US20080227119A1 - ELISA for VEGF - Google Patents

ELISA for VEGF Download PDF

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US20080227119A1
US20080227119A1 US11/866,560 US86656007A US2008227119A1 US 20080227119 A1 US20080227119 A1 US 20080227119A1 US 86656007 A US86656007 A US 86656007A US 2008227119 A1 US2008227119 A1 US 2008227119A1
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vegf
antibody
detectable
kit
elisa
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Yu-Ju G. Meng
Kyu H. Hong
Johnny Gutierrez
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Genentech Inc
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Genentech Inc
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Assigned to GENENTECH, INC. reassignment GENENTECH, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GUTIERREZ, JOHNNY, HONG, KYU H, MENG, YU-JU G
Publication of US20080227119A1 publication Critical patent/US20080227119A1/en
Priority to US12/817,827 priority patent/US8449885B2/en
Priority to US13/660,563 priority patent/US9146245B2/en
Priority to US14/814,139 priority patent/US9625462B2/en
Priority to US15/456,846 priority patent/US20170276683A1/en
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475Assays involving growth factors

Definitions

  • This invention relates to immunoassays for detecting certain populations of VEGF that can be used as diagnostic and prognostic methods for patients with cancer, cardiovascular, or other pathologies.
  • angiogenesis is implicated in the pathogenesis of a variety of disorders. These include solid tumors, intra-ocular neovascular syndromes such as proliferative retinopathies or age-related macular degeneration (AMD), rheumatoid arthritis, and psoriasis (Folkman et al. J. Biol. Chem. 267:10931-10934 (1992); Klagsbrun et al. Annu. Rev. Physiol. 53:217-239 (1991); and Garner A, Vascular diseases. In: Pathobiology of ocular disease. A dynamic approach . Garner A, Klintworth G K, Eds.
  • VEGF vascular endothelial growth factor
  • VEGF has been shown to be a key mediator of neovascularization associated with tumors and intra-ocular disorders (Ferrara et al., supra).
  • the VEGF mRNA is overexpressed by the majority of human tumors examined (Berkman et al. J. Clin Invest 91:153-159 (1993); Brown et al. Human Pathol. 26:86-91 (1995); Brown et al. Cancer Res. 53:4727-4735 (1993); Mattern et al. Brit. J. Cancer. 73:931-934 (1996); and Dvorak et al. Am J. Pathol. 146:1029-1039 (1995)).
  • VEGF vascular endothelial growth factor
  • VEGF is produced by tissues and does not have to enter the circulation to exert its biological effect, but rather acts locally as a paracrine regulator.
  • several studies have tried to correlate levels of circulating VEGF with tumor burden and have suggested VEGF levels as a potential prognostic marker (Ferrari and Scagliotti Eur. J. Cancer 32A:2368 (1996); Gasparini et al. J. Natl. Cancer Inst.
  • VEGF vascular endothelial growth factor
  • An ELISA kit for VEGF detection is commercially available from R&D Systems (Minneapolis, Minn.).
  • the R&D VEGF ELISA kit has been used in sandwich assays wherein a monoclonal antibody is used to capture the target VEGF antigen and a polyclonal antibody is used to detect the VEGF.
  • a monoclonal antibody is used to capture the target VEGF antigen
  • a polyclonal antibody is used to detect the VEGF.
  • VEGF 165/165 can be proteolytically cleaved into three other forms: a 165/110 heterodimer, a 110/110 homodimer, and a 55-amino-acid C-terminal fragment (Keyt et al. J. Biol. Chem. 271:7788-7795 (1996); Keck et al. Arch. Biochem. Biophys. 344:103-113 (1997)).
  • Antibody-sandwich ELISA methods for VEGF as an antigen were developed to detect VEGF forms in biological samples.
  • the VEGF ELISA provided herein is capable of detecting VEGF isoforms and fragments of VEGF greater than 110 (“VEGF 110+ ”). Kits thereof are also provided.
  • methods for detecting selective vascular endothelial growth factor (VEGF) forms greater than 110 amino acids (VEGF 110+ ) in a biological sample comprise the steps of: (a) contacting and incubating the biological sample with a capture reagent immobilized to a solid support, wherein the capture reagent is an antibody that recognizes same epitope as antibody 5C3 against human VEGF, said monoclonal antibody binding specifically to residues greater than 110 of human VEGF; (b) separating the biological sample from the immobilized capture reagents; (c) contacting the immobilized capture reagent-target molecule complex with a detectable antibody that binds to the KDR and/or FLT1 receptor binding domains of VEGF; and (d) measuring the level of VEGF 110+ bound to the capture reagents using a detection means for the detectable antibody.
  • a capture reagent immobilized to a solid support wherein the capture reagent is an antibody that recognizes same epitope as antibody 5C3 against
  • the detectable antibody binds to an epitope in VEGF 1-110.
  • comparison ELISA can be performed to detect different types of VEGF.
  • the biological sample e.g., tumor samples or tumor lysates, plasma, serum, or urine, etc.
  • the biological sample is isolated from a human subject.
  • the capture reagent is the 5C3 monoclonal antibody. In one embodiment, the immobilized capture reagent is coated on a microtiter plate. In certain embodiments, the detectable antibody is a monoclonal antibody. In one embodiment, the detectable antibody is a murine monoclonal antibody. In one embodiment, the immobilized monoclonal antibody is MAb 5C3 and the detectable antibody is MAb A4.6.1. In certain embodiments, the detectable antibody is directly detectable. In one embodiment, detectable antibody is amplified by a calorimetric reagent. In one embodiment, the detectable antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3′,5,5′-tetramethyl benzidine.
  • the human subject is a vascular, diabetic, or cancer patient and the measuring step (d) further comprises a comparison with a standard curve to determine the level of VEGF compared to a normal individual.
  • kits are also provided.
  • an immunoassay kit for detecting vascular endothelial growth factor (VEGF) forms greater than 110 amino acids (VEGF 110+ ) in a biological sample can comprise: (a) as capture reagent, an antibody against human VEGF, wherein the monoclonal antibody binds specifically to the residues greater than 110 of human VEGF; and (b) as detection reagent, a detectable antibody that binds to the KDR and/or FLT1 receptor binding domains of VEGF.
