Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • 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
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/08—Vasodilators for multiple indications
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to the use of VEGF, and variants thereof, VEGF receptor agonists, and other agents to modulate the endothelial nitric oxide synthase (eNOS) activity.
• eNOS endothelial nitric oxide synthase
• modulation of eNOS activity is used to treat or prevent mammalian diseases or disorders associated with vascular endothelial cell dysfunction.
• the two ma or cellular components of the vasculature are the endothelial and smooth muscle cells.
• the endothelial cells form the lining of the inner surface of all blood vessels and constitute a nonthrombogenic interface between blood and tissue.
• endothelial cells are an important component for the development of new capillaries and blood vessels.
• endothelial cells proliferate during the ang ogenesis, or neovascularization, associated with tumor growth and metastasis, as well as a variety of non-neoplastic diseases or disorders.
• polypeptides reportedly induce the proliferation of endothelial cells.
• FGF basic and acidic fibroblast growth factors
• PD-ECGF platelet-derived endothelial cell growth factor
• VEGF vascular endothelial growth factor
• VEGF has been reported as a key regulator of angiogenesis and vasculogenesis .
• VEGF is unique in its high specificity for endothelial cells. It is important not only for normal physiological processes such as wound healing, the female reproductive tract, bone/cartilage formation and embryonic formation, but also during the development of conditions or diseases that involve pathological angiogenesis, for example, tumor growth, age-related macular degeneration (AMD) and diabetic retinopathy.
• AMD age-related macular degeneration
• VEGF In addition to being an angiogenic factor in angiogenesis and vasculogenensis, VEGF, as a pleiotropic growth factor, exhibits multiple biological effects in other physiological and pathological processes, such as endothelial cell survival, vessel permeability and vasodilation, monocyte chemotaxis and calcium influx. Ferrara and Davis-Smyth (1997), supra . VEGF was first identified in media conditioned by bovine pituitary follicular or folliculostellate cells. Biochemical analyses indicate that bovine VEGF is a di e ⁇ c protein with an apparent molecular mass of approximately 45,000 Daltons and with an apparent mitogenic specificity for vascular endothelial cells. DNA encoding bovine VEGF was isolated by screening a cDNA library prepared from such cells, using oligonucleotides based on the ammo-terminal ammo acid sequence of the protein as hybridization probes.
• Human VEGF was obtained by first screening a cDNA library prepared from human cells, using bovine VEGF cDNA as a hybridization probe. One cDNA identified thereby encodes a 165-am ⁇ no acid protein having greater than 95% homology to bovine VEGF; this 165-am ⁇ no acid protein is typically referred to as human VEGF (hVEGF) or VEGF 165 .
• hVEGF human VEGF
• the mitogenic activity of human VEGF was confirmed by expressing the human VEGF cDNA in mammalian host cells. Media conditioned by cells transfected with the human VEGF cDNA promoted the proliferation of capillary endothelial cells, whereas control cells did not. [ See Leung et al . , Science, 246:1306 (1989)].
• VEGF vascular endothelial cell growth factor
• VEGF 121 is a soluble mitogen that does not bind hepann; the longer forms of VEGF bind heparin with progressively higher affinity.
• the hepa ⁇ n-binding forms of VEGF can be cleaved m the carboxy terminus by plasmin to release a diffusible form(s) of VEGF.
• Amino acid sequencing of the carboxy terminal peptide identified after plasmm cleavage is Argno-Alam.
• VEGF Ammo terminal "core” protein, VEGF (1-110) isolated as a homodimer, binds neutralizing monoclonal antibodies (such as the antibodies referred to as 4.6.1 and 3.2E3.1.1) and soluble forms of FLT-1 and KDR receptors with similar affinity compared to the intact VEGF 165 homodimer.
• Certain VEGF-related molecules have also been identified. Ogawa et al . described a gene encoding a polypeptide (called VEGF-E) with about 25% amino acid identity to mammalian VEGF.
• the VEGF-E was identified n the genome of Orf virus (NZ-7 strain) , a parapoxvirus that affects sheep and goats and occasionally, humans, to generate lesions with angiogenesis.
• VEGF vascular endothelial growth factor
• Binding studies were also reported. A competition experiment was conducted by incubating cells that overexpressed either the KDR receptor or the FLT-1 receptor with fixed amounts of 125 I-labeled human VEGF or VEGF-E and then adding increasing amounts of unlabeled human VEGF or VEGF-E. The investigators reported that VEGF-E selectively bound KDR receptor as compared to FLT-1. [Ogawa et al . J. Biological Chem . 273:31273-31281 (1998)].
• VEGF-E vascular endothelial growth factor-E
• the VEGF-E molecule reported by Meyer et al . was identified in the genome of Orf virus strain D1701. In vi tro, the VEGF-E was found to stimulate release of tissue factor and stimulate proliferation of vascular endothelial cells. In a rabbit m vivo model, the VEGF-E stimulated angiogenesis in the rabbit cornea. Analysis of the binding properties of the VEGF-E molecule reported by Meyer et al . , in certain assays revealed the molecule selectively bound to the KDR receptor as compared to the FLT-1 receptor.
• VEGF-B a protein referred to as "VEGF-B” selectively binds FLT-1.
• the investigators disclose a mutagenesis experiment wherein the Asp63, Asp64, and Glu67 residues in VEGF-B were mutated to alanine residues. Analysis of the binding properties of the mutated form of VEGF-B revealed that the mutant protein exhibited a reduced affinity to FLT-1.
• VEGF contains two sites that are responsible respectively for binding to the KDR (kinase domain region) and FLT-1 (FMS-like tyrosine kinase) receptors. These receptors are believed to exist primarily on endothelial (vascular) cells.
• KDR kinase domain region
• FLT-1 FLT-1-like tyrosine kinase receptors.
• KDR kinase domain region
• FLT-1 FLT-1 receptor-like tyrosine kinase
• VEGF production increases in cells that become oxygen-depleted as a result of, for example, trauma and the like, thereby allowing VEGF to bind to the respective receptors to trigger the signaling pathways that give rise to a biological response.
• the binding of VEGF to such receptors may lead to increased vascular permeability, causing cells to divide and expand to form new vascular pathways - i.e., vasculogenesis and angiogenesis.
• vascular permeability causing cells to divide and expand to form new vascular pathways - i.e., vasculogenesis and angiogenesis.
• VEGF-induced signaling through the KDR receptor is responsible for the mitogenic effects of VEGF and possibly, to a large extent, the angiogenic activity of VEGF. [Waltenberger et al . , J. Biol . Chem . , 269:26988-26995 (1994)].
• the biological role(s) of FLT-1 is less well understood.
• the KDR receptor has been found to bind VEGF predominantly through the sites on a loop which contains arginme (Arg or R) at position 82 of VEGF, lysine (Lys or K) at position 84, and histidine (His or H) at position 86.
• the FLT-1 receptor has been found to bind VEGF predominantly through the sites on a loop which contains aspartic acid (Asp or D) at position 63, glutamic acid (Glu or E) at position 64, and glutamic acid (Glu or E) at position 67. [Keyt et al . , J. Biol . Chem . , 271:5638-5646 (1996)].
• the wild type VEGF protein has been used as the starting point for introduction of mutations in specific receptor-bindmg sites, in attempts to create VEGF variants selectively bind to one receptor such as KDR. Keyt et al . , supra ; Shen et al. (1998) J. Biol . Chem . 273:29979-29985.
• the resulting VEGF variants showed moderate receptor selectivity. More recently, based on the crystal structure of VEGF and functional mapping of the KDR binding site of VEGF, it has further been found that VEGF engages KDR receptors using two symmetrical binding sites located at opposite ends of the molecule.
• Each site is composed of two "hot spots” for binding that consist of residues from both subunits of the VEGF homodimer. Muller et al . , Structure, 5:1325- 1338 (1997). Two of these binding determinants are located within the dominant hot spot on a short, 3-stranded beta-sheet that is conserved n transforming growth factor beta2 (TGF-beta) and platelet-derived growth factor (PDGF) .
• TGF-beta n transforming growth factor beta2
• PDGF platelet-derived growth factor
• NO endothelium-derived nitric oxide
• eNOS endothelial NO synthase
• NO is produced from conversion of L-arginme to citrullme by NO-synthase, an enzyme which consists of 3 isoforms denominated endothelial nitric oxide synthase (eNOS), nducible NOS (iNOS) and neuronal NOS (nNOS).
• eNOS denominated endothelial nitric oxide synthase
• iNOS nducible NOS
• nNOS neuronal NOS
• VEGF has been shown to induce rapid release of NO from rabbit, pig, bovine and human vascular endothelial cells. (see, e.g., vanderZee et al., Circula tion, 95 : 1030-1037
• VEGF was found to stimulate human endothelial cells to grow n a NO-dependent manner and promote the NO-dependent formation of vessel-like structures m the 3-D collagen gel model (Papapetropoulis et al., supra ) .
• eNOS inhibitors were reported to inhibit VEGF- prised mitogenic and angiogenic effects. (Papapetropoulis et al . , supra ) .
• mactivation of eNOS expression impaired VEGF- mduced angiogenesis in an eNOS knockout mouse model. (Murohara et al., J. Cl in . Inves t .
• VEGF induces vasodilation in vitro in a dose- dependant fashion and produces transient tachicardia, hypotension and a decrease in cardiac output when injected intravenously n conscious, instrumented rats.
• Such acute hemodynamic effects appear to be caused by a decrease in venous return, mediated primarily by endothelial cell-derived NO. Yang et al., supra ; Hariawala et al., J. Surg. Res . , 63 : 11-82 (1996) . Wu et al . , Am. J. Physiol .
