WO1987004171A1 - Process for preparing antibody complex - Google Patents

Abstract

A process for preparing an antibody complex which comprises modifying a part of amino groups of an antibody, whose ability of binding with antigen is reduced by modifying the amino groups, or of the fragment thereof with a reversible modifier for a proteinic amino group, reacting the remaining amino groups of the antibody or the fragment thereof with a compound having a functional group capable of reacting with an amino group, then removing the reversible modifier residue from the product. This process enables to obtain an antibody complex suffering less reduction in an antigen-binding ability, and has a possibility of being used in affinity chromatography or as a diagnostic agent or cancer-treating agent.

Description

 M m Production method of antibody conjugate Technical field

 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.

Background art

Complexes in which an antibody having a function of binding specifically to an antigen and various compounds are covalently bound are widely used. For example, 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. To be able to In addition, when 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, is administered to the living body, the complex is radiated to the tissue. Integrated activity Therefore, they can be used for the diagnostic imaging of those patients. Furthermore, a complex in which a biologically active substance is bound to an antibody becomes a highly selective drug. For example, 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. In addition, 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 ¾ 製造 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. There is a method of binding to a carboxyl group (containing glutamate or aspartic acid) or a carboxyl group (containing glutamate or aspartate), and particularly to the amino group. The way to do this is In particular, it is heavily used. For example, 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. (See Makoto Yamazaki, Shinichi Ishii, Ishizaki-'1¾, “Affinity Chromatography,” 3rd δ¾, 1975, p. 19, p. 87). In addition, 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.

Fallon, H. W. Strauss, E. Haber, Science, 209_, 295). In the production of a complex of an antibody and a biologically active substance, for example, a method for binding an anticancer drug mesoplex (P.N.

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)). In particular, first of all, a cross-linking agent, for example, a cross-linking agent for introducing an active disulfide group, N—succinimidyl 3— (2—pyridyldimethy) propio The net (abbreviated as SPDP) is introduced by reacting it with the amino group of the antibody, and thus A new disulfide bond is formed by reacting a thiol group containing a ricin A chain with the introduced active disulfide group. The antibody ♦

An A-chain complex is being produced.

However, the conventional methods for producing various conjugates by reacting with an amino group of an antibody have disadvantages that are common to them. This is because, when a substance binds to 〜 to several of a large number of amino groups present in an antibody protein, those amino groups are bound to an antibody. The fact is that they are indiscriminately used for binding substances regardless of whether they are involved in the binding activity to the antigen. Therefore, when amino groups involved in antigen binding are susceptible to a chemical reaction, particularly those amino groups are modified, and as a result, For example, the antigen-binding activity of the resulting complex is significantly reduced. For example, the monoclonal antibody 225.28S, which is a monoclonal antibody against a high-molecular-weight antigen on the cell membrane of melanoma, can 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. A. Fawazaz, T.S. Wang, A. E. st abrok, J. M. Rosen, A. Hardy, P. 0. AI derson, S. C.

S r i vastava, P. R i cards, and S.

Ferrone, J.NucIearMedicine, 26, 488-92 (1985)) ₀

Disclosure of invention

The present inventors have used the material Amino ^ V * -¾3 □ for the purpose of widespread and general use without causing any inevitable or inconvenient disadvantages with conventional methods. As a result of intensive research to develop 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. However, 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.

 That is, the present invention provides an antigen by modifying an amino group. □ After 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.

More specifically, the present invention is divided into the following two embodiments. (1) 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. After modification with an amino group reversible modifying agent, the remaining amino group of the antibody or its fragment is reacted with a compound having an amino group-reactive functional group. Thereafter, 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), and (2) 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.

 Danihi

After reacting with a cross-linking agent having a functional group, a compound having a cross-linking agent-reactive group at the cross-linking agent residue thus introduced into the antibody or its fragment is obtained. 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. BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, 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. In the present invention, 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.

 Further, the method of the present invention can be applied to any class or subclass of antibody. That is, 171 bodies have IgG, IgA, IgM, IgD and IgD.

Classes of IgE are known, and IgG has subclasses of IgG1, IgG2a, IgG2b, and IgG3. However, IgA has IgA and IgA2 subclasses, and 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.

In the present invention, 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), and then an amino-reactive functional group is added to the ίτ-isomer or the remaining amino group of the fragment. Immediately after the reaction of the compound having the compound (A), or after further subjecting the product to a further chemical reaction, the product (A) Reversible modifier) Any compound capable of removing a residue and regenerating an amino group may be used. Examples of 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

 Among succinic anhydrides, tetrafluorosuccinic acid

Exhaust system 3, 6—End key

Sodium delta hydrophthalic anhydride Exocis — 3.6 — End

Xoxohexahydrophthalic anhydride

Of porino and oral carboxylic acids

 It is necessary to introduce 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.

It has a reactive functional group 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. 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]

1 CH 2-G-R ²

 ! !

 0

[The definition of R ² is the same as in formula [I]. ]

 The acetyl group compound represented by the general formula [H] is used as a disulphide group-introducing agent.

 R S-R 1 ― C R [I J

 I!

0 ³ is a divalent organic group which forms a disulfide group together with the sulfur atom bonded thereto. The definitions of R ¹ and R ² are the same as in formula [I].

The disulphide compound represented by the general formula [IV],

R ³ -SRC one RC IV

 II

N H

 Residues up to 1 [m] Unit-based system

List

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. Also, 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. For example, 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.

In the formulas [I], [m〗 and [IV], 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

 — Dicarboxymidyl group

In the formulas [I] and [IV], 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. Thus, specific examples thereof include, for example, a 2-pyridylthio group (S—), 4— Pyridylthio group

(S-), N-year old 2-pyridylthio group S-), 2-difluorothio group

.

 〇 N H N

(S-), 4-Nitroguchi 1 2-Pyridylthio

Group (0 ₂ N —, ku-S —), 2 — benzothiazolylthio group (S-I) 9 benzoimida

Zylthio group (S-) and N-phenylamino-N'-fluoroiminothio group

— S — ノ etc. can be mentioned.

In the formula [IV], 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. When R- ⁴ is an amidin residue

R ⁴ represents, for example, a diamino group (H 2)

As a specific example of the maleimide group-introducing agent represented by the formula [I], for example, meta- (N-maleimide) benzoic acid N-hydroxy 2,4-Dimethyl phenyl ester, 3-(ιΝ 一 マ レ ミ ド) Pro Pionic acid N—Hydroxysuccinimide ester, 4— (N—maleimide) butyric acid 2,4—Dimethyl phenyl ester, N — Male middle dairy Nilroisin N — Hydroxy one 5 — Norboso is ren — Two, three-diphenyl benzene N-hydroxyl-succinimide ester, 4- (N-maleide) butyrate imida Lido, N- (N-malemidroisylaralanylloisyl) (xazolidinone) is converted to a acetyl acetyl group-introducing agent represented by the formula [H]. Specific examples include, but are not limited to, 2,4-dinitrophenylester, and acetic acid N-hydroxysimid es. Tell in 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-pyridyldithioa As a specific example of the active disulfide group-introducing agent represented by the formula [IV], cetylaralanylloisylararanyl-opening isocyanidazolide is exemplified. For example, 3 - (2 one pin lysyl autonomy old) profile pin on-I Mi de 馥 methylation E scan Te Le,

3- (2-pyridyldiethyl) butyl imidate ester, 41- (pyridyldithio) butylimido acid methyl ester, 21-pi Rigid roysylalanylloisylaminopropionic acid methyl ester, 3- (2-pyridyldithio) pulp amide, etc. There are many things to list.

