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WO1987004171A1 - Procede de preparation d'un complexe d'anticorps - Google Patents

Procede de preparation d'un complexe d'anticorps Download PDF

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Publication number
WO1987004171A1
WO1987004171A1 PCT/JP1986/000628 JP8600628W WO8704171A1 WO 1987004171 A1 WO1987004171 A1 WO 1987004171A1 JP 8600628 W JP8600628 W JP 8600628W WO 8704171 A1 WO8704171 A1 WO 8704171A1
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WIPO (PCT)
Prior art keywords
antibody
group
producing
agent
antibody conjugate
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PCT/JP1986/000628
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English (en)
Japanese (ja)
Inventor
Noriaki Endo
Naoji Umemoto
Yoshinori Kato
Takeshi Hara
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Teijin Ltd
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Teijin Ltd
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Publication of WO1987004171A1 publication Critical patent/WO1987004171A1/fr
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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

Definitions

  • the present invention relates to a method for producing an antibody conjugate comprising an antibody and a specific compound. More specifically, the present invention is based on the use of the amino group of an antibody to link it to a compound having a specific function. The present invention relates to a method for producing an antibody conjugate with a small decrease in antibody activity.
  • a complex in which a specific antibody is bound to an insoluble support is used in an affinity matrix to separate a specific substance from a mixture.
  • a complex obtained by coupling a radioactive substance to a specific tissue in a living body for example, an antibody having specificity for a tumor or thrombus
  • the complex is radiated to the tissue. Integrated activity Therefore, they can be used for the diagnostic imaging of those patients.
  • a complex in which a biologically active substance is bound to an antibody becomes a highly selective drug.
  • a complex in which an antitumor antibody and an anticancer drug or other cytotoxic substance are bound is a cancer therapeutic agent that selectively acts on a tumor.
  • a complex in which a fluorescent substance and an enzyme are bound to an antibody is used, for example, as an immunochemical reagent or a diagnostic reagent.
  • new complexes may be devised for future needs.
  • a common requirement for the production of these conjugates is that the required substance must be satisfactorily expressed in the antibody as a TO 3 ⁇ 4 ⁇ production method. This is a production method that does not impair the property of specifically binding to the original function.
  • a commonly used method for binding a substance to an antibody is to use an amino group (lysine, N-terminal amino) containing the amino acid that constitutes the antibody.
  • the way to do this is In particular, it is heavily used.
  • affinity chromatography or a complex of an antibody and an insoluble support used for separation and purification using the principle can be used as a support.
  • Reactive groups with amide-blocks ie, epoxy groups, carboxylic acid ester groups, acid azide groups, bromoacetyl groups, and cyanogen bromide activation groups These are produced by a method of introducing a reactive group such as, and then reacting the antibody with the amino group to bind the antibody to a support.
  • a reactive group such as, and then reacting the antibody with the amino group to bind the antibody to a support.
  • a complex in which a radioactive substance is bound to an antibody is, as exemplified by the reaction between the antibody and a Portton-Hunter reagent which is a reagent for introducing a radioactive element, as shown in FIG. It is produced by the reaction of a compound containing an active ester of liponic acid with an amino group of an antibody (A.E.B 0 I ton and W. Hunter, Biocheoi). J. 133, 529-539 (1973)), as well as in the genetic preamplifier acetic acid (abbreviated DTPA).
  • a new type of chelating agent is contained in a carboxy-containing chelating agent. In above attached to ⁇ Mi amino group of an antibody with sill group, it is prepared in a way you combine your bets-out radionuclides / I n the key record over Bok agent (B. A. K haw, J.T.
  • K u I karni A. H. Blair, T, I. G hose, Cancer Research, 1, 2700-2706 (1931)). It is produced by linking the amino group of an antibody with a lipoxyl group, and for example, by the method of linking the A chain of lysotoxin lysin (K. A. Krolick, C. Vi II emez , P. I sakson, J. W. Uhr. And E, S. Vitet ta Proc. A 1. A cad. Sci, USA,, 77, 5419-5423 (1980)).
  • a cross-linking agent for example, a cross-linking agent for introducing an active disulfide group, N—succinimidyl 3— (2—pyridyldimethy) propio
  • SPDP a cross-linking agent for introducing an active disulfide group
  • An A-chain complex is being produced.
  • the monoclonal antibody 225.28S which is a monoclonal antibody against a high-molecular-weight antigen on the cell membrane of melanoma
  • the antibody activity of the complex obtained by equimolar reaction of the intramolecular acid anhydride of the agent DTPA was reduced to about 1/2 that of the original antibody.
  • the present inventors have used the material Amino ⁇ V * -3 ⁇ 43 ⁇ for the purpose of widespread and general use without causing any inevitable or inconvenient disadvantages with conventional methods.
  • a method for producing an antibody-substance conjugate capable of producing an antibody-substance complex first, one or more amino acids containing amino The amino group is reacted with a reversible modifier to protect these amino groups, and then the remaining amino group is combined with necessary substances and introduced if necessary.