• mice lacking the eNOS gene showed impaired endothelium-dependent vasodilation, angiogenesis, and hypertension (Murohara et.al., 1998 J. Clm . Inves t . 101:2567-2578; Yang et . Al . , 1996, supra ) , and over-expression of eNOS in mice by gene transfer was shown to increase nitric oxide production and significantly attenuated MAP and neomtima formation (Sase et . al., supra ; Drummond and Harrison, 1998, J. Cl m . Inves t . 102:2033-2044; Ohashi et.al., 1998 J. Cl m . Inves t . 102:2061-2071) .
• Up-regulation of eNOS expression by physiological or pharmacological approaches may provide a useful therapeutic approach to the treatment of diseases associated with endothelial cell dysfunction, for example, by increasing the production and sustained release of endogenous NO.
• the present mvention is based on the observation that prolonged VEGF treatment effectively up-regulates eNOS expression and activity, thereby enhancing sustained nitric oxide production, and that the eNOS upregulation by VEGF requires the VEGF-KDR receptor, activation of the KDR-associated tyrosine kinase (TK) and a downstream PKC-dependent pathway. Modulation of eNOS expression/activity is clinically useful, for example, in the treatment of disorders characterized by endothelial cell dysfunction including eNOS dysfunction and/or defects in nitric oxide production.
• the invention provides as claimed a method of treating disorders in mammals wherein nitric oxide is an important regulator, such as hypertension, diabetes, atherosclerosis, thrombosis, angina and heart failure, by modulating eNOS expression or activity, for example, by the administration of an effective amount of VEGF, a receptor- selective VEGF variant, or an agent or molecule which acts as an agonist of VEGF receptor activation.
• nitric oxide is an important regulator, such as hypertension, diabetes, atherosclerosis, thrombosis, angina and heart failure
• eNOS expression or activity for example, by the administration of an effective amount of VEGF, a receptor- selective VEGF variant, or an agent or molecule which acts as an agonist of VEGF receptor activation.
• the invention provides a method for protecting a mammal from conditions associated with endothelium dysfunction by providing VEGF or VEGF receptor agonist.
• the invention provides a method of stimulating a sustained production of endogenous NO in an endothelial cell by providing a receptor selective VEGF variant.
• a KDR selective VEGF variant is used for specifically binding the KDR receptor, which n turn activates the pathway leading to the upregulation of eNOS and sustained NO production.
• Figure 1A-1D depict the eNOS upregulation activity of VEGF.
• Figure 1A is a representative Western blot showing inducement of a time-dependent increase in eNOS expression by VEGF treatment. The arrow indicates eNOS.
• Figure 1C is a bar diagram illustrating the chronic effect of VEGF on eNOS activity, as expressed by the activity ratio of VEGF-treated cells (after 2-day VEGF exposure) over untreated controls (at Day 0)
• Figure ID is a bar diagram illustrating the acute effect of VEGF on eNOS activity, as expressed by the percentage of activity increases of the VEGF-treated cells ( treated for 0 to 60 mm) over the untreated control.
• Figure 2 is a graph showing the fold increase in eNOS protein following treatment with VEGF, L-NAME, SNAP (alone) or a combination of VEGF with L-NAME or SNAP for 0-5 days.
• Figures 3A-3C depict the VEGF receptor specificities for eNOS regulation.
• Figure 3A is a bar diagram showing that VEGF 165 , VEGFuo, and a KDR selective binding variant ("KDR-sel") induce eNOS up-regulation, whereas FLT-1 receptor selective variants ("FLT-1-sel” and PLGF) did not.
• Figure 3B is a Western blot showing VEGF-induced eNOS up-regulation in KDR-transfected PAE cells but not in FLT-1 containing PAE cells.
• Figure 3C is a Western blot showing dose-dependent prevention of VEGF-induced eNOS up-regulation by KDR tyrosine kinase selective inhibitor SU1498.
• Figures 4A-4B depict the inhibition of VEGF modulation on eNOS.
• Figure 4A is a Western blot showing the effects on eNOS expression in ACE cells treated with VEGF in combination with various specific inhibitors of tyrosine kinase, PLC-gamma or PKC.
• Figure 4B is a bar diagram illustrating analysis of eNOS levels by densitometry .
• Figures 5A-5C depict PKC activity n eNOS modulation by VEGF.
• Figure 5A is a Western blot showing that VEGF treatment resulted in a rapid redistribution of PKC- alpha, gamma and epsilon from cytosolic to membrane fractions.
• Figure 5B shows that VEGF increased PKC activity.
• Figure 5C shows that activation of PKC with PMA increased eNOS levels.
• Figure 6 is a bar diagram illustrating effects of various angiogenic factors on eNOS expression.
• the histograms show the fold increase in eNOS protein in growth factor treated cells normalized to untreated control cells .
• Figure 7 depicts the ELISA assay titration curve for the native VEGF (8-109) .
• Figure 8 depicts the KIRA assay titration curve for the native VEGF (8-109) .
• Figure 9 depicts the HUVEC proliferation assay titration curve for the native VEGF (8-109) .
• Figure 10 is a Western blot showing the eNOS expression levels in endothelial cells treated by VEGF 165 , VEGF 110 , two KDR selective VEGF variants (KDR-full and KDR-short), or a Fit selective variant (Fit-short).
• Figure 11 is a Western blot data showing the m vivo eNOS expression affected by a VEGF antagonist (MuFlt-IgG) .
• Figure 12 is a Western blot showing the phosphotyros e levels of eNOS in endothelial cells treated with VEGF for different time courses.
• VEGF vascular endothelial cell growth factor
• VEGF native vascular endothelial cell growth factor
• 121-, 189-, and 206- am o acid vascular endothelial cell growth factors as described by Leung et al . , Science, 246:1306 (1989), and Houck et al . , Mol . Endocrm . , 5:1806 (1991), together with the naturally occurring allelic and processed forms thereof.
• VEGF and "native VEGF” are also used to refer to truncated forms of the polypeptide comprising amino acids 8 to 109 or 1 to 109 of the 165-am ⁇ no acid human vascular endothelial cell growth factor. Reference to any such forms of VEGF may be identified in the present application, e.g., by "VEGF (8-109),” “VEGF (1-109)” or “VEGF 165 .”
• the amino acid positions for a "truncated” native VEGF are numbered as indicated in the native VEGF sequence. For example, ammo acid position 17 (methionme) in truncated native VEGF is also position 17 (methionme) in native VEGF.
• the truncated native VEGF has binding affinity for the KDR and FLT-1 receptors comparable to native VEGF.
• VEGF variant refers to a VEGF polypeptide which includes one or more ammo acid mutations in the native VEGF sequence and preferably, has selective binding affinity for either the KDR receptor or the FLT-1 receptor.
• the VEGF variant includes one or more am o acid mutations in any one of positions 17 to 25 and/or 63 to 66 of the native VEGF sequence.
• the one or more ammo acid mutations include amino acid substitution (s) .
• VEGF variants include one or more ammo acid mutations and exhibit binding affinity to the KDR receptor which is equal or greater than the binding affinity to the KDR receptor by native VEGF, and preferably, exhibit less binding affinity to the FLT-1 receptor than the binding affinity of native VEGF for FLT-1.
• binding affinity of the VEGF variant for the KDR receptor is approximately equal (unchanged) or greater than (increased) as compared to native VEGF, and the binding affinity of the VEGF variant for the FLT-1 receptor is less than or nearly eliminated (as compared to native VEGF)
• the binding affinity of the VEGF variant is "selective" for the KDR receptor.
• the VEGF variants include one or more ammo acid mutations and exhibit binding affinity to the FLT-1 receptor which is equal or greater than the binding affinity to the FLT-1 receptor by native VEGF, and preferably, exhibit less binding affinity to the KDR receptor than the binding affinity of native VEGF for KDR.
• binding affinity of the VEGF variant for the FLT-1 receptor is approximately equal (unchanged) or greater than (increased) as compared to native VEGF, and the binding affinity of the VEGF variant for the KDR receptor is less than or nearly eliminated (as compared to native VEGF)
• the binding affinity of the VEGF variant is "selective" for the FLT-1 receptor.
• Preferred VEGF variants of the mvention will have at least 10- fold less binding affinity to FLT-1 receptor (as compared to native VEGF) , and even more preferably, will have at least 100-fold less binding affinity to FLT-1 receptor (as compared to native VEGF) .
• the respective binding affinity of the VEGF variant for KDR or FLT-1 may be determined by ELISA, RIA, and/or BIAcore assays, known in the art and described further in the Examples below.
• Preferred VEGF variants of the invention will also exhibit activity in KIRA assays reflective of the capability to induce phosphorylation of the KDR receptor.
• Preferred VEGF variants of the invention will additionally or alternatively induce endothelial cell proliferation (which can be determined by known art methods such as the HUVEC proliferation assay) . Induction of endothelial cell proliferation is presently believed to be the result of signal transmission by the KDR receptor.
• VEGF variants described herein For purposes of shorthand designation of VEGF variants described herein, it is noted that numbers refer to the am o acid residue position along the am o acid sequence of the putative native VEGF (provided in Leung et al . , supra and Houck et al . , supra . ) . Amino acid identification uses the single-letter alphabet of amino acids, i.e.,
• VEGF receptor refers to a cellular receptor for VEGF, ordinarily a cell-surface receptor found on vascular endothelial cells, as well as fragments and variants thereof which retain the ability to bind VEGF (such as fragments or truncated forms of the extracellular domain) .
• VEGF receptor is the fms-like tyrosine kinase (FLT or FLT-1), a transmembrane receptor in the tyrosine kinase family.
• FLT-1 FLT-1 receptor
• the full length FLT-1 receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain with tyrosine kinase activity.
