EP1151002A1 - Anticorps specifiques au kdr et leurs utilisations - Google Patents

Anticorps specifiques au kdr et leurs utilisations

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Publication number
EP1151002A1
EP1151002A1 EP00913280A EP00913280A EP1151002A1 EP 1151002 A1 EP1151002 A1 EP 1151002A1 EP 00913280 A EP00913280 A EP 00913280A EP 00913280 A EP00913280 A EP 00913280A EP 1151002 A1 EP1151002 A1 EP 1151002A1
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EP
European Patent Office
Prior art keywords
acid sequence
kdr
seq
nucleic acid
encodes
Prior art date
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EP00913280A
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German (de)
English (en)
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EP1151002A4 (fr
Inventor
Zhenping Zhu
Larry Witte
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ImClone LLC
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ImClone Systems Inc
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Publication of EP1151002A1 publication Critical patent/EP1151002A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Angiogenesis is the process of developing new blood vessels. The process involves the proliferation, migration and tissue infiltration of capillary endothelial cells from pre-existing blood vessels. Angiogenesis is important in normal physiological processes including embryonic development, follicular growth, and wound healing as well as in pathological conditions involving tumor growth and metastasis. (Folkman, J. and Klagsbrun, M. Science 235:442-447 (1987)).
  • vascular endothelium is usually quiescent in adults, and its activation is tightly regulated during angiogenesis.
  • TGFb transforming growth factor
  • aFGF and bFGF acidic and basic fibroblast growth factor
  • PDGF platelet derived growth factor
  • VEGF vascular endothelial growth factor
  • VEGF is a homodimeric glycoprotein consisting of two 23 kD subunits with structural similarity to PDGF.
  • the isoforms include two membrane bound forms (VEGF 0 and VEGF 18 ) and two soluble forms (VEGF ⁇ 65 and VEGF ⁇ j).
  • VEGF 165 is the most abundant isoform.
  • VEGF is expressed in embryonic tissues (Breier et al., Development (Camb.) 114:521 (1992)), macrophages, proliferating epidermal keratinocytes during wound healing (Brown et al., J. Exp. Med., 176:1375 (1992)), and may be responsible for tissue edema associated with inflammation (Ferrara et al., Endocr. Rev. 13:18 (1992)). In situ hybridization studies have demonstrated high VEGF expression in a number of human tumor lines including glioblastoma multiforme, hemangioblastoma, central nervous system neoplasms and AIDS-associated Kaposi's sarcoma (Plate, K.
  • VEGF receptors typically are class III receptor-type tyrosine kinases characterized by having several, typically five or seven, immunoglobulin-like loops in their amino- terminal extracellular receptor ligand-binding domains (Kaipainen et al., J. Exp. Med. 178:2077-2088 (1993)).
  • VEGF receptors include FLT-1, sequenced by Shibuya M. et al., Oncogene 5, 519-524 (1990); KDR (kinase insert domain-containing receptor), described in PCT US92/01300, filed February 20, 1992, and in Terman et al., Oncogene 6:1677-1683 (1991); and FLK-1, sequenced by Matthews W. et al. Proc. Natl. Acad. Sci. USA, 88:9026-9030 (1991).
  • KDR is the human homolog of the mouse FLK-1 receptor.
  • the KDR and FLK-1 receptors are also known as VEGFR2.
  • Equivalent receptors having homologous amino acid sequences, as defined above, occur in all mammals, e.g. human, mouse.
  • the binding of an antibody to one VEGF receptor does not necessarily imply binding to another VEGF receptor, and binding to a VEGF receptor in one mammal does not necessarily imply binding to the equivalent receptor in another mammal.
  • VEGF receptors are expressed mainly on endothelial cells and hematopoietic cells. After VEGF is synthesized and secreted by tumor cells, VEGF binds to VEGFR2 receptors and stimulates the growth of new blood vessels. It has been found that tumors are incapable of growing beyond a certain size unless they have a dedicated blood supply.
  • monoclonal antibodies against the murine VEGF receptor, FLK-1, by hybridoma technology was described in PCT application US95/01678. These monoclonal antibodies were said to inhibit receptor activation by interferring with the interaction between VEGF and its receptor.
  • the object of the present invention is to provide highly effective antibodies that neutralize the interaction between VEGF and its human receptor, KDR, with affinities higher than those known in the prior art.
  • Immunoglobulin molecules include monovalent single chain antibodies, multivalent single chain antibodies, diabodies, triabodies, antibodies, humanized antibodies and chimerized antibodies.
  • the invention further provides nucleic acid molecules that encode these immunoglobulin molecules.
