30 min. shaking.
  • the infected TGI cells were then plated onto 2YTAG plates and incubated overnight at 30°C as provided above for making ofthe phage stock.
  • Successive rounds ofthe screening procedure were employed to further enrich for displayed scFv having the desired binding specificity.
  • individual bacterial colonies were screened individually to identify clones having desired KDR binding characteristics. Identified clones were further tested for blocking of VEGF binding.
  • DNA fingerprinting of clones was used to differentiate unique clones. Representative clones of each digestion pattern were picked and subject to DNA sequencing. Human antibodies specific for KDR.
  • a large human Fab phage display library containing 3.7 x 10 10 clones (DeHaard et al., J. Biol. Chem. 274: 18218-30 (1999)) was used for the selection.
  • the library consists of combinations of PCR-amplified antibody variable light chain genes fused to human constant chain genes (K and ⁇ ) and variable heavy chain genes fused to DNA encoding the human IgGl heavy chain C H 1 domain. Both heavy and light chain constructs are preceded by a signal sequence - pel for the light chain and gene HI signal sequence for the heavy chain.
  • Heavy chain constructs further encode a portion ofthe gene HI protein for phage display, a hexahistidine tag, and an 11 amino-acid-long c-myc tag, followed by an amber codon (TAG).
  • TAG amber codon
  • the hexahistidine and c-myc tags can be used for purification or detection.
  • the amber codon allows for phage display using suppressor hosts (such as TGI cells) or production of Fab fragments in soluble form when transformed into a nonsupressor host (such as HB2151 cells).
  • the library stock was grown to log phase, rescued with M13-KO7 helper phage and amplified overnight in 2YTAK medium (2YT containing 100 ⁇ g/ml of ampicillin and 50 ⁇ g/ml of kanamycin) at 30°C.
  • the phage preparation was precipitated in 4% PEG/0.5M NaCl, resuspended in 3% fat-free milk/PBS containing 500 ⁇ g/ml of AP protein and incubated at 37°C for 1 h to capture phage displaying anti-AP Fab fragments and to block other nonspecific binding.
  • KDR-AP (10 ⁇ g/ml in PBS) coated Maxisorp Star tubes were first blocked with 3% milk/PBS at 37°C for 1 h, and then incubated with the phage preparation at RT for 1 h. The tubes were washed 10 times with PBST (PBS containing 0.1%) Tween-20) followed by 10 times with PBS. Bound phage were eluted at RT for 10 min with 1 ml of a freshly prepared solution of 100 mM triethylamine (Sigma, St. Louis, MO).
  • the eluted phage were incubated with 10 ml of mid-log phase TGI cells at 37°C for 30 min stationary and 30 min shaking.
  • the infected TGI cells were pelleted and plated onto several large 2YTAG plates and incubated overnight at 30°C. All the colonies grown on the plates were scraped into 3 to 5 ml of 2YTA medium, mixed with glycerol (10% final concentration), aliquoted and stored at -70°C.
  • 100 ⁇ l of the phage stock was added to 25 ml of 2YTAG medium and grown to mid-log phase.
  • the culture was rescued with M13K07 helper phage, amplified, precipitated, and used for selection followed the procedure described above, with reduced concentrations of KDR-AP immobilized on the immunotube and increased number of washes after the binding process.
  • D1F7 V H nucleotide and amino acid sequences in SEQ ED NOS:71 and 72; V L nucleotide and amino acid sequences in SEQ ID NOS:73 and 74.
  • D2C6 V H nucleotide and amino acid sequences in SEQ ID NOS:75 and 76; V L nucleotide and amino acid sequences in SEQ ID NOS:77 and 78.
  • D2H2 V H nucleotide and amino acid sequences in SEQ ID NOS: 82 and 83; V L nucleotide and amino acid sequences in SEQ ED NOS: 84 and 85.
  • D1H4 V H nucleotide and amino acid sequences in SEQ ED NOS:79 and 76; V L nucleotide and amino acid sequences in SEQ ED NOS: 80 and 81.