• the extracellular domain is involved in the binding of VEGF, whereas the intracellular domain is involved in signal transduction.
• Another example of a VEGF receptor is the KDR receptor (also referred to as FLK-1) .
• the term "KDR receptor" used in the application refers to the VEGF receptor described, for instance, by Matthews et al . , Proc . Na t . Acad. Sci .
• a receptor "agonist” is an agent that has affinity to and activate a receptor normally activated by a naturally occurring ligand, thus triggering a biochemical response.
• the receptor activation capability of the agonist will be at least qualitatively similar (and may be essentially quantitatively similar) to a native ligand of the receptor.
• Non-limitmg examples of a receptor agonist include ligand variant, antibody against the receptor and antibody against the receptor-ligand complex.
• 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 antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
• the monoclonal antibodies herein specifically include "chime ⁇ c" antibodies n 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. Patent No. 4,816,567; and Morrison et al . , Proc. Na tl . Acad. Sci . USA 81:6851-6855 (1984)).
• a "human antibody” is one which possesses an ammo acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein.
• This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-bindmg residues.
• Treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down
• Those m need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
• a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
• a therapeutically effective amount of the antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual.
• a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects.
• a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
• Chronic administration refers to administration of the agent (s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. Intermittent administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
• 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, cats, cows, horses, sheep, pigs, etc. Preferably, the mammal is human.
• Administration "in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
• Pathologic conditions and disorders "associated with” NO or “characterized by” eNOS dysfunction and/or wherein NO is an "important regulator” are those conditions and disorders where nitric oxide insufficiency or excess s correlated with disease. Such disorders and conditions include, for example, hypertension, diabetes, thrombosis, angina, atherosclerosis, and heart failure, wherein nitric oxide levels in mammalian cells or tissues are present in insufficient quantities as compared to normal or healthy mammalian cells or tissues. Further applications in which the use of VEGF will be beneficial include the use of VEGF to increase eNOS or NO production prior to, concurrent with or subsequent to angioplasty to prevent restenosis or neointima formation.
• hypertension or “hypertensive condition” as used herein refer to a physiological state or syndrome in mammals typically characterized by increased peripheral vascular resistance or cardiac output, or both.
• “hypertension” or “hypertensive condition” may optionally be indicated by blood pressure measurements equal to or greater than approximately 140 mm Hg systolic and approximately 90 mm Hg Hg diastolic. Hypertension is further characterized in Oates,
• eNOS activity is used in a broad sense and refers to the ability to induce or enhance, inhibit or decrease, or maintain eNOS protein expression and/or activity.
• the invention provides methods for treating a NO associated disorder or condition such as hypertension, diabetes, thrombosis, angina, heart failure or atherosclerosis.
• a NO associated disorder or condition such as hypertension, diabetes, thrombosis, angina, heart failure or atherosclerosis.
• Applicants have observed that some coronary artery disease patients (humans) treated with rhVEGF (either by mtracoronary or intravenous infusion administration at 0.05 microgram/Kg body weight/minute) can have a dose-rate dependent reduction in mean arterial pressure. This type of reduction in mean arterial pressure was acute and typically was observed during the first 20 minutes of infusion.
• some cancer patients humans
• recombinant humanized monoclonal antibodies against VEGF can have a dose-dependent increase in mean arterial pressure.
• VEGF vascular homeostasis
• VEGF or molecules modulating VEGF receptor activation, as described herein may be employed to treat conditions or disorders associated with NO or eNOS dysfunction.
• the invention also provides methods for protecting a mammalian subject from conditions associated with endothelium dysfunction such as thrombosis.
• endothelium dysfunction such as thrombosis.
• VIVA clinical trial VEGF in Ischemia for Vascular Angiogenesis
• VEGF treated angina patients show trends of improvement compared to placebo group, in such measures as angina class, angina frequency, and treadmill times at a prolonged time point.
• the VEGF or VEGF receptor agonist of the invention provides important vascular protective effects through up-regulatmg eNOS and NO in endothelial cells.
• the prolonged treatment of target subject with VEGF or VEGF receptor agonist according to the invention provides chronic effects on eNOS upregulation and sustained production of NO, which are more beneficial for therapeutic treatments or prophylactic measures wherein a sustained level of NO is desired.
• the methods of the invention are also applicable in treatment or prevention wherein the patients currently rely on exogenous nitrate sources, such as angina patients. Luscher TF (1992) Br. J. Clm . Pharmacol . 34 Suppl . 1.29S-35S.
• the invention further provides methods of using a KDR selective VEGF variant for stimulating a sustained production of endogenous NO in an endothelial cell.
• Native VEGF is a pleiotropic growth factor having multiple biological effects in regulating physiological and pathological vascular functions. When used in vitro or in vivo, native VEGF may cause unwanted adverse effects in addition to the targeted function (s), a problem often complicating the therapeutic applications of VEGF. Accordingly, the invention provides methods of using the receptor-selective variants as alternative therapeutic agents that may have fewer side effects than the native VEGF protein.
• the VEGF or VEGF receptor agonist of the invention is capable of upregulatmg the expression level of eNOS in an endothelial cell.
• the eNOS expression level can be upregulated by increasing the transcription of the eNOS gene, or alternatively, by preventing the degradation of the transcribed eNOS mRNAs, or the combination of both.
• the VEGF or VEGF receptor agonist of the invention is capable of upregulatmg the activity of endogenous eNOS. It has been shown that a number of proteins are associated with eNOS and regulate the eNOS activity via protein-protein interactions. For example, it has been shown that caveolm and PLC- ⁇ decrease the eNOS activity, whereas HSP-70 and HSP-90 increase the eNOS activity, when associated with eNOS.
• VEGF treatment induces dissociation of caveolm and PLC- ⁇ from eNOS and increase association of eNOS to HSP-70 and HSP-90. Furthermore, prolonged VEGF treatment is shown to reduce the phosphotyrosme level of eNOS, another indication of eNOS activation (Example 14).
• VEGF and VEGF variants for use in the disclosed methods may be prepared by a variety of methods well known in the art.
• the VEGF employed in the methods of the present invention comprises recombinant VEGF 165 .
• Ammo acid sequence variants of VEGF can be prepared by mutations in the VEGF DNA. Such variants include, for example, deletions from, insertions into or substitutions of residues within the ammo acid sequence shown in Leung et al . , supra and Houck et al . , supra . Any combination of deletion, insertion, and substitution may be made to arrive at the final construct having the desired activity.
• the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure [ see EP 75,444A].
• the VEGF variants optionally are prepared by site-directed mutagenesis of nucleotides in the DNA encoding the native VEGF or phage display techniques, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
• the mutation per se need not be predetermined.
• random mutagenesis may be conducted at the target codon or region and the expressed VEGF variants screened for the optimal combination of desired activity.
• Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well-known, such as, for example, site-specific mutagenesis.
• VEGF variants described herein are preferably achieved by phage display techniques, such as those described in the Examples .
• the mutated protein region may be removed and placed in an appropriate vector for protein production, generally an expression vector of the type that may be employed for transformation of an appropriate host.
• Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably 1 to 10 residues, and typically are contiguous.
• Ammo acid sequence insertions include amino- and/or carboxyl-termmal fusions of from one residue to polypeptides of essentially unrestricted length as well as intrasequence insertions of single or multiple amino acid residues.
• Intrasequence insertions i.e., insertions within the native VEGF sequence
• An example of a terminal insertion includes a fusion of a signal sequence, whether heterologous or homologous to the host cell, to the N- terminus to facilitate the secretion from recombinant hosts.
• Additional VEGF variants are those in which at least one ammo acid residue in the native VEGF has been removed and a different residue inserted in its place. Such substitutions may be made in accordance with those shown in Table 1.
• Lys K arg; gin; glu
• Changes in function or lmmunological identity may be made by selecting substitutions that are less conservative than those in Table 1, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
• substitutions that in general are expected to produce the greatest changes in the VEGF variant properties will be those in which (a) glycine and/or prolme (P) is substituted by another ammo acid or is deleted or inserted; (b) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) a cysteine residue is substituted for (or by) any other residue; (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) a residue having an electronegative charge, e.g., glutamyl or aspartyl; (e) a residue having an electronegative side chain is substituted for (or by) a residue having an electropositive charge
• a phage display-selected VEGF variant may be expressed in recombinant cell culture, and, optionally, purified from the cell culture.
• the VEGF variant may then be evaluated for KDR or FLT-1 receptor binding affinity and other biological activities, such as those disclosed in the present application.
• the binding properties or activities of the cell lysate or purified VEGF variant can be screened in a suitable screening assay for a desirable characteristic.
• a change in the immunological character of the VEGF variant as compared to native VEGF, such as affinity for a given antibody may be desirable. Such a change may be measured by a competitive-type lmmunoassay, which can be conducted in accordance with techniques known in the art.
• the respective receptor binding affinity of the VEGF variant may be determined by ELISA, RIA, and/or BIAcore assays, known in the art and described further m the Examples below.
• Preferred VEGF variants of the invention will also exhibit activity in KIRA assays (such as described n the Examples) reflective of the capability to induce phosphorylation of the KDR receptor.
• Preferred VEGF variants of the invention will additionally or alternatively induce endothelial cell proliferation (which can be determined by known art methods such as the HUVEC proliferation assay in the Examples) .
• the VEGF variants described in Keyt et al., J. Biol . Chem. , 271 : 5638-5646 (1996) are also contemplated for use in the present invention.
• VEGF variants may be prepared by techniques known in the art, for example, recombinant methods. Isolated DNA used in these methods is understood herein to mean chemically synthesized DNA, cDNA, chromosomal, or extrachromosomal DNA with or without the 3'- and/or 5 '-flanking regions.
