Method for preparing antibody-drug conjugate having high homogeneity

Abstract

The present invention relates to a method for preparing an antibody-drug conjugate (ADC). Specifically, the present invention relates to a method for preparing an antibody-drug conjugate (ADC) having high homogeneity, especially a method for preparing an antibody-drug conjugate (ADC) having a high D4 percentage.

Description

Methods of making antibody-drug conjugates with high homogeneity

[0001] Related Applications

[0002] This application claims the benefit of PCT Patent Application No. PCT / CN2024 / 107864, filed July 26, 2024, and Chinese Patent Application No. 202511014693.4, filed July 22, 2025, which are incorporated by reference in their entirety. TECHNICAL FIELD

[0003] The present invention relates to methods of making antibody-drug conjugates (ADCs). In particular, the present invention relates to methods for making antibody-drug conjugates (ADCs) with high homogeneity, especially methods of making antibody-drug conjugates (ADCs) with high percentage of D4. BACKGROUND

[0004] Antibody-drug conjugates (ADCs) contain an antibody for targeting, a linker for drug attachment, and a payload (e.g., a drug) as an effector. Current common ADC drugs include Adcetris, Kadcyla (trastuzumab-mertansine conjugate), Besponsa (inotuzumab ozogamicin), Mylotarg (gemtuzumab ozogamicin), Polivy (polatuzumab vedotin-piiq), Blenrep (mabellumab), Enhertu (deruxteumab), Padcev (enfortumab vedotin), Trodelvy (sacituzumab govitecan), Tivdak (tisotumab vedotin), Zynlonta (loncastuximab tesirine), Akalux, Lumoxiti (pixantrone), or Libtayo, etc. Meanwhile, there are more drugs in the process of research in clinical stage.

[0005] Among the existing antibody-drug conjugate preparation technologies, the most widely used is cysteine-based conjugation, which includes the following two steps:

[0006] In the first step, a reducing agent is used to reduce the interchain disulfide bond of cysteine on the antibody to two cysteines. In the second step, an organic solvent and a linker-drug are added to the reaction system, so that the thiol group of cysteine reacts with the reactive group in the linker to generate a mixture solution of antibody-drug conjugates, and the final product is obtained after purification.

[0007] The methods for synthesizing antibody-drug conjugates (ADCs) with certain homogeneity in the prior art, such as the method disclosed in WO2020164561A1, need to be carried out at low reaction temperature (4°C), and the reduction reaction step therein consumes a long time, thus being not conducive to application in large-scale production, and poor in economy.

[0008] The number of drugs conjugated to a single antibody molecule is an important factor in obtaining efficacy and safety of ADCs. For example, in conjugation methods based on reduction of native interchain disulfide bonds, the interchain S-S bonds are more accessible to the solvent than other disulfide bonds. Thus, the interchain disulfide bonds can be used as binding sites for conjugation of drugs (or drug-linkers) to the antibody. Typically, one therapeutic antibody molecule belonging to the IgGl or IgG4 subclass has 4 interchain S-S bonds, each formed by two -SH groups, thus the number of drugs conjugated to a single antibody molecule is 2, 4, 6 or 8. If the number of drugs conjugated to a single antibody molecule is 0, the product is called D0. Thus, D2 refers to an ADC in which two drug molecules are conjugated to one single antibody molecule, wherein the two drug molecules can be conjugated to -SH groups generated by reduction of S-S bonds between heavy and light chains, or can be conjugated to -SH groups generated by reduction of S-S bonds between heavy and heavy chains. D4 refers to an ADC in which four drug molecules are conjugated to one single antibody molecule. D6 refers to an ADC in which six drug molecules are conjugated to one single antibody molecule. And D8 refers to an ADC in which eight drug molecules are conjugated to one single antibody molecule, i.e. all four 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 generally unstable and have low immunogenicity. Among them, D0 has no ADC efficacy, and D6+D8 are considered to be the cause of instability and low immunogenicity due to their hydrophobicity induced by payload (i.e. drug) molecules. Although antibody-drug conjugate efficacy in vitro has been shown to depend directly on drug loading (Hamblett KJ et al., Clin Cancer Res. 2004 Oct. 15; 10(20): 7063-70), the in vivo antitumor activity of antibody-drug conjugates with four drugs per molecule (D4) is comparable to that of conjugates with eight drugs per molecule (D8) at the same mAb dose, even though the D8 conjugate contains only half the amount of drugs per mAb as the D4 conjugate. Drug-loading also affects plasma clearance, with the clearance of D8 conjugates being 3 times faster than that of D4 conjugates and 5 times faster than that of D2 conjugates. Typically, the level of D4 represents the homogeneity of antibody-drug conjugates. That is, if the content of D4 in the mixture is high, the ADC is considered to have high homogeneity. Antibody-drug conjugates with improved homogeneity provide therapeutic benefits, such as higher therapeutic index, improved efficacy and reduced drug toxicity. Homogeneous antibody conjugates also provide more accurate and consistent measurements in diagnostic and imaging applications. Thus, new methods of preparing ADCs with high homogeneity are highly desirable and long pursued.