  • the invention also provides a method of making the immunoglobulin molecules mentioned above.
  • the invention further provides a method of neutralizing the activation of KDR, a method of inhibiting angiogenesis in a mammal and a method of inhibiting tumor growth in a mammal with such immunoglobulin molecules.
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • KDR kinase insert domain-containing receptor (also known as VEGF receptor 2);
  • FLK-1 fetal liver kinase 1;
  • scFv single chain Fv;
  • HUVEC human umbilical vein endothelial cells
  • PBS 0.01M phosphate buffered saline (pH 7.2);
  • PBST PBS containing 0.1% Tween-20;
  • AP alkaline phosphatase
  • EGF epidermal growth factor
  • V H and V L variable domain of immunogloblin heavy and light chain, respectively
  • Figure 1 is a graph showing the direct binding of different scFv antibodies (plCl 1, plF12, p2A6 and p2A7) to immobilized KDR.
  • Figure 2 is a graph showing the inhibition of binding of KDR to immobilized VEGF 165 by different scFv antibodies (plCl 1, plF12, p2A6 and p2A7).
  • Figure 3 is a graph showing the inhibition of VEGF-induced HUVEC proliferation by scFv antibodies (p2A6 and plCl 1).
  • Figure 4 is the nucleotide and deduced amino acid sequence of V H and V L chains of c-plCl l.
  • Figure 5 is a graph showing the direct binding of antibodies (c-plCl 1, plcl 1, p2A6) to immobilized KDR.
  • Figure 6 is a graph showing the FACS analysis of c-plCl 1 binding to KDR- expressing HUVEC.
  • Figure 8 is a graph showing the inhibition of binding of radiolabeled VEGF 165 to immobilized KDR receptor by c-plCl 1 and cold VEGF 165 .
  • Figure 9 is a graph showing the inhibition of VEGF-induced HUVEC proliferation by anti-KDR antibodies (c-plCl 1, plCl 1).
  • the present invention provides immunoglobulin molecules which bind specifically to an extracellular domain of KDR with an affinity comparable to that of VEGF. As a result of such binding, the immunoglobulin molecules are able to neutralize activation of the receptor more effectively than immunoglobulin molecules previously described.
  • An extracellular domain of a VEGF receptor is herein defined as a ligand-binding domain on extracellular region of the receptor.
  • Immunoglobulin molecules are proteins that recognize and bind to a specific antigen or substance. Immunoglobulin molecules include naturally occuring antibodies, monovalent single chain antibodies, multivalent single chain antibodies, diabodies, triabodies, chimerized antibodies, humanized antibodies and other molecules which bind specifically with antigens.
  • the immunoglobulin molecules of the present invention bind KDR with an affinity comparable to that of the natural ligand.
  • Affinity represented by the equilibrium constant for the association of an antigen with an immunoglobulin molecule (K) measures the binding strength between an antigenic determinant and an immunoglobulin molecule, irrespective of the number of binding sites.
  • An antigenic determinant also known as an epitope, is the site on an antigen at which a given immunoglobulin molecule binds.
  • Typical values of K are 10 5 to 10 11 liters/mol. Any K less than 10 4 liters/mol is considered to indicate binding which is nonspecific.
  • the reciprocal of K is designated as K . (K d also may be referred to as the dissociation constant.) The lesser the value of the K d , the stronger the binding strength between an antigenic determinant and the antibody binding site.
  • the natural ligand of KDR is human VEGF.
  • VEGF binds KDR with the affinity (K d ) of 0.93 nM.
  • an anti- KDR antibody needs to bind KDR with an affinity comparable to VEGF.
  • the anti- DR antibody needs to successfully compete with VEGF with respect to binding KDR.
  • An antibody with a K d of at most 5 nM is considered to bind with an affinity comparable to the natural ligand.
  • the antibodies of the invention preferably bind KDR with an affinity of at most about 4nM, more preferably with an affinity of at most about 3 nM, most preferably with an affinity of at most about 2 nM, and optimally with an affinity of about InM.
  • the antibodies of the invention neutralize KDR.
  • neutralizing a receptor means diminishing and/or inactivating the intrinsic kinase activity of the receptor to transduce a signal.
  • a reliable assay for KDR neutralization is the inhibition of receptor phosphorylation.
  • the present invention is not limited by any particular mechanism of KDR neutralization.
  • the mechanism of KDR neutralization by antibodies is not well understood, and the mechanism followed by one antibody is not necessarilly the same as that followed by another antibody.
  • Some possible mechanisms include preventing binding of the VEGF ligand to the extracellular binding domain of the KDR, and preventing dimerization or oligomerization of receptors. Other mechanisms cannot, however, be ruled out.