  • An attempt was made to develop a method for producing an antibody conjugate, characterized in that the reversible modifier introduced after the further processing of the added substance was removed from the amino group of the antibody.
  • the conjugate thus obtained has a reduced antibody activity when produced by a conventional method. And maintain sufficient antibody activity
  • the inventor of the present invention has reached the present invention.
  • the present invention provides an antigen by modifying an amino group.
  • a part of the amino group of the antibody or its fragment whose activity is reduced is modified with a reversible modifying agent for the protein amino group, then the antibody or its fragment is modified.
  • Reacting a compound having an amino group-reactive functional group with the remaining amino group in the remaining fragment, and then immediately after the product Is characterized in that after further subjecting the product to a chemical reaction, the reversible modifier residue of the protein amino group is removed from the product after the chemical reaction. It is a manufacturing method.
  • the present invention is divided into the following two embodiments.
  • a part of the amino group of an antibody or its fragment whose antigen-binding activity is reduced by wedding of the amino group is replaced with a protein amino group.
  • the remaining amino group of the antibody or its fragment is reacted with a compound having an amino group-reactive functional group.
  • a method for producing an antibody complex comprising removing a reversible modulator residue of a protein amino group from a product (hereinafter, referred to as the present invention).
  • Akira-Method A) the amino group of an antibody or its fragment whose antigen-binding activity is reduced by modifying the amino group. Is modified with a protein amino group reversible modifier, and then the remaining amino group of the antibody or its fragment reacts with the amino group.
  • a method for producing an antibody conjugate which comprises removing a reversible modifier residue of a protein amino group after binding and then completing the method (hereinafter referred to as method B of the present invention). It is.
  • an antibody whose antigen-binding activity is reduced by modifying an amino group is an antibody against any antigen or octapeptide with respect to its specificity. It is okay. That is, for example, antibodies against cancer, bacteria, viruses, viz., Mycoplasma, parasites, pathogens, antibodies against tumor-related substances, tumor-associated antigens, Antibodies, toxins, enzymes, and antibodies against cell differentiation antigens, histocompatibility antigens, and other cell membrane antigens Antibodies to lugen, hormones, drugs, and other biologically active substances can be used.
  • the antibody may be, for example, a polyclonal antibody produced by immunizing an animal with an antigen / hapten, or a cell fusion or EB antibody.
  • a monoclonal antibody produced by a method for transforming antibody-producing cells using a virus may be used.
  • Monoclonal antibodies can produce large amounts of high-purity antibodies with specificity for various antigens by cell fusion and virus transformation. It has the advantage of breaking.
  • 171 bodies have IgG, IgA, IgM, IgD and IgD.
  • IgG has subclasses of IgG1, IgG2a, IgG2b, and IgG3.
  • IgA has IgA and IgA2 subclasses
  • IgM has IgM1 and IgM2 subclasses.
  • the body of any of these classes and subclasses can be used as the antibody of the present invention.
  • Antibodies can be used in whole The fragment containing the antigen-binding portion can be used. Such fragments include, for example, the degradation of monomeric IgMs or rgA antibody molecules of IgM antibodies by apcin.
  • the divalent fragment F (ab ') 2 obtained, and the monovalent fragment Fab obtained by decomposing with papain can be mentioned.
  • the reversible modifying agent for a protein amino group refers to an amino group that is deeply involved in the binding of an antibody or a fragment thereof to an antigen.
  • a reversible modifier ; covalent bonding of residues
  • an amino-reactive functional group is added to the ⁇ -isomer or the remaining amino group of the fragment.
  • the product (A) Reversible modifier Any compound capable of removing a residue and regenerating an amino group may be used.
  • the reversible crowning agent for such a protein amino group include maleic anhydrides, succinic anhydrides, polycarboxylic acid, and lipogenic acid. Derivatives, diketen, 2—Hydroxyacetic acid aldehyde is a specific example. For example, in the case of maleic anhydrides, anhydrous
  • trifluoroacetic anhydride without an amino-reactive functional group antibody or its fragment. It can be a compound, it can be a wide range of cross-linking agents, bioactive substances, substances, fluorescent substances and insoluble branches.
  • a compound that has a functional group that reacts with a substance that is desired to be bound to an antibody or its fragment by a cross-linking agent at the time of circumstance Particularly preferred are a sulfide group-introducing agent, an acetyl acetyl group-introducing agent, an active disulfide group-introducing agent, and a thiol agent-introducing agent.
  • a sulfide group-introducing agent As the maleimide group-introducing agent, the general formula [e],
  • the maleimide compound represented by the general formula [II] is a compound of the general formula [II]
  • the acetyl group compound represented by the general formula [H] is used as a disulphide group-introducing agent.
  • R 1 and R 2 are the same as in formula [I].
  • the divalent organic group represented by R 1 in the formulas [I ⁇ , [K] and [IV] is an organic group that is stable under the chemical conditions of the method of the present invention.
  • the simplest examples are the alkylene and phenylene groups with or without branching.
  • the organic groups I Table R 1 is not good at a divalent organic group containing a pair flops Chi de residues inside the peptidase Dozanmoto or Waso.