Ten

Fifteen

Use

20

Okay. The method of introducing a reactive functional group introduced by a cross-linking agent into another functional group and then removing the reversible modifier residue of the protein amino group is also provided by the present invention. Included in Method A.

 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.

 Particularly preferred as such 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.

Nitrosoprea derivatives

0

NO

 0

Chlorambucil derivative

0

0

Mitomycin c derivative

0

 N

 C

Daunomycin-induced rest

CH

5-Fluoro-2'-derivative xylidine derivative 〇

 0

Desacetylvin blastine azide derivative

Vinblastine active ester derivative

 C〇2 CH3 0

Mesoplex active ester derivative

 〇

NH;

 Y H

NN CH: 0- ■ CiN-CH (CH2) 2 CO2 N

〇

Actinomycin-doxazinone induction rule.

L-N-Me Val L-N-Me Val

Sar Sar

0 L One Pro Shi One Pro 〇

GH: CH:

Further, specific examples of the biologically active substance as a compound having an amino group-reactive functional group suitably used in the method A of the present invention include an antibacterial substance. Is an isoazide derivative

 で For anti-viral substances,

 Acicloguanosine derivatives

〇

Η ₂

Then 0 C c 〇

〇 For antifungal substances,

 Fluorocytosine derivative

0

NHC 0 C 0 ₂ N

0

 〇 to N-

In the toxin or its fragment, 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

CH ₃₀

Examples of the enzyme include ceratiopeptidase and the like. In addition, 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,

 2, 6— 才 才 ((ボ (('/ Ϊ

 0

0 NC 0 ₂ H

 >

 〇

Ethylene diamine lactic acid derivative

For radioactive substances, the Volunter-Hunter reagent

Η 0

 [I * represents a radioactive iodine atom. ]

For fluorescent materials, full-length resin

"Ν = C = S

Η

Η 〇 〇

Examples of the insoluble support 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. In the case of the protein toxin ricin, its fragment A chain, etc., it is possible to react with the antibody or the antibody without specifically leading to the reactive derivative. The reaction is carried out using a condensing agent such as water-soluble rubodiimide or glutaraldehyde in the opening for the reaction with the amino group of the fragment. Close . In this case, the amino-reactive functional group of ricin and its' A chain is a carboxyl group or an amino group. In addition, 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.

In the method B of the present invention, a part of the amino group is used. Has an amino group-reactive functional group on the remaining amino group of an antibody or its fragment modified with a protein amino group reversible modifier. After reacting the cross-linking agent, a compound having a cross-linking agent-reactive functional group is bound to the cross-linking agent residue introduced into the antibody or its fragment. Thereafter, the reversible modifier residue of the protein amino group is removed. As the cross-linking agent having a functional group capable of reacting with a mino group, the same as the cross-linking agent used in the method A of the present invention can be used. They are as already described.

In the method (2) of the present invention, 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.

 To the crosslinker residue introduced into the antibody or its fragment:? In order to bond a compound having a reactive agent-reactive functional group, a compound having a crosslinking agent-reactive functional group is immediately reacted with the reactive group of the introduced crosslinking agent residue. In some cases, a compound having a cross-linking agent-reactive functional group may be reacted after converting the reactive group of the remaining cross-linking agent into another reactive group. You can do it. An example of converting a reactive group of a crosslinking agent residue to another reactive group is to convert a disulfide group to a 2-mercaptoethanol dithiolate plate. Example of conversion to a thiol group by a mercapto reagent such as thiol group. Conversely, 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. For example, when a reactive group originally contained in a wood ira agent is used for a reaction with a compound having a cross-linking agent-reactive functional group, 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; When the im agent is a chiral group-introducing agent, the crosslinking agent-reactive functional group is preferably, for example, a maleimide group, a door acetyl group, or an active sulfide group.

Further, in order to bind a compound having a cross-linking agent-reactive functional group to a cross-linking agent residue introduced into the body or its fragment, both of them must be used in addition to the first. Bonding using the cross-linking agent of No. 2 is also included in the method B of the present invention. For example, the introduced crosslinker residue (a reactive group that the original crosslinker had or a reactive group after undergoing a transformation) is a thiol group. And if it is a crosslinker-reactive functional group or a thiol group, for example, IN '-(1,2-phenylene) dimale ^, iN, N ' -C 1, 4 -phenylene) There is a method that uses dimaleimide as a second crosslinking agent. In this case, the reaction is carried out stepwise, for example, by adding 架橋, Ν '(1, 2, 2-phenylene) to the crosslinker residue introduced into the antibody or its fragment. ) After reacting with dimaleimide, remove excess N, N '-(1, 2-phenylene) dimaleide and then have a crosslinker-reactive functional group. It is desirable to react certain compounds or vice versa.

 -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 . Further, in the case of a compound having a disulfide group therein, a compound which originally generated a chiral group from the disulfide group was originally generated. It goes without saying that a compound having a thiol group itself can be used as a compound having a crosslinker-reactive functional group.

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.

Bioactive substance

 Antitumor biomaterial

 ♦ Neocartinostantin (abbreviated as NCS) derivative

HS 0 NH-NCS

ICH 2 CONH — NCS

NH described adjacent to NCS is an imino group derived from NCSj's own amino group.

No Downomycin derivative

Might mycin and methotrexin, vinblastine, or bindesin conjugated to human serum albumin (abbreviated HSA)

H S-H S A-N H-D) n

 ヽ

S and N written adjacent to HSA represent the 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

No For mitomycin C derivatives;:

In the case of mesoplex derivative

For vinplastin derivatives

For vindesine derivatives

One D

o

Antibacterial substances

 Isonia Hide derivative

Anti-viral substances Cycloguanosine derivatives

\ 〇 C 0 SH Antifungal substance

Toxin or its fragment

 Ricin A chain fragment

 Thiol group-introducing agent N-acetylsomocysteine A ricin A-like anti-viral protein obtained from Acacia magna pond, treated with lactone. In white holmon

 Estrone derivative

CH 30

enzyme

 Thiol group-introducing agent 2 — Cellulase peptide treated with iminothiirane Radioactive guest

Volton — Hunter reagent derivative H 0 — C-CH 2 CH 2 G 0 NHSH

I

 [I * represents a radioactive iodine atom. ]

In the case of fluorescent substances, full-length resin derivatives

Insoluble support

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)) As a raw material in the method of the present invention. 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. Further, 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 By processing There are a wide variety of insoluble supports such as gels, etc., which have been industrially supplied as useful substances, or are easily supplied industrially. 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 $ α, 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. It is preferable that 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. Regarding 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. In the case of purification, 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. First, in the reaction of a compound having an amino group-reactive functional group, 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. Furthermore, conditions that promote the elimination of the modifying agent residue bound to the amino group of the antibody are not preferred, ie, maleic anhydride, exo- — Cis 3, 6-endoxo — ⁴ -methyl phthalic anhydride, exocis 3 ′, S — endoxo The 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.