[0009] Therefore, there is a need to find a new method for preparing antibody-drug conjugates with simplified steps, mild conditions, shortened time consumption, low cost and / or further improved homogeneity of ADC products. SUMMARY

[0010] In view of the drawbacks in the prior art, the present application introduces a combination of different metal ions (e.g., Zn 2+ and Ca 2+ ) in the process of preparing ADCs. Compared with the method for preparing antibody-drug conjugates (ADCs) with improved homogeneity disclosed in WO2020164561A1, the method according to the present application is more simplified, the reaction temperature is increased to be closer to room temperature and the reduction reaction time is shortened, it is easier to realize scale-up and production, and the homogeneity of ADC products is improved. In addition, the method of the present application has a longer reaction plateau, is easier to operate for large-scale production, and leaves more operation time for post-treatment.

[0011] According to the present application, the method for preparing antibody-drug conjugates (ADCs) with improved homogeneity comprises the following steps:

[0012] (a) incubating a reducing agent and an antibody to be conjugated in the presence of an effective amount of a first metal ion and a second metal ion to perform a reduction reaction, to obtain a reaction mixture.

[0013] In some embodiments, the method according to the present application further comprises the following step: (b) reacting a linker-payload carrying a reactive group with the reaction mixture obtained in step (a).

[0014] In some embodiments, the method according to the present application further comprises the following step: (c) oxidizing the reaction mixture obtained in step (b) with an oxidizing agent.

[0015] In some embodiments, the method according to the present application further comprises the following step: (d) recovering the antibody-drug conjugate obtained in step (c).

[0016] In some embodiments, the reducing agent of step (a) is selected from an organic or inorganic reducing agent, such as a sulfur-containing reducing agent, a phosphine-containing reducing agent, a sugar-containing reducing agent, an alkali metal-containing reducing agent, and the like. In some embodiments, the reducing agent in step (a) can be selected from one or more of tris(2-carboxyethyl)phosphine (TCEP) and its hydrochloride salt, diphenylphosphinoacetic acid, 2-[2-(diphenylphosphino)ethyl]pyridine, 3-(diphenylphosphino)benzenesulfonic acid, 4-(diphenylphosphino)benzoic acid, 2-(diphenylphosphino)ethylamine, 3-(diphenylphosphino)propylamine, 3-(diphenylphosphino)propanoic acid, 2-(diisopropylphosphino)ethylamine, 2-(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphine, 1,3,5-triaza-7-phosphanorbornane, n-butyldi(1-adamantyl)phosphine, tris(3-hydroxypropyl)phosphine (THPP), 2-(diphenylphosphino)benzenesulfonic acid (diPPBs), dithiothreitol (DTT), 2-(diphenylphosphino)acetic acid (DPAA), dithioerythritol (DTE), β-mercaptoethanol, LiAlH4, Na2S2O3, KBH4, or hydrazine.

[0017] In some embodiments, the pH of the buffer system in step (a) is about 5.5 to 9. In some embodiments, the pH of the buffer system used in step (a) is about 5.5 to 8; preferably, the pH of the buffer system used in step (a) is about 7.

[0018] In some embodiments, step (a) is performed in a buffer system. In various embodiments, the buffer system of step (a) can include HEPES (containing N-(2-hydroxyethyl)piperazineethanesulfonic acid), PBS (containing phosphate), MES (containing morpholinoethanesulfonic acid), Tris (containing tris(hydroxymethyl)aminomethane or its hydrochloride salt), TAPS (containing tris(hydroxymethyl)aminomethane), Bicine (containing N,N-dihydroxyethylglycine), Tricine (containing tris(hydroxymethyl)methylglycine), TES (containing N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), MOPS (containing 3-morpholino propanesulfonic acid), PIPES (containing 1,4-piperazine diethanesulfonic acid), citric acid / sodium citrate, or histidine buffer, and the like.