  • a preferred immunoglobulin molecule is an antibody fragment called a monovalent single chain antibody (scFv).
  • Monovalent single chain antibodies include an antibody variable heavy-chain fragment (V H ) linked to an antibody variable light- chain fragment (V L ) by a peptide linker which allows the two fragments to associate to form a functional antigen binding site (see, for example U.S. Pat. No. 4,946,778, Ladner et al., (Genex); WO 88/09344, Creative Biomolecules, Inc/Huston et al.).
  • WO 92/01047 Cambridge Antibody Technology et al./McCafferty et al., describes the display of scFv fragments on the surface of soluble recombinant genetic display packages, such as bacteriophage.
  • a single chain antibody with a linker (L) can be represented as VL-L-V H or VH -L-VL.
  • Valency refers to the number of antigen binding sites which an immunoglobulin molecule has for a particular epitope.
  • a monovalent antibody has one binding site for a particular epitope.
  • Avidity is the measure of the strength of binding between an immunoglobulin molecule with its antigen. Avidity is related to both the affinity between an epitope with its antigen binding site on the immunoglobulin molecule, and the valency of the immunoglobulin molecule.
  • Single chain antibodies lack some or all of the constant domains of the whole antibodies from which they are derived. Therefore, they may overcome some of the problems associated with the use of whole antibodies. For example, single-chain antibodies tend to be free of undesired interactions between biological molecules and the heavy-chain constant region, or other unwanted biological activity. Additionally, single-chain antibodies are considerably smaller than whole antibodies and may therefore have greater capillary permeability than whole antibodies, allowing single- chain antibodies to localize and bind to target antigen-binding sites more efficiently. Also, single chain antibodies can be produced on a relatively large scale in prokaryotic cells, thus facilitating their production. Furthermore, the relatively small size of single-chain antibodies makes them less likely to provoke an unwanted immune response in a recipient than whole antibodies.
  • the peptide linkers used to produce the single chain antibodies may be flexible peptides selected to assure that the proper three-dimensional folding of the V L and V H domains may occur once they are linked so as to maintain the target molecule binding- specificity of the full length anti-KDR antibody.
  • the carboxyl terminus of the V L or V H sequence may be covalently linked by such a peptide linker to the amino acid terminus of a complementary V H or V L sequence.
  • the linker is generally 10 to 50 amino acid residues.
  • the linker is 10 to 30 amino acid residues. More preferably the linker is 12 to 30 amino acid residues. Most preferably is a linker of 15 to 25 amino acid residues.
  • An example of such linker peptides include (Gly-Gly-Gly- Gly-Ser) 3.
  • Multivalent single chain antibodies each having one V H and one V L domain covalently linked by a first peptide linker, can be covalently linked by at least one more peptide linker to form a multivalent single chain antibody.
  • Multivalent single chain antibodies allow for the construction of antibody fragments which have the specificity and avidity of whole antibodies, but lack the constant regions of the full length antibodies.
  • Multivalent immunoglobulin molecules may be monospecific or multispecific. The term specificity refers to the number of different types of antigenic determinants to which a particular immunoglobulin molecule can bind. If the immunoglobulin molecule binds to only one type of antigenic determinant, the immunoglobulin molecule is monospecific. If the immunoglobulin molecule binds to different types of antigenic determinants then the immunoglobulin molecule is multispecific.
  • a bispecific multivalent single chain antibody allows for the recognition of two different types of epitopes.
  • the epitopes may both be on KDR.
  • one epitope may be on KDR, and the other epitope may be on another antigen.
  • Each chain of a multivalent single chain antibody includes a variable light- chain fragment and a variable heavy-chain fragment, and is linked by a peptide linker to at least one other chain.
  • the peptide linker is composed of at least fifteen amino acid residues. The maximum number of amino acid residues is about one hundred. In a preferred embodiment, the number of V L and V H domains is equivalent.
  • the peptide linker (IN) joining the V H and V L domains to form a chain and the peptide linker (L ) joining two or more chains to form a multivalent scFv have substantially the same amino acid sequence.
  • bivalent single chain antibody can be represented as follows:
  • Multivalent single chain antibodies which are trivalent or greater have one or more antibody fragments joined to a bivalent single chain antibody by additional peptide linkers.
  • One example of a trivalent single chain antibody is:
  • Diabodies have two chains. Each chain of the diabody includes a V H domain connected to a V L domain. The domains are connected with linkers that are short enough to prevent pairing between domains on the same chain, thus driving the pairing between complementary domains on different chains to recreate the two antigen-binding sites.