  • a second library consisting of combinations ofthe single heavy chain of D2C6 with a diverse population of light chains derived from the original library, was created and screened. Ten additional Fabs were identified, designated SA1, SA3, SB10, SB5, SC7, SD2, SD5, SF2, SF7, and 1121.
  • Complete V L nucleotide and amino acid sequences are presented in the Sequence Listing as follows.
  • S Al V L nucleotide and amino acid sequences in SEQ ED NOS: 86 and 87.
  • SA3 V L nucleotide and amino acid sequences in SEQ ED NOS:88 and 89.
  • SB10 V L nucleotide and amino acid sequences in SEQ ED NOS:90 and 91.
  • SB5 V L nucleotide and amino acid sequences in SEQ ED NOS:92 and 93.
  • SC7 V L nucleotide and amino acid sequences in SEQ ED NOS:94 and 95.
  • SD2 V L nucleotide and amino acid sequences in SEQ ED NOS:96 and 97.
  • SD5 V L nucleotide and amino acid sequences in SEQ ED NOS:98 and 99.
  • SF2 V L nucleotide and amino acid sequences in SEQ ID NOS:100 and 101.
  • SF7 V L nucleotide and amino acid sequences in SEQ ED NOS:102 and 103.
  • 1121 V L nucleotide and amino acid sequences in SEQ ED NOS: 104 and 105.
  • V L CDR sequences are presented in Table 2.
  • variable domains of scFv p 1 C 11 and scFv 6.12 were used for PCR-directed assembly to create the expression plasmid, pDAB-KFl (Fig. 1 A).
  • the following gene fragments were generated by PCR from the V L and V H domains of plCll and MAB6.12: the V L domain of plCl 1 followed by a segment encoding a 5 amino-acid-linker, GGGGS; the V H domain of MAB6.12 preceded by a segment encoding the GGGGS linker; the V L domain of MAB6.12 preceded by a segment encoding the E.
  • coli heat stable enterotoxin H (stH) signal sequence (Picken, R. N., et al, 1983, Infect. Immun. 42:269-275) and followed by a segment encoding the GGGGS linker; and the V H domain of plCl 1 preceded by a segment encoding the GGGGS linker.
  • Cross-over scFv, pLH-lCl 1-6.12 and pLH-6.12-lCl 1 were constructed by annealing of PCR fragments plCl 1 V L and MAB6.12 V H , and MAB6.12 V L and plCl 1 V H , respectively, followed by PCR amplification to introduce appropriate restriction sites for subsequent cloning.
  • the expression plasmid, pDAB-KFl, for co-secretion ofthe two cross-over scFv was constructed by ligation ofthe Sfil/Nhel and the Nhel/Notl fragments from pLH-lCl 1-6.12 and pLH-6.12-lCl 1, respectively, into vector pCANTAB 5E. All sequences encoding the cross-over scFv fragments were verified by DNA sequencing. Expression and purification ofthe diabody.
  • the diabody was prepared from E. coli strain HB2151 containing the expression plasmid grown at 30°C in a shaker flask following the procedure previously described (Lu, D. et al, 1999, J. Immunol. Methods 230:159-171).
  • a periplasmic extract of the cells was prepared by resuspending the cell pellet in 25 mM Tris (pH 7.5) containing 20% (w/v) sucrose, 200 mM NaCl, 1 mM EDTA and 0.1 mM PMSF, followed by incubation at 4°C with gentle shaking for 1 h.
  • the soluble diabody was purified from the supernatant by anti-E tag affinity chromatography using the RPAS Purification Module (Amersham Pharmacia Biotech). To examine the purity ofthe diabody preparation, both the E. coli periplasmic extract and the purified diabody were electrophoresed in an 18% polyacrylamide gel (Novex, San Diego, CA) and visualized by staining with Colloidal Blue Stain kit (Novex). Dual specificity ofthe diabody to KDR and Flt-1.
  • Two assays were carried out to determine the dual antigen binding capability ofthe diabody.
  • a cross-linking assay was used to investigate whether the diabody is capable of binding both of its target antigens simultaneously.
  • the diabody or its parent scFv were first incubated in a 96-well Maxi-sorp microtiter plate (Nunc, Roskilde, Denmark) precoated with Flt-l-Fc fusion protein (1 ⁇ g/ml x 100 ml per well overnight at 4°C) at room temperature (RT) for I h.
  • the plate was washed three times with PBS containing 0.1 % Tween (PBST), followed by incubation with KDR-AP fusion protein at RT for additional 1 h.