[0019] In some embodiments, the first metal ion and the second metal ion in step (a) are independently selected from the following ions: Zn 2+ , Cd 2+ , Hg 2+ , Mg 2+ , Sr 2+ , Ba 2+ , Ti 2+ , Ti 3+ , Zr 2+, Cr 2+ , Cr 3+ , Mo 2+ , Mn 2+ , Mn 3+ , Fe 2+ , Fe 3+ , Co 2+ , Co 3+ , Ni 2+ , Pd 2+ , Pt 2+ , Cu + , Cu 2+ , Ag + , and / or Ca 2+ . In some embodiments, the first metal ion and the second metal ion in step (a) are independently selected from the group consisting of: Zn 2+ , Mn 2+ , Ni 2+ , Fe 2+ , Fe 3+ , Cu 2+ , and / or Ca 2+ . Preferably, the first metal ion is Zn 2+ . Preferably, the second metal ion is Ca 2+ or Mn 2+ . More preferably, the first metal ion and the second metal ion are selected from the combination of Zn 2+ and Ca 2+ . More preferably, the first metal ion and the second metal ion are selected from the combination of Zn 2+ and Mn 2+ . More preferably, the first metal ion and the second metal ion are selected from the combination of Zn 2+ and Ni 2+ . More preferably, the first metal ion and the second metal ion are selected from the combination of Zn 2+ and Fe 2+ . More preferably, the first metal ion and the second metal ion are selected from the combination of Zn 2+ and Fe 3+ . More preferably, the first metal ion and the second metal ion are selected from the combination of Zn 2+ and Cd 2+ . More preferably, the first metal ion and the second metal ion are selected from the combination of Zn 2+ and Cu 2+ . More preferably, the first metal ion and the second metal ion are selected from the combination of Mn 2+ and Ni 2+ . More preferably, the first metal ion and the second metal ion are selected from the combination of Mn 2+ and Fe 2+ . More preferably, the first metal ion and the second metal ion are selected from the combination of Mn2+ and Fe 3+ . More preferably, the first metal ion and the second metal ion are selected from the group consisting of Mn 2+ and Cu 2+ . More preferably, the first metal ion and the second metal ion are selected from the group consisting of Mn 2+ and Ca 2+ . More preferably, the first metal ion and the second metal ion are selected from the group consisting of Ni 2+ and Fe 2+ . More preferably, the first metal ion and the second metal ion are selected from the group consisting of Ni 2+ and Fe 3+ . More preferably, the first metal ion and the second metal ion are selected from the group consisting of Ni 2+ and Cu 2+ . More preferably, the first metal ion and the second metal ion are selected from the group consisting of Ni 2+ and Ca 2+ . More preferably, the first metal ion and the second metal ion are selected from the group consisting of Fe 2+ and Fe 3+ . More preferably, the first metal ion and the second metal ion are selected from the group consisting of Fe 2+ and Cu 2+ . More preferably, the first metal ion and the second metal ion are selected from the group consisting of Fe 2+ and Ca 2+ . More preferably, the first metal ion and the second metal ion are selected from the group consisting of Fe 3+ and Cu 2+ . More preferably, the first metal ion and the second metal ion are selected from the group consisting of Fe 3+ and Ca 2+ . More preferably, the first metal ion and the second metal ion are selected from the group consisting of Cu 2+ and Ca 2+ . Preferably, the first metal ion is different from the second metal ion.

[0020] A first metal ion suitable for use in step (a) of the method of the present application can include, but is not limited to, Zn 2+ , Cd 2+ , Hg 2+ , Mg 2+ , Sr 2+ , Ba 2+ , Ti 2+ , Ti 3+ , Zr 2+ , Cr 2+ , Cr 3+ , Mo 2+ , Mn 2+ , Mn 3+ , Fe2+ Fe 3+ Co 2+ Co 3+ Ni 2+ Pd 2+ Pt 2+ Cu + Cu 2+ Ag + and / or Ca 2+ Among these, Zn is used due to its easy availability and low cost. 2+ For example, in step (a), a suitable salt containing a metal ion and / or a complex containing a metal ion, such as a metal cation-amino chelate / complex / complex / coordination, may be added. Examples of metal cation-amino chelates / complexes / complexes / coordinations include, for example, those disclosed in PCT Patent Publication WO2022 / 078524A2, particularly those disclosed on page 21, line 22 to page 24, line 2. Those skilled in the art will understand that these examples, although specific to Zn, are not exhaustive. 2+ List, but include Zn 2+ Replace with the above Cd 2+ Hg 2+ Mg 2+ 、Sr 2+ Ba 2+ Ti 2+ Ti 3+ Zr 2+ Cr 2+ Cr 3+ Mo 2+ Mn 2+ Mn 3+ Fe 2+ Fe 3+ Co 2+ Co 3+ Ni 2+ Pd 2+ Pt 2+ Cu + Cu 2+ Ag + and / or Ca 2+Examples of corresponding amino chelates / complexes / complexes / complexes containing these ions will be obtained; these examples of corresponding amino chelates / complexes / complexes / complexes can likewise be used in the present application. In some embodiments, the salts containing metal ions can be used as long as they are soluble in the reaction solution so that free transition metal ions can be released into the reaction solution. In this regard, as suitable zinc salts can include, but are not limited to, ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, zinc formate, and zinc tetrafluoroborate. In some embodiments, ZnCl2, ZnI2, ZnBr2, Zn(NO3)2, or ZnSO4is used. Likewise, mention can be made of salts that are soluble in the reaction solution and can release free Cd 2+ or Hg 2+ ions, which can include, but are not limited to: CdCl2, Cd(NO3)2, CdSO4, Cd(CH3COO)2, CdI2, CdBr2, cadmium formate, and cadmium tetrafluoroborate; HgCl2, Hg(NO3)2, HgSO4, Hg(CH3COO)2, HgBr2, mercury(II) formate, and mercury(II) tetrafluoroborate, etc.

