WO1991008220A1 - Procede de synthese regulee par etapes d'especes chimiques, notamment de peptides, de produits couples obtenus selon le procede, et emploi de ces produits couples, par exemple en tant que vaccins - Google Patents

Procede de synthese regulee par etapes d'especes chimiques, notamment de peptides, de produits couples obtenus selon le procede, et emploi de ces produits couples, par exemple en tant que vaccins Download PDF

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Publication number
WO1991008220A1
WO1991008220A1 PCT/DK1990/000311 DK9000311W WO9108220A1 WO 1991008220 A1 WO1991008220 A1 WO 1991008220A1 DK 9000311 W DK9000311 W DK 9000311W WO 9108220 A1 WO9108220 A1 WO 9108220A1
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group
peptide
bsa
hsa
ppd
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PCT/DK1990/000311
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English (en)
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Gunnar Houen
Arne Holm
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Gunnar Houen
Arne Holm
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Publication of WO1991008220A1 publication Critical patent/WO1991008220A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier

Definitions

  • the present invention concerns a method for the stepwise, controlled synthesis of chemical species, particularly peptides, using a proteinaceous substance such as a protein as the synthesis substrate.
  • Peptides are synthesized on amino or carboxyiic acid groups of the proteinaceous substrate, either directly or via a linker group which has been attached to amino or carboxyiic acid groups or to other functional groups in the substrate.
  • the method is well-suited for building-up on the proteinaceous substrate a number of peptide chains having the same primary sequence, and to be presented for a given biological or serological system, e.g. for immunization, antibody assay etc.
  • carboxyiic acid groups on the proteinaceous substrate may be used in either unprotected or protected form, or may be functionalized before or after peptide synthesis in a desired manner to allow special chemical or biological properties of the conjugates to be obtained.
  • the synthesis of peptides on the amino or carboxyiic acid groups can be followed up by synthesis of another peptide on suitably modified, initially unexploited carboxyiic acid, amino or other groups on the substrate or by conjugation to other molecules such as sugars, lipids, etc.
  • peptide conjugates may be obtained with a high degree of peptide/substrate substitution.
  • the method provides well-defined protein conjugates at a far lower cost than any conventional approach, and it eliminates a number of otherwise time consuming and expensive reactions.
  • the method is suited, for example, to the preparation of peptide/protein conjugates and free peptides in amounts varying from milligrams to kilograms.
  • SPPS solid-phase peptide synthesis
  • J.Am.Chem.Soc. 85, 2149 (1963)) is generally far better than synthesis in homo ⁇ geneous solution with respect to product yield and the amount of work necessary, and for automating the process of synthesis.
  • the original polymeric support introduced by Merrifield is a functionalized cross-linked styrene/divinylbenzene copolymer. It is normally provided in the form of spheres or particles, often with a predominant particle size of 20-80 ⁇ m.
  • the functionalization originally preferred was functionalization of the aromatic rings of the copolymer with chloromethyl groups. Subsequently, well over 50 functionalization methods have been described among which Friedel-Crafts reactions to introduce chloromethyl, aminomethyl, and benzhydrylamino groups are the most widely applied [see, e.g., G.Barany et al., Int .Peptide Protein Res. 30 r 705-739 (1987)].
  • the Merrifield resin has been used for numerous syntheses and has undergone further development. Reduction of the divinylbenzene content of the polymerization mixture from 2 to 1 % resulted in a resin that swells better in commonly employed solvents such as dichloromethane or dimethylformamide. Good swelling properties of synthetic resins are of great importance, as a small fraction of the functionalized molecules otherwise remains hidden in certain areas of the synthetic substrate such that it is inaccessible for acylation by activated derivatives of hindered amino acids such as threonine and isoleucine.
  • the purpose of the functionality is to provide an anchoring bond between the support and the C-terminal of the first amino acid which it is desired to couple to the support.
  • Other refinements of the Merrifield technique include incorporation of a bifunctional "linker 1 * group (also called a "spacer” or “handle” group) between a functionality (e.g. one of the above-mentioned func ⁇ tionalities) on the polymer chain of the support and the C-terminal of the first amino acid to be coupled, the linker being tailored, inter alia, to fulfil certain requirements with regard to the coupling of the first amino acid to the support and/or with regard to the ease with which the completed synthesized peptide chain is cleaved from the support.
  • dimethylformamide is generally well-suited as a solvent for protected peptides, and is a preferred solvent for many peptide bond formation reactions (see, e.g., R.C.Sheppard, Chemistry in Britain. 402 (1983)).
  • polyamide type, make use of two different types of amino acid protecting groups, and their use thus entails two chemically different methodologies.
  • the conventional acid-labile t-butoxycarbonyl protecting groups are replaced with other, base-labile N-protecting groups such as Fmoc, which is cleaved off within a few seconds upon treatment with secondary amines such as piperidine.
  • the benzyl-based side-chain protecting groups employed in the traditional Merrifield methodology are replaced with, e.g., t-butyl- based side-chain protecting groups.
  • the "polyamide” deprotection scheme makes peptide synthesis possible under considerably milder conditions than in the Merrifield strategy.
  • Useful designs of solid-phase peptide synthesis methods which employ the above described "polyamide" substrate are normally those in which the growing peptide chain is attached to the support during the synthesis by means of an acid- or base-cleavable "linker", e.g. 4-hydroxymethylphenoxyacetic acid, 3-methoxy-4- hydroxymethyl-hydroxymethylphenoxyacetic acid (both of which are acid-clea- vable) or 4-hydroxy-methylbenzoic acid (which is base-cleavable).
  • an acid- or base-cleavable “linker” e.g. 4-hydroxymethylphenoxyacetic acid, 3-methoxy-4- hydroxymethyl-hydroxymethylphenoxyacetic acid (both of which are acid-clea- vable) or 4-hydroxy-methylbenzoic acid (which is base-cleavable).
  • the free amino groups in the ethylenediamine units are then acylated with the "linker", after which the first amino acid is coupled to the hydroxymethyl group of the "linker” in the form of an Fmoc-protected amino acid anhydride; the coupling process is catalyzed by DMAP.
  • the first amino acid can be coupled to the support, after packing the latter in suitable columns, wells or other reaction containers, by the reaction of the Fmoc-protected amino acid Pfp ester catalyzed by DMAP.
  • the synthesis cycle continues with cleavage of the Fmoc group from the coupled, first amino acid followed by coupling of the next Fmoc-protected amino acid with its carboxyl group suitably activated, e.g. by introducing the Fmoc- protected amino acid in the form of a symmetrical anhydride or an activated ester.
  • the product from this step can be a C-terminal peptide acid, amide, ester or hydrazide (see, e.g., J.M.Stewart and J.D.Young (1984) in Solid Phase Peptide Synthesis, 2nd edn., Pierce Chemical Co., Rockford IL).
  • Appropriate reagents for cleavage from Merrifield type supports include acids such as neat, anhydrous liquid hydro ⁇ gen fluoride (HF) [see e.g.
  • Reagents for cleavage of peptides from "polyamide" resins include trifluoroacetic acid and the above mentioned nucleophi ⁇ les (see, e.g., R.Sheppard; vide supra).
  • nucleophi ⁇ les see, e.g., R.Sheppard; vide supra.
  • peptides or their analogues have been synthesized using the original method, and recent developments has led to an increase of several orders of magnitude in the output of synthetic peptides for use in the elucidation of biological proceses (see e.g. Barany et. al. vide supra).
  • One important aspect is the use of synthetic peptides as vaccines, e.g.
  • BSA bovine serum albumin
  • Glutaraldehyde which is frequently used as coupling agent between a peptide and a carrier protein has the disadvantage of leading to unspecific coupling and not always giving reproducible results.
  • More specific methods use bifunctional linkers such as the N-hydroxysuccinimide ester of -(4-carboxycyclohexylmethyl)- maleimide (see e.g. S.Yoshitake et al. J.Biochem.
  • EP-A1 -0329994 describes an immunogen and its use in recovering antibodies against hemoglobin A1 C.
  • An immunogenic peptide is coupled to a protein by means of a spacer A with the formula
  • X is S or NH and B is an organic residue containing a succinimide group.
  • B is an organic residue containing a succinimide group.
  • the peptide is prepared separatley following a known synthesis protocol. It is then coupled to a protein in a convenient way. The method does not comprise stepwise peptide synthesis on a protein and it does not differ essentially from well-known conjugating methods.
  • a peptide containing the amino acid residues 13 to 24 of the VP1 structural HAV protein is conjugated chemically to a carrier protein.
  • the method of conjugation is the well-known glutaraldehyde method.
  • EP-A1 -0284492 describes novel peptide fractions which induce the formation of protective antibodies against bovine leukemia virus. A process for the preparation of these peptide fractions and their use, e.g. as vaccines, is also described.
  • the peptide is prepared separately according to a known synthesis protocol and conjugated chemically to a carrier protein by well-known methods using hetero- bifunctional reagents.
  • EP-A2-0243929 concerns polypeptide and protein derivatives and a process for their preparation.
  • the method comprises protein and polypeptide derivatives and salts thereof, which are characterized in that a protein or a polypeptide is conjugated via a selected group to the same or a different protein or polypeptide.
  • proteinaceous substances e.g. proteins
  • SPPS solid- phase peptide synthesis
  • the lack of such a methodology may have many causes but is probably due to a general belief that the proteinaceous substance would be lost during the numerous essential washes with solvents and treatments with reagents.
  • the solvents for SPPS generally include solvents such as dichloromethane and N.N-dimethylformamide, and the reagents often comprise trifluoroacetic acid and piperidine. It is therefore imperative that the proteinaceous substrates in question are of very low solubility or are virtually insoluble in media comprising such solvents/reagents in order to permit the carrying out of a satisfactory number of washing steps. This prerequisite can, of course, be verified by very simple weig- hing washing experiments before attempting any actual synthesis, and this constitutes part of the technique of the present invention.
  • albumin The best-described class of proteins among the common carrier proteins is the serum albumins.