When further processing is performed on the substance bound to the antibody, the reaction conditions are also similar. In this case, 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

 ·> This is not limited to the case where 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. Of the so-called cross-linking 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. When adding purification, perform column chromatography such as gel opening and ion exchange, and dialysis.

 Prior to the reaction for removing the reversible modifying agent residue from the amino group, if the reversible modifying agent for the amino group remains in the system without being reacted. Can be inactivated, if necessary, by treating it with a compound having an amino group such as lysine, 2-aminoethanol, etc.

Next, preferred conditions for the reaction for removing the reversible modifier residue from the amino group of the antibody will be described. 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. In addition, tetrasulfonyl succinyl group, anhydrous trisyl rosyl group and acetoacetyl group are removed under weak alkaline conditions of PH810. Is preferred. In addition, 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. Also, 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.

 Next, examples will be described in order to explain the present invention in more detail, but the present invention is not limited to these examples. 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.

 This example is intended to illustrate the principles of the method of the present invention as set forth in claims 1 and 31.

R A:

CONH Ι9 + NHCO- (CH ₂ ) ₃ —

 0-,

H

 PH 6.0

1-2 1-1 Anti-human melanoma monoclonal antibody

Preparation of Complex (111) by Reaction of 96.5 (IgG2a) and N-Succinimidyl 41- (N-maleimide) Peptide-Antibody 96.5 of 6.5 / ¾ dissolved in 50 mM sodium borate buffer (PH 8.5) 1 ^ was added to dimethyl maleamide anhydride (dimethylformamide). The solution (0.56Μ) 10 ^^ was added under ice-cooling and stirring, and reacted for 30 minutes. During this time, 0.2 iM NaOH was added dropwise to keep the pH of the reaction solution at 8 to 9. Subsequently, 15 ^ JI of a DMF solution (0.054M) of N-succinimidyl-41 (N-maleimide) plate was added and allowed to react for 30 minutes. . After the completion of the reaction, the reaction solution was dialyzed against 0.1 iVI sodium phosphate buffer (PH 6.0) at 4Ό for 3S hours, and the reaction to remove the dimethyl maleyl group was allowed to proceed. A solution 1.1 of the anti-body (1-1) into which the target substance (N-maleimide) butylyl group was introduced was obtained.

1-2. Quantitative determination of complex Using a part of the complex solution obtained in 1-,

The concentration of the absorption by Ri antibody蛋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 ^ .

 In addition, an excess amount of N- (2,4-diphenyl phenyl) cysteine that reacts with the maleimide group introduced into the antibody is added and reacted. The solution was passed through a column of Sephadex G-25 (1.0 X 40C, 0.1 M phosphate buffer (pH 7.0)) to remove low molecular weight substances, and obtained. The absorbance at 280 nm due to the protein fraction of the protein fraction and the absorbance at 360 nm due to the introduced 2,4-dinitrophenyl group were measured. Then, the maleimide group introduced into the antibody molecule was quantified. As a result, the antibody concentration was IxlO ^ M, and the number of maleimide groups bound to the antibody was 3.2 on average per antibody molecule.

— 3. Quantification of the binding activity (antibody activity) of the complex to the antigen

Human melanoma cell line HiVIT — 2 suspension (1 ^× 10 ^s cells /) Q.2 mi was mixed with various concentrations of the test sample 0.1, mixed, and allowed to react at room temperature for 1 hour with occasional stirring. In addition, a constant concentration of I-labeled 96.5 antibody (I-96.5) 0.1

Add and mix at room temperature with occasional stirring.

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.

The concentration of the complex sample (1 C a) at which the complex inhibited 50% of the ^{| 2S} I-labeled 96.5 binding to HMT-12 cells was determined, and the original 96.5 antibody was determined.

As a result of calculating the antigen-binding activity of the complex from the comparison with IC 50, the antigen-binding activity of the complex was 63.2% of that of the 96.5 antibody.

— 4. 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.

 11-Dimethyl maleic anhydride and cross-linking agent N-succinimidyl 41- (N-maleimide) butyrate He reacted. Next, an aqueous solution (0.1M) 40 ^ A of N- (2,4-dinitrophenyl) cysteine is added to the reaction solution as a compound having a cross-linking agent-reactive functional group. , And react for 30 minutes.

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. .

I. 5. Quantification of antibody activity of the complex

 11. The antibody activity of the complex obtained in

It was quantified by the same method as in 1-3. Antibody activity of complex The sex was 65% of the original 96.5 antibody.

-6. Produce a complex of an antibody and a crosslinker having an amino group-reactive functional group without protecting a part of the amino group of the antibody with a reversible modifier. Comparison

 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).

¾5 formula: 0

96.5 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

 (95.7%) 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.

 13 As shown in 13, 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. Example 2 Production of Complex of Antibody and Crosslinking Agent

Anti:

 F

I g

— 1. Preparation of composite

1 mM ethylenediaminetetraacetic acid in which a monoclonal antibody (IgM) 90 / ^ with specific binding ability to mouse breast cancer cells MM46 is dissolved 0.1 M sodium phosphate buffer (ID 7.5) containing EDTA (abbreviated as EDTA) was added to tetrahydrofuran succinic anhydride under water cooling and stirring. 100 M 1 of a 0.1 M dioxane solution was added and reacted for 30 minutes. During this time, the pH of the solution was kept at 7 to 8 by dropwise addition of 0.1 M Na〇H. Next, 0.01-year-old N-succinimidyl 3- (2-pyridyl zircino) propionate (abbreviated as SPDP) Sun solution 15 J1 was added to the reaction solution and reacted for 60 minutes. Next, with stirring, add 0.1 NN a to the reaction solution.

0 H was added dropwise, the H was adjusted to 9.5, and the mixture was allowed to stand at 4 ° C for 4 days to remove tetrazoloxazyl radical. After completion of the reaction, the solution was washed with a column of Sephadex G-25 (10 mM sodium phosphate buffer (pH 7.4) containing ΊcXΊmM EDTA) (pH 7.4)-0.14 MN The solution was passed through a C) to remove low-molecular substances to obtain a solution of the antibody into which the objective 3 — (2-pyridyldithiyl) propynyl group was introduced.

— 2.

