WO1988007200A1 - Conjugaison d'anticorps monoclonaux - Google Patents

Conjugaison d'anticorps monoclonaux Download PDF

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Publication number
WO1988007200A1
WO1988007200A1 PCT/GB1988/000215 GB8800215W WO8807200A1 WO 1988007200 A1 WO1988007200 A1 WO 1988007200A1 GB 8800215 W GB8800215 W GB 8800215W WO 8807200 A1 WO8807200 A1 WO 8807200A1
Authority
WO
WIPO (PCT)
Prior art keywords
mab
hrpo
spdp
siab
monoclonal antibody
Prior art date
Application number
PCT/GB1988/000215
Other languages
English (en)
Inventor
Gwynfor Rees Williams
Original Assignee
Perry, Robert, Edward
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB878706513A external-priority patent/GB8706513D0/en
Priority claimed from GB878706512A external-priority patent/GB8706512D0/en
Priority claimed from GB878706515A external-priority patent/GB8706515D0/en
Priority claimed from GB878706514A external-priority patent/GB8706514D0/en
Application filed by Perry, Robert, Edward filed Critical Perry, Robert, Edward
Publication of WO1988007200A1 publication Critical patent/WO1988007200A1/fr

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Classifications

    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates

Definitions

  • This invention relates to enzyme-conjugated monoclonal antibodies, and to a conjugation method for their preparation.
  • a monoclonal antibody can be used, owing to its high specificity for a particular epitope, as the basis of a highly sensitive assay for, say, an infectious disease. AIDS and infectious hepatitis are two hazards for which diagnosis is extremely important.
  • a monoclonal antibody is conjugated to an appropriate enzymatic label, such as horseradish peroxidase.
  • Cross-linking reagents are useful for joining two molecules which, by themselves, would not normally bind to each other.
  • Ml and 2 will be used to designate the two molecules to be conjugated.
  • a cross-linking reagent has two or more reactive groups, one of which will bind to a complementary reactive site on Ml, and another which will bind to M2.
  • Homobifunctional cross-linking reagents are known. Examples are glutaraldehyde, bis-imidates (bis- imidoesters) , diisocyanates, bis (N-hydroxysuccinimide) esters, phenyl azides, and dimaleimides .
  • the most common method of conjugation using these types of reagents is to mix Ml and M2 and the cross-linker under appropriate reaction conditions.
  • the products that form from this reaction, at a low degree of polymerisation are a mixture of free (monomeric) Ml, free M2 , Ml-Ml conjugate, M2-M2 conjugate and the desired M1-M2 conjugate.
  • high polymeric conjugates are produced of the form (Ml) -(M2) where n and m are from about 5-20.
  • heterobifunctional cross-linking agents have been developed.
  • the general structure of these types of reagents resembles a dumbbell, in which two different types or reactive groups are separated by some inert spacer chain of atoms.
  • examples of such reagents include succinimidylpyriodyl- dithiopropionate (SPDP) , maleimidobenzoylsuccinimide
  • MBS succinimidyliodoacetamidobenzoate
  • SIAB succinimidyliodoacetamidobenzoate
  • Ml label Ml with SPDP
  • SH free suifhydryl
  • Conjugation is via a disulfide bond. See US-A-4232119.
  • One problem with this method is that the disulfide bond is labile to reducing agents. Thio-disulfide exchange can also occur with other SH-containing proteins.
  • SIAB can be used according to the method of US-A-4251445 ⁇ Ml is labelled with SIAB; this is then added to an enzyme which apparently must contain a natural SH group. The method does not allow labelling enzyme with SIAB and reacting with Ml-SH.
  • the method of the present invention is used to form a stable conjugate from two different proteins or macromolecules. More specifically, the method involves (a) introducing a reactive group into one protein (Ml) which will not react with functional groups present in that molecule, and (2) introducing a functional group into a second protein (M2) which will react with the reactive group introduced into Ml but not with functional groups in M2. If the functional group is naturally present in M2 it need not be introduced. If the functional group is naturally present in both Ml and M2, the functional group should be blocked in one of the proteins and that one labelled with the reactive group.
  • Ml one protein
  • M2 second protein
  • the functional group is preferably SH. Some proteins contain this naturally, some may be modified to contain it (e.g. FAb' fragments) , and others may require its introduction.
  • the reactive group is preferably iodoacetyl, 2-alkoxypyridyl or vinylsulfone. In general, it can be any reactive group whose reactivity to thiols is greater (about 100-fold greater) than its reactivity to amines in nucleophilic displacement or addition reactions.
  • the reactive and functional groups must each be present in a heterobifunctional cross-linking reagent which also contains a reactive group- that will react with a common functional group of proteins.