  • the peptide linker includes at least five amino acid residues and no more than ten amino acid residues, e.g. (Gly-Gly-Gly-Gly-Ser), (Gly-Gly-Gly- Gly-Ser) .
  • the diabody structure is rigid and compact. The antigen-binding sites are at opposite ends of the molecule. Diabodies may be monospecfic or bispecific.
  • Triabodies are constructed with the amino acid terminus of a V L or V H domain directly fused to the carboxyl terminus of a V L or V H domain, i.e., without any linker sequence.
  • the triabody has three Fv heads with the polypeptides arranged in a cyclic, head-to-tail fashion.
  • a possible conformation of the triabody molecule is planar with the three binding sites located in a plane at an angle of 120 degrees from one another.
  • Triabodies may be monospecific, bispecific or trispecific.
  • the antibodies of this invention contain all six complementarity determining regions of the whole antibody, although antibodies containing fewer than all of such regions, such as three, four or five CDRs, are also functional.
  • the present invention also provides chimerized and humanized antibodies which bind specifically to an extacellular domain of KDR and neutralize activation of the receptor.
  • Chimeric antibodies include variable regions from the mouse antibody and constant regions from a human antibody. A chimeric antibody retains its binding specificity but more closely resembles a natural human antibody.
  • the only part of humanized antibodies which contain mouse amino acids are those which are necessary to recognize KDR , i.e. the CDRs.
  • DNA encoding chimerized antibodies may be prepared by recombining DNA substantially or exclusively encoding human constant regions and DNA encoding variable regions derived substantially or exclusively from the sequence of the variable region of a mammal other than a human.
  • DNA encoding humanized antibodies may be prepared by recombining DNA encoding constant regions and variable regions, other than the CDRs, derived substantially or exclusively from the corresponding human antibody regions and DNA encoding CDRs derived substantially or exclusively from a mammal other than a human.
  • Suitable mammals include any mammal from which monoclonal antibodies may be made. Examples of such mammals include a rabbit, rat, mouse, horse, goat, or primate. Mice are preferred.
  • Suitable sources of DNA molecules that encode fragments of antibodies include any cell, such as hybridomas and spleen cells, that express the full length antibody.
  • Another source is single chain antibodies produced from a phage display library as is known in the art.
  • the antibodies of this invention may be or may combine members of any immunoglobulin class, such as IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof.
  • the KDR used to make the immunoglobulin molecule is usually bound to a cell, such as an endothelial cell.
  • the KDR may also be bound to a non-endothelial cell, such as a tumor cell.
  • the KDR may be free from the cell, preferably in soluble form.
  • high affinity anti-RTJR scFv antibodies which block VEGF binding to KDR, were isolated from a phage display library constructed from mice immunized with a soluble form of the human VEGF receptor. Over 90% of recovered clones after two rounds of selection are specific to KDR. The binding affinities for KDR of these scFvs are in the nM range, which are as high as those of several bivalent anti-ROR monoclonal antibodies produced using hybridoma technology. A human phage library may also be used to generate such high affinity anti- KDR scFvs.
  • Each domain of the antibodies of this 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 occuring 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 al./Griffiths et al.). For instance, it is possible to join together domains corresponding to antibody variable domains which are missing at least one amino acid.
  • the important characterizing feature is the ability of each domain to associate with a complementary domain to form an antigen binding site. Accordingly, the terms "variable heavy/light chain fragment" should not be construed to exclude variants which do not have a material effect on how the invention works.
  • Substantially the same amino acid sequence is defined herein as a sequence with at least 70%, preferably at least about 80%, and more preferably at least about 90% homology to another amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988).
  • the antibodies of this invention may be fused to additional amino acid residues.
  • residues may be a peptide tag, perhaps to facilitate isolation, or they may be a signal sequence for secretion of the polypeptide from a host cell upon synthesis.
  • secretory leader peptides are used, being amino acids joined to the N-terminal end of a polypeptide to direct movement of the polypeptide out of the cytosol.
  • the present invention also provides nucleic acid molecules which comprise a sequence encoding a polypeptide according to the invention, and diverse repertoires of such nucleic acid.
  • DNA deletions and recombinations described above may be carried out by known methods, such as those described in PCT applications WO 93/21319, WO 89/09622, European Patent applications 239,400, 338,745 and 332,424 and/or other standard recombinant DNA techniques, such as those described below.
  • c-p 1 C 11 was produced from p 1 c 11.
  • Chimeric-p 1 C 11 binds specifically to the extracellular domain of soluble as well as cell surface-expressed KDR.
