Reducing agent for removing cap of immunoglobulin or antigen-binding fragment thereof

Abstract

A reducing agent containing, as an active ingredient, a compound in which a plurality of water-soluble functional groups are bonded to a triarylphosphine-based compound represented by chemical formula (I) can be used as a reducing agent for selectively removing a cysteine residue cap at the amino acid position 80 according to the Kabat numbering scheme in a light chain variable region of an immunoglobulin or antigen-binding fragment thereof. (In the formula, R1, R2, and R3 are each independently an aryl group or a heteroaryl group.)

Description

Reducing agent for cap removal of immunoglobulins or their antigen-binding fragments

[0001] The present invention relates to a reducing agent for selectively removing the cap of the cysteine ​​residue at amino acid position 80 by Kabat numbering in the light chain variable region of an immunoglobulin or its antigen-binding fragment, a method for producing a decapped immunoglobulin or its antigen-binding fragment using the same, and a method for producing a conjugate of an immunoglobulin or its antigen-binding fragment and a functional substance.

[0002] Antibody-drug conjugates (ADCs), which combine immunoglobulins (antibodies) that bind to antigens characteristically expressed in cancer with anticancer drugs that possess potent cytotoxic activity, are drugs that minimize side effects on normal cells caused by the administration of anticancer drugs alone while also possessing strong killing power against cancer cells. The manufacturing method mainly involves reducing disulfide bonds in immunoglobulins, which have high solvent accessibility, to generate cysteine ​​or lysine residue side chains, and then attaching anticancer drugs that have functional groups (e.g., maleimides or active esters) that can bind to these residues.

[0003] On the other hand, it has been found that ADCs produced using this method yield a heterogeneous mixture with a wide distribution of drug-to-antibody ratios (DARs). Among these widely distributed DARs, ADC species with high DARs are more easily cleared from the bloodstream than ADC species with low DARs, which may lead to a deterioration in therapeutic index and pharmacokinetics (Non-Patent Literature 1).

[0004] Therefore, ADCs with a uniform DAR distribution are expected to exhibit less variability in clearance rates from the blood compared to heterogeneous ADCs, leading to improvements in therapeutic index and pharmacokinetics. Against this backdrop, in recent years, with the development of protein engineering and antibody modification technologies, numerous examples of the creation of regioselective and highly uniform ADCs that utilize the structural characteristics of immunoglobulins have been reported. One of the main methods involves selectively introducing drugs to unpaired cysteine ​​residues in immunoglobulins.

[0005] These unpaired cysteine ​​residues are generally known to form disulfide bonds with external cysteine ​​or glutathione through post-translational modification during immunoglobulin production by mammalian cells (Non-Patent Literature 1). Molecules introduced through such post-translational modification are called "caps," and in order to utilize unpaired cysteine ​​as a modification site, it is necessary to selectively reduce the disulfide bond between it and this cap (cap removal).

[0006] Specific examples of selective cap removal of unpaired cysteine ​​include cap removal using thiol-based reducing agents, such as the removal of the cysteine ​​residue at amino acid position 80 by Kabat numbering in the variable region of the light chain of immunoglobulins using an L-cysteine ​​solution contained in a buffer (Patent Document 1), and the removal of unpaired cysteine ​​artificially introduced into immunoglobulins (Patent Document 2).

[0007] In addition to thiol-based reducing agents, there are also methods for removing caps using triarylphosphine-based reducing agents. Specifically, there is a method (Patent Document 3) in which an artificially introduced cap that is easily reduced due to electron deficiency is artificially introduced into an unpaired cysteine ​​in an immunoglobulin, and a triarylphosphine-based reducing agent is used to selectively remove the artificially introduced cap while preventing the destruction of the naturally occurring interchain disulfide. There is also a method (Patent Document 4) in which a naturally occurring cap derived from a naturally occurring component that is bound to an unpaired cysteine ​​is used to selectively remove the cap derived from a naturally occurring component that is bound to the other cysteine ​​by using a triarylphosphine-based reducing agent.

[0008] Special table 2018-526975 Publication Special table 2017-506262 Publication Special table 2018-525990 Publication Special table 2008-546670 Publication WO2019 / 191630

[0009] Nature Biotechnology, 2008, Vol. 26, No. 8, p. 925 - 932

[0010] The method of cap removal using a thiol - based reducing agent destroys the native inter - chain disulfide (native inter - chain disulfide) present in a part of immunoglobulins. Therefore, it requires an oxidation process for the reconstruction of disulfide bonds and takes a long time until a series of cap removals is completed. Further, as shown in the chemical reaction formula represented by the following formula (I), in the cap removal by a thiol - based reducing agent (R - SH; 1), the cap of immunoglobulin asymmetric cysteine (Ab - S - S - CAP; 2) is removed to generate immunoglobulin (Ab - SH; 3), but the cap structure (R - S - S - Cap; 4) removed from the immunoglobulin by the equilibrium reaction becomes the cap agent of the immunoglobulin. Therefore, in order to efficiently proceed with the decapping reaction of immunoglobulin over this equilibrium state, an open - system chemical reaction formula (ii) in which a reducing agent is continuously flowed from the outside to remove the cap structure removed from the immunoglobulin from the reaction field was essential.

[0011]

[0012] When using a triarylphosphine - based reducing agent, the problem of the equilibrium reaction during decapping that occurs when using a thiol - based reducing agent can be solved. However, special and complicated processes such as artificially introducing a cap into the immunoglobulin in advance or substituting one cysteine of the native inter - chain disulfide in the immunoglobulin with a different amino acid residue in advance were required. Also, when using a triarylphosphine - based reducing agent, a method for selectively removing the cap from the cysteine residue at position 80 of the amino acid in the Kabat numbering in the variable region of the light chain of immunoglobulin had not been established.

[0013] The inventor of the present invention focused on the cysteine residue at position 80 of the amino acid in the Kabat numbering in the variable region of the light chain of immunoglobulin as a binding site for functional substances such as immunoglobulin and various drugs, and conducted intensive studies to solve the problem of simply realizing selective cap removal at this site in a short time. As a result, it was found that the above problem can be solved by a triarylphosphine-based reducing agent having a plurality of water-soluble functional groups, and the present invention was completed.

[0014] That is, the present invention is composed of the following (1) to (20).

[0015] (1) A reducing agent for selectively removing the cap of the cysteine residue at position 80 of the amino acid in the Kabat numbering in the variable region of the light chain of immunoglobulin or its antigen-binding fragment, comprising as an active ingredient a compound in which a plurality of water-soluble functional groups are bonded to a triarylphosphine-based compound represented by the following formula (II).

[0016]

[0017] (R １ , R ２ and R ３ are each independently an aryl group or a heteroaryl group.) (2) The reducing agent according to (1), wherein the water-soluble functional group is a sulfonic acid group or a sulfonate group.

[0018] (3) The reducing agent according to (1) or (2), wherein 2 or 3 water-soluble functional groups are bonded to the triarylphosphine-based compound, and 0 or 1 is directly bonded to any one of the above R １ , R ２ and R ３ .

[0019] (4) The reducing agent according to any one of (1) to (3), wherein the compound to which a plurality of water-soluble functional groups are bonded to the triarylphosphine compound is potassium 4,4'-(phenylphosphinediyl)dibenzenesulfonate, triphenylphosphine-3,3',3''-trisulfonic acid trisodium salt, or 3,3'-(phenylphosphinediyl)dibenzenesulfonic acid disodium salt.

[0020] (5) The reducing agent according to any one of (1) to (4), wherein the cap is cysteine, glutathione, or cysteinylglycine.

[0021] (6) A method for removing the cap from an immunoglobulin or an antigen-binding fragment, wherein the cap of the cysteine ​​residue at amino acid position 80 in the light chain variable region of the immunoglobulin or antigen-binding fragment is selectively removed by Kabat numbering using a reducing agent described in any of (1) to (5).

[0022] (7) The method according to (6), further comprising the step of mixing the reducing agent with a solution containing the immunoglobulin or an antigen-binding fragment thereof, and then allowing it to stand.

[0023] (8) The method according to (7), wherein the solvent of the solution is a buffer solution with a pH of 4 to 9.

[0024] (9) The method according to (7) or (8), wherein the solvent of the solution is a buffer solution at a pH under physiological conditions.

[0025] (10) The method according to any one of claims (7) to (9), wherein the step is to let the mixture stand at 30 to 40°C after mixing.

[0026] A method for producing decapped immunoglobulin or antigen-binding fragments, comprising the step of selectively removing the cap of the cysteine ​​residue at amino acid position 80 in the light chain variable region of immunoglobulin or antigen-binding fragment by Kabat numbering in any of the methods of (11)(6) to (10).

[0027] (12) The method for producing immunoglobulin according to (11), wherein the immunoglobulin is a rabbit-derived chimeric immunoglobulin.

[0028] (13) The method for producing a human immunoglobulin according to (12), wherein the rabbit-derived chimeric immunoglobulin is a humanized immunoglobulin containing a complementarity-determining region of human-rabbit chimeric immunoglobulin or rabbit immunoglobulin.

[0029] (14) The method for producing immunoglobulin according to any one of (11) to (13), wherein the isotype of the immunoglobulin is IgG.

[0030] (15) The method for producing the immunoglobulin or its antigen-binding fragment according to any one of (11) to (14), wherein the immunoglobulin or its antigen-binding fragment has immunoreactivity with the CAPRIN-1 protein.

[0031] (16) An immunoglobulin or antigen-binding fragment thereof that is immunologically reactive with the CAPRIN-1 protein, wherein the cap of the cysteine ​​residue at amino acid position 80 is selectively removed by Kabat numbering in the light chain variable region.

[0032] A method for producing a conjugate, comprising the step of attaching a functional substance to a cysteine ​​residue of a decapped immunoglobulin or its antigen-binding fragment obtained by any of the methods described in (17)(11) to (15).

[0033] (18) The method for producing the functional substance according to (17), wherein the functional substance is one or more selected from the group consisting of drugs, fluorophores, fluorescent dyes, polypeptides, immunoglobulins, antibiotics, nucleic acids, radionuclides, chemical linkers, small molecules, chelating agents, and lipids.

[0034] (19) The method for producing according to (17) or (18), wherein the decapping immunoglobulin or its antigen-binding fragment has immunoreactivity with the CAPRIN-1 protein.

[0035] (20) A conjugate in which a functional substance is bound to a cysteine ​​residue in which the cap at amino acid position 80 has been removed by Kabat numbering in the light chain variable region of an immunoglobulin or antigen-binding fragment that is immunoreactive with CAPRIN-1 protein.

[0036] This specification includes the disclosures of Japanese Patent Application No. 2024-167986, which forms the basis of the priority claim of this application.

[0037] The present invention enables the selective removal of a cap at the cysteine ​​residue at amino acid position 80 in the Kabat numbering of the light chain variable region of immunoglobulin or its antigen-binding fragment, and the binding of a functional substance via the cysteine ​​residue, to be carried out quickly and easily.

[0038] In this specification, "immunoglobulin" and "antibody" refer to living molecules that specifically bind to any one or more target antigens or epitopes, and the terms are used interchangeably. Representative examples of the animal species of the living organism include humans, rodents (mice, rats, etc.), rabbits, sheep, and chickens. Typical immunoglobulin structures consist of two pairs of polypeptides, totaling four polypeptide chains: two full-length light-chain polypeptides and two full-length heavy-chain polypeptides. These polypeptides are generally linked to each other by innate disulfide bonds.

[0039] In this specification, "antigen-binding fragment" refers to a fragment of a molecule among the immunoglobulins that has specific binding ability to any one or more target antigens or epitopes, and includes at least one light chain variable region. Examples include those that bind to the target in monovalent form (e.g., Fab, F(ab'), and Fv fragments, etc.) and those that bind to the target in divalent form (e.g., F(ab)'). ２ Examples include fragments, etc. Other examples of antigen-binding fragments artificially modified by linker linking through genetic engineering or chemical transformation include those that bind to the target in a monovalent state (e.g., scFv antibody), those that bind to the target in a bivalent state (e.g., diabody), and those that bind in a polyvalent state (triabody or higher) (e.g., triabody, tetrabody).

[0040] In this specification, "light chain variable region" refers to the variable region portion of an immunoglobulin light chain, which consists of complementarity determining regions (CDRs) that are directly involved in the recognition of target antigens or epitopes, and framework regions (FRs) that provide appropriate stereochemistry for the CDRs and support the recognition of target antigens or epitopes.

[0041] In this specification, "the cysteine ​​residue at position 80 in Kabat numbering in the light chain variable region" refers to a cysteine ​​residue that is the 80th amino acid or an equivalent position when counted from the N-terminus of the light chain using the Kabat numbering method. This cysteine ​​may be naturally occurring or artificially introduced by obvious methods by those skilled in the art. In addition, leader sequences (or signal peptide sequences) that promote translocation across the endoplasmic reticulum membrane may be added for efficient cellular production of immunoglobulins, but these are not included in the Kabat numbering count.

[0042] In this specification, "cap" refers to a disulfide bond formed by an external monothiol compound (R-SH) through post-translational modification of an unpaired cysteine ​​side chain (Ab-SH) in immunoglobulin during immunoglobulin production by mammalian cells. Examples of monothiol compounds include, but are not limited to, cysteine, glutathione, and cysteinylglycine.

[0043] In this specification, "selective cap removal" or "selective cap removal" refers to the specific cleavage of the disulfide bond between the cap and the immunoglobulin by reducing it, while maintaining the intrinsic disulfide bond present in the immunoglobulin.

[0044] In this specification, "decapped immunoglobulin or its antigen-binding fragment" refers to immunoglobulin or its antigen-binding fragment obtained by selective cap removal of immunoglobulin or its antigen-binding fragment.

[0045] As used herein, the term "rabbit-derived immunoglobulin" refers to rabbit immunoglobulin or an immunoglobulin derived from rabbit immunoglobulin. Rabbit immunoglobulin is an immunoglobulin obtained by administering an antigen to a rabbit for immunization, fusing spleen cells and myeloma cells isolated from the rabbit, and selecting a clone that produces an antibody that binds to the target antigen from the resulting immortalized fused cells (hybridomas). An immunoglobulin derived from rabbit immunoglobulin refers to an immunoglobulin obtained by using genetic engineering techniques or the like based on the amino acid sequence information of rabbit immunoglobulin.

[0046] Hereinafter, the details of the present invention will be described.

[0047] <Reducing agent> The present inventors have found that a compound in which a plurality of water-soluble functional groups are bonded to a triarylphosphine-based compound represented by the following formula (III) (hereinafter referred to as "triarylphosphine-based reducing agent") can be used as a reducing agent that can easily and selectively remove the cap of the cysteine residue at amino acid position 80 (hereinafter referred to as "light chain Cys80") in the Kabat numbering in the variable region of the light chain of an immunoglobulin or its antigen-binding fragment (hereinafter, these are collectively referred to as "immunoglobulin" or "antibody") in a short time. The caps that can be removed by the triarylphosphine-based reducing agent include all caps that can be present at light chain Cys80, preferably cysteine, glutathione or cysteinylglycine, more preferably cysteine or glutathione.

[0048]

[0049] R of the triarylphosphine-based compound １ , R ２ and R ３ are each independently an aryl group or a heteroaryl group, preferably each independently an aryl group or a heteroaryl group having 5 to 7 members, more preferably each independently an aryl group or a heteroaryl group having 6 members, and still more preferably, R １ , R ２ and R ３All of them are 6-membered ring aryl groups, and particularly preferably R １ , R ２ and R ３ All of them are phenyl groups.

[0050] The triarylphosphine-based reducing agent is the R of the triarylphosphine-based compound. １ , R ２ and R ３ It is characterized by having a total of two or more water-soluble functional groups, by having a water-soluble functional group attached to any of the elements.

[0051] The aforementioned water-soluble functional group is not particularly limited as long as it does not hinder the effects of the present invention, but as a specific example, a functional group having a proton charge (e.g., SO) ３ H, COOH, PO ３ H ２ OH, NH ２ Examples include (etc.) or salts thereof, functional groups having no proton charge (e.g., quaternary ammonium salts), and uncharged water-soluble functional groups (e.g., PEG). Preferably, the functional group having a proton charge or a salt thereof is preferred, and more preferably, SO ３ H, COOH, PO ３ H ２ OH or NH ２ or a salt thereof, more preferably SO ３ A sulfonic acid group or sulfonic acid base represented by R (where R is hydrogen or a monovalent positively charged counterion), and particularly preferably SO ３ The sulfonic acid base is represented by R (where R is a monovalent positively charged counterion). The monovalent positively charged counterion constituting the sulfonic acid base is preferably an alkali metal ion, and more preferably a potassium ion or a sodium ion.

[0052] The number of water-soluble functional groups in the triarylphosphine-based reducing agent may be multiple, i.e., two or more, but preferably two to six, more preferably two or three, in this case, R １ , R ２ and R ３Each of these preferably has 0 to 5, more preferably 0 or 1, water-soluble functional groups attached to it. The multiple water-soluble functional groups may be the same or different, but preferably they are the same.

[0053] The aforementioned water-soluble functional group is R １ , R ２ and R ３ Each of them may be directly bonded, or spacers such as alkyl chains or PEG chains may be interposed between them, but direct bonding is preferred.

[0054] Among the aforementioned triarylphosphine-based reducing agents, the R of the triarylphosphine-based compound is particularly preferred. １ , R ２ and R ３ All of them are phenyl groups, and the water-soluble functional group is SO ３ K or SO ３ It is Na, the number of water-soluble functional groups is 2 or 3, and the water-soluble functional group is R １ , R ２ and R ３Each of these has zero or one directly bonded to it, and the compound has a chemical structure in which all of the water-soluble functional groups are identical. Representative examples of compounds with such chemical structures include: 2-(phenyl(3-sulfophenyl)phosphinyl)benzenesulfonic acid, 2-(phenyl(4-sulfophenyl)phosphinyl)benzenesulfonic acid, 3,3'-(phenylphosphinediyl)dibenzenesulfonic acid, 3-(phenyl(4-sulfophenyl)phosphinyl)benzenesulfonic acid, 4,4'-(phenylphosphinediyl)dibenzenesulfonic acid, 3,3'-((2-sulfophenyl)phosphonediyl)dibenzenesulfonic acid, 2-((3-sulfophenyl)(4-sulfophenyl)phosphinyl)benzenesulfonic acid, 4,4'-((2-sulfophenyl)phosphonediyl)dibenzenesulfonic acid, 3,3',3''-(phosphinetriyl)tribenzenesulfonic Examples include compounds in which at least one of the sulfonic acid groups of acid, 3,3'-((4-sulfophenyl)phosphonedyl)dibenzenesulfonic acid, 4,4'-((3-sulfophenyl)phosphonedyl)dibenzenesulfonic acid or 4,4',4''-(phosphonatetriyl)tribenzenesulfonic acid is a sodium salt, a potassium salt, or a double salt of both.Among these, preferred are compounds in which at least one of the sulfonic acid groups of 3,3'-(phenylphosphonediyl)dibenzenesulfonic acid, 4,4'-(phenylphosphonediyl)dibenzenesulfonic acid, 3,3',3''-(phosphonatetriyl)tribenzenesulfonic acid, or 4,4',4''-(phosphonatetriyl)tribenzenesulfonic acid is a sodium salt, a potassium salt, or a double salt of both, and particularly preferred is a compound represented by the following formula (IV), which is potassium 4,4'-(phenylphosphonatedyl)dibenzenesulfonate (BSPP, [1]) or triphenylphosphonate-3,3',3''-trisulfonic acid trisodium salt (TSPP, [2]) or 3,3'-(phenylphosphonatedyl)dibenzenesulfonic acid disodium salt [3].

[0055]

[0056] The aforementioned triarylphosphine reducing agent can be prepared according to chemical synthesis methods well known to those skilled in the art, in which case whether it has the desired chemical structure can be confirmed by NMR (using radionuclides such as 1H, 13C, 31P, etc.), mass spectrometry, X-ray crystallography, etc.

[0057] Furthermore, some of the aforementioned triarylphosphine reducing agents are available as reagents. For example, preferred specific examples of the triarylphosphine compounds, such as potassium 4,4'-(phenylphosphinediyl)dibenzenesulfonate, triphenylphosphine-3,3',3''-trisulfonic acid trisodium salt, and 3,3'-(phenylphosphinediyl)dibenzenesulfonic acid disodium salt, are commercially available as reagents from companies such as Sigma-Aldrich.

[0058] <Immunoglobulins> There are no particular restrictions on the immunoglobulins that are subject to selective cap removal, as long as they are immunoglobulins that have a cap on the light chain Cys80. Some rabbit immunoglobulins naturally have the structure of a light chain Cys80, and in the process of chimeric immunoglobulinization of rabbit immunoglobulins that have a light chain Cys80, the light chain Cys80 becomes unpaired cysteine, and a cap is formed on the light chain Cys80, making them subject to selective cap removal. Preferably, the immunoglobulins that have a cap on the light chain Cys80 are rabbit-derived chimeric immunoglobulins.

[0059] Specific examples of rabbit-derived chimeric immunoglobulins include human-rabbit chimeric immunoglobulins and humanized immunoglobulins containing the complementarity-determining region of rabbit immunoglobulins, but preferably humanized immunoglobulins containing the complementarity-determining region of rabbit immunoglobulins.

[0060] Human-rabbit chimeric immunoglobulins are chimeric immunoglobulins that combine all or part of the variable region of an immunoglobulin obtained from a rabbit with the constant region of a human immunoglobulin. Human-rabbit chimeric immunoglobulins can be produced using well-known methods; for example, they can be obtained by ligating the DNA encoding the variable region of an antibody with the DNA encoding the constant region of a human antibody, incorporating this into an expression vector, and introducing it into a host to induce production.

[0061] Humanized immunoglobulins are modified immunoglobulins, also known as reshaped human antibodies. Humanized immunoglobulins are constructed by transplanting the complementarity-determining region of an antibody derived from an immunized animal into the framework regions of human immunoglobulin. For example, the humanized immunoglobulin containing the complementarity-determining region of rabbit immunoglobulin is a humanized immunoglobulin in which the complementarity-determining region of a rabbit-derived antibody is transplanted into the framework regions of human immunoglobulin. Genetic recombination is also a well-known technique for constructing humanized immunoglobulins. Specifically, for example, DNA designed to link the DNA sequences encoding the complementarity-determining regions of mouse immunoglobulin and rabbit immunoglobulin with the DNA sequence encoding the framework region of human immunoglobulin is first synthesized by PCR from several oligonucleotides that have overlapping portions at their respective ends. Next, the obtained DNA is ligated with DNA encoding the constant region of human immunoglobulin, incorporated into an expression vector, and introduced into a host to induce production (see European Patent No. 239400, WO96 / 02576). The framework region of the human immunoglobulin ligated via the complementarity-determining region is selected such that the complementarity-determining region forms a good antigen-binding ability. If necessary, amino acids in the framework region of the variable region of the reconstituted human immunoglobulin may be substituted so that the complementarity-determining region of the reconstituted human immunoglobulin forms an appropriate antigen-binding ability (Sato K. et al., Cancer Research 1993, 53:851-856). Alternatively, the framework region may be substituted with one derived from various human immunoglobulins (see WO99 / 51743).

[0062] There are no particular limitations on the immunoglobulin isotypes targeted for selective cap removal. Specific examples include IgG, IgE, IgM, IgA, IgD, or IgY, or any of their subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. However, IgG is preferred, and IgG1 is more preferred. If the target of selective cap removal is an antigen-binding fragment, it is preferably an antigen-binding fragment of IgG, and more preferably an antigen-binding fragment of IgG1.

[0063] The antigen-binding fragments subject to selective cap removal are not particularly limited as long as they include a light chain variable region containing Cys80 or a fragment thereof, but antigen-binding fragments having disulfide bonds between light chains and heavy chains or between heavy chains are preferred, and specific examples include Fab, F(ab') ２ These are some examples.

[0064] While there are no particular restrictions on the immunoreactive target antigens of the immunoglobulins targeted for selective cap removal, cancer antigens are preferred because the conjugates described below are favorably applied in cancer treatment and prevention.

[0065] Specific examples of cancer antigens include GD2, PDGFRα (platelet-derived growth factor receptor α), CD22, HER2, EPCAM, fibronectin, CD33, GPNMB, CD27, DEC-205, folate receptor, CD37, CD19, TROP2, CEACAM5, HER3, IGF-1R, TNT-1 / B, PSMA, CD20, ICAM-1, CD30, CD38, MUC1, EGFR, and Tenascin- C, Mesothelin, CD138, c-MET, CD79b, TEM-1, GM2, Glypican 3, CD74, NOTCH1, NOTCH2, NOTCH3, CD3, CSF-1R, FGFR2b, HLA-DR, EPHA3, B7-H3, CD123, GPA33, Frizzled7 receptor, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD25, MET, BST1(C D157), P-cadherin, GITR, CD73, FGFR2, CXCR4, LAG-3, FucosylGM1, FGFR3, OX40, BCMA, ERBB3, CD137 (4-1BB), PTK 7, EFNA4, FAP, DR5, CA6, LAMP1, EPHA2, FGFR4, α2-PI, GDF15, CA9, CD166, ROR1, TBA, TIM-3, CD200, AXL, Thomsen Examples include, but are not limited to, Friedenreich antigen, CD39, CLEC12A, LGR3, transferrin receptor, 5T4, RTK, NaPi2b, Lewis blood group B antigen, A34, Lysyl-Oxide, DLK-1, α9 integrin, TAG-72 (CA72-4), CD45, MCEMP1, CD179b, CSPG5, MRAP2, and CAPRIN-1. Preferably, CAPRIN-1, whose usefulness as a target for cancer treatment and prevention has been confirmed in WO2010 / 016526, etc.

[0066] <Decapping> The present invention relates to a method for decapping the light chain Cys80 of immunoglobulin using the triarylphosphine reducing agent, and to a method for producing decapped immunoglobulin in which the light chain Cys80 of immunoglobulin has been decapped.

[0067] Decapping of the light chain Cys80 of immunoglobulins is carried out by adding the triarylphosphine reducing agent to a solution of the immunoglobulin to be decapped. During the decapping reaction, chemical substances that directly or indirectly promote the reaction may be added or reaction byproducts may be removed, but it is preferable not to perform these operations, i.e., to allow the decapping reaction to proceed while standing. Note that the movement of the reaction vessel itself, including shaking or inverting the reaction vessel, is included in "standing".

[0068] The solvent for the immunoglobulin solution to be decapped is not particularly limited as long as it does not inhibit the decapping reaction, but a buffer solution is preferred. A buffer solution is an aqueous solution containing a pH buffer that has the ability to suppress pH fluctuations (buffering capacity). Specific examples of pH buffers include phosphates, borates, acetates, and citrates, as well as pH buffers that contain both an amine and an acidic functional group in a single molecule (e.g., Good's buffer, Tris, amino acids, etc.).

[0069] Furthermore, the buffer solution may contain not only a pH buffer but also additives that support the desired chemical reaction, such as substrate stabilization and improved solubility. Typical examples of additives include salts (e.g., sodium chloride, potassium chloride, calcium chloride), denaturants (e.g., urea, guanidine hydrochloride), surfactants (e.g., Tween), cosolvents miscible with water (e.g., dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, acetonitrile, alcohols, polyols, acetone), protein stabilizers (including monosaccharides such as glucose, disaccharides such as sucrose, and polysaccharides), and chelating agents (those that adsorb metal ions, e.g., EDTA). A buffer solution that satisfies the above conditions is preferably a phosphate buffer solution, and more preferably a phosphate-buffered saline solution.

[0070] The preferred pH range of the buffer solution is about 4.0 to about 9.0, more preferably about 5.0 to about 8.5, even more preferably about 6.0 to about 8.0, and most preferably about 7.4, which is the physiological condition.

[0071] The preferred reaction temperature for the decapsulation reaction is about 30°C to about 40°C, more preferably about 35°C to about 40°C, and most preferably about 37°C.

[0072] The preferred reaction time for the decapsulation reaction is within approximately 10 hours, and more preferably within approximately 4 hours. By using the aforementioned triarylphosphine-based reducing agent, the reaction time can be significantly reduced compared to conventional methods.

[0073] In this specification, the term "approximately" indicates that a variation of up to ±10% is permitted from the numerical value or range associated with that term.

[0074] After the decapsulation reaction, the decapsulated immunoglobulin contained in the buffer may be purified and recovered by protein purification methods well known to those skilled in the art, such as gel filtration chromatography, ultrafiltration, or affinity chromatography, or it may be subjected to the conjugate manufacturing method described later in its original state.

[0075] When an immunoglobulin containing a light chain Cys80 that is to be decapped is divalent or higher, the immunoglobulin may contain multiple light chain Cys80s, up to the number of valencies of the immunoglobulin. By decapping immunoglobulins containing multiple light chain Cys80s, it is possible to remove the caps from at least one, preferably two or more, light chain Cys80s on each light chain Cys80, and more preferably all of the caps that the immunoglobulin to be decapped may have.

[0076] Whether the caps on the light chain Cys80 of immunoglobulins have been removed by the decapping treatment can be determined by LC-MS analysis. Specifically, by comparing the LC-MS peaks of immunoglobulins before and after decapping treatment, if a change in the peaks corresponding to the loss of mass due to the caps can be observed before and after the decapping treatment, it can be determined that the caps have been removed. Furthermore, to determine whether the caps have been selectively removed, if the relative intensity ratio of the LC-MS peaks observed when fragmentation occurs due to the reduction of disulfide bonds between the light chain and heavy chain or between heavy chains of immunoglobulins is kept below 5% of the peak derived from the raw material antibody, it can be determined that the caps on the light chain Cys80 have been selectively removed by the decapping treatment.

[0077] By decapping immunoglobulins having a light chain Cys80, 50% or more, preferably 70% or more, more preferably 90% or more, and even more preferably 100% of the immunoglobulin subjected to the treatment becomes decapped immunoglobulin, in which the light chain Cys80 has been removed. The ratio (percentage) of decapped immunoglobulin can be calculated by the relative ratio of the LC-MS peak intensity. Furthermore, when a conjugate is prepared by combining decapped immunoglobulin with a functional substance described later, the ratio of the conjugate can be considered as the ratio of decapped immunoglobulin.

[0078] <Conjugate> A conjugate of immunoglobulin and a functional substance can be created by attaching the functional substance via the thiol group of the cysteine ​​residue from which the cap has been removed in the light chain Cys80 of the decapping immunoglobulin.

[0079] Functional substances refer to one or more compounds that enhance the function of immunoglobulins or confer new functions. Examples include fluorophores, fluorescent dyes, polypeptides, immunoglobulins, antibiotics, nucleic acids, radionuclides, chemical linkers, small molecules, chelating agents, lipids, and drugs. There are no particular restrictions, but drugs are preferred.

[0080] In some embodiments, the drug is an anticancer agent. Suitable anticancer agents include, but are not limited to, antitubulin agents, DNA ligators, DNA replication inhibitors, DNA alkylating agents, antifolate agents, antimetabolites, chemotherapy sensitizers, and topoisomerase inhibitors.

[0081] A preferred method for bonding a functional substance to a cysteine ​​residue from which the cap of the light chain Cys80 has been removed is one that utilizes a thiol-reactive functional group. Examples of thiol-reactive functional groups include, but are not limited to, maleimide, haloacetamide, pyridyl disulfide, thiosulfone, vinylsulfone, haloacetyl, aziridine, acryloyl, and aryl.

[0082] Furthermore, the thiol reaction functional group and the functional substance may be connected by a linker or spacer. The linker or spacer may be non-cleavable or cleavable. Specific examples of non-cleavable linkers or spacers include, but are not limited to, polyethylene glycol (PEG) and alkyl groups. Specific examples of cleavable linkers or spacers include, but are not limited to, enzymatic cleavage type linkers (e.g., valine-citrulline-para-aminobenzyl), acid dissociation type linkers (acetal linkers, ketal linkers, hydrazide linkers), and reductive cleavage type linkers (disulfide-containing linkers).

[0083] By adding a stock solution to the detached immunoglobulin, in which the functional substance is dissolved in water, a water-miscible organic solvent, or a mixture thereof, a conjugate can be prepared in which the functional substance is bound via the thiol group of the cysteine ​​residue of the light chain Cys80 of the detached immunoglobulin. Specific examples of water-miscible organic solvents suitable as solvents for the functional substance include dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, acetonitrile, alcohols, polyols, and acetone. After the binding reaction is complete, the conjugate contained in the buffer can be purified and recovered by protein purification methods well known to those skilled in the art, such as gel filtration chromatography, ultrafiltration, and affinity chromatography.

[0084] The following examples illustrate important features of the present invention; however, these examples are illustrative of the disclosed embodiments and are not intended to limit them.

[0085] In this example, the antibody to be decapsulated was human-rabbit chimeric anti-CAPRIN-1 antibody #1 (hereinafter referred to as "light chain Cys80-containing antibody") obtained in Example 9(2) of WO2013 / 125654. This antibody was obtained by introducing two recombinant expression vectors into mammalian cells according to a conventional method, which were prepared by inserting the genes encoding the light chain variable region and heavy chain variable region of a rabbit anti-CAPRIN-1 monoclonal antibody containing the light chain Cys80 into a mammalian cell expression vector containing the light chain constant region of human IgG1 and a mammalian cell expression vector containing the heavy chain constant region of human IgG1, respectively, as described in Example 9 of WO2013 / 125654. Furthermore, based on the amino acid sequence information of the light chain variable region and heavy chain variable region of the rabbit anti-CAPRIN-1 monoclonal antibody (SEQ ID NOs. 54 and 52 in the WO2013 / 125654 specification), as well as the well-known amino acid sequence information of the light chain constant region and heavy chain constant region of human IgG1, this antibody is presumed to have a light chain Cys80 and a cap structure at that position.

[0086] (Reference Example 1) LC-MS analysis of antibodies containing light chain Cys80 or its F(ab') ２ A 10 μg sample of antibody that had undergone a decapping reaction on a fragment was collected and diluted to 50 μl with a 0.1% formic acid aqueous solution to prepare the sample. 10 μl of this sample was then added to a Q exactive column linked to an ACQUITY UPLC protein BEH C4 column (Waters). ＴＭ Analysis was performed using a Plus quadrupole / orbitrap hybrid mass spectrometer (Thermo Fisher Scientific). The eluents used were Solution A, an aqueous solution containing 0.02% (v / v) trifluoroacetic acid and 0.08% (v / v) formic acid, and Solution B, an acetonitrile solution containing 0.02% (v / v) trifluoroacetic acid and 0.08% (v / v) formic acid. Analysis was performed under gradient conditions by mixing Solutions A and B. The obtained MS data were deconvoluted using the analysis software BioPharma Finder (Thermo Fisher Scientific) to determine the molecular weight range (F(ab')) from 70,000 to 170,000. ２ For fragments, analysis was performed at a resolution of 20,000 to 110,000.

[0087] (Reference Example 2) LC-MS analysis of antibody-drug conjugates and calculation of antibody-drug binding count (DAR) of the antibody or its F(ab') ２ A 10 μg sample of the fragment-drug conjugate was collected and diluted to 50 μl using PBS(-) to prepare the sample. 10 μl of this sample was then added to a Q exactive PLC column linked to an ACQUITY UPLC protein BEH C4 column (Waters). ＴＭAnalysis was performed using a Plus quadrupole / orbitrap hybrid mass spectrometer (Thermo Fisher Scientific). The eluents used were Solution A, an aqueous solution containing 0.02% (v / v) trifluoroacetic acid and 0.08% (v / v) formic acid, and Solution B, an acetonitrile solution containing 0.02% (v / v) trifluoroacetic acid and 0.08% (v / v) formic acid. Analysis was performed under gradient conditions by mixing Solutions A and B. The obtained MS data were deconvoluted using the analysis software BioPharma Finder (Thermo Fisher Scientific), and molecular weights ranging from 70,000 to 170,000 (F(ab')) were analyzed. ２ For fragments, the analysis was performed in the range of 20,000 to 110,000.

[0088] The MS intensity corresponding to the drug binding rate (DAR) per antibody (or antigen-binding fragment) molecule in the antibody (or antigen-binding fragment)-drug conjugate obtained from the above analysis was applied to the following formula to calculate the average DAR.

[0089]

[0090] (Example 1) Reduction treatment of light chain Cys80-containing antibody with the triarylphosphine reducing agent potassium 4,4'-(phenylphosphinediyl)dibenzenesulfonate (BSPP) The light chain Cys80-containing antibody was dissolved in Dulbecco's phosphate-buffered saline (Ca, Mg-free) (Nacalai Tesque) (hereinafter referred to as "PBS(-) solution") to obtain a light chain Cys80-containing antibody solution (antibody concentration 3 mg / ml), to which potassium, a triarylphosphine-containing reducing agent, was added to the PBS(-) solution. A reduction treatment was performed by adding a BSPP solution containing 4,4'-(phenylphosphonediyl)dibenzenesulfonate (BSPP; Sigma-Aldrich) to the light chain Cys80-containing antibody in an amount of 8 molar equivalents of BSPP. This mixture was left to stand at 37°C for 4 hours, and Zeba... ＴＭThe light chain Cys80-containing antibody was purified and recovered after reduction treatment using the Spin Desalting Column (40K MWCO; Thermo Fisher Scientific).

[0091] LC-MS analysis was performed according to Reference Example 1, and the LC-MS peaks of the light chain Cys80-containing antibody before and after the reduction treatment were compared. The results showed a mass change of approximately 242 Da, equivalent to about two capping cysteine ​​molecules, a mass change of approximately 612 Da, equivalent to two capping glutathione molecules, and a mass change of approximately 427 Da, equivalent to one capping cysteine ​​molecule and one capping glutathione molecule, before and after the reduction treatment. On the other hand, no peaks indicating antibody fragmentation due to reduction of disulfide bonds between light chains and heavy chains were observed, indicating that cap removal proceeded selectively. Furthermore, the LC-MS peak before the reduction treatment was not observed in the LC-MS peak after the reduction treatment, and only shifted LC-MS peaks due to the removal of cysteine ​​and glutathione were observed. These results confirm that the light chain Cys80-containing antibody before the reduction treatment had a cysteine ​​or glutathione cap structure on the light chain Cys80, and that the reduction treatment selectively reduced and decapsulated the cap structure of the light chain Cys80.

[0092] (Example 2) Preparation of antibody-drug conjugate using BSPP-reduced antibody To the detached antibody obtained in Example 1, a solution of mc-vc-PAB-MMAE (trade name: VcMMAE; Chemscene LLC), a payload consisting of monomethyl auristatin E (MMAE), an anticancer drug and antitubulin agent, with an enzyme-cleaved linker (mc-vc-PAB) attached, was added in N,N-dimethylacetamide to a concentration of 10 mM. After reacting at room temperature for 1 hour, Zeba ＴＭ The antibody-drug conjugate was purified and recovered using the Spin Desalting Column (40K MWCO).

[0093] LC-MS analysis was performed according to Reference Example 2, and a peak was observed in the LC-MS peak of the light chain Cys80-containing antibody after the reduction treatment, which increased by approximately 2634 Da, representing the mass of two mc-vc-PAB-MMAE molecules. Conjugation of the unconjugated DAR0 antibody and DAR1, in which mc-vc-PAB-MMAE was introduced to only one of the light chain Cys80 molecules, was not observed in the LC-MS chart, and the average DAR was 2.0.

[0094] (Example 3) Reduction treatment of light chain Cys80-containing antibody with triarylphosphine-3,3',3''-trisulfonic acid trisodium salt (TSPP) The light chain Cys80-containing antibody was reduced according to the conditions of Example 1, except that triarylphosphine-3,3',3''-trisulfonic acid trisodium salt (TSPP; Tokyo Chemical Industry Co., Ltd.), a triarylphosphine-based reducing agent, was used as the reducing agent. As shown in Reference Example 1, LC-MS analysis was performed, and the LC-MS peaks of the light chain Cys80-containing antibody before the reduction treatment and the LC-MS peaks of the light chain Cys80-containing antibody after the reduction treatment were compared. The intensity ratio of the peaks indicating antibody fragmentation due to reduction of disulfide bonds between the light chain and heavy chain and between heavy chains was approximately 0.6% of the full-length antibody, confirming that the caps were selectively removed.

[0095] (Example 4) Preparation of antibody-drug conjugates using TSPP-reduced antibody Antibody-drug conjugates were prepared using the decapping antibody obtained in Example 3 according to the method described in Example 2. LC-MS analysis was performed according to Reference Example 2, and MS peaks corresponding to the DAR0 antibody, in which decapping had not progressed at the time of Example 3, and the DAR1 or DAR2 conjugate, in which mc-vc-PAB-MMAE was introduced to one or both sides of the light chain Cys80 of the decapping antibody, were observed. The MS intensity ratios for DAR0, DAR1, and DAR2 were approximately 44%, 44%, and 12%, respectively, and the average DAR was 0.68. These results indicate that the antibody-drug conjugates obtained using TSPP-reduced antibodies primarily consisted of conjugates in which the drug was bound to one or both of the decapped light chain Cys80 molecules. However, because a certain amount of antibody was not decapped, the drug did not bind to the antibody, resulting in an average DAR of less than 1.

[0096] (Comparative Example 1) Reduction treatment of light chain Cys80-containing antibody with non-triarylphosphine reducing agent Tris(2-carboxyethyl)phosphine (TCEP) The light chain Cys80-containing antibody was reduced according to the conditions of Example 1, except that Tris(2-carboxyethyl)phosphine (TCEP; Nacalai Tesque), which is not a triarylphosphine compound, was used as the reducing agent. When comparing the LC-MS peaks of the light chain Cys80-containing antibody before and after the reduction treatment, the MS peak indicating the full-length antibody disappeared, and MS peaks corresponding to the light chain-heavy chain fragment (HL fragment), the heavy chain-heavy chain fragment (HH fragment), and the heavy chain-heavy chain-light chain fragment (HHL fragment) resulting from the reduction of the disulfide bond between the heavy chains of the antibody were observed. From these results, it was determined that selective reduction of light chain Cys80 is not possible with the non-triarylphosphine reducing agent TCEP.

[0097] (Example 5) F(ab)' antibody possessing light chain Cys80 ２The solvent for the full-length antibody containing fragmented light chain Cys80 (20 mg / ml, PBS(-) solution) is Zeba ＴＭ The solvent was replaced with 0.1 M citrate buffer (pH 3.5, citrate purchased from Fujifilm Wako Pure Chemical Industries) using a Spin Desalting Column (40K MWCO). To this, 6 units of pepsin solution, prepared with the same 0.1 M citrate buffer (pH 3.5) used for solvent replacement of the antibody to achieve a pepsin concentration of 0.5 mg / ml, were added per 1 mg of antibody. The mixture was reacted at 37°C for 1 hour while inverting and mixing. Half the volume of 1 M Tris·HCl (pH 9.0, Tris·HCl purchased from Fujifilm Wako Pure Chemical Industries) was added to inactivate the pepsin in the reaction mixture. Subsequently, the reaction mixture was concentrated at 4000 G at 10°C for 5 minutes using an Amicon Ultra-15 centrifugal filter unit (10K MWCO; Merck), and Aekta Pure was obtained. ＴＭ Hiload connected to port 150 (Cytiva) ＴＭ 26 / 600, Superdex ＴＭ Reaction impurities were separated using 200 pg (Cytiva) to obtain F(ab)' ２ The fragments were purified and recovered.

[0098] (Example 6) Light chain Cys80-containing antibody F(ab)' induced by the triarylphosphine-based reducing agent BSPP ２ The fragment reduction treatment was performed on F(ab)' obtained in Example 5. ２ Except for using the fragment (3 mg / ml, PBS(-) solution), F(ab)' is prepared according to the method described in Example 1. ２ The fragment was subjected to reduction treatment. F(ab)' before the reduction treatment. ２ LC-MS peaks of the fragment and F(ab)' after the reduction treatment. ２Comparison of LC-MS peaks of the fragments revealed mass changes of approximately 242 Da, equivalent to two capping cysteine ​​molecules, 612 Da, equivalent to two capping glutathione molecules, and 427 Da, equivalent to approximately one capping cysteine ​​and one glutathione molecule, before and after the reduction treatment. On the other hand, no peaks indicating mass changes due to reduction of disulfide bonds between light chains and heavy chains, or between heavy chains and heavy chains, were observed. These results confirm that the light chain Cys80-containing antibody before the reduction treatment had a cap structure of cysteine ​​or glutathione on the light chain Cys80, and that the cap structure of the light chain Cys80 was selectively reduced and decapped by the reduction treatment.

[0099] (Example 7) BSPP reduction treatment F(ab)' ２ Decapped F(ab)' obtained in Example 6 of antibody fragment-drug conjugate preparation using fragments ２ Using the fragments, antibody fragment-drug conjugates were prepared according to the method described in Example 2. LC-MS analysis was performed according to Reference Example 2, and the detached F(ab)' before the reduction treatment was analyzed. ２ From the LC-MS peaks of the fragment, only a peak with an increased mass of approximately 2634 Da, representing the mass of the two molecules of mc-vc-PAB-MMAE, was observed. The unconjugated DAR0 F(ab)' ２ F(ab)' of DAR1 in which mc-vc-PAB-MMAE is introduced only in the fragment or in one of the light chains, Cys80. ２ No fragment conjugates were observed in the LC-MS chart, and the mean DAR was 2.0.

[0100] (Example 8) Reduction treatment of light chain Cys80-containing antibody with triarylphosphine-based reducing agent 3,3'-(phenylphosphinediyl) dibenzenesulfonic acid disodium salt The light chain Cys80-containing antibody was reduced according to the conditions of Example 1, except that 3,3'-(phenylphosphinediyl) dibenzenesulfonic acid disodium salt (BLD Pharma), a triarylphosphine-based reducing agent, was used as the reducing agent. As shown in Reference Example 1, LC-MS analysis was performed, and the LC-MS peaks of the light chain Cys80-containing antibody before the reduction treatment and the LC-MS peaks of the light chain Cys80-containing antibody after the reduction treatment were compared. The intensity ratio of the peaks indicating antibody fragmentation due to reduction of disulfide bonds between the light chain and heavy chain and between heavy chains was approximately 1.2% of the full-length antibody, confirming that the caps were selectively removed.

[0101] (Example 9) Preparation of antibody-drug conjugate using 3,3'-(phenylphosphonediyl)dibenzenesulfonic acid disodium salt-reduced antibody An antibody-drug conjugate was prepared using the decapsulated antibody obtained in Example 8 according to the method described in Example 2. LC-MS analysis was performed according to Reference Example 2, and only a peak with an increase of approximately 2634 Da, representing the mass of two mc-vc-PAB-MMAE molecules, was observed from the LC-MS peak of the light chain Cys80-containing antibody after the reduction treatment. The unconjugated DAR0 antibody and the DAR1 conjugate, in which mc-vc-PAB-MMAE was introduced to only one of the light chain Cys80 molecules, were not observed from the LC-MS chart, and the average DAR was 2.0.

[0102] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A reducing agent comprising a compound in which multiple water-soluble functional groups are bonded to a triarylphosphine compound shown in the following formula (I), for selectively removing the cap of the cysteine ​​residue at the 80th amino acid position in Kabat numbering in the light chain variable region of immunoglobulin or its antigen-binding fragment. (R １ , R ２ and R ３ These are, independently, aryl groups or heteroaryl groups.

2. The reducing agent according to claim 1, wherein the water-soluble functional group is a sulfonic acid group or a sulfonic acid base.

3. Two or three of the water-soluble functional groups are bonded to the triarylphosphine compound, and the R １ , R ２ and R ３ The reducing agent according to claim 1, wherein zero or one of the following is directly bonded.

4. The reducing agent according to claim 1, wherein the compound to which a plurality of water-soluble functional groups are bonded to the triarylphosphine compound is potassium 4,4'-(phenylphosphinediyl)dibenzenesulfonate, triphenylphosphine-3,3',3''-trisulfonic acid trisodium salt, or 3,3'-(phenylphosphinediyl)dibenzenesulfonic acid disodium salt.

5. The reducing agent according to claim 1, wherein the cap is cysteine, glutathione, or cysteinylglycine.

6. A method for removing a cap from an immunoglobulin or an antigen-binding fragment, comprising using a reducing agent according to any one of claims 1 to 5 to selectively remove the cap from the cysteine ​​residue at amino acid position 80 in the light chain variable region of the immunoglobulin or its antigen-binding fragment by Kabat numbering.

7. The method according to claim 6, comprising the step of mixing the reducing agent with a solution containing the immunoglobulin or an antigen-binding fragment thereof, and then allowing it to stand.

8. The method according to claim 7, wherein the solvent of the solution is a buffer solution with a pH of 4 to 9.

9. The method according to claim 7, wherein the solvent of the solution is a buffer solution with a pH under physiological conditions.

10. The method according to claim 7, wherein the step is to allow the mixture to stand at 30 to 40°C after mixing.

11. A method for producing decapped immunoglobulin or an antigen-binding fragment thereof, comprising the step of selectively removing the cap of the cysteine ​​residue at amino acid position 80 by Kabat numbering in the light chain variable region of the immunoglobulin or antigen-binding fragment thereof, according to any one of claims 6 to 10.

12. The manufacturing method according to claim 11, wherein the immunoglobulin is a rabbit-derived chimeric immunoglobulin.

13. The method for producing a human immunoglobulin according to claim 12, wherein the rabbit-derived chimeric immunoglobulin is a humanized immunoglobulin containing a complementarity-determining region of human-rabbit chimeric immunoglobulin or rabbit immunoglobulin.

14. The manufacturing method according to claim 11, wherein the isotype of the immunoglobulin is IgG.

15. The method for producing the immunoglobulin or its antigen-binding fragment having immunoreactivity with CAPRIN-1 protein, according to claim 11.

16. An immunoglobulin or its antigen-binding fragment having immunological reactivity with CAPRIN-1 protein, in which the cap of the cysteine ​​residue at amino acid position 80 is selectively removed by Kabat numbering in the light chain variable region.

17. A method for producing a conjugate, comprising the step of attaching a functional substance to a cysteine ​​residue of a decapped immunoglobulin or its antigen-binding fragment obtained by the method of claim 11.

18. The manufacturing method according to claim 17, wherein the functional substance is one or more selected from the group consisting of drugs, fluorophores, fluorescent dyes, polypeptides, immunoglobulins, antibiotics, nucleic acids, radionuclides, chemical linkers, small molecules, chelating agents, and lipids.

19. The method for producing immunoglobulin according to claim 17, wherein the decapping immunoglobulin or its antigen-binding fragment has immunoreactivity with CAPRIN-1 protein.

20. A conjugate in which a functional substance is bound to a cysteine ​​residue whose cap at amino acid position 80 has been removed by Kabat numbering in the light chain variable region of an immunoglobulin or antigen-binding fragment that has immunological reactivity with CAPRIN-1 protein.

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