  • the detectable antibody binds to an epitope in VEGF 1-110.
  • the kit further comprises a solid support for the capture reagents.
  • the capture reagents can be immobilized on the solid support (e.g., a microtiter plate).
  • the kit further comprises a detection means (e.g., colormetric means, fluorimetric means, etc.) for the detectable antibodies.
  • the kit further comprises purified VEGF as an antigen standard.
  • an additional VEGF ELISA or more can be provided for comparison studies with the VEGF 110+ ELISA.
  • the kit includes a capture reagent monoclonal antibody, which is murine monoclonal antibody MAb 5C3, and a detectable antibody, which is MAb A4.6. 1.
  • the invention provides an anti-VEGF antibody 5C3 (obtainable from or produced by hybridoma deposited under ATCC number PTA-7737).
  • the invention also provides an antibody that does not bind VEGF 1-110 and binds to the same VEGF 110+ epitope as the monoclonal antibody produced by hybridoma cell line PTA-7737.
  • an antibody of the invention is conjugated to a detectable label.
  • the hybridoma 5C3.1.1 deposited under ATCC deposit number PTA-7737 is provided.
  • FIG. 1 Panels A, B and C illustrates the detection of recombinant VEGF165, VEGF121(1) (truncated, likely missing approximately 9 amino acids from the carboxy-terminus according to the manufacturer, R&D systems), VEGF121 (2) (from Pepro Tech), VEGF110 (N-terminal fragment generated by plasmin digestion of VEGF) and VEGF8-109 (artificial VEGF with amino acids 8-109 of VEGF165) molecules by different VEGF ELISAs.
  • A ELISA A using 3.5F8 for coat and biotinylated A4.6.1 for detection.
  • B ELISA B using A4.6.1 for coat and biotinylate 2E3 for detection.
  • C ELISA C using 5C3 for coat and biotinylated A4.6.1 for detection.
  • FIG. 2 illustrates protein blotting of VEGF produced by A673 cells, using 3.5F8 (left) or A4.6.1 (right) for probing.
  • Samples are VEGF purified from conditioned medium of A673 cells using an A4.6.1 Affinity column (lane 1) and recombinant VEGF proteins VEGF 165 , VEGF 121 (likely missing approximately 9 amino acids from the carboxy-terminus according to the manufacturer, R&D systems) and VEGF 8-109 produced by E. coli (lanes 2, 3 and 4, respectively).
  • FIG. 3 illustrates a diagram of VEGF 165 , VEGF 121 and VEGF 110 (N-terminal fragment generated by plasmin digestion of VEGF) showing the proposed binding sites of the antibodies used in the three VEGF ELISAs.
  • VEGF refers to the 165-amino acid vascular endothelial cell growth factor, and related 121-, 145-, 189-, and 206-amino acid vascular endothelial cell growth factors, as described by Leung et al. Science 246:1306 (1989), Houck et al. Mol. Endocrin. 5:1806 (1991), and Neufeld et al., supra, together with the naturally occurring allelic and processed forms of those growth factors. See also, e.g., FIG. 1A and B of U.S. Pat. No. 6,057,428.
  • Active VEGF fragment can be released from ECM-bound VEGF by plasmin cleavage, generating the first 110 amino acids (see, e.g., Keyt B A, et al.,: The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem. 271: 7788-7795 (1996)).
  • VEGF 110+ refers to VEGF fragments that are greater than 110 amino acids (from the N-terminal), but do not include the first 110 amino acids or smaller fragments (e.g., VEGF 8-109 ).
  • detecting is used in the broadest sense to include both qualitative and quantitative measurements of a target molecule.
  • the detecting method as described herein is used to identify the mere presence of VEGF 110+ or VEGF in a biological sample.
  • the method is used to test whether VEGF 110+ or VEGF in a sample is at a detectable level.
  • the method can be used to quantify the amount of VEGF 110+ or VEGF in a sample and further to compare the VEGF 110+ or VEGF levels from different samples.
  • biological sample refers to a body sample from any animal, but preferably is from a mammal, more preferably from a human.
  • biological sample is from a vascular, diabetic, or cancer patient.
  • samples include biological fluids such as serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, as well as tissue extracts such as homogenized tissue, tumor tissue, and cellular extracts.
  • the sample is a body sample from any animal, in one embodiment it is from a mammal, in one embodiment from a human subject. In one embodiment, such biological sample is from clinical patients.
  • detectable antibody refers to an antibody that is capable of being detected either directly through a label amplified by a detection means, or indirectly through, e.g., another antibody that is labeled.
  • the antibody is typically conjugated to a moiety that is detectable by some means.
  • the detectable antibody is biotinylated antibody.
  • detection means refers to a moiety or technique used to detect the presence of the detectable antibody in the ELISA herein and includes detection agents that amplify the immobilized label such as label captured onto a microtiter plate.
  • the detection means is a colorimetric detection agent such as avidin or streptavidin-HRP.
  • capture reagent refers to a reagent capable of binding and capturing a target molecule in a sample such that under suitable condition, the capture reagent-target molecule complex can be separated from the rest of the sample.
  • the capture reagent is immobilized or immobilizable.
  • the capture reagent is preferably an antibody or a mixture of different antibodies against a target antigen.
  • antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • Antibody fragments comprise a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof.
  • antibody fragments include Fab, Fab′, F(ab′) 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • an “intact antibody” is one comprising heavy- and light-chain variable domains as well as an Fc region.
  • “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • V H variable domain
  • Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
  • the monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences
  • Chimeric antibodies of interest herein include “primatized” antibodies comprising variable-domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant-region sequences (U.S. Pat. No. 5,693,780).
  • a non-human primate e.g. Old World Monkey, such as baboon, rhesus or cynomolgus monkey
  • human constant-region sequences U.S. Pat. No. 5,693,780
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made further to refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a humanized 5C3 antibody is provided and used the methods provided herein.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs).
  • the variable domains of native heavy and light chains each comprise four FRs, largely adopting a sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service , National Institutes of Health, Bethesda, Md. (1991)).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′) 2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
  • “Fv” is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy-chain and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy-chain CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear at least one free thiol group.