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 メ of the generated 2—mercapto pyridine and its absorption at 343 nm (ε 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 Ί molecule was 3.9 on average. Example 3 Production of Complex of Antibody and Crosslinking Agent Reaction:

ig

96.5 Antibody

Solution of dimethyl maleic anhydride (0.5M) 20 ^ in solution 2 of 5 OmM sodium borate buffer (PH 9.0>) containing 1 niM EDTA in which The mixture was added under ice-cooling and stirring with the mixture, and reacted for 30 minutes, during which 0.2 M NaOH was added dropwise to maintain the pH of the reaction solution at 9. Next, 21 im After adding 15fflg of Notchi-lane hydrochloride and dissolving it, it was reacted for Ί hour.Next, it was added to 0.1 0.1 sodium phosphate buffer containing ΊοιΜ EDTA (Ρ Ρ6). versus The solution was dialyzed at 4 C for 3 δ hours excluding the dimethyl maleyl group to obtain a solution of the antibody with a thiol group.The number of thiol groups introduced into the antibody was EII. man method

(Refer to Kozo Narita and Takashi Murachi, “Chemistry of Protein I (Biochemical Experiment Course 1)”, Tokyo Chemical Ambassador, 1976, p. 84).

It was determined that 7.2 units had been introduced. Example 4. Production of complex of antibody and cross-linking agent

 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 IgιΜ antibody by the same method as in Example 2. Example 5. Production of complex of antibody and anticancer drug Reaction formula:

CONH-19 MTX) n '

Ig MTX) _n 〇 ₂ Hno n PH 6

5-1. Production of composite

A 0.5 δ MD ιΜ F solution of dimethyl maleic anhydride was added to a 1.0 solution of 0.1 Μ borate buffer (ρ Η 8.8) in which 6.5 of the 96, 5 antibody was dissolved, and 10 ^ of a 0.5 MD MD Μ F solution of dimethyl maleic anhydride was stirred under ice-cooling. In addition to the following, the reaction was carried out for 30 minutes. During that time, 0.2Μ Na 0Η was added dropwise to the reaction solution.

PH was kept at 8-9. Next, the N-hydroxysuccinimide ester (P.KuI karn i et aI.) Of the method mix (hereinafter abbreviated as MTX) is used. , 〇 ancer R esearc, _1_, 2700-2706 (1981). Were synthesized. ) Was added thereto and reacted for 4 hours. After the reaction is completed, add 0.02 μl phosphate buffer to 0.14

 4 for Ma Ci (PH 6.0). After dialysis for 36 hours in C, the dimethyl maleyl group was eliminated to obtain a solution 1.2 / ^ of the complex, which was the target substance.

-2, Quantification of complex

 Using a part of the solution of the complex obtained in 5-1 and measuring the absorbance at 280 nm and 372 nm, the mass of the antibody protein and the amount of MTX in the complex are measured based on the measured absorbance. Was asked. At 280 nm

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

96.5 NTX of MTX was added to 2,8 / ώ of a 0.1 M phosphoric acid solution (PH 7,5) in which 5.6 ^ antibody was dissolved. HID '□ 0.104 iVl

5 ^ DF solution (the same as that used in Example 6-1) was added and allowed to react for 4 hours. Reaction mixture was added to 0,02 M phosphate buffer 0.14

 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.

5-4. Quantitative determination of binding activity of complex to antigen

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. In addition, 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 6

 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

 Production of complex of antibody and protein anticancer drug neocalcinostatin (abbreviated as NCS) Reaction formula:

NHAC

SH no n

7— 1. Production of IgG with a Thiol Group Introduced Anti-Melanoma Monoclonal Antibody ZM E018

(IgG2a) 50 mM sodium borate containing 1 mM EDTA in which 30 ^ was dissolved-0.14 Na CJ buffer (PH 9.0) 3 was added with dimethyl maleate A 0.5 MDMF solution of anhydrous 32 ^ was added with stirring under ice-cooling and reacted for 30 minutes. During this time, the pH of the reaction solution was kept at 8 to 9 by dropwise addition of 0.2 M Na aH. Subsequently, 1 S ^ i of a 1.0-MDMF solution of N-acetylphosphonic acid lactone was added, and the mixture was reacted for 1 hour. The 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.

-2. N-CS with a acetyl group

10 M U sodium phosphate buffer with NCS 0.9 / ¾ 0.14 M Na C (PH 7.4)

After adding 0.9 MDF solution of N-succinimidyl acetate to the 0.9 MDF and adding 8 ^ JI to the mixture for 1 hour, Sephadex G-25 was added. (1 ^ X4 an, 0.1 iM in sodium phosphate buffer) to remove the low molecular weight substance, and remove the solution of NCS into which the cedoacetyl group was introduced. Obtained .

-3, Production of complex of antibody and NGS

In the solution of the antibody into which the thiol group obtained in 7-1 was introduced, the solution of NCS in which the acetyl acetyl group obtained in 7-1 was introduced 1.5 / ^ And reacted for 24 hours. Then, 0.2 MHC Δ was added, and the reaction solution was allowed to react for 24 hours at a pH of 6, thereby removing the dimethyl maleyl group. Separate the reaction mixture to Sephadex G — 150 sp a (1.5 ^ X100 in 0.1 M sodium phosphate buffer) to obtain a solution of the complex of the antibody and NCS. As a result of analysis by disk electrophoresis, 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 . Example 8.

 Production of a complex of human serum albumin (abbreviated to HS A) in which an antibody is combined with an anticancer drug

Reaction formula

Ig

NH (HS) -HSA ^ HD) n 麵

La

, C0 ₂ Hno n

 NH

II

Now Ig C NHC SS— HSA C NH—D) n〃) n In the formula, D represents the anticancer drug mitomycin〇 derivative, and is represented by the following formula _c

8- 1. Introduction of 2-pyridyldithio group into antibody

 96.5 Antibody 15ff¾ was used as a solution of 50 nVI sodium borate buffer (ΡH9), 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

The pH of the 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

 Using EDTA-containing sodium phosphate buffer (H7.5), tightly tighten the gel mouth column of Cephadex G-125 (1.0 ^ X40 an). A solution 5. of the antibody into which the 2-pyridyldithiyl group was introduced except for the low molecular substance was obtained. The number of 2-pyridyl dithiol groups introduced into the antibody molecule can be determined by:-Excessive dithiothreitol acting on the sample containing a fixed amount of antibody; The absorbance at the absorption maximum (343 nm) of the pyridone was measured, and the amount of 21-pyridyldithio group contained in the sample was determined. The molar ratio to the antibody amount was calculated and determined, and the average was 4.8.

-2, Preparation of HSA by combining M. Machine C (M.VIC and Omission)

0.1M sodium phosphate buffer (H7.0) 2.0 ^ containing 1ED ED-A in which HSA 19.5 «st was dissolved was added to Ίa-1 [4— (N-succinimi Dokishikaruboniru) Butyril] Might Machine C

Dissolve into DMF 50, 1-add and stir After allowing the mixture to stand at 4 ° C for 6 hours to react, the cells were dialyzed against the buffer at 4 ° C for 36 hours. 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..3Λ¾. 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. Was determined by the-EI 1 man method, that is, a portion of the solution contained 'excess 5,5'-dithiopis-mono (2-ditorobenzoic acid) (abbreviated as DTNB). ), And the mixture was reacted at 4 for 30 minutes. Then, the reaction solution was passed through Sephadex G-25, and the protein was pooled. The protein concentration was determined by the Lowry method and 5-melka was added to a part of the solution by adding an excess of dithylate slat. Plot 2—Nitro- mouth To release benzoic acid, measure the absorption at 412 nm, and determine the concentration of the free substance, that is, the concentration of the thiol group contained in HSA. did . Based on the ratio to the protein concentration determined by the Lowry method, the number of thiol groups on iVIMC-HSA was calculated to be an average of 0.58 per HSA molecule. -3. 2-A conjugate with a dimethylmaleylated antibody that has introduced a pyridyldiethyl group and the HSA that has been introduced with MMC, thus removing the dimethylmaleyl group. Production of.