  • the plate-bound KDR-AP was then quantified by the addition of AP substrate, p-nitrophenyl phosphate (Sigma, St. Louis, MO), followed by reading ofthe absorbance at 405nm (Lu, D. et al, 1999).
  • AP substrate p-nitrophenyl phosphate
  • the second, direct binding assay various amounts of diabody or scFv were added to KDR or Flt-1 coated 96-well plates and incubated at RT for 1 h, after which the plates were washed 3 times with PBST.
  • the substrate for AP was added, followed by reading ofthe absorbance at 405nm to quantify the plate-bound KDR-AP.
  • the Flt-l-Fc assay the plate was incubated with a mouse anti-human Fc-HRP conjugate to quantify the plate-bound Flt-l-Fc.
  • the IC 50 i.e., the antibody concentration required for 50% inhibition of KDR or Flt-1 binding to VEGF or PIGF, was then calculated. Analysis of binding kinetics.
  • HUVEC 5 x 10 3 cells/well
  • VEGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • Various amounts ofthe antibodies were added to duplicate wells and pre-incubated at 37°C for 1 h, after which VEGF 165 was added to a final concentration of 16 ng/ml.
  • 0.25 ⁇ Ci of [ 3 H]-TdR was added to each well and incubated for an additional 4 h.
  • the cells were washed once with PBS, trypsinized and harvested onto a glass fiber filter (Printed Filtermat A, Wallach) with a cell harvester (Harvester 96, MACH HI M, TOMTEC, Orange, CT). The membrane was washed three times with H 2 O and air-dried. Scintillation fluid was added and DNA incorporated radioactivity was determined on a scintillation counter (Wallach, Model 1450 Microbeta Liquid Scintillation Counter). Leukemia migration assay.
  • HL60 and HEL cells were washed three times with serum-free plain RPMI 1640 medium and suspended in the medium at 1 x 10 6 /ml. Aliquots of 100 ⁇ l cell suspension were added to either 3- ⁇ m-pore transwell inserts (for HL60 cells), or 8- ⁇ m-pore transwell inserts (for HEL cells) (Costar®, Corning Incorporated, Corning, NY) and incubated with the antibodies for 30 min at 37°C. The inserts were then placed into the wells of 24-well plates containing 0.5 ml of serum-free RPMI 1640 with or without VEGF165.
  • the migration was carried out at 37°C, 5% CO 2 for 16-18 h for HL60 cells, or for 4 h for HEL cells.
  • Migrated cells were collected from the lower compartments and counted with a Coulter counter (Model Zl, Coulter Electronics Ltd., Luton, England).
  • EXAMPLE 2 anti-KDR x anti-Flt-1 diabody Diabody structure.
  • An anti-KDR x anti-Flt-1 diabody made according to Example I was purified and analyzed by SDS-PAGE.
  • the two component polypeptides were resolved under the electrophoretic conditions and gave rise to two major bands with mobility close to that anticipated (Fig. IB); the lower band represents the first polypeptide (m.w., 25179.6 daltons), and the upper band correlates with the second polypeptide with E-tag (m.w., 26693.8 daltons) (Fig. 1A). Dual specificity.
  • a cross-linking assay to investigate whether the anti-KDR x anti-Flt-1 diabody was capable of simultaneously binding to both of its target antigens To test the capability ofthe Flt-1 -bound diabody to capture soluble KDR, the diabody was first allowed to bind to immobilized Flt-1, followed by incubation with KDR-AP. As shown in Fig. 2A, the diabody, but not the parent monospecific scFv, efficiently cross-linked the soluble KDR to the immobilized Flt-1, as demonstrated by the plate-bound AP activity.
  • the antigen binding efficiency of the diabody was determined on immobilized KDR and Flt-1.
  • the diabody bound as efficiently as the parent scFv plCl 1 to KDR (Fig. 2B). Binding the diabody to Flt-1 was slightly reduced, compared to the parent scFv 6.12 (Fig. 2C). As expected, the KDR-specific scFv plCl 1 did not bind to Flt-1 (Fig. 2B), and Flt-1 -specific scFv 6.12 did not bind to KDR (Fig. 2C). Data shown in Fig. 2 represent the mean ⁇ SD of triplicate samples.