  • F(ab′) 2 antibody fragments originally were produced as pairs of Fab′ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
  • antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • Single-chain Fv or “scFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains that enables the scFv to form the desired structure for antigen binding.
  • hypervariable region when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding.
  • the hypervariable region comprises amino acid residues from a “complementarity-determining region” or “CDR” (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service , National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop” (e.g.
  • “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic, and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, sheep, pigs, cows, etc. Preferably, the mammal is human.
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • examples of cancer include but are not limited to, carcinoma including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (e.g., hepatic carcinoma and hepatoma), bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, rectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, basal cell carcinoma, testicular cancer, esophageal cancer, hepatic carcinoma, soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, mesothelioma, multiple myelom
  • vascular and cardiac are used interchangeably and describe patients with indications that stimulate angiogenesis and/or cardiovascularization, and those that inhibit angiogenesis and/or cardiovascularization.
  • disorders include, for example, arterial disease, such as atherosclerosis, hypertension, inflammatory vasculitis, Reynaud' s disease and Reynaud's phenomenon, aneurysms, and arterial restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema; and other vascular disorders such as peripheral vascular disease, AMD, cancer such as vascular tumors, e.g., hemangioma (capillary and cavernous), glomus tumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma, and lymphangiosarcoma, tumor
  • diabetes refers to a progressive disease of carbohydrate metabolism involving inadequate production or utilization of insulin and is characterized by hyperglycemia and glycosuria. This term includes all forms of diabetes, such as type I and type II diabetes and insulin-resistant diabetes, such as Mendenhall's Syndrome, Werner Syndrome, leprechaunism, lipoatrophic diabetes, and other lipoatrophies.
  • affinity purified refers to purifying a substance by eluting it through an affinity chromatography column.
  • VEGF Vascular endothelial growth factor
  • VEGF Vascular endothelial growth factor
  • the human VEGF gene has eight exons separated by introns.
  • RNA splicing results in the generation of at least four major isoforms having 121, 165, 189 and 206 amino acids in monomer (see, e.g., Houck K A, et al.,: The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol 5: 1806-1814 (1991); and, Tischer E, et al.,: The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 266: 11947-11954 (1991)).
  • isoforms including those having 145 (see, e.g., Poltorak Z., et al.,: VEGF 145, a secreted vascular endothelial growth factor isoform that binds to extracellular matrix. J Biol Chem 272: 7151-7158 (1997)) and 183 (see, e.g., Jingjing L, et al.,: Human Muller cells express VEGF 183, a novel spliced variant of vascular endothelial growth factor. Invest Ophthalmol Vis Sci 40:752-759 (1999)) amino acids in monomer have also been reported.
  • VEGFR-1 receptor tyrosine kinases
  • VEGFR-2 receptor tyrosine kinases
  • VEGF 165 also interacts with neuropilin (see, e.g., Soker S.
  • VEGF 189 and VEGF 206 bind to heparin with a high affinity and are mostly sequestered in the extracellular matrix (ECM).
  • ECM extracellular matrix
  • VEGF 165 binds to heparin with intermediate affinity and is partially soluble and partially bound to cell surface and ECM.
  • VEGF 121 does not bind to heparin and is freely soluble.
  • VEGF 121 and VEGF 165 were found to be the most dominantly expressed variants in breast and ovarian cancer tumor specimens and cell lines by reverse transcription-PCR analysis, while VEGF 206 expression was not detected.
  • VEGF 183 and VEG 189 expression was found to be non-detectable or at low levels in the cell lines and were detected in some of the tumor specimens (see, e.g., Stimpfl M, et al.,: Vascular Endothelial growth factor splice variants and their prognostic value in breast and ovarian cancer. Clinical Cancer Research 8: 2253-2259 (2002)).
  • Active VEGF fragment can be released from ECM-bound VEGF by plasmin cleavage, generating the first 110 amino acids (see, e.g., Keyt B A, et al., The carboxyl - terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem. 271: 7788-7795 (1996)). This could be a mechanism to locally regulate the bioavailability of VEGF during physiological and pathological processes of angiogenesis. See, e.g., Houck K A, et al. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms.
  • Enzyme-linked immunosorbent assays for various antigens include those based on colorimetry, chemiluminescence, and fluorometry. ELISAs have been successfully applied in the determination of low amounts of drugs and other antigenic components in plasma and urine samples, involve no extraction steps, and are simple to carry out.
  • the assay described herein is an ELISA that utilizes antibodies as capture reagents and detectable antibodies for VEGF and VEGF 110+ . In certain embodiments, the ELISA is cell-based.
  • the biological sample suspected of containing VEGF or containing VEGF 110+ is contacted and incubated with the capture (or coat) antibodies so that the capture antibodies capture or bind to the VEGF or VEGF 110+ so that it can be detected in a detection step.
  • the detection step involves use of the detectable antibody, which, when contacted with any of the bound VEGF or VEGF 110+ binds to the protein of interest, if present, and a detection means is used to detect the label on the antibody and hence the presence or amount of VEGF or VEGF 110+ present.
  • This ELISA can be compared with an ELISA that recognizes total VEGF (e.g., U.S. Pat. No. 6,855,508; those described herein, and those known in the art) or isoforms of VEGF to determine the type of VEGF present.
  • the assay utilizes the following steps.
  • the biological sample is contacted and incubated with the immobilized capture (or coat) reagent, which is an anti-VEGF monoclonal antibody.
  • This antibody may be from any species, but preferably the monoclonal antibody is a murine or rat monoclonal antibody, more preferably murine, and most preferably MAb 5C3 derived from the hybridoma identified herein.
  • the immobilized monoclonal antibody is a murine monoclonal antibody, most preferably MAb 5C3. Immobilization conventionally is accomplished by insolubilizing the capture reagent either before the assay procedure, as by adsorption to a water-insoluble matrix or surface (U.S. Pat.
