Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
• • 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

Definitions

• the invention relates to therapeutic antibodies and methods for inhibiting angiogenesis.
• Plasma high molecular weight kininogen is a 120 kD glycoprotein encoded by a single gene (Kitamura et al., J. Bio. Chem. 1985; 260:8610-8617).
• BK bradykinin
• Mediation of the biological effects of HK requires prior cell binding.
• Domain 3 (D3) inhibits thrombin action on platelets (Puri etal., Blood 1991 ; 77:500-507) as well as cell binding to platelets (Kunapuli etal., J. Bio. Chem.
• HK a undergoes major conformational changes detected by electron microscopy (Weisel etal., J. Bio. Chem.1994; 269:10100-10106) and circular dichroism (Villanueva etal., Biochem. Biophys. Res. Commun. 1989; 158:72-79), and acquires the ability to bind to anionic surfaces (Scott etal., J. Clin. Invest.1984; 73:954-962).
• the HK light chain consists of the following sequence of HK amino acids 372-626 (SEQ ID NO:1):
• HK light chain amino acids (Ser)372 to Thr(383) form the C- terminal portion of HK domain 4 remaining after bradykinin liberation.
• HK amino acids Val(384) to Lys(502) form the HK domain 5 (D5).
• HK amino acids Thr(503) to Ser(626) form the HK domain 6 (D6).
• critical amino acids His(441)-His(457) in a histidine-glycine-rich region are responsible for binding to an artificial negatively-charged surface (DeLa Cadena etal., Prot. Sci.1992; 1:151-160).
• HK a formation results in the exposure of D5.
• a histidine-glycine-lysine-rich region (residues His(475)- Lys(502)) which also supports binding to an anionic surface (Kunapuli et al., J. Biol. Chem. 1993; 268:2486-2492).
• Hasan et al. (Hasan et al., J. Biol. Chem. 1995; 270:19256-19261) have mapped the endothelial cell binding domain on D5 to the histidine-glycine-lysine-rich region, specifically residues His(471 )-Gly(496), which could represent a second region for cell interaction. Khan et al.
• HK a inhibits the adhesion and spreading of human osteosarcoma cells on vitronectin-coated polystyrene plates, and the spread of bovine aortic endothelial cells on vitronectin.
• Monoclonal antibody C11 C1 of U.S. Pat.4,908,431 to Colman etal. is produced by hybridoma ATCC HB 8964 and recognizes the HK light chain.
• the antibody inhibits the coagulant activity of HK (Schmaier et al., J. Biol. Chem. 1987; 262:1405-1411), a property of the light chain, but not the ability of HK to inhibit the cysteine protease, calpain (Bradford et al., Biochem. J. 1990; 270:83-90), a property of the heavy chain.
• MAb C11C1 inhibits binding of 125 I-HK to the anionic surface of kaolin (DeLa Cadena et al, Prot. Sci. 1992; 1 :151-160), and inhibits binding of kininogen to neutrophils (Wachtfogei et al., J. Bio. Chem. 1994; 269:19307-19312).
• MAb C11C1 is desc ⁇ bed in Pat. 4,908,431 as being useful for measuring kininogen levels in plasma and other specimens of interest. The antibody is also described as being useful in removing kininogen from blood or other body fluids, for the treatment of diseases characterized by elevated kininogen levels. No other therapeutic uses for MAb C11 C1 are disclosed in Pat. 4,908,431.
• MAb 115-21 which is specific for the prekallikrein binding site on HK domain 6.
• MAb 115-21 is disclosed as being possibly useful in delineating the role of HK and prekallikrein in contact activation and kinin- related human pathology.
• Angiogenesis is the process in which new blood vessels grow into an area which lacks a sufficient blood supply. Angiogenesis commences with the erosion of the basement membrane surrounding endothelial cells and pericytes forming capillary blood vessels. Erosion of the basement membrane is triggered by enzymes released by endothelial cells and leukocytes. The endothelial cells then migrate through the eroded basement membrane when induced by angiogenic stimulants. The migrating cells form a "sprout" off the parent blood vessel. The migrating endothelial cells proliferate, and the sprouts merge to form capillary loops, thus forming a new blood vessel.
