CN116099009A - Antibody conjugate and preparation method thereof - Google Patents

Abstract

The invention discloses an antibody conjugate and a preparation method thereof, wherein the preparation method of the antibody conjugate comprises the following steps: an antibody reduction step, wherein the antibody and a reducing agent are subjected to a reduction reaction under a first reaction condition, so that disulfide bonds of the antibody are reduced to generate sulfhydryl groups, and a reduction product is obtained, wherein the first reaction condition comprises: the first reaction temperature is 4-37 ℃, the first reaction pH is 4-11, and the molar ratio of the reducing agent to the antibody is 2-11; and a step of coupling the antibody and the drug, wherein the antrodia camphorata is added into the reduction product to carry out coupling reaction under a second reaction condition to obtain an antibody conjugate, and the second reaction condition comprises: the second reaction temperature is 37 ℃, the second reaction pH is 6.9-8.9, and the molar ratio of the reduction product to the antrodia camphorate is 4.34-6.14.

Description

Antibody conjugate and preparation method thereof

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to an antibody conjugate and a preparation method thereof.

Background

Antrodia camphorate is an expensive mushroom growing in the inner cavity of an endangered indigenous tree species in Taiwan, i.e. the inner cavity of the evergreen broad-leaved tree Antrodia camphorate (Cinnamomum kanehi rai Hayata). Antrodia camphorate has been used as a traditional drug in Taiwan, and is known to have a variety of health benefits including scavenging free radicals, anti-inflammatory, antibacterial, liver-protecting, neuroprotection, antidiabetic and free radical-induced DNA damage protective activities. Antrodia ethanol extract has been shown to trigger the growth of rat fibroblasts and to participate in wound healing. In the case of human leukemia, it has been shown that tumor progression can be prevented by inducing cell cycle arrest in the G1 phase. Antrodia camphorate can store a large amount of active compounds including terpenes, benzene, lignans, benzoquinone derivatives, succinic acid and maleic acid derivatives, polysaccharides, sterols, nucleotides, fatty acids, etc. The majority of active compounds found mainly in fruiting bodies of Antrodia camphorata are terpenoids, and many studies have been carried out to evaluate the biological and pharmacological properties of this group of specific compounds. However, despite the many health benefits, it is difficult to mass-produce these compounds as natural drugs due to the very slow natural growth rate and host specificity of the fruiting body. These special features of fruiting bodies require identification of other compounds abundant in mycelium and can be mass produced by artificial cultivation of Antrodia camphorata.

An Zhuokui Nol (Antroquinone) is a tetrahydroubiquinone derivative, which is mainly present in Antrodia camphorata mycelia, and has various biological and pharmacological activities. To date, 7 anthraquinones and their related compounds have been identified in Antrodia camphorata, including anthraquinone, anthraquinone B, C, D, L, M and 4-acetyl anthraquinone B. Two main methods for producing anthraquinone alcohol are chemical total synthesis and biological synthesis in the artificial fermentation process of Antrodia camphorate. Studies on anthraquinone biosynthesis have shown that sorbic acid formed from acetyl-CoA and malonyl-CoA via polyketide pathways is an indispensable benzoquinone ring precursor for natural synthesis of anthraquinone by Antrodia camphorata. The knockout of phthalic acid synthase (orsel l inic acid synthase) in Antrodia camphorata has been shown to prevent An Zhuokui Norl biosynthesis. Several studies have been conducted to assess the efficacy of this compound in the treatment of a number of diseases, including diabetes, liver and kidney diseases, neurodegenerative diseases and cancer, and a number of clinical trials to assess the safety and efficacy of anthraquinone therapy for hypercholesterolemia, hyperlipidemia, metastatic pancreatic cancer and non-small cell lung cancer.

Many studies have been conducted to explore the traditional health benefits of Antrodia camphorate for its biological properties against a variety of physiological and pathological conditions. Among many therapeutic functions, the effect of Antrodia camphorate extract on tumor growth and metastasis has been widely studied. For example, antrodia camphorate fermentation broths have been shown to inhibit breast cancer cell proliferation by inducing G1 phase cell cycle arrest. The treatment also showed that tumor progression in nude mice injected with breast cancer cells could be delayed. In addition, dose-dependent reduction of ovarian cancer cell growth was also observed after Antrodia camphorata treatment. Experimental results show that Antrodia camphorate plays an anti-proliferation role by inhibiting HER2 phosphorylation and inhibiting PI3K/AKT signal pathway. Since the biological activity of crude Antrodia camphorate extract is mainly dependent on its bioactive compounds, the effect of individual compounds on cancer progression is also emphasized. Among these bioactive compounds, anthraquinones are of particular interest to the scientific community for their health benefits and relatively easy biosynthetic processes. In many cancer cells, including lung cancer, liver cancer and leukemia, anthraquinone has been demonstrated to exert anticancer effects by inhibiting Ras and other Ras-related small GTP binding proteins (Rho). Mechanistically, antroqu inono l has been shown to alter post-translational modifications of farnesyl transferase and geranylgeranyl transferase-I by direct binding to these enzymes and subsequent inhibition of their biological functions. This results in the production and accumulation of unprocessed Ras and related proteins in cancer cells. The mode of interaction between anthraquinone and farnesyl transferase, molecular docking analysis indicated that the isoprenoid antroqu inono l moiety bound to the hydrophobic cavity of farnesyl transferase and the loop structure of antroqu inono l was located close to the Ras-CAAX motif binding site on farnesyl transferase. Anthraquinone induces the autophagy pathway by increasing the conversion of LC3B-1 to LC3B-I I and increasing the level of LC3B-I I-associated autophagosomes in lung cancer cells.

Antibody drugs have high specificity, and since 1960, tumor treatment using antibodies has been started. Mouse monoclonal antibodies were initially used for the assay, but due to immunogenicity, serum half-life was short and lack of effective interaction with human immune effector cells, progress was very limited. Therefore, humanized antibodies, which can be produced by protein engineering, have been used, and the key to therapeutic action of humanized monoclonal antibodies is to specific single epitopes. However, the antibody drugs used in clinic at present have a plurality of defects, such as poor penetrability of complete antibody molecules due to large molecular weight, and difficulty in penetrating connective tissues to reach solid tumor sites. In addition, the clinical dosage is large, and the production cost is high, so that patients are difficult to bear. At the same time, the antigen heterogeneity of tumor cell population and the antigen modulation of target cells affect the curative effect of the monoclonal antibody medicine, etc. Humanized antibodies are produced using complementarity determining region grafting or by phage, yeast or ribosomal expression. Humanized antibodies have reduced immunogenicity and greater killing capacity against tumor cells compared to murine antibodies. Increasing the concentration of the antibody drug in the tumor cells can be addressed by increasing the permeability of the monoclonal antibody drug. In general, the circulation period of an intact antibody is long, which can usually take several days to a week, but, because the intact antibody molecule is large, has a molecular weight of 150kDa, which makes it difficult to reach the inside of tumor cells through the extracellular space or capillary cortex, and thus it is not the optimal form of tissue transfer in vivo. Studies of different sizes of antibody fragments by radioimmunoconjugate liquid flashing showed that low molecular weight antibody fragments showed good cell penetration and more uniform distribution in tissues. The use of low molecular weight antibody fragments can also reduce antibody responses in humans. Therefore, the research of miniaturized antibodies has important significance for improving the curative effect of antibody medicines.

Methods of preparing antibody drug conjugates involve the random modification of antibody amino acid residues and the selective introduction of drug molecules onto antibody molecules. Wherein the random modification process comprises amination of carboxylic acid, acylation of amino group of lysine side chain, and the like. The antibody molecule contains a number of groups that can be used to modify the cross-linking groups, including amino and carboxyl groups, the amino groups being the s-amino groups of lysine side chains and the amino groups at the N-terminus of the antibody. Carboxyl groups are typically carbon-terminal and aspartic acid and glutamic acid residues are used. Amino and carboxyl groups are widely present in antibodies and most proteins, and the coupling of these groups randomly cross-links the individual parts of the antibody molecule, which results in randomness of the antibody drug coupling sites, and often also blocks the antigen binding sites of the protein molecules, resulting in a decrease in the activity of the antibody drug conjugates.

Disclosure of Invention

Accordingly, the present invention is directed to improving the problem of decreased activity of an antibody conjugate in the existing methods for preparing antibody conjugates.

In order to achieve the above object, the present invention also provides a method for preparing an antibody conjugate, comprising:

an antibody reduction step, wherein the antibody and a reducing agent are subjected to a reduction reaction under a first reaction condition, so that disulfide bonds of the antibody are reduced to generate sulfhydryl groups, and a reduction product is obtained, wherein the first reaction condition comprises: the first reaction temperature is 4-37 ℃, the first reaction pH is 4-11, and the molar ratio of the reducing agent to the antibody is 2-11;

and a step of coupling the antibody and the drug, wherein the antrodia camphorata is added into the reduction product to carry out coupling reaction under a second reaction condition to obtain an antibody conjugate, and the second reaction condition comprises: the second reaction temperature is 37 ℃, the second reaction pH is 6.9-8.9, and the molar ratio of the reduction product to the antrodia camphorate is 4.34-6.14.

Preferably, in the method for preparing an antibody conjugate, the step of coupling the antibody to the drug comprises:

dissolving antrodia camphorata in an organic solvent, and then adding the antrodia camphorata into the reduction product to perform coupling reaction under a second reaction condition to obtain the antibody conjugate.

Preferably, in the method for preparing an antibody conjugate, the second reaction condition includes: the second reaction temperature is 37 ℃, and the second reaction pH is 7-8.

Preferably, in the method for preparing an antibody conjugate, the first reaction temperature is 37 ℃, and the first reaction pH is 7-8.

Preferably, in the method for preparing an antibody conjugate, before the step of coupling the antibody with the drug, after the step of reducing the antibody, the method further comprises:

the degree of reduction of the antibody is detected and the first reaction conditions are adjusted according to the degree of reduction.

Preferably, in the method for preparing an antibody conjugate, the step of detecting the degree of reduction of the antibody and adjusting the first reaction condition according to the degree of reduction comprises:

performing experiments at different levels on three factors of the first reaction condition through a response surface method experiment design;

and selecting the optimal first reaction temperature in the first reaction conditions of the test and the optimal first reaction pH condition, and optimizing the molar ratio of the reducing agent to the antibody.

Preferably, in the method of preparing an antibody conjugate, the reducing agent comprises one or more of mercaptoethanol, mercaptoethylamine, acetylcysteine and tris (2-formylethyl) phosphine hydrochloride.

Preferably, in the method for preparing an antibody conjugate, after the step of coupling the antibody with the drug, the method further comprises:

and a purification step, namely removing superfluous antrodia camphorata anthraquinone joints and organic solvents after the reaction of the antibody and the drug coupling step is terminated.

Preferably, in the method of preparing an antibody conjugate, the purifying step comprises:

after the reaction of the antibody and the drug coupling step is terminated, determining a purification method according to the scale of the reaction;

when the reaction scale is a laboratory scale, purification is performed by one or more of ion exchange chromatography, affinity chromatography, gel filtration method, centrifugal ultrafiltration method, and ion exchange chromatography and affinity chromatography.

In order to achieve the above object, the present invention further provides an antibody conjugate, which is prepared by the preparation method of the antibody conjugate.

The invention has the following beneficial effects:

the preparation method of the antibody conjugate provided by the invention comprises the steps of carrying out a reduction reaction on an antibody and a reducing agent under a first reaction condition so as to reduce disulfide bonds of the antibody to generate sulfhydryl groups, thereby obtaining a reduction product, wherein the first reaction condition comprises the following steps: the first reaction temperature is 4-37 ℃, the first reaction pH is 4-11, the mol ratio of the reducing agent to the antibody is 2-11, then the linalool anthraquinone is added into the reduction product for coupling reaction under the second reaction condition to obtain the antibody conjugate, wherein the second reaction condition comprises: the second reaction temperature is 37 ℃, the second reaction pH is 6.9-8.9, the molar ratio of the reduction product to the antrodia camphorata is 4.34-6.14, and thus, each antibody is coupled with the optimal reaction condition close to the drug, and the activity of the antibody conjugate prepared in this way is optimal;

further, the composition of the antibody conjugate prepared by the preparation method of the antibody conjugate is reasonable, so that the preparation method is beneficial to the cancer treatment of people, and the preparation process is simple and convenient for industrial production.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic flow chart of an embodiment of a method for preparing an antibody conjugate according to the present invention.

The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.

Detailed Description

In the embodiment of the invention, the term "and/or" describes the association relation of the association objects, which means that three relations can exist, for example, a and/or B can be expressed as follows: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.

It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order.

The term "plurality" in embodiments of the present invention means two or more, and other adjectives are similar.

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. However, it will be understood by those of ordinary skill in the art that in various embodiments of the present invention, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, the claimed technical solution of the present invention can be realized without these technical details and various changes and modifications based on the following embodiments. The following embodiments are divided for convenience of description, and should not be construed as limiting the specific implementation of the present invention, and the embodiments can be mutually combined and referred to without contradiction.

The invention provides a preparation method of an antibody conjugate, which comprises the following steps:

an antibody reduction step S100, wherein the antibody and a reducing agent are subjected to a reduction reaction under a first reaction condition, so that disulfide bonds of the antibody are reduced to generate sulfhydryl groups, and a reduction product is obtained, wherein the first reaction condition comprises: the first reaction temperature is 4-37 ℃, the first reaction pH is 4-11, and the molar ratio of the reducing agent to the antibody is 2-11.

The disulfide bond of the antibody is used for carrying out drug coupling to generate an antibody drug conjugate, and firstly, the disulfide bond of the antibody is required to be reduced to generate sulfhydryl. In this example, the antibodies selected for the immunoglobulin G (IgG), which is the most abundant immunoglobulin in human and animal serum, were produced mainly by plasma cells of spleen and lymph nodes. The unique structural features of antibody molecules are that antibodies can retain antigen activity to the maximum extent. The basic unit of an antibody is a symmetrical structure consisting of two heavy and two light chains, with a molecular weight of about 150kDa. Wherein the light chain has a molecular weight of about one sixth of the total antibody molecule, about 25kDa, and consists of two domains, a variable region (VL) and a constant region (CL). The light chain is of two types, namely K and X, the K type in the human light chain accounts for 60% and the human type accounts for 40%; the mouse light chain has 95% of K-type and 5% of human-type. The heavy chain molecular weight is one third of the whole antibody molecule, about 50kDa. One end of the light and heavy chains is called the carboxy-terminus and the other end is called the amino-terminus, and the IgG antibody molecule is covalently cross-linked by interchain disulfide bonds from two heavy chains and two light chains, the light and heavy chains being linked by disulfide bonds in the CL and CH1 regions, respectively. The heavy chain and heavy chain are linked by disulfide bonds in the hinge region. Disulfide bonds are critical to the structure, stability and biological properties of IgG molecules.

Depending on the heavy chain constant region, there are five main types of antibody molecules, designated IgG, igM, ig, igE and IgD, respectively. Wherein the three types of antibody molecules, igG, igE and IgD, all comprise Ig monomers consisting of two heavy chains and two light chains. IgA and IgM, which are more than a small unit and are commonly called J chains, are acidic peptide fragments rich in saccharides and have a molecular weight of about 15kDa. The heavy chain of an immunoglobulin is usually glycosylated, especially in the region of the Fc fragment. However, there are also saccharides in the antigen binding region. There are two antigen binding sites on each Ig monomer, formed by regions of hypervariability near the N-terminus of the heavy and light chains.

In particular, in the antibody reduction step S100, the reduction of disulfide bonds of the antibody may be chemically controlled. For example, one or more of thiol-based reducing agents (dithiothreitol, mercaptoethanol, mercaptoethylamine, cysteine, acetylcysteine, glutathione, etc.), phosphines (tricarboxyethylphosphine, soluble tris (2-carboxyethyl) phosphine (Tris (2-carboxyyethy l) phosphine, TCEP for short) and solid phases TCEP, TSNPP, etc.), borohydrides, NADPH, and other reducing agents that can function in aqueous phase-based solutions (liquid phase/organic phase greater than 9:1), etc. can be used as the reducing agent.

The first reaction conditions include: the first reaction temperature is 4-37 ℃, the first reaction pH is 4-11, and the molar ratio of the reducing agent to the antibody is 2-11. In addition, the reaction time may be determined according to whether the reduction reaction is complete. For control of the reaction time, the reaction can be terminated by adding the same volume of ice bath buffer and removing unreacted reducing agent, i.e., the reaction can be terminated by adding the same volume of ice bath buffer when the reaction is complete.

More specifically, the antibody reduction step S100 includes:

in step S110, different pH buffers are prepared to form ice bath buffers.

Step S120, dissolving a reducing agent in a buffer solution, and dissolving an antibody in the buffer solution;

step S130, reduction reaction.

In step S140, the reducing agent is removed after the reaction is completed.

For example:

(1) Different pH buffers are prepared, namely 20mM acetic acid-sodium acetate buffer pH 4.0, 20mM acetic acid-sodium acetate buffer pH 5.0,20mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer pH 6.0, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer pH 7.0, 20mM tris (hydroxymethyl) aminomethane hydrochloride (Tri-HCl) buffer pH8.0 and glycine-sodium hydroxide buffer pH 9.0, the solution is subjected to vacuum filtration by adopting 0.45pm acetate fiber membranes, and the solution is placed in a refrigerator at 4 ℃ for refrigeration for standby.

(2) Different reducing agents were dissolved in different pH buffers to prepare 10mM solutions, and IgG was dissolved in the corresponding buffers to prepare 0.25mM solutions.

(3) mu.L of 0.25mM IgG was taken, 50. Mu.L of 1mM reducing agent was added, and finally the corresponding buffer was added to 200. Mu.L, and the mixture was left to react for 1 hour in a shaking table at 37 ℃.

(4) The reduced IgG was centrifuged at 4000g with a 3kDa molecular weight cut-off membrane at least 5 times for removal of the reducing agent.

(5) Samples of each time period were taken out in 20ml, added with 5ml of 5X sample buffer, boiled at 100℃for 5 minutes, centrifuged at 8000g for 5min and loaded for electrophoresis.

(6) After the electrophoresis is finished, the gel is dyed by coomassie brilliant blue staining solution for 5 hours, and then is decolorized by a decolorizing solution for 4-24 hours, wherein the decolorizing solution is required to be changed for 3-4 times until protein strips are clear, and the background color is basically removed.

(7) The eluted gel was scanned by using a tan software (gel imager analysis software), and a gel image was analyzed by using a tan GIS analysis software (digital gel image processing system). The processing system analyzes the percentage content of various reduction reaction products of the gel to obtain the proportion of each product.

Methods for selectively binding drug molecules to antibodies include gene recombination techniques to express fusion proteins, reductive amination of oxidized polysaccharides of antibodies, reductive alkylation of disulfide bonds between antibody chains. Antibodies and drug molecules often retain activity better after coupling if the number of functional groups utilized is limited and at some independent site of the molecule. Among these, reductive alkylation by interchain disulfide bonds between the heavy and light chains of the antibody is one of the most amenable to mass production of these several selective modifications. The disulfide bonds on the antibody molecule are limited in number and relatively fixed in position, and the antigen binding site cannot be shielded after the drug is coupled, so that the disulfide bonds on the antibody become optimal sites for the coupled drug. However, the reduction of these endogenous disulfide bonds is nonspecific, i.e., both the hinge region disulfide bonds and the disulfide bonds between the heavy and light chains are reduced, resulting in a compositionally diverse mixture of coupled products. The properties, selectivity, drug substitution/efficacy, stability, toxicity, etc. of the individual components are not clear. There is therefore a need to chemically control the reduction of disulfide bonds, preferentially reducing disulfide bonds in the hinge region.

A reduction detection step S200 of the antibody, wherein the reduction degree of the antibody is detected, and the first reaction condition is adjusted according to the reduction degree;

taking immunoglobulin G (IgG) as an example, the antibody can generate 8 sulfhydryl groups at most, and the fully reduced antibodies can be connected with equivalent drugs with the antrodia camphorata to generate uniform antibody drug conjugate, so that a great amount of drug-conjugated antibodies and uniformity of the drugs can seem to achieve good curative effects, however, the fact is not the case, the drugs can cause the reduction of in vivo clearance rate and the enhancement of toxicity, when 4 drugs are connected to each antibody molecule, and the drug effect is most obvious and the in vivo survival rate is highest when the drugs are conjugated to a hinge region. It is therefore necessary to chemically control the antibody reduction reaction to reduce disulfide bonds in the antibody reduction hinge region, resulting in four sulfhydryl groups for coupling drugs with linkers.

Disulfide bonds in the antibody may be reduced under the first reaction conditions using a reducing agent. The following six possible ways may occur during the reduction:

1. an intact antibody;

2. all disulfide bonds between heavy chains of the heavy chains are reduced;

3. all disulfide bonds between the light chain and the heavy chain are reduced;

4. a disulfide bond between the heavy and light chains is reduced;

5. all disulfide bonds are reduced simultaneously;

6. the heavy chain-heavy chain and the heavy chain-light chain are each reduced one.

Since the number of drug carriers on an antibody conjugate depends on the number of thiol groups contained after reduction of each antibody, it is necessary to detect the number of thiol groups after reduction of the antibody, to optimize the parameters of the reduction reaction, to obtain a first reaction condition suitable for achieving the desired thiol groups, and to obtain the amount of drug with a linker required for the coupling reaction. The method for detecting the number of the sulfhydryl groups after the reduction of the antibody can be a direct method, and is commonly used as an E l lman reagent method, and the method can be used for directly detecting the concentration of the sulfhydryl groups; the method can also be an indirect method, wherein the reduction degree of the antibody is detected to obtain different fragment numbers, and the number of sulfhydryl groups is calculated according to the different number of sulfhydryl groups contained in different fragment numbers. Other reference methods for detecting the reduction degree of the antibody can be adopted, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis, size Exclusion Chromatography (SEC) and size exclusion chromatography-mass spectrometry (SEC-MS) and the like.

In this embodiment, when the reducing agent reduces IgG, SDS-PAGE may be used as a rapid and stable detection and analysis technique for the product after antibody reduction, or an optimal reducing agent may be selected from the reducing agents such as dithiothreitol (DTT for short), TCEP, cysteine, acetylcysteine, mercaptoethanol, sodium borohydride, etc., and the first reaction condition is subjected to a response surface test design, so that the first reaction condition is optimized, thereby obtaining a high-efficiency reducing agent. These reducing agents can be used to attach drugs with antrodia camphorata and to perform antibody drug conjugate preparation experiments. The method specifically comprises the following steps:

(1) Experiments at different levels were performed for three factors of the first reaction conditions by the response surface method experimental design.

In this example, the most significant factor affecting the yield of antibody sassafras coupling is the ratio of antibody to reducing agent, followed by the first reaction temperature, followed by the first reaction pH, with optimization of the model by Des ign-Expert, preferably 37℃and the first reaction pH of

(2) And selecting the optimal first reaction temperature in the first reaction conditions of the test and the optimal first reaction pH condition, and optimizing the molar ratio of the reducing agent to the antibody.

For example, the optimal first reaction temperature is 37 ℃, and the first reaction pH is 7.0; the molar ratio of reducing agent (DTT, TCEP, ac-Cys, golu, cysteine, mercaptoethylamine, sodium borohydride, TP, 2-aminoethylthiol hydrochloride) to antibody was optimized at a first reaction temperature of 37 ℃, at a first reaction pH of 7.0, in this example DTT, mercaptoethylamine, ac-Cys and TCEP showed a certain selectivity when reducing IgG in a certain ratio, and the corresponding antibody sassafras coupling ratios were 30.4%, 26.5%, 25.8% and 29.5% when DTT/igg=2, mercaptoethylamine/igg=50, TCEP/igg=0.5 and Ac-Cys/igg=3.

(3) IgG was reduced on the solid medium. Since protein a can be selected to bind to the Fc fragment of IgG, protein a was used to adsorb IgG, which was then selectively reduced with TCEP. The results showed that the yield of antibody sassafras coupling was 21.0%. Therefore, the reduction with solid phase medium is not as easy as in solution, and the reduction with solid phase medium cannot be performed during the preparation of the drug conjugate.

Coupling the antibody with the drug, wherein step S300 is carried out, the antrodia camphorata is added into the reduction product for coupling reaction under the second reaction condition, so as to obtain the antibody conjugate, and the second reaction condition comprises: the second reaction temperature is 37 ℃, the second reaction pH is 6.9-8.9, and the molar ratio of the reduction product to the antrodia camphorate is 4.34-6.14.

The antibody is reduced to a suitable extent and a coupling reaction with the drug can be performed. The reaction between maleimide groups and mercapto groups is mild, requiring only a slight excess of maleimide, in

The reaction is carried out for 1h at about 37℃for example if the antibody is reduced to 4 thiol groups per antibody, usually +.>

Double Antrodia camphorata is reacted for 30min.

It should be noted that, when the water solubility of the drug is good, the coupling reaction can be rapidly performed, and when the antrodia camphorata anthraquinone is added into the reduced protein and then mixed uniformly. When the water solubility of the Antrodia camphorata anthraquinone linker is poor, proper organic solvent is needed to dissolve the Antrodia camphorata anthraquinone, so that the antibody and the Antrodia camphorata anthraquinone linker are both in solution, and the whole reaction is carried out in solution. Typically, the antrodia camphorate anthraquinone is dissolved by adopting Dimethylformamide (DMF) or Dimethylacetamide (DMAC) and then reacts with the reduced product in an isovolumetric manner.

And a purification step S400, wherein after the reaction of the antibody and the drug coupling step is terminated, the superfluous antrodia camphorata anthraquinone joint and the organic solvent are removed.

In particular, a specific purification method may be selected according to the size of the reaction, for example, if the reaction size is laboratory scale such as microgram or less, purification methods such as ion exchange chromatography, affinity chromatography, gel filtration, centrifugal ultrafiltration, ion exchange chromatography and affinity chromatography may be used for spring bloom; a combination of ultrafiltration/dialysis may also be used. For gel filtration, which is a simple and reliable chromatographic technique for separation according to the differences in the size and shape of protein molecules, desalting of proteins is often performed using a gel filtration column of G25. Dialysis is to put the mixture to be separated into a dialysis bag by utilizing the property that protein molecules cannot freely pass through a semipermeable membrane made of a high polymer material, and then put the dialysis bag into a buffer solution. Under the action of pressure, substances with molecular radius smaller than the pore diameter of the membrane in the solution pass through the ultrafiltration membrane through the sieving function, and substances with molecular radius larger than the pore diameter of the membrane in the solution are trapped on the ultrafiltration membrane, so that the separation of molecules with different molecular weights is realized. The ultrafiltration process can also realize the concentration effect on protein solution, and because protein molecules are easy to adsorb on the membrane surface of the ultrafiltration membrane, membrane pollution is caused, the ultrafiltration speed is reduced, and protein loss can be caused, so that when ultrafiltration is applied to protein separation or concentration, the ultrafiltration membrane needs to be periodically cleaned or replaced.

The antibody conjugate prepared by the preparation method of the antibody conjugate provided by the invention is obtained by identifying a product by adopting hydrophobic chromatography and reversed phase liquid chromatography analysis, and the optimal reaction condition close to a drug is coupled on average on each antibody by adopting the antibody conjugate prepared by the preparation method of the antibody conjugate. The invention has reasonable composition, is beneficial to the cancer treatment of people after scientific test according to the antibody conjugate, has simple preparation process, is convenient for industrialized production and has wide prospect.

To further verify the effect of the first reaction conditions on the yield of antibody conjugate coupling in the antibody reduction step S100, the effect of the first reaction conditions of examples 1 to 20 on the yield of antibody coupling (in this example, the yield of antibody coupling is the yield of antibody sassafras coupling) assuming that the other step conditions are identical is as shown in table 1.

TABLE 1 influence of examples 1 to 20 on the antibody coupling ratio under the first reaction conditions

The three-dimensional quadratic equation of the coupling yield of the antibody sassafras and the variable of each factor of each reaction is to be described: y= -124.122+1.69460 x ₁ +34.00161X ₂ +1.82750X ₃ -0.070838X ₁ X ₂ -0.045720X ₁ X ₃ +

0.31226X ₂ X ₃ -0.013744X ₁ ² -2.33758X ₂ ² -0.26149X ₃ ² 。

As can be seen from Table 1, the molar ratio of the reducing agent to the antibody had the most significant effect on the yield of the coupling of the antibody Cinnamomum kanehirae, and the coupling ratio of the antibody increased and then decreased with increasing molar ratio of the reducing agent to the antibody. Next to the first reaction temperature, the yield of antibody coupling yield increases with increasing first reaction temperature.

In order to further verify the effect of the second reaction conditions in the antibody-drug coupling step S300 on the yield of antibody conjugate coupling, the effect of the second reaction conditions of examples 21 to 30 on the antibody coupling yield (in this example, the antibody coupling yield is the antibody sassafras coupling yield) assuming that the other step conditions are identical is as shown in table 2. The antibody coupling yield was set at the target value and the maximum limit was 100%.

TABLE 2 influence of examples 21 to 30 on the antibody coupling ratio under the second reaction conditions

With increasing temperature, the optimal temperature for the second reaction temperature was chosen to be 37℃because the temperature had some effect on the protein, and it can be seen from Table 2 that with increasing ratio of reduced product (i.e., molar ratio of reduced product to antrodia camphorata), the yield of antibody coupling increased and then decreased.

Since the composition and uniformity of antrodia camphorate are closely related to the safety of the drug and the therapeutic effect of the drug, analysis of the composition and non-uniformity of the antibody conjugate is increasingly important. The hydrophobic chromatography column TSKge l Buty l-NPR (4.6 mm X3.5 cm) is important for high-speed, high-resolution separation performance of proteins. The filler is a non-porous hydrophilic resin with a particle diameter of 2.5 mu m and a butyl group bonded to the surface.

Hydrophobic interaction chromatography is based on the principle of high-salt adsorption and low-salt elution. The influence of salt concentration on adsorption increases the hydrophobic area of the mobile phase exposed to the protein surface, thereby enhancing the binding capacity of the protein to the hydrophobic medium. Therefore, under the condition that the loading concentration and the column temperature are kept unchanged, the binding force between the protein and the medium can be stronger by increasing the salt concentration in the mobile phase. However, when the salt concentration in the mobile phase is too high, components having weak binding ability with the medium may be eluted, thereby causing leakage of the sample. The salt concentration can directly influence the binding capacity of components in a sample and a medium, and the salt concentration is too high, so that the hydrophobicity is strong, and the components are difficult to elute; while salt concentrations that are too low are poorly hydrophobic, the components do not readily bind to the medium. Thus, a suitable salt concentration is selected.

The flow rate has great influence on the analysis result, the flow rate is low, the consumed time is long, the peak shape is bad, the flow rate is increased, the separation degree is reduced, the peak area is reduced, the peak shape is better, but the column pressure is increased, so that the selection of proper flow rates is important for the detection result, and the factors of 0.2, 0.4, 0.6, 0.8 and 1mL/min flow rate, comprehensive retention time, separation degree, peak shape and the like are tested respectively, and the optimal flow rate is selected.

Reversed phase chromatography analysis was performed after excess DTT treatment of the antibody conjugate, where the heavy and light chains of the antibody were separated and not covalently bound together. The higher the drug loading, the longer the retention time, so the heavy and light chains are eluted separately. The eluted products in turn, based on the average drug loading per antibody and the antibody composition of two heavy chains and two light chains, the heavy chain and light chain carrying different amounts of each can be calculated separately.

To facilitate detection of the antibody conjugates prepared in accordance with the present invention, the following experiments may be employed:

(1) IgG 75. Mu.L with a concentration of 26.6mg/mL was dispensed into centrifuge tubes, TCEP and DTT were prepared as 10mM solutions with PB7.0 buffer, then diluted to 1mM, and then linalool was dissolved in N, N-dimethylformamide to prepare a solution with a concentration of 2 mM.

(2) Reduction reaction: the reducing agent was added to the IgG solution in the proportions (TCEP: igG) and volumes (V (TCEP)/μl) shown in Table 3, buffer was added to a total volume of 150 μl, and the mixture was blown and mixed with a pipette, and the reaction system was placed in a constant temperature shaking table at 37deg.C and reacted for 2 hours at 180 rpm.

(3) After the reaction, SDS-PAGE was used to examine whether the reaction was reduced. 5. Mu.L of the sample was taken out of the reaction system and diluted to 20. Mu.L, and SDS-PAGE was performed with the non-reducing electrophoresis loading buffer solution to examine the degree of the reduction reaction.

(4) The unreacted reducing agent was removed by centrifugation at least 5 times with a 3K ultrafiltration tube ultrafiltration membrane at 4000 g. At the same time, the whole reaction is replaced by Tr is-HCl 8.0 buffer system.

(5) Different concentrations of linalool (mM) were added, the ratio of linalool to antibody (IgG) (e.g., ratios of 4, 5, 6, respectively) and the volume (V (linalool)/μl) were as shown in Table 4, tr i s-HCl pH8.0 was added to the reaction system of 200 μl. After being uniformly mixed by a mixer, the reaction system is placed into a constant temperature shaking table at 37 ℃ for reaction for 1h at 180 rotational speeds.

(6) Cysteine was added to the final reaction system to a concentration of 1mM, and the unreacted drug was quenched. The protein concentration of the reacted sample was measured by BCA method.

TABLE 3 parameters of antibody reduction reactions

TABLE 4 Antrodia camphorata anthraquinone coupling reaction parameters

Selection of the concentration of ammonium sulfate by hydrophobic interaction chromatography

The salt concentration directly influences the hydrophobic adsorption capacity, and the salt concentration is too high and has strong hydrophobicity and is not suitable for elution; and the salt concentration is too low, the hydrophobicity is weak, and the adsorption is not suitable. Thus, a suitable salt concentration is selected. When the samples respectively contain ammonium sulfate with different salt concentrations of 0.5, 0.8, 1.0, 1.3 and 1.50M, the HIC analysis of the hydrophobic chromatography is carried out, when the salt concentrations are 0.5, 0.8 and 1.0M, the samples cannot be adsorbed on a medium, and basically all the samples flow through, and when the salt concentration is 1.5M, different antibody drug conjugates appear in elution peaks, so that the salt concentration is 1.5M when the samples are selected for loading.

Selection of hydrophobic interaction chromatography flow rates

In the separation process by adopting the hydrophobic chromatography HIC, the flow velocity can influence the separation degree between different Antrodia camphorata anthraquinone conjugates, and the flow velocity is generally equal to

Is varied between, at a flow rate->

When the flow rate exceeds 0.8mL/min, the system pressure reaches 10MPa, and the pressure is close to the tolerance pressure of an AKTA chromatographic system. At a flow rate of 0.6mL/min, the system pressure was 8MPa, and the pressure tolerance of the chromatographic system and analytical column was within the range, however, the peak separation of the different Antrodia camphorata anthraquinone conjugates was poor and could not be measuredThe individual products are well separated. When the flow rate is 0.2mL/min, the analysis time is prolonged, and the optimal flow rate is selected to be 0.4mL/min by comprehensively considering various factors.

Through different flow rates, the sample contains different salt concentrations to optimize the hydrophobic chromatography method, and the analysis conditions for analyzing the antibody drug conjugate by adopting the hydrophobic chromatography are determined: the mobile phase was Buffer A1.5M (NH ₄ ) ₂ SO ₄ ，50mM KHPO ₄ ，pH 7.0，Buffer B:50mM KHPO ₄ 20% isopropyl alcohol was added at pH 7.0 at a flow rate of 0.4mL/min, and the salt concentration in the sample was 1.5M ammonium sulfate and the protein concentration was 1mg/mL. Loading 50 mu L and detecting at 280 nm.

Detection by reverse phase chromatography column PLRP-S, analytical method: buffer A: H ₂ O+0.1％ TFA Buffer B:CH ₃ Cn+0.1% tfa gradient: 30-45% B30 min,95% B3 min,30% B5 min sample treatment 100. Mu.L 2mg/mL IgG was reacted with 100. Mu.L 50mM DTT in PBS solution at 37℃for 20min and then loaded with 20. Mu.L, 280nm for detection.

Antibody reduction was performed by varying the ratio of reducing agents, followed by coupling with safrole, hydrophobic interaction chromatography and reversed phase high performance chromatography, for example when IgG: DTT/TCEP: drug=1:3:6, and the average Drug coupled to each antibody was 3.56 as calculated by analysis.

Screening experiments were performed on different reducing agents under different reaction parameters, and SDS-PAGE was used to detect the different degrees of reduction of the antibodies, thereby determining the selective reducing agent. The optimal reducing agent is selected to be reduced under proper reducing reaction conditions, and the optimal reducing agent and the medicine with the antrodia camphorata are subjected to coupling reaction to synthesize the antibody-medicine conjugate with specificity and carrier effect. The products were identified by hydrophobic chromatography and reversed phase liquid chromatography analysis, and the optimal reaction conditions for the formation of the average of the coupling of antrodia camphorate on each antibody were determined.

The products after the reduction of the antibody are detected by two methods of size exclusion chromatography and polyacrylamide gel electrophoresis (SDS-PAGE), and the coupling of the IgG and the antibody sassafras can be better separated by adopting a non-reduction SDS-PAGE method, so that the method can be used as a detection method for optimizing the reduction reaction condition of the antibody. Meanwhile, the sequence of disulfide bond reduction in the antibody reduction process is obtained through detection of two reducing agents of DTT and TCEP under different reaction times.

And (3) carrying out response surface test design on the reaction temperature, the reaction pH and the reduction ratio of the reducing agent to the antibody, and optimizing the reaction conditions to obtain the efficient reducing agent which is used for connecting the medicine with the antrodia camphorata and carrying out an antibody medicine conjugate preparation experiment. The optimal temperature is determined to be 37 ℃ through the experimental design of a response surface method, and the reaction pH is 7-8. The reaction molar ratio of DTT, TCEP, ac-Cys, G l u, cysteine, mercaptoethylamine, sodium borohydride, TP, 2-aminoethylthiol hydrochloride was optimized, DTT, mercaptoethylamine, ac-Cys and TCEP showed a certain selectivity when IgG was reduced in a certain proportion, and when DTT/igg=2, mercaptoethylamine/igg=50, TCEP/igg=0.5 and Ac-Cys/igg=3, the corresponding antibody sassafras coupling ratios were 30.4%, 26.5%, 25.8% and 29.5%.

The analysis conditions of the hydrophobic chromatography analysis antibody drug conjugate are determined by optimizing the hydrophobic chromatography method through different flow rates and different salt concentrations: the mobile phase was Buffer A1.5M (NH ₄ ) ₂ SO ₄ ，50mM KHPO ₄ ，pH 7.0，Buffer B:50mM KHPO ₄ 20% isopropyl alcohol was added at pH 7.0 at a flow rate of 0.4mL/min, and the salt concentration in the sample was 1.5M sulfuric acid per protein concentration of 1mg/mL. Loading 50 mu L and detecting at 280 nm.

The analysis method is determined by PLRP-S detection of a reversed phase chromatographic column: buffer A, H2O+0.1%TFA Buffer B:CH ₃ Cn+0.1% tfa gradient: 30-45%B 30min,95%B 3min,30%B 5min. Sample treatment: after 100. Mu.L of 2mg/mL IgG was reacted with 100. Mu.L of 50mM DTT in PBS for 20min at 37℃20. Mu.L was loaded for detection at 280 nm.

Antibody reduction was performed by different reducing agent ratios, followed by Drug coupling, hydrophobic interaction chromatography and reversed-phase high performance chromatography, and when IgG: DTT/TCEP: drug=1:3:6, the average Drug coupled to each antibody was 3.56 as calculated by the analysis.

By X-ray diffraction analysis, the antibody molecule consists essentially of three parts, two identical antibody fragments-an antigen binding arm (generally written as Fab (fragment ant igen bind ing arms)) and one fragment that is readily crystallized (Fc (fragment that crysta l l izes)). The two fabs are joined by a hinge region. The Fab can rotate about the hinge region up to a maximum angle of 123 deg. where the angle between two fabs can be varied from 115 deg. to 172 deg. between Fab and Fc from 66 deg. to 123 deg.. This allows the antibody to bind to the antigen to the maximum.

It will be apparent that the embodiments described above are merely some, but not all, embodiments of the invention. Based on the embodiments of the present invention, those skilled in the art may make other different changes or modifications without making any creative effort, which shall fall within the protection scope of the present invention.

Claims (10)

1. A method of preparing an antibody conjugate comprising:

an antibody reduction step, wherein the antibody and a reducing agent are subjected to a reduction reaction under a first reaction condition, so that disulfide bonds of the antibody are reduced to generate sulfhydryl groups, and a reduction product is obtained, wherein the first reaction condition comprises: the first reaction temperature is 4-37 ℃, the first reaction pH is 4-11, and the molar ratio of the reducing agent to the antibody is 2-11;

and a step of coupling the antibody and the drug, wherein the antrodia camphorata is added into the reduction product to carry out coupling reaction under a second reaction condition to obtain an antibody conjugate, and the second reaction condition comprises: the second reaction temperature is 37 ℃, the second reaction pH is 6.9-8.9, and the molar ratio of the reduction product to the antrodia camphorate is 4.34-6.1.

2. The method of preparing an antibody conjugate of claim 1, wherein the step of coupling the antibody to the drug comprises:

dissolving antrodia camphorata in an organic solvent, and then adding the antrodia camphorata into the reduction product to perform coupling reaction under a second reaction condition to obtain the antibody conjugate.

3. The method of preparing an antibody conjugate of claim 2, wherein the second reaction conditions comprise: the second reaction temperature is 37 ℃, and the second reaction pH is 7-8.

4. The method of preparing an antibody conjugate according to claim 1 or 2, wherein the first reaction temperature is 37 ℃ and the first reaction pH is 7-8.

5. The method of preparing an antibody conjugate of claim 1 or 2, wherein the method further comprises, prior to the step of coupling the antibody to the drug, after the step of reducing the antibody:

the degree of reduction of the antibody is detected and the first reaction conditions are adjusted according to the degree of reduction.

6. The method of claim 5, wherein the step of detecting the degree of reduction of the antibody and adjusting the first reaction condition based on the degree of reduction comprises:

performing experiments at different levels on three factors of the first reaction condition through a response surface method experiment design;

and selecting the optimal first reaction temperature in the first reaction conditions of the test and the optimal first reaction pH condition, and optimizing the molar ratio of the reducing agent to the antibody.

7. The method of making an antibody conjugate of claim 6, wherein the reducing agent comprises one or more of mercaptoethanol, mercaptoethylamine, acetylcysteine, and tris (2-formylethyl) phosphine hydrochloride.

8. The method of preparing an antibody conjugate of claim 2, wherein after the step of coupling the antibody to the drug, the method further comprises:

and a purification step, namely removing superfluous antrodia camphorata anthraquinone joints and organic solvents after the reaction of the antibody and the drug coupling step is terminated.

9. The method of preparing an antibody conjugate of claim 8, wherein the purifying step comprises:

after the reaction of the antibody and the drug coupling step is terminated, determining a purification method according to the scale of the reaction;

when the reaction scale is a laboratory scale, purification is performed by one or more of ion exchange chromatography, affinity chromatography, gel filtration method, centrifugal ultrafiltration method, and ion exchange chromatography and affinity chromatography.

10. An antibody conjugate prepared by the method of any one of claims 1 to 9.

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