  • the binding affinity of the c-plCl 1 is 0.82 nM. It effectively neutralizes activation of KDR and MAP kinases p44/p42 of human endothelial cells.
  • c-plCl 1 efficiently neutralizes VEGF-induced mitogenesis of human endothelial cells.
  • the c-plCl 1 binds more efficiently to KDR than its parent scFv, plCl 1, and is more potent in neutralizing activity of KDR interaction and in inhibiting VEGF- stimulated mitogenesis of HUVEC.
  • the affinity of the c-plCl 1 for binding to KDR is approximately 2.5-fold higher than that of the parent scFv, mainly due to the slower off-rate of the bivalent c-plCl 1 (See Table 2.)
  • VEGF ⁇ 65 protein was expressed in baculovirus and purified.
  • cDNA encoding the extracellular domain of KDR was isolated by RT-PCR from human fetal kidney mRNA and subcloned into the Bgl II and BspE I sites of the vector AP-Tag. In this plasmid the cDNA for KDR extracellular domain is fused in-frame with the cDNA for human placental AP. The plasmid was electroporated into NIH 3T3 cells together with the neomycin expression vector pSV-Neo and stable cell clones were selected with G418. The soluble fusion protein KDR-A ⁇ was purified from cell culture supernatant by affinity chromatography using immobilized monoclonal antibodies to AP.
  • Example I Mice immunization and construction of single chain antibody phage display library
  • mice Female BALB/C mice were given two intraperitoneal (i.p.) injections of 10 ug KDR- AP in 200 ul of RIBI Adjuvant System followed by one i.p. injection without RIBI adjuvant over a period of two months. The mice were also given a subcutaneous (s.c.) injection of 10 ug KDR- AP in 200 ul of RIBI at the time of the first immunization. The mice were boosted i.p. with 20 ug of KDR- AV three days before euthanasia. Spleens from donor mice were removed and the cells were isolated. RNA was extracted and mRNA was purified from total RNA of splenocytes. A scFv phage display library was constructed using the mRNA which was displayed on the surface of the filamentous phage M 13.
  • antibody V H and V L domains are joined together by a 15 amino-acid-long linker (GGGGS) 3 and fused to the N-terminal of phage protein III.
  • a 15 amino-acid-long E tag which is followed by an amber codon (TAG) was inserted between the C-terminal of V L and the protein III for detection and other analytic purposes.
  • TAG amber codon
  • the amber codon positioned between the E tag and the protein III enables the construct to make scFv in surface-displaying format when transformed into a suppressor host (such as TGI cells), and scFv in soluble form when transformed into a nonsupressor host (such as HB2151 cells).
  • the assembled scFv DNA was ligated into the pCANTAB 5E vector.
  • the transformed TGI cells were plated onto 2YTAG plates and incubated. The colonies were scraped into 10 ml of 2YT medium, 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 Ml 3K07 helper phage and amplified overnight in 2 YTAK medium (2YT containing 100 ug/ml of ampicillin and 50 ug/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 ug/ml of AP protein and incubated at 37 C for 1 h to capture phage displaying anti-AP scFv and to block other nonspecific binding.
  • Phage of individual clones were used to infect a nonsuppressor E. coli host HB2151 and the infectant selected on 2YTAG-N plates.
  • Expression of scFv in HB2 151 cells was induced by culturing the cells in 2YTA medium containing 1 mM isopropyl- 1-thio-B-D-galactopyranoside at 30 C.
  • 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. After centrifugation at 15,000 rpm for 15 min., the soluble scFv was purified from the supernatant by affinity chromatography using the RPAS Purification Module (Pharmacia Biotech).
  • Two assays were employed to examine quantitatively the binding of purified soluble scFv to KDR.
  • Four different clones including the two VEGF blockers, plCl 1 and plF12, one nonblocker, the dominant clone p2A6 and the nonbinder p2A7, were expressed in shaker flasks using a nonsuppressor host E.coli HB2151 cells.
  • the soluble scFv was purified from the periplasmic extracts of E.coli by anti-E-tag affinity chromatography. The yield of purified scFv of these clones ranged from 100 - 400 ug / liter culture.
  • soluble scFv were mixed with a fixed amount of KDR-AP (50 ng) and incubated at room temerature for 1 h. The mixture were then transferred to 96-well microtiter plates coated with VEGF 165 (200 ng/well) and incubated at room temperature for an additional 2 h, after which the plates were washed 5 times and the substrate for AP was added to quantify the bound KDR-AP molecules.
  • IC 50 i.e., the scFv concentration required for 50% inhibition of KDR binding to VEGF, was then calculated.
