CN117838879B - Preparation method for improving homogeneity of antibody-drug conjugate and application thereof - Google Patents

Preparation method for improving homogeneity of antibody-drug conjugate and application thereof Download PDF

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CN117838879B
CN117838879B CN202311769186.2A CN202311769186A CN117838879B CN 117838879 B CN117838879 B CN 117838879B CN 202311769186 A CN202311769186 A CN 202311769186A CN 117838879 B CN117838879 B CN 117838879B
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CN117838879A (en
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王泽坤
蔡永凤
李雨鑫
范启睿
李志强
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Shanghai Yaoming Helian Biotechnology Co ltd
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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Abstract

The invention provides a preparation method for improving the homogeneity of an antibody-drug conjugate and application thereof, wherein the preparation method comprises the following steps: (1) Incubating a reducing agent and an antibody to be conjugated in a buffer system containing transition metal ions, reducing interchain disulfide bonds in the antibody to produce reduced sulfhydryl groups; (2) Covalently coupling a drug-linker complex carrying two reactive groups to the reduced sulfhydryl group obtained in step (1); thereby producing an antibody-drug conjugate mixture. The method selectively modifies two drug molecules at the hinge region of an antibody using any given reactive linker without modifying the antibody sequence. The method can produce ADCs with improved homogeneity, and simultaneously the method is simple to operate and can realize cost reduction and efficiency improvement.

Description

Preparation method for improving homogeneity of antibody-drug conjugate and application thereof
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to a preparation method for improving the homogeneity of an antibody-drug conjugate and application thereof.
Background
ADCs (antibody-drug conjugates) contain antibodies for targeting, linkers for drug attachment, and high potency payloads (e.g., drugs) as effectors. Since US FDA approves Adcetris (vitamin b) in 2011, ADC drug speeds have gradually increased, and are now widely expanding for the treatment of cancer. Currently included are drugs such as Adcetris, kadcyla (trastuzumab-maytansinoid conjugate), besponsa (oxtuzumab), mylotarg (gemtuzumab), polivy (poltuzumab), blenrep (mar Bei Tuoshan antibody), enhertu (detrastuzumab), padcev (enrolment mab), trodelvy (go Sha Tuozhu mab), tivdak (tixomab), zynlonta (rituximab), akalux, lumoxiti (pecumomab) or alidi. At the same time, more drugs are in the course of clinical stage of research.
Kadcyla and Enhertu both used trastuzumab (trastuzumab), but DM1 (maytansinoid DM1, MERTANSINE DM 1) and DXD (DNA topoisomerase I inhibitor) as the loads, respectively, whose DAR values (small molecule toxin antibody ratio DAR, drug-to-anti ratio) were chosen to be 3.5 and 8, respectively. This illustrates that the DAR value is selected to be appropriate for the type of load. Of the drugs on the market, zynlonta (rituximab) is a more specific case, which uses the small molecule drug PBD (pyrrolobenzodiazepine)Dimer, PBD Dimer) has the highest toxicity among the loads of all ADCs. Corresponding thereto is that it has the lowest drug-to-antibody ratio of 2 in the existing on-market ADC: 1. an important parameter to consider in the selection of DAR values in the design of this drug is the toxicity of the load. The higher the toxicity, the lower the DAR value should be adapted to avoid unnecessary damage to the human body when the load is released non-specifically.
There is no absolute standard within the industry for the DAR value to be high or low, but rather to float over a range. Taking ADC with load Exatecan (escitalopram Kang Jia sulfonate) analogue as an example, exatecan analogue is a lower toxicity load, DAR value in Enhertu is 8; but the DAR value in SHR-a1811 is 5.7. Their DAR values are all higher than 4.0 (this is the usual DAR value for toxic loading MMAE (monomethyl auristatin E, monomethyl auristatin E)), but their DAR value differences are as high as 2.3. Therefore, in the development process of the ADC, the attempt of multiple DAR values is a necessary path.
For average DAR values, the differences in the distribution of DAR values also need to be considered. For example, an ADC with a DAR value of 2, a product obtained by mixing 75% non-toxic antibodies without drug with 25% high-toxicity ADC with a DAR value of 8; unlike a clean DAR value of 2 ADC, it does not perform as well in vivo. Because the two groups of substances actually involved in toxicity comparison are ADC of DAR2 and ADC of 1/4 of DAR8 when the dosage is low. Thus, in trying multiple DAR values, attempts should be made with ADCs having DAR values that are as specific as possible to ensure that the results of pharmacological experiments are more reference value.
To meet this need, various coupling techniques have been developed in an effort to better produce ADCs with better homogeneity. For drug attachment, coupling is performed using functional groups that are highly reactive and stable to both the antibody and linker-payload (i.e., linker-drug) to form stable covalent bonds. Conventional means of attachment, i.e., covalent attachment of the drug moiety to the antibody via a linker, typically results in a heterogeneous molecular mixture in which the drug moiety is attached at several sites on the antibody. For example, cytotoxic drugs have typically been conjugated to antibodies through multiple lysine residues commonly contained in antibodies, resulting in heterogeneous antibody-drug conjugate mixtures.
For example, antibody-drug conjugates are typically produced by two conventional chemical strategies: lysine (Lys) -based conjugation and cysteine-based conjugation, the cysteines (Cys) resulting from interchain disulfide bond (S-S bond) reduction. For the reaction of primary amine groups on lysine residues, the most widely used reactive group on the linker-payload is the NHS ester (i.e., N-hydroxysuccinimide). However, the use of NHS esters in the production of antibody-drug conjugates is limited by their inherent properties, e.g. the reaction between NHS ester and primary amine is very slow under acidic conditions, so conjugation needs to be performed in a buffer with a high pH (i.e. > 7.0), however high pH is sometimes not friendly to antibodies and NHS tends to hydrolyze under alkaline conditions, which makes purification and identification of the free drug after conjugation more complicated. Furthermore, due to the low reactivity of NHS esters with primary amines on antibodies, the reaction needs to be carried out at high temperature (i.e. 22 ℃). Furthermore, due to the low solubility, the linker-payload prepared by NHS ester (i.e. SMCC-DM 1) requires more organic solvent to be completely dissolved in the reaction system, which increases the risk of antibody aggregation.
For conjugation of cysteines from interchain disulfide bond reduction, it includes a step of opening interchain disulfide bonds in the presence of various reducing agents such as TCEP (tris (2-carboxyethyl) phosphine), DTT (dithiothreitol) and the like, followed by nucleophilic reaction of sulfhydryl groups. In this conjugation process, the antibody-drug conjugate is typically formed as follows: one or more antibody cysteine sulfhydryl groups are conjugated to one or more linker moieties that bind to a drug, thereby forming an antibody-linker-drug complex. Unlike most amines (which are protonated near pH7 and low in nucleophilicity), cysteine sulfhydryl groups are reactive at neutral pH. Since free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteines often exist in their oxidized form as disulfide-linked oligomers or have internally bridged disulfide bonds. The antibody cysteine thiol is typically more reactive, i.e., more nucleophilic, to the electrophilic conjugation reagent than the antibody amine or hydroxyl. However, engineering cysteine sulfhydryl groups by mutating multiple amino acid residues of a protein to cysteine can be problematic. In concentrated protein solutions, whether in the periplasm of E.coli, culture supernatant, or in partially or fully purified proteins, unpaired Cys residues on the protein surface can pair and oxidize to form intermolecular disulfide bonds, and thus can form protein dimeric or multimeric forms. Disulfide dimer formation renders the new Cys unreactive for conjugation with drugs, ligands or other labels. Furthermore, if the protein oxidizes between a newly engineered Cys and an existing Cys residue to form an intramolecular disulfide bond, neither Cys group can be used for active site participation and interaction. In addition, by misfolding or loss of tertiary structure, deproteinized or nonspecific proteins can be made free.
Therefore, it is very important to develop new ADCs as therapeutic agents. However, conventional conjugation methods always result in heterogeneous molecular mixtures in which the drug moiety is attached to several sites on the antibody. Under different reaction conditions, heterogeneous mixtures typically contain antibody distributions with linked drug moieties ranging from 0 to about 8 or more. In addition, in each conjugate subclass having a specific integer ratio of drug moiety/single antibody, there is a potentially heterogeneous mixture of drug moieties linked to individual sites of the antibody. The analytical and preparative methods are insufficient to isolate and characterize the antibody-drug conjugate species in the heterogeneous mixture obtained from the conjugation reaction. The heterogeneous mixture is so complex to facilitate that characterization and purification are difficult and expensive. Each conjugation product in such a mixture may have different pharmacokinetic, distribution, toxicity, and efficacy profiles, and non-specific conjugation also often affects the function of the antibody. An antibody is a large, complex and structurally diverse biomolecule with many reactive functional groups. The reactivity of antibodies with linker reagents and drug-linker intermediates depends on factors such as pH, concentration, salt concentration, and co-solvent. In addition, the reproducibility of the multi-step conjugation process is difficult to guarantee due to the difficulty in controlling the reaction conditions and characterizing the reactants and intermediates.
Controlling the number of drugs conjugated to a single antibody molecule is an important factor in the efficacy and safety of the resulting ADC. For example, in conjugation methods based on reduction of natural interchain disulfide bonds, interchain S-S bonds are more accessible to solvents than other disulfide bonds. Thus, the interchain disulfide bond may serve as a binding site for drug (or drug-linker) coupling to an antibody. Typically, one therapeutic antibody molecule belonging to the subclass IgG1 or IgG4 has 4 interchain S-S bonds, each formed by two-SH groups (sulfhydryl groups), so the number of drugs coupled to a single antibody molecule is 2, 4, 6 or 8. If the number of drugs coupled to a single antibody molecule is 0, the product is referred to as D0. Thus, D2 refers to an ADC in which two drug molecules are coupled to one single antibody molecule, wherein both drug molecules may be coupled to-SH groups generated by reducing an S-S bond between a heavy chain and a light chain, or may be coupled to-SH groups generated by reducing an S-S bond between a heavy chain and a heavy chain. D4 refers to an ADC in which four drug molecules are conjugated to one single antibody molecule. D6 refers to ADCs in which six drug molecules are conjugated to a single antibody molecule. And D8 refers to an ADC in which eight drug molecules are coupled to one single antibody molecule, i.e. all four inter-chain S-S bonds in one antibody molecule are reduced to eight-SH groups and each-SH group is linked to one drug molecule.
Typically, the heterogeneous mixture of ADC molecules produced by conventional conjugation methods is a mixture of D0, D2, D4, D6 and D8. It is well known in the art that heterogeneous ADC products are often unstable and highly immunogenic. It is important to ensure that the number of conjugated drug moieties per antibody is the same and that each moiety is specifically conjugated to the same amino acid residue in each antibody for optimal efficacy and to ensure constant dosing. Thus, methods have been developed to improve the homogeneity of antibody-drug conjugates. Among them, the schemes for selectively preparing D4 and D2 have been studied more widely.
Techniques for selectively preparing D4, such as a sulphur bridge scheme in which an antibody molecule of the IgG1 or IgG4 subclass has 4 interchain S-S bonds, the 4S-S bonds are fully reduced and the linker in its drug-linker is reacted with both sulfhydryl groups. Thus, when the drug-linker reaction is optimized to the ideal state. The product of the full reaction with the fully reduced antibody molecule is the D4 product. Since a bridging molecule is a design concept, we can understand it as a bridging molecule as long as it has two molecules that can react with thiol groups.
Patent WO2016064749A2 discloses a dibromomaleimide-based linker using two bromine atoms instead of the two hydrogen atoms on conventional maleimides. In terms of reaction mechanism, the reaction of maleimide and mercapto groups is an addition reaction, while the reaction of dibromomaleimide and mercapto groups is an addition-elimination reaction, and may occur twice. In terms of the reaction results, the substitution reaction of the mercapto group with bromine is performed once per reaction.
The reactive site of the current bridging molecule is a sulfhydryl group, the reaction mode of which is similar to that of the traditional linker, and the reaction is divided into addition, substitution or addition-elimination in principle. The problems with bridged molecules are also relatively uniform: the possibility of mismatch exists in the mercapto ligation reaction process, which is mainly represented by that more semi-antibody products can be generated; in addition, the target DAR value of the series of molecules is only 4, and when the DAR value of the target changes, the bridging molecules cannot keep high selectivity; in view of the reaction process, since the reaction always occurs stepwise, nucleophilic substitution of sulfhydryl groups requires electron deficiency of the substrate, but in the existing linker design, conjugation between multiple reaction centers and the linker precursor often occurs, which means that the activity of the reactants is passivated after the first step of reaction occurs, which requires controlling the reaction by other methods to avoid half-ligation of the bridged molecules.
Patent WO2020164561A1 provides another alternative to the selective preparation of D4, which uses metal ions to lock the two S-S bonds of the hinge region of the antibody, rendering it incapable of undergoing reduction under the action of a typical reducing agent. Under this action, only two disulfide bonds on one antibody can be opened, releasing 4 sulfhydryl groups, so that it can selectively prepare the product of D4. As with the bridging molecule, this approach, while capable of accommodating a variety of different linkers, is limited to target DAR values of 4.
One of the techniques for selectively preparing D2, the site-specific labelling technique, has been developed and applied to the preparation of ADCs for preclinical and clinical studies. For example, improved antibody-drug conjugates THIOMAB TM have been developed that provide site-specific conjugation of drugs to antibodies by cysteine substitutions at specific sites. The engineered cysteines at the sites can be used to conjugate cytotoxic drugs but do not interfere with immune protein folding and assembly or alter antigen binding and effector function. However, site-specific labelling techniques involve protein engineering and/or enzymatic catalysis and thus suffer from disadvantages such as low levels of antibody expression, complex purification and high cost.
Other techniques for inserting unnatural amino acids into sequences, which are also used to prepare D2 conjugates, react orthogonal to the natural antibodies, except that when two unnatural amino acids are inserted in total on the antibody, homogeneous antibody-drug 1 can be prepared: 2, the multi-drug ADC can also be prepared using conventional reactions. Similar to cysteine-edited proteins, unnatural amino acid editing is also limited by low expression levels and complex purification. In addition, it is necessary to create a set of tRNA systems for translating unnatural amino acids that are more expensive and complex than cysteine-inserted antibodies.
To achieve the preparation of homogeneous antibody-drug 1:2, and can also be prepared by performing region recognition on antibody sequences by using affinity peptides, and coupling the recognized regions, thereby realizing the preparation of antibody-drug conjugates with homogeneity. For example, reaction with methionine under 365nm light using Fc-III affinity peptides with p-benzoylphenyl groups; however, the peptide fragments used in the affinity peptide technology need to be purified and removed after the coupling is completed, and the purification difficulty and cost are higher because the molecular weight of the polypeptide is larger than that of a common small molecular medicine.
There are a great deal of research and development and production demands on D2-type antibody-drug conjugates in the market at present, but the current scheme for selectively producing D2 has the problems of sequence modification, complicated and expensive purification process, and the like, so that the scheme for preparing D2 ADC with higher homogeneity, which is simple to operate and low in cost, has important application in the preparation process of ADC drugs.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a preparation method for improving the homogeneity of an antibody-drug conjugate and application thereof. The present invention developed a novel bioconjugate process for selectively producing D2, selectively modifying two drug molecules at the hinge region of an antibody, using any given reactive linker, without modifying the antibody sequence. The method can produce ADCs with improved homogeneity, and simultaneously the method is simple to operate and can realize cost reduction and efficiency improvement.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method of preparing an antibody-drug conjugate for improving homogeneity, the method comprising:
(1) Incubating a reducing agent and an antibody to be conjugated in a buffer system containing transition metal ions, reducing interchain disulfide bonds in the antibody to produce reduced sulfhydryl groups;
(2) Covalently coupling a drug-linker complex carrying two reactive groups to the reduced sulfhydryl group obtained in step (1); thereby producing an antibody-drug conjugate mixture.
The invention develops a novel bioconjugate method which can produce ADC with improved homogeneity, and the preparation method is simple to operate and can achieve the effects of reducing cost and enhancing efficiency. The ADC produced by the bioconjugate method has high safety and better efficacy. The invention has simple operation process, and can finish purification under simple operation by using a commercial product.
In particular, the method of the present invention may result in significantly improved homogeneity of the antibody-drug conjugate (ADC) compared to conventional conjugation methods using nucleophilic reactions of reducing agents and sulfhydryl groups. The D2 content is generally higher than 60%, while the D2 content of the traditional process is generally lower than 40%.
In the present invention, the presence of transition metal ions enables the selective reduction of disulfide bonds. In the presence of transition metal ions, the two interchain S-S bonds in the Fab region are selectively reduced. Thus, two linker-payloads carrying two reactive groups are linked to one antibody to form D2. The high content of D2 in the obtained ADC clearly improves the homogeneity of the ADC.
Preferably, in step (1), the reducing agent is selected from: any one or a combination of at least two of tris (2-carboxyethyl) phosphine (TCEP), diphenylphosphinoacetic acid, 2- [2- (diphenylphosphino) ethyl ] pyridine, 3- (diphenylphosphino) benzenesulfonic acid, 4- (diphenylphosphino) benzoic acid, 2- (diphenylphosphino) ethylamine, 3- (diphenylphosphino) propylamine, 3- (diphenylphosphino) propionic acid, 2- (diisopropylphosphino) ethylamine, 2- (diphenylphosphino) benzoic acid, (2-hydroxyphenyl) diphenylphosphine, 1,3, 5-triaza-7-phosphatricyclo [3.3.1.13.7] decane, n-butylbis (1-adamantyl) phosphine or a salt thereof, sodium triacetoxyborohydride or sodium cyanoborohydride.
Preferably, in step (1), the final concentration of the reducing agent in the incubation system is 0.04-0.4mM, for example, 0.04mM, 0.08mM, 0.1mM, 0.12mM, 0.15mM, 0.2mM, 0.25mM, 0.3mM, 0.35mM or 0.4mM, etc.
Preferably, in step (1), the transition metal ion is selected from: any one or a combination of at least two of Zn 2+、Cd2+ or Hg 2+.
Preferably, in step (1), the transition metal ion is added to the buffer system in the form of a transition metal salt selected from ：ZnCl2、Zn(NO3)2、ZnSO4、Zn(CH3COO)2、ZnI2、ZnBr2、Zn(HCOO)2、Zn(BF4)2、CdCl2、Cd(NO3)2、CdSO4、Cd(CH3COO)2、CdI2、CdBr2、Cd(HCOO)2、Cd(BF4)2、HgCl2、Hg(NO3)2、HgSO4、Hg(CH3COO)2、HgBr2、Hg(HCOO)2 or Hg (BF 4)2, any one or a combination of at least two).
In the present invention, transition metal ions suitable for use in the bioconjugation method of the present invention may include, but are not limited to, zn 2+、Cd2+、Hg2+ and the like. Among them, zn 2+ can be used because Zn 2+ is relatively easily available and low cost. For example, suitable transition metal salts may be added in step (1) as long as they are soluble in the reaction solution so that free transition metal ions can be released into the reaction solution.
In the present invention, a transition metal salt of Zn 2+ is exemplified, and zinc ZnCl2、Zn(NO3)2、ZnSO4、Zn(CH3COO)2、ZnI2、ZnBr2、 formate, zinc tetrafluoroborate, or the like is included. Also, other transition metal salts that are soluble and can release free Cd 2+ or Hg 2+ ions, which may include, but are not limited to ：CdCl2、Cd(NO3)2、CdSO4、Cd(CH3COO)2、CdI2、CdBr2、Zn(HCOO)2、Zn(BF4)2、HgCl2、Hg(NO3)2、HgSO4、Hg(CH3COO)2、HgBr2、Hg(HCOO)2 or Hg (BF 4)2, etc.), may be added to the reaction solution.
Preferably, in step (1), the final concentration of the transition metal ion in the incubation system is 0.01-0.2mM, for example, 0.01mM, 0.02mM, 0.04mM, 0.05mM, 0.06mM, 0.08mM, 0.1mM, 0.12mM, 0.14mM, 0.15mM, 0.16mM, 0.18mM or 0.2mM, etc.
In the present invention, transition metal ions are key factors responsible for the high level of D2 and the low levels of D0, D1, D3 and D4 in the resulting ADC.
Preferably, in step (1), the buffer system is selected from: any one or a combination of at least two of HEPES buffer, histidine buffer, PBS buffer or MES buffer.
In the present invention, the buffer solution system needs to have buffering capacity in a specified pH range, and the corresponding buffer system should not cause precipitation of transition metal ions and cannot have strong chelation with transition metal ions.
Preferably, in step (1), the pH of the buffer system is 5-8, e.g. 5, 5.5, 6, 6.5, 7, 7.5 or 8, etc., preferably 5.5-7.5.
Preferably, in step (1), the temperature of the incubation is: the temperature of-10℃to 37℃may be, for example, -10℃or-5℃or-0℃or-5℃or-10℃or-15℃or-20℃or-30℃or 37℃and is preferably 0 to 20 ℃.
Preferably, in step (1), the incubation time is 8-24 hours, for example, 8, 10, 12, 14, 16, 18, 20, 22 or 24, etc.
Preferably, in step (2), the linker moiety of the conjugated antibody in the linker in the complex contains at least two reactive groups, each covalently coupled to two sulfhydryl groups on the antibody.
In the present invention, the antibody conjugated with the linker-drug using the bioconjugation method is not particularly limited. The choice of antibody depends on the disease or disorder (e.g., cancer) being treated by the antibody-drug conjugate (ADC). The antibodies can specifically bind to a corresponding antigen expressed on cancer cells (also known as a Tumor Associated Antigen (TAA)), viral antigen, or microbial antigen, have antibody-dependent cell-mediated phagocytosis (ADCP) activity, and have in vivo anti-tumor, anti-viral, or anti-microbial activity. The interchain S-S bond in an antibody is the site of attachment of the drug-linker complex.
In some embodiments, the antibody may include, but is not limited to, a monoclonal antibody or a polyclonal antibody. Specific examples of antibodies include: a human, humanized or chimeric antibody.
In some embodiments, the antibody is a monoclonal antibody, e.g., a human antibody or a humanized antibody.
Preferably, in step (2), the reactive groups in the complex are selected from: dibromomaleimide, 4, 5-dibromo-1, 2-dihydro-click-Any one or a combination of at least two of 3, 6-diketones, maleimides, organic bromides, iodides, sulfones or alkenes with electron-deficient groups.
In the present invention, for the desired drug and the selected linker, one skilled in the art can select an appropriate method to couple them together. Since the ratio of the drug to the reactive group according to the invention is 1:2, the linker moiety in its linker that is coupled to the antibody molecule may thus be a specific double reactive group linker such as: dibromomaleimides, and the like. Or any combination of conventional linkers. The most common reactive group capable of bonding to a thiol in ADC preparation is maleimide. In addition, organic bromides, iodides, sulfones, and alkenes and alkynes having electron-deficient groups are often used.
In the invention, the method has higher tolerance to the types of the connectors and can be effectively adapted to a plurality of different connectors.
Preferably, in step (2), the drug in the complex has a cytotoxic, antitumor or labelling effect.
Preferably, in step (2), the covalent coupling conditions are: the concentration of the substance in the covalently coupled system is at least 2 times or more greater than the concentration of the antibody, the reaction temperature is-10℃to 37℃and may be, for example, -10℃to-5℃to 0℃to 5℃to 10℃to 15℃to 20℃to 30℃to 37℃and the reaction time is 0.5 to 24 hours and may be, for example, 0.5, 1,6, 12, 18 or 24.
Preferably, the reaction temperature is from 0℃to 20 ℃.
Preferably, the covalently coupled system further comprises an organic solvent which keeps the system clear.
Preferably, the covalently coupled system further comprises a metal chelator that modulates the reactivity of the drug.
Preferably, the metal chelator comprises any one or a combination of at least two of EDTA, DPTA or DOTA.
Preferably, the concentration of the metal chelator is 0-3 times the concentration of the antibody.
In the present invention, the concentration of the substance of the drug is at least 2 times or more greater than the concentration of the antibody when the covalent coupling is performed; according to the solubility of the drug, an organic solvent can be added into the reaction system during covalent coupling to keep the system clear; depending on the reactivity of the drug, a metal chelator such as EDTA, DPTA or DOTA may be added to the reaction system at a concentration of 0-3 times the concentration of the antibody at the time of covalent coupling to adjust the reactivity of the drug. The specific reaction temperature and time are determined according to the reactivity of the drug, and the reaction temperature is usually-10 to 37 ℃, preferably 0 to 20 ℃, and the reaction time is usually 0.5 to 24 hours.
The invention is not particularly limited with respect to the drugs and linkers that can be used in the bioconjugation method of the invention, and the drug molecules need to have the desired (e.g., cytotoxic, anti-tumor or labeling, etc.) effect and to have at least one substituent group or partial structure that allows attachment to the linker structure, and the linker moiety of the linker that is coupled to the antibody contains at least two reactive groups, each covalently coupled to two sulfhydryl groups on the antibody.
A wide variety of diagnostic, therapeutic and labeling agents known in the art may be conjugated to the antibody molecule. For example, in the broadest sense, the drug to be conjugated may include a diagnostic agent, a drug molecule, e.g., a cytotoxic agent, a toxin, a radionuclide, a fluorescent agent. Wherein the fluorescent agent is, for example, an amine-derived fluorescent probe such as 5-dimethylaminonaphthalene-1- (N- (2-aminoethyl)) sulfonamide-dansyl ethylenediamine, or Oregon488 Cadaverine (cadaverine) (catalog number O-10465,Molecular Probes), or dansyl cadaverine, or N- (2-aminoethyl) -4-amino-3, 6-dithio-L, 8-naphthalimide, or dipotassium salt (fluorescent Huang Yi diamine), or rhodamine B ethylenediamine (catalog number L-2424, molecular Probes), or thiol-derivatized fluorescent Probes, e.g.FLL-cysteine (catalog number B-20340,Molecular Probes).
Preferably, the method further comprises the step of reoxidizing the unreacted mercapto groups in step (2) with an oxidizing agent.
Preferably, the oxidizing agent is selected from: any one or a combination of at least two of dehydroascorbic acid, 5-dithiobis (2-nitrobenzoic acid), oxygen or air.
Preferably, in step (3), the final concentration of the oxidizing agent is 0.04-0.8mM, for example 0.04mM、0.06mM、0.08mM、0.1mM、0.15mM、0.2mM、0.25mM、0.3mM、0.35mM、0.4mM、0.45mM、0.5mM、0.55mM、0.6mM、0.65mM、0.7mM、0.75mM or 0.8mM, etc.
Preferably, in step (3), the reaction conditions of the oxidation are: the concentration of the oxidizing agent is at least higher than the concentration of the antibody, and the temperature is: -10 ℃ to 37 ℃, preferably 0 ℃ to 20 ℃; the reaction time is 0.5-24 hours.
Preferably, in step (2), a purification step is further included, the purification comprising: and removing the transition metal ions by using EDTA as a chelating agent.
Preferably, the purification method comprises: purification is performed using any one or a combination of at least two of a desalting column, size exclusion chromatography, hydrophobic interaction chromatography, ultrafiltration tube, or tangential flow filtration system.
In the present invention, the oxidation step is further followed by a purification step, and the transition metal ions can be removed in a subsequent purification step, thereby reducing the influence on the quality of the final product. Illustratively, the transition metal ions are removed in a purification step by using EDTA as a chelating agent that will be filtered out in a subsequent dialysis, ultrafiltration or gel filtration.
Preferably, the preparation method comprises the following steps:
(1) Adding a transition metal salt comprising a transition metal ion of either Zn 2+、Cd2+ or Hg 2+ to a buffer system selected from the group consisting of: any one or a combination of at least two of HEPES buffer solution, histidine buffer solution, PBS buffer solution or MES buffer solution, wherein the pH value of the buffer system is 5-8; incubating a reducing agent and an antibody to be conjugated in a buffer system containing transition metal ions at-10 ℃ to 37 ℃ for 8-24 hours, selectively reducing inter-chain disulfide bonds of the antibody; the final concentration of the reducing agent in the incubation system is 0.04-0.4mM, and the final concentration of the transition metal ion in the incubation system is 0.01-0.2mM;
(2) Covalently binding a drug having cytotoxic, anti-tumor or labelling effects to a linker comprising at least two reactive groups to obtain a drug-linker complex carrying two reactive groups; the reactive groups are respectively and covalently coupled with two sulfhydryl groups on the antibody; covalently coupling an excess of the drug-linker complex carrying two reactive groups to the reduced sulfhydryl group obtained in step (1); the covalent coupling conditions are as follows: the mass concentration of the drug in the covalent coupling system is at least 2 times or more than that of the antibody, the reaction temperature is-10 ℃ to 37 ℃ and the reaction time is 0.5 to 24 hours;
Re-oxidizing unreacted sulfhydryl groups with an oxidizing agent, the final concentration of the oxidizing agent being 0.04-0.8mM; the reaction conditions of the oxidation are as follows: the concentration of the oxidizing agent is at least higher than the concentration of the antibody, and the temperature is: -10-37 ℃; the reaction time is 0.5-24 hours; the antibody-drug conjugate obtained is recovered by purification using any one or a combination of at least two of a desalting column, size exclusion chromatography, hydrophobic interaction chromatography, ultrafiltration tube or tangential flow filtration system.
In a second aspect, the invention provides the use of a method of preparation of the first aspect for improving homogeneity of an antibody-drug conjugate in the preparation of an ADC medicament.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
Compared with the prior art, the invention has the following beneficial effects:
(1) The present invention allows selective preparation of homogeneous antibody-drug 1:2 conjugates, which successfully demonstrates that transition metal ions are the key factors responsible for the high levels of D2 and low levels of D0, D1, D3 and D4 in the resulting ADCs. Furthermore, the present invention demonstrates that the method produces an ADC product with high Fab preference. The method has been demonstrated with several commercial therapeutic antibodies and shows great consistency.
(2) The antibody-drug 1:2 conjugate of the present invention accounts for more than 60% wt of the total composition of the product, and by using the method for producing the antibody-drug conjugate of the present invention, the homogeneity of the antibody-drug conjugate is higher than that of the product produced by the conventional conjugation method. Specifically, in the ADC prepared by the method of the present invention, the content of d0+d4 is less than 20wt%, and the content of D3 is less than 20wt%. Furthermore, the content of D2 is generally more than 60% by weight, and for example more than 70% by weight. Whereas the content of D2 in ADCs prepared by conventional conjugation methods is typically below 40wt%.
(3) The connecting part of the medicine and the antibody is concentrated on disulfide bonds between light chains and heavy chains of the antibody, and the disulfide bonds between the heavy chains are reduced to be opened after the metal complex hinge region is used. The method avoids protein modification or enzyme catalysis, is based on natural inter-chain disulfide bonds, only needs transition metal ions, does not use expensive enzyme catalysts, and further reduces the cost. Thus, the process of the present invention is less complex, simple to operate, and the homogeneity of the resulting antibody-drug conjugate is significantly improved and the cost is greatly reduced compared to conventional processes for preparing ADCs.
Drawings
FIG. 1 is HIC of Herceptin-DBM-MMAF conjugate prepared by the method of the present invention.
FIG. 2 is HIC of Herceptin-DBM-MMAF conjugate prepared by using conventional method.
FIG. 3 is a Lys-C digested LC-MS of a Herceptin-DBM-MMAF conjugate prepared by the method of the invention.
FIG. 4 is a Lys-C digested LC-MS of a Herceptin-DBM-MMAF conjugate prepared by conventional methods.
FIG. 5 is HIC of Rituxan-MMAF conjugates prepared by the method of the present invention.
FIG. 6 is HIC of Rituxan-MMAF conjugates prepared by using conventional methods.
FIG. 7 is HIC of an Erbitux-MMAF conjugate prepared by the method of the present invention.
FIG. 8 is HIC of Erbitux-MMAF conjugates prepared by using conventional methods.
FIG. 9 is HIC of Herceptin-diBrPD-MMAE conjugate prepared by using the method of the present invention.
FIG. 10 is HIC of Rituxan-diBrPD-MMAE conjugates prepared by using the method of the present invention.
FIG. 11 is HIC of an Erbitux-diBrPD-MMAE conjugate prepared by using the method of the invention.
FIG. 12 is HIC of Herceptin-bismaleimide-MMAE conjugate prepared by using the method of the present invention.
FIG. 13 is HIC of Rituxan-bismaleimide-MMAE conjugates prepared by using the method of the present invention.
FIG. 14 is HIC of Erbitux-bismaleimide-MMAE conjugates prepared by using the method of the invention.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
EXAMPLE 1 preparation of Herceptin-DBM-MMAF conjugates and detection of homogeneity of said conjugates
The Herceptin-DBM-MMAF conjugate is prepared by a one-pot reaction, and the specific steps are as follows:
(1) ZnCl 2 (0.12 mM) and TCEP (0.098 mM) were added to a solution of Herceptin (prepared from WuXi Biologics, 0.03mM, in phosphate buffer, pH 7, 20mM according to the standard procedure for the preparation of monoclonal antibodies, according to the corresponding protein sequences disclosed) and the reaction mixture was allowed to stand overnight at 4 ℃.
(2) DBM-MMAF (commercially available from WuXi Applec., 0.12 mM) in DMA (dimethylacetamide, commercially available from ALDRICH SIGMA) was introduced and the reaction was continued at 4℃for 2 hours.
(3) N-acetylcysteine (0.24 mM) was added to deplete excess DBM-MMAF.
(4) EDTA (0.24 mM) was added to capture Zn 2+ and DHAA (commercially available from ALDRICH SIGMA,0.24 mM) was added to oxidize excess sulfhydryl groups.
(5) The reaction mixture was purified using a desalting column (type: 40K,0.5mL, REF:87766, lot#SJ251704, manufacturer: thermo).
As a control, the reaction was carried out in the same procedure in the presence of 0.06mM TCEP but without ZnCl 2.
Homogeneity determination was performed. Drug/antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification of D0, D1, D2, D3 and D4 by Hydrophobic Interaction Chromatography (HIC) was performed at Tosoh TSKgel Butyl-NPR 4.6mm I.D.×3.5cm,2.5 μm at a flow rate of 0.6mL/min at ambient temperature. The loading was 50. Mu.g, solvent A was 1.5M (NH 4)2SO4 and 50mM sodium phosphate pH 7. Solvent B was 75% v/v 50mM sodium phosphate pH 7 and 25% v/v isopropyl alcohol. The different drug loaded materials were eluted by sequential fractionation gradients. The results of the assays are shown in Table 1 and FIGS. 1-2, FIG. 1 being HIC of the Herceptin-DBM-MMAF conjugate prepared by the method of the invention, and FIG. 2 being HIC of the Herceptin-DBM-MMAF conjugate prepared by using conventional methods.
TABLE 1
As can be seen from Table 1, the two herceptins prepared by the methods of the present invention and by conventional methods have very close HIC DAR values, representing that on average, each antibody in the two products was coupled with approximately two MMAF. In contrast, the D2 component content of the sample synthesized by the method of the invention is 71.43%, which is significantly higher than 39.32% in the sample of the conventional method. Thus, the samples synthesized by the methods of the present invention more reflect the properties of the D2 component, while the D2 component is the ideal sample, and the component with two drugs per antibody is referred to as the D2 component, and thus the samples synthesized by the methods of the present invention are more nearly ideal. On the other hand, it can be seen that the image of the D2 component in fig. 2 is more complex than in fig. 1. Since there are 4 pairs of available thiol groups on the antibody for coupling, it is possible to generate a plurality of isomers coupled at different sites, of which there are 3 isomers which are greatly different, corresponding to three small peaks of the D2 component in fig. 2 which are difficult to separate. The D2 peak in fig. 1 is a single peak, which means that the D2 sample synthesized by the method of the present invention has a good site selectivity, and in order to prove this, the binding site was measured in this example.
Binding site assays were performed. Using a combination of HPLC-MS and Lys-C kit (commercially available and Wako pure chemical industries, ltd.) 1. Mu.L of Lys-C enzyme (10 units dissolved in distilled water) and 3. Mu.L of 1M Tris buffer (pH=8.0) were added to 15. Mu.g of Herceptin-DBM-MMAF solution prepared by the method of the present invention and the conventional method, respectively, and the solution was then added to 15. Mu.L of ultrapure water, incubated at 37℃for 15 minutes, and then analyzed on HPLC-MS (column type: AGILENT PLRP-S1000A,8 μm, 50X 2.1 mm). The results are shown in Table 2 and FIGS. 3-4. FIG. 3 is a Lys-C digested LC-MS of a Herceptin-DBM-MMAF conjugate prepared by the method of the present invention, and FIG. 4 is a Lys-C digested LC-MS of a Herceptin-DBM-MMAF conjugate prepared by a conventional method.
TABLE 2
The Lys-C enzyme will selectively hydrolyze the intact antibody into two Fab's containing one reducible disulfide bond and one Fc containing two reducible disulfide bonds. Table 2 is a summary of the raw data of fig. 3 and 4, and from the results of table 2, it is clear that Herceptin-DBM-MMAF synthesized by the method of the present invention, whose coupling reaction modification sites are concentrated in the Fab region of the antibody, has no signal of the coupled compound visible by mass spectrometry in the Fc region; the reaction is highly specific in regioselectivity. The coupling sites of the drug are randomly distributed in the Fc region and the Fab region of the antibody only by the Herceptin-DBM-MMAF synthesized by the conventional method. According to the general knowledge of antibody coupling drug development, drugs are coupled at different sites of antibodies, and the properties of the final drug are different. If the coupling site-specific sample can be used at the early stage of the study, great convenience is provided for subsequent sample selection. It can also be seen from fig. 3 and 4 that the spectral peaks of fig. 3 are significantly fewer and more uniform.
EXAMPLE 2 preparation of DBM-MMAF conjugates of other antibodies and homogeneity detection of said conjugates
To demonstrate that the present method is applicable to a larger number of antibody classes, two additional commercially available antibodies (Rituxan/Erbitux) were selected for the present method as well as coupling by conventional methods, and the selection of antibodies in this example was based solely on the commercial availability of antibodies.
The DBM-MMAF conjugate of the antibody was prepared in a one-pot reaction, the specific procedure is as follows:
(1) ZnCl 2 (0.12 mM) and TCEP (0.098 mM) were added sequentially to a solution of Rituxan/Erbitux (prepared by standard methods for monoclonal antibody preparation according to the corresponding protein sequences disclosed, from WuXi Biologics, 0.03mM in phosphate buffer, pH 7, 20 mM) and the reaction mixture allowed to stand overnight at 4 ℃.
(2) DBM-MMAF (commercially available from WuXi Applec., 0.12 mM) was introduced in DMA (commercially available from ALDRICH SIGMA) and the reaction was continued at 4℃for 2 hours.
(3) N-acetylcysteine (0.24 mM) was added to deplete excess DBM-MMAF.
(4) EDTA (0.24 mM) was added to capture Zn 2+ and DHAA (commercially available from ALDRICH SIGMA,0.24 mM) was added to oxidize excess sulfhydryl groups.
(5) The reaction mixture was purified using a desalting column (type: 40K,0.5mL, REF:87766, lot#SJ251704, manufacturer: thermo).
As a control, the reaction was carried out in the same procedure in the presence of 0.06mM TCEP but without ZnCl 2.
Homogeneity determination was performed. Drug/antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification of D0, D1, D2, D3 and D4 by Hydrophobic Interaction Chromatography (HIC) was performed at Tosoh TSKgel Butyl-NPR 4.6mm I.D.×3.5cm,2.5 μm at a flow rate of 0.6mL/min at ambient temperature. The sample loading was 50. Mu.g, solvent A was 1.5M (NH 4)2SO4 and 50mM sodium phosphate pH 7. Solvent B was 75% v/v 50mM sodium phosphate pH 7 and 25% v/v isopropyl alcohol. Different drug loaded materials were eluted by sequential fractionation gradients. The results are shown in Table 3 and FIGS. 5-8. FIG. 5 is HIC for Rituxan-DBM-MMAF conjugates prepared by the method of the invention, FIG. 6 is HIC for Rituxan-DBM-MMAF conjugates prepared by using the conventional method. FIG. 7 is HIC for Erbitux-DBM-MMAF conjugates prepared by the method of the invention, and FIG. 8 is HIC for Erbitux-DBM-MMAF conjugates prepared by using the conventional method.
TABLE 3 Table 3
As can be seen from the detection results in the above examples 1-2, as shown in tables 1, 3 and fig. 1, 5 and 7, the results prove that: the content of D2 is generally more than 60% by weight, for example more than 70% by weight (FIGS. 1, 5 and 7). In contrast, the D2 content in ADCs prepared by the control conjugation method in the absence of transition metal ions is typically less than 40wt% (fig. 2, 6 and 8), while the corresponding chromatograms also indicate that the D2 product produced is a mixture of multiple reaction sites without the participation of transition metal ions.
It can also be seen from Table 2 and FIGS. 3-4 that Herceptin-DBM-MMAF prepared by the conventional method, although having similar DAR values as Herceptin-MMAF prepared by the method of the present invention, had a random distribution of the linked drug on a single antibody, and did not have homogeneity. The distribution of the drug of the product prepared by the method on the antibody is highly homogeneous.
The above results clearly demonstrate that ADCs prepared by the method of the invention using transition metal ions have significantly improved homogeneity, in terms of the homogeneity of the DAR value of the product, and in terms of the homogeneity of the distribution of the drug on the antibody.
EXAMPLE 3 preparation of diBrPD-VC-MMAE conjugates of antibodies and detection of homogeneity of said conjugates
In order to further illustrate that the method of the present invention is effective for a variety of bridging heads in addition to having universality for the coupling of a variety of proteins, another bridging linker-drug diBrPD-VC-MMAE was selected in this example, and the conjugate was prepared using the method.
The diBrPD-VC-MMAE conjugate of the antibody was prepared by one pot reaction, and the specific steps are as follows:
(1) ZnCl 2 (0.12 mM) and TCEP (0.105 mM) were added to a solution of Herceptin/Rituxan/Erbitux (prepared from WuXi Biologics, 0.03mM in phosphate buffer, pH 7, 20mM according to the standard procedure for monoclonal antibody preparation, according to the corresponding protein sequences disclosed) and the reaction mixture was allowed to stand overnight at 12 ℃.
(2) DiBrPD-VC-MMAE (commercially available from syncabio., 0.45 mM) in DMA (dimethylacetamide, commercially available from ALDRICH SIGMA), and EDTA (0.09 mM) were introduced to capture part of the zinc ions and the reaction was continued at 12 ℃ for 24 hours.
(3) N-acetylcysteine (0.24 mM) was added to deplete excess diBrPD-VC-MMAE.
(4) EDTA (0.24 mM) was added to completely capture Zn 2+ and DHAA (commercially available from ALDRICH SIGMA,0.24 mM) was added to oxidize excess sulfhydryl groups.
(5) The reaction mixture was purified using a desalting column (type: 40K,0.5mL, REF:87766, lot#SJ251704, manufacturer: thermo).
Homogeneity determination was performed. Drug/antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification of D0, D1, D2, D3 and D4 by Hydrophobic Interaction Chromatography (HIC) was performed at Tosoh TSKgel Butyl-NPR 4.6mm I.D.×3.5cm,2.5 μm at a flow rate of 0.6mL/min at ambient temperature. The sample loading was 30 μg, solvent A was 1.5M (NH 4)2SO4 and 50mM sodium phosphate pH 7. Solvent B was 75% v/v 50mM sodium phosphate pH 7 and 25% v/v isopropyl alcohol. The different drug loaded materials were eluted by sequential fractionation gradients. The results are shown in Table 4 and FIGS. 9-11, FIG. 9 is HIC for the Herceptin-diBrPD-VC-MMAE conjugate prepared by using the method of the present invention, FIG. 10 is HIC for the Rituxan-diBrPD-VC-MMAE conjugate prepared by the method of the present invention, and FIG. 11 is HIC for the bitux-diBrPD-VC-MMAF conjugate prepared by the method of the present invention.
TABLE 4 Table 4
As can be seen from Table 4, the D2 component content of the diBrPD-VC-MMAE conjugate of the antibody can be effectively controlled to be more than 60% by using the method of the invention, and the reaction process is not required to be greatly modified.
Example 4 preparation of antibody-bismaleimide-VC-MMAE conjugates and detection of homogeneity of the conjugates
In addition to the above examples, to further demonstrate the universality of this patent, this example additionally selects a linker-drug: bismaleimide-VC-MMAE to demonstrate the versatility of the present method. It should be noted that, the drug linker most widely used in the field of maleimide (maleimide) antibody-conjugated drugs, bismaleimide (bismaleimide) has two maleimide linkers on the molecule to achieve the bridging function, and can be applied to the largest range of bridging requirements without any problem.
The antibody-bismaleimide-VC-MMAE conjugate was prepared by one-pot reaction, and the specific steps are as follows:
(1) ZnCl 2 (0.12 mM) and TCEP (0.105 mM) were added to a solution of Herceptin/Rituxan/Erbitux (prepared from WuXi Biologics, 0.03mM in phosphate buffer, pH 7, 20mM according to the standard procedure for monoclonal antibody preparation, according to the corresponding protein sequences disclosed) and the reaction mixture was allowed to stand overnight at 12 ℃.
(2) Bismaleimide-VC-MMAE (commercially available from Quanta biosign, 0.45 mM) in DMA (dimethylacetamide, commercially available from ALDRICH SIGMA) was introduced and the reaction was continued at 12 ℃ for 24 hours.
(3) N-acetylcysteine (0.24 mM) was added to deplete excess Bismaleimide-VC-MMAE.
(4) EDTA (0.24 mM) was added to capture Zn 2+ and DHAA (commercially available from ALDRICH SIGMA,0.24 mM) was added to oxidize excess sulfhydryl groups.
(5) The reaction mixture was purified using a desalting column (type: 40K,0.5mL, REF:87766, lot#SJ251704, manufacturer: thermo).
Homogeneity determination was performed. Drug/antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification of D0, D1, D2, D3 and D4 by Hydrophobic Interaction Chromatography (HIC) was performed at Tosoh TSKgel Butyl-NPR 4.6mm I.D.×3.5cm,2.5 μm at a flow rate of 0.6mL/min at ambient temperature. The sample loading was 30 μg, solvent A was 1.5M (NH 4)2SO4 and 50mM sodium phosphate pH 7. Solvent B was 75% v/v 50mM sodium phosphate pH 7 and 25% v/v isopropyl alcohol. The different drug loaded materials were eluted by sequential fractionation gradients. The results are shown in Table 5 and FIGS. 12-14, FIG. 12 is HIC for the Herceptin-bismaleimide-VC-MMAE conjugate prepared by using the method of the present invention, FIG. 13 is HIC for the Rituxan-bismaleimide-VC-MMAE conjugate prepared by using the method of the present invention, and FIG. 14 is HIC for the Erbitux-bismaleimide-VC-MMAE conjugate prepared by using the method of the present invention.
TABLE 5
As can be seen from Table 5, the process of the present invention is still applicable to bismaleimide-VC-MMAE developed based on the maleimide most commonly used in the art today. The selection of the coupling drug by the method of the invention is proved to be only required that the coupling drug has reactivity with sulfhydryl groups, and is not limited by the reaction type and the kind of functional groups of the linker.
In summary, the present invention developed a novel bioconjugation method that selectively modifies two drug molecules in the Fab region of an antibody using any given reactive linker, while not modifying the antibody sequence. The method can produce the ADC with improved homogeneity, has simple operation steps and can reduce the cost and enhance the efficiency. Antibody-drug 1 in the present invention: 2 the conjugate comprises more than 60% wt of the total composition of the product, the homogeneity of the antibody-drug conjugate being higher than the product produced by conventional conjugation methods by using the method of producing an antibody-drug conjugate of the invention.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.

Claims (10)

1. A method of making an improved homogeneity of an antibody-drug conjugate DAR2, comprising:
(1) Incubating a reducing agent and an antibody to be conjugated in a buffer system containing transition metal ions, reducing interchain disulfide bonds in the antibody to produce reduced sulfhydryl groups;
(2) Covalently coupling a drug-linker complex carrying at least two reactive groups to the reduced sulfhydryl group obtained in step (1); thereby producing an antibody-drug conjugate mixture;
In the step (2), the linker part of the coupling antibody in the linker in the complex contains at least two reactive groups, and the two sulfhydryl groups on the antibody are respectively and covalently coupled;
In the step (1), the transition metal ion is Zn 2+;
in step (2), the reactive groups in the complex are independently selected from: at least two of maleimide, organic bromide, iodide, sulfone compounds or alkene and alkyne compounds with electron-deficient groups;
in step (2), the covalent coupling conditions are: the concentration of the substance of the drug in the covalently coupled system is at least 2 times greater than the concentration of the antibody.
2. The method of claim 1, wherein in step (1), the reducing agent is selected from the group consisting of: any one or a combination of at least two of tris (2-carboxyethyl) phosphine, diphenylphosphinoacetic acid, 2- [2- (diphenylphosphino) ethyl ] pyridine, 3- (diphenylphosphino) benzenesulfonic acid, 4- (diphenylphosphino) benzoic acid, 2- (diphenylphosphino) ethylamine, 3- (diphenylphosphino) propylamine, 3- (diphenylphosphino) propionic acid, 2- (diisopropylphosphino) ethylamine, 2- (diphenylphosphino) benzoic acid, (2-hydroxyphenyl) diphenylphosphine, 1,3, 5-triaza-7-phosphatricyclo [3.3.1.13.7] decane, n-butylbis (1-adamantyl) phosphine, sodium triacetoxyborohydride or sodium cyanoborohydride;
in step (1), the final concentration of the reducing agent in the incubation system is 0.04-0.4mM.
3. The method of claim 1, wherein in step (1), the transition metal ion is added to the buffer system in the form of a transition metal salt selected from ：ZnCl2、Zn(NO3)2、ZnSO4、Zn(CH3COO)2、ZnI2、ZnBr2、Zn(HCOO)2 or Zn (BF 4)2, or a combination of at least two;
In step (1), the final concentration of the transition metal ion in the incubation system is 0.01-0.2mM.
4. The method of claim 1, wherein in step (1), the buffer system is selected from the group consisting of: any one or a combination of at least two of HEPES buffer, histidine buffer, PBS buffer or MES buffer;
In step (1), the pH of the buffer system is 5.5-7.5.
5. The method of claim 1, wherein in step (1), the incubation is at a temperature of 0 ℃ to 20 ℃;
in step (1), the incubation time is 8-24 hours.
6. The method of claim 1, wherein in step (2), the drug in the complex has a cytotoxic, anti-tumor or labeling effect;
in the step (2), the reaction temperature is between-10 and 37 ℃ and the reaction time is between 0.5 and 24 hours;
the reaction temperature is 0-20 ℃;
The covalent coupling system also comprises an organic solvent, and the organic solvent keeps the system clear;
the covalently coupled system further comprises a metal chelator that modulates the reactivity of a drug;
The metal chelating agent comprises any one or a combination of at least two of EDTA, DPTA or DOTA;
The concentration of the metal chelating agent is 0-3 times of the concentration of the antibody.
7. The method of preparing an antibody-drug conjugate according to any one of claims 1-6, further comprising the step of reoxidizing unreacted thiol groups of step (2) with an oxidizing agent;
the oxidizing agent is selected from: any one or a combination of at least two of dehydroascorbic acid, 5-dithiobis (2-nitrobenzoic acid), oxygen or air;
The final concentration of the oxidant is 0.04-0.8mM;
the reaction conditions of the oxidation are as follows: the concentration of the oxidant is at least higher than that of the antibody, and the temperature is 0-20 ℃; the reaction time is 0.5-24 hours.
8. The method of claim 7, further comprising a purification step after step (2), said purification comprising: removing transition metal ions by using EDTA as a chelating agent;
The purification method comprises the following steps: purification is performed using any one or a combination of at least two of a desalting column, size exclusion chromatography, hydrophobic interaction chromatography, ultrafiltration tube, or tangential flow filtration system.
9. The method of preparing for improving the homogeneity of an antibody-drug conjugate DAR2 of claim 8, comprising:
(1) Adding a transition metal salt containing a transition metal ion of Zn 2+ to a buffer system selected from the group consisting of: any one or a combination of at least two of HEPES buffer solution, histidine buffer solution, PBS buffer solution or MES buffer solution, wherein the pH value of the buffer system is 5-8; incubating a reducing agent and an antibody to be conjugated in a buffer system containing transition metal ions at-10 ℃ to 37 ℃ for 8-24 hours, selectively reducing inter-chain disulfide bonds of the antibody to produce reduced sulfhydryl groups; the final concentration of the reducing agent in the incubation system is 0.04-0.4mM, and the final concentration of the transition metal ion in the incubation system is 0.01-0.2mM;
(2) Covalently binding a drug having cytotoxic, anti-tumor or labelling effects to a linker comprising at least two reactive groups to obtain a drug-linker complex carrying at least two reactive groups; the two reactive groups in the linker are respectively and covalently coupled with two sulfhydryl groups on the antibody; covalently coupling an excess of the drug-linker complex carrying at least two reactive groups to the reduced sulfhydryl group obtained in step (1); the covalent coupling conditions are as follows: the mass concentration of the drug in the covalent coupling system is at least 2 times or more than that of the antibody, the reaction temperature is-10 ℃ to 37 ℃ and the reaction time is 0.5 to 24 hours;
Re-oxidizing unreacted sulfhydryl groups with an oxidizing agent, the final concentration of the oxidizing agent being 0.04-0.8mM; the reaction conditions of the oxidation are as follows: the concentration of the oxidizing agent is at least higher than the concentration of the antibody, and the temperature is: -10-37 ℃; the reaction time is 0.5-24 hours; the antibody-drug conjugate obtained is recovered by purification using any one or a combination of at least two of a desalting column, size exclusion chromatography, hydrophobic interaction chromatography, ultrafiltration tube or tangential flow filtration system.
10. Use of the method of preparation of any one of claims 1-9 to improve the homogeneity of an antibody-drug conjugate DAR2 in the preparation of an ADC medicament.
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