  • the preferred functional group in this case is NH_, . All proteins contain this amine group.
  • the reactive group preferred is an active ester such as N-hydroxysuccinimidyl, para-nitrophenyl, dinitrophenyl, or hydroxybenzo- triazole.
  • the preferred structure of the heterobifunctional reagent containing the SH functional group is Y-S-A-B-COO-X wherein A and B are spacer moieties and may be C, _ alkylene or phenylene; X is an active ester as described above; Y is H or a sulfhydryl-protecting group such as 2-thiopyridyl, 4-thiopyridyl or acetyl.
  • the preferred structure of the heterobifunctional reagent containing the group reactive to thiols is Z-D-COO-E wherein D is a spacer moiety and may be C. regularly alkylene or phenylene; E is an active ester as described above; and Z is a thiol-reactive group as described above.
  • sulfo-SIAB the reagent containing the thiol-reactive group
  • SPDP the reagent containing the thiol group
  • SPDP can be used to introduce thiol groups in proteins that do not contain them naturally, e.g. IgG or alkaline phosphatase.
  • SIAB is used to introduce the thiol-reactive group (iodoacetyl) into the other molecule in the conjugate.
  • Ml is preferably horseradish peroxidase.
  • M2 is a monoclonal antibody, preferably against infectious hepatitis or the env, gag or pol vival gene product of human immunodeficiency virus.
  • monoclonal antibodies can be prepared by fusing spleen cells from a mammal (such as a mouse, rabbit or goat) which has been immunized against the antigen with an appropriate myeloma cell line. The resultant product is then cultured in, say, a standard HAT (hypoxanthine, aminopterin and thymidine) medium. The fused cells yielding an antibody which give a positive response to the presence of the antigen are removed and cloned using one of the standard techniques. The monoclonal antibodies from the clones are then tested against standard antigens to determine their specificity for the antigen. The monoclonal antibodies selected can then be conjugated to an appropriate label, such as horseradish peroxidase.
  • an appropriate label such as horseradish peroxidase.
  • Example illustrates the invention.
  • the apparatus and techniques used in the preapration of reagents and/or performance or evaluation of the method of this invention are standard or as described above.
  • PBS phosphate-buffered saline
  • HRPO horseradish peroxidase
  • SPDP the reagent containing the thiol group
  • HRPO (2.5 mg/ml in 100 mM PBS, pH 7.5) is added a 20-fold molar excess of SPDP.
  • the SPDP is prepared immediately Before use as a 5.0 mg/ml solution in dimethyl sulfoxide. The reaction is allowed to proceed for 30 minutes at room temperature.
  • HRPO-SPDP is separated from free SPDP by chromatography on Superose 6. The conditions typically result in 1-3 moles SPDP per mole HRPO. This intermediate can be stored for 3 months at 4°C.
  • HRPO-SPDP (2.5 mg/ml in 100 mM PBS, pH 7.5) is added a 30-fold molar excess of dithioerythritol (2.0 mg/ml in PBS) . Reaction is allowed to proceed at room temperature for 15 minutes. HRPO-SH is separated from dithioerythritol by chromatography on Superose 6.
  • MAb directed against hepatitis B antigen (2.5 mg/ml in 100 mM PBS, pH 7.5) is added a 20-fold molar excess of sulfo-SIAB (3.0 mg/ml in 100 mM PBS, pH 7.5) . reaction is allowed to proceed at room temperature for 30 minutes. MAb-SIAB is separated from free SIAB by chromatography on Superose 6. These conditions typically result in 1-3 moles SPDP per mole MAb. This intermediate must be used immediately.
  • the procedure of the Example may be repeated using a MAb directed against env, gag or pol virai gene product.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Virology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Un procédé permettant de conjuguer la peroxydase de raifort (HRPO) avec un anticorps monoclonal (MAb), qui consiste à (a) combiner le HRPO avec du succinimidylpyridyldithiopropionate (SPDP), afin de former une liaison covalente HRPO-SPDP; (b) combiner le MAb avec du sulfosuccinimidyliodoacétamidobenzoate (SIAB), afin de former une liaison covalente SIAB-MAb; (c) combiner le SPDP-HRPO avec le MAb-SIAB pour former un conjugué de HRPO-SPDP-SIAB-MAb.
PCT/GB1988/000215 1987-03-19 1988-03-21 Conjugaison d'anticorps monoclonaux WO1988007200A1 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
GB8706514 1987-03-19
GB878706513A GB8706513D0 (en) 1987-03-19 1987-03-19 Conjugating horse-radish peroxidase to monoclonal antibody
GB878706512A GB8706512D0 (en) 1987-03-19 1987-03-19 Conjugating horse-radish peroxidase to monoclonal antibody
GB8706515 1987-03-19
GB8706513 1987-03-19
GB878706515A GB8706515D0 (en) 1987-03-19 1987-03-19 Conjugating horse-radish peroxidase to monoclonal antibody
GB878706514A GB8706514D0 (en) 1987-03-19 1987-03-19 Conjugating horse-radish peroxidase to monoclonal antibody
GB8706512 1987-03-19