• Angiogenesis can occur under certain normal conditions in mammals such as in wound healing, in fetal and embryonic development, and in the formation of the corpus luteum, endometrium and placenta. Angiogenesis also occurs in certain disease states such as in tumor formation and expansion, or in the retina of patients with certain ocular disorders. Angiogenesis can also occur in a rheumatoid joint, hastening joint destruction by allowing an influx of leukocytes with subsequent release of inflammatory mediators.
• angiogenesis The evidence for the role of angiogenesis in tumor growth was extensively reviewed and presented by O'Reilly and Folkman in U.S. Pat. 5,639,725, the entire disclosure of which is incorporated herein by reference. It is now generally accepted that the growth of tumors is critically dependent upon angiogenesis. It has been proposed to inhibit angiogenesis with peptides (e.g., angiostatin and endostatin), recombinant proteins, and monoclonal antibodies directed to angiogenic proteins such as basic fibroblast growth factor (FGF-2) or vascular endothelial growth factor (VEGF) or their cellular receptors.
• FGF-2 basic fibroblast growth factor
• VEGF vascular endothelial growth factor
• Additional therapeutic agents are desired for inhibition of angiogenesis.
• the inhibition of angiogenesis with antibodies against HK has not been proposed prior to the present invention.
• angiogenesis is meant the generation of new blood vessels into a tissue or organ.
• a method for inhibiting angiogenesis is provided.
• An effective amount of an antibody against an antigenic determinant of high molecular weight kininogen domain 5 is administered to a mammal to inhibit angiogenesis.
• a method for inhibiting endothelial cell proliferation comprising contacting endothelial cells with an effective concentration of an antibody against an antigenic determinant of high molecular weight kininogen domain 5.
• a method for inhibiting tumor growth or formation in a mammal comprises administering to the mammal an effective amount of an antibody against an antigenic determinant of high molecular weight kininogen domain 5.
• BK bradykinin.
• CAM chorioallantoic membrane.
• EGF endothelial cell growth medium.
• FGF-2 is human basic fibroblast growth factor.
• GST glutathione-S-transferase.
• HK means the mature form of high molecular weight kininogen, and any allelic variations thereof.
• mature is meant the post- translationally-modified form of HK which results from cleavage of an eighteen amino acid leader from the initially translated molecule. All numbering with respect to amino acid positions of HK is from the N- terminus of the mature form as position 1.
• HK is synonymous with “single chain HK", that is, the mature form of high molecular weight kininogen prior to cleavage by kaliikrein and the formation of two-chain high molecular weight kininogen.
• HK a means two-chain high molecular weight kininogen, the product of kaliikrein cleavage of mature high molecular weight kininogen, and any allelic variations thereof.
• VEC human umbilical vein endothelial cell
• MAb monoclonal antibody
• PBS phosphate buffered saline
• VEGF vascular endothelial cell growth factor
• Fig. 1A is a plot of the effect of MAb C11C1 on FGF-2-induced human umbilical vein endothelial cell (HUVEC) proliferation. The antibody is directed against HK domain 5.
• Fig. 1 B is a plot of the effect of MAb B10-17 on FGF-2-induced
• the antibody is directed against the light chain of factor V and serves as a unrelated control antibody.
• Fig. 2 is a graph of the inhibition of endothelial cell tube formation in w ' .ro by MAb C11C1.
• Fig. 3 is a plot of the percent inhibition of endothelial cell tube formation in vitro by MAb C11C1 in terms of tube length/area and tube area.
• Fig. 4A shows the microscopic appearance of endothelial cells stimulated with FGF-2.
• Fig. 4C shows FGF-2-simulated endothelial cells treated with 400 nM MAb C11C1.