  • non-covalent or covalent coupling for example, using glutaraldehyde or carbodiimide cross-linking, with or without prior activation of the support with, e.g., nitric acid and a reducing agent as described in U.S. Pat. No. 3,645,852 or in Rotmans et al. J. Immunol. Methods 57:87-98 (1983)), or afterward, e.g., by immunoprecipitation.
  • the solid phase used for immobilization may be any inert support or carrier that is essentially water insoluble and useful in immunometric assays, including supports in the form of, e.g., surfaces, particles, porous matrices, etc.
  • supports in the form of, e.g., surfaces, particles, porous matrices, etc.
  • commonly used supports include small sheets, Sephadex, polyvinyl chloride, plastic beads, and assay plates or test tubes manufactured from polyethylene, polypropylene, polystyrene, and the like including 96-well microtiter plates, as well as particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides.
  • reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos.
  • the immobilized capture reagent is coated on a microtiter plate, and in particular the preferred solid phase used is a multi-well microtiter plate that can be used to analyze several samples at one time, e.g., a microtest 96-well ELISA plate such as that sold as Nune Maxisorb or Immulon.
  • the plate is a MICROTESTTM or MAXISORPTM 96-well ELISA plate such as that sold as NUNC MAXISORBTM or IMMULONTM.
  • the solid phase is coated with the capture reagent as defined above, which may be linked by a non-covalent or covalent interaction or physical linkage as desired. Techniques for attachment include those described in U.S. Pat. No. 4,376,110 and the references cited therein. If covalent, the plate or other solid phase is incubated with a cross-linking agent together with the capture reagent under conditions well known in the art, e.g., such as for 1 hour at room temperature.
  • cross-linking agents for attaching the capture reagent to the solid phase substrate include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxy-succinimide esters, for example, esters with 4-azido-salicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis-(succinimidyl-propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane.
  • Derivatizing agents such as methyl-3-[(p-azidophenyl)-dithio]pro-pioimi-date yield photoactivatable intermediates capable of forming cross-links in the presence of light.
  • 96-well plates are typically coated with the capture reagent (typically diluted in a buffer such as 0.05 M sodium carbonate by incubation for at least about 10 hours, more preferably at least overnight, at temperatures of about 4-20° C., or about 4-8° C., and at a pH of about 8-12, or about pH 9-10, or about pH 9.6). If shorter coating times are desired, one can coat, e.g., 96-well plates at room temperature for two hours. The plates may be stacked and coated long in advance of the assay itself, and then the assay can be carried out simultaneously on several samples in a manual, semi-automatic, or automatic fashion, such as by using robotics.
  • the capture reagent typically diluted in a buffer such as 0.05 M sodium carbonate by incubation for at least about 10 hours, more preferably at least overnight, at temperatures of about 4-20° C., or about 4-8° C., and at a pH of about 8-12, or about pH 9-10, or about pH 9.6. If shorter coating times are
  • the coated plates are then typically treated with a blocking agent that binds non-specifically to and saturates the binding sites to prevent unwanted binding of the free ligand to the excess sites on the wells of the plate.
  • a blocking agent that binds non-specifically to and saturates the binding sites to prevent unwanted binding of the free ligand to the excess sites on the wells of the plate.
  • appropriate blocking agents for this purpose include, e.g., gelatin, bovine serum albumin, egg albumin, casein, and non-fat milk.
  • the blocking treatment typically takes place under conditions of ambient temperatures for about 1-4 hours, preferably about 1 to 3 hours, or overnight at 0-4° C.
  • VEGF standard.(purified VEGF) or the biological sample to be analyzed, appropriately diluted, is added to the immobilized phase.
  • the preferred dilution rate is about 1-15%, preferably about 10%, by volume.
  • Buffers that may be used for dilution for this purpose include (a) PBS containing 0.5% BSA, 0.05% TWEEN 20 TM detergent (P20), 0.05% PROCLINTM 300 antibiotic, 5 mM EDTA, 0.25% Chaps surfactant, 0.2% beta-gamma globulin, and 0.35M NaCl, pH 7.4; (b) PBS containing 0.5% bovine serum albumin, 0.05% polysorbate 20, 5 mM EDTA, 0.25% CHAPS, 0.2% bovine ⁇ -globulins, and 0.35 M NaCl; pH 7.4 (c) PBS containing 0.5% BSA, 0.05% polysorbate 20 (P20), and 0.05% PROCLINTM 300, pH 7; (d) PBS
  • concentration of the capture reagents will generally be determined by the concentration range of interest of the VEGF taking any necessary dilution of the biological sample into account, the final concentration of the capture reagent will normally be determined empirically to maximize the sensitivity of the assay over the range of interest.
  • the conditions for incubation of sample and immobilized capture reagent are selected to maximize sensitivity of the assay and to minimize dissociation.
  • the incubation is accomplished at fairly constant temperatures, ranging from about 0C to about 40° C., preferably from about 20 to 25 ° C.
  • the time for incubation depends primarily on the temperature, being generally no greater than about 10 hours to avoid an insensitive assay.
  • the incubation time is from about 0.5 to 3 hours, and more preferably 1.5-3 hours at room temperature to maximize binding of free VEGF 110+ or VEGF to capture reagents.
  • the duration of incubation may be longer if a protease inhibitor is added to prevent proteases in the biological fluid from degrading the VEGF.
  • the pH of the incubation mixture will ordinarily be in the range of about 4-9.5, preferably in the range of about 6-9, more preferably about 7-8, and most preferably the pH of the assay (ELISA) diluent is pH 7.4.
  • the pH of the incubation buffer is chosen to maintain a significant level of specific binding of the capture reagent to the VEGF 110+ or VEGF being captured.
  • Various buffers may be employed to achieve and maintain the desired pH during this step, including borate, phosphate, carbonate, Tris-HCl or Tris-phosphate, acetate, barbital, and the like.
  • the particular buffer employed is not critical to the invention, but in individual assays one buffer may be preferred over another.
  • the biological sample is separated (preferably by washing) from the immobilized capture reagent to remove uncaptured molecules.
  • the solution used for washing is generally a buffer (“washing buffer”) with a pH determined using the considerations and buffers described above for the incubation step, with a preferable pH range of about 6-9.
  • the washing may be done three or more times.