• the VEGF variants herein are made by synthesis in recombinant cell culture. For such synthesis, it is first necessary to secure nucleic acid that encodes a VEGF or VEGF variant.
• DNA encoding a VEGF molecule may be obtained from bovine pituitary follicular cells by (a) preparing a cDNA library from these cells, (b) conducting hybridization analysis with labeled DNA encoding the VEGF or fragments thereof (up to or more than 100 base pairs in length) to detect clones in the library containing homologous sequences, and (c) analyzing the clones by restriction enzyme analysis and nucleic acid sequencing to identify full-length clones.
• full-length clones are not present in a cDNA library, then appropriate fragments may be recovered from the various clones using the nucleic acid sequence information disclosed herein for the first time and ligated at restriction sites common to the clones to assemble a full-length clone encoding the VEGF.
• genomic libraries will provide the desired DNA.
• a VEGF-encod g gene is expressed in a cell system by transformation with an expression vector comprising DNA encoding the VEGF. It is preferable to transform host cells capable of accomplishing such processing so as to obtain the VEGF n the culture medium or periplasm of the host cell, i.e., obtain a secreted molecule .
• Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaP0 4 and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.
• Transformation refers to introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells.
• the calcium treatment employing calcium chloride as described by Cohen, Proc. Na tl . Acad. Sci . (USA) , 69: 2110 (1972) and Mandel et al . , J. Mol . Biol . , 53: 154 (1970), is generally used for prokaryotes or other cells that contain substantial cell-wall barriers.
• the vectors and methods disclosed herein are suitable for use in host cells over a wide range of prokaryotic and eukaryotic organisms.
• prokaryotes are preferred for the initial cloning of DNA sequences and construction of the vectors useful in the invention.
• E. col i K12 strain MM 294 ATCC No. 31,446
• Other microbial strains that may be used include E. coli strains such as E. coli B and E. coli X1776 (ATCC No. 31,537). These examples are, of course, intended to be illustrative rather than limiting.
• Prokaryotes may also be used for expression.
• the aforementioned strains, as well as E. col i strains W3110 (F-, lambda-, prototrophic, ATCC No. 27,325), K5772 (ATCC No. 53,635), and SR101, bacilli such as Bacill us subtilis, and other enterobacteriaceae such as Salmonella typhimurium or Serra tia marcesans, and various pseudomonas species, may be used.
• plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with these hosts.
• the vector ordinarily carries a replication site as well as marking sequences that are capable of providing phenotypic selection in transformed cells.
• E. coli is typically transformed using pBR322, a plasmid derived from an E . coli species (see, e . g. , Bolivar et al . , Gene, 2:95 (1977)].
• pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
• the pBR322 plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters that can be used by the microbial organism for expression of its own proteins .
• promoters most commonly used in recombinant DNA construction include the ⁇ -lactamase (penicillmase) and lactose promoter systems [Chang et al . , Na ture, 375:615 (1978); Itakura et al . , Science, 198:1056 (1977); Goeddel et al . , Na ture, 281:544 (1979)] and a tryptophan (trp) promoter system [Goeddel et al . , Nucleic Acids Res . , 8:4057 (1980); EPO Appl . Publ . No. 0036,776] .
• eukaryotic microbes such as yeast cultures
• Saccharomyces cerevisiae or common baker's yeast
• Saccharomyces cerevisiae is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available.
• the plasmid YRp7 for example [Stmchcomb et al . , Na ture, 282:39 (1979); Kmgsman et al . , Gene, 7:141 (1979); Tschemper et al . , Gene, 10:157 (1980)], is commonly used.
• This plasmid already contains the trpl gene that provides a selection marker for a mutant strain of yeast lacking the ability to grow m tryptophan, for example, ATCC No. 44,076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
• the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
• Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase [Hitzeman et al . , J. Biol . Chem . , 255:2073 (1980)] or other glycolytic enzymes [Hess et al . , J. Adv. Enzyme Reg. , 7:149 (1968); Holland et al .
• enolase such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokmase, pyruvate decarboxylase, phosphofructokmase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, t ⁇ osephosphate isomerase, phosphoglucose isomerase, and glucokinase.
• the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
• promoters which have the additional advantage of transcription controlled by growth conditions, are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
• Any plasmid vector containing yeast-compatible promoter, origin of replication and termination sequences is suitable.
• cultures of cells derived from multicellular organisms may also be used as hosts.
• any such cell culture is workable, whether from vertebrate or invertebrate culture.
• interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure n recent years [ Tissue Cul ture, Academic Press, Kruse and Patterson, editors (1973)].
• useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7, 293, and MDCK cell lines.
• Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located m front of the gene to be expressed, along with any necessary ⁇ bosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences .
• control functions on the expression vectors are often provided by viral material.
• promoters are derived from polyoma, Adenov ⁇ rus2, and most frequently Simian Virus 40 (SV40) .
• the early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment that also contains the SV40 viral origin of replication [Fiers et al . , Na ture, 273:113 (1978)].
• Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250-bp sequence extending from the Hmdlll site toward the Bgll site located in the viral origin of replication.
• promoter or control sequences normally associated with the desired gene sequence provided such control sequences are compatible with the host cell systems .
• An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
• an exogenous origin such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
• One secondary coding sequence comprises dihydrofolate reductase (DHFR) that is affected by an externally controlled parameter, such as methotrexate (MTX) , thus permitting control of expression by control of the methotrexate concentration.
• DHFR dihydrofolate reductase
• MTX methotrexate
• a preferred host cell for transfection by the vectors of the invention that comprise DNA sequences encoding both VEGF and DHFR protein it is appropriate to select the host according to the type of DHFR protein employed. If wild-type DHFR protein is employed, it is preferable to select a host cell that is deficient in DHFR, thus permitting the use of the DHFR coding sequence as a marker for successful transfection in selective medium that lacks hypoxanthine, glycine, and thymidine.
• An appropriate host cell in this case is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub and Chasm, Proc. Na tl . Acad. Sci . (USA) , 77:4216 (1980).
• DHFR protein with low binding affinity for MTX is used as the controlling sequence, it is not necessary to use DHFR- deficient cells. Because the mutant DHFR is resistant to methotrexate, MTX- containing media can be used as a means of selection provided that the host cells are themselves methotrexate sensitive. Most eukaryotic cells that are capable of absorbing MTX appear to be methotrexate sensitive.
• One such useful cell line is a CHO line, CHO-K1 (ATCC No. CCL 61) .
• Plasmid DNA fragments are cleaved, tailored, and religated in the form desired to prepare the plasmids required. If blunt ends are required, the preparation may be treated for 15 minutes at 15°C with 10 units of Polymerase I (Klenow) , phenol-chloroform extracted, and ethanol precipitated.
• Size separation of the cleaved fragments may be performed using, by way of example, 6 percent polyacryiamide gel described by Goeddel et al . , Nucleic Acids Res . , 8:4057 (1980).
• the ligation mixtures are typically used to transform E . col i K12 strain 294 (ATCC 31,446) or other suitable E. col i strains, and successful transformants selected by ampicillin or tetracycline resistance where appropriate. Plasmids from the transformants are prepared and analyzed by restriction mapping and/or DNA sequencing by the method of Messing et al . , Nucleic Acids Res . , 9:309 (1981) or by the method of Maxa et al . , Methods of Enzymology, 65:499 (1980).
• amplification of DHFR-prote - codmg sequences is effected by growing host cell cultures in the presence of approximately 20,000-500,000 nM concentrations of methotrexate (MTX), a competitive inhibitor of DHFR activity.
• MTX methotrexate
• the effective range of concentration is highly dependent, of course, upon the nature of the DHFR gene and the characteristics of the host. Clearly, generally defined upper and lower limits cannot be ascertained. Suitable concentrations of other folic acid analogs or other compounds that inhibit DHFR could also be used.
• MTX itself is, however, convenient, readily available, and effective.
• Antibodies against the KDR receptor or FLT-1 receptor may also be employed in the methods of the present invention.
• the KDR receptor or FLT-1 receptor antibody is a monoclonal antibody.
• the KDR receptor antibody is an agonist antibody which, preferably, is capable of up-regulat g eNOS levels and/or activity.
• a mouse or other appropriate host animal is immunized with antigen by subcutaneous, mtraperitoneal, or intramuscular routes to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein (s) used for immunization.
• lymphocytes may be immunized in vitro.
• Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
• a suitable fusing agent such as polyethylene glycol
• the antigen may be KDR receptor or FLT-1 receptor or optionally, a fragment or portion or epitope or variant thereof having one or more ammo acid residues that participate in the binding of hVEGF to its receptors.
• immunization with an extracellular domain sequence of KDR may especially be useful in producing antibodies that are agonists or antagonists of hVEGF, since it is region (s) within or spanning the extracellular domain that are involved in hVEGF binding.
• region (s) within or spanning the extracellular domain that are involved in hVEGF binding.
• the use of chimeric, anti-idiotypic, humanized or human antibodies against KDR or FLT-1 are contemplated for use in the present invention and may be prepared using techniques known to the skilled artisan.
• the VEGF receptor monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
• DNA encoding the monoclonal antibodies of the mvention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of mu ⁇ ne antibodies).
• the hybridoma cells of the invention serve as a preferred source of such DNA.
• the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulm protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
• the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous munne sequences [U.S. Patent No. 4,816,567; Morrison et al., supra] or by covalently joining to the immunoglobulm coding sequence all or part of the coding sequence for a non-immunoglobulm polypeptide.
• non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combinmg site of an antibody of the invention to create a chimeric bivalent antibody.
• the VEGF receptor antibodies may be monovalent antibodies.
• Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulm light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslmkmg. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslmkmg. In vi tro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art .
• the VEGF receptor antibodies may further comprise humanized antibodies or human antibodies.
• Humanized forms of non-human (e.g., munne) antibodies are chimeric lmmunoglobulms, immunoglobulm chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-bmdmg subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulm.
• Humanized antibodies include human lmmunoglobulms (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• CDR complementary determining region
• humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulm and all or substantially all of the FR regions are those of a human immunoglobul consensus sequence.
• the humanized antibody optimally also will comprise at least a portion of an immunoglobulm constant region
• a humanized antibody has one or more ammo acid residues introduced into it from a source which is non-human. These non-human ammo acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain.
• Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Na ture, 321:522-525 (1986); Riechmann et al., Na ture, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol . Biol . , 227:381 (1991); Marks et al . , J. Mol . Biol . , 222:581 (1991)].
• the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal
• human antibodies can be made by introducing of human immunoglobulm loci into transgemc animals, e.g., mice in which the endogenous immunoglobulm genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
• the therapeutic methods of the present invention include administering VEGF to a mammal to treat hypertension.
• Hypertension is a relatively common cardiovascular disease in mammals. For instance, elevated arterial pressures can cause pathological changes in the vasculature or hypertrophy of the left ventricle of the heart. Hypertension can result in stroke in some mammals, as well as lead to disease of the coronary arteries or myocardial infarction.
• the VEGF of the invention may be formulated and dosed n a fashion consistent with good medical practice taking into account the specific hypertensive condition to be treated, the condition of the individual patient, the site of delivery of the agent, the method of administration, and other factors known to practitioners.
• "An effective amount" of VEGF includes amounts that prevent, lessen the worsening of, alleviate, or cure the condition being treated or symptoms thereof.
• “an effective amount” of VEGF is that amount which enhances or up-regulates nitric oxide production in the mammal.
• the VEGF may be used to treat acute conditions of hypertension, as well as for treating patients suffering from chronic hypertension.
• the VEGF may be prepared for storage or administration by mixing the VEGF having the desired degree of purity with physiologically acceptable carriers, excipients, or stabilizers.
• physiologically acceptable carriers excipients, or stabilizers.
• suitable carrier vehicles and their formulation, inclusive of other human proteins, for example, human serum albumin, are described, for example, in Remington ' s Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al .
• an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation lsotonic.
• the carrier include buffers such as saline, Ringer's solution, and dextrose solution.
• the pH of the solution is preferably from about 5.0 to about 8.0.
• the VEGF is water soluble, it may be formulated in a buffer such as phosphate or other organic acid salt at a pH of about 7.0 to 8.0. If a VEGF is only partially soluble in water, it may be prepared as a microemulsion by formulating it with a nonionic surfactant such as Tween, Pluronics, or PEG, e.g., Tween 80, in an amount of 0.04-0.05% (w/v) , to increase its solubility.
• Further carriers include sustained release preparations which include the formation of microcapsular particles and implantable articles.
• sustained release preparations include, for example, semipermeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles .
• a suitable material for this purpose is a polylactide, although other polymers of poly- (beta-hydroxycarboxylic acids), such as poly-D- (-) -3-hydroxybutyr ⁇ c acid [EP 133, 988A] , can be used.
• Other biodegradable polymers such as, for example, poly (lactones) , poly (acetals) , poly (orthoesters) , or poly(ortho- carbonates) are also suitable.
• sustained release compositions see U.S. Patent No. 3,773,919, EP 58,481A, U.S. Patent No. 3,887,699, EP 158,277A, Canadian Patent No. 1176565, Sidman et al . , Biopoly ers , 22:547 (1983), and Langer et al . , Chem . Tech . , 12:98 (1982).
• antioxidants e.g., ascorbic acid
• low molecular weight (less than about ten residues) polypeptides e.g., polyarginine or t ⁇ peptides
• proteins such as serum albumin, gelatin, or lmmunoglobulms
• hydrophilic polymers such as polyvinylpyrrolidone
• amino acids such as glycine, glutamic acid, aspartic acid, or arginme
• monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrms
• chelatmg agents such as EDTA
• sugar alcohols such as mannitol or sorbitol.
• excipients, carriers, stabilizers, or other additives may result in the formation of salts of the VEGF.
• the selected compound (s) and corresponding degradation products should be nontoxic and avoid aggravating the condition treated and/or symptoms thereof. This can be determined by routine screening in animal models of the target disorder or, if such models are unavailable, in normal animals.
• the VEGF to be used for therapeutic administration should be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). The VEGF ordinarily will be stored in lyophilized form or as an aqueous solution.
• Administration to a mammal may be accomplished by injection (e.g., intravenous, mtraperitoneal, subcutaneous, intramuscular) or by other methods such as infusion that ensure delivery to the bloodstream in an effective form.
• a mammal may be accomplished by injection (e.g., intravenous, mtraperitoneal, subcutaneous, intramuscular) or by other methods such as infusion that ensure delivery to the bloodstream in an effective form.
• therapeutic compositions containing the VEGF generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
• the VEGF of the invention may be administered by intravenous infusion at a dose of approximately up to 0.05 microgram/Kg/mmute for about 4 hours on a daily schedule. Such VEGF may be administered at 48 hour or 72 hour intervals.
• the VEGF may be administered intramuscularly or subcutaneously in a sustained release formulation at a dose of approximately 0.25 to about 2.5 mg/Kg, preferably, approximately 0.3 to about 1.0 mg/Kg.
• the VEGF may be administered to the mammal via a plasmid or viral vector to provide a sustained expression of the VEGF gene product to improve endothelial cell function or eNOS expression.
• VEGF in stent implantation.
• Local delivery of VEGF coated STENT provides for local eNOS upregulation and is beneficial to, for example, inhibit restenosis after balloon injury, since NO is a potent antithrombotic agent and has also been shown to inhibit smooth muscle cell (SMC) proliferation and restenosis.
• SMC smooth muscle cell
• the VEGF of the invention can also be used in a topical application for treating indications such as wound healing.
• VEGF can upregulate local eNOS and or iNOS production so as to enhance healing and prevent infection.
• the VEGF can be suitably combined with additives, such as carriers, adjuvants, stabilizers, or excipients.
• additives such as carriers, adjuvants, stabilizers, or excipients.
• suitable topical formulations include ointments, creams, gels, or suspensions, with or without purified collagen.
• the compositions also may be impregnated into transdermal patches, plasters, and bandages, preferably n liquid or semi- liquid form.
• a gel formulation having the desired viscosity may 'be prepared by mixing VEGF with a water-soluble polysaccharide, such as a cellulose derivative, or synthetic polymer, such as polyethylene glycol.
• a water-soluble polysaccharide such as a cellulose derivative, or synthetic polymer, such as polyethylene glycol.
• water soluble as applied to the polysaccharides and polyethylene glycols is meant to include colloidal solutions and dispersions.
• the solubility of, for example, cellulose derivatives is determined by the degree of substitution of ether groups, and the stabilizing derivatives useful herein should have a sufficient quantity of such ether groups per anhydroglucose unit in the cellulose chain to render the derivatives water soluble.
• a degree of ether substitution of at least 0.35 ether groups per anhydroglucose unit is generally sufficient.
• the cellulose derivatives may be in the form of alkali metal salts, for example, Li, Na, K, or Cs salts.
• suitable polysaccharides include, for example, cellulose derivatives such as etherified cellulose derivatives, including alkyl celluloses, hydroxyalkyl celluloses, and alkylhydroxyalkyl celluloses, for example, methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose, and hydroxypropyl cellulose; starch and fractionated starch; agar; alginic acid and alginates; gum arable; pullullan; agarose; carrageenan; dextrans; dextrms; fructans; nulin; mannans; xylans; arabmans; chitosans; glycogens; glucans; and synthetic biopolymers; as well as gums such as xanthan gum; guar gum; locust bean gum; gum arable; traga
• the polysaccharide is an etherified cellulose derivative, more preferably one that is well defined, purified, and listed in USP, for example, methylcellulose and the hydroxyalkyl cellulose derivatives, such as hydroxypropyl cellulose, hydroxyethyl cellulose, and hydroxypropyl methylcellulose. Most preferred herein is methylcellulose.
• a gel formulation comprising methylcellulose preferably comprises about 2-5% methylcellulose and 300-1000 mg of VEGF per milliliter of gel. More preferably, the gel formulation comprises about 3% methylcellulose.
• the polyethylene glycol useful for a gel formulation is typically a mixture of low and high molecular weight polyethylene glycols to obtain the proper viscosity.
• a mixture of a polyethylene glycol of molecular weight 400-600 with one of molecular weight 1500 would be effective for this purpose when mixed in the proper ratio to obtain a paste.
• other novel or conventional therapies e.g., growth factors such as aFGF, bFGF, PDGF, IGF, NGF, HGF, anabolic steroids, EGF or TGF-beta
• co-treatment drugs be included per se m the compositions of this invention, although this will be convenient where such drugs are proteinaceous .
• Such admixtures are suitably administered in the same manner as the VEGF.
• VEGF receptor selective variants VEGF receptor antibodies
• Effective dosages and schedules for such administration may be determined empirically, and making such determinations is within the skill
• VEGF Up-Regulation of eNOS Expression bovine adrenal cortex endothelial cells (ACE) cells were incubated with rhVEGF for 0-5 days. At the end of incubation, total cell lysates were prepared, and eNOS protein levels were determined by Western blot analysis. Materials :
• rhVEGF 165 Recombinant human VEGF 165 (rhVEGF 165 ) was produced in E. Col i [see, Sieffle et al . , Biochem. Biophys. Res. Comm. , 222 : 249-255 (1996); also available from R & D Systems] .