  • the peptide residue may be a peptide residue that is stable under the chemical conditions of the method of the present invention.
  • AI — AI a -Leu-, -Leu-Ala- L eu-, one AI a— s eu— A la— s eu—,-AI a- AI a-,-AI a- AI a- A la-,-A la- II e-, II e- AI e -II e,-A la- II e- A la- II e-,-A 1 e-V a I—,-V al-A la- V al-, one A la— V a I-AI a- V a I — and the like can be mentioned as preferable examples.
  • R 2 is an alcohol residue of the active ester or an amide residue of the active amide.
  • examples of such alcohol residue include 2,4-dinitrophenoxy group. , N — Succinid
  • a monovalent organic group represented by R 3 which forms a disulfide group together with a bonding sulfur atom is Those which form an active disulfuride group are particularly preferred.
  • specific examples thereof include, for example, a 2-pyridylthio group (S—), 4— Pyridylthio group
  • R + is an alcohol residue of an imidic acid ester or an amino acid residue of an amidin. Specific examples of the residue include a methoxy group and an ethoxy group.
  • R- 4 is an amidin residue
  • R 4 represents, for example, a diamino group (H 2)
  • maleimide group-introducing agent represented by the formula [I]
  • a specific example of the disulphide group-introducing agent to be expressed is, for example, 3— (2-pyridyldithiyl) propionic acid N—hydroxysiloxane Thin I Mi de et scan ether, 4 i (2 - pin lysyl di Chi old) butyric acid N - arsenide mud key sheet succinate down I Mi de et scan Tenore, 4 one (4 one pin 1 Li di Honoré di Cho)) Severe acid 2,4—Dimethyl mouth ester, 2—Pyridyldithioalanylloisylararanylloisin N—Hydroxy Cis-similar-do-ester, N- [3- (2-pyridyldithio) propyl] nil] xazolidinone, 4-pyridyldi
  • the biologically active substance as a compound having an amino group-reactive functional group in the method A of the present invention includes an antitumor substance, an antibacterial substance, an antiviral substance, and an antifungal substance. Substances, anti-mycoplasma substances, anti-parasitic substances, cytotoxic substances, toxins or their fragments, hormones, enzymes and the like.
  • an antitumor substance is an anticancer drug or an anticancer drug derivative having a group capable of reacting with and binding to an amino group, and is a specific example thereof.
  • Can but are not limited to, compounds represented by the following chemical formulas.
  • the castor seed toxin ricin an anti-toxin A-like protein obtained from its A-chain, American burdock For viral proteins and hormones, the ester derivatives
  • Examples of the enzyme include ceratiopeptidase and the like.
  • specific examples of the compound having an amino group-reactive functional group suitably used in the present invention method A include a chelating agent, Thiamine triampenteneacetic acid derivative,
  • [I * represents a radioactive iodine atom. ]
  • the insoluble support examples include cyanogen bromide-activated cellulose 4B gel.
  • the compounds (substances) of which specific examples have been described above are mostly reactive functional groups, that is, active esters, aldehyde groups, acid azide groups, acid anhydride groups. It has an amino group of the antibody or its amino acid group without the help of a condensing agent.
  • a condensing agent such as water-soluble rubodiimide or glutaraldehyde in the opening for the reaction with the amino group of the fragment. Close .
  • the amino-reactive functional group of ricin and its' A chain is a carboxyl group or an amino group.
  • the reaction with an amino group of an antibody or its fragment is induced by induction of a daunomycin derivative having an aldehyde group as shown in the specific example above.
  • the Schiff base generated by the reaction is, if necessary, subjected to hydrogenation by using sodium hydrogenated cyanide.
  • the compound having a cross-linking agent-reactive functional group needs to be introduced into an antibody or its fragment without impairing antigen-binding activity.
  • Any compound that has a cross-linking agent-reactive functional group may be used, and may be a wide range of compounds. Examples of such compounds include biologically active substances (antitumor substances and antibacterial substances). Substance, anti-viral substance, anti-fungal substance, anti-mycoplasma substance, anti-parasitic substance, cytotoxic substance, mother element or its fragment, formol , Enzymes, etc.), chelating agents, radioactive substances, fluorescent substances, insoluble supports, and the like.
  • a thiol group is converted to 2-pyridyldisulfide, 41-pyridyldisulfate.
  • An example of conversion to active disulphide by using a feed, 5,5'-dithiothiopice (2-nitrobenzoic acid) or the like can be given.
  • the cross-linking agent reactive functional group is the reactive group of the cross-linking agent residue introduced into the antibody or its fragment (the reactive group originally contained in the original cross-linking agent). Or as described above A different one is selected corresponding to the reactive group after the conversion.
  • the cross-linking agent is In the case of a carboxylic acid group-introducing agent, a thioacetyl group-introducing agent, or an active disulfide group-introducing agent, a crosslinking agent-reactive functional group is, for example, a chiral group;
  • the crosslinking agent-reactive functional group is preferably, for example, a maleimide group, a door acetyl group, or an active sulfide group.