  • the binding kinetics of the diabody to KDR and Fit- 1 were determined by surface plasmon resonance using a BIAcore instrument (Table 3) and are consistent with the ELISA results of Fig. 2.
  • the diabody binds to KDR with kinetics similar to its parent scFv plCl 1 with a Kd of 1.4 nM.
  • the binding affinity ofthe diabody to Flt-1 was moderately reduced compared to scFv 6.12, mainly due to a slower on-rate ofthe diabody (Table 3).
  • Fig. 3 A shows that the diabody blocks KDR from binding to immobilized VEGF, in a dose-dependent manner as efficiently as scFv plCl 1, with an IC 50 of approximately 2 nM.
  • the diabody also blocks Flt-1 from binding to VEGF with an IC 50 of about 15 nM, which is about 10-fold less potent than the parent scFv 6.12 (Fig. 3B).
  • the diabody blocks PIGF, a Flt-1 -specific ligand, from binding to immobilized Flt-1 with an IC 50 of approximately 4 nM (Fig. 3C).
  • scFv plCl 1 had no effects on Flt-1/VEGF and Flt-1/PlGF interaction, whereas scFv 6.12 had no effects on KDR/VEGF interaction.
  • Data shown represent the mean ⁇ SD of triplicate samples.
  • EXAMPLE 3 Biological activity Inhibition of VEGF-induced migration of leukemia cells and mitogenesis of HUVEC.
  • the diabody was first tested for its activity in inhibiting VEGF and PlGF-induced cell migration. Both VEGF and PIGF induced migration of human leukemia cells, HL60 and HEL, in a dose-dependent manner (Fig. 4A and 4D). scFv plCl 1 and scFv 6.12 effectively inhibited VEGF and PlGF-induced cell migration (Fig. 4B, 4C, 4E and 4F). Data shown are representative of at least three separate experiments and represent the mean ⁇ SD of triplicate determinations.
  • scFv pi CI 1 is a stronger inhibitor of VEGF-induced cell migration
  • scFv 6.12 is slightly more potent in inhibiting PlGF-induced cell migration.
  • the diabody is equally effective in blocking cell migration induced by both VEGF and PIGF.
  • VEGF-neutralizing activity ofthe bifunctional diabody was further determined using a HUVEC mitogenic assay. Data shown are the means of duplicates and are the representative of at least three separate experiments. As previously seen, scFv plCl 1 effectively inhibited VEGF-stimulated HUVEC mitogenesis (measured by [ 3 H]-TdR incorporation) in a dose-dependent manner with an IC 50 of approximately 2 nM. Anti-Flt-1 scFv 6.12 showed a very weak anti-mitogenic effect in this assay.
  • the bifunctional diabody demonstrated a much stronger inhibitory effect than either scFv plCIl and scFv 6.12 at every antibody concentration tested, with an IC S0 of approximately 0.5 nM (Fig. 5). Data shown are the means of duplicates and are the representative of at least three separate experiments.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention se rapporte à la production de protéines de liaison d'antigènes qui se lient spécifiquement aux domaines extra-cellulaires de deux récepteurs VEGF différents. L'activation en bloc des protéines de liaison d'antigènes bispécifiques des récepteurs VEGF sert à réduire ou à inhiber des fonctions cellulaires dépendant de VEGF telles que la mitogenèse de cellules endothéliales vasculaires et la migration des cellules leucémiques. Les protéines de liaison d'antigènes de la présente invention peuvent être monovalentes ou multivalentes; elles possèdent des sites de liaison d'antigènes constitués de domaines variables de chaînes lourdes et de chaînes légères d'immunoglobulines et peuvent aussi comprendre des domaines constants d'immunoglobulines.
PCT/US2002/041372 2001-06-26 2002-12-24 Anticorps bispecifiques qui se lient aux recepteurs de vegf WO2004003211A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US10/520,026 US20090028859A1 (en) 2001-06-26 2002-12-24 Bispecific antibodies that bind to vegf receptors
AU2002368062A AU2002368062A1 (en) 2002-06-26 2002-12-24 Bispecific antibodies that bind to vegf receptors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/US2002/020332 WO2003002144A1 (fr) 2001-06-26 2002-06-26 Anticorps bispecifiques se liant aux recepteurs vegf
USPCT/US02/20332 2002-06-26