• the VEGF 110 hepa ⁇ n binding domain-deficient variant was made from VEGF 165 by limited proteolytic digestion with plasmm as described previously (Keyt et al., 1996, J. Biol . Chem . , 271:7788-7795) .
• VEGF receptor selective mutants FLT-sel R82E/K84E/H86E, deficient in KDR binding
• KDR-sel D63A/E64A/E67A, deficient in FLT-1 binding
• the heterodimeric form of recombinant human hepatocyte growth factor (HGF) was produced in and isolated from Chinese hamster ovary cells as previously described (Shen et al., 1997, Am. J. Physiol . , 272 : L1115-L1120) .
• FGF human fibroblast growth factor
• PLGF recombinant human placental growth factor
• TGF-betal recombinant human transforming growth factor
• EGF human epidermal growth factor
• Monoclonal anti-eNOS antibody was purchased from BIOMOL (Plymouth Meeting, PA)
• Monoclonal anti-PKC antibodies were purchased from Transduction Lab (Lexington, KY) .
• SU1498 was purchased from CALBIOCHEM (San Diego, CA) .
• PP1, PD98059, staurosporme, herbimycin A, geissem, phorbol 12-myr ⁇ state 13-acetate (PMA), wortmannin, Ly294002, L-N G -Nitroargmme methyl ester (L- NAME) were ordered from BIOMOL (Plymouth Meeting, PA) .
• Sodium nitroprusside (SNAP) was from Sigma (St. Louis, MO).
• NOSdetect Assay kit was purchased from STRATAGENE (La Jolla, CA) .
• L- [2, 3, 4 , 5- 3 H] Argmme monohydrochlo ⁇ de was purchased from Amersham Phamacia Biotech. Geneticm (G480) was obtained from Life Technologies (Gaithersburg, MD) . All reagents were prepared as 1000X stock solution unless otherwise specified.
• Cell Cultures :
• Bovine adrenal cortex capillary endothelial cells were prepared and maintained as previously described (Ferrara et.al., 1989 Biochem . Biophys . Res . Commun . 161:851-858). Briefly, cells were plated onto 6-well tissue culture plates (Costar) and grown in low glucose Dulbecco's modified Eagle's medium, supplemented with 2 mM L-Glutamate (Life Technologies), 10% bovine calf serum (HyClone Lab., Inc.) and 100 ⁇ g/ml Penicillin/Streptomycin (Life Technologies). ACE cells were used between passages 4 and 8.
• Porcine aorta endothelial (PAE) cells, and receptor transfected PAE cells were provided by Orlandoe Ferrara (Genentech, Inc.) and cultured in Ham's F-12 medium containing 10% FBS (for PAE), or plus 250 ⁇ g/ml G480 (for PAE/KDR and PAE/FLT-1) .
• cells were incubated in the medium containing 10% FBS supplemented with VEGF or other drugs as specified. Medium was changed every 24 hours.
• eNOS activity in ACE cells treated with or without rhVEGF for 2 days was determined by measuring the formation of [ 3 H] citrullme from [ 3 H] argmme. Briefly, ACE cells were homogenized in a buffer containing 25 M T ⁇ s-HCl, pH 7.4 , 1 mM EDTA and 1 mM EGTA, and then subjected to microcent ⁇ fugation at 14,000 rpm for 5 minutes.
• eNOS activity can also be determined by measuring the production of cyclic GMP (cGMP) , a down-stream product following NO release as a result of eNOS activation. Papapetropoulis et al .
• BioMol EIA cGMP kit is a competitive lmmunoassay for quantitative determination of cGMP in samples treated with 0.1m HC1.
• the kit uses a polyclonal antibody to cGMP to bind, in a competitive manner, the cGMP in sample or an alkaline phosphatase molecule which has cGMP covalently attached to it. After incubation, the excess reagents are washed away and substrate added. Then enzyme reaction is stopped and the yellow color generated is read on a microplate reader at 405nm. The intensity of the bound yellow color is inversely proportional to the concentration of cGMP in samples. Resul ts :
• FIG. 1A shows the results of a time-course experiment, indicating that prolonged VEGF treatment induced a transient increase in eNOS expression.
• the peak expression (5.5 fold) was observed at 2 days (48 hours) post exposure to 500 pM VEGF ( Figure IB) .
• eNOS activity in cell lysates from cells incubated with or without VEGF for 0-60 mm or 2 days was measured using a NOS detection kit.
• Figure IC shows that eNOS activity in VEGF-treated cells was about 5 fold greater than that in untreated control cells, which was proportional to the increased eNOS protein levels (5.5 fold) .
• Figure ID shows that acute VEGF treatment (0-60 min) resulted in a time-dependent increase in eNOS activity.
• the total NOS activity measured in ACE cells likely represents eNOS activity because (1) the assay measured Ca +2 dependent NOS activity and (2) there was no detectable eNOS proteins by Western blot in either VEGF treated or untreated cells (data not shown) . Together, these data demonstrate that VEGF increases both eNOS expression and activity in cultured endothelial cells .
• nitric oxide in VEGF-induced eNOS expression was investigated by co-mcubation of VEGF with L-NAME (2mM) , an eNOS inhibitor, with SNAP, a nitric oxide donor, or with L-arginine, the substrate of eNOS.
• ACE cells were incubated with VEGF alone or in combination with an eNOS inhibitor, L-NAME.
• eNOS protein was detected by Western blot. The materials and methods used in this study were as described above for Example 1.
• Figure 2 shows that VEGF alone induced a time-dependent but transient increase in eNOS expression with a maximal effect at 2 days post-exposure.
• co-mcubation of L-NAME with VEGF resulted in a sustained increase in eNOS expression
• NO donor, SNAP prevented both transient and sustained increase in eNOS (Figure 2) .
• Example 3 KDR Receptor Activation is Required for VEGF-induced eNOS Expression
• VEGF receptor-selective variants which bind preferentially to either one of the VEGF receptors, KDR or FLT-1-, at equal concentrations, were incubated with ACE cells for two days.
• the specific agents used in this study and shown in Figure 3A were: lane 1, control; lane 2, VEGF 165 ; lane 3, VEGFn 0 ; lane 4, KDR-sel; lane 5, FLT-sel; lane 6, PLGF.
• eNOS protein was detected using the methods described for Example 1. The materials and methods used in this study were as described above for Example 1.
• FIG. 3A shows that both VEGF ⁇ 65 and VEGFno, a heparin binding domam- deficient mutant with normal binding to KDR and FLT-1, induced a similar degree of eNOS expression.
• the KDR selective variant (KDR-sel) binds to KDR receptor normally but with reduced binding to FLT-1.
• the KDR-selective variant up-regulated eNOS expression, whereas the FLT-1 selective binding variant (FLT-sel) failed to do so.
• Placental growth factor (PLGF) which is known to only bind to FLT-1 receptor, had no effect on eNOS expression.
• KDR receptor in eNOS up-regulation was further confirmed by experiments in which receptor transfected porcine aorta endothelial (PAE) cells were used.
• PAE, PAE/KDR and PAE/Flt-1 cells were incubated with or without 500 pM rhVEGF for 2 days.
• eNOS was detected as described above for Example 1.
• Other materials and methods used in this study were as described above for Example 1.
• VEGF-induced eNOS Expression Various inhibitors were tested to investigate the role of down-stream signaling molecules following receptor activation in VEGF-induced eNOS expression. The materials and methods used in this study were as described above for Example 1. ACE cells were treated with specific tyrosine kinase inhibitors m combination with 500 pM rhVEGF for 2 days. eNOS protein was detected by Western blot.
• Lane 1 control (no VEGF or inhibitors); lane 2, VEGF (500 pM) ; lane 3, VEGF and herbimycm A ("Her"; 2 ⁇ M) ; lane 4, VEGF + PP1 (10 ⁇ M) ; lane 5, VEGF and staurosporin
• Calphostm C (Cal”, 10 ⁇ M) ; lane 8, VEGF and cheleryth ⁇ ne chloride ("che",
• FIG 4B shows the respective quantitation of eNOS levels using a densitometer .
• ACE cells were treated with rhVEGF (500 pM) for 10 minutes. Cytosolic and membrane fractions were prepared to investigate the re-distribution or PKC isoforms.
• VEGF induced a rapid re-distribution of PKC-alpha, -gamma, and -epsilon.
• Figure 5A See Figure 5A
• VEGF treatment time-dependently increased PKC activity ( Figure 5B) .
• Example 5 Effect of Other Angiogenic Factors on eNOS Expression
• ACE cells were treated with equal molar concentrations (500 pM) of VEGF, HGF, FGF, EGF, TGF-betal or TGF-beta2 for 2 days.
• the materials and methods used in this study were as described above for Example 1.
• FIG. 6 shows that in addition to VEGF, HGF is also capable of increasing eNOS expression, whereas FGF, EGF had no significant effect on eNOS expression. TGF-betal and TGF-beta2 reduced endogenous eNOS expression. These data indicate that VEGF and HGF are distinct from the other angiogenic growth factors in their ability to increase eNOS expression vi tro .
• KDR-Specific VEGF Variants To generate KDR-specific variants, two phage libraries were constructed in which residues of VEGF(1-109) found to be important for Flt-1 binding but not KDR binding were randomly mutated. Phagemid Construction
• Phagemid vector pB2105 (Genentech, Inc.) was produced by PCR amplification of the cDNA encoding residues 1-109 of VEGF, using primers that allowed subsequent ligation of Nsi I / Xba I restriction fragment into the phagemid vector, phGHam-g3 (Genentech, Inc.). This introduced an amber codon immediately following residue 109 and fused the VEGF 1-109 cDNA to the C-terminal half of gill encompassing residues 249 through 406.
