EP0000938A1 - Complexes covalents macromoléculaires, procédés de préparation et compositions pharmaceutiques les contenant - Google Patents

Complexes covalents macromoléculaires, procédés de préparation et compositions pharmaceutiques les contenant Download PDF

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Publication number
EP0000938A1
EP0000938A1 EP78100717A EP78100717A EP0000938A1 EP 0000938 A1 EP0000938 A1 EP 0000938A1 EP 78100717 A EP78100717 A EP 78100717A EP 78100717 A EP78100717 A EP 78100717A EP 0000938 A1 EP0000938 A1 EP 0000938A1
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group
macromolecules
reactive
acyl
macromolecule
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EP0000938B1 (fr
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Walter Charles Joseph Ross
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National Research Development Corp UK
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National Research Development Corp UK
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • 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
    • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/06—Enzymes or microbial cells immobilised on or in an organic carrier attached to the carrier via a bridging agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/82—Proteins from microorganisms
    • Y10S530/825—Bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/866—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving immunoglobulin or antibody fragment, e.g. fab', fab, fv, fc, heavy chain or light chain

Definitions

  • the present invention relates to macromolecular complexes, a method of making them and pharmaceutical compositions containing them. More specifically the method of making the complexes involves linking two macromolecules through a linking agent having two or more reactive groups of different reactivity.
  • enzymes may be linked to water-insoluble carriers and thereby insolubilised; drugs may be linked to carrier proteins (e.g. specific antibodies) to enable them to be localised at the site where the drug is required; or carrier proteins may be linked to liposomes containing entrapped drugs and derived from phospholipids having a reactive end group, again permitting localisation of drugs at specific sites.
  • carrier proteins e.g. specific antibodies
  • a common method of linking macromolecules comprises the reaction, in a single step, of the two macromolecules and the linking agent.
  • dimers or polymers of the macromolecules used are formed as well as the desired adduct. This dimerisation or polymerisation is disadvantageous because:-
  • single step coupling can lead to the formation of intramolecular bonds, thus inhibiting or destroying the biological activity of the macromolecule.
  • steps may be expressed as:- in which M 1 , M 2 , A, B and L are as defined above and L' may be the same as L or a modified version of L (derived from A and L) and L" may be the same as L' or a modified version of L' (derived from L' and B).
  • L is used solely but should be considered to include the possibilities of L' and L" as the context requires.
  • Linking agents suitable for use in the present invention include those in which the first group, A, will undergo reaction at a temperature, pH or level of irradiation at which the second group, B, is not reactive.
  • Reactive groups suitable for use as group A (the more reactive group) in such linking agents include (a) activated carboxyl groups, for example those of formula -COX in which X is a halogen atom, e.g. chlorine or bromine, (to give acid halides); an azido group -N 3 ; the residue of an organic acid, e.g. (to give acid anhydrides) or the residue of an alcohol e.g.
  • Suitable reactive groups B are activated halogen groups i.e. halogen groups two carbon atoms removed from a heteroatom carrying a lone pair of electrons (electron releasing group) for example halogen groups in radicals such t as -S-CH 2 CH 2 -Cl or N-CH 2 CH 2 -Cl (mustards) (which are unreactive at the lower temperatures at which the aforementioned activated carboxyl groups may be made to react) or halogen atoms in an a-position relative to a carbonyl group e.g.
  • a-halocarboxylic acids in a-halocarboxylic acids; the heterocyclic aziridine ring -N which is stable at neutral oralkaline pH but which undergoes ring opening at acid pH; and an arylazido group which becomes reactive on irradiation with light.
  • Linking agents which are preferably used in the method of the invention are:-
  • linking agents include acyl derivatives of 2,4-dinitro-S-aziridinyl-benzoic acid, for example the mixed anhydride: in which the acid anhydride group will react at alkaline or neutral pH with primary or secondary amino groups on the macromolecule M 1 to give amide links, with the aziridine ring being opened at acid pH by carboxyl groups on the macromolecule M ; and acyl derivatives of 5-azido-2,4-dinitrobenzoic acid, e.g. in which the acid chloride group reacts with amino groups under conditions of low radiation intensity, e.g. in a dark room, the azide group then reacting, after elimination of a molecule of nitrogen, by insertion into a C-H bond when irradiated with visible light of a suitable higher intensity.