[0021] The second metal ions suitable for use in step (a) of the process of the present application can include, but are not limited to, soluble salts and / or complexes or complexes of Ca 2+ , Mn 2+ , Mg 2+ , Sr 2+ , Ba 2+ , Ti 2+ , Ti 3+ , Zr 2+ , Cr 2+ , Cr 3+ , Mo 2+ , Mn 3+ , Fe 2+ , Fe 3+ , Co 2+ , Co 3+ , Ni 2+ , Pd 2+ , Pt 2+ , Cu + , Cu 2+ , Ag + . Here, as anions of soluble salts and / or complexes or complexes, anions including the following can be used: Cl - , Br - , I - , SO4 2- , HSO4 - , SO3 2- , HSO3- CH3SO3 - C6H5SO3 - NO2 - PO4 3- HPO4 2- H2PO4 - CO3 2- HCO3 - HCOO - CH3COO - CF3COO - CCl3COO - CHF2COO - CHCl2COO - CH2FCOO - CH2ClCOO - BF4 - C6H5COO - C6H5O - C6H 11 O7 - C3H5O3 - and the like.

[0022] In this regard, salts that can include, but are not limited to, CaCl2, CaBr2, CaI2, Ca(HSO4)2, Ca(HSO3)2, Ca(CH3SO3)2, Ca(C6H5SO3)2, Ca(NO3)2, Ca3(PO4)2, CaHPO4, Ca(H2PO4)2, Ca(HCO3)2, Ca(HCOO)2, Ca(CH3COO)2, Ca(CF3COO)2, Ca(CCl3COO)2, Ca(CHF2COO)2, Ca(CHCl2COO)2, Ca(CH2FCOO)2, Ca(CH2ClCOO)2, Ca(BF4)2, Ca(C6H5COO)2, Ca(C6H5O)2, Ca(C6H 11O7)2, Ca(C3H5O3)2; MnCl2, MnBr2, MnI2, Mn(HSO4)2, MnSO3, Mn(HSO3)2, Mn(CH3SO3)2, Mn(C6H5SO3)2, Mn(NO3)2, Mn3(PO4)2, MnHPO4, Mn(H2PO4)2, Mn(HCO3)2, Mn(HCOO)2, Mn(CH3COO)2, Mn(CF3COO)2, Mn(CCl3COO)2, Mn(CHF2COO)2, Mn(CHCl2COO)2, Mn(CH2FCOO)2, Mn(CH2ClCOO)2, Mn(BF4)2, Mn(C6H5COO)2, Mn(C6H5O)2, Mn(C6H 11 O7)3, Mn(C3H5O3)3; MgCl2, MgBr2, MgI2, Mg(HSO4)2, MgSO3, Mg(HSO3)2, Mg(CH3SO3)2, Mg(C6H5SO3)2, Mg(NO3)2, Mg3(PO4)2, MgHPO4, Mg(H2PO4)2, Mg(HCO3)2, Mg(HCOO)2, Mg(CH3COO)2, Mg(CF3COO)2, Mg(CCl3COO)2, Mg(CHF2COO)2, Mg(CHCl2COO)2, Mg(CH2FCOO)2, Mg(CH2ClCOO)2, Mg(BF4)2, Mg(C6H5COO)2, Mg(C6H5O)2, Mg(C6H 11 O7)3, Mn(C3H5O3)3; MgCl2, MgBr2, MgI2, Mg(HSO4)2, MgSO3, Mg(HSO3)2, Mg(CH3SO3)2, Mg(C6H5SO3)2, Mg(NO3)2, Mg3(PO4)2, MgHPO4, Mg(H2PO4)2, Mg(HCO3)2, Mg(HCOO)2, Mg(CH3COO)2, Mg(CF3COO)2, Mg(CCl3COO)2, Mg(CHF2COO)2, Mg(CHCl2COO)2, Mg(CH2FCOO)2, Mg(CH2ClCOO)2, Mg(BF4)2, Mg(C6H5COO)2, Mg(C6H5O)2, Mg(C6H 11O7)2, Mg(C3H5O3)2; SrCl2, SrBr2, SrI2, Sr(HSO4)2, SrSO3, Sr(HSO3)2, Sr(CH3SO3)2, Sr(C6H5SO3)2, Sr(NO3)2, Sr3(PO4)2, SrHPO4, Sr(H2PO4)2, Sr(HCO3)2, Sr(HCOO)2, Sr(CH3COO)2, Sr(CF3COO)2, Sr(CCl3COO)2, Sr(CHF2COO)2, Sr(CHCl2COO)2, Sr(CH2FCOO)2, Sr(CH2ClCOO)2, Sr(BF4)2, Sr(C6H5COO)2, Sr(C6H5O)2, Sr(C6H 11 O7)2, Ba(C3H5O3)2; TiCl2, TiBr2, TiI2, Ti(HSO4)2, TiSO3, Ti(HSO3)2, Ti(CH3SO3)2, Ti(C6H5SO3)2, Ti(NO3)2, Ti3(PO4)2, TiHPO4, Ti(H2PO4)2, Ti(HCO3)2, Ti(HCOO)2, Ti(CH3COO)2, Ti(CF3COO)2, Ti(CCl3COO)2, Ti(CHF2COO)2, Ti(CHCl2COO)2, Ti(CH2FCOO)2, Ti(CH2ClCOO)2, Ti(BF4)2, Ti(C6H5COO)2, Ti(C6H5O)2, Ti(C6H 