Publications (1)

Publication Number Publication Date
WO2004003211A1 true WO2004003211A1 (fr) 2004-01-08

Family

ID=29998733

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/041372 WO2004003211A1 (fr) 2001-06-26 2002-12-24 Anticorps bispecifiques qui se lient aux recepteurs de vegf

Country Status (2)

Country Link
AU (1) AU2002368062A1 (fr)
WO (1) WO2004003211A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005020972A2 (fr) 2003-08-27 2005-03-10 (Osi) Eyetech, Inc. Polytherapie pour le traitement de troubles neovasculaires oculaires
WO2006086586A2 (fr) * 2005-02-10 2006-08-17 Baylor Research Institute Anticorps monoclonaux anti-interferon alpha et procedes d'utilisation
US7888481B2 (en) 2005-02-10 2011-02-15 Baylor Research Institute Anti-interferon alpha monoclonal antibodies and methods for use
EP2395017A2 (fr) 2003-05-30 2011-12-14 Merus B.V. Conception et utilisation de régions variables appariées de molécules de liaison spécifiques
US9758805B2 (en) 2012-04-20 2017-09-12 Merus N.V. Methods and means for the production of Ig-like molecules
USRE47770E1 (en) 2002-07-18 2019-12-17 Merus N.V. Recombinant production of mixtures of antibodies
US10934571B2 (en) 2002-07-18 2021-03-02 Merus N.V. Recombinant production of mixtures of antibodies
US10973920B2 (en) 2014-06-30 2021-04-13 Glykos Finland Oy Saccharide derivative of a toxic payload and antibody conjugates thereof
US11237165B2 (en) 2008-06-27 2022-02-01 Merus N.V. Antibody producing non-human animals
US12123043B2 (en) 2020-07-21 2024-10-22 Merus N.V. Methods and means for the production of Ig-like molecules

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LU D.: "Acquired antagonistic activity of a bispecific diabody directed against two different epitopes on vascular endothelial growth factor receptor 2", J. IMMUNOL. METHODS, vol. 230, no. 1-2, November 1999 (1999-11-01), pages 159 - 171, XP002141909 *
LU D.: "Complete inhibition of vascular endothelial growth factor (VEGF) activities with a bifunctional diabody directed against both VEGF kinase receptors, fms-like tyrosine kinase receptor and kinase insert domain-containing receptor", CANCER RESEARCH, vol. 61, 1 October 2001 (2001-10-01), pages 7002 - 7008, XP002958189 *