  • the temperature of washing is generally from refrigerator to. moderate temperatures, with a constant temperature maintained during the assay period, typically from about 0-40° C., more preferably about 4-30° C.
  • the wash buffer can be placed in ice at 4° C. in a reservoir before the washing, and a plate washer can be utilized for this step.
  • a cross-linking agent or other suitable agent may also be added at this stage to allow the now-bound VEGF 110+ or VEGF to be covalently attached to the capture reagent if there is any concern that the captured VEGF 110+ or VEGF may dissociate to some extent in the subsequent steps.
  • the immobilized capture reagent is contacted with detectable antibodies, preferably at a temperature of about 20-40° C., more preferably about 20-25° C., with the exact temperature and time for contacting the two being dependent primarily on the detection means employed.
  • detectable antibodies preferably at a temperature of about 20-40° C., more preferably about 20-25° C.
  • the exact temperature and time for contacting the two being dependent primarily on the detection means employed.
  • the contacting is carried out (e.g., about 1 hour or more) to amplify the signal to the maximum.
  • a molar excess of an antibody with respect to the maximum concentration of free VEGF 110+ or VEGF expected (as described above) is added to the plate after it is washed.
  • This antibody is directly or indirectly detectable. While the detectable antibody may be a polyclonal or monoclonal antibody, e.g., in certain embodiments, it is a monoclonal antibody, in one embodiment murine, and in one embodiment MAb A4.6. 1. Also, the detectable antibody can be directly detectable, and in one embodiment has a colorimetric label, and in another embodiment has a flurometric label.
  • the detectable antibody is biotinylated and the detection means is avidin or streptavidin- peroxidase and 3,3′,5,5′-tetramethyl benzidine.
  • the readout of the detection means can be fluorimetric or calorimetric.
  • the affinity of the antibody must be sufficiently high that small amounts of the free VEGF 110+ or VEGF can be detected, but not so high that it causes the VEGF 110+ or VEGF to be pulled from the capture reagents.
  • the measuring step preferably comprises comparing the reaction that occurs as a result of the above three steps with a standard curve to determine the level of VEGF 110+ or VEGF compared to a normal individual, or preferably comprises comparing the reaction that occurs as a result of the above three steps with an another VEGF ELISA recognizing different isoforms or total VEGF to determine the level of the types of VEGF when the ELISAs are compared, and optionally compared to a normal individual.
  • Polyclonal antibodies to the VEGF generally are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the VEGF and an adjuvant. It may be useful to conjugate the VEGF or a fragment containing the target amino acid sequence to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOC12, or R1N ⁇ C ⁇ NR, where R and R1 are different alkyl groups.
  • a protein that is immunogenic in the species to be immunized e.g., keyhole limpe
  • the antibodies used as the coat or detectable antibodies may be obtained from any convenient vertebrate source, such as murine, primate, lagomorpha, goat, rabbit, rat, chicken, bovine, ovine, equine, canine, feline, or porcine.
  • Chimeric or humanized antibodies may also be employed, as described, e.g., in U.S. Pat. No. 4,816,567; Morrison et al. Proc. Natl. Acad. Sci. USA 81:6851 (1984); Neuberger et al. Nature 312: 604 (1984); Takeda et al. Nature 314:452 (1985); and WO 98/45331 published Oct. 15, 1998, as well as in those additional references set forth above.
  • Animals may be immunized against the immunogenic conjugates or derivatives by combining 1 mg or 1 ⁇ g of conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • the animals are boosted with 1 ⁇ 5 to 1/10 the original amount of conjugate in Freund's incomplete adjuvant by subcutaneous injection at multiple sites.
  • 7 to 14 days later animals are bled and the serum is assayed for anti-VEGF titer. Animals are boosted until the titer plateaus.
  • the animal is boosted with the conjugate of VEGF, but conjugated to a different protein and/or through a different cross-linking agent.
  • Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are used to enhance the immune response. Methods for the production of polyclonal antibodies are described in numerous immunology textbooks, such as Davis et al. Microbiology, 3 rd Edition , (Harper & Row, New York, N.Y., 1980).
  • Monoclonal antibodies are prepared by recovering spleen cells from immunized animals and immortalizing the cells in conventional fashion, e.g. by fusion with myeloma cells or by Epstein-Barr virus transformation, and screening for clones expressing the desired antibody. See, e.g., Kohler and Milstein Eur. J. Immunol. 6:511 (1976). Monoclonal antibodies, or the antigen-binding region of a monoclonal antibody, such as Fab or (Fab) 2 fragments, may alternatively be produced by recombinant methods.
  • Suitable antibodies include those already utilized in known RIAs for the protein in question, e.g., those antibodies directed against VEGF as described in the references given in the introduction herein.
  • the invention also provides an antibody that does not bind VEGF 1-110 and binds to the same VEGF 110+ epitope as the monoclonal antibody produced by hybridoma cell line PTA-7737.
  • a hybridoma 5C3.1.1 deposited under ATCC deposit number PTA-7737 is provided.
  • the antibody added to the immobilized capture reagents will be either directly labeled, or detected indirectly by addition, after washing off of excess first antibody, of a molar excess of a second, labeled antibody directed against IgG of the animal species of the first antibody.
  • indirect assay, labeled antisera against the first antibody are added to the sample so as to produce the labeled antibody in situ.
  • the label used for either the first or second antibody is any detectable functionality that does not interfere with the binding of free VEGF 110+ or VEGF to the antibody.
  • suitable labels are those numerous labels known for use in immunoassay, including moieties that may be detected directly, such as fluorochrome, chemiluminscent, and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected.
  • radioisotopes 32 P, 14 C, 125 I, 3 H, and 131 I examples include the radioisotopes 32 P, 14 C, 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No.
  • luciferin 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, biotin/streptavidin, biotin/Streptavidin- ⁇ -galactosidase with MUG, spin labels, bacteriophage labels, stable free radicals, and the like.
  • HRP horseradish peroxidase
  • coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like may be used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. See, for example, U.S. Pat. Nos. 3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et al. Nature 144:945 (1962); David et al. Biochemistry 13:1014-1021 (1974); Pain et al. J. Immunol. Methods 40:219-230 (1981); and Nygren J. Histochem.