  • Figure 1 shows the dose-dependent binding of scFv to immobilized KDR as assayed by a direct binding ELISA.
  • Data shown in Figure 2 are the means ⁇ SD of triplicate determinations.
  • Clone plCl 1 the dominant clone after each round of panning, showed the highest KDR binding capacity and the highest potency in blocking VEGF binding to KDR (Table 1).
  • KDR-AV fusion protein was immobilized onto a sensor chip and soluble scFv were injected at concentrations ranging from 62.5 nM to 1000 nM. Sensorgrams were obtained at each concentration and were evaluated using a program, BIA Evaluation 2.0, to determine the rate constant kon and koff Kd was calculated from the ratio of rate constants koff/kon.
  • Table 1 shows the results of the surface plasmon resonance on a BIAcore instrument.
  • Kd 6fold weaker affinity
  • p2A7 did not bind to the immobilized KDR on the BIAcore.
  • HUVEC Phosphorylation assays were performed with early passage HUVEC following a protocol described previously. Briefly, HUVEC were incubated in serum free EBM- 2 base medium supplemented with 0.5% ⁇ bovine serum albumin at room temperature for 10 min. in the presence or absence of scFv antibodies at 5 ug/ml, followed by stimulation with 20 ng/ml VEGF 165 at room temperature for an additional 15 min. The cells were lysed and the KDR receptor was immunoprecipitated from the cell lysates with Protein A Sepharose beads coupled to a rabbit ant -KDR polyclonal antibody (ImClone Systems Incorporated).
  • the beads were washed, mixed with SDS loading buffer, and the supernatant subjected to Western blot analysis.
  • blots were probed with an anti-phosphotyrosine Mab, 4G10.
  • MAP kinase activity assay cell lysates were resolved with SDS-PAGE followed by Western blot analysis using a phospho-specific MAP kinase antibody. All signals were detected using ECL.
  • VEGF-blocking scFv plCl 1 but not the non-blocking scFv p2A6, was able to inhibit KDR receptor phosphorylation stimulated by VEGF. Further, plCl 1 also effectively inhibited VEGF-stimulated activation of MAP kineses p44/p42. In contrast, neither plCl 1, nor p2A6 inhibited FGF-stimulated activation of MAP kinases p44/p42.
  • HUVEC 5 x 103 cells/well
  • VEGF vascular endothelial growth factor
  • bFGF vascular endothelial growth factor
  • EGF vascular endothelial growth factor
  • the cells were placed on ice, washed twice with serum-containing medium, followed by a 10 minute incubation at 4 C with 10% TCA. The cells were then washed once with water and solubilized in 25 ul of 2% SDS. Scintillation fluid (150 ul/well) was added and DNA incorporated radioactivity was determined on a scintillation counter (Wallach, Model 1450 Microbeta Scintillation Counter).
  • HUVEC is shown in Fig. 3.
  • the VEGF-blocking scFv plCl 1 strongly inhibited VEGF induced DNA synthesis in HUVEC with an EC 50 , i.e., the antibody concentration that inhibited 50% of VEGF-stimulated mitogenesis of HUVEC, of approximately 5 nM.
  • the non-blocking scFv p2A6 showed no inhibitory effect on the mitogenic activity of VEGF.
  • Neither p 1 C 11 nor p2A6 inhibited bFGF-induced DNA synthesis in HUVEC (not shown).
  • Data shown in Fig. 3 are representative of at least three separate experiments. ( ) VEGF only; ( ) no VEGF.
  • VEGF 165 and RTJR-alkaline phosphatase fusion proteins were expressed in baculovirus and NIH 3T3 cells, respectively, and purified following the procedures described above.
  • the ant -KDR scFv plCl 1 and scFv p2A6, an antibody that binds to KDR but does not block RDR-VEGF interaction, were isolated from a phage display library constructed from a mouse immunized with KDR as described above.
  • C225 is a chimeric IgGl antibody directed against epidermal growth factor (EGF) receptor.
  • variable domains of the light (V L ) and the heavy (V H ) chains of plCl 1 were cloned from the scFv expression vector by PCR using primers 1 and 2, and primers 3 and 4, respectively.
  • the leader peptide sequence for protein secretion in mammalian cells was then added to the 5' of the V L and the V H by PCR using primers 5 and 2, and primers 5 and 4, respectively.