  • Several reviews on albumins have appeared in the past few decades (see, e.g., T.Peters, Jr. Adv.Protein Chemistry 37 (1985) 161 -245).
  • albumin the fraction which remained soluble upon dialysis against water was named albumin, and the precipitated proteins were named globulins.
  • albumin is generally taken to mean serum albumin or plasma albumin isolated from man (HSA), cow (BSA) and several other animal species.
  • Albumin's chief traits are those of an acidic, very water-soluble, stable protein. Its isoelectric point when carrying fatty acids is about pH 4.8.
  • Albumins are characterized by having a low number of tryptophan and methionine moieties and a large number of cysteine moieties and of the charged amino acids aspartic acid (41 and 36 in BSA and HSA, respectively), glutamic acid (59 and 62 in BSA and HSA, respectively), lysine (59 in BSA and HSA) and arginine (23 and 24 in BSA and HSA, respectively).
  • the glycine and isoleucine contents are lower than in many other common proteins.
  • Albumins are proteins consisting of a single chain of about 580 residues, cross-linked by 17 disulfide bridges of nine loops - six large and three small - and consist of three alfa-helical domains.
  • T e presence of functional groups such as amino, carboxyiic, and disulfide groups has allowed a number of, mainly single step, chemical modifications to be carried out with serum albumin, keyhole limpet hemocyanin and certain other proteins.
  • modifications include conjugation of peptides to carrier proteins using glutaraldehyde or the above-mentioned bifunctional agents, conjugation of proteins and antibodies with fiuorochromes such as isothiocyanate derivatives of fiuore- scein or rhodamine (see, e.g., J. W.
  • One aspect of the invention relates to a method for the stepwise, controlled synthesis of chemical species, particularly peptides, the method comprising
  • proteinaceous substance conforming to at least one of the following criteria: A. it is a polypeptide, oligopeptide or protein, as defined by the material comprising:
  • amino acid moieties especially at least 50 amino acid moieties, at least two of which are different,
  • B It comprises, as a significant part of its molecular structure, a polypeptide, oligopeptide, or protein as defined under A, the substance being, e.g., a glycoprotein, a lipoprotein, a protein conjugated with nucleic acids, or a substancecomprising mixtures thereof, e.g. a bacterial cell, a bacterial cell wall fragment, or cell extract,
  • any other molecule which is reactive towards available functionalities on the proteinaceous substance so as to form a bond between the proteinaceous substance and the molecule the molecule being, e.g. an amino sugar, a carboxylated sugar, an activated acid, an amine, an aldehyde or a ketone,
  • the reactant in the first step optionally being selected from:
  • reactants which permanently or reversibly blocking selected functional groups, in particular carboxyiic acid groups, in the proteinaceous substance, such as primary or secondary amines, e.g. dialkylamines, e.g. diethylamine, and lower alkanolamines such as 2-ethanolamine, optionally combined with an activating reagent such as diisopropyl carbodiimide, dicyclohexyicarbodiimide or BOP reagent.
  • primary or secondary amines e.g. dialkylamines, e.g. diethylamine
  • lower alkanolamines such as 2-ethanolamine
  • the proteinaceous substance is either a protein or a molecule a significant proportion of which is a protein.
  • the protein (or the protein part of another molecule) is used directly as the substrate for the synthesis, the synthesis either being carried out directly on a native functionality present on the protein, or on a functionality introduced by modification of such a native func ⁇ tionality.
  • Such functionalities comprise amino, carboxyl, hydroxy, and thiol.
  • the gist of the present invention is to introduce, on a specific functionality on the protein, a coupled group via a specific functionality on the coupling reagent, thereby coupling a desired chemical entity to the protein in a functionality-specific manner, as contrasted to the known conjugation of a molecule to a protein by use of high concentrations of bifunctional reagents.
  • the method permits highly specific attachment of e.g. protected amino acids, peptides and other reactants.
  • the method has been found to be very effective and to permit the production of high yields of synthesis product.
  • the method permits highly specific attachment of several different peptides or reactants by controlled use of different functionalities on the protein as explained in the following.
  • the reactant may be an activated amino acid or an activated peptide, or the reactant may, in principle, be any molecule which can react with a functionality (native or provided) on the protein, e.g., linker molecules for establis ⁇ hing cleavable linking and/or spacing linking for later reaction cycles, or active molecules such as therapeutically active molecules, e.g. molecules of a me- dicament, such as, e.g. a steroid.
  • a functionality e.g., linker molecules for establis ⁇ hing cleavable linking and/or spacing linking for later reaction cycles
  • active molecules such as therapeutically active molecules, e.g. molecules of a me- dicament, such as, e.g. a steroid.
  • One of the critical features of the invention is use of the proteinaceous substance as the "solid-phase" substrate for the synthesis.
  • This function of the proteinaceous substance is conditioned by the substance being capable of being retained on a filter during the steps of reagent removal and washing, and preferably during the coupling steps.
  • Most proteins are substantially insoluble in a large number of solvents useful in these steps of the method of the invention, such as DCM, DMF, piperidine/DMF, ethyl acetate, etc., and this substantial insolubility is thus utilized according to the invention to allow separation of the reactants from the reaction products using the proteinaceous substance itself as the insoluble substrate.
  • suitable molecular weight exclusion limits are, e.g., from 500 to 300,000, such as, e.g., 1000, 5000, 10,000, 30,000, 50,000, 100,000.
  • the ex ⁇ clusion limit employed for a given proteinaceous substance will, of course, be chosen in accordance with the molecular weight of the substance and the reac ⁇ tants.
  • the "blocking" reagents are typically amines, such as primary or secondary amines.
  • the effect of the "blocking” is either ascribable to raising the pH in the microen- vironment adjacent to the proteinaceous substance, in which case addition of non- reacting base such a trialkylamine such as triethylamine should produce a similar effect, or ascribable to a certain "denaturing" involving modification of the confor ⁇ mation of the protein.
  • the protein part of the proteina- ceous substance is selected from naturally occurring proteins and modified naturally occurring proteins, such as:
  • albumins in particular serum albumins (SA), including human serum albumin (HSA) and bovine serum albumin (BSA), parvoalbumin, ⁇ -lactalbumin, ovalbumin, gelatin, lactoferrin, ⁇ -lactoglobulin, histones, keratins, keyhole limpet hemocyanin (KLH), tetanous toxoid (TNT), cholera toxin, hirudin, purified protein derivative (PPD), prolamins, hemocyanin, globins, hemoglobins, including methe- moglobin, orosomucoid, transferrin, ferritin, apoferritin, ferritoxin, ceruloplasmin, haptoglobin, fibrinogen, fibrin, blood coagulation factors, Cohn blood fractions, agglutinins, plasmin, avidin, globulins, including ⁇ -globuiins, e.g., ⁇ -globul
  • the reactant is an amino acid as defined under (2) (i) or a peptide as defined under (2) (ii) above
  • the cycle comprising steps (2)-(4) is performed two or more times with either such an amino acid or such a peptide in each step, the amino acid being the same or different in each step, and the peptide being the same or different in each step, to build up peptide chains on amino groups on the proteinaceous substance, the following steps being performed after each respective step (4):
  • amino groups of the proteinaceous substance may be first reacted with a linker reactant which, when coupled to the proteinaceous substan ⁇ ce, provides a linker group useful as reaction target for an amino acid as defined under (2) (i) or a peptide as defined under (2) (ii) above, the linker group being a group which is capable of being cleaved under conditions which substantially do not deteriorate the peptide formed on the linker group, the method optionally further comprising:
  • the proteinaceous substance may be subjected, in a first step, to a treatment or treatments converting functional groups other than amino groups to linker groups useful as reaction target for a reactant selected from those defined under (2) (i), (2) (ii) and (2) (iii), the linker groups being selected from:
  • the groups thus converted may be, e.g.:
  • carboxyiic acid groups which are converted into an amino-terminated group by treatment with a bifunctional compound, such as a diamine, e.g. ethylenediamine, capable of forming an amide bond with the carboxyiic acid group and providing a free amino target group,
  • a bifunctional compound such as a diamine, e.g. ethylenediamine, capable of forming an amide bond with the carboxyiic acid group and providing a free amino target group
  • hydroxy groups which, e.g., are reacted with an ⁇ -haloacid to form a carboxy- terminated group which is subsequently converted into an amino-terminated group by treatment with a bifunctional compound, such as a diamine, e.g. ethylenediami ⁇ ne, capable of forming an amide bond with the carboxyiic acid group and providing a free amino target group, disulfide bonds, which are first reduced to SH groups and then reacted with an haloacid to form a carboxy-terminated group which is subsequently converted i an amino-terminated group by treatment with a bifunctional compound, such a diamine, e.g. ethylenediamine, capable of forming an amide bond with the car ⁇ boxyiic acid group and providing a free amino target group.
  • a bifunctional compound such as a diamine, e.g. ethylenediami ⁇ ne, capable of forming an amide bond with the carboxyiic acid group and providing a free amino
  • the method of the invention makes it possible to prepare a large number of coupled products comprising a proteinaceous substance conforming to at least of the following criteria:
  • amino acid moieties preferably at least 20 amino acid moieties, of which preferably at least two are different, in particular at least 30 amino acid moieties, of which preferably at least two are different,
  • amino acid moieties especially at least 50 amino acid moieties, at least two of which are different,
  • B It comprises, as a significant part of its molecular structure, a polypeptide, oligopeptide, or protein as defined under A, the substance being, e.g., a glycoprotein, a lipoprotein, a protein conjugated with nucleic acids, or a substance comprising mixtures thereof, e.g. a bacterial cell, wall fragment, or a cell extract,
  • the multitude of coupled groups may be a number of groups less than or equal to the number of the relevant functional groups on the polypeptide, oligopeptide or protein part of the proteinaceous substance.
  • the extent of coupling may be controlled in each particular case by adjustment of the reaction conditions, such as the concentration of the reactant, the duration of the treatment, etc.