  • labels herein are fluorescent to increase amplification and sensitivity to 8 pg/ml, more preferably biotin with streptavidin- ⁇ -galactosidase and MUG for amplifying the signal.
  • a calorimetric label is used, e.g., where the detectable antibody is biotinylated and the detection means is avidin or streptavidin- peroxidase and 3,3′,5,5′-tetramethyl benzidine.
  • the amount of bound antibody is determined by removing excess unbound labeled antibody through washing and then measuring the amount of the attached label using a detection method appropriate to the label, and correlating the measured amount with the amount of free VEGF 110+ or VEGF in the biological sample.
  • the amount of color developed and measured will be a direct measurement of the amount of VEGF 110+ or VEGF present.
  • HRP is the label
  • the color is detected using the substrate 3,3′,5,5′-tetramethyl benzidine at 450 nm absorbance.
  • color or chemiluminiscence is developed and measured by incubating the immobilized capture reagent with a substrate of the enzyme. Then the amount of free VEGF 110+ or VEGF concentration is calculated by comparing with the color or chemiluminescence generated by the standard VEGF run in parallel.
  • kits are packaged combinations including the basic elements of:
  • the kit further comprises a solid support for the capture reagents, which may be provided as a separate element or on which the capture reagents are already immobilized.
  • the capture antibodies in the kit may be immobilized on a solid support, or they may be immobilized on such support that is included with the kit or provided separately from the kit.
  • the capture reagents are coated on a microtiter plate.
  • the detection reagent may be labeled antibodies detected directly or unlabeled antibodies that are detected by labeled antibodies directed against the unlabeled antibodies raised in a different species.
  • the label is an enzyme
  • the kit will ordinarily include substrates and cofactors required by the enzyme, and where the label is a fluorophore, a dye precursor that provides the detectable chromophore.
  • the kit may further comprise a detection means for the detectable antibodies, such as the labeled antibodies directed to the unlabeled antibodies, preferably in a fluorimetric-detected format.
  • the kit will ordinarily include substrates and cofactors required by the enzyme, where the label is a fluorophore, a dye precursor that provides the detectable chromophore, and where the label is biotin, an avidin such as avidin, streptavidin, or streptavidin conjugated to HRP or ⁇ -galactosidase with MUG.
  • the capture reagent is monoclonal antibody, preferably rodent, more preferably murine or rat, still more preferably murine, and most preferably MAb 5C3.
  • the detectable antibody is a biotinylated monoclonal antibody
  • the monoclonal antibody is rodent, more preferably murine or rat, still more preferably murine, yet still more preferably MAb A4.6. 1.
  • the capture reagent is immobilized in this kit.
  • the kit can contain multiple ELISA for comparison studies as described herein for detecting various forms of VEGF and VEGF 110+ .
  • the kit also typically contains instructions for carrying out the assay, and/or VEGF as an antigen standard (e.g., purified VEGF, preferably recombinantly produced VEGF, and VEGF110), as well as other additives such as stabilizers, washing and incubation buffers, and the like.
  • an antigen standard e.g., purified VEGF, preferably recombinantly produced VEGF, and VEGF110
  • other additives such as stabilizers, washing and incubation buffers, and the like.
  • VEGF vascular endothelial growth factor
  • mammalian cells available from Genentech, Inc., South San Francisco, Calif., and from those companies and processes described herein.
  • the components of the kit will be provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to provide for concentrations in solution of the reagents that substantially maximize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentration for combining with the sample to be tested.
  • VEGF Vascular endothelial growth factor
  • VEGF 165-206 ELISA detects VEGF 165 and longer isoforms but not VEGF 121 .
  • ELISA B detects VEGF110-206 ELISA detects VEGF 165 and isoforms, VEGF 121 and VEGF 110 .
  • ELISA C detects VEGF 165 and longer isoforms, VEGF 121 and VEGF fragments with molecular weight larger than VEGF 110 but not VEGF 110 (referred to herein as “VEGF 110+ ”).
  • VEGF 165 Genentech
  • VEGF 121 Progenitor 121
  • VEGF 8-109 Consisting of amino acids 8-109 of VEGF 165
  • truncated VEGF 121 R&D Systems, Minneapolis, Minn.
  • Truncated VEGF 121 has an intact N-terminus by mass spectrometry but has a mass of 26 KDa, consistent with truncation of approximately nine amino acids from the carboxy-terminus according to the manufacturer. It migrated between VEGF 110 and VEGF 121 when analyzed by SDS-PAGE under reducing conditions.
  • VEGF 110 was prepared by plasmin digestion of VEGF 165 (Keyt B A, et al.,: The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem. 271: 7788-7795 (1996)). The molecular weight measured by mass spectrometry was 25390, matching the theoretical mass of 25389. The concentration was determined using bicinchorinic acid method (Pierce, Rockford, Ill.). Molecular weights used for concentration calculation of VEGF 8-109 , VEGF 121 and VEGF 165 were 23.8, 28.9 and 38.2 KDa, respectively.
  • Monoclonal anti-VEGF antibodies A4.6.1, 3.5F8, 2E3 and 5C3 were generated by immunizing mice with VEGF 165 produced in CHO cells (Kim K J, et al.,: The vascular endothelial growth factor proteins: Identification of biologically relevant regions by neutralizing monoclonal antibodies. Growth Factors 7: 53-64 (1992)).
  • A673 cells (American Type Culture Collection,) were grown in 50:50 F12/DMEM, 2 mM L-glutamine and 5% FBS to 60% confluency and then in serum free medium (Genentech) till confluency.
  • VEGF was purified from the supernatants using an A4.6. 1-Sepharose column that was prepared with CNBr activated Sepharose (Amersham Biosciences, Piscataway, N.J.).