In the solution 6 ^ of the dimethyl maleylated antibody into which the 2-pyridyldiethyl group obtained in 8-1 was introduced, the MMC-introduced HSA obtained in 14-2 was added. The solutions were mixed and allowed to react at 4 ° C for 15 hours. Next, the reaction solution was dialyzed against 0.1 M sodium phosphate buffer (PH 6.1) for 48 hours to remove the dimethyl maleyl group, and the target antibody was obtained. A solution 7. of a complex of 96.5 and MMC-bound HSA was obtained. Dodecyl sulfate sodium-polyacrylamide was found to be unreacted by gel electrophoresis. The product was found to have 1-3 MMC-bound HSAs bound. It was also found that unreacted MMC-bound HSA remained, so the use of a Cephadex G-150 superfine Except for this, the complex was purified by chromatography.

Example 9. Production of antibody / antibacterial complex

Inversion:

 V CONH 19 <NHC0 ^ ~ VCH〇),

Person C C 0 ₂ _ H LI ノ CONHNH;

PH 5.5 PH 5.5

Escherichia coli antibody (I0G fraction) against Mycobacterium tuberculosis 60 ^ was added to 50 mM borate buffer (PH δ.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. Next, 0.5M of N-succinimidyl 4-ho and Lumirubenzoret

After adding DMF solution 10 and reacting for 30 minutes with T, the reaction solution was applied to Sephadex G—25 power column (2.0 × 40 ^ 0.1 M sodium acetate buffer (PH 5.5 )) To obtain a protein fraction.

Add isoninicotinic acid t-dazide 10 / ¾ to the above solution, react for 15 hours, and further add 0.1 iVl sodium acetate buffer (· 5.5) to 24. Time dialysis By removing the dimethylmaleyl group, a solution of a complex of the target antibody and isonicotinate hydrazide was obtained. Example 10. Production of a conjugate of an antibody and an antiviral drug

式 ceremony

now

 PH 6 Ig

A mouse monoclonal antibody against IgG (IgG), 60 / ^, dissolved in 50m borate buffer (PH 9.0) 6 under water cooling, Under stirring, a DMF solution (0.5 iV1) of 66 · Jl of dimethylmaleic anhydride. Was added and reacted for 30 minutes. in the meantime , The pH of the reaction solution was kept at around 9 by dropwise addition of 0.2 NaOH. Next, add a DMF solution (0.5Μ) 24 ^ — of an ester of N-hydroxysuccinimide active acid of a cycloguanosine compound and a citrate derivative, N-hydroxysuccinimide. The reaction mixture was dialyzed against 0.1! VI sodium phosphate buffer solution (PH 6.0) for 36 hours, and then reacted with the target antibody. A solution of a complex of acyclo and guanosine was obtained. Example 11

 Production of complex of antibody and metal chelating agent

n '

ig

-1. Complex of anti-MM46 antibody and gold and chelating agent 0.2 M sodium borate buffer in which anti-MM46 monoclonal antibody (IgG) 15 is dissolved. Solution (PH 9.0) 3 was added with glycol aldehyde 1 ^ and dissolved, and then added with 0.1 M aqueous solution of sodium cyanoborohydride for 30 minutes. Reacted. The reaction solution was dialyzed against 0.1 M sodium phosphate buffer solution at 4 ° C for 24 hours, and the recovered solution was 2,6-di-aged N- (Calbox Chill) Morpholine (abbreviated as DCH) 0.1 M dilute hexane solution 5 J1 was added and reacted for 30 minutes Pa. Finally, 0.1 molar aqueous solution of sodium periodate To a final concentration of 1 mM!] And react for Ί hour to remove 2 — hydroxyethyl groups. The reaction mixture was diluted with Sephadex G — 25 (1.5 ^ X 40 ai 1 OmiM sodium phosphate buffer solution — 0.14 M

 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.

Example 12 Production of Complex of Antibody and Metal Chelating Agent

Reaction formula

: 3 CCO ヽ

 〇

 : 3 CCO

Ig now (CF3 C〇 子 n IS `` C0 ₂ H 0

-I

 N ~ N ^ N

C〇 ₂ H 0 PH10.5 「0> Οι H La la Ig C〇 N ~ NN

 CO2 H

H0 ₂ C -1. Complex of anti-MM46 monoclonal antioxidant (IgG) with the metal chelating agent diethyltriamminepentanoic acid DTPA and abbreviated Production of.

 Anti-MM46 monoclonal antibody (IgG) 15/72? Dissolved in 50 mM sodium phosphate buffer (H9) 2 containing 0.14 iVla CJ A solution of 0.1 iVi dioxane in acetic acid at low mouth was added at 4 ° C with stirring and reacted for 30 minutes. During that time, 0.1 M Na0H was added dropwise to the reaction solution.

The pH was adjusted to around 9 = Next, a 0.01 M dioxane solution of DTPA in a 0.01 M dioxane solution was added, and the mixture was reacted for 30 minutes. Next, after the reaction, 1 M sodium carbonate was added dropwise to adjust the pH to 10.5, and the mixture was allowed to stand for 4 hours to release the trifluoroacetyl group. . 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. A solution of wInC13 dissolved in sodium acetate buffer, ΊmCi, was added, the mixture was stirred, and the mixture was allowed to stand for 30 minutes. Excess InC 13 through a column of Sephadex G—150 column) 1.0 X 60 ^, 0.1M sodium phosphate buffer (f) H 5.5))) Except for the above, a protein fraction showing radioactivity was pooled. The specific activity of the obtained In binding antibody was 1.1 Ci / Ig. Example 13. Production of antibody-fluorescent complex

50 mM NHaOH S

 PH 8.5 II

(CH ₃ COCH2 NH ^ -Ig> lg NHC H

C0 ₂ Hno n '0 ₂ Hno n' 96.5 Conjugation of Fluorescein to Antibody A

 Monoclonal antibody 96.5 10 m $ dissolved

0.25 M sodium carbonate buffer (PH 8.5) 1 M, water-cooled and stirred, 0.1 M diketene

The DMF solution was added and the mixture was allowed to react for 30 minutes, and then 50 ^ g of a full-length resincintiocyanate DiV1F solution 50 was added. After 5 minutes, the stirring was stopped, and the mixture was further reacted at 4 with 15 hours. Then, add ΊMNH ₂₀ H to make the final concentration 5001 and leave it at room temperature for 15 minutes to remove the acetyl group and remove the reaction solution. After passing through column G (25 × 1.5 on sodium phosphate buffer solution: 0.14 MNaC), pool the protein fraction to achieve the desired results. Example 14. Production of complex of antibody and insoluble support obtained antibody labeled with full-length resin Example 14

Reaction formula:

• CONH Ig-NH

-C〇 ₂ H / n

Mouse monoclonal antibody against oncofetal protein (abbreviated as GEA) · (IgG) 50 mM borate buffer (PH 8.5) in which ZS is dissolved To this, 38 Jl of a DlVIF solution of citraconic anhydride (0.5 M) was added under ice-cooling while stirring the mixture, and reacted for 30 minutes. During this time, the pH of the reaction solution was maintained at around 8.5 by dropwise addition of 0.2 M aOH.