  • the introduced crosslinker residue (a reactive group that the original crosslinker had or a reactive group after undergoing a transformation) is a thiol group.
  • -A compound having a maleimide group as a cross-linking agent-reactive functional group is, for example, a maleimide group represented by the formula [e] in a compound having an amino group. Can be obtained by reacting a metal compound.
  • Compounds having a cross-linking agent-reactive group as an acetyl group are, for example, compounds having an amino group as a compound having an acetyl group represented by the formula [II]. It can be obtained by reacting a chilled compound.
  • a compound having a thiol group as a crosslinking agent-reactive functional group is, for example, a compound having a 7-mino group and a 2-iminothiolane or peracetase in a compound having a 7-mino group.
  • Chilho Moshi Station After reacting with dithiolactone or reacting with a disulfide compound represented by the formula [III] or [IV].
  • the active disulfide group can be obtained by reducing it to a thiol group with 2-methylethanol, dithiophene-thanol or the like. it can .
  • Compounds having an active disulfide group as a cross-linking agent-reactive functional group may be, for example, a compound having an amino group and having a 2— imino group or N—Acetylphos moistin lactone reacts with the introduced thiol group to convert the introduced thiol group to 2—pyridyldisulfide, 4-pyridyldisulfide. , 5, 5 '-Activated disulfide group by dimethyl bis (2_nitrobenzoic acid)' etc. or have an amino group
  • the compound is a disulfide compound represented by the formula [I] or [IV]. It can be obtained by reacting things.
  • the compounds having a cross-linking agent-reactive functional group suitably used in the method B of the present invention include the following compounds.
  • NCS Neocartinostantin
  • NCSj's own amino group is an imino group derived from NCSj's own amino group.
  • HSA human serum albumin
  • HSA sulfur and nitrogen atoms of HSA, respectively.
  • D binds to the amino group of HS A and represents an antitumor substance. Represents the number of D connected to H S A and is ⁇ 50 50
  • N-acetylsomocysteine A ricin A-like anti-viral protein obtained from Acacia magna pond, treated with lactone.
  • white holmon N-acetylsomocysteine A ricin A-like anti-viral protein obtained from Acacia magna pond, treated with lactone.
  • Thiol group-introducing agent 2 Cellulase peptide treated with iminothiirane Radioactive guest
  • [I * represents a radioactive iodine atom. ]
  • Cyanogen bromide activated sepharose gel treated with glutathione-2-pyridyl disulfide K. B rock Iehurst, J. Carl. sson, M.P.J.K.Kierstan and E.M.C.Crook, Bioceni. J 133_, 573-584 (1-973)
  • glutathione-2-pyridyl disulfide K. B rock Iehurst, J. Carl. sson, M.P.J.K.Kierstan and E.M.C.Crook, Bioceni. J 133_, 573-584 (1-973)
  • Maleic anhydride citraconic anhydride, 2,3-dimethyl maleate, which is a reversible modifier of the protein amino group used
  • Maleic anhydrides such as acid anhydride, cis-aconitic acid anhydride, etc., tetrafluorosuccinic acid, exo-cis 1-3,6-endokis Sodium + — tetrahydric anhydride, ⁇ phthalic anhydride, exocyssitol 3, 6-endoxohexahydrhydric anhydride, etc.
  • Reactive derivatives of polyhalogenated carboxylic acids such as succinic acids, trifluoroacetic anhydride, etc., diketenes, 2-hydroxyacetic aldehydes Substances such as, are supplied as industrial products.
  • a compound having an amino group-reactive functional group to be bound to an antibody used as a raw material in the method of the present invention is a maleimide group-introducing agent, Cross-linking agents such as acetyl group-introducing agents, disulfide group-introducing agents, thionyl group-introducing agents, various antitumor agents, antibacterial agents, antiviral agents, anticancer agents Drugs and their derivatives, bioactive substances such as toxins or fragments, hormones, nitrogen, etc., and ethylene amide acetic acid derivatives.
  • Metal chelating agents such as entrapped aminopentanoic acid derivatives, radioactive substances such as Porton-Hunter reagents, fluorescent substances such as full-length resin, polysaccharides
  • insoluble supports such as gels, etc.
  • the resulting material is reacted with the amino group of the antibody, either as is, or in a manner that is self-evident or has been previously disclosed. It can be used as a raw material of the present invention, in addition to its purpose.
  • the method of the present invention is usually carried out by a continuous operation of the following three step reactions. That is, 1) protection of the amino group of the body or its fragment by a reversible decorating agent of the amino group of the protein 2) protection of the amino group-reactive functional group A reaction to add a compound to the remaining amino group by $ ⁇ , and if necessary, to further add the bound compound 3) Reversible modifier from the amino group This is a reaction for removing residues.
  • the amount of the protein amino reversible modifier used in the first reaction will vary depending on the antibody and / or decorating agent used. Preferably, it is used in an amount of 1 to 500 times the amount of the antibody or the fragment.
  • the reaction is based on antibody activity and protein.
  • the pH of the reaction solution be in the range of 4 to 10 and the reaction temperature be 1435, under mild conditions that do not impair the properties of the reaction.