  • the mixed anhydride in which the acid anhydride group will react at alkaline or neutral pH with primary or secondary amino groups on the macromolecule M 1 to give amide links, with the aziridine ring being opened at acid pH by
  • the linking group L of the linking agent serves as a carrier for the two reactive groups A and B and may be any group which serves such a purpose, provided that the differential activity between the groups A and B (as herein defined) is retained.
  • the linking agent portion L may advantageously contribute to the differential reactivity of groups A and B.
  • the group L used will generally be dictated by the availability or ease of synthesis of the linking agent A-L-B and conveniently the two groups A and B are attached to an aromatic ring or rings, preferably a benzene ring.
  • the linking agent contains only one of the more reactive groups A in order to prevent dimer or polymer formation in the first step of the reaction. It is further preferable that only one group B is present in the linking agent, although the use of linking agents having two or more of such groups is not precluded.
  • stepwise reaction it is preferred to isolate and purify (for example by chromatography) the product of the first step before the second linking reaction is performed, to remove unreacted linking agent and, if desired, any unreacted macromolecule or any by-product, the presence of which is undesirable. It will in general also be necessary to purify the final product to remove any unreacted starting materials or undesirable side product.
  • the products obtained by the method of the invention will not consist of a single chemical entity but will comprise a series of conjugates falling within a molecular weight band, by which they may be characterised.
  • the degree of variation will depend upon the number and variety of functional groups in the macromolecules M and M 2 .
  • the products obtained by the method of the present invention have a ratio of 1:1 with respect to 2 the two macromolecules M 1 and M 2 .
  • the intermediate complex obtained is of the form M 1 -(L-B) n , where n is greater than 1, and thus the intermediate complex itself will not be a single chemical entity but a series of complexes comprising macromolecules having a varying number of linking groups attached thereto. It is advisable to produce intermediate complexes of this type as in the second step not all of the groups B in the linking molecules will react with the second macromolecule M 2 because of side reactions, for example with the solvent or with other components in the reaction mixture.
  • Control over the ratio of M 1 to M 2 is further complicated by the fact that in the second step two or more of the intermediate complexes represented by M 1 -L-B may react together, rather than with M 2 , to give further intermediates of the type represented by M 1 -L-M 1 -L-B which may themselves react with either M 2 or a further molecule M 1- L - B .
  • reaction techniques known in the art for controlling undesirable side reactions e.g. using an excess of one component or by adding a component to the reaction mixture continuously during the course of the reaction, may be employed to try to obtain the desired ratio.
  • Any macromolecules containing suitable functional groups may be linked by using linking agents containing the aforementioned groups, although the method is particularly applicable to macromolecules of biological origin, such as peptides e.g. proteins, especially antigens, antibodies, enzymes, lipoproteins, serum albumins, toxins and toxoids.
  • the macromolecule M 1 usually reacts via its amino groups and macromolecule W reacts for example via its amino or carboxyl groups, or C-H bonds.
  • the method of the present invention is particularly useful for linking together a chemotherapeutic agent, e.g. diphtheria toxin, to an antibody, i.e. immunoglobulin, since the complex so produced is thereby rendered site specific and the chemotherapeutic agent may be concentrated at the required point, thereby reducing the dose required and hence side-effects whilst at the same time improving the therapeutic effect of the therapeutic agent.
  • a chemotherapeutic agent e.g. diphtheria toxin
  • an antibody i.e. immunoglobulin
  • the complexes of the present invention may be used for a variety of purposes.
  • anti-cancer agents of improved selectivity for treatment of the disease in a mammal, including man may be obtained by linking a tumour-specific antibody to the original anti-cancer agent.
  • Improved immunosuppressive agents for treatment or prophylaxis in a mammal, including man of for example, graft versus host (GVH) disease or transplant (graft) rejection by a host mammal and autoimmune diseases may be obtained, for example, by linking one or more toxins to an anti-lymphocytic globulin.
  • anti-parasitic agents for treatment or prophylaxis of diseases in a mammal, including man, caused by parasites may be obtained by linking an antibody for the parasite e.g. Trypanosoma cruzi, to a toxin, e.g. diphtheria toxin.
  • a toxin e.g. diphtheria toxin.