11 O7)2, Ba(C3H5O3)2; TiCl2, TiBr2, TiI2, Ti(HSO4)2, TiSO3, Ti(HSO3)2, Ti(CH3SO3)2, Ti(C6H5SO3)2, Ti(NO3)2, Ti3(PO4)2, TiHPO4, Ti(H2PO4)2, Ti(HCO3)2, Ti(HCOO)2, Ti(CH3COO)2, Ti(CF3COO)2, Ti(CCl3COO)2, Ti(CHF2COO)2, Ti(CHCl2COO)2, Ti(CH2FCOO)2, Ti(CH2ClCOO)2, Ti(BF4)2, Ti(C6H5COO)2, Ti(C6H5O)2, Ti(C6H 11O7)2, Ti(C3H5O3)2; TiCl3, TiBr3, TII3, Ti(HSO4)3, Ti2(SO3)3, Ti(HSO3)3, Ti(CH3SO3)3, Ti(C6H5SO3)3, Ti(NO3)3, TiPO4, Ti2(HPO4)3, Ti(H2PO4)3, Ti(HCO3)3, Ti(HCOO)3, Ti(CH3COO)3, Ti(CF3COO)3, Ti(CCl3COO)3, Ti(CHF2COO)3, Ti(CHCl2COO)3, Ti(CH2FCOO)3, Ti(CH2ClCOO)3, Ti(BF4)3, Ti(C6H5COO)3, Ti(C6H 11 O7)3, Ti(C3H5O3)3; ZrCl2, ZrBr2, ZrI2, Zr(HSO4)2, ZrSO3, Zr(HSO3)2, Zr(CH3SO3)2, Zr(C6H5SO3)2, Zr(NO3)2, Zr3(PO4)2, ZrHPO4, Zr(H2PO4)2, Zr(HCO3)2, Zr(HCOO)2, Zr(CH3COO)2, Zr(CF3COO)2, Zr(CCl3COO)2, Zr(CHF2COO)2, Zr(CHCl2COO)2, Zr(CH2FCOO)2, Zr(CH2ClCOO)2, Zr(BF4)2, Zr(C6H5COO)2, Zr(C6H 11 O7)2, Zr(C3H5O3)2; CrCl2, CrBr2, CrI2, CrSO4, Cr(HSO4)2, CrSO3, Cr(HSO3)2, Cr(CH3SO3)2, Cr(C6H5SO3)2, Cr(NO3)2, Cr3(PO4)2, CrHPO4, Cr(H2PO4)2, Cr(HCO3)2, Cr(HCOO)2, Cr(CH3COO)2, Cr(CF3COO)2, Cr(CCl3COO)2, Cr(CHF2COO)2, Cr(CHCl2COO)2, Cr(CH2FCOO)2, Cr(CH2ClCOO)2, Cr(BF4)2, Cr(C6H5COO)2, Cr(C6H5O)2, Cr(C6H 11O7)2, Cr(C3H5O3)2; CrCl3, CrBr3, CrI3, Cr2(SO4)3, Cr(HSO4)3, Cr2(SO3)3, Cr(HSO3)3, Cr(CH3SO3)3, Cr(C6H5SO3)3, Cr(NO3)3, CrPO4, Cr2(HPO4)3, Cr(H2PO4)3, Cr(HCO3)3, Cr(HCOO)3, Cr(CH3COO)3, Cr(CF3COO)3, Cr(CCl3COO)3, Cr(CHF2COO)3, Cr(CHCl2COO)3, Cr(CH2FCOO)3, Cr(CH2ClCOO)3, Cr(BF4)3, Cr(C6H5COO)3, Cr(C6H 11 O7)3, MoCl2, MoBr2, MoI2, Mo(HSO4)2, MoSO3, Mo(HSO3)2, Mo(CH3SO3)2, Mo(C6H5SO3)2, Mo(NO3)2, Mo3(PO4)2, MoHPO4, Mo(H2PO4)2, Mo(HCO3)2, Mo(HCOO)2, Mo(CH3COO)2, Mo(CF3COO)2, Mo(CCl3COO)2, Mo(CHF2COO)2, Mo(CHCl2COO)2, Mo(CH2FCOO)2, Mo(CH2ClCOO)2, Mo(BF4)2, Mo(C6H5COO)2, Mo(C6H 11 O7)2, Mo(C3H5O3)2; FeCl2, FeBr2, FeI2, FeSO4, Fe(HSO4)2, FeSO3, Fe(HSO3)2, Fe(CH3SO3)2, Fe(C6H5SO3)2, Fe(NO3)2, Fe3(PO4)2, FeHPO4, Fe(H2PO4)2, Fe(HCO3)2, Fe(HCOO)2, Fe(CH3COO)2, Fe(CF3COO)2, Fe(CCl3COO)2, Fe(CHF2COO)2, Fe(CHCl2COO)2, Fe(CH2FCOO)2, Fe(CH2ClCOO)2, Fe(BF4)2, Fe(C6H5COO)2, Fe(C6H5O)2, Fe(C6H 11O7)2, Fe(C3H5O3)2; FeCl3, FeBr3, FeI3, Fe(HSO4)3, Fe2(SO3)3, Fe(HSO3)3, Fe(CH3SO3)3, Fe(C6H5SO3)3, Fe(NO3)3, FePO4, Fe2(HPO4)3, Fe(H2PO4)3, Fe(HCO3)3, Fe(HCOO)3, Fe(CH3COO)3, Fe(CF3COO)3, Fe(CCl3COO)3, Fe(CHF2COO)3, Fe(CHCl2COO)3, Fe(CH2FCOO)3, Fe(CH2ClCOO)3, Fe(BF4)3, Fe(C6H5COO)3, Fe(C6H 