  • the reaction conditions when the reversible decorating agent for the amino group used is maleic anhydride, the reaction is preferably carried out in a weak carboxylic acid of 8 to 10%. Trifluoric acid and polyhalogenating ability H7 to 9 are preferred as reactive derivatives of rubic acid.
  • the reaction of PH 4 to 9 is preferred for diketen, 2-hydroxyhydrazine aldehyde, and the like.
  • the reaction time is usually 10 minutes to 24 hours, but often ends in 30 minutes to 5 hours.
  • the antibody whose amino group has been protected with a reversible modifier can be purified once and used in the next step, or the reaction solution can be used in the next step.
  • the latter operation is preferably used when the reversible modifier is no longer present in the reaction system after a certain period of time after being hydrolyzed, such as maleic anhydride. Acid anhydrides generally apply.
  • this can be achieved, for example, by simple gel filtration column operations using Sephadex G-125 or by dialysis under 4. .
  • a compound having an amino group-reactive functional group is bonded to the remaining amino group of the antibody or its fragment, and if necessary, a compound bonded thereto is further added.
  • the reaction is carried out under conditions that do not impair the antibody, that is, the reaction is carried out.
  • the pH is preferably in the range of 4 to 10 and the reaction temperature is preferably in the range of -4 ° to 35 ° C.
  • reaction to the antibody adorned with dehydrofuran anhydride, etc. is preferably carried out in a weak alkaline solution of pH 7 to 10 ⁇ , and tetraflu. Reaction with antibodies modified with succinic acid, triflic anhydride, diketene, etc. is preferably performed with PH 4-8.
  • reaction conditions are also similar.
  • the reaction against the antibody modified with 2-hydroxy aldehyde If the amino-protecting group still needs to be retained, do not use peroxide acid.
  • the amount of the compound having an amino group-reactive functional group used in the compound varies depending on the properties of the compound, but the molar ratio of the antibody or its fragment is one. It is preferably up to 100 times. If the water solubility of the compound is low, the antibody may be insolubilized by binding to the antibody in a large amount.In such a case, the amount of the substance used in the reaction is limited. Use. However, fcfc A
  • the substance to be used is a substance that is used as an inherently insoluble substance such as an insoluble gel.
  • Compounds having an amino group-reactive functional group can be used as a maleimide group-introducing agent, a metal acetyl group-introducing agent, a disulfide group-introducing agent, or a thiol group-introducing agent.
  • a maleimide group-introducing agent such as an agent
  • a metal acetyl group-introducing agent such as an agent
  • a disulfide group-introducing agent such as an agent
  • thiol group-introducing agent such as an agent
  • binding them to the antibody or its fragment and then introducing it into the antibody or its fragment
  • a processing reaction such as a reaction of a compound having a crosslinking agent-reactive functional group with the cross-linking agent residue to form a bond can be performed, but in this case, Preferred reaction conditions for the reaction are also above and around
  • the reaction for removing the reversible modifier residue from the amino group may be performed after the previous reaction, but after purifying the single product, but in many cases, the purification is carried out. Instead, proceed immediately to the reaction to remove the reversible modifier residue on the amino group.
  • purification When adding purification, perform column chromatography such as gel opening and ion exchange, and dialysis.
  • reaction for removing the reversible modifier residue from the amino group of the antibody Male base, exo-system 3, 6-endoxo Delta 4- hydroxy phthalate group, exo-system 1, 3, 6-ene Doxohexahydrophthalyl group, etc. It is preferable to remove the weakly acidic or neutral PH 48 by treatment under near neutral conditions.
  • the reaction temperature is Ru Oh one 4 4 35 good or teeth rather, between hours reaction time is usually from 0.5 to 96 hours.
  • tetrasulfonyl succinyl group, anhydrous trisyl rosyl group and acetoacetyl group are removed under weak alkaline conditions of PH810. Is preferred.
  • a basic substance such as hydroxylamine, morpholine or the like can be added as an auxiliary agent at a concentration of 10 mlOOmM to promote the reaction.
  • the reaction temperature is preferably 435 ° C, and the reaction time is usually 0.5 to 96 hours.
  • the 2-hydroxyethyl group can be removed by the action of 110 mM boric acid.
  • Purification of the antibody complex produced as described above is performed by dialysis, gel permeation, ammonium sulfate precipitation, ethanol precipitation, acetate precipitation to remove low molecular weight substances. Conventional methods such as precipitation can be used. If the complex is an insoluble substance, a simple method such as filtration and washing can be used. Separation of high molecular weight materials is carried out by gel permeation or ion exchange chromatography. It can be done using the usual methods, such as fission and isoelectric focusing.
  • Example 1 Example 1
  • a complex of an antibody and a crosslinking agent having an amino group-reactive functional group, and a complex of the antibody and a compound having a crosslinking agent-reactive functional group via the crosslinking agent is a complex of an antibody and a crosslinking agent having an amino group-reactive functional group, and a complex of the antibody and a compound having a crosslinking agent-reactive functional group via the crosslinking agent.