  • a particularly interesting coupled product is a product carrying a multitude of groups coupled thereto, the groups comprising at least two different species selected from:
  • an interesting coupled product is one in which the coupled group or groups is/are a peptide group or peptide groups selected from one or several peptide species, in particular where the peptide group or groups is/are selected from the group consisting of peptide hormones, and precursors, fragments, derivatives, antago ⁇ nists and agonists thereof, the peptide hormones being, in particular, peptide hormones selected from the group consisting of
  • ⁇ -endorphin ⁇ -lipotropin, ⁇ -endorphin, gastrin releasing peptide, growth hormone releasing factor, neurotensin, ⁇ -melanocyte stimulating hormone, adrenocorticotropic hormone, vasopressin, angiotensin, substance P, dynorphin, bradykinin, luteinising hormone releasing hormone, parathyroid hormone, atrial natriuretic factor, atriopeptin, calcitonin, calcitonin gene related peptide, dermo hin, kyotorpin, auriculin, ⁇ -casomo ⁇ hin, tachykinin, bombesin, caerulein, chorionic gonadotropin, cholecystokinin, casomo ⁇ hin, corticotropin, corticotropin releasing factor, corticotropin inhibiting peptide, somatostatin, eukephalin, gastric inhibitory peptide, gastrin,
  • peptide group or groups is/are selected from the group consisting of peptide growth factors, and precursors, fragments, derivatives, antagonists and agonists thereof, the peptide growth factors being, in particular, peptide growth factors selected from the group con ⁇ sisting of
  • fibroblast growth factor transforming growth factor ⁇ , transforming growth factor ⁇ I and II, insulin, cachetin, epidermal growth factor, bursin, insulin-like growth factor I, insulin-like growth factor II, inhibin, liver cell growth factor (Gly His Lys), nerve growth factor, platelet derived growth factor, tuftsin, and interleukins.
  • Another interesting coupled product is one in which the peptide group or groups is/are selected from the group consisting of synthetic fragments of proteins with defined biological effect, and derivatives, fragments and analogues thereof, the proteins being, in particular, proteins selected from the group consisting of
  • fibronectin cell attachment sequence Gly Asp Arg
  • Ser Gly Asp Arg Gly leucine zipper sequence of, e.g., c-fos or c-jun, helix-turn helix DNA binding sequence, a protease inhibitor, a kinase inhibitor, a muramyl peptide, zinc finger sequence,
  • nuclear translocation signal such as Lys Lys Lys Arg Lys.memrane translocation signal and glycosylation sequence (Asn X Ser/Thr), homeobox sequences,
  • pro ⁇ teinaceous substance moiety is an albumin, especially human serum albumin or bovine serum albumin.
  • Coupled products are products in which the protein is purified protein derivative (PPD) or tetanus toxoid (TNT). Specifically intersting classes of coupled products are:
  • a coupled product which is human serum albumin (HSA) coupled to a hormone, and in particular being selected from the group consisting of
  • HSA-B-endo ⁇ hin HSA- ⁇ -lipotropin
  • HSA- ⁇ -endorphin HSA-gastrin releasing peptide
  • HSA-growth hormone releasing factor HSA-neurotensin
  • HSA- ⁇ -melanocyte stimulating hormone HSA-adrenocorticotropic hormone
  • HSA-vasopressin HSA-vasopressin
  • HSA-parathyroid hormone HSA-atrial natriuretic factor, HSA-atriopeptin, HSA-calcitonin, HSA-calcitonin gene related peptide,
  • HSA-dermo ⁇ hin HSA-kyotorpin
  • HSA-auriculin HSA- ⁇ -casomo ⁇ hin
  • HSA-tachykinin HSA-tachykinin
  • HSA-bombesin HSA-caerulein, HSA-chorionic gonadotropin, HSA-cholecystokinin, HSA-casomo ⁇ hin, HSA-corticotropin,
  • HSA-corticotropin inhibiting peptide HSA-somatostatin
  • HSA-gastrin related peptide HSA-thyrotropin releasing hormone
  • HSA-thyroid stimulating hormone HSA-proIactin
  • HSA-neuromedin B, C, K, L, and U HSA-neuromedin B, C, K, L, and U
  • HSA-neurotensin HSA-oxytocin
  • the peptide species coupled to the HSA is a precursor, a fragment, a derivative or an analogue (e.g. an antagonist or an agonist) of the hormone;
  • HSA human serum albumin
  • HSA-insulin HSA-cachetin
  • HSA-insulin-like growth factor II HSA-inhibin
  • HSA-liver cell growth factor Gly His Lys
  • a functional group in HSA an amino group, a carboxy group, or a hydroxy group converted into a derivative containing an amino group or a carboxy group
  • the HSA molecule carrying one or,
  • HSA human serum albumin
  • HSA-fibronectin cell attachment sequence (Gly Asp Arg)
  • HSA-leucine zipper sequence of e.g. c-fos or c-jun
  • HSA-protease inhibitor HSA-kinase inhibitor
  • HSA-nuclear translocation signal such as Lys Lys Lys Arg Lys, HSA-memrane translocation signal,
  • the coupled peptide is a derivative, fragment or analog thereof; a coupled product which is bovine serum albumin (BSA) coupled to a hormone, and in particular being selected from the group consisting of:
  • BSA bovine serum albumin
  • BSA- ⁇ -lipotropin BSA- ⁇ -endorphin, BSA-gastrin releasing peptide, BSA-growth hormone releasing factor, BSA-neurotensin,
  • BSA- ⁇ -melanocyte stimulating hormone BSA-adrenocorticotropic hormone, BSA-vasopressin, BSA-angiotensin, BSA-substance P,
  • BSA-luteinising hormone releasing hormone BSA-parathyroid hormone, BSA-atrial natriuretic factor,
  • BSA-calcitonin gene related peptide BSA-dermorphin, BSA-kyotorpin,
  • BSA-auriculin BSA- ⁇ -casomorphin, BSA-tachykinin, BSA-bombesin, BSA-caerulein,
  • BSA-chorionic gonadotropin BSA-cholecystokinin, BSA-casomo ⁇ hin, BSA-corticotropin, BSA-corticotropin releasing factor, BSA-corticotropin inhibiting peptide,
  • BSA-eukephalin BSA-gastric inhibitory peptide
  • BSA-neurokinin A and B BSA-neuromedin B, C, K, L, and U,
  • BSA-pancreastatin BSA-vasoactive intestinal peptide
  • a functional group in BSA an amino group, a carboxy group, or a hydroxy group converted into a derivative containing an amino group or a carboxy group
  • the BSA molecule carrying one or, preferably,
  • BSA bovine serum albumin
  • BSA-fibroblast growth factor BSA-transforming growth factor a
  • BSA-epidermal growth factor BSA-bursin
  • BSA-liver cell growth factor Gly His Lys
  • BSA-nerve growth factor BSA-nerve growth factor
  • the BSA molecule carrying one or, preferably, a multitude of the groups coupled thereto, the multitude being a number less than or equal to the number of the relevant functional groups on the BSA molecule,
  • the peptide species coupled to the HSA is a precursor, a fragment, a derivative or an analogue (e.g. an antagonist or an agonist) of the growth factor;
  • BSA bovine serum albumin
  • BSA-fibronectin cell attachment sequence Ser Gly Asp Arg Gly
  • BSA-leucine zipper sequence e.g. c-fos or c-jun
  • BSA-nuclear translocation signal such as Lys Lys Lys Arg Lys
  • the hyphen designating a coupling amide bond between a functional group in BSA an amino group, a carboxy group, or a hydroxy group converted into a derivative containing an amino group or a carboxy group
  • a functional group of the synthetic fragment in question an amino group, a carboxy group, or a hydroxy group converted into a derivative containing an amino group or a carboxy group
  • the BSA molecule carrying one or, preferably, a multitude of the groups coupled thereto, the multitude being a number less than or equal to the number of the relevant functional groups on the BSA molecule
  • a coupled product which is purified protein derivative (PPD) coupled to a hormo- ne, and in particular being selected from the group consisting of: PPD- ⁇ -endor ⁇ hin,
  • PPD- ⁇ -endorphin PPD-gastrin releasing peptide
  • PPD-adrenocorticotropic hormone PPD-vasopressin
  • PPD-bradykinin PPD-luteinising hormone releasing hormone
  • PPD- ⁇ -casomorphin PPD-tachykinin
  • PPD-corticotropin inhibiting peptide PPD-somatostatin
  • PPD-gastric inhibitory peptide PPD-gastrin
  • PPD-growth hormone PPD-growth hormone releasing factor
  • PPD-neuromedin B, C, K, L, and U PPD-neuropeptide Y
  • PPD-vasoactive intestinal peptide PPD-secretin
  • the PPD molecule carrying one or, preferably, a multitude of the hormone groups coupled thereto, the multitude being a number less than or equal to the number of the relevant functional groups on the PPD molecule, and the corresponding coupled products where the peptide species coupled to the PPD is a precursor, a fragment, a derivative or an analogue (e.g. an antagonist or an agonist) of the hormone;
  • a coupled product which is purified protein derivative (PPD) coupled to a peptide growth factor, and in particular being selected from the group consisting of:
  • PPD-fibroblast growth factor PPD-transforming growth factor ⁇
  • PPD-epidermal growth factor PPD-bursin
  • PPD-liver cell growth factor Gly His Lys
  • PPD-nerve growth factor PPD-nerve growth factor
  • the PPD molecule carrying one or, preferably, a multitude of the groups coupled thereto, the multitude being a number less than or equal to the number of the relevant functional groups on the PPD molecule,
  • the peptide species coupled to the PPD is a precursor, a fragment, a derivative or an analogue (e.g. an antagonist or an agonist) of the growth factor;
  • PPD purified protein derivative
  • PPD-fibronectin cell attachment sequence (Gly Asp Arg)
  • PPD-fibronectin cell attachment sequence Ser Gly Asp Arg Gly
  • PPD-leucine zipper sequence e.g. c-fos or c-jun
  • PPD-nuclear translocation signal such as Lys Lys Lys Arg Lys
  • TNT tetanus toxoid
  • TNT-growth hormone releasing factor TNT-neurotensin
  • TNT- ⁇ -melanocyte stimulating hormone TNT-adrenocorticotropic hormone
  • TNT-vasopressin TNT-vasopressin
  • TNT-angiotensin TNT-substance P, TNT-dynorphin, TNT-bradykinin, TNT-luteinising hormone releasing hormone,
  • TNT-parathyroid hormone TNT-atrial natriuretic factor
  • TNT-atriopeptin TNT-calcitonin
  • TNT-calcitonin gene related peptide TNT-calcitonin gene related peptide
  • TNT-dermorphin TNT-kyotorpin
  • TNT-auriculin TNT- ⁇ -casomo ⁇ hin
  • TNT-auriculin TNT-auriculin
  • TNT- ⁇ -casomo ⁇ hin TNT-tachykinin
  • TNT-bombesin TNT-caerulein, TNT-chorionic gonadotropin, TNT-cholecystokinin, TNT-casomo ⁇ hin,
  • TNT-corticotropin TNT-corticotropin releasing factor, TNT-corticotropin inhibiting peptide, TNT-somatostatin, TNT-eukephalin, TNT-gastric inhibitory peptide,
  • TNT-gastrin related peptide TNT-thyrotropin releasing hormone
  • TNT-thyroid stimulating hormone TNT-prolactin
  • TNT-neuromedin B, C, K, L, and U TNT-neuromedin B, C, K, L, and U
  • TNT-neurotensin TNT-oxytocin
  • TNT-galanin TNT-pancreatic polypeptide
  • the peptide species coupled to the TNT is a precursor, a fragment, a derivative or an analogue (e.g. an antagonist or an agonist) of the hormone; a coupled product which is tetanus toxoid (TNT) coupled to a peptide growth factor, and in particular being selected from the group consisting of:
  • TNT-cachetin TNT-epidermal growth factor
  • TNT-inhibin TNT-liver cell growth factor (Gly His Lys)
  • the TNT molecule carrying one or, preferably, a multitude of the hormone groups coupled thereto, the multitude being a number less than or equal to the number of the relevant functional groups on the TNT molecule,
  • TNT is a precursor, a fragment, a derivative or an analogue (e.g. an antagonist or an agonist) of the growth factor;
  • TNT tetanus toxoid
  • TNT-fibronectin cell attachment sequence (Gly Asp Arg) TNT-fibronectin cell attachment sequence (Ser Gly Asp Arg Gly),
  • TNT-leucine zipper sequence of e.g. c-fos or c-jun
  • TNT-kinase inhibitor TNT-muramyl peptide
  • TNT-nuclear translocation signal such as Lys Lys Lys Arg Lys
  • TNT-memrane translocation signal TNT-memrane translocation signal
  • TNT-gfycosylation sequence Asn X Ser/Thr
  • the above-mentioned "polyamide"-type or Merrifi- eld-type reagent strategies, or any other solid-phase peptide synthesis reagent strategy using Fmoc, Boc or any other alfa-amino protecting group or side-chain protecting group may be employed using albumins or other proteinaceous substances as solution- or solid-phase substrates to synthesize peptides in a free state or attached (conjugated) to the proteinaceous macromolecular substrate.
  • Any appropriate kind of chemical method for further substitution of the conjugate product with other functions such as lipid functionalities, alcohol groups, etc., may be used before or after peptide synthesis.
  • One aspect of a method for solid-phase peptide-protein synthesis according to the invention affords peptides directly attached to amino groups of the protein.
  • polyamide polyamide
  • Second, third, etc. amino acids to be coupled are introduced in the form of Fmoc-protected amino acid O-Dhbt or O-Pfp esters or Fmoc-protected amino acids activated with dicyclohexylcarbodiimide, BOP-reagent or a similar activation agent.
  • the Dhbt- ester derivatives have the great advantage that the liberated Dhbt-OH reacts with remaining amino groups present with the formation of the strongly yellow-coloured anion Dhbt-O ⁇ - which disappears gradually as the amino groups become acylated.
  • Dhbt indicator A detailed description of the chemistry involved in this "Dhbt indicator" reaction is given by Atherton et al. (J.Chem.Soc. Perkin Trans. 1 (1988) 2887)].
  • the amino groups of the macromolecule may be substituted with a non-cleavable linker, such 6-aminohexanoic acid or any other suitable linker, attached e.g. by the carboxyiic group of the aminohexanoic acid to the amino groups of the macromolecule and the amino group of the aminohexanoic acid then used for peptide synthesis.
  • Carboxyiic acid groups may be protected e.g. by transformation into a diethylamido group by treatment with diethylamine in the presence of dicyclohexylcarbodiimide as activating agent.
  • a cleavable linker group is coupled to the amino groups of the protein.
  • the above-mentioned Fmoc-strategy or any other solid-phase or solution chemistry method may be used.
  • the above-mentioned Fmoc-protected amino acid anhydride/DMAP strategy is employed for coupling of the first amino acid, and piperidine dissolved in DMF is used as base for cleavage of the Fmoc group (i.e. deprotection) from this and the subsequent amino acids.
  • amino acids to be coupled are introduced in the form of Fmoc-protected amino acid ester as described above.
  • treament with trifiouroacetic acid causes cleavage of the side-chain protecting groups and cleavage of the peptide from the protein support.
  • the mixture of peptide and protein dissolves in the reagent and the products are recovered by addition of ether or simple evaporation of the solvent.
  • the peptide is separated from the protein carrier by conventional gel filtration or dialysis.
  • carboxyiic acid groups may be protected, e.g. by diethyla midation with diethylamine/dicyclohexylcarbodiimide.
  • a protein substrate in which disulfide groups are reduced to thiol groups followed by treatment with iodoacetic acid to give a S- carboxymethylated protein.
  • Peptide synthesis may then be performed according to the invention.
  • a proteinaceous substrate is used in which amino and/or carboxyiic groups are reversibly protected followed by conversion of other func ⁇ tional groups such as -OH (Ser, Tyr, Thr, etc.), -SH (Cys, etc.), guanidino (Arg, etc) or other groups in the substrate into amino or carboxyiic groups which can then be used for stepwise solid-phase synthesis.
  • the above mentioned aspects are used in combination to build up several different peptide chains with or without biological activity, such as hormones, growth factors, cytokines, cell attachment sequences, etc.
  • a protein, glycoprotein, lipoprotein, or combination of proteins including cell extracts etc. may be used as starting point for the chemical stepwise synthesis of, e.g., sugars, vitamins, lipids, steroids, cytotoxic compounds or other chemical compounds.
  • one aspect made possible through the invention is a method of producing an antibody specific for a peptide such as a peptide hormone, a peptide growth factor, or a peptide with another defined biological effect, which comprises immunizing an animal with a coupled product according to the invention in which the molecule coupled to the proteinaceous substance is the peptide to which the specific antibody is to be raised, or an immunogenically effective part of the peptide, to make cells in the animal produce an antibody specific for said peptide, and obtaining such cells from the animal, or isolating the antibody from serum or plasma of the animal.
  • a coupled product according to the invention in which the molecule coupled to the proteinaceous substance is the peptide to which the specific antibody is to be raised, or an immunogenically effective part of the peptide
  • This method may further comprise:
  • the animal immunized may be selected from the group consisting of rabbit, monkey, sheep, goat, mouse, rat, pig, horse, and guinea pig.
  • the cells producing the antibody are normally spleen or lymph cell.
  • Another interesting aspect of the invention is solid phase enzyme immunoassay or radioimmunoassay using as the solid phase coupled protein-peptide products according to the invention.
  • Coupled products of the invention are alse highly interesting as vaccines, in particular when the protein is a serum protein.
  • one aspect of the invention is a method of immunising an animal, including a human being, e.g. against a disease caused by a microorganism or a virus, comprising parenterally admini ⁇ stering a coupled product according to the invention in which the coupled group is an immunogenic peptide sequence of a protein, e.g. a protein of a microorganism or virus.
  • a further aspect of the invention is a method of purifying antibodies against a peptide, comprising applying a medium containing the antibodies to a suspension or solution of a coupled product according to the invention carrying a coupled peptide, to bind the peptide-specific antibodies to the peptide moiety of the coupled product, washing to remove unbound antibodies, cleaving the antibodies from the peptide moiety, and separating the antibodies from the medium, e.g. by dialysis.
  • the present invention relates to the use of the FS- coupled product (Functionality-Selectively coupled product) of the invention for immunizing purposes.
  • the preparation of the FS-coupled product should be made so as to allow an optimal stimulation of the relevant parts of the immune system, i.e to present the immunogenic bound group for a period of time and in a form being optimal with respect to the recognization, the uptake or any other interaction or processing necessary for the stimulation.
  • the FS-coupled product of the invention may solely comprise T-cell epitopes or solely B-cell epitopes or a combination of these.
  • the composition of the FS- coupled groups of the invention may be tailored to their intended use, e.g. their use as a vaccine component.
  • B-cell epitopes are advantageous for most applica tions as they are required for eliciting an antibody production.
  • T-cell epitopes are extremely advantageous as they enhance and accelerate the immune response and the production of antibodies.
  • the memory function of the immune system resides in the T-cells. By stimulating this part of the immune system, the antibody production is significant after approximately 5 days. If the memory function is not participating, e.g.
  • the antibody production is significant after several weeks, consisting primarily of low avidity IgM antibodies and after several months consisting of IgG antibodies of higher avidity.
  • the FS-coupled product of the invention is contemplated to be useful as a prophylactic or therapeutic agent, e.g. a vaccine, or in a diagnostic kit and may be used in the manufacture thereof. This will be explained below.
  • the term "vaccine” is to be understood to comprise any preparation containing an immunologically effective part of a FS-coupled product suited for administration to living organisms for the prevention, amelioration or treatment of e.g. a bacterial or viral infection or hormonal disturbance.
  • immunological is understood to comprise the process of evoking a specific immunologic response with the expectation that this will result in humoral, and/or secretory, and/or cell-mediated immunity to the infection, i.e. immunity is to be understood to comprise the ability of the individual to resist or overcome infection or to overcome infection "more easily” compared to individuals who have not been immunized or to tolerate the infection without being clinically affected or to block transmission.
  • the immunization according to the present invention is a process of increasing re ⁇ sistance to the bacterial or viral infection.
  • An overall aspect in the preparation of the vaccines of the invention is the physiological acceptability of the components and of the total composition of the vaccine.
  • the final formulation of the vaccine should be a mixture of substances supporting and enhancing the immune respon ⁇ se induced by the specific immunogenic component.
  • a multivalent vaccine is for ⁇ mulated, i.e. several immunologically effective components are incorporated into a single vaccine being effective in reducing infection, and/or transmission - all in all inducing an effective protective immunity.
  • the vaccine may comprise one or more selected specific molecules in order to provide the multivalent nature of the vaccine.
  • Especially interesting additional molecules are immunologically active molecules obtained from pathogenic organisms which give rise to a vaccine being effective in reducing infection or providing immunity for one or more pathogenic organisms.
  • Routine methods for vaccine production involve risks of obtaining unwanted side effects, e.g. due to the vaccine containing unwanted (or even unidentified) conta ⁇ minants.
  • the methods of preparation of vaccines according to the present in ⁇ vention are designed to ensure that the identity and immunological effectiveness of the specific molecules are maintained and that no unwanted microbial contami ⁇ nants are introduced.
  • the final products are distributed under aseptic conditions into preferably sterile containers which are then sealed to exclude extraneous microorganisms.
  • the vaccine may further comprise an adjuvant in order to increase the immunoge- nicity of the vaccine preparation.
  • the adjuvant may serve the purpose of enhan- cing the stimulatory properties of the polypeptide by stimulating the production of cytokines or lymphokines from the cells of the immune system in a non-specific way.
  • the adjuvant may be selected from the group consisting of Freund's in ⁇ complete adjuvant, aluminium hydroxide, a saponin, a muramyl peptide, a lipopo- lysaccharide, a T-cell immunogen, interleukin-2, interferon-gamma, an oil, such as a vegetable oil, e.g. peanut oil, or a mineral oil, e.g. silicone oil, and B.C.G.
  • the bound group may advantageously remain coupled to the proteinaceus substance, which may be any carrier usually employed in the preparation of vaccines.
  • the proteinaceus substance may be a e.g. a polypeptide such as a serum protein to which the selected immunologically interesting group or groups are covalently or non-covalently bound.
  • the carrier should preferably be non-toxic and non-allergenic such as serum albumin.
  • the bound group may be multivalently bound to the macromolecular carrier as this provides an increased immunogenicity of the vaccine preparation. In this regard, it may prove advantageous to couple several different groups to the carrier so as to obtain a vaccine comprising a variety of different immunogenic determinants, i.e. a cocktail vaccine, which may be employed for the immunization of diseases caused by a variety of different organisms.
  • immunization schedules may be employed when using the vaccine of the invention: In some instances it may be appropriate to provide active immunization early in life. Furthermore, it may be desirable to employ repeated administrations, e.g. at regular or prolonged intervals, optionally - as far as injections are con- cerned - at various body sites, e.g. at the same time. Any immunization schedule which may be contemplated or shown to produce an appropriate immune response can be employed in accordance with the principles of the present invention.
  • the vaccine should be administered in a way which ensures an efficient stimula- tion of the immune system. This means that the vaccine should be brought into contact with the cells of the immune system for a sufficient period of time and in a form capable of functioning as an immunogen.
  • Several ways are possible. Of these the most conventional are the parenteral ways, i.e., the subcutaneous, intradermal, intramuscular or the intravenous route.
  • the aerosol vaccine is in most cases administered via the nasal route. It is known that peptides can be transported intactly through the nasal mucosa to reach the blood. When transported further down the respiratory tract, the antigen is taken up by the macrophages functioning as scavengers and is in this way potentially presented to the immune system. Some of the material administered as an aerosol may possibly reach the intestines and stimulate the immune system present in the intestines and this way stimulate the immune system of the body, or may be taken up by the intestinal mucosa in intact form and liberated to the blood stream where it will be presented for the immune system.
  • the vaccine may also be administered strictly via the nasal route. This way simplifies the administration and circumvents the problems associated with spreading of infectious diseases through multiple use of syringes.
  • Another aspect of the invention is a monoclonal or polyclonal antibody reactive with a bound group on the FS-coupled product, or an immunologically subsequ ⁇ ence thereof, and a method for the preparation thereof.
  • antibody refers to a substance which is produced by a mammal or a more precisely a cell of mammalian origin belonging to the immune system as a response to exposure to the FS-coupled products of the invention.
  • the antibodies of the present invention may be produced by a method which comprises administering to an animal the FS-coupled product of the invention thereby presenting the bound groups of the FS-coupled product in an immunoge ⁇ nic form so as to make cells producing antibodies reactive with said bound groups, and isolating the ceils producing the antibodies or serum or plasma containing the antibodies from the animal.
  • the methods of producing the antibodies of the present invention will be explained further below.
  • the antibody is preferably a monospecific antibody.
  • the monospecific antibody may be prepared by injecting to a suitable animal a substantially pure preparation of the FS-coupled product of the invention followed by one or more booster injections at suitable intervals (e.g. one or two weeks to a month) up to four or five months before the first bleeding.
  • suitable intervals e.g. one or two weeks to a month
  • the established immunization schedule is continued, and the animals are bled about one week after each booster immu ⁇ nization, and antibody is isolated from the serum in a suitable manner (cf. e.g. Harboe and Ingild, Scand. J. Immun. 2 (Suppl. 1), 1973, pp.
  • the antibody may be a polyclo ⁇ nal antibody.
  • Polyclonal antibodies may be obtained, e.g. as described in Harboe and Ingild, see above. More specifically, when polyclonal antibodies are to be obtained, the FS-coupled product is, preferably after addition of a suitable adju ⁇ vant, such as Freund's incomplete or complete adjuvant such as a muramyl peptide, injected into an animal.
  • a suitable adju ⁇ vant such as Freund's incomplete or complete adjuvant such as a muramyl peptide
  • the animals may be rabbits.
  • the animals are bled regularly, for instance at weekly intervals, and the blood obtained is separated into an antibody contai- ning serum fraction, and optionally said fraction is subjected to further conventional procedures for antibody purification, and/or procedures involving use of a purifica ⁇ tion procedure in which the FS-coupled product in question is used.
  • monoclonal antibodies are obtained.
  • the monoclonal antibody may be raised against or directed substantially against an essential component of the bound group of the FS-coupled product, i.e. an epitope.
  • the monoclonal antibody may be produced by conventional techniques (e.g. as described by Kohler and Milstein, Nature 256, 1975, p. 495) e.g. by use of a hybridoma cell line, or by clones or subclones thereof or by cells carrying genetic information from the hybridoma cell line coding for said monoclonal antibody.
  • the monoclonal antibody may be produced by fusing cells producing the monoclonal antibody with cells of a suitable cell line, and selecting and cloning the resulting hybridoma cells producing said monoclonal antibody.
  • the monoclonal antibody may be produced by immortalizing an unfused cell line producing said monoclonal antibody, subsequently growing the cells in a suitable medium to produce said antibody, and harvesting the monoclonal antibody from the growth medium.
  • the immunized animal used for the preparation of antibodies of the invention is preferably selected from the group consisting of rabbit, monkey, sheep, goat, mouse, rat, pig, horse and guinea pigs.
  • the cells producing the antibodies of the invention may be spleen cells or lymph cells, e.g. peripheric lymphocytes.
  • hybridoma cells When hybridoma cells are used in the production of antibodies of the invention, these may be grown in vitro or in a body cavity of an animal.
  • the antibody- producing cell is injected into an animal such as a mouse resulting in the formation of an ascites tumour which releases high concentrations of the antibody in the ascites of the animal.
  • the animals will also produce normal antibodies, these will only amount to a minor percentage of the monoclonal antibodies which may be purified from ascites by standard purification procedures such as centrifu- gation, filtration, precipitation, chromatography or a combination thereof.
  • a purifi ⁇ cation procedure using the FS-coupled product in question is especially preferred.
  • An example of a suitable manner in which the monoclonal antibody may be produced is as a result of fusing spleen cells from immunized mice (such as Balb/c mice) with myeloma cells using conventional techniques (e.g. as described by R. Dalchau, J. Kirkley, J.W. Fabre, "Monoclonal antibody to a human leukocyte — specific membrane glycoprotein probably homologous to the leukocyte-common (L-C) antigen of the rat", Eur. J. Immunol. 10, 1980, pp. 737-744).
  • the antibodies resulting from the obtained fusions are screened by conventional techniques such as binding assays e.g. an ELISA assay employing the FS-coupled product in question.
  • the FS-coupled products of the invention are useful in e.g. a diagnostic agent in which the presence of antibodies against the bound group of the FS-product in question is to be determined.
  • the diagnostic agent may be one which is suited for use in an agglutination assay in which the solid particles to which the FS-coupled product is coupled agglutinate in the presence of antibodies against the bound group of the FS-coupled product in the sample subjected to testing. In this type of testing, no labelling of the antibody is necessary. For most uses it is, however, preferred that the FS-coupled product is provided with a label for the detection of bound antibody.
  • the substance used as label may be selected from any sub ⁇ stance which is in itself detectable or which may be reacted with another substan- ce to produce a detectable product.
  • the label may be selected from radioac ⁇ tive isotopes, enzymes, chromophores, fluorescent or chemiluminescent substan ⁇ ces, and complexing agents.
  • enzymes useful as labels are ⁇ -galactosidase, urease, glucose oxidase, carbonic anhydrase, peroxidases (e.g. horseradish peroxidase), phospha tases (e.g. alkaline or acid phosphatase), glucose-6-phosphate dehydrogenase and ribonuclease.
  • peroxidases e.g. horseradish peroxidase
  • phospha tases e.g. alkaline or acid phosphatase
  • glucose-6-phosphate dehydrogenase ribonuclease.
  • Enzymes are not in themselves detectable, but must be combined with a substrate to catalyze a reaction the end product of which is detectable.
  • a substrate may be added to the reaction mixture resulting in a coloured, fluorescent or chemiluminescent product or in a colour change or in a change in the intensity of the colour, fluorescence or chemiluminescence. Examples of substrates which are useful in the present method as substrates for the enzymes mentioned above are
  • the substrate may be combined with, e.g. a chromophore which is either a donor or acceptor.
  • Fluorescent substances which may be used as labels for the detection of the components as used according to the of invention may be 4-methylumbelliferyl-p- hosphate, 4-methylumbell.feryl-D-galactopyranoside, and 3-(p-hydroxyphenyl) propionic acid. These substances may be detected by means of a fluorescence spectrophotometer. Chemiluminescent substances which may be peroxidase/eo- sin/EDTA, isoluminol/-EDTA/H 2 O 2 and a substrate therefor.
  • Chromophores may be o-phenylenediamine or similar compounds. These sub ⁇ stances may be detected by means of a spectrophotometer. Radioactive isotopes may be any detectable and in a laboratory acceptable isotope, e.g. 125 l, 131 l, 3 H, "P, ⁇ S or 14 C. The radioactivity may be measured in a laboratory acceptable isotope, e.g. 125 l, 131 l, 3 H, "P, ⁇ S or 14 C. The radioactivity may be measured in a laboratory acceptable isotope, e.g. 125 l, 131 l, 3 H, "P, ⁇ S or 14 C. The radioactivity may be measured in a laboratory acceptable isotope, e.g. 125 l, 131 l, 3 H, "P, ⁇ S or 14 C. The radioactivity may be measured in a laboratory acceptable isotope, e.g. 125 l, 131 l, 3
  • Complexing agents may be Protein A, Protein G (which forms a complex with immunoglobulins), biotin (which forms a complex with avidin and streptavidin), and lectin (which forms a complex with carbohydrate determinants, e.g. receptors).
  • the complex is not in itself directly detectable, necessitating labelling of the substance with which the complexing agent forms a complex.
  • the marking may be performed with any of the labelling substances described above.
  • an antibody or a FS-coupled product of the invention may be coupled to a bridging compound coupled to a solid support.
  • the bridging compound, which is designed to link the solid support and the antibody may be hydrazide, Protein A, glutaraldehyde, carbodiimide, or lysine.
  • the solid support employed is e.g. a polymer or it may be a matrix coated with a polymer.
  • the matrix may be of any suitable solid material, e.g. glass, paper or plastic.
  • the polymer may be a plastic, cellulose such as specially treated paper. nitrocellulose paper or cyanogenbromide-activated paper.
  • E ⁇ xamples of suitable plastics are latex, a polystyrene, polyvinylchloride, polyurethane, polyacrylamide, polyvinylacetate and any suitable copolymer thereof.
  • E ⁇ xamples of silicone poly ⁇ mers include siloxane.
  • the solid support may be in the form of a tray, a plate such as a mitrotiter plate, e.g. a thin layer or, preferably, strip, film, threads, solid particles such as beads, including Protein A-coated bacteria, or paper.
  • a mitrotiter plate e.g. a thin layer or, preferably, strip, film, threads, solid particles such as beads, including Protein A-coated bacteria, or paper.
  • the FS-coupled product and the antibody raised against the bound group of the FS-coupled product according to the present invention may be used in an assay for the identification and/or quantification of said FS-coupled product or antibody reacting with at least part of the FS-coupled product present in a sample. Both a qualitative and a quantitative determination may be obtained according to the use of the present invention.
  • the identification and/or quantification of said FS-coupled product or antibody reacting with at least part of the FS-coupled product may be performed for both a scientific, a clinical and an industrial purpose.
  • the antibody used in the present method is preferably in substantially pure form (purified according to suitable techniques or by the methods of the invention, see below) in order to improve the precision and/or accuracy of the assays of the invention.
  • the determination of antibodies reactive with the FS-coupled product of the invention and being present in a sample may be carried out by use of a method comprising contacting the sample with the FS-coupled product of the invention and detecting the presence of bound antibody resulting from said contacting and correlating the result with a reference value.
  • the FS-coupled product of the invention When the FS-coupled product of the invention is to be employed in an assay for determining the presence of antibodies against the FS-coupled product in a sample, it may be in the form of a diagnostic reagent or a diagnostic agent. As will be apparent to a person skilled in the art several techniques may be applied in connection with such diagnostic reagents.
  • the present invention relates to administration of the various above-mentioned functionality-specifically coupled products to a mammal, preferably a human being, may be performed by any administration method which is suitable for administering proteins or peptides or antibodies.
  • Typical admini- stration routes are parenteral, oral, nasal, topical or rectal administration, in each case, the functionality-specifically coupled products to be administered should be formulated in a manner which will protect the functionality-specifically coupled products against degradation, in particular by enzymes.
  • the parenteral administration is the safest way of administering proteins and peptides.
  • the parenteral administration route should be selected dependent on where the functionality-specifically coupled products is to be released, e.g. intravenously, intramuscularly or subcutaneously, etc. It is also important to consider the neces ⁇ sity of "packing" the functionality-specifically coupled products in a suitable manner in order to
  • suitable measures should be taken to protect the functionality-specifically coupled products from enzymatic degradation in the gastrointestinal tract, e.g. by packing the functionality-specifically coupled products in such a way that it will not be released from the formulation (i.e. the pharmaceutical composition) until it has reached the site where either the functionality-specifically coupled products is to exert its activity locally (i.e. in the gastrointestinal tract) or from where the ab ⁇ so ⁇ tion may take place (e.g. M-cells in the colon).
  • enhancers which are capable of making functionality-specifically coupled pro- ductss of the peptide type pass the rectal mucosa and thereby become absorbed.
  • Nasal administration is an administration form which is presently intensively investigated in order to provide abso ⁇ tion of substances of the peptide type from the nasal cavity. In principle, this may take place in two ways, firstly by using enhancers, and secondly by using the bioadhesion principle in which the func ⁇ tionality-specifically coupled products may be maintained for a long period of time at a suitable domain in the nose.
  • Topical administration may be performed by formulating the functionality-specifi- cally coupled products in a salve, an ointment, a lotion, a creme, etc.
  • Formulations for parenteral use may be presented in unit dose forms, e.g. ampou ⁇ les, or in multi-dose containers with an added suitable preservative.
  • the composi ⁇ tion may be in form of a solution, a suspension, an emulsion or a delivery device for implantation or may be presented as a dry powder to be reconstituted with water or another suitable solvent before use.
  • the compositions may comprise suitable pharmaceutically acceptable carriers and/or exipients.
  • the composition may, in addition, conveniently comprise suspending, stabilizing, pH-adjusting agents and/or dispersing agents.
  • compositions for oral or rectal use may be formulated according to conventional pharmaceutical practice and may be in the form of tablets, capsules, pills, pow ⁇ ders, ampoules, granulates, drag ⁇ es, pastes, gels, suppositories or enemas; or liquid formulations such as suspensions (oily or aqueous), solutions, elixirs, emulsions or drenches and the like.
  • the auxiliary additives of the pharmaceutical compositions may be any con ⁇ ventional pharmaceutical additives and carriers:
  • Binding agents such as cellulose derivatives, starch, gelatin and polyvinyipyrrolidi- ne; fillers such as sugar, mannitol, lactose, microcrystalline cellulose, potato starch, calcium phosphate; suitable abso ⁇ tion promotors; disintegrants such as potato starch, alginic acid; lubricants such as magnesium stearate, stearic acid, talc; emulsifying agents such as lecithin, sorbitan monooleate; wetting agents such as lecithin, poiyoxyethylene esters; or buffering agents such as acetate, citrate, phosphate.
  • fillers such as sugar, mannitol, lactose, microcrystalline cellulose, potato starch, calcium phosphate
  • suitable abso ⁇ tion promotors such as potato starch, alginic acid
  • lubricants such as magnesium stearate, stearic acid, talc
  • emulsifying agents such as lecit
  • the solid compositions may by means of specially adapted coating techniques be provided with a coating adapted to protect the composition from unwanted chemi- cal changes prior to the release of the active compound.
  • the coating may be adapted to release the active compound in a predetermined pattern e.g. in order to achieve a controlled release formulation.
  • compositions suitable for topical use such as compositions suitable for application to the skin according to the present invention are suitably creams, gels, ointments, lotions, liniments, suspensions, solutions, pastes, sticks, sprays, soaps, shampoos, powders, suppositories and enemas.
  • the topical administration should be onto or close to the pathological changes in the body.
  • the compositions may be any suitable medicated mass adapted for direct application or for introduction into the relevant orifice of the body, e.g. the rectal, urethral or vaginal orifice.
  • the compositions may simply be applied directly onto the diseased part, e.g. the skin or mucosa.
  • Other relevant formulation adaptations for application to the eye may e.g.
  • eye lotions eye ointments, eye-drops or drug delivery systems adapted for administration to the eye such as compositions suitable for implantation admini- stration.
  • drug delivery systems adapted for administration to the eye such as compositions suitable for implantation admini- stration.
  • it may be applied by means of special medical devices such as dressings including occlusive dressings, or alternatively plasters, pads, sponges, strips, or other forms of suitable flexible pieces of material.
  • Formulations for topical administration may comprise pharmaceutically acceptable carriers and/or exipients such as ointment bases (e.g. paraffin, polyethylene glycols, Tween p, Span p , vegetable oils), suspending or emulsifying agents (e.g. lecithin, sorbitan monooleate, gelatin, methyl cellulose, gum acacia, sorbitan monooleate derivatives), gelforming agents (e.g. Carbopol, alginates, gelatin), preservatives (e.g. methyl or propyl p-hydroxybenzoates, benzalkonium chloride), antioxidants (e.g. tocopherol, ascorbic acid, butylated hydroxy anisol), humectants (e.g. glycerin, propylene glycol, urea), and perfumes and skin protective agents.
  • ointment bases e.g. paraffin, polyethylene glycols, Tween p, Span p
  • Formulations for topical administration to the eye may comprise pharmaceutically inert vehicles or be in form of a suitable carrier system.
  • Pharmaceutically accep ⁇ table carriers and/or exipients for the preparation of eyedrops include for example buffering agents such as boric acid or bo rates, pH adjusting agents to obtain optimal stability or solubility of the actuve drug substance, agents to adjust the tonicity such as sodium chloride or borates, viscosity altering agents such as hydroxypropyl cellulose, methylcellulose, polyvinylpyrrolidone, polyvinyl alcohols or polyacrylamide, oily vehicles such as vehicles comprising arachis oil, castor and mineral oils.
  • buffering agents such as boric acid or bo rates
  • agents to adjust the tonicity such as sodium chloride or borates
  • viscosity altering agents such as hydroxypropyl cellulose, methylcellulose, polyvinylpyrrolidone, polyviny
  • Eyedrops presented as emulsions or suspensions may furthermore comprise stabilizing, dispersing, wetting, emulsifying and/or suspending agents.
  • Eye lotions and eye ointments may comprise pharmaceutically acceptable carriers and/or exipients such as used in an eyedrop composition or in other relevant topical composition, e.g. ointments, creams, lotions and the like.
  • antibodies highly specific for the N-terminal part or the C-terminal part of the coupled peptide or amino acid are obtained which may be used for the determination of the C- or N-terminal amino acids in other peptides or proteins before and after proteolytic or chemical fragmentation and separation of the fragments.
  • BSA proteinaceous synthesis substrate
  • Procedures making use, according to the invention, of a proteinaceous substrate for solid-phase peptide synthesis may be carried out in equipment of the type used for conventional solid-phase peptide synthesis with synthetic polymer substrates.
  • a preferred type of reaction vessel has the form of a separating funnel, in the lower part of the inside of which there is a sintered glass filter which separates the inside of the "funnel" into upper and lower compartments and upon which a weighed amount of the appropriate proteinaceous substrate is placed.
  • the upper end of the vessel is equipped with a screw stopper permitting the introduc ⁇ tion of activated and protected amino acids.
  • the lower end of the vessel is equipped with a two-way stopcock, e.g.
  • the glass parts are immersed in a 10% solution of dimethyldichlorosilane (DMCS) in dry toluene for 20 min, followed by washing with toluene (3 times), immersion in dry methanol for 20 min They are then washed with methanol and finally with dry acetone. The glass parts are then dried at 120 °C overnight.
  • DMCS dimethyldichlorosilane
  • BSA contains 59 lysine units, of which 29 have been reported to be exposed for conjugation to peptides (L. Aldwin and D. Nitekci Anat.Biochem. 1 (1987) 494).
  • the molecular weight of the protein is approximately 70,000 dalton, corresponding to 0.43 mequiv. of exposed amino groups per gram of BSA.
  • Piperidine was used to effect removal of the Fmoc protecting group.
  • a 20% solution of piperidine in DMF (5 ml) was added to Fmoc-Val-BSA (0.271 g) and the mixture shaken for 5 min. After draining, the process was repeated.
  • the product was washed with DMF (5 x 3 ml) and DCM (2 x 5 ml) and the product dried in vacuo. Weight: 0.251 g.
  • the peptide was cleaved from the protein using 95% aqueuos TFA followed by work-up of the product. Cleavage and work-up were carried out according to either of the following two procedures: ⁇ . Peptide-HMPA-BSA diethylamide (0.125 g) was dissolved in 95% TFA (2.5 ml) and left for 2 hours at room temperature in a closed flask. The resulting gelatinous product was dissolved in additional 95% TFA (5 ml) and the solvent then removed in vacuo on a rotary evaporatoer. Dry diethyl ether (20 ml) was added, and the mixture was left for 10 min at room temperature and the ether decanted from the solid product. This process was repeated twice.
  • Peptide-HMPA-BSA diethylamide 0.125 g was dissolved in 95% TFA (2.5 ml) and left for 2 hours at room temperature in a closed flask. The resulting gelatinous product was dissolved in additional 95% TFA (5 ml) and the solvent then removed in vacuo on a rotary evaporator. Dry diethyl ether (20 ml) was added, and the mixture was left for 10 min at room temperature and the ether decanted from the solid product. This process was repeated twice. The product was dried with a slow stream of nitrogen for ca. 30 min. Weight of the crude product: 0.082 g. The crude product was dissolved in water (2 ml) and dialyzed (exclusion limit ca. 30.000) (twice, 24 hours each time, 4°C) against water (50 ml). The two aqueous extracts gave on freeze drying 0.016 g and 0.001 g, respectively, of the pure peptide. Table 1.
  • Weight increase in grams in each coupling step before and after removal of Fmoc protecting groups.
  • the peptide was synthesized on a conventional "polyamide" resin [Macrosorb SPR 100 flow resin (Sterling Organics Ltd.)] for the purpose of comparison with the peptide synthesized on BSA in example 3.
  • the following amino acids were coupled as the Dhbt-esters using standard procedures (e.g. as in example 3): Fmoc-Gly-Dhbt, 73 mg; Fmoc-Ala-Dhbt, 75 mg; Fmoc-Leu-Dhbt, 82 mg, and Fmoc-Leu-Dbht, 82 mg.
  • the resin was washed with ether and dried.
  • the peptide was cleaved from the resin (0.250 g) by adding 5 ml of 95% aqueous TFA and leaving the mixture at room temperature for 2 hours.
  • the solution was filtered from the resin and the TFA removed by evaporation with a slow stream of nitrogen.
  • the product was treated with several small amounts of dry ether to give the crude peptide.
  • the peptide was subjected to gel-filtration
  • the peptide H-Phe-Leu-Glu-Glu-Val-OH was obtained by cleavage of the conjugate (0.125 g) with 95% TFA as described in example 3.
  • the TFA treatment caused at the same time side-chain deprotection of giutamic acid.
  • Weight of peptide/protein mixture 0.093 g.
  • the peptide (from 0.125 g of the conjugate) may be purified as described in example 3A: Weight after "centrifree” purification and lyophiiization: 0.027 g. Weight after gelfiltration and lyophiiization: 0,025 g.
  • the peptide (from 0.125 g) may be purified as described in example 3B: Weight after dialysis and lyophiiization: 0.015 g.
  • Weight increase in grams in each coupling step before and after removal of Fmoc protecting groups.
  • 0.43 x 442 x 3 0.118 g
  • H-Leu-Leu-Ala-Gly- Val-BSA (0.125 g) was transferred to a centrifuge tube and dissolved in DCM/TFA (1 :1 v/v) (4 ml) at 0°C. After 5 min, dry diethyl ether (4 ml) was added, causing precipitation of the conjugate. The solvent was removed after centrifugation of the product, which was further washed three times with ether (3 x 4 ml) followed by drying in a stream of nitrogen. Weight: 0.116 g.
  • Weight increase in grams in each coupling step before and after removal of Fmoc protecting groups.
  • BSA (0.196 g) was placed in the silylated reaction vessel and the carboxyiic acid groups diethylamidated as described in example 2. Coupling of the first amino acid as Fmoc-protected Dhbt ester, blocking of remaining amino groups with acetic anhydride and coupling of the subsequent amino acids in the sequence in the form of Fmoc-protected Dhbt esters was performed as described in Example 6.
  • H-Ser(tBu)-Met-Asp(tBu)-Thr(tBu)-Ser(tBu)- Lys(Boc)-Glu(tBu)-BSA diethylamide (0.109 g) was transferred to a centrifuge tube and dissolved in DCM/TFA (1 :1 v/v) (4 ml) at 0°C. After 5 min, dry diethyl ether (4 ml) was added, causing precipitation of the conjugate. The solvent was removed after centrifugation of the product, which was further washed three times with ether (3 x 4 ml) followed by drying in a stream of nitrogen.
  • the amino acid analysis of this conjugate is given in Table 6, together with the resultant amino acid composition of the peptide chain. The results indicate an average of 35 synthesized peptide chains per BSA molecule.
  • An immunization mixture was prepared which had the following composition: 1 ml of a 1 mg/ml suspension of H-Ser-Met-Asp-Thr-Ser-Lys-Glu-BSA (prepared as in example 7), 12.8 ml of 0.9% (w/v) NaCl solution (Merck, 6404), 2.2 ml of AI(OH) 3 solution (Alhydrogel, 6.9 mg/ml, Statens Seruminstitut, Copenhagen) and
  • the collected blood was left to coagulate at room temperature for 2 hours, after which the clot was loose ⁇ ned by a wooden spatula.
  • the resulting coagulated blood was kept overnight at 4°C and centrifuged at 2,000 x g for 20 minutes.
  • the blood serum was removed and stored until use at -20°C.
  • Microtitre plates (Nunc, Roskilde, Denmark, 439454) were coated with 100 ⁇ l of H-Ser-Met-Asp-Thr-Ser-Lys-Glu-BSA in 50% TFA (0.2 mg/ml) by incubation overnight at room temperature.
  • To each well was then added 50 ⁇ l of 10 M NaOH and 100 ⁇ l of 3 M TRIS.HCI (pH 8.8) and the plate was again incubated overnight at room temperature.
  • the wells were then rinsed with distilled water and incubated with 0.1 M phosphate buffered saline (PBS) (pH 7.2) containing 1 mg BSA/ml and 0.1% Tween 20 (Merck) for 1 hour.
  • PBS phosphate buffered saline
  • the thus prepared plates were next incubated with 100 ⁇ l/well of sera from the immunized rabbits or control serum diluted in PBS supplemented with 0.1% (w/v) bovine serum albumin (BSA, Sigma, A-4503) overnight at room temperature, using the following dilutions: 1:200, 1:400, 1 :800, 1:1600, 1:3200 and 1:6400.
  • BSA bovine serum albumin

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Abstract

On a synthétisé des espèces chimiques, notamment des peptides, selon un procédé régulé par étapes, dans lequel on utilise une substance protéique comme substrat de synthèse. Les produits obtenus par le procédé peuvent être utilisés, par exemple, en tant que vaccins contre diverses maladies et en tant que matières matricielles ou molécules porteuses.
PCT/DK1990/000311 1989-12-01 1990-11-30 Procede de synthese regulee par etapes d'especes chimiques, notamment de peptides, de produits couples obtenus selon le procede, et emploi de ces produits couples, par exemple en tant que vaccins WO1991008220A1 (fr)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0503301A2 (fr) * 1991-02-14 1992-09-16 Fuji Photo Film Co., Ltd. Dérivés peptidiques et leur emploi
WO1993008842A1 (fr) * 1991-11-08 1993-05-13 Somatogen, Inc. Hemoglobines utilisees comme agents administrateurs de medicaments
WO1994008616A1 (fr) * 1992-09-22 1994-04-28 Regents Of The University Of Minnesota Procede d'augmentation de la production d'×ufs chez des especes aviaires par immunisation active contre un peptide intestinal vasoactif
WO1996000087A2 (fr) * 1994-06-24 1996-01-04 Akzo Nobel N.V. Materiel de preciblage et nouveaux conjugues de preciblage
WO1998010794A2 (fr) * 1996-09-11 1998-03-19 Felix Kratz Conjugues de transferrine, d'albumine et de polyethyleneglycol a action antineoplastique
WO1999024074A2 (fr) * 1997-11-07 1999-05-20 Conjuchem, Inc. Nouveaux conjugues opioides et supports endogenes
EP1167383A1 (fr) * 1997-11-07 2002-01-02 Conjuchem, Inc. Nouveaux conjugués opioides et supports endogènes
US6437092B1 (en) 1998-11-06 2002-08-20 Conjuchem, Inc. Conjugates of opioids and endogenous carriers
US6706892B1 (en) 1999-09-07 2004-03-16 Conjuchem, Inc. Pulmonary delivery for bioconjugation
US7268113B2 (en) 2001-02-02 2007-09-11 Conjuchem Biotechnologies Inc. Long lasting growth hormone releasing factor derivatives
US7737251B2 (en) 2001-02-16 2010-06-15 Conjuchem Biotechnologies Inc. Long lasting glucagon-like peptide 2 (GLP-2) for the treatment of gastrointestinal diseases and disorders
US7741286B2 (en) 1999-05-17 2010-06-22 Conjuchem Biotechnologies Inc. Long lasting anti-angiogenic peptides
US8076288B2 (en) 2004-02-11 2011-12-13 Amylin Pharmaceuticals, Inc. Hybrid polypeptides having glucose lowering activity
US8642542B2 (en) 1995-12-30 2014-02-04 Novozymes Biopharma Dk A/S Recombinant fusion proteins to growth hormone and serum albumin
US8993517B2 (en) 2001-12-21 2015-03-31 Human Genome Sciences, Inc. Albumin fusion proteins

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EP0175613A2 (fr) * 1984-09-11 1986-03-26 Merck & Co. Inc. Peptides HAV immunogéniques
EP0243929A2 (fr) * 1986-04-30 1987-11-04 Robin Ewart Offord Dérivés de polypeptides et de protéines et procédé pour leur préparation
EP0284492A1 (fr) * 1987-03-20 1988-09-28 Rhone Merieux Nouvelles fractions peptidiques inductrices d'anticorps protecteurs contre le virus de la leucémie bovine, procédé pour l'obtention de telles fractions, leurs séquences codantes et vaccins réalisés à partir de ces fractions
US4859765A (en) * 1983-10-17 1989-08-22 Syntex (U.S.A.) Inc. Synthetic peptide sequences useful in biological and pharmaceutical applications and methods of manufacture
EP0329994A1 (fr) * 1988-02-04 1989-08-30 Roche Diagnostics GmbH Imminogène et son utilisation pour préparer des anticorps contre l'hémoglobine A1C

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EP0175613A2 (fr) * 1984-09-11 1986-03-26 Merck & Co. Inc. Peptides HAV immunogéniques
EP0243929A2 (fr) * 1986-04-30 1987-11-04 Robin Ewart Offord Dérivés de polypeptides et de protéines et procédé pour leur préparation
EP0284492A1 (fr) * 1987-03-20 1988-09-28 Rhone Merieux Nouvelles fractions peptidiques inductrices d'anticorps protecteurs contre le virus de la leucémie bovine, procédé pour l'obtention de telles fractions, leurs séquences codantes et vaccins réalisés à partir de ces fractions
EP0329994A1 (fr) * 1988-02-04 1989-08-30 Roche Diagnostics GmbH Imminogène et son utilisation pour préparer des anticorps contre l'hémoglobine A1C

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Cited By (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0503301A3 (en) * 1991-02-14 1993-06-16 Fuji Photo Film Co., Ltd. Peptide derivatives and application thereof
US5436221A (en) * 1991-02-14 1995-07-25 Fuji Photo Film Co., Ltd. Tumor metastasis inhibiting compounds and methods
EP0503301A2 (fr) * 1991-02-14 1992-09-16 Fuji Photo Film Co., Ltd. Dérivés peptidiques et leur emploi
US5679777A (en) * 1991-11-08 1997-10-21 Somatogen, Inc. Hemoglobins as drug delivery agents
WO1993008842A1 (fr) * 1991-11-08 1993-05-13 Somatogen, Inc. Hemoglobines utilisees comme agents administrateurs de medicaments
US5759517A (en) * 1991-11-08 1998-06-02 Somatogen, Inc. Hemoglobins as drug delivery agents
WO1994008616A1 (fr) * 1992-09-22 1994-04-28 Regents Of The University Of Minnesota Procede d'augmentation de la production d'×ufs chez des especes aviaires par immunisation active contre un peptide intestinal vasoactif
US5557033A (en) * 1992-09-22 1996-09-17 Regents Of The University Of Minnesota Method of increasing egg production in avian species by active immunization against vasoactive intestinal peptide
WO1996000087A3 (fr) * 1994-06-24 1996-03-07 Akzo Nobel Nv Materiel de preciblage et nouveaux conjugues de preciblage
WO1996000087A2 (fr) * 1994-06-24 1996-01-04 Akzo Nobel N.V. Materiel de preciblage et nouveaux conjugues de preciblage
US8642542B2 (en) 1995-12-30 2014-02-04 Novozymes Biopharma Dk A/S Recombinant fusion proteins to growth hormone and serum albumin
WO1998010794A2 (fr) * 1996-09-11 1998-03-19 Felix Kratz Conjugues de transferrine, d'albumine et de polyethyleneglycol a action antineoplastique
WO1998010794A3 (fr) * 1996-09-11 1998-08-06 Felix Kratz Conjugues de transferrine, d'albumine et de polyethyleneglycol a action antineoplastique
US6709679B2 (en) 1996-09-11 2004-03-23 Felix Kratz Antineoplastic conjugates of transferin, albumin and polyethylene glycol
US6310039B1 (en) 1996-09-11 2001-10-30 Felix Kratz Antineoplastic conjugates of transferrin, albumin and polyethylene glycol
AU750387B2 (en) * 1997-11-07 2002-07-18 Conjuchem Biotechnologies Inc. Novel conjugates of opioids and endogenous carriers
WO1999024074A2 (fr) * 1997-11-07 1999-05-20 Conjuchem, Inc. Nouveaux conjugues opioides et supports endogenes
US6500918B2 (en) 1997-11-07 2002-12-31 Conjuchem, Inc. Conjugate comprising an antinociceptive agent covalently bonded to a blood component
US6602981B2 (en) 1997-11-07 2003-08-05 Conjuchem, Inc. Antinociceptive agent derivative
US6610825B2 (en) 1997-11-07 2003-08-26 Conjuchem, Inc. Method for alleviating pain or providing an analgesic effect in a patient
WO1999024074A3 (fr) * 1997-11-07 1999-08-19 Conjuchem Inc Nouveaux conjugues opioides et supports endogenes
EP1167383A1 (fr) * 1997-11-07 2002-01-02 Conjuchem, Inc. Nouveaux conjugués opioides et supports endogènes
US6437092B1 (en) 1998-11-06 2002-08-20 Conjuchem, Inc. Conjugates of opioids and endogenous carriers
US7741286B2 (en) 1999-05-17 2010-06-22 Conjuchem Biotechnologies Inc. Long lasting anti-angiogenic peptides
US6706892B1 (en) 1999-09-07 2004-03-16 Conjuchem, Inc. Pulmonary delivery for bioconjugation
US7268113B2 (en) 2001-02-02 2007-09-11 Conjuchem Biotechnologies Inc. Long lasting growth hormone releasing factor derivatives
US7737251B2 (en) 2001-02-16 2010-06-15 Conjuchem Biotechnologies Inc. Long lasting glucagon-like peptide 2 (GLP-2) for the treatment of gastrointestinal diseases and disorders
US8993517B2 (en) 2001-12-21 2015-03-31 Human Genome Sciences, Inc. Albumin fusion proteins
US9221896B2 (en) 2001-12-21 2015-12-29 Human Genome Sciences, Inc. Albumin fusion proteins
US9296809B2 (en) 2001-12-21 2016-03-29 Human Genome Sciences, Inc. Albumin fusion proteins
US8076288B2 (en) 2004-02-11 2011-12-13 Amylin Pharmaceuticals, Inc. Hybrid polypeptides having glucose lowering activity
US8697647B2 (en) 2004-02-11 2014-04-15 Odile Esther Levy Hybrid polypeptides with selectable properties
US9453063B2 (en) 2004-02-11 2016-09-27 Amylin Pharmaceuticals, Llc. Hybrid polypeptides with selectable properties

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