• Fig. 4B shows other FGF-2-simulated endothelial cells. The cells were treated with 40 nM MAb C11C1 in Fig.
• Fig. 5A shows the chorioallantoic membrane (CAM) of a control chicken egg treated with phosphate-buffered saline (PBS).
• Fig. 5B shows the membrane of an egg treated with FGF-2 and PBS.
• Fig. 5C shows the membrane of an egg treated with FGF-2 and MAb C11 C1.
• Fig. 6 quantifies the CAM neovascularization according to the number of branch points of eggs treated with PBS (Fig. 5A), FGF-2 (Fig.
• Fig. 7 is a plot of the number of branch points in the CAM neovascularization of eggs treated with FGF-2 versus FGF-2 plus MAb
• the present invention is based upon the discovery that antibodies which bind to HK domain 5 inhibit endothelial cell proliferation, tube formation and neovascularization. This activity confers upon such antibodies the ability to inhibit cytokine-d riven angiogenesis in vivo. According to the assays utilized herein, antibodies against HK domain 5 potently inhibit the proliferation of human endothelial cells in vitro in response to a typical endothelial cell growth factor, FGF-2. Such antibodies also inhibit endothelial tube formation, another benchmark of angiogenesis.
• antibodies against HK domain 5 are effective in an in vivo model of angiogenesis.
• the antibodies inhibit the growth of new blood vessels in the chorioallantoic membrane of intact living chicken eggs stimulated with FGF-2.
• the mature human HK light chain amino acid sequence is set forth herein as SEQ ID NO:1.
• domain 5 comprises amino acids 372 to 502, but the segment 372-419 can be removed by kaliikrein cleavage between Arg(419) and Lys(420) without prejudice to the function of the remaining portion.
• the antibodies used in the practice of the present invention are selected to bind to an antigenic determinant located in the HK segment Gly(440)-Lys(502).
• the antibodies are selected to bind to an antigenic determinant located in the HK segment Gly(440)- His(455).
• the antibodies are selected to bind to an antigenic determinant in the HK segment Gly(486)- Lys(502). According to a most preferred embodiment of the invention, the antibodies bind to the same antigenic determinant recognized by MAb C11 C1 , the product of hybridoma ATCC HB-8964.
• the antibodies used in the practice of the present invention may be polyclonal or monoclonal, although monoclonal antibodies are preferred.
• the antibody is preferably a chimeric antibody.
• the antibody is preferably a humanized chimeric antibody.
• Antibodies may be prepared against HK domain 5 by conventional immunologic techniques. Such antibodies, which are inhibitors of angiogenesis, may be used to treat any condition characterized by undesirable angiogenesis. Such conditions include, for example, retinopathy, such as retinopathy caused by diabetes; rheumatoid arthritis; and atherosclerotic plaques. The antibody may be administered to prevent plaque rupture leading to thrombotic occlusion of coronary or cerebral arteries. Antibodies against HK domain 5 may also be used to inhibit the growth or spread of tumors at any site in the body.
• the therapeutic antibody may be taken up in a pharmaceutically acceptable carrier suitable for proteinaceous drugs.
• carries include but are not limited to water, saline, dextrose, mannitol, lactose, or other sugars, lecithin, albumin, sodium glutamate cysteine hydrochloride, ethanol, polyols (propyleneglycol, ethylene, polyethyleneglycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, agar-agarand tragacanth, or mixtures of these substances, and the like.
• compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like). If desired, absorption enhancing or delaying agents (such as liposomes, aluminum monostearate, or gelatin) may be used.
• auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like).
• absorption enhancing or delaying agents such as liposomes, aluminum monostearate, or gelatin
• the compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
• Antibody against HK domain 5 is administered either locally or systemically to inhibit undesired angiogenesis in mammals, inclusive of humans.