  • the concentration of the absorption by Ri antibody ⁇ white matter of 280 nm was determined Me (you and E 2 .0>. As a result, the antibody concentration is Ri 5.5 Roh / a Oh, antibody times yield One A de 6.1 ⁇ .
  • the reaction was performed for 1 hour.
  • the above cells and reagents were prepared by using a phosphate buffered saline solution containing 10% normal serum serum and 0.02% sodium azide, and then using the above saline solution. After the cells were washed three times by centrifugation, the radioactivity bound to the cells was measured with an instrument for measuring the radioactivity.
  • 2S I-labeled 96.5 binding to HMT-12 cells was determined, and the original 96.5 antibody was determined.
  • the antigen-binding activity of the complex was 63.2% of that of the 96.5 antibody.
  • a compound having a crosslinker reactive functional group is bound to the crosslinker residue introduced into the antibody, and then a reversible modifying agent for the protein amino group is added. Except for the production of the composite.
  • the conjugate was dialyzed against 0.1 M sodium phosphate buffer (pH 6.0) at 40 ° C. for 30 hours to obtain a target solution of the complex.
  • the absorbance at 280 and 360 nm was measured to determine the concentration of the antibody protein and the number of N- (2,4-di-2-nitrophenyl) cysteine bound to the antibody. .
  • the antibody recovery was 6.1 fflg, indicating that an average of 3.4 molecules of N- (2,4-dinitrophenyl) cysteine were bound to one molecule of the antibody. .
  • This comparative example shows that when the complex was produced by the conventional production method, the obtained complex had significantly lower antibody activity (antigen binding activity).
  • Antibody 9.3 / ⁇ was dissolved in O. liVl sodium phosphate buffer (PH 7.40) 1 under ice cooling with N-succinimidyl 4 — (N-maleimi D) A butyrate DMF solution (0.43M> 23 ⁇ ) was added and reacted for 30 minutes. The reaction solution was added to 0.1 iVI sodium phosphate buffer (PH 6.0). On the other hand, dialysis was performed for 4 hours at 4 ° C to dissolve the target complex. Liquid 1.2 was obtained. By performing the same operations as in Example 1-2, the recovered amount of the antibody and the maleimide group bound to the antibody molecule were quantified. Antibody recovery yield
  • the average number of maleimide groups introduced into one antibody molecule was 3.5.
  • the antigen-binding activity of the complex was measured by the same method as in Example 13 to 13. The complex showed 42.6% of the activity of the original 96.5 antibody.
  • the antibody activity of the conjugate produced by the method of the present invention is 63.2% of the 96.5 antibody, which is clearly the same as the conventional method. It is higher than the activity of the obtained complex.
  • the protein amount of the obtained complex was determined from the absorption at 280 nm. 6 (recovery 70%).
  • the number of 2-pyridyldithiyl groups introduced into the antibody was determined by the following procedure. For a part of the solution of the complex, the amount of the protein is determined from the absorbance at 280 nm, and then an excess of dithylate sleat is added, and the mixture is reacted at room temperature for 20 minutes. Therefore, the ⁇ of the generated 2—mercapto pyridine and its absorption at 343 nm ( ⁇ 7.06 x 10 3 ) It was determined by measurement. From these two quantifications, it was determined that the average number of 2-pyridyldithiyl groups introduced into the antibody protein ⁇ molecule was 3.9 on average.
  • a crosslinking agent containing a peptide bond represented by the following formula was used instead of the SPDP in Example 2.
  • the antibody was introduced into the Ig ⁇ antibody by the same method as in Example 2.
  • Example 5 Production of complex of antibody and anticancer drug Reaction formula:
  • a 0.5 ⁇ MD ⁇ F solution of dimethyl maleic anhydride was added to a 1.0 solution of 0.1 ⁇ borate buffer ( ⁇ ⁇ 8.8) in which 6.5 of the 96, 5 antibody was dissolved, and 10 ⁇ of a 0.5 MD MD ⁇ F solution of dimethyl maleic anhydride was stirred under ice-cooling.
  • the reaction was carried out for 30 minutes. During that time, 0.2 ⁇ Na 0 ⁇ was added dropwise to the reaction solution.
  • PH was kept at 8-9.
  • N-hydroxysuccinimide ester P.KuI karn i et aI.
  • MTX N-hydroxysuccinimide ester
  • the absorption derived from MTX was experimentally determined to be 2.52 times the absorption of iVI TX at 372 nm, and the absorbance of the complex at 280 was determined by the absorption derived from MTX. After correction, the antibody protein mass was determined. As a result, it was found that the complex was composed of an antibody molecule with an average of 7.9 molecules of MTX bound to the antibody molecule.-3, Comparative Example
  • the solution was dialyzed against NaCi (PH 7.0) at 4 ° C. for 48 hours to obtain a solution 2.5 of the complex.
  • the composition of the complex was determined in the same manner as in 5-2, and as a result, it was found that an average of 7.3 molecules of iVlTXX was bound to one molecule of the antibody.
  • the binding activity to the antigen of the 96.5 antibody and MTX conjugate prepared according to 5-1 was measured by the same method as in 13 using the same method.