11 O7)3, Fe(C3H5O3)3; CoCl2, CoBr2, CoI2, CoSO4, Co(HSO4)2, CoSO3, Co(HSO3)2, Co(CH3SO3)2, Co(C6H5SO3)2, Co(NO3)2, Co3(PO4)2, CoHPO4, Co(H2PO4)2, Co(HCO3)2, Co(HCOO)2, Co(CH3COO)2, Co(CF3COO)2, Co(CCl3COO)2, Co(CHF2COO)2, Co(CHCl2COO)2, Co(CH2FCOO)2, Co(CH2ClCOO)2, Co(BF4)2, Co(C6H5COO)2, Co(C6H 11 O7)2, Co(C3H5O3)2; CoCl3, CoBr3, CoI3, Co2(SO4)3, Co(HSO4)3, Co2(SO3)3, Co(HSO3)3, Co(CH3SO3)3, Co(C6H5SO3)3, Co(NO3)3, CoPO4, Co2(HPO4)3, Co(H2PO4)3, Co(HCO3)3, Co(HCOO)3, Co(CH3COO)3, Co(CF3COO)3, Co(CCl3COO)3, Co(CHF2COO)3, Co(CHCl2COO)3, Co(CH2FCOO)3, Co(CH2ClCOO)3, Co(BF4)3, Co(C6H5COO)3, Co(C6H 11O7)2, Pd(C3H5O3)2; PtCl2, PtBr2, PtI2, Pt(HSO4)2, PtSO3, Pt(HSO3)2, Pt(CH3SO3)2, Pt(C6H5SO3)2, Pt(NO3)2, Pt3(PO4)2, PtHPO4, Pt(H2PO4)2, Pt(HCO3)2, Pt(HCOO)2, Pt(CH3COO)2, Pt(CF3COO)2, Pt(CCl3COO)2, Pt(CHF2COO)2, Pt(CHCl2COO)2, Pt(CH2FCOO)2, Pt(CH2ClCOO)2, Pt(BF4)2, Pt(C6H5COO)2, Pt(C6H5O)2, Pt(C6H 11 O7)2, Pd(C3H5O3)2; PtCl2, PtBr2, PtI2, Pt(HSO4)2, PtSO3, Pt(HSO3)2, Pt(CH3SO3)2, Pt(C6H5SO3)2, Pt(NO3)2, Pt3(PO4)2, PtHPO4, Pt(H2PO4)2, Pt(HCO3)2, Pt(HCOO)2, Pt(CH3COO)2, Pt(CF3COO)2, Pt(CCl3COO)2, Pt(CHF2COO)2, Pt(CHCl2COO)2, Pt(CH2FCOO)2, Pt(CH2ClCOO)2, Pt(BF4)2, Pt(C6H5COO)2, Pt(C6H5O)2, Pt(C6H 11 O7)2, Pd(C3H5O3)2; PtCl2, PtBr2, PtI2, Pt(HSO4)2, PtSO3, Pt(HSO3)2, Pt(CH3SO3)2, Pt(C6H5SO3)2, Pt(NO3)2, Pt3(PO4)2, PtHPO4, Pt(H2PO4)2, Pt(HCO3)2, Pt(HCOO)2, Pt(CH3COO)2, Pt(CF3COO)2, Pt(CCl3COO)2, Pt(CHF2COO)2, Pt(CHCl2COO)2, Pt(CH2FCOO)2, Pt(CH2ClCOO)2, Pt(BF4)2, Pt(C6H5COO)2, Pt(C6H5O)2, Pt(C6H 11O7)2, Pt(C3H5O3)2; CuCl2, CuBr2, CuI2, CuSO4, Cu(HSO4)2, CuSO3, Cu(HSO3)2, Cu(CH3SO3)2, Cu(C6H5SO3)2, Cu(NO3)2, Cu3(PO4)2, CuHPO4, Cu(H2PO4)2, Cu(HCOO)2, Cu(CH3COO)2, Cu(CF3COO)2, Cu(CCl3COO)2, Cu(CHF2COO)2, Cu(CHCl2COO)2, Cu(CH2FCOO)2, Cu(CH2ClCOO)2, Cu(BF4)2, Cu(C6H5COO)2, Cu(C6H 11 O7)2, Pt(C3H5O3)2; CuCl2, CuBr2, CuI2, CuSO4, Cu(HSO4)2, CuSO3, Cu(HSO3)2, Cu(CH3SO3)2, Cu(C6H5SO3)2, Cu(NO3)2, Cu3(PO4)2, CuHPO4, Cu(H2PO4)2, Cu(HCOO)2, Cu(CH3COO)2, Cu(CF3COO)2, Cu(CCl3COO)2, Cu(CHF2COO)2, Cu(CHCl2COO)2, Cu(CH2FCOO)2, Cu(CH2ClCOO)2, Cu(BF4)2, Cu(C6H5COO)2, Cu(C6H 11 O7, CuC3H5O3; AgCl, AgBr, AgI, AgHSO4, Ag2SO3, AgHSO3, AgCH3SO3, AgC6H5SO3, AgNO3, Ag3PO4, Ag2HPO4, AgH2PO4, AgHCOO, AgCH3COO, AgCF3COO, AgCCl3COO, AgCHF2COO, AgCHCl2COO, AgCH2FCOO, AgCH2ClCOO, AgBF4, AgC6H5COO, AgC6H5O, AgC6H 11 O7, AgC3H5O3.