Bromyl bromide suspended in buffer solution 6 above II

 UJ 1

11 W \ Γ \ 1

C v I!

 ノ l 11 VII Em \ J I

 J 1 1

 1 rn¾ hH = v It v 1 ― ^.

 1 Appendix Γ 1 BOJ rr

 Bird

 G A— s 1 i Γ

V V J 1 r \ y o ΠΊ

 I to * ?! 1 Y \ 1

 J c 1

 丄 (\ ni.

 ο> ro n: (

Θ

 Joy rt

 , C ~ a c

a □ H <:: “'

 1 '

ffl] V

Reaction formula:

I

2 OH

96.5 exo-cis-1,3-6-ene in 50 oiM sodium borate buffer (purified H8.5) 2 in which Fab fragment 12 /? Doxo-Tetrahydrophthalic anhydride in 0.1 M dioxane solution

Was added under ice-cooling and stirring, and reacted for 30 minutes. During that time, the pH of the reaction solution was maintained at 8 to 9 by dropwise addition of 0.2 M NaOH. Subsequently, the Volunteer-Hunter reagent (labeled p-hydroxifene) To a solution of rupropionic acid N (hydroxysuccinimide ester) (2000Gi / mmoIe) in 20% A (20mCi) The reaction was performed for 30 minutes. Reaction solution was added to 0.1 M sodium phosphate buffer

After dialysis against (pH 5.0) at 4 for 36 hours, exo-cis-1,6-endoxosome ⁴ -tetrahydrophthalyl group is removed and removed. Then, a solution containing the target, radiation-labeled Fab fragment

3, 7 were obtained (1.3 mci). Example 16.

 Production of complex of antibody and antibacterial agent

Anti, I heart formula:

In the production method in Jeongjeong · Row 5, instead of 96.5 anti-^, a rabbit egret antibody (IgG fraction) against Mycobacterium tuberculosis was used as a mesotrophic method. X-N-hydroxysuccinimide ester instead of antimicrobial isonizid p- (N-succinimide Using a hydrazide derivative with cis carbonyl) benzyl aldehyde, the others are subjected to similar operations to give a complex suspension of 複合 ゥ ギ 钴 イ and Isoni-azide. I got it. Example 17.

 Production of complex of antibody and hormone

Reaction formula:

Ai C0 ₂ H La

In the production method in Example 5, an anti-alkaline phosphotase monoclonal antibody (IgG) was used instead of the 96.5 antibody. Instead of the trexette, use the Estrone's succinometer N-Hydroxy succinimide ester, and perform other operations in the same manner. As a result, an anti-alkaline phosphonase monoclonal antibody-estrone complex was obtained. Example 18. Preparation of conjugate of antibody and antifungal agent

g

γΟΟΝΗ-Ig NHCO ^ CONH PH 6, C0 ₂ HJ n

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 19. Production of antibody-enzyme conjugate

Reaction formula:

^ • GONH Ig NHCOCHz ί HS-3-galactosidase π, ^

C〇 ₂ H No n

H

 La IS-NHCOCH2 S-3-galactosidase

In the preparation of 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. Using the enzyme, a complex of the antibody and 3-galactosidase was obtained. Example 20. Production of complex of antibody and chelating agent

 NHAc

 ONH 10 NHCO

 SH ノ n '

〇 ₂ H No n

zM In the production of Example 7, a chelating agent (S) was used instead of the NCS into which a acetyl group was introduced.

 1-(BromoAcet Amid Benzil)

 Using EDTA, the conditions for the elimination reaction of the dimethyl maleyl group were set to PH = 7.0, 36 hours at room temperature, and the other procedures were repeated to obtain the ZME018 antibody.

A complex of the reducing agent was obtained. Example 21 Production of Complex of Antibody and Fluorescent Substance

 ¾5 formula:

U i

NO:

In the preparation of Example 7, instead of reacting the NCS having a introduced cetyl group with PH 5, the fluorescent substance 7—chloro 4—2 Benzo — 2 year old Ί, 3 — diazole is reacted at pH 7.0, and the conditions for the elimination reaction of the dimethylmalein group are adjusted to pH = 7.0 at room temperature. The operation was performed in the same manner as above except for 30 hours to obtain a complex of the ZME018 antibody and a fluorescent substance. Example 22 Preparation of a conjugate of an antibody and a toxin

 Reaction formula:

NHAc

 Ig † NHCO

 PH 7 SS CONH-PAP

 n "In the production of Example 7, the protein toxin PAP was replaced by NCS having a acetyl acetyl group introduced into it. Fluoride

 (See Y. Masuho, K. Kisshida, and T. Hara, Bi0chem. Bi0pys. Res. Commun., 105, 462-469 (1982)). The conditions for the elimination reaction of the dimethyl maleyl group were PH 7.0, 36 hours at room temperature, and the other procedures were the same.

A complex of ZE018 antibody and toxin was obtained. Example 23. Preparation of a conjugate of an antibody and an insoluble support Formula:

, C〇 ₂ H n

A buffer solution (1 oiM) containing the Μ Μ 018 018 antibody which was protected with a dimethyl maleyl group and introduced a thiol group using the process of 7-Ί in the production of Example 7 Activated 5.0 M sodium phosphate solution (H 7.4) containing ED 5.0 Τ 5.0 ^ Activated chitin propylserose 4B gel (Pharmacia) In addition to the suspension in the buffer solution, 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 24, Production of complex of antibody and radioactive substance

γ

义

Instead of the human serum albumin bound to MMC in the production of Example 8, I-labeled N— (2—mercaptoethyl) 13— (4—hydroxy Shi Hue Nyl) Propionamide was used to produce a complex of an antibody and a radioactive substance. Example 25. Production of complex of antibody and anticancer drug

ig

Ig So MTX) n

25-1. 96.5 Production of complex of antibody and MTX

96.5 Antibodies 7.15 ^ dissolved in 50 mM boric acid buffer solution — 0. M sodium phosphate (ρH 8.6) 1.1 DMF solution (95.3 mM) of dimethylmaleic anhydride in 0.2 NN a Keep the pH of the solution at 8.6 to 9.0 with OH and gradually add dropwise. The reaction was carried out for 30 minutes. Next, the reaction solution was dispensed into three test tubes at a rate of 0.35 i, and a DMF solution (17.4 m) of the N—hidden succinimide ester of MTX was added to each tube. The reaction was carried out at 4 ° C. for 4 hours in addition to the MTX activity ester IgG molar ratio of 10, 20, 40. The three reaction solutions were dialyzed for 2 days against 10 mM phosphate buffer-0.14 M sodium chloride (f) H 7.2) at 4 ° C for 2 days. g G —. TX complex was obtained. The average molar binding ratio of MTX / IgG and the antigen-binding activity of each complex were determined by the methods described in 5-2 and 113, respectively. The result was as follows

Note that the antibody recovery in this method is not available. In each case, it is over 70%.

-2. Comparative example

 96.5 Antibody dissolved in 0.05 M sodium phosphate buffer (H 8.0) at 4 ° C.

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.

Measured by the method described in 5-2 and 1-3. The results were as follows.

MTX / IgG molar binding ratio Antigen binding activity

 2-4 51%

 4.6 25%

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.

-3. Preparation of complex of Ζ Μ 018 antibody and Μ Τ X 0.9 0.9% sodium chloride solution with 21.2 ^ Ε 018 antibody dissolved in 1.

 (PH 8.8) 0.2 / ^, then 1 N

a) While maintaining the H of the solution at 8.8 to 9.0 at 0 H, gradually add 60 Jl of DMF solution of dimethylmaleic anhydride (498 fflM), and stir at 0 ° C with stirring. The reaction took place for a few minutes. Next, add 25 ^ of a DMF solution (83.4 mM) of M-N-hydroxysuccinimide ester of MTX to the reaction mixture and incubate at 4 ° C for 4 hours at room temperature. I did it. The reaction was dialyzed at 4 ° C against 10 mM phosphate buffer-0.14 M sodium chloride (PH 7.2) for 2 days to give a TX / I9G average molar binding ratio of 5.9. It is. The IgG-MTX complex was obtained. The antigen binding activity of the obtained complex was 60%. The antibody recovery was 80%. As described above, 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

26— Manufacture of antibody-MTX complex

A. Production by the method of the present invention

 An antibody produced according to the reaction formula described in Example 25 and an anti-human melanoma antibody 96.5 or ZME013) 10-20 /? ¾! Dissolved in 50 mM borate buffer-0.14 M sodium chloride (PH 8.6).. Dissolved in DMF 50 ^ to 200-fold moles of anhydrous dimethyl maleate antibody. Acidic acid, N.

a The pH was added while maintaining the pH at 8.5-9.0 with 〇H, and the reaction was carried out for 30 minutes at 0. Next, add 10-30 fold moles of MTX N-succinimide ester to the antibody dissolved in DMF 15-20 ^ H. Let react for hours. The reaction mixture was folded at 4 for 2 to 3 days to obtain a 96,5-^^ or ∑E018-MTX complex. Each complex The MTXZIgG average molar binding ratio of the product was determined by the method described in Example 512. The results were as shown in the table.

Β Manufacturing by conventional methods

 Antibodies (anti-human melanoma antibodies 96, 5 or ZM

(Ε 018) 0.1 M phosphate buffer (ΡΗ 8.0) in which 1-10 was dissolved was dissolved in DMF 10, and N-succinate of MTX was dissolved in DMF 10 at 10-15 times the molar amount of the antibody. The reaction was carried out at 4 ° C for 4 hours with the addition of a dimidyl ester. After the formed precipitate was removed by centrifugation, the reaction solution was dialyzed at 4 G for 2 to 3 days to obtain 96.5-ΜTX or ZE018—MTX complex. The MTX / I0G average molar binding ratio of each complex was determined by the method described in Example 5-2. The results were as shown in the table. 6-2 Measurement of antigen-binding activity of the complex

 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

^I2S I-labeled 96.5 as an I-labeled antibody and Z In the case of the ME 018 complex, SK and a non-Bok ra node on Ma cells棕- MEL - 28, and have use of the ^'25 as the 〖labeled antibody' ^2S I-labeled Z iVl E 018, It was determined by the method described in Examples 13 to 13. Incubation of the cells with the complex or I-labeled antibody was performed at 2 (TC. The antigen binding activity obtained was as shown in the table. It was.

From the table, it can be seen that in the case of 96.5—ΜX-complex and ZiVl018018-c-X-complex, the composite produced by the method of the present invention is different from the composite produced by the conventional method. It also shows that the antigen binding activity is high. Industrial applicability

 According to the method of the present invention, an antibody complex retaining antibody activity can be efficiently produced. The obtained antibody conjugate can be used for various purposes. For example, 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. In addition, 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. Furthermore, 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.

Claims

By modifying the amino group, antigen binding activity can be improved.

After a part of the amino group of the antibody or its fragment that reduces the activity of the antibody or its fragment is decorated with a reversible modifying agent for the protein amino group, the antibody or its fragment The remaining amino group in the fragment is reacted with a compound having an amino group-reactive functional group, and the reversible modification of the protein amino group from the metabolite is carried out. A method for producing an antibody conjugate, comprising removing an agent residue.

The method for producing an anti-fundamental compound stand according to claim 5, wherein the antibody is a monoclonal antibody.

4. The method for producing an antibody conjugate according to claim 3, wherein the reversible modifying agent for the protein amino group is maleic anhydride.

The method for producing an antibody conjugate according to claim 1, wherein the reversible enamel of the protein amino group is succinic anhydride.

The method according to claim, wherein the reversible modifying agent for the protein amino group is a reactive derivative of polycarboxylic acid carboxylic acid. 2. The method for producing an antibody conjugate according to claim 1.

 6. The method for producing an antibody conjugate according to claim 1, wherein the reversible protein amino group is used, and the decorating agent is diketen.

 7. The method for producing an antibody conjugate according to claim 1, wherein the reversible modifying agent for the protein amino group is 2-hydroxyacetic acid aldehyde.

 8. The method for producing an antibody complex according to claim 1, wherein the compound having an amino group-reactive functional group is a crosslinking agent.

 9. The method for producing an antibody conjugate according to claim 8, wherein the crosslinking agent is a maleimide group-introducing agent.

0. The maleimide group-introducing agent has the general formula [I],

Lu Mi

Is a maleimide compound represented by the formula: Item 10. The method for producing an antibody conjugate according to Item 9.

9. The method for producing an antibody conjugate according to claim 8, wherein the cross-linking agent is a pseudoacetyl group-introducing agent. Is a general formula [2],

I C H 2 -C-R 2 …… [J]

 II

 0

[R ² was or A Le co-Lumpur residues in the active E scan Te Lumpur is Ru Oh In A Mi down residues in the active A Mi de.

A method for producing the antibody conjugate according to claim 11, which is a chloride compound represented by the formula: wherein the crosslinking agent is a disulfide group-introducing agent. The method for producing an antibody conjugate according to item 8; wherein the disulfide group-introducing agent has the general formula [H],

R ³ -S-R ¹ CR ² I 1

 0

R ¹ is a divalent organic group, R ² is the alcohol residue of the active ester or the residual S of the active amide S, and R ³ is the sulfur atom to to

And form a dissolfide group. Is a disulfide compound represented by

 14. The method for producing an antibody conjugate according to claim 13. 15. The disulfide group-introducing agent has the general formula [W

R ³ one S one R ¹ one C - R [W]

 N H

Is a divalent radical, R 3 is a monovalent radical which combines with the (/) atom to form a disulfide group, and R ⁺ is an imido acid group. Steal's alcohol residue or amidine's amidine residue

 14. The method for producing an antibody conjugate according to claim 13, which is a disulfide compound represented by the following formula:

16. The method for producing an antibody conjugate according to claim 8, wherein the cross-linking agent is a thiol group-introducing agent.