  • the antigen-binding activity of the 96.5-TX complex prepared by the conventional method in which a part of the amino group of the antibody is not protected by a reversible modifying agent, was also measured by the method described below. Specified. When iVlTX was bound to the 96.5 antibody molecule by an average of 7.31 according to the conventional production method, the antigen-binding activity was reduced to 4.2% of the original antibody, whereas the method of the present invention was used. If MTX is bound to 7.91 on one antibody molecule on average, The binding activity is 19.6%, and it is clear that the complex produced by the method of the present invention has higher antigen binding activity than the complex produced by the conventional method.
  • Example 7 Using the anti-L1210 egret antibody (IgG fraction) in place of the 96.5 antibody in Example 5, the same procedure as in Example 5 was performed to produce an antibody-MTX complex.
  • Example 7 Using the anti-L1210 egret antibody (IgG fraction) in place of the 96.5 antibody in Example 5, the same procedure as in Example 5 was performed to produce an antibody-MTX complex.
  • NCS protein anticancer drug neocalcinostatin
  • reaction solution is passed through a column of Sephadex G-25 (2nX40 an, in 0.1 mM sodium phosphate buffer containing 1m EDTA) to remove low molecular weight substances. Quality Then, a solution of the antibody into which a thiol group was introduced was obtained.
  • the product was found to be a mixture of a complex ⁇ in which one or three molecules of NCS were bound to one antibody molecule, and a mixture of antibodies in which NCS did not bind. Met .
  • D represents the anticancer drug mitomycin ⁇ derivative, and is represented by the following formula c
  • Antibody 15ff3 ⁇ 4 was used as a solution of 50 nVI sodium borate buffer ( ⁇ H9), 2 and dimethyl maleic anhydride :? ) 0.5M DMF solution 20 was added under ice-cooling and stirring, and reacted for 30 minutes. in the meantime
  • reaction solution was kept around 9 by dropwise addition of 0.2 M NaOH. Then, 5 — of a 0.5 M methanol solution of 3-(2-pyridyl) propyl imidate acid methyl ester hydrochloride was added, and After reacting for 30 minutes, anti-f solution 1 oi M
  • the amount of MMC-bound HSA contained in the collected liquid 2.6 was determined by the Lowry method (Nobuo Ui, Nobuo Tamiya, edited by Kozo Narita, “Protein Chemistry (Biochemical Experiment Course I)”) , 1976, 51), the result was 16..3 ⁇ 3 ⁇ 4.
  • the number of MMCs bound to one molecule of HSA was determined for some samples by determining the amount of MiMC from the absorbance at the maximum absorption of MMC of 360, and by the Lo / ry method. As a result of calculating the ratio to the fc protein amount, the average was 28.4. "In addition, the number of thiol groups originally contained in the HSA portion of the MMC-bound HSA was calculated.
  • the number of thiol groups on iVIMC-HSA was calculated to be an average of 0.58 per HSA molecule.
  • Escherichia coli antibody (I0G fraction) against Mycobacterium tuberculosis 60 ⁇ was added to 50 mM borate buffer (PH ⁇ .7) solution 6 under ice-cooling and dimethyl methylamine.
  • a DMF solution of phosphoric anhydride () 140 was added and reacted for 30 minutes. During that time, the pH of the reaction solution was kept at around 9 by dropwise addition of 0.2 M NaOH.
  • the protein fractions were collected by passing through NaCl (H7.4)) to obtain a solution of a complex of the target antibody and the chitolating agent DCH.
  • the reaction solution was dialyzed against 0.1 iVl sodium acetate buffer (pH 5.5) for 36 hours to obtain an antibody-DTPA complex solution 4.1 as a target substance. One part of the obtained solution (containing 2.0 / ⁇ of the antibody) was diluted to 1.0 with the same buffer, and the solution was diluted to 0.1 U'i.
  • exo-cis-1,6-endoxosome 4 -tetrahydrophthalyl group is removed and removed. Then, a solution containing the target, radiation-labeled Fab fragment
  • Example 18 Preparation of conjugate of antibody and antifungal agent
  • Example 5 In the production method in Example 5, instead of the 96.5 antibody, a monoclonal monoclonal antibody (19G) against the script coccus was replaced with a meso-method.
  • Trixetone N Anti-fungal agent instead of hydroxysuccinimide ester 5 — Glutaramide N- The hydroxysuccinimide ester derivative is used, and the others are subjected to the same procedures as described above to obtain a monoclonal antibody and 5-to-year-old rosin. A complex of was obtained.
  • Example 8 3-(2-pyridyl dithiocyanate) propyl acetate N-hydrochloroacetic acid was substituted for methyl imido ester.
  • the xysuccinimide ester is replaced with a thiol-containing enzyme 3—galactosyl instead of the human serum albumin bound to mitosisin C.
  • a complex of the antibody and 3-galactosidase was obtained.
  • Example 20 Production of complex of antibody and chelating agent
  • Example 7 In the production of Example 7, a chelating agent (S) was used instead of the NCS into which a acetyl group was introduced.