• L-528 CAC GAA GTG GTG AAG TTC NNS GAT GTC NNS NNS CGC AGC NNS TGC CAT CCA ATC GAG (SEQ ID NO:l) L- 530:GGG GGC TGC TGC AAT NNS GAG NNS NNS GAG TGT GTG CCC ACT (SEQ ID NO: 2) .
• Heteroduplex DNA was synthesized according to a procedure adapted from Kunkel et al . , Meth . Enzym . 204:125-139 (1991). Through this method, a mutagenic oligonucleotide was incorporated into a biologically active, covalently closed circular DNA (CCC-DNA) molecule. The procedure was carried out according to the following steps.
• the oligonucleotides described above were 5 ' -phosphorylated.
• each 5' -phosphorylated oligonucleotide was annealed to a phagemid template (single-strand DNA purified from a du t -/ung- E . col i strain CJ-236) . This was done by first combining 1 ⁇ g single strand DNA template, 0.12 ⁇ g phosphorylated oligonucleotide, and 2.5 ⁇ l lOx TM buffer (500 mM Tris-HCl, 100 mM MgCl 2 , pH 7.5), adding water to a total volume of
• the DNA quantities provided an oligonucleotide to template molar ratio of 3:1, assuming that the oligonucleotide to template length ratio is
• Each 5' -phosphorylated oligonucleotide was then enzymatically extended and ligated to form a CCC-DNA molecule by adding the following reagents to the annealed mixture: 1 ⁇ l 10 mM ATP, 1 ⁇ l 25 mM dNTPs, 1.5 ⁇ l 100 mM DTT, 3 units T4 DNA ligase, and 3 units T7 DNA polymerase. The mixture was then incubated at 20°C for at least 3 hours. The DNA was purified by ethanol precipitation and resuspended in 15 ⁇ l of water. E. coli Electroporation
• the library phage were produced in a supressor strain of E . col i known as E. col i XLl-blue (Stratagene, LaJolla, CA) by E. coli electroporation.
• purified heteroduplex DNA first was chilled in a 0.2-cm gap electroporation cuvet on ice, and a 100 ⁇ l aliquot of electrocompetent E . coli XLl-blue was thawed on ice. The E. col i cells were added to the DNA and mixed by pipetting several times . The mixture was transferred to the cuvet and electroporated using a Gene Pulser (Bio-rad, Hercules, CA) with the following settings: 2.5 kV field strength, 200 ohms resistance, and 25 mF capacitance.
• Gene Pulser Bio-rad, Hercules, CA
• SOC media 5 g bacto-yeast extract, 20 g bacto-tryptone, 0.5 g NaCl, 0.2 g KCI; add water to 1 liter and adjust pH to 7.0 with NAOH; autoclave; then add 5 mL of autoclaved 2 M MgCl 2 and 20 mL of filter sterilized 1 M glucose was added and the mixture was transferred to a sterile culture tube and grown for 30 minutes at 37°C with shaking.
• SOC media 5 g bacto-yeast extract, 20 g bacto-tryptone, 0.5 g NaCl, 0.2 g KCI; add water to 1 liter and adjust pH to 7.0 with NAOH; autoclave; then add 5 mL of autoclaved 2 M MgCl 2 and 20 mL of filter sterilized 1 M glucose
• serial dilutions were plated on 2YT (10 g bacto-yeast extract, 16 g bacto-tryptone, 5 g NaCl; add water to 1 liter and adjust pH to 7.0 with NaOH; autoclaved) plates (supplemented with
• the culture was transferred to a 250-ml baffled flask containing 25 ml 2YT, 25 mg/ml ampicillin, M13-VCS (10 10 pfu/mL) (Stratagene, LaJolla, CA) , and incubated overnight at 37°C with shaking. The culture was then cent ⁇ fuged for 10 minutes at 10 krpm, 2°C, in a Sorvall GSA rotor (16000g). The supernatant was transferred to a fresh tube and 1/5 volume of PEG-NaCl solution (200 g/L PEG-8000, 146 g/L NaCl; autoclaved) was added to precipitate the phage. The supernatant/PEG-NaCl solution was incubated for 5 minutes at room temperature and centrifuged again to obtain a phage pellet.
• PEG-NaCl solution 200 g/L PEG-8000, 146 g/L NaCl; autoclaved
• the supernatant was decanted and discarded.
• the phage pellet was recentrifuged briefly and the remaining supernatant was removed and discarded.
• the phage pellet was resuspended in 1/20 volume of PBT buffer (PBS, 0.2% BSA, 0.1% Tween 20), and insoluble matter was removed and discarded by centrifugmg the resuspended pellet for 5 minutes at 15 krpm, 2
• VEGF variants by their binding affinities.
• VEGF (1-109) variants-gill fusion protein were expressed and displayed on the phage surface, allowing the phage to bind to KDR and/or Flt-1 receptors .
• Each library was sorted for binding to KDR (1-3) monomer using a competitive binding technique similar to a method used by H. Jin, J. Clm . Inves t . , 98: 969 (1996), and shown to be useful for generating receptor- selective variants.
• each library was sorted for binding to immobilized KDR (1-3) monomer (Genentech, South San Francisco, California) in the presence of a high concentration (100 nM) of competing Flt-1 (1-3) monomer (Genentech, Inc.) in solution. This was accomplished by first coating Maxisorp immunoplate wells (Nalge Nunc
• the wells were washed eight times with PT buffer (PBS, 0.05% Tween 20) to remove the block buffer. Aliquots of 100 ⁇ l of library phage solution (10 12 phage/ml) in PBT buffer (PBS, 0.2% BSA, 0.1% Tween 20) were then added to each of the coated and uncoated wells. The Flt-1 (1-3) monomer was added with the phage solution. The wells were incubated at room temperature for 2 hours with gentle shaking.
• PT buffer PBS, 0.05% Tween 20
• KDR-bound phage was eluted from the wells by incubating the wells with 100 ⁇ l of 0.2 mM glycine at pH 2 for 5 minutes at room temperature. To collect the KDR-bound phage, the glycine solution was transferred to an eppendorf tube and neutralized with 1.0 M Tris-HCl at pH 8.0.
• the KDR-bound phage were then repropagated by adding half of the eluted phage solution to 10 volumes of actively growing E . coli XLl-blue
• the culture from the plates was transferred to 10 volumes of 2YT/amp/VCS (2 YT being supplemented with 50 mg/ml ampicillin and 10 10 pfu/ml
• the affinity sort procedure was monitored by calculating the enrichment ratio and was repeated until the enrichment ratio reached a maximum (about 5 to 6 sorting cycles) .
• the enrichment ratio is the number of phage eluted from a well coated with KDR (1-3) monomer divided by the number of phage binding to an uncoated control well.
• a ratio greater than one is usually indicative of phage binding specifically to the KDR (1-3) protein, .thereby indicating resistance to binding to added Flt-1 (1-3) monomer.
• the enrichment ratio reached a maximum, individual clones were analyzed for specific binding.
• Phage ELISA Specific binding of phage having VEGF 1-109 variant-gill protein on its surface to the KDR (1-3) monomer was measured using a phage ELISA according to Muller et al., PNAS, 94: 7192 (1997).
• microtiter plates (Maxisorp, Nunc-Immunoplate, Nalge Nunc International, Rochester, New York) were coated with purified KDR (1-3) monomer or Flt-1 (1-3) monomer (5ug/ml) in 50mM sodium carbonate at pH 9.6 and incubated at 4
• VEGF 1-109 variant proteins were isolated as retractile bodies from the shake flask culture of E. coli ⁇ 21 cl ) .
• the refolding of the mutant proteins was performed as described by Yihai et al., J. Biol . Chem . , 271: 3154-3162 (1996) .
• the variants were mixed and unfolded with 6 M guanidine HCL plus 1 mM oxidized glutathione at pH 6, and dialyzed against 10 volumes of 2 M urea with 2 mM reduced glutathione and 0.5 mM of oxidized glutathione in 20 M Tris-HCL at pH 8 for 10 hours.
• Table 2 shows the VEGF variant identifier name, the amino acid substitutions introduced, and the codon encoding the respective substituted ammo acids.
• the asterisk (*) next to certain variant identifiers indicates various VEGF variants which demonstrated particularly preferred binding affinities and/or biological activities.
• the variant identifiers which contain an " s" indicate VEGF variant polypeptides which consisted of the 1-109 truncated form of VEGF and contained the recited mutations provided in the Table.
• the variant identifiers which contain an " f" indicate VEGF variant polypeptides which consisted of the full length 1-165 form of VEGF and contained the recited mutations provided in the Table.
• the naming and identification of the mutations in the variant sequences is in accord with naming convention. For example, for the first entry in Table 2, the mutation is referred to as "M18E”. This means that the 18 position of the native VEGF sequence (using the numbering in the amino acid sequence for native human VEGF as reported in Leung et a l . , s upra and Houck et a l .
• EXAMPLE 7 Binding of VEGF Variants to KDR Receptor
• Receptor binding assays were performed in 96-well immunoplates (Maxisorp, Nunc-Immunoplate, Nalge Nunc International, Rochester, New York) .
• Each well was coated with 100 ⁇ l of a solution containing 8 ⁇ g/ml of a monoclonal antibody to KDR known as MAKD5 (Genentech, South San Francisco,
• VEGF(8-109), native VEGF (1-165), native VEGF (1-109) variants, or VEGF165 variants (0.16-168 nM in monomer) were incubated with biotinylated native VEGF (8-109) (84 nM) and KDR (1-3) (1 ⁇ g/ml) for 2 hours at room temperature in assay buffer (0.5% BSA, 0.05% Tween 20 in PBS). Aliquots of this mixture (100 ⁇ l) were added to the precoated microtiter wells and the plate was incubated for 1 hour at room temperature.