• antibody may be administered by any convenient route which will result in delivery to the circulation of an amount of antibody sufficient to inhibit angiogenesis in the target tissue. Any route acceptable for the delivery of proteinaceous pharmaceuticals may be employed.
• the HK domain 5 antibodies may be given by subcutaneous, intravenous, intraarterial, intraperitoneal or intramuscular injection. Parenteral administration is preferred, most particularly intravenous administration.
• the antibodies may be dissolved in any appropriate intravenous delivery vehicle containing physiologically compatible substances, such as sodium chloride, glycine, and the like, having a buffered pH compatible with physiologic conditions.
• the vehicle is a sterile saline solution.
• antibody may be given by direct local injection, by continuous infusion via infuser pumps, by implantation of controlled release device, and the like. Local administration is preferred for treatment of tumors.
• the antibody may be injected into the tumor site.
• the dosage of antibody administered depends on the relative affinity of the antibody for domain 5, the stability of the antibody, the size and weight of the subject, the extent of the disease lesion, and the like.
• An "effective amount” is any amount of antibody useful in inhibiting the undesired angiogenesis in the target tissue.
• the amount of antibody delivered should be sufficient to result in a concentration of the antibody in the blood in the range of from about 1 to about 10 ⁇ M.
• the selection of the appropriate dosage to result in this blood concentration may be readily determined by those skilled in the art using only routine skill. Side effects are believed to be minimal, as patients with HK deficiency are known to be asymptomatic.
• Preparation of polyclonal antisera against domain 5 involves stimulating an immune response against the appropriate immunogen in a suitable host. Serum is taken from the host and then subjected to affinity purification to isolate polyclonal antibodies against the immunogen. Monoclonal antibodies are prepared by well-known techniques, as exemplified by the protocol leading to the preparation of MAb C11C1 set forth in U.S. Pat.4,908,432, incorporated herein by reference.
• MAb C11C1 is the product of the hybridoma HB-8964. The hybridoma was deposited in the American Type Culture Collection, 10801 University Boulevard., Manassas, VA 20110-2209 on December 6, 1985.
• Antibody C11C1 belongs to sub-class IgG ⁇ Antibody C11C1 may be purified from the HB- 8964 culture supernatant according to the ammonium sulfate precipitation/Protein A affinity chromatography method set forth in Pat. 4,908,432. The same method may be used to purify any other IgG antibody against HK domain 5.
• the immunogen for preparing polyclonal or monoclonal antibodies against HK domain 5 may comprise, in decreasing size, HK, HK a , isolated domain 5, or fragment of domain 5.
• the immunogen is a D5 fragment which comprises the HK segment Gly(440)- Lys(502).
• the fragment comprises HK segment Gly(440)-His(455) or HK segment Gly(486)-Lys(502).
• Other peptides from the HK domain 5 region Gly(440)-Lys(502) may be selected as the immunogen.
• D5 peptides may be coupled by virtue of their free cysteine sulfhydryl (either in the peptide sequence or added to the carboxy or amino terminus where the native sequence did not contain a cysteine) to keyhole limpet hemocyanin (KLH; Sigma) using the coupling agent m- maleimidobenzyol-N-hydroxysuccinimide ester (MBS; Pierce) as described by Liu et al., Biochemistry 18:690-697 (1979).
• KLH keyhole limpet hemocyanin
• D5 peptides for use as immunogens in the generation of D5 antibodies may be prepared by both manual and automated techniques employing commercially available peptide synthesizers.
• D5 peptides may be prepared by recombinant DNA techniques as glutathione- S-transferase (GST) fusion proteins according to the procedure of Kunapuli et al., J. Biol. Chem. 268:2486-2492 (1993), the entire disclosure of which is incorporated herein by reference.
• the fusion proteins are generated by deletion mutagenesis using plasmid pSK931 , the derivation of which is described by Kanapuli etal. Plasmid pSK931 was generated as described by Kunapuli et al.
• Plasmids capable of expressing the desired fragments of HK light chain are constructed by digestion of pSK931 with a combination of restriction enzymes and religating the plasmid with oligonucleotide linkers in order to retain the reading frame.
• the recombi- nant proteins are then purified by glutathione-Sepharose affinity chromatography, eluting bound recombinant protein with glutathione.
• the recombinant fusion proteins contain GST at their N-terminus.
• the GST portion may be cleaved off. Alternatively, the GST portion may be permitted to remain attached, to serve as a carrier for the D5 peptide for immunization.
• mice are immunized with the appropriate immunogen.
• BALB/cAnSkh mice are preferred, although other strains may be used.
• the immunization schedule and concentration of immunogen administered should be such so as to produce useful quantities of suitably primed splenocytes.
• the mice Upon completion of the immunization regimen, more fully described below, the mice are sacrificed and their spleens removed. A suspension of splenocytes in a suitable medium is prepared. Approximately 2.5-5 ml of medium per spleen is sufficient. The protocols for in vitro cell suspension are well established.
• the spleen cells are fused with mouse myeloma cells by means of a fusion promoter.
• the preferred fusion promoter is polyethylene glycol ("PEG"), molecular weight 1 ,300-1 ,600 kDa. Other known fusion promoters may be used.
• the mouse myeloma cell line is preferably one of the "drug-resistant" types, to enable selection of hybrids. The most frequently used class of myelomas are the 8-azaguanine-resistant cell lines, which are widely known and available. These cell lines lack the enzyme hypoxanthine guanine phosphoribosyl transferase and therefore do not survive in "HAT" (hypoxanthine-aminopterinthymidine) medium.
• HAT hypoxanthine-aminopterinthymidine
• myeloma cells with different genetic deficiencies e.g., other enzyme deficiencies, drug sensitivities, etc.
• myeloma cell line should not itself produce any antibody, although in some circumstances secreting myeloma cell lines may be used.
• the preferred fusion promoter is PEG of average molecular weight 1 ,300-1 ,600 kDa (available from ATCC), other known fusion promoters may be used.
• Fusion of cells may be carried out in an adherent monolayer, such as according to the method described by T.J. McKearn, "Fusion of Cells in an Adherent Monolayer” in Monoclonal Antibodies: Hybridomas: A New Dimension In Biological Analysis, (Kennett, R.H., McKearn, T.J., and Bechtol, K.B., eds., Plenum Press, New York and London, 10368-369, 1980) .
• Other fusion techniques may be employed.
• a cell ratio of 2-5:1 spleen cells per myeloma cell may be used. This ratio may be varied depending on the source of spleen or myeloma cells.
• a mixture of unfused myeloma cells, unfused spleen cells and fused cells are distributed for culturing in separate compartments (e.g., the wells of microtiter plates) in a selective medium in which the unfused myeloma cells will not survive. Distribution of the cells may be by resuspension in a volume of diluent which is statistically calculated to isolate a desired number of cells per compartment. See, McKearn, T.J., "Cloning of Hybridoma Cell Lines by Limiting Dilution in Fluid Phase" in Monoclonal Antibodies, p. 374.
• unfused 8-azaguanine-resistant myeloma cells When HAT is used as the medium, unfused 8-azaguanine-resistant myeloma cells will not grow. Unfused spleen cells will normally die after a few days, since they are non-malignant. Culturing proceeds for a time sufficient to allow their death. Fused cells continue to reproduce and grow in the selective medium.
• the supernatant in each container or compartment having hybrid cell growth is screened and evaluated for the presence of antibody to the immunogen.
• Any suitable antibody-binding detection method may be used, e.g., ELISA, radioimmunoassay, etc.
• the desired monoclonal antibody may be produced by in vitro culturing of the hybridomas or by in vivo peritoneal exudate induction in mice.
• the in vitro culturing method will yield monoclonal antibody of higher purity.
• the antibody is recovered from the culture supernatant essentially free of undesired immunoglobulin.
• Antibody concentrations of 25-50 micrograms/ml are possible by this method.
• growth media containing serum such as fetal calf serum
• a small amount of other immunoglobulin is present.
• In vivo peritoneal exudate induction in mice may be less preferred for generating D5 antibody, as the anti- angiogenic effect of the antibody may inhibit ascites formation.
• Antibodies may be screened for ability to inhibit angiogenesis by any of the known angiogenesis assays, such as any of the assays described in the hereinafter Examples. According to one such assay, antibody is tested for its ability to inhibit endothelial cell proliferation, such as HUVEC proliferation.
• the antibody inhibits HUVEC proliferation with an IC 50 of no more than 10 ⁇ M, preferably no more than 1 ⁇ M, when HUVEC are stimulated by an endothelial cell growth medium containing the following, per 500 ml volume: 2.0 ml bovine brain extract (Clonetics® catalogue #CC-4092, Clonetics Corp., Walkersville, MD) 3 mg/ml; 0.5 ml human EGF, 10 mg/ml; 0.5 ml hydrocortisone, 1 mg/ml; 10 ml fetal bovine serum (FBS, Clonetics® catalogue #CC-4092); 50 mg/ml gentamicin and 50 ⁇ g/ml amphotericin-B, 0.5 ml.
• an endothelial cell growth medium containing the following, per 500 ml volume: 2.0 ml bovine brain extract (Clonetics® catalogue #CC-4092, Clonetics Corp., Walkersville, MD) 3 mg/ml; 0.5 ml human EGF
• the aforementioned endothelial cell growth medium is available from Clonetics Corp., Walkersville, MD as EGM CC-3024. Proliferation is scored by counting the number of cells.
• the monoclonal antibodies may be advantageously cleaved by proteolytic enzymes to generate fragments retaining the antigen-binding site. For example, proteolytic treatment of IgG antibodies with papain at neutral pH generates two identical so-called "Fab" fragments, each containing one intact light chain disulfide-bonded to a fragment of the heavy chain (Fc). Each Fab fragment contains one antigen-combining site.
• the remaining portion of the IgG molecule is a dimer known as "Fc".
• fragments are known to those skilled in the art. See, Goding, Monoclonal Antibodies Principles and Practice, Academic Press (1983), p. 119-123. Fragments of the monoclonal antibodies containing the antigen binding site, such as Fab and F(ab') 2 fragments, may be preferred in therapeutic applications, owing to their reduced immunogenicity. Such fragments are less immunogenic than the intact antibody, which contains the immunogenic Fc portion.
• the term "antibody” includes intact antibody molecules and fragments thereof which retain antigen binding ability.
• the effects of sensitization in the therapeutic use of animal origin monoclonal antibodies in the treatment of human disease may be diminished by employing a hybrid molecule generated from the same Fab fragment, but a different Fc fragment, than contained in MAbs previously administered to the same subject. It is contemplated that such hybrid molecules formed from the D5 monoclonal antibodies may be used in therapy.
• the effects of sensitization are further diminished by preparing animal/human chimeric antibodies, e.g., mouse/human chimeric antibodies, or humanized (i.e. CDR-grafted) antibodies.
• Such monoclonal antibodies comprise a variable region, i.e., antigen binding region, and a constant region derived from different species.
• chimeric antibody an antibody which comprises elements partly derived from one species and partly derived form at least one other species, e.g., a mouse/human chimeric antibody.
• Chimeric animal-human monoclonal antibodies may be prepared by conventional recombinant DNA and gene transfection techniques well known in the art. The variable region genes of a mouse antibody- producing myeloma cell line of known antigen-binding specificity are joined with human immunoglobulin constant region genes. When such gene constructs are transfected into mouse myeloma cells, antibodies are produced which are largely human but contain antigen-binding specificities generated in mice. As demonstrated by Morrison et al., Proc. Natl. Acad. Sci.
• both chimeric heavy chain V region exon (V H )-human heavy chain C region genes and chimeric mouse light chain V region exon (V ⁇ )-human K light chain gene constructs may be expressed when transfected into mouse myeloma cell lines.
• V H chimeric heavy chain V region exon
• V ⁇ mouse light chain V region exon
• Patents 5,292,867; 5,091 ,313; 5,204,244; 5,202,238; and 5,169,939. The entire disclosures of these patents, and the publications mentioned in the preceding paragraph, are incorporated herein by reference. Any of these recombinant techniques are available for production of rodent/human chimeric monoclonal antibodies against human HK domain 5.
• “humanized” antibodies have been constructed in which only the minimum necessary parts of the mouse antibody, the complementarity-determining regions (CDRs), are combined with human V region frameworks and human C regions (Jones etal., Nature 321 , 522-525 (1986); Verhoeyen et al., Science 239, 1534-1536 (1988); Reichmann et al., 322, 323-327 (1988); Hale et al. Lancet 2, 1394-1399 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029-10033, (1989)).
• CDRs complementarity-determining regions
• Rodent antigen binding sites are built directly into human antibodies by transplanting only the antigen binding site, rather than the entire variable domain, from a rodent antibody. This technique is available for production of chimeric rodent human anti-D5 antibodies of reduced human immunogenicity.
• MAb C11C1 product of hybridoma ATCC HB-8964
• MAb C11C1 was produced and characterized as previously described (U.S. Pat. 4,908,431 ; Schmaier et al., J. Biol. Chem. 1987; 262:1405-1411).
• MAb C11C1 did not produce ascites and therefore was produced in culture supernatant.
• the IgG fraction was isolated by affinity chromatography (Affi-Gel Protein A, BioRad Corp., Richmond, CA).
• a second MAb, B-10-17, a non-neutralizing antibody directed to the B domain of factor V was produced in ascites and purified by affinity chromatography on Protein A-agarose.
• HUVEC HUVEC were cultured as previously described (Lin et al., Blood 1997; 90:690-697). Endothelial cell proliferation was measured by using the cell titer 96 AQueous Non-Radioactive Proliferation Assay (ProMega, Madison, Wl). The basis for the assay is the use of a novel tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium inner salt (MTS).
• MTS novel tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium inner salt
• HUVEC The compound isbioreduced by cells into a formazan (which absorbs at 490 nm) soluble in tissue culture medium in the presence of an electron coupling reagent, phenothiazone methosulfate (PMS).
• PMS phenothiazone methosulfate
• HUVEC were trypsinized and centrifuged and subsequently washed twice in phosphate-buffered saline. After counting, HUVEC were plated at 10,000 cells/well, incubated with endothelial cell growth medium (EGM, Clonetics® No. CC-3024 with or without MAb C11 C1 or MAb B-10-17 at a final volume of 100 ⁇ l in 96-well plate at 5% C0 2 and 95% humidity.
• EMM endothelial cell growth medium
• MTS-PMS solution were combined with 100 ⁇ l of PMS using aseptic technique. Twenty ⁇ l of the combined MTS-PMS solution was pipetted into each well of the 96 well assay plate containing the sample. The plate was incubated for 2 hours at 37 °C in a humidified 5% C0 2 atmosphere and the absorbance was read at 490 nm using an ELISA plate reader (Baltrop, Bioorg & Med. Chem. Letters 1991 ; 1 :611).
• MAb C11C1 The ability of MAb C11C1 to inhibit endothelial cell tube formation was determined by the method of Grant & Kleiman, EXS 1997; 79:317-33, as follows. Matrigel (Becton Dickinson Co., Franklin Lakes, NJ) stored at 4°C, was added to each of the wells of a 24 well microplate (250 ⁇ l containing 3 mg) after incubation at 37°C for 20 min. HUVEC (40,000 cells/well) were then added with or without MAb C11C1 (4-400 nM). The positive control was an endothelial growth medium containing 2% fetal bovine serum (EGM CC-3121 , Clonetics Corp., Walkersville, MD).
• the negative control was endothelial cell basal medium (EBM CC-3121 , Clonetics Corp.). The plate was incubated at 37 °C for 18 hours, fixed with Lucostate (Fisher Scientific), and stained with methylene blue in methanol. For quantitation, tube length/area or tube area/mm 2 was quantified by an inverted Nikon microscope and Image Pro-Plus software (Media Cybernetics, Silver Spring, MD).
• MAb C11C1 inhibited tube formation in EGM + 10% FCS stimulated endothelial cells in Matrigel whether measured by tube length/area or tube area/field (Fig. 2).
• degree of inhibition was plotted versus log of the concentration of MAb C11 C1.
• an approximately linear relationship was found between 12-400 nM with an IC 50 of 85 nM for tube area/field and 101 nM for tube length/area (Fig. 3).
• the microscopic appearance of typical experiments is shown in Fig. 4. Two experiments are seen.
• Fig. 4A a Matrigel preparation containing endothelial cells was stimulated with EGM + 10% FCS. Multiple tube formation is visible.
• Fig. 4C 400 nM MAb C11 C1 produced marked inhibition in the same system.
• Fig.4B another study, tube formation in Matrigel is seen with marked stimulation.
• 40 nM MAb C11C1 produced marked inhibition of tube formation.
• Ten-day-old chicken embryos were purchased from Spafas, Inc. (Preston, CT) and were incubated at 37°C with 55% humidity. A small hole was punctured in the shell concealing the air sac with a hypodermic needle. A second hole was punctured in the shell on the broad side of the egg directly over an avascular portion of the embryonic membrane, as observed during candling. A false air sac was created beneath the second hole by the application of negative pressure to the first hole, which caused the chorioallantoic membrane (CAM) to separate from the shell.
• CAM chorioallantoic membrane
• Filter disks of #1 filter paper (Whatman International, United Kingdom) were soaked in 3 mg/ml cortisone acetate (Sigma, St. Louis, MO) in a solution of 95% ethanol and water and subsequently air dried under sterile conditions.
• FGF-2 Life Technologies, Gaithersburg, MD was used to grow vessels on the CAMs of 10-day old chick embryos.
• CAM tissue directly beneath FGF-2-saturated filter disk was resected from embryos treated 48 hours prior with compounds or controls. Tissues were washed three times with PBS. Sections were placed in a 35 mm petri dish (Nalge Nunc, Rochester, NY) and examined under a SV6 stereo microscope (Karl Zeiss, Thornwood, NY) at 50x magnification. Digital images of CAM sections adjacent to filters were collected using a 3- CCD color video camera system (Toshiba America, New York, NY) and analyzed with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). The number of vessel branch points contained in a circular region equal to the area of a filter disk (angiogenesis index) was counted for each section. Percent inhibition data is expressed as the quotient of the experimental value minus the negative control value divided by the difference between the positive control value and the negative control value.
• Fig. 5A shows a control egg (PBS-treated).
• Fig. 5B shows an egg treated with FGF-2 in PBS. Extensive vascularization is apparent. The extent of FGF-2-induced vascularization was sharply reduced by treatment with MAb C11C1 (2.38 ⁇ g) in Fig. 5C.
• Fig. 6 quantifies the neovascularization by measuring the number of branch points.
• MAb C11C1 inhibited the FGF-2-stimulated neovascularization to a level only moderately above that of the buffer control.
• Fig. 7 presents the data with the number of branch points with buffer substituted to measure the degree of inhibition.
• the inhibition observed was 71.3 ⁇ 8.2% SEM.
• 60.8 ⁇ 9.6% inhibition was found (data not shown).
• At least six embryos were used per treatment group. Each experiment was performed twice.
• the mean ⁇ SEM in Figure 7 is based on 12 separate observations.

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