  • Complexes of the present invention may be used as reagents in enzyme linked immunosorbent assay (E.L.I.S.A.) reactions
  • E.L.I.S.A. enzyme linked immunosorbent assay
  • an antibody to the substance being detected is labelled by being linked to an enzyme suitable for catalysing a readily monitored reaction.
  • the complexes obtained by the present process generally have greater activity than complexes produced by earlier methods.
  • a suitable dose for a mammal of a canplex of the invention will lie in the range of 10 -6 to 10 molar equivalents of the amount of the particular chemotherapeutic agent in the complex which would be required in its unbound form for the therapeutic effect obtained.
  • the carrier used in a pharmaceutical formulation may be any that is acceptable, i.e. does not have any substantially deleterious effect on the recipient.
  • the carrier used for a particular administration will depend upon the route used, which would usually be parenteral; for example a liquid, e.g. aqueous, carrier would be suitable for parenteral administration, e.g.. intravenous injection.
  • a suitable aqueous carrier is water for injections.
  • formulations of the present invention in liquid media may be freeze-dried for convenient storage.
  • Chlorambucil (25 mg) was dissolved in solution (B) (1.0 ml) and to an aliquot (0.3 ml) of the solution obtained was added solution (A) (0.3 ml). The reaction mixture was then stirred in an ice bath for 30 minutes.
  • the reaction mixture was added to a solution of horse anti-human lymphocytic globulin (ALG; 50 mg) in a mixture of saline borate (SB) buffer (0.05 M borate buffer containing 1.7% sodium chloride, fungicidal and bacteriostatic agents; pH 8.9-9.1) (5 ml) and dioxan (1.4 ml) and stirred at 4°C for 90 minutes to form a conjugate,
  • SB saline borate
  • the protein band (detected on an LBK flow-through Spectrophotometric cell at 281 nm) (20 ml) was collected and concentrated to 2.5 ml in an Amicon ultrafiltration cell. A sample (0.1 ml) of the concentrate was diluted for spectrophotometric estimation of the level of binding of chlorambucil to the ALG (all done in a cold room at 4°C).
  • the concentrated conjugate solution (1 ml) was added to diphtheria toxin (1.05 ml) (25 mg/ml + 0.05 ml of I 125 -radior labelled toxin) and stirred at room temperature for 30 hours.
  • the high molecular weight fraction having M.wt. of 180,000 to 250,000 comprised a series of conjugates of ALG and diphtheria toxin conjugated through chlorambucil, there being an average of from 1.0 to 1.5 moles of diphtheria toxin conjugated, through a chlorambucil residue, to each mole of ALG.
  • a second high molecular weight fraction (M. wt >250,000) was also obtained.
  • the fluorescent titre for ALG-chlorambucil-diphtheria toxin (M.wt 180,000 to 250,000) 1:1 was 3.0 x 10 -5 mM which is insignificantly different from the titre for ALG alone (2.6 x 10 -5 mM).
  • the linkage of diphtheria toxin to ALG did not affect the antigen binding capacity of the antibody.
  • the conjugate of M.wt >250,000 did show a somewhat reduced binding capacity, with a titre of 6.6 x 10 -5 mM.
  • ALG-chlorambucil-diphtheria toxin (M.wt 180,000 to 250,000) was as toxic to the macrophages as was diphtheria toxin alone, killing down to 10 -12 M, or about 50 molecules per cell. Thus the coupling of diphtheria toxin to ALG using chlorambucil does not inactivate the toxic properties of the toxin.
  • CLA 4 cells (2 x 10 cells per 200 1 culture) in Hanks balanced salt solution containing 10% foetal calf serum were exposed to diphtheria toxin for 1 hour before washing five times. Cell survival was measured at the end of the 24 hours incubation by the ability of the cells to incorporate 3 H-leucine into protein.
  • diphtheria toxin was not very effective at killing CLA 4 cells compared with other sensitive cells such as guinea pig macrophages and human fibroblasts.
  • diphtheria toxin would only kill CLA 4 cells at concentrations down to 10 -9 M, and on short exposure no cell death was apparent even at the maximum concentration used of 10 -8 M.
  • the ALG-chlorambucil-diphtheria toxin conjugate (M.W. 180,000 to 250,000) was very potent and killed CLA 4 cells at concentrations down to 10 -11 M for both 1 hour and 24-hour exposures.
  • diphtheria toxin By linking diphtheria toxin to ALG its cytotoxic effect on 1 hour exposure to CLA 4 cells was improved by a factor of about 1000. ALG alone did not kill CLA 4 cells, and did not modify the toxicity of diphtheria toxin when presented simultaneously to the cells.
  • diphtheria toxin when diphtheria toxin was coupled to a non-immune immunoglobulin which had no ability to bind to CLA 4 cells, the conjugate was 50 fold less toxic than diphtheria toxin alone so that the improved toxicity of ALG-chlorambucil-diphtheria toxin seems to be specific for the target cell. This is supported by the finding that ALG-chlorambucil-diphtheria toxin was about twenty-fold less efficient at killing human fibroblasts, a cell to which ALG does not bind, than was diphtheria toxin alone.
  • BSA bovine serum albumin