  • Example 22 Preparation of a conjugate of an antibody and a toxin
  • Example 23 Preparation of a conjugate of an antibody and an insoluble support Formula:
  • Activated chitin propylserose 4B gel (Pharmacia)
  • the reaction was carried out at 4 for 2 hours.
  • the gel was filtered off, resuspended in a solution of PH 6 and reacted at room temperature for 48 hours, and then the gel was filtered off and washed to obtain a gel to which the antibody was bound.
  • Example 25 Production of complex of antibody and anticancer drug
  • the reaction solution is added with 4 C of iVl TX N-hydroxysuccinimide ester, and the reaction mixture is treated with 0.01 M sodium phosphate buffer-0.14 MN aa. (pH 7.2) to prepare a 96.5 antibody-MTX complex.
  • the MTXZIg average molar binding ratio and antigen-binding activity of the complex were respectively determined.
  • the antigen-binding activity of the 96.5 antibody-MTX complex produced by the method of the present invention described in 25-1 was compared with the 96.5 antibody-M produced by the conventional method described in 25-2. Compared with the antigen binding activity of the TX complex, it is clear that the complex produced by the method of the present invention has a higher antigen binding activity.
  • the IgG-MTX complex was obtained.
  • the antigen binding activity of the obtained complex was 60%.
  • the antibody recovery was 80%.
  • the method of the present invention has made it possible to produce a complex retaining antibody activity. In addition, according to this method, the antibody recovery is also good.
  • Example 26 Production of conjugate of antibody and anticancer drug
  • Antibodies (anti-human melanoma antibodies 96, 5 or ZM
  • the antigen-binding activity of the 96.5—MTX and ZME018 complex described above was determined to be HMT-2 in the case of the 96.5 complex, as a human melanoma cell line.
  • I2S I-labeled 96.5 as an I-labeled antibody and Z
  • an antibody complex retaining antibody activity can be efficiently produced.
  • the obtained antibody conjugate can be used for various purposes.
  • a complex in which a specific antibody and an insoluble support are bound to each other is used for affinity chromatography, and an antibody having specificity for a tumor or thrombus is used.
  • the complex obtained by binding radioactive material to the tissue can be used for diagnostic imaging of those tissues.
  • a complex in which an antitumor antibody and an anticancer drug or other cytotoxic substance are bound to each other is a cancer therapeutic agent that selectively acts on a tumor.
  • the complex obtained by linking a fluorescent enzyme to an antibody can be used as a reagent or diagnostic agent in the field of immunology and biochemistry.

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Abstract

Le procédé ci-décrit consiste à modifier une partie des groupes amino d'un anticorps dont la capacité de liaison avec un antigène est réduite par modification des groupes amino, ou bien du fragment dudit anticorps avec un modificateur réversible d'un groupe amino protéinique, à faire réagir les groupes amino restants de l'anticorps ou du fragment de ce dernier avec un composé possédant un groupe fonctionnel capable de réagir avec un groupe amino, puis à éliminer le résidu du modificateur réversible contenu dans le produit. Ce procédé permet d'obtenir un complexe d'anticorps subissant une réduction moindre de sa capacité de liaison avec un antigène et pouvant être utilisé dans la chromatographie par affinité ou comme agent de diagnostic ou encore comme agent thérapeutique contre le cancer.
PCT/JP1986/000628 1985-12-27 1986-12-11 Procede de preparation d'un complexe d'anticorps Ceased WO1987004171A1 (fr)

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WO1988007553A1 (fr) * 1987-03-26 1988-10-06 Teijin Limited Procede de preparation d'un complexe d'anticorps
US5612034A (en) * 1990-10-03 1997-03-18 Redcell, Inc. Super-globuling for in vivo extended lifetimes
CN109996527A (zh) * 2016-10-10 2019-07-09 阿布拉科斯生物科学有限公司 纳米颗粒制剂及其制备和使用方法

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Publication number Priority date Publication date Assignee Title
JPS5161640A (en) * 1974-09-20 1976-05-28 Searle & Co Koshuyozaino seizoho
JPS51126281A (en) * 1975-02-04 1976-11-04 Searle & Co Cytootoxic substance
JPS51144723A (en) * 1975-05-27 1976-12-13 Yeda Res & Dev Antiitumor agent
JPS5877662A (ja) * 1981-11-04 1983-05-11 Hitachi Ltd 免疫定量用試薬
JPS6089433A (ja) * 1983-10-21 1985-05-20 Green Cross Corp:The 制癌作用物質複合体の製造法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5161640A (en) * 1974-09-20 1976-05-28 Searle & Co Koshuyozaino seizoho
JPS51126281A (en) * 1975-02-04 1976-11-04 Searle & Co Cytootoxic substance
JPS51144723A (en) * 1975-05-27 1976-12-13 Yeda Res & Dev Antiitumor agent
JPS5877662A (ja) * 1981-11-04 1983-05-11 Hitachi Ltd 免疫定量用試薬
JPS6089433A (ja) * 1983-10-21 1985-05-20 Green Cross Corp:The 制癌作用物質複合体の製造法

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