  • the column eluate and recombinant VEGF controls (0.2 ⁇ g per lane) were run on a 18% Tris-Glycine gels (Invitrogen, Carlsbad, Calif.) under reducing conditions and were blotted unto nitrocellulose. The blot was blocked with 0.5 M Tris-HCl, pH 7.5, 1.5 M NaCl, 50 mM EDTA, 0.5% Trition100 containing 3% bovine serum albumin and probed with 200 ng/ml of 3.5F8 or A4.6.1 followed by 2 ng/ml of goat anti-mouse Fc-HRP (Jackson ImmunoResearch). Signals were developed using SuperSignal West Dura (Pierce) and recorded on X-ray film.
  • VEGF ELISAs for measuring VEGF concentrations
  • VEGF165-206 ELISA a fluorometric ELISA A was used for measuring VEGF in samples.
  • the fluorimetric ELISA A used 3.5F8 for coat and biotinylated A4.6.1 followed by streptavidin- ⁇ galactosidase for detection and 4-methylumbelliferyl- ⁇ -D galactoside as the substrate (Rodriguez C R, et al.,: A sensitive fluorometric enzyme - linked immunosorbent assay that measures vascular endothelial growth factor 165 in human plasma. J Immunol Methods 219: 45-55 (1998)).
  • the VEGF 165 standards were 1-128 pg/mL, or 0.026-3.35 pM.
  • the colorimetric ELISA A used 3.5F8 for coat and biotinylated A4.6.1 for detection, following the protocol used for the ELISA C described below.
  • the VEGF 165 standards were 1.6-200 pg/mL.
  • ELISA B (VEGF110-206 ELISA) (previously named VEGF121-206 ELISA, Konecny G E, et al.,: Association between HER -2/ neu and Vascular Endothelial Growth Factor Expression Predicts Clinical Outcome in Primary Breast Cancer Patients. Clinical Cancer Research, 10: 1706-1716 (2004)): MaxiSorp 96-well microwell plates were coated with 0.5 ⁇ g/ml antibody A4.6.1 in 50 mM carbonate buffer, pH 9.6 at 100 ⁇ l/well at 4° C. overnight. Plates were washed after this step and between the subsequent room temperature incubation steps with PBS, pH 7.4, containing 0.05% polysorbate 20.
  • VEGF standards 1.56-200 pg/ml VEGF 165 or 0.0409-5.24 pM VEGF in twofold serial dilution
  • serially diluted samples minimum 1:10 dilution
  • PBS pH 7.4
  • bovine serum albumin 0.05% polysorbate 20
  • 5 mM EDTA 0.25% CHAPS
  • 0.2% bovine ⁇ -globulins Sigma, St.
  • VEGF vascular endothelial growth factor
  • streptavidin-HRP Amersham, Copenhagen, Denmark
  • biotinyl-tyramide ELAST ELISA amplification System, Perkin Elmer Life Sciences Inc., MA
  • the substrate TMB (3,3′,5,5′-tetramethyl benzidine) (Kirkegaard & Perry Laboratories) was added and the reaction was stopped by adding 1 M phosphoric acid. Absorbance was read at 450 nm on a Titertek stacker reader (ICN, Costa Mesa, Calif.). The titration curves were fit using a four-parameter regression curve-fitting program (KaleidaGraph, Synergy software, Reading, Pa.). Data points which fell in the range of the standard curve were used for calculating the putative VEGF concentrations in the samples.
  • ELISA C (VEGF121-206 ELISA): Microwell plates were coated with 1 ⁇ g/ml anti-VEGF 5C3 antibody and blocked as described above. VEGF standards (4.00-512 pg/ml VEGF 165 or 0.105-13.4 pM VEGF in 2-fold serial dilution) and serially diluted samples in sample buffer were added to the plates. The plates were incubated for 2 h. Bound VEGF was detected by adding biotinylated A4.6.1 followed by streptavidin-HRP and TMB as the substrate. Plates were read and data were analyzed as described above. The recovery of 4.00-512 pg/ml VEGF 165 in 10% plasma was 77-101% after subtracting 1.6 pg/ml putative endogenous VEGF in the 10% plasma used for this study.
  • VEGF ELISAs The previously described ELISA A uses 3.5F8 for coat and biotinylated A4.6.1 for detection (Rodriguez C R, et al.,: A sensitive fluorometric enzyme - linked immunosorbent assay that measures vascular endothelial growth factor 165 in human plasma. J Immunol Methods 219: 45-55, 1998). It detects VEGF165 (VEGF 165 ) but not VEGF121(1) (VEGF 121 (1)), which is from R&D systems and missing approximately 9 amino acids from the carboxy-terminus, and VEGF121(2) (VEGF 121 (2)), which is from PeproTech ( FIG. 1A ).
  • 3.5F8 binds VEGF 165 but not VEGF 121 by BIAcore.
  • A4.6. 1 binds to the receptor binding domain (Kim K J, et al.,: The vascular endothelial growth factor proteins: Identification of biologically relevant regions by neutralizing monoclonal antibodies. Growth Factors 7: 53-64, 1992) that is present in all isoforms and in VEGF 110 .
  • 3.5F8 likely binds near amino acids 116 and 118, which are not present in VEGF 121 .
  • 5C3 likely binds near amino acids 111-113, which are not present in VEGF 110 ( FIG. 3 ).
  • ELISA A can likely detect VEGF isoforms which contained VEGF 165 sequences including VEGF 183 , VEGF 189 and VEGF 206 (see, e.g., Stimpfl M, et al.,: Vascular Endothelial growth factor splice variants and their prognostic value in breast and ovarian cancer. Clinical Cancer Research 8: 2253-2259, 2002).
  • ELISA B (previously named VEGF121-206 ELISA, Konecny G E, et al., Association between HER -2/ neu and Vascular Endothelial Growth Factor Expression Predicts Clinical Outcome in Primary Breast Cancer Patients.
  • A4.6.1 and 2E3 bind to the receptor binding domain that is present in all three molecules. See, e.g., Kim K J, et al.
  • the vascular endothelial growth factor proteins Identification of biologically relevant regions by neutralizing monoclonal antibodies. Growth Factors 7:53-64 (1992); and, Muller Y A, et al. Vascular endothelial growth factor: Crystal structure and functional mapping of the kinase domain receptor binding site. Proc Natl Acad Sci USA 94:7192-7197 (1997). Other antibodies that bind in these regions can also be used.