  • Primer 2 5' TCG ATC TAG AAG GAT CCA CTC ACG TTT TAT TTC CAG3' Bam I
  • Primer 4 5' TCG AAG GAT CCA CTC ACC TGA GGA GAC GGT3'
  • V H gene was digested with Hind III and BamH I and ligated into the vector pGID105 containing the human IgGl ( ⁇ ) heavy chain constant domain (C H ) to create the expression vector for the chimeric plCl 1 heavy chain, c-plCl 1-H. Both constructs were examined by restriction enzyme digestion and verified by dideoxynucleotide sequencing.
  • both the V H and the V L domains are precisely fused on their 5' ends to a gene segment encoding a leader peptide sequence as marked.
  • the V H and the V L domains are ligated via Hind 111/ BamHI sites into expression vector pGlD105, which contains a cDNA version of the human ⁇ l constant region gene, and pKNIOO, which contains a cDNA version of the human K chain constant region gene, respectively.
  • expression is under control of the HCMVi promoter and terminated by an artificial termination sequence.
  • the light and the heavy chain complimentarily determining region (CDR) residues defined according the hypervariable sequence definition of Kabat et al., are underlined and labeled CDR-H1 to H3 and CDR-L1 to L3, respectively.
  • COS cells were co-transfected with equal amounts of c-plCl 1-L and c-plCl 1- H plasmids for transient IgG expression.
  • Subconfluent COS cells grown in DMEM / 10%) FCS in 150 mm culture dishes were rinsed once with 20 ml of DMEM containing 40 mM Tris (pH 7.4), followed by incubation at 37 C for 4.5 h with 4 ml of DMEM / DEAE-Dextran / DNA mixture (DMEM containing 40 mM Tris, 0.4 mg/ml of DEAE-Dextran (Sigma), and 20 ug each of c-plCl 1-L and c-plCl 1 -H plasmids).
  • the cells were incubated at 37 C for 1 h with 4 ml of DMEM / 2% FCS containing 100 nM of chloroquine (Sigma), followed by incubation with 1.5 ml of 20% glycerol / PBS at room temperature for 1 min.
  • the cells were washed twice with DMEM / 5% FCS and incubated in 20 ml of the same medium at 37 C overnight.
  • the cell culture medium was changed to serum-free DMEM / HEPES after the cells were washed twice with plain DMEM.
  • the cell culture supernatant was collected at 48 h and 120 h after the transfection.
  • the chimeric IgG was purified from the pooled supernatant by affinity chromatography using Protein G column following the protocol described by the manufacturer (Pharmacia Biotech). The IgG-containing fractions were pooled, buffer exchanged into PBS and concentrated using Centricon 10 concentrators (Amicon Corp., Beverly, MA). The purity of the IgG was analyzed by SDS-PAGE. The concentration of purified antibody was determined by ELISA using goat anti-human y chain specific antibody as the capture agent and HRP- conjugated goat anti-human k chain antibody as the detection agent. Standard curve was calibrated using a clinical grade antibody, C225.
  • HUVEC cells were grown in growth factor-depleted EBM-2 medium overnight to induce the expression of KDR.
  • the cells were harvested and washed three times with PBS, incubated with c-plCl 1 IgG (5 ug/ml) for 1 h at 4 C, followed by incubation with a FITC labeled rabbit anti-human Fc antibody (Capper, Organon Teknika Corp., West Chester, PA) for an additional 60 min.
  • the cells were washed and analyzed by a flow cytometer (Model EPICS®, Coulter Corp., Edison, NJ).
  • Figure 6 is a graph showing the FACS analysis of c-plCl 1 binding to KDR- expressing HUVEC. As previously seen with the parent scFv plCl 1, c-plCl 1 binds specifically to KDR expressed on early passage HUVEC.
  • Figure 5 is a graph showing the direct binding of antibodies to immobilized KDR.
  • C-plCl l is shown to bind more efficiently to immobilized KDR receptor than the parent scFv.
  • KDR-AP fusion protein was immobilized onto a sensor chip, and antibodies or VEGF were injected at concentrations ranging from 25 nM to 200 nM. Sensorgrams were obtained at each concentration and were evaluated using a program, BIA Evaluation 2.0, to determine the rate constants kon and koff . Kd was calculated as the ratio of rate constants koff /kon.
  • BIAcore analysis reveals that c-plCl 1 bind to KDR with higher affinity than the parent scFv (Table 2).
  • the Kd of c-plCl 1 is 0.82 nM, compared to 2.1 nM for the scFv.
  • the increased affinity of c-plCl 1 is mainly due to a slower dissociation rate (koff) of the bivalent chimeric IgG. It is important to note that the affinity (Kd) of c- plCl 1 for binding to KDR is similar to that of the natural ligand VEGF for binding to KDR, which is 0.93 nM as determined in our BIAcore analysis (Table 2).