• the complex of KDR (1-3) and biotinylated native VEGF that was bound to the microtiter plate was detected by incubating the wells with peroxidase- labeled streptavidm (0.2 mg/ml, Sigma, St. Louis, Missouri) for 30 minutes at room temperature. The wells were then incubated with 3, 3', 5, 5'- tetramethyl benz dine (0.2 gram/liter; Kirkegaard & Perry Laboratories, Gaithersburg, Maryland) for about 10 minutes at room temperature. Absorbance was read at 450 nm on a Vmax plate reader (Molecular Devices, Menlo Park, California) .
• Titration curves were fit with a four-parameter nonlinear regression curve-fitting program (KaleidaGraph, Synergy Software, Reading, Pennsylvania). Concentrations of VEGF variants corresponding to the midpoint absorbance of the titration curve of the native VEGF (8-109) were calculated and then divided by the concentration of the native VEGF corresponding to the midpoint absorbance of the native VEGF titration curve. (See Figure 7)
• the binding affinities determined for the VEGF (1-109) variants and VEGF165 variants are shown m Table 3. Many of the VEGF variants exhibited binding to KDR receptor that was within about two-fold of the binding of native VEGF (8-109) .
• EXAMPLE 8 Binding of VEGF Variants to Flt-1 Receptor
• the binding of the VEGF (1-109) variants and VEGF165 variants (described in Example 6) to Flt-1 receptor was evaluated by measuring the ability of the variants to inhibit binding of biotinylated native VEGF (8- 109) to Flt-1 receptor.
• the VEGF variants evaluated contained the mutations shown in Table 2.
• Receptor binding assays were performed in 96-well immunoplates (Maxisorp, Nunc-Immunoplate, Nalge Nunc International, Rochester, New York) .
• Each well was coated with 100 ⁇ l of a solution containing 2 ⁇ g/ml of rabbit F(ab')2 to human IgG Fc (Jackson ImmunoResearch, West Grove, Pennsylvania) in 50 mM carbonate buffer at pH 9.6 and incubated at 4°C overnight. The supernatant was then discarded, the wells were washed three times in washing buffer (0.05% Tween 20 in PBS), and the plate was blocked (150 ⁇ l per well) with block buffer (0.5% BSA, 0.01% thimerosal in PBS) at room temperature for one hour. The supernatant was discarded, and the wells were washed.
• the wells were filled with 100 ⁇ l of a solution containing Flt-IgG (a chimeric Fit-human Fc molecule) at 50 ng/ml in assay buffer (0.5% BSA, 0.05% Tween 20 in PBS) .
• the wells were incubated at room temperature for 1 hour and then washed three times in wash buffer (0.05% Tween 20 in PBS).
• VEGF(8-109), native VEGF165, VEGF (1-109) variants, or VEGF165 variants (0.03-33 nM in monomer) were mixed with biotinylated native VEGF (8-109) (0.21 nM) or biotinylated native VEGF165
• Titration curves were fit with a four-parameter nonlinear regression curve-fitting program (KaleidaGraph, Synergy Software, Reading, Pennsylvania) . Concentrations of VEGF variants corresponding to the midpoint absorbance of the titration curve of the native VEGF (8-109) were calculated and then divided by the concentration of the native VEGF corresponding to the midpoint absorbance of the native VEGF titration curve.
• the binding affinities determined for the VEGF (1-109) variants and VEGF165 variants are shown in Table 3. Many of the VEGF variants exhibited binding to Flt-1 receptor that was more than 2, 000-fold less than the binding of native VEGF (8-109) .
• the relative binding affinity data reported in Table 3 for certain VEGF variants (for instance, LK-VRB-7s* and LK-VRB- 8s*) to FLT-1 receptor is not reported in nM values since the amount of detectable binding was beyond the sensitivity of the ELISA assay.
• EXAMPLE 9 Induction of KDR Receptor Phosphorylation by VEGF (1-109) Variants
• the VEGF variants evaluated contained the mutations found in Table 2. Specifically, the following VEGF (1-109) variants were studied: LK-VRB-ls*; LK-VRB-2s*; LK-VRB-3s; LK-VRB-4s; LK-VRB-5s; and LK-VRB-6s.
• VEGF (1-109) variants (0.01-10 nM) were added to CHO cells that express the KDR receptor with a gD tag at the N-termmus (Genentech, South San Francisco, California). Cells were lysed by 0.5% Triton-XlOO, 150 mM NaCl, 50 mM Hepes at pH 7.2, and phosphorylated gD-KDR receptor n the lysate was quantified by conducting an ELISA. For the ELISA, 96-well immunoplates (Maxisorp, Nunc-Immunoplate, Nalge Nunc International, Rochester, New York) were used.
• Each well was coated with 100 ⁇ l of a solution containing 1 ⁇ g/ml of a mouse monoclonal antibody to gD known as 3C8 (Genentech, South San Francisco, California) m 50 mM carbonate buffer at pH 9.6 and incubated overnight at 4°C. The supernatant was discarded, the wells were washed three times in washing buffer (0.05%
• the phosphorylation-mducing activity of the VEGF variants are provided in Table 4.
• the VEGF variants generally exhibited phosphorylation- lnducing activity that was within two-fold of the activity of native VEGF (8-109) .
• VEGF (1-109) or VEGF165 variants were determined by using human umbilical vein endothelial cells (HUVEC) (Cell Systems, Kirkland, Washington) as target cells.
• the VEGF variants evaluated contained the mutations in Table 2. Specifically, the following VEGF (1-109) variants were studied: LK-VRB-ls*; LK-VRB-2s*; LK-VRB-7s*; and LK-VRB- ⁇ s*.
• HUVEC is a primary cell line that is maintained and grown with growth factors such as acidic FGF in CS-C Complete Growth media (Cell Systems, Kirkland, Washington) .
• growth factors such as acidic FGF in CS-C Complete Growth media (Cell Systems, Kirkland, Washington) .
• CS-C Complete Growth media
• VEGF variants at several concentrations (about 10 nM to 0.01 nM) diluted in the same fasting media were added to the wells to bring the volume to 150 ⁇ l per well and incubated for 18 hours.
• 3 H- thymidine (Amersham Life Science, Arlington Heights, IL) was added to each well at 0.5 ⁇ Ci per well and incubated for another 24 hours for the cells to take up the radioactivity. The cells were then harvested onto another 96- well filter plate and the excess label was washed off before loading the plates on the Topcount (Packard, Meriden, Connecticut) .
• the cell proliferation capabilities of the VEGF variants are shown in Table 5.
• the VEGF variants generally exhibited cell proliferation capability that was within two-fold of the capability of native VEGF (8- 109) .
• VEGF variants identified and characterized in Examples 6-11 were used to illustrate the specific correlation of KDR receptor activity with eNOS upregulation. Methods and materials used in this study were as described above in Example 1. ACE cells were treated with 500 pM specific VEGF variants for 2 days. eNOS protein was detected by Western blot. Figure 10 shows that both VEGF ⁇ 65 and VEGF 110 , a hepa ⁇ n binding domain- deficient mutant with normal binding to KDR and FLT-1, induced a similar degree of eNOS expression.
• the VEGF variants LK-VRB-2f (KDR-full) and LK- VRB-2s* (KDR-short) showed highly specific binding to the KDR receptor (Table 6).
• FIG 11 shows that the eNOS expression level in mice treated with MuFlt-IgG is significantly reduced.
• VEGF antagonist down-regulates eNOS expression in vivo, and implies a role for endogenous VEGF in the regulation of eNOS.
• EXAMPLE 14 Modulation of eNOS activity by VEGF
• VEGF vascular endothelial growth factor
• Endothelial cells were first treated with VEGF for 0 (control), 1, 5, 15, 30 or 60 minutes.
• Cell lysates were immunoprecipitated with an anti-eNOS antibody, and then immunoblotted with an antibody against ⁇ -caveolm, PLC- ⁇ , Hsp90 or Hsp70 for Western blot analysis.
• the results show that VEGF significantly reduces eNOS association levels of caveolm and PLC- ⁇ , even at short exposure time (1 minute). Meanwhile, VEGF can increase the levels of Hsp90 and Hsp70 associated with eNOS.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Medicinal Chemistry (AREA)
• Animal Behavior & Ethology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmacology & Pharmacy (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Cardiology (AREA)
• Heart & Thoracic Surgery (AREA)
• Diabetes (AREA)
• Toxicology (AREA)
• Hematology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Molecular Biology (AREA)
• Genetics & Genomics (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• Gastroenterology & Hepatology (AREA)
• Zoology (AREA)
• Immunology (AREA)
• Cell Biology (AREA)
• Urology & Nephrology (AREA)
• Vascular Medicine (AREA)
• Emergency Medicine (AREA)
• Endocrinology (AREA)
• Obesity (AREA)
• Hospice & Palliative Care (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=22588624

Family Applications (1)

Country Status (6)

Families Citing this family (2)

Family Cites Families (7)

• 2000
  • 2000-11-02 AU AU17565/01A patent/AU782158B2/en not_active Expired
  • 2000-11-02 JP JP2001535394A patent/JP2003513105A/ja active Pending
  • 2000-11-02 EP EP00980281A patent/EP1225910A2/en not_active Withdrawn
  • 2000-11-02 CA CA002385665A patent/CA2385665A1/en not_active Abandoned
  • 2000-11-02 WO PCT/US2000/030294 patent/WO2001032695A2/en not_active Application Discontinuation
  • 2000-11-02 IL IL14867400A patent/IL148674A0/xx unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events