  • the protein band (detected as described in Example 1) (in 13 ml of eluant) was collected and a sample was diluted for spectrophotometric determination of the level of binding of 2-4-dinitro-5-aziridinyl benzoic acid to the BSA.
  • the protein band (conjugate) was concentrated to 2.0 ml in an Amicon ultra- filtration cell and the concentrate so obtained was added to HyG (50 mg) in SB buffer (2 ml). The pH at this point was 9.1.
  • reaction mixture was stirred rapidly while the pH was adjusted to 5.3 by slow addition of 1N hydrochloric acid from a microsyringe.
  • hydrochloric acid was complete the reaction mixture was stirred in a water bath at 40 0 C and the reaction followed spectrophotometrically, small samples being taken every 5 hours, diluted and examined by UV spectroscopy. After 20 hours a shift of ⁇ max from 336 nm to 356 nm indicated that the reaction was complete and the pH was adjusted to 9.1 by slow addition, to the rapidly stirred mixture, of 1N sodium hydroxide.
  • the high molecular weight fraction (M.wt >150,000) comprised a series of conjugates of bovine serum albumin and human (H G) having an average of 9 moles of 2,4-dinitro-5- aziridinyl benzoic acid bound to each mole of BSA and to which conjugates was bound 80% of the labelled BSA. It was estimated that the conjugate band contained 1 to 2 molecules of BSA for each molecule of H ⁇ G.
  • reaction mixture was allowed to cool and applied to a dry silica gel column which was washed with chloroform (200 ml) to elute 2 minor bands.
  • the solvent was then changed to chloroform:methanol:water (70:30:5), and the product first being adsorbed on to the top of the column and then eluted as two major bands; when the second band started to elute the solvent was changed to pure ethanol and all the material was eluted from the column.
  • the conjugate band (the first major band eluted) was dried by evaporation under reduced pressure at less than 40°C, redissolved in chloroform (20 ml), filtered through a Millipore filter (0.22 ⁇ m pore size) to remove silica fines and dried by rotary evaporation (reduced pressure and ambient temperature). A yellow liquid residue (presumed to be largely water) remained and was removed by successive addition and evaporation under reduced pressure of dry benzene.
  • the product travels as a single spot (Rf. 0.6) in the above mentioned TLC systems.
  • Liposomes are prepared from egg lecithin (10 mg) and dipalmitoyl phosphatidyl ethanolamine (2 mg) (obtained as described above) by the following method.
  • the lipids are mixed and dried as a thin film under reduced pressure on a rotary evaporator at ambient temperature and then suspended in sodium chloride (1 ml, 73 ml) and phosphate buffer (1 mM; pH 7.0); (at this pH the aziridine ring of the 2,4-dinitro-5-aziridinyl benzoic acid residue is poorly reactive).
  • a solution (2 ml; 15 mg/ml) of bovine serum albumin (BSA) in sodium chloride (73 mM) and acetate buffer (5 mM) is then added and the pH of the mixture is adjusted to 4.1 with aqueous hydrochloric acid.
  • BSA bovine serum albumin
  • acetate buffer 5 mM
  • BSA binds electrostatically to the liposomes (the liposomes are negatively charged, the BSA is net positively charged), and the 2-,4-dinitro-5-aziridinyl benzoic acid residue reacts with suitable residues on the liposome.
  • the pH is adjusted to 7.0 with aqueous sodium hydroxide and the liposomes are separated from unbound BSA by gel chromatography on Sepharose 6B.
  • Chlorambucil (7.5 mg) was dissolved in 0.3 ml solution (B) and to the stirred, cooled (2°C) solution was added 0.3 ml solution (A). After 30 minutes a pre-cooled solution of protein-1 (5 ml 10 mg protein-1 per ml in borate:NaCl (0.05 M:1.7%, pH 9) and dioxan (1.4 ml) was added quickly and stirring continued at 2°C for 11 ⁇ 2 to 2 hours. The solution was then passed through a jacketed (2°C) column of Sephadex SG 25 and eluted with the same borate-saline buffer.
  • Fractions were collected and the molecular weights were calculated from the elution volumes.
  • the protein-2 content of the fractions was estimated from the gamma-radiation counts and the protein-1 content from the U.V. absorbance at 280 nm after deducting the contribution from protein-2.
  • fractions containing unconjugated protein-1 and protein-2 fractions of M.wt 180,000 to 250p00 and >250,000 were obtained.
  • the fraction of M.wt 180,000 to 250,000 consisted of conjugates of protein-1 and protein-2, there being an average of 1 to 1.5 molecules of prbtein-2 coupled to each molecule of protein-1.
  • protein-1 it is possible to use normal horse and normal bovine immunoglobulin, horse antimouse thymocytic globulin, horse anti-human lymphocytic globulin and F(ab) 2 fragments (obtained by pepsin digestion) of normal horse immunoglobulin and of horse anti-human lymphocytic globulin.
  • protein-2 it is possible to use bovine serum albumin, diphtheria toxin and abrin.
  • Sephadex SG 150 superfine for the final chromatographic separation and in this case the fraction of M.wt 130,000 to 200,000 contained predominantly a 1:1 protein-l:protein-2 conjugate.
  • a portion of each of the complexes formed in Examples 1 to 13 is dispersed into separate aliquots of water for injections so as to form respective solutions suitable for injection.