[0023] In some embodiments, the ratio of the first metal ion to antibody in step (a) is in the range of about 1 : 1 to 30: 1 by molar ratio, preferably in the range of about 1 : 1.5 to 20: 1, preferably in the range of about 2: 1 to 16: 1, preferably in the range of about 4: 1 to 8: 1, for example about 10: 1. In some embodiments, the ratio of the second ion to antibody in step (a) is above 0: 1, preferably in the range of about 1 : 1 to 30: 1 by molar ratio, preferably in the range of about 1.5: 1 to 20: 1, preferably in the range of about 2: 1 to 16: 1, preferably in the range of about 4: 1 to 8: 1, for example about 10: 1.

[0024] In some embodiments, the ratio of the sum of the first metal ion and the second metal ion in step (a) to the antibody is in the range of about 1:1 to 30:1, preferably in the range of about 2:1 to 20:1, preferably in the range of about 4:1 to 16:1, more preferably in the range of about 8:1 to 12:1, for example about 10:1, by molar ratio.

[0025] In some embodiments, the ratio of the first metal ion to the second metal ion in step (a) is in the range of about 1:0.2 to 1:15, preferably in the range of about 1:0.5 to 1:8, preferably in the range of about 1:1 to 1:4, more preferably in the range of about 1:2 to 1:4, by molar ratio.

[0026] In some embodiments, the incubation temperature in step (a) is about 0 °C to room temperature (about 20-25 °C). In some embodiments, the incubation temperature in step (a) is about 12 °C to 15 °C, 10 °C to 20 °C, or 12 °C to 25 °C, or 10 °C to 25 °C. In some embodiments, the incubation time in step (a) is about 2-24 hours, preferably about 4-20 hours, more preferably about 8-20 hours, still more preferably about 12-20 hours, most preferably about 12, 16, 20 hours. In some embodiments, the incubation condition in step (a) is incubation at a temperature of about 12 °C to about 20 °C for 12-24 hours.

[0027] In some embodiments, the antibody in step (a) is selected from monoclonal antibodies and polyclonal antibodies, antibody fusion proteins. In some embodiments, the antibody is selected from human antibodies, humanized antibodies, and chimeric antibodies. In some embodiments, the antibody specifically binds to a corresponding antigen expressed on cancer cells (also known as tumor associated antigen (TAA)), viral antigen or microbial antigen, and has antibody-dependent cell-mediated phagocytosis (ADCP) activity, in vivo anti-tumor, anti-viral or anti-microbial activity. In some embodiments, the monoclonal antibody is selected from trastuzumab, pertuzumab, racotumomab, abciximab, adalimumab, alfaferone, alemtuzumab, basiliximab, belimumab, belotufosumab, canakinumab, pexidartinib, cetuximab, daclizumab, denosumab, efalizumab, golimumab, infliximab, ipilimumab, isibamumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, sucralfate, and ustekinumab.

[0028] In some embodiments, the drug in the linker-drug moiety is selected from the group consisting of a diagnostic agent, a therapeutic agent, and a labeling agent. In some embodiments, the drug in the linker-drug moiety is selected from the group consisting of a cytotoxic agent, a toxin, a radionuclide, a fluorescent agent (e.g., an amine derivatized fluorescent probe such as 5-dimethylaminonaphthalene-l-(N-(2-aminoethyl))sulfonamide-dansyl ethylenediamine, Oregon Green® 488cadaverine (catalog number O-10465, Molecular Probes), dansyl cadaverine, N-(2-aminoethyl)-4-amino-3,6-dithio-l,8-naphthalimide, dipotassium salt (fluorescent yellow ethylenediamine), rhodamine B ethylenediamine (catalog number L-2424, Molecular Probes), or a thiol derivatized fluorescent probe such as Alexa Fluor® FLL-cysteine (catalog number B-20340, Molecular Probes)), a chemotherapeutic agent, an immunotherapeutic agent, an antiviral agent, an antimicrobial agent, a molecular degrading agent, an immune agonist, and a nuclear pharmaceutical chelator. In some embodiments, the drug in the linker-drug moiety is selected from the group consisting of an Auristatin, a topoisomerase inhibitor, a Maytansinoid, and a PBD.

[0029] In another aspect, the present application provides an antibody-drug conjugate prepared by the method of the present application, which has a higher homogeneity than those prepared by conventional conjugation methods. In some embodiments, the content of antibody-drug conjugates having a DAR value of 4 in the antibody-drug conjugate prepared according to the method of the present application is greater than 60%, preferably greater than 65%, more preferably greater than 70%, for example greater than 71%, 72%, 73%, or 74%. In some embodiments, the sum of the content of antibody-drug conjugates having a DAR value of 0 (DAR0 or D0) and a DAR value of 8 (DAR8 or D8) in the antibody-drug conjugate prepared according to the method of the present application is less than 20%, preferably less than 15%, more preferably less than 10%. In some embodiments, the content of antibody-drug conjugates having a DAR value of 6 (DAR6 or D6) in the antibody-drug conjugate prepared according to the method of the present application is less than 20%, preferably less than 15%, more preferably less than 10%.

[0030] In another aspect, the present application provides a pharmaceutical composition containing an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier or excipient.

[0031] In another aspect, the present application provides the use of an antibody-drug conjugate described herein or a pharmaceutical composition described herein in the manufacture of a medicament for treating a disease selected from the group consisting of a cancer, an autoimmune disease, an inflammation, and a metabolic disease. ​

[0032] In another aspect, the present application provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate described herein or a pharmaceutical composition described herein, the disease is selected from the group consisting of cancer, autoimmune disease, inflammation and metabolic disease. DETAILED DESCRIPTION

[0033] In the present application, the transition metal ion generates selectivity in the reduction of disulfide bonds. In the presence of the transition metal ion, the two interchain S-S bonds in the Fab region are selectively reduced. Thus, four payload-linker complexes carrying reactive groups (i.e. four drug-linker complexes) are attached to one antibody to form D4. The high content of D4 in the obtained ADC undoubtedly improves the homogeneity of the ADC.

[0034] The present application is further illustrated in the following specific embodiments and examples. It is to be understood that these embodiments and examples are merely illustrative of the present application and do not limit the scope of the present application.

[0035] Definitions

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that there is a plurality of definitions for a term herein, those in this section prevail. Unless otherwise required by context, the terms "about" and "substantially" are used herein to describe and account for small fluctuations, such as due to variations in manufacturing or testing conditions.

[0037] In this document, the terms "comprise" and "comprising" can be used interchangeably with "include" and "including". "Comprise" and "comprising" are to be interpreted as specifying the presence of the stated features or components, but not to preclude the presence or addition of one or more other features, components, or groups thereof. In addition, "comprise" and "comprising" are intended to include examples of the terms "consist of" and "consisting of". Thus, the terms "consist of" and "consisting of" can be substituted for the "comprise" and "comprising" terms to provide more specific embodiments.

[0038] As used herein, the terms "antibody-drug conjugate", "antibody-drug conjugate", "antibody conjugated drug" and "ADC" have the same meaning and refer to a drug composed of an antibody, a drug ((effective) payload) and a linker connecting the antibody and the drug.

[0039] As used herein, the term "(cytotoxic) drug" can refer to any cytotoxic molecule having, for example, an antitumor effect, an anti-infective or an anti-inflammatory effect and having at least one substituent group or moiety structure that allows attachment to a linker structure. The drug can kill cells (e.g., cancer cells) and / or inhibit the growth, proliferation, or metastasis of cells (e.g., cancer cells), thereby reducing, alleviating, or eliminating one or more symptoms of a disease or