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10934571B2 (en) 2002-07-18 2021-03-02 Merus N.V. Recombinant production of mixtures of antibodies
USRE47770E1 (en) 2002-07-18 2019-12-17 Merus N.V. Recombinant production of mixtures of antibodies
US9738701B2 (en) 2003-05-30 2017-08-22 Merus N.V. Method for selecting a single cell expressing a heterogeneous combination of antibodies
US10670599B2 (en) 2003-05-30 2020-06-02 Merus N.V. Method for selecting a single cell expressing a heterogeneous combination of antibodies
US10605808B2 (en) 2003-05-30 2020-03-31 Merus N.V. Antibody producing non-human animals
EP2395017A2 (fr) 2003-05-30 2011-12-14 Merus B.V. Conception et utilisation de régions variables appariées de molécules de liaison spécifiques
EP2395016A2 (fr) 2003-05-30 2011-12-14 Merus B.V. Conception et utilisation de régions variables appariées de molécules de liaison spécifiques
EP2281885A1 (fr) 2003-08-27 2011-02-09 Ophthotech Corporation Combination thérapeutique pour le traitement des troubles oculaires néovasculaires
WO2005020972A2 (fr) 2003-08-27 2005-03-10 (Osi) Eyetech, Inc. Polytherapie pour le traitement de troubles neovasculaires oculaires
EP3168304A1 (fr) 2003-08-27 2017-05-17 Ophthotech Corporation Thérapie combinée pour le traitement des troubles néovasculaires oculaires
NO343054B1 (no) * 2005-02-10 2018-10-22 Baylor Res Inst Anti-interferon  monoklonale antistoffer og anvendelser
WO2006086586A3 (fr) * 2005-02-10 2006-10-12 Baylor Res Inst Anticorps monoclonaux anti-interferon alpha et procedes d'utilisation
US8333965B2 (en) 2005-02-10 2012-12-18 Baylor Research Institute Anti-inteferon alpha monoclonal antibodies and methods for use
WO2006086586A2 (fr) * 2005-02-10 2006-08-17 Baylor Research Institute Anticorps monoclonaux anti-interferon alpha et procedes d'utilisation
US8080638B2 (en) 2005-02-10 2011-12-20 Baylor Research Institute Anti-interferon alpha monoclonal antibodies and methods for use
US7888481B2 (en) 2005-02-10 2011-02-15 Baylor Research Institute Anti-interferon alpha monoclonal antibodies and methods for use
US11237165B2 (en) 2008-06-27 2022-02-01 Merus N.V. Antibody producing non-human animals
US10337045B2 (en) 2012-04-20 2019-07-02 Merus N.V. Methods and means for the production of Ig-like molecules
US10752929B2 (en) 2012-04-20 2020-08-25 Merus N.V. Methods and means for the production of ig-like molecules
US9758805B2 (en) 2012-04-20 2017-09-12 Merus N.V. Methods and means for the production of Ig-like molecules
US10329596B2 (en) 2012-04-20 2019-06-25 Merus N.V. Methods and means for the production of Ig-like molecules
US11926859B2 (en) 2012-04-20 2024-03-12 Merus N.V. Methods and means for the production of Ig-like molecules
US10973920B2 (en) 2014-06-30 2021-04-13 Glykos Finland Oy Saccharide derivative of a toxic payload and antibody conjugates thereof
US12123043B2 (en) 2020-07-21 2024-10-22 Merus N.V. Methods and means for the production of Ig-like molecules

Also Published As

Publication number Publication date
AU2002368062A1 (en) 2004-01-19

Similar Documents

Publication Publication Date Title
US20090028859A1 (en) Bispecific antibodies that bind to vegf receptors
US8057791B2 (en) Human antibodies specific to KDR and uses thereof
US20040259156A1 (en) Bispecific immunoglobulin-like antigen binding proteins and method of production
KR101287777B1 (ko) 가용성 ｃｅａ에 대한 내성을 가지는 약학적 조성물
AU2009299787B2 (en) Anti CXCR4 antibodies and their use for the treatment of cancer
JP2009526857A (ja) 機能性抗体
JP2008512352A (ja) 新規な四価の二重特異性抗体
WO2004003211A1 (fr) Anticorps bispecifiques qui se lient aux recepteurs de vegf
US20240141061A1 (en) Novel antibody against cd55 and use thereof
Putelli Human antibody phage technology-isolation and characterization of monoclonal antibodies for the targeting of cancer and rheumatoid arthritis

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 10520026

Country of ref document: US

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP