GB2191200A - Process for preparing conjugates of metalloproteins and derivatives thereof - Google Patents
Process for preparing conjugates of metalloproteins and derivatives thereof Download PDFInfo
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- GB2191200A GB2191200A GB08613315A GB8613315A GB2191200A GB 2191200 A GB2191200 A GB 2191200A GB 08613315 A GB08613315 A GB 08613315A GB 8613315 A GB8613315 A GB 8613315A GB 2191200 A GB2191200 A GB 2191200A
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- gly
- val
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- ala
- lys
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
-
- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
Description
GB 2 191 200 A 1
SPECIFICATION
Process for preparing conjugates of metalloproteins and novel derivatives thereof This invention relates to a process for preparing conjugates of biologically active metal loproteins, without 5 incurring loss of activity, comprises preparing novel conjugates of the corresponding apoproteins, purifying them and reconstituting the biological activity by metal addition.
Field of invention
The present invention relates to a convenient preparation of biologically active metalloprotein conjugates 10 without incurring loss of biological activity.
Such novel conjugates are particularly useful fortargeting the metalloprotein to a specific tissue or organ fortherapeutic purposes, to decrease the immunogenicity and antigenicity of the said metalloprotein andlor to increase their bioavailability and therapeutic efficacy.
More specifically, the novel conjugates prepared by the process of the present invention, are conjugates 15 between metalloproteins and purified human albumin and optionally purified immunoglobulin of the IgG type or suitable fragments thereof. In particular conjugates are obtained with fragments with antigen binding (Fab) of tissue specific antibodies such as the antimyosin antibody. The corresponding metal loprotein-albu min antimyosin conjugate is particularly useful fortargeting the metalloprotein conjugate to myosin, a protein of the heart muscle. 20 The metalloprotein conjugates of the present invention possess useful therapeutic activities, in particular scavenging properties against toxic oxygen free radicals. Such useful properties are evidenced by a decrease of harmful effects attributed to oxygen free radicals, such as arrhythmias, tissue necrosis, and tissue damage due to radiation and poisoning due to certain chemicals (paraquat) of certain cytotoxic drugs (anthracyclines).
The scope of the present invention is not limited to specific metalloproteins conjugates, but relates 25 particularly to metalloproteins known as superoxydismutases. The preferred metals embodied in this invention are copper and zinc. The structure of the corresponding apoproteins, exemplified by their monomeric polypeptide chains, is detailed in the following sections.
Description of prior art 30
There is a very extensive description of metalloproteins pertinent to the present invention both in the scientific and in the patent literature. See for instance "Superoxide Dismutase", Larry W. OBERLEY, ed., CRC Press Inc., Vol. (1982,1985), and "Biological and Clinical Aspects of Superoxide and Superoxide Dismutase", W. H. BANNISTER and J. V. BANNISTER, eds., 1980 by Elsevier North Holland Inc., and J. T.
JOHANSEN, U.S. Pat. 4,340,675, (July 20th, 1982), and M. J. POZNANSKY, Can. Pat. 1168150, (May 29th, 35 1984).
In particular, Copper-Zinc Dismutases have been shown to be dimers of approximate molecular weight 32,000, containing 2 g-atoms of copper and 2 g-atoms of zinc per mole. The dimers are made of two identical subunits associated by noncovalent interaction, the structure of the subunits being known to be specific to the species or the origin of the metalloprotein. 40 It is further known that the biological activity is lost if the metals, particularly copper, are removed to form the corresponding apoproteins. It is also known that total inactivation and metal depletion occurs at a low pH (_-3.5) in presence of chelating agents, and moreover that even in absence of chelating agents, partial loss of biological (dismutase) activity occurs at low pH and during dialysis, (J. C. DUNBAR and J. T. JOHANSEN, Carisberg Res. Commun. 47, 163, (1982). 45 It is therefore not surprising that considerable loss of activity (30 to 40%) was described by K. WONG et aL, Agents and Actions, 10, 231 (1980), during the preparation of an albumin conjugate, presumably due to a dialysis purification step andlor other process conditions when a metalloprotein of bovine origin (Cu-Zn superoxide dismutase) was used.
Other examples of biological inactivation of metalloproteins due to loss of metals are known to the skilled in 50 the art (c.f. K. H. BEEM et aL, Biochemistry 16,1931,1977).
The desirability of obtaining metalloprotein conjugates (either with albumin, antibodies or other agents such as Ficoll, polyethyleneglycol etc.) has been reviewed by M. J. POZNANSKY and R. L. JULIANO, Pharmac.
Reviews 36, 277-336, (1984).
In all prior cases a suitable metalloprotein dimer has been used to obtain the desired conjugate, in presence 55 of a cross-1 inking reagent and the appropriate carrier protein (albumin) andlor the suitable antibody followed by extensive purification steps to remove unreacted proteins or reagents.
Detailed description of the invention
The invention consists in a novel process whereby the monomeric or dimeric apoprotein (without metals) is 60 subjected to a cross-linking procedure in presence of the carrier protein (albumin) and, optionally, the desired Fab (fragment with antigen binding) antibody and a cross-linking agent selected from the group of glutaraldehyde, carbodiimides (particularly EDC1, 1-ethyl-3- (3- dimethyiaminopropyi) carbodUrnide), cisaconi tic anhydride, a mixed anhydride, cyanuric chloride or an imidoester to form the corresponding apoprotein- albumin or optionally the mixed apoprotein-albumin-antibody conjugate, usually ander mild condition, at 65 2 GB 2191200 A 2 neutral pH and even in the cold. According to the present process the conjugate thus obtained, after quenching the cross-linking reaction with a suitable reagent (glycine for the glutaraldehyde reaction), is purified by dialysis or by ultrafiltration through molecular sieve filters or by gel exclusion chromatography.
The purified a po protein-conju gate thus obtained has a molecular weight which is a function of the amount of cross-linking agent used and the length of time the conjugation reaction is allowed to proceed. 5 After determination of the average molecular weight, the purified conjugate is then reconstituted with appropriate solution of metals, copper followed by zinc in case of Cu-Zn metal loproteins, at carefully controlled pH according to the procedure of BEEM et al. (J. Biol. Chem. 249,7298; 1974: Biochemistry 16, 1931; 1977). The reconstituted metalloprotein-conjugate complex is biologically active and it is ready for therapeutic application. 10 A surprising and unforeseen aspect of the present invention is the possibility of utilizing apoproteins as starting materials, since it is well known that metals, particularly zinc, have a stabilizing effect on the protein active site (c.L H. M. STEINMAN, Chapter 2, Vol 1 of "Superoxide Dismutase-, loc. cit.), and that reconstitution of biological activity is possible with the a poprotei n-co nju gate complex.
Another advantageous aspect of the present invention is that apoproteins are directly available from 15 reconbinant biotechnology processes and can be used as starting material without extensive purification.
Another advantage of the present process is the possibility of reconstituting the biological activity of the final product with metals other than zinc, for instance cobalt or radiolabeled 65 Zn, which are needed to study the pharmacokinetic properties of the conjugate (c.f. S. N. GIRI and H. P. MISRA, Med. Biology 62,285,1984).
20 Examples
1) A peptide of the formula R-Ala-Thr-Lys-Ala-Val-Cys-Val-Leu-Lys-Gly-Asp-Gly-Pro-ValGin-Gly-Ser-lie-Asn-Phe-Glu-Gin-Lys-Glu-Ser-Asp-Gly-ProVal-Lys-Val-Trp-Gly-Ser-lie-Lys-Gly-Leu-Thr-Glu-Gly-LeuHis-Gly-Phe-His-Val-His-Gin-Phe-Gly-Asn-Asp-Thr-Ala-GlyCys-Thr-Ser-Ala-Gly-Pro-His-Phe-Asn-Pro-Leu-Ser-Arg-LysHis-Gly-Gly-Pro-Lys-Asp-Glu-Glu-Arg-His-Val-Gly-Asp-LeuGly-Asn-Val-Thr-Ala-Asp-Lys-Asp-Gly-Val-Ala-Asp-Vai-Valfle-Glu-Asp-Ser-Val-lie-Ser-Leu-Ser-Gly-Asp-His-Cys-lielle-Gly-Arg-Thr-Leu-Vai-Val-His-Glu-Lys-Ala-Asp-Asp-LeuGly-Lys-Gly-Gly-Asn-Glu-Glu-Ser-Thr-Lys-Thr-Gly-Asn-AlaGly-Ser-Arg-Leu-Ata-Cys-Gly-Val-lie-Gly-lie-Ala-Gin-OH 35 in which R = H (obtained from genetically engineered E. coil by recombinant technology) or in which R =Ac (obtained from human erythrocytes after dialisis against 1 X 10' M EDTA at pH 3.8 and purified through a Sephadex G-25 column), (there is a disulfide bond between the two underlined Cys), (10 mg) and human purified (defatted) albumin (500 mg) were added to 10 m] of 0.02 M sodium phosphate buffer, pH 7.4 followed by 100 K[ of a solution of glutaraldehyde (25%) in distilled water. 40 The reaction was allowed to proceed at 40C for 4 hours, then quenched with 120 mg of glycine, and the mixture dialyzed against several changes of 0.13 M NaCI - 0.16 M glycine, followed by gel filtration on a Bio-Gei A agarose column with an exclusion limit of 500,000 daltons and the product eluted with 0.02 M sodium phosphate buffer pH 7.4 containing 0.13 M NaCI, then freeze-dried in dry ice-ethanol and lyophilized.
The average apparent molecular weight of the apoprotein-albumin conjugate was 300,000 (range 100,000 45 500,000) determined by gel filtration analysis. The ratio dimer-albumin is approximately 1: 10.
For metal reconstitution, two equivalent concentrations of Cu and Zn 21 were added per mole of the apoprotein-albumin conjugate and stored for 2 hours at 4'C. The pH was adjusted to 7.8 by adding enough 0.1 M KOH containing two equivalents Of CU2' and Zn 21 per mole of conjugate. The solution was passed through a Sephadex column G-25 which was equilibrated with phosphate buffered saline, Ph 7.8. 50 The metal loprotein-albumin thus contained was Iyophilized and assayed for SOD activity which was u/mg of dried material.
11) A peptide of the formula 55 H-Val-Gin-Ala-Val-Ala-Val-Leu-Lys-Gly-Asp-Ala-Gly-Val-SerGiy-Vai-Val-Lys-Phe-Glu-Gin-Ala-Ser-Glu-Ser-Glu-Pro-ThrThr-Val-Ser-Tyr-Glu-iie-Ala-Gly-Asn-Ser-Pro-Asn-Ala-GluArg-Gly-Phe-His-fle-His-Glu-Phe-Gly-Asp-Ala-Thr-Asn-GlyCys-Val-Ser-Ala-Gly-Pro-His-Phe-Asn-Pro-Phe-Lys-Lys-ThrHis-Gly-Ala-Pro-Thr-Asp-Glu-Val-Arg-His-Val-Gly-Asp-MetGly-Asn-Val-Lys-Thr-Asp-Glu-Asn-Gly-Val-Ala-Lys-Gly-SerPhe-Lys-Asp-Ser-Leu-iie-Lys-Leu-lie-Gly-Pro-Thr-Ser-ValVal-Gly-Arg-Ser-Vai-Val-lie-His-Ala-Gly-Gin-Asp-Asp-LeuGly-Lys-Gly-Asp-Thr-Glu-Glu-Ser-Leu-Lys-Thr-Gly-Asn-Ala- 65 3 GB 2 191 200 A 3 Gly-Pro-Arg-Pro-Ala-Cys-Gly-Val-lie-Gly-Leu-Thr-Asn-OH (in which there is a disulfide bond between the two underlined CYS), obtained according to the process of US Pat 4,340,675 (July 20th, 1982) and subsequent dialysis against 1 X 103 M EDTA at pH 3.8 as in Example 1, was similarly cross-linked with glutaraidehyde in presence of purified human albumin according to the procedure 5 of Example 1. The apoprotein-albumin conjugate was similarly purified and the product incubated in presence Of CU21, Zn 2' as in Example 1 to yield a biologically active metal loprotein-albu min conjugate of approximately 800 ulmg of dried material.
111) A metal-free peptide as exemplified in Example 1 or 11 were used as starting material. 2 mg of peptide, 50 10 mg human albumin, and 2 mg of antimyosin antibody from which the Fc portion was removed using pepsin (c.f. L. HUDSON and F. C. HAY, "Practical Immunology% Oxford, Blackwell Scientific Publications, 1976), and 50 KI of glutaraidehyde (25%) were stirred gently at 4Cfor 4 hr afterwhich 300 mg of glycine is added to quench the reaction. Following an overnight dialysis at 4C against 1 % NaCI and 1 % glycin, u n reacted peptide and small polymers are separated by ultrafiltration using Amicon XM 300 filters. A metal 15 reconstituting step is then carried out as in Example 1 to yield a biologically active metal loprotein-albu min antimyosin complex of 500 units dismutase activity per mg of dried material.
It will be readily apparent to those'skilled in the art that the foregoing description has been for purpose of illustration and that a number of modifications and changes may be made without departing from the spirit and scope of the present invention. For example different kinds of antibodies than those of Example Ill can be 20 used, or different cross-linking agents and conditions other than those described, may be used. Therefore, it is intended that the invention will not be limited except by the claims which follow:
Claims (11)
- 25 1. A process for obtaining biologically active metal loprotein-al bumin conjugates or metal loprotein albumin-antibody conjugates in high yield and without loss of biological activity, comprising cross-linking a metal-free apoprotein by means of a cross-linking agent in presence of human albumin and optionally an antibody fragment, purifying said metal-free conjugates and reconstituting the biological activity by adding the desired metals to said conjugates. 30
- 2. A process according to claim 1, characterized by the fact that the metal-free apoprotein is selected from the group of peptides having the following amino acid sequence R-Ala-Thr-Lys-Ala-Val-Cys-Val-Leu-Lys-Gly-Asp-Gly-Pro-ValGin-Gly-Ser-lie-Asn-Phe-Glu-Gin-Lys-Glu-Ser-Asp-Gly-ProVal-Lys-Val-Trp-Gly-Ser-lie-Lys-Gly-Leu-Thr-Glu-Gly-LeuHis-Gly-Phe-His-Val-His-Gin-Phe-Gly-Asn-Asp-Thr-Ala-GlyCys-Thr-Ser-Ala-Gly-Pro-His-Phe-Asn-Pro-Leu-Ser-Arg-LysHis-Gly-Gly-Pro-Lys-Asp-Glu-Glu-Arg-His-Val-Gly-Asp-LeuGly-Asn-Val-Thr-Ala-Asp-Lys-Asp-Gly-Val-Ala-Asp-Val-Vallle-Glu-Asp-Ser-Val-lie-Ser-Leu-Ser-Gly-Asp-His-Cys-lielle-Gly-Arg-Thr-Leu-Vai-Val-His-Glu-lys-Ala-Asp-Asp-LeuGly-Lys-GiyGiy-Asn-Glu-Glu-Ser-Thr-Lys-Thr-Gly-Asn-AlaGly-Ser-Arg-Leu-Ala-Cys-Gly-Val-lie-Gly-lie-Ala-Gin-OH 45 where R is H or Acetyl and there is a disulfide bond between the two underlined Cys.
- 3. A process according to claim 1, characterized by the fact that the metal-free apoprotein has the following amino acid sequence:H-Val-Gin-Ala-Val-Ala-Val-Leu-Lys-Gly-Asp-Ala-Gly-Val-SerGly-Vai-Val-Lys-Phe-Glu-Gin-Ala-Ser-Glu-Ser-Glu-Pro-ThrThr-Val-Ser-Tyr-Glu-1le-Ala-Gly-Asn-Ser-Pro-Asn-Ala-GluArg-Gly-Phe-His-lie-His-Glu-Phe-Gly-Asp-Ala-Thr-Asn-GlyCys-Val-Ser-Ala-Gly-Pro-His-Phe-Asn-Pro-Phe-Lys-Lys-ThrHisGiy-Ala-Pro-Thr-Asp-Glu-Val-Arg-His-Val-Gly-Asp-MetGly-Asn-Val-Lys-Thr-Asp-Glu-Asn-Gly-Val-Ala-Lys-Gly-SerPhe-Lys-Asp-Ser-Leu-1le-Lys-Leu-lle-Gly-Pro-Thr-Ser-ValVal-Gly-Arg-Ser-Vai-Val-lie-His-Ala-Gly-Gin-Asp-Asp-LeuGly-Lys-Gly-Asp-Thr-Glu-Glu-Ser-Leu-Lys-Thr-Gly-Asn-AlaGly-Pro-Arg-Pro-Ala-Cys-Gly-Val-lie-Gly-Leu-Thr-Asn-OH 60 and there is a disulfide bond between the two underlined Cys.
- 4. A process according to claim 1, characterized by the fact that the optional antibody fragment with antigen binding (Fab) is an antimyosin antibody fragment.
- 5. A process according to claim 1, characterized by the fact that the cross-] inking agent is selected from the 65 4 GB 2 191200 A 4 group of glutaraldehyde, a carbodiimide, cis-aconitic anhydride, cyanuric chloride and an imidoester.
- 6. A process according to claim 1 or 5, characterized by the fact that the cross-linking agent is preferably glutaraidehyde.
- 7. A process according to claim 1, characterized by the fact that the metals are Cu2+ and Zn 21.
- 8. A novel, biologically active metal loprotein-albu min conjugate or metal loprotein-albumin antibody 5 conjugate, prepared according to the process of claim 1, of the following monomer structure:R-Ala-Thr-Lys-Ala-Val-Cys-Val-Leu-Lys-Gly-Asp-Gly-Pro-ValGin-Gly-Ser-lie-Asn-Phe-Glu-Gin-Lys-Glu-Ser-Asp-Gly-ProVal-Lys-Val-Trp-Gly-Ser-lie-Lys-Gly-Leu-Thr-Glu-Gly-LeuHis-Gly-Phe-His-Val-His-Gin-Phe-Gly-Asn-Asp-Thr-Ala-GlyCys-Thr-Ser-Ala-Gly-Pro-His-Phe-Asn-Pro-Leu-Ser-Arg-LysHis-Gly-Gly-Pro-Lys-Asp-Glu-Glu-Arg-His-Vai-Gly-Asp-LeuGly-Asn-Val-Thr-Ala-Asp-Lys-Asp-Gly-Val-Ala-Asp-Vai-Vallle-Glu-Asp-Ser-Val-fle-Ser-Leu-Ser-Gly-Asp-His-Cys-lielle-Gly-Arg-Thr-leu-Vai-Val-His-Glu-Lys-Ala-Asp-Asp-LeuGly-Lys-Gly-Gly-Asn-Glu-Glu-Ser-Thr-Lys-Thr-Gly-Asn-AlaGiySer-Arg-Leu-Ala-Cys-Gly-Val-lie-Gyi-iie-Ala-Gin-OH Where R is H or Acetyl and there is a disulfide bond between the two underlined Cys, the metals are CU2'and 20 Zn 2+ and the human albumin: metalloprotein content is comprised between 20: 1 and 1: 1, optionally the fragment with antigen binding is an antimyosin antibody fragment.
- 9. A novel, biologically active metalloprotein or metal loprotein-al bumin-antibody conjugate, prepared according to the process of claim 1, of the following monomer structure 25 H-Val-Gin-Val-Ala-Val-Ala-Val-Leu-Lys-Gly-Asp-Ala-Gly-Val-SerGly-Vai-Val-Lys-Phe-Glu-Gin-Ala-Ser-Glu-Ser-Glu-Pro-ThrThr-Val-Ser-Tyr-Glu-lie-Ala-Gly-Asn-Ser-Pro-Asn-Ala-GluArg-Gly-Phe-His-lie-His-Glu-Phe-Gly-Asp-Ala-Thr-Asn-GlyCys-Val-Ser-Ala-Gly-Pro-His-Phe-Asn-Pro-Phe-Lys-Lys-ThrHis-Gly-Ala-Pro-Thr-Asp-Glu-Val-Arg-His-Val-Gly-Asp-MetGly-Asn-Val-Lys-Thr-Asp-Glu-Asn-Gly-Val-Ala-Lys-Gly-SerPhe-Lys-Asp-Ser-Leu-iie-Lys-Leu-iie-Gly-Pro-Thr-Ser-ValVal-Gly-Arg-Ser-Vai-Val-fle-His-Ala-Gly-Gin-Asp-Asp-LeuGly-Lys-Gly-Asp-Thr-Glu-Glu-Ser-Leu-Lys-Thr-Gly-Asn-AlaGly-Pro-Arg-Pro-Ala-Cys-Gly-Val-lie-Gly-Leu-Thr-Asn-OH and there is a disulfide bond between the two underlined Cys, where the metals are Cu 2+ and Zn 2' and the human albumin: metalloprotein content is comprised between 20: 1 and 1: 1 and, optionally, the fragment with antigen binding is an antimyosin antibody fragment. 40
- 10. Novel copper-zinc-protein-albumin and copper-zinc-protein-albumin- antibody conjugates where the protein has the following structure:R-Ala-Thr-Lys-Ala-Val-Cys-Val-Leu-Lys-Gly-Asp-Gly-Pro-ValGin-Gly-Ser-fle-Asn-Phe-Glu-Gin-Lys-Glu-Ser-Asp-Gly-ProVal-Lys-Val-Trp-Gly-Ser-lie-Lys-Gly-Leu-Thr-Glu-Gly-LeuHis-Gly-Phe-His-Val-His-Gin-Phe-Gly-Asn-Asp-Thr-Als-GlyCys-Thr-Ser-Ala-Gly-Pro-His-Phe-Asn-Pro-Leu-Ser-Arg-LysHis-Gly-Gly-Pro-Lys-Asp-Glu-Glu-Arg-His-Val-Gly-Asp-LeuGly-Asn-Val-Thr-Ala-Asp-Lys-Asp-Gly-Val-Ala-Asp-Vai-Val- so lle-Glu-Asp-Ser-Val-lie-Ser-Leu-Ser-Gly-Asp-His-Cys-lielle-Gly-Arg-Thr-Leu-Vai-Val-His-Glu-Lys-Ala-Asp-Asp-LeuGly-Lys-Gly-Gly-Asn-Glu-Glu-Ser-Thr-Lys-Thr-Gly-Asn-AlaGly-Ser-Arg-Leu-Ala-Cys-Gly-Val-lie-Gly-lie-Ala-Gin-OH 55 where R is H or Acety], and there is a disulfide bond between the two underlined Cys, the optional antibody is antimyosin antibody and the human albumin: metalloprotein content is comprised between 20: 1 and 1: 1.