Publications (1)

Publication Number Publication Date
WO1988007200A1 true WO1988007200A1 (fr) 1988-09-22

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1988/000215 WO1988007200A1 (fr) 1987-03-19 1988-03-21 Conjugaison d'anticorps monoclonaux

Country Status (1)

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WO (1) WO1988007200A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0579130A2 (fr) * 1992-07-17 1994-01-19 SCLAVO DIAGNOSTICI S.r.l. Des protéines fonctionalisées et leur utilisation pour la préparation de composés conjugués marqués
EP3095467A1 (fr) * 2005-11-23 2016-11-23 Ventana Medical Systems, Inc. Conjugué moléculaire

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2382695A1 (fr) * 1977-03-04 1978-09-29 Pharmacia Diagnostics Ab Reactif pour la mise en oeuvre de procedes d'analyse immunochimiques en presence d'un liquide aqueux et son application
US4251445A (en) * 1978-03-24 1981-02-17 Weltman Joel K N-succinimidyl haloacetyl aminobenzoates as coupling agents
EP0186371A2 (fr) * 1984-12-11 1986-07-02 Medical And Scientific Research Associates Anticorps monoclonaux spécifiques pour les antigènes du virus de l'hépatite-B

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2382695A1 (fr) * 1977-03-04 1978-09-29 Pharmacia Diagnostics Ab Reactif pour la mise en oeuvre de procedes d'analyse immunochimiques en presence d'un liquide aqueux et son application
US4251445A (en) * 1978-03-24 1981-02-17 Weltman Joel K N-succinimidyl haloacetyl aminobenzoates as coupling agents
EP0186371A2 (fr) * 1984-12-11 1986-07-02 Medical And Scientific Research Associates Anticorps monoclonaux spécifiques pour les antigènes du virus de l'hépatite-B

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Biological Abstracts, Reports, Reviews and Meetings, vol. 26, abstract no. 51519, 1984, (Philadelphia, PA, US) T.S-F. Wang et al.: "DNA primase from human KB cells", see the computer print-out, & Fed. Proc. (USA), 1983, 42(7), abstract 1327 *
Chemical Abstracts, vol. 96, no. 15, 12 April 1982, (Columbus, Ohio, US), E. ress et al.: "Endogenous murine leukemia virus-encoded proteins in radiation leukemias of BALB/C mice", see page 156, column 2, abstract no. 116702s, & Virology 1982, 117 (1), 207-218 *
Chemical Abstracts, vol. 99, no. 11, 12 September 1983 (Columbus, Ohio, US), F. Veronese et ak.: "Monoclonal antibodies to feline sarcoma virus gag and fes gene translational products" see pages 432, 433, abstract no 86433x, & J. Cell. Biochem. 1983, 21(1), 9-18 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0579130A2 (fr) * 1992-07-17 1994-01-19 SCLAVO DIAGNOSTICI S.r.l. Des protéines fonctionalisées et leur utilisation pour la préparation de composés conjugués marqués
EP0579130A3 (fr) * 1992-07-17 1994-11-30 Sclavo Diagnostici S R L Des protéines fonctionalisées et leur utilisation pour la préparation de composés conjugués marqués.
EP3095467A1 (fr) * 2005-11-23 2016-11-23 Ventana Medical Systems, Inc. Conjugué moléculaire

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