 17. The claim according to claim 16, wherein the agent for introducing a thiol group is 2-iminothiamine or N-acetylthiomethylene thiolactone. Of antibody conjugate

18. The antibody according to claim 1, wherein the compound having an amino group-reactive functional group is a biologically active substance. Manufacture of coalescence ¾ &.

 19. The method for producing an antibody conjugate according to claim 18, wherein the biologically active substance is an antitumor substance.

 20. The method for producing an antibody complex according to claim 18, wherein the biologically active substance is an antibacterial substance.

 21. The method for producing an antibody conjugate according to claim 18, wherein the biologically active substance is an antiviral substance.

22. The method for producing an antibody conjugate according to claim 18, wherein the biologically active substance is an antifungal substance.

 23. The method for producing an antibody conjugate according to claim 18, wherein the biologically active substance is a cytotoxic substance.

 24. The method for producing an antibody conjugate according to claim 18, wherein the biologically active substance is a toxin or a fragment thereof.

 25. The method for producing an antibody conjugate according to claim 18, wherein the biologically active substance is formone.

26. bioactive agent Ru Oh enzyme, method for producing antibody complex range囲第18 claim of claim ₃

27. The antibody according to claim 1, wherein the compound having an amino group-reactive functional group is a chelating agent. Manufacturing method of composite.

4. The method for producing an antibody conjugate according to claim 3, wherein the compound having an amino group-reactive functional group is a radioactive substance.

2. The method for producing an antibody conjugate according to claim 1, wherein the compound having an amino group-reactive functional group is a fluorescent substance.

4. The method for producing an antibody conjugate according to claim 1, wherein the compound having an amino group-reactive functional group is an insoluble support, and the range of qf is determined.

It 's time to marry Aminoki! 9 Antibodies with reduced antigen-packing activity or some of the 7 mino groups in the fragment are modified with a protein amino group reversible modifier, Alternatively, the remaining amino group of the fragment is reacted with a crosslinking agent having an amino group-reactive functional group, and thus, the antibody or the antibody. After a compound having a cross-linking agent-reactive functional group is bound to the cross-linking agent residue introduced into the fragment, the reversible modifier residue of the protein amino group is removed. Method for producing antibody conjugate characterized by

32. The method for producing an antibody complex according to claim 31, wherein the antibody is a monoclonal antibody.

 33. The method for producing an antibody conjugate according to claim 31, wherein the reversible modifier for the protein amino group is maleic anhydride.

 34. The method for producing an antibody conjugate according to claim 32, wherein the reversible modifying agent for the protein amino group is succinic anhydride.

 10 35. The method for producing an antibody conjugate according to claim 31, wherein the reversible modifying agent for a protein amino group is a reactive derivative of a polyhalogenated carboxylic acid.

 36. The method for producing the antibody conjugate according to claim 31, wherein the reversible modifying agent for the protein amino group is diketen.

15 jano iS.

 37. The method for producing an antibody conjugate according to claim 31, wherein the reversible crowning agent for the protein amino group is 2-hydroxyacetic acid aldehyde.

 38. Crosslinking agent with amino group reactive functional group

20. The claim 31 which is an imido group-introducing agent. For producing antibody conjugates

The maleimide group-introducing agent has the general formula [I]

[RCI] organic group, R ^2. Is an active ester group or an amide of an active amide.

Is a maleimide compound represented by the formula:

35. The method for producing an antibody conjugate according to claim 38, wherein:

32. The method for producing an antibody conjugate according to claim 31, wherein the crosslinking agent having an amino group-reactive functional group is a chodoacetyl group-introducing agent.

The acetyl group-introducing agent has the general formula [I],

IC Η 2-C-R ² ...... [! I]

 II

 0

[R ² is an alcohol residue of the active ester or an amide residue of the active amide. 41. The method for producing an antibody conjugate according to claim 40, wherein the method is a pseudoacetyl compound represented by the formula:

32. The method for producing an antibody conjugate according to claim 31, wherein the cross-linking agent having an amino group-reactive functional group is a dissolving group-introducing agent. .

The disulphide group-introducing agent has the general formula [H],

R ³ -S-R ¹ C 1 R ² …… []

 II

 0

1 is a divalent organic group, R ² is the active ester alcohol residue or the amide residue of the active amide, and R ³ is the same as the bound sulfur atom. It is a monovalent organic group that forms a disulphide group.

43. The method for producing an antibody conjugate according to claim 42, which is a disulphide compound represented by the formula: The disulphide group-introducing agent has the general formula [IV],

R ^3- one S-one R ^1- one

R 1 is divalent organic

 With sulfur atoms

 To be formed

Stealth alcohol residue or amidine It is a min residue. 43. The method for producing a well body composite according to claim 42, wherein the method is a disulfide compound represented by I.

45. The method for producing an antibody conjugate according to claim 31, wherein the crosslinking agent having an amino group-reactive functional group is a titanium group-introducing agent.

 46. The antibody according to claim 45, wherein the thylamine group-introducing agent is 2-iminothiolane or N-acetyltyl homocysteine lactone. Composites> a ¾ ¾ o

 47. The method for producing an antibody conjugate according to claim 31, wherein the compound having a crosslinker-reactive functional group is a bioactive substance.

 48. The method according to claim 47, wherein the biologically active substance is an antitumor substance.

 49. The method according to claim 47, wherein the biologically active substance is an antibacterial substance.

 50. The method for producing an antibody conjugate according to claim 47, wherein the biologically active substance is an antiviral substance.

5 1. The claim that the biologically active substance is an anti-capillary substance. πη

 50. The method for producing an antibody conjugate according to item 47.

 52. The method for producing an antibody conjugate according to claim 47, wherein the biologically active substance is a cytotoxic substance.

 53. The method according to claim 47, wherein the biologically active substance is a toxin or a fragment thereof.

 54. The method for producing an antibody conjugate according to claim 47, wherein the biologically active substance is formone.

 55. The method for producing an antibody conjugate according to claim 47, wherein the biologically active substance is an enzyme.

 56. The method for producing an antibody conjugate according to claim 31, wherein the compound having a crushing agent-reactive functional group is a chelating agent.

 57. The method for producing an antibody conjugate according to claim 31, wherein the compound having a crosslinking agent-reactive functional group is a radioactive substance.

 58. The method for producing an antibody conjugate 'according to claim 31, wherein the compound having a crosslinker-reactive functional group is a fluorescent substance.

59 · y ^ z | Compounds with α-agent-reactive functional group are insoluble 32. The method for producing an antibody conjugate according to claim 31, which is a support.