- 11. Novel copper-zinc-protein-a 1 bum in and copper-zinc-protei n-a 1 bum i n-antibody conjugates where the protein has the following structure 60 H-Val-Gin-Ala-Val-Ala-Val-Leu-Lys-Gly-Asp-Ala-Gly-Val-SerGIV-Vai-Val-Lys-Phe-Glu-Gin-Ala-Ser-Glu-Ser-Glu-Pro-ThrThr-Val-Ser-Tyr-Glu-lie-Ala-Gly-Asn-Ser-Pro-Asn-Ala-GluArg-Gly-Phe-His-lie-His-Glu-Phe-Gly-Asp-Ala-Thr-Asn-GlyCys-Val-Ser-Ala-Gly-Pro-His-Phe-Asn-Pro-Phe-Lys-Lys-Thr- 65 GB 2 191 200 A 5 His-Gly-Ala-Pro-Thr-Asp-Glu-Val-Arg-His-Val-Gly-Asp-MetGly-Asn-Val-Lys-Thr-Asp-Glu-Asn-Gly-Val-Ala-Lys-Gly-SerPhe-Lys-Asp-Ser-Leu-lle-Lys-Leu-ile-Gly-Pro-Thr-Ser-ValVal-Gly-Arg-Ser-Vai-Val-lie-His-Ala-Gly-Gin-Asp-Asp-LeuGly-Lys-Gly-Asp-Thr-Glu-Glu-Ser-Leu-Lys-Thr-Gly-Asn-Ala- 5 Gly-Pro-Arg-Pro-Ala-Cys-Gly-Val-lie-Gly-Leu-Thr-Asn-OH and there is a disulfide bond between the two underlined Cys, the optional antibody is an antimyosin antibody and the human albumin: metalloprotein content is comprised between 20: 1 and 1: 1.Printed for Her Majesty's Stationery Office by Croydon Printing Company (UK) Ltd, 10187, D8991685. Published by The Patent Office, 25 Southampton Buildings, London WC2A lAY, from which copies may be obtained.
Priority Applications (10)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB8613315A GB2191200B (en) | 1986-06-02 | 1986-06-02 | Process for preparing conjugates of metalloproteins and novel derivatives thereof |
CH2040/87A CH674939A5 (en) | 1986-06-02 | 1987-05-25 | |
NL8701254A NL8701254A (en) | 1986-06-02 | 1987-05-26 | PROCESS FOR PREPARING CONJUGATES OF METALLOPROTEINS AND NEW DERIVATIVES THEREOF. |
JP62137600A JPS62292800A (en) | 1986-06-02 | 1987-05-29 | Biologically active metal protein composite and manufacture |
ES8701587A ES2004734A6 (en) | 1986-06-02 | 1987-05-29 | Process for preparing conjugates of metalloproteins and derivatives thereof |
IT8705187A IT1207805B (en) | 1986-06-02 | 1987-06-01 | PROCEDURE FOR PREPARING METALLOPROTEIN CONJUGATES AND NEW DERIVATIVES OF THE CONJUGATES themselves. |
FR878707639A FR2603287B1 (en) | 1986-06-02 | 1987-06-01 | PROCESS FOR THE PREPARATION OF METALLOPROTEIN COMPLEXES AND NEW DERIVATIVES THEREOF |
DK280187A DK280187A (en) | 1986-06-02 | 1987-06-01 | METHOD OF PREPARING METALOPROTEIN CONJUGATES |
BE8700605A BE1002130A3 (en) | 1986-06-02 | 1987-06-01 | PROCESS FOR THE PREPARATION OF METALLOPROTEIN COMPLEXES AND NEW DERIVATIVES THEREOF. |
DE19873718476 DE3718476A1 (en) | 1986-06-02 | 1987-06-02 | METHOD FOR PRODUCING CONJUGATES OF METAL PROTEINS AND NEW DERIVATIVES THEREOF |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB8613315A GB2191200B (en) | 1986-06-02 | 1986-06-02 | Process for preparing conjugates of metalloproteins and novel derivatives thereof |
Publications (3)
Publication Number | Publication Date |
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GB8613315D0 GB8613315D0 (en) | 1986-07-09 |
GB2191200A true GB2191200A (en) | 1987-12-09 |
GB2191200B GB2191200B (en) | 1990-07-11 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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GB8613315A Expired - Fee Related GB2191200B (en) | 1986-06-02 | 1986-06-02 | Process for preparing conjugates of metalloproteins and novel derivatives thereof |
Country Status (10)
Country | Link |
---|---|
JP (1) | JPS62292800A (en) |
BE (1) | BE1002130A3 (en) |
CH (1) | CH674939A5 (en) |
DE (1) | DE3718476A1 (en) |
DK (1) | DK280187A (en) |
ES (1) | ES2004734A6 (en) |
FR (1) | FR2603287B1 (en) |
GB (1) | GB2191200B (en) |
IT (1) | IT1207805B (en) |
NL (1) | NL8701254A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002012278A2 (en) * | 2000-08-10 | 2002-02-14 | Universita' Degli Studi Di Napoli 'federico Ii' | Pseudo-metalloproteins, corresponding preparation and use as biosensors |
Families Citing this family (1)
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MX354530B (en) * | 2013-02-27 | 2018-02-19 | Univ Autonoma Del Estado De Morelos | Compositions comprising immp for contributing to the development, growth and improvement of the productive parameters of animals of zootechnical interest, and use of same. |
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CA1168150A (en) * | 1981-12-18 | 1984-05-29 | The Governors Of The University Of Alberta | Targeting conjugates of albumin and therapeutic agents |
-
1986
- 1986-06-02 GB GB8613315A patent/GB2191200B/en not_active Expired - Fee Related
-
1987
- 1987-05-25 CH CH2040/87A patent/CH674939A5/fr not_active IP Right Cessation
- 1987-05-26 NL NL8701254A patent/NL8701254A/en not_active Application Discontinuation
- 1987-05-29 ES ES8701587A patent/ES2004734A6/en not_active Expired
- 1987-05-29 JP JP62137600A patent/JPS62292800A/en active Pending
- 1987-06-01 FR FR878707639A patent/FR2603287B1/en not_active Expired - Fee Related
- 1987-06-01 BE BE8700605A patent/BE1002130A3/en not_active IP Right Cessation
- 1987-06-01 DK DK280187A patent/DK280187A/en not_active Application Discontinuation
- 1987-06-01 IT IT8705187A patent/IT1207805B/en active
- 1987-06-02 DE DE19873718476 patent/DE3718476A1/en not_active Withdrawn
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002012278A2 (en) * | 2000-08-10 | 2002-02-14 | Universita' Degli Studi Di Napoli 'federico Ii' | Pseudo-metalloproteins, corresponding preparation and use as biosensors |
WO2002012278A3 (en) * | 2000-08-10 | 2002-06-13 | Univ Napoli Federico Ii | Pseudo-metalloproteins, corresponding preparation and use as biosensors |
Also Published As
Publication number | Publication date |
---|---|
DK280187A (en) | 1987-12-03 |
BE1002130A3 (en) | 1990-07-24 |
NL8701254A (en) | 1988-01-04 |
GB2191200B (en) | 1990-07-11 |
DE3718476A1 (en) | 1987-12-03 |
FR2603287A1 (en) | 1988-03-04 |
DK280187D0 (en) | 1987-06-01 |
FR2603287B1 (en) | 1991-02-08 |
CH674939A5 (en) | 1990-08-15 |
JPS62292800A (en) | 1987-12-19 |
IT8705187A0 (en) | 1987-06-01 |
GB8613315D0 (en) | 1986-07-09 |
ES2004734A6 (en) | 1989-02-01 |
IT1207805B (en) | 1989-06-01 |
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Legal Events
Date | Code | Title | Description |
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PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 19920602 |