  • This ELISA detects VEGF 165 , VEGF 121 , truncated VEGF 121 (missing approximately 9 amino acids from the carboxy-terminus), VEGF 110 and VEGF 8-109 equally well ( FIG. 1B ).
  • This ELISA can detect total VEGF, including fragments larger than VEGF 110 generated by matrix metalloproteinase digestion.
  • ELISA C described herein, which uses 5C3 for coat and biotinylated A4.6.1 for detection, detects VEGF 165 , VEGF 121 , and truncated VEGF 121 equally well but does not detect VEGF 110 or VEGF 8-109 ( FIG. 1 , C).
  • 5C3 binds VEGF 121 but not VEGF 8-109 by BIAcore. This ELISA can detect all the VEGF molecules detected by the VEGF 110-206 except VEGF 110 and smaller fragments.
  • ELISA A, ELISA B and ELISA C were 10, 16 and 40 pg/ml VEGF 165 (or 0.26, 0.41 and 1.05 pM for different VEGF isoforms and fragments) for VEGF in samples using a minimum 1:10 dilution, respectively.
  • ELISA B and ELISA C were reproducible (Table 1 & 2).
  • ELISA B and ELISA C were specific to VEGF (VEGF-A).
  • Insulin-like growth factor 1 growth hormone, recombinant nerve growth factor, tumor necrosis factor (Genentech), platelet-derived growth factor AB, placenta growth factor, transforming growth factor ⁇ 1 (R&D Systems) (up to 200 ng/ml) only gave background signals. Heparin (Leo Laboratories, Bucks, UK and Dublin, Ireland) (up to 100 U/ml) did not have a significant effect on the assay.
  • ELISA B (VEGF 110-206 ELISA): The standard range was 1.56-200 pg/ml VEGF 165 (0.0409-5.24 pM VEGF) in buffer. The OD ratio of 1.56 pg/ml standard relative to the blank was 1.37 ⁇ 0.11. CV is coefficient of variation. Mean Control a (pg/ml) Inter % CV Intra % CV Low 3.07 17.7 13.5 Middle 38.0 9.50 6.54 High 127 9.11 6.95 a The middle and high controls were made by spiking recombinant VEGF 165 into human EDTA plasma. The low control was made by spiking VEGF 165 into 70% plasma since plasma contained endogenous VEGF. Controls were diluted 1:10 and assayed in duplicate in 34 independent assays.
  • VEGF 121-206 ELISA The standard range was 4.00-512 pg/ml VEGF 165 (0.105-13.4 pM VEGF). The OD ratio of 4 pg/ml standard relative to the blank was 2.72 ⁇ 0.37. CV is coefficient of variation. Mean Control a (pg/ml) Inter % CV Intra % CV Low 3.28 20.6 8.35 Middle 11.7 6.56 2.39 High 56.5 2.57 1.37 a The controls were made by spiking recombinant VEGF 165 into human EDTA plasma. They were diluted 1:10 and assayed in duplicate in 15 independent assays.
  • VEGF in conditioned media of cell lines Conditioned media from six stable CHO clones transfected with VEGF 165 cDNA (Meng et al., 2000) were measured by the three ELISAs, which used non-glycosylated VEGF produced in E. coli as standard. Glycosylated recombinant VEGF 165 in conditioned media from six stable CHO clones gave very similar concentrations in the three ELISAs. Concentrations measured by ELISA B were 28, 63, 64, 43, 3.8 and 3.2 nM, respectively. Ratios of VEGF concentrations measured by ELISA A and ELISA C compared to those by ELISA B were 0.90 ⁇ 0.08 and 1.08 ⁇ 0.10, respectively. Therefore, the three ELISAs quantitated glycosylated VEGF equally well and there was little proteolysis of VEGF 165 under the culture conditions.
  • VEGF concentrations in A673 cell conditioned medium measured by ELISA A, ELISA B and ELISA C were 0.15, 0.29 and 0.24 nM VEGF, respectively.
  • the concentration measured by ELISA A was lower, indicating VEGF 121 was present.
  • VEGF was purified from conditioned medium using an A4.6.1 affinity column and analyzed by protein blotting, two bands, likely glycosylated and non-glycosylated VEGF 165 were detected by 3.5F8.
  • the lower band had the same mobility as the purified VEGF 165 produced in E. coli ( FIG. 2 , left). N-glycanase treatment converted the upper band to the lower band.
  • VEGF concentrations in conditioned media from breast cell lines SK-BR-3, BT-474, T-47D and MCF-7 measured by ELISA B were 3.6, 16, 13, and 13 pM, respectively.
  • Ratios of VEGF concentrations measured by ELISA A to those by ELISA B were 0.49, 0.42, 0.43 and 0.38 (or 49%, 42%, 43% or 38%), respectively, in agreement with 43, 35, 40 and 41% of VEGF 165 expression in these respective cell lines (Stimpfl M, et al.,: Vascular Endothelial growth factor splice variants and their prognostic value in breast and ovarian cancer. Clinical Cancer Research 8: 2253-2259, 2002).
  • Ratios of VEGF concentrations measured by ELISA C to those by ELISA B were 1.1-1.2 for these cell lines, indicating that little VEGF 110 was present.
  • VEGF concentrations in conditioned media from ovarian cell lines ES-2, OVCAR-3 and SK-OV-3 measured by ELISA B were 32, 11 and 20 pM, respectively.
  • Ratios of VEGF concentrations measured by ELISA A to those by ELISA B were 0.24, 0.20, and 0.32 (or 24%, 20% and 32%), respectively, compared to 38, 42 and 24% of VEGF 165 expression in these respective cell lines (Stimpfl et al., supra).
  • Ratios of VEGF concentrations measured by ELISA C to those by ELISA B were 0.64-0.79 for these cell lines, indicating VEGF 110 (or smaller fragments) may be present.
US11/866,560 2006-10-04 2007-10-03 ELISA for VEGF Abandoned US20080227119A1 (en)

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US13/660,563 US9146245B2 (en) 2006-10-04 2012-10-25 ELISA for VEGF
US14/814,139 US9625462B2 (en) 2006-10-04 2015-07-30 Elisa for VEGF
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