  • Example V(d) Competitive VEGF binding assay.
  • EC 50 i.e., the antibody concentration required for 50% inhibition of KDR binding to VEGF, was then calculated.
  • Figure 7 shows that c-plCl 1 block KDR receptor from binding to immobilized VEGF in a dose-dependent manner.
  • the chimeric antibody is more potent in blocking YEGF-KDR interaction with an IC 50 (i.e., the antibody concentrations required to inhibit 50% of KDR trom binding to VEGF) of 0.8 nM, compared to that of 2.0 nM for the scFv.
  • the control scFv p2A6 also binds KDR (Fig. 5) but does not block VEGF-RDR interaction (Fig. 7).
  • c-plCII antibody or cold VEGFj 65 protein were mixed with a fixed amount of 1251 labeled VEGF 165 and added to 96- well microtiter plates coated with KDR receptor. The plates were incubated at room temperature for 2h, washed 5 times and the amounts of radiolabeled VEGF 165 that bound to immobilized KDR receptor were counted. Concentrations of c-pICII and cold VEGFj 65 required to block 50% of binding of the radiolabeled VEGF to immobilized KDR receptor were determined.
  • Subconfluent HUVEC cells were grown in growth factor depleted EBM-2 medium for 24 to 48h prior to experimentation. After pretreatment with 50 nM sodium orthovanadate for 30 min, the cells were incubated in the presence or absence of antibodies for 15 min, followed by stimulation with 20 ng/ml of VEGF ⁇ 65 , or 10 ng/ml of FGF at room temperature for an additional 15 min.
  • Proteins were resolved with SDS-PAGE and subjected to Western blot analysis.
  • blots were probed with an antiphosphotyrosine Mab, PY20 (ICN Biomedicals, Inc. Aurora, OH).
  • PY20 ICN Biomedicals, Inc. Aurora, OH.
  • MAP kinase activity assay cell lysates were resolved with SDS-PAGE followed by Western blot analysis using a phospho-specific MAP kinase antibody (New England BioLabs, Beverly, MA). All signals were detected using ECL (Amersham, Arlington Heights, IL). In both assays, the blots were reprobed with a polyclonal an ⁇ -KDR antibody (ImClone Systems) to assure that equal amount of protein was loaded in each lane of SDS-PAGE gels.
  • a polyclonal an ⁇ -KDR antibody ImClone Systems
  • C-plCl 1 effectively inhibits VEGF-stimulated phosphorylation of KDR receptor and activation of p44/p42 MAP kineses.
  • C225 does not show any inhibition of VEGF-stimulated activation of KDR receptor and MAP kineses.
  • c-p 1 C 11 nor C225 alone has any effects on the activity of KDR receptor and p44/p42 MAP kineses.
  • c-plCl l does not inhibit FGF-stimulated activation of p44/p42 MAP kineses (not shown).
  • neither scFv p2A6, nor the chimeric IgG form of p2A6 (c-p2A6) inhibits VEGF-stimulated activation of KDR receptor and MAP kineses (not shown).
  • HUVEC human endothelial cells
  • HUVEC 5 x 10 cells/well
  • EBM-2 medium without VEGF, bFGF or EGF
  • Various amounts of antibodies were added to duplicate wells and pre-incubated at 37 C for 1 hour, after which VEGF ⁇ 6 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 hours.

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Abstract

La présente invention concerne une molécule d'immunoglobuline se liant au KDR avec une affinité comparable au VEGF humain, et neutralisant l'activation de KDR. Les molécules d'immunoglobulines contiennent des anticorps à chaîne unique monovalents, des anticorps à chaîne unique multivalents, des dianticorps, trianticorps, anticorps, et des anticorps humanisés et chimérisés. Par ailleurs, cette invention concerne des molécules d'acide nucléique codant ces molécules d'immunoglobulines. De plus, cette invention concerne un procédé de préparation des molécules d'immunoglobulines précitées. L'invention concerne également un procédé de neutralisation de l'activation de KDR, un procédé d'inhibition de l'angiogenèse chez un mammifère ainsi qu'un procédé d'inhibition de croissance tumorale chez un mammifère utilisant ces molécules d'immunoglobulines.
EP00913280A 1999-01-29 2000-01-28 Anticorps specifiques au kdr et leurs utilisations Withdrawn EP1151002A4 (fr)

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EP1151002A4 (fr) 2002-05-02
IL144578A0 (en) 2002-05-23
CN1345334A (zh) 2002-04-17
AU3475100A (en) 2000-08-18
JP2002536968A (ja) 2002-11-05
CA2361553A1 (fr) 2000-08-03

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