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EP78100717A 1977-08-22 1978-08-21 Complexes covalents macromoléculaires, procédés de préparation et compositions pharmaceutiques les contenant Expired EP0000938B1 (fr)

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GB3517377 1977-08-22
GB3517377 1977-08-22

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EP0000938A1 true EP0000938A1 (fr) 1979-03-07
EP0000938B1 EP0000938B1 (fr) 1984-10-17

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US (1) US4275000A (fr)
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JP (1) JPS5470384A (fr)
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0017507A2 (fr) * 1979-04-09 1980-10-15 Teijin Limited Protéine hybride anti-tumeur et son procédé de préparation
EP0031999A2 (fr) * 1979-12-14 1981-07-15 Teijin Limited Protéine hybride antitumeur et procédé pour sa préparation
EP0044167A2 (fr) * 1980-07-14 1982-01-20 The Regents Of The University Of California Agent cytotoxique dirigé par un anticorps
EP0055115A1 (fr) * 1980-12-22 1982-06-30 Teijin Limited Hybride protéique cytotoxique et son procédé de préparation
EP0055575A1 (fr) * 1980-12-22 1982-07-07 Teijin Limited Hybride protéique cytotoxique et procédé pour sa préparation
EP0056322A1 (fr) * 1981-01-12 1982-07-21 Lilly Industries Limited Conjugués d'immunoglobulines
FR2523311A1 (fr) * 1982-03-12 1983-09-16 Pasteur Institut Produit de couplage entre une albumine et un ligand specifique, obtention et applications dans le domaine biologique
EP0123114A1 (fr) * 1983-03-28 1984-10-31 Miles Laboratories, Inc. Immunogènes fixés par une liaison urée, anticorps et une méthode préparatoire
EP0161195A1 (fr) * 1984-04-10 1985-11-13 Société Anonyme dite: IMMUNOTECH Procédé pour le dosage immunologique des monoamines
EP0321604A1 (fr) * 1986-06-16 1989-06-28 Sanko Junyaku Co., Ltd. Complexe d'antigène-anticorps et méthodes d'utilisation
EP0407390A1 (fr) * 1987-12-24 1991-01-16 Bio Metric Systems Inc Revetements biocompatibles.
EP0485749A2 (fr) * 1990-11-15 1992-05-20 Research Corporation Technologies, Inc. Modification chimique d'anticorps pour la création d'immunoconjugués

Families Citing this family (31)

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EP0044167A2 (fr) * 1980-07-14 1982-01-20 The Regents Of The University Of California Agent cytotoxique dirigé par un anticorps
EP0044167A3 (fr) * 1980-07-14 1982-04-21 The Regents Of The University Of California Agent cytotoxique dirigé par un anticorps
EP0055115A1 (fr) * 1980-12-22 1982-06-30 Teijin Limited Hybride protéique cytotoxique et son procédé de préparation
EP0055575A1 (fr) * 1980-12-22 1982-07-07 Teijin Limited Hybride protéique cytotoxique et procédé pour sa préparation
EP0056322A1 (fr) * 1981-01-12 1982-07-21 Lilly Industries Limited Conjugués d'immunoglobulines
FR2523311A1 (fr) * 1982-03-12 1983-09-16 Pasteur Institut Produit de couplage entre une albumine et un ligand specifique, obtention et applications dans le domaine biologique
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EP0161195A1 (fr) * 1984-04-10 1985-11-13 Société Anonyme dite: IMMUNOTECH Procédé pour le dosage immunologique des monoamines
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EP0485749A2 (fr) * 1990-11-15 1992-05-20 Research Corporation Technologies, Inc. Modification chimique d'anticorps pour la création d'immunoconjugués
EP0485749A3 (en) * 1990-11-15 1992-09-09 Brunswick Corporation Chemical modification of antibodies for creating of immunoconjugates

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DE2862449D1 (en) 1984-11-22
JPS5470384A (en) 1979-06-06
IT1107772B (it) 1985-11-25
EP0000938B1 (fr) 1984-10-17
US4275000A (en) 1981-06-23
IT7850797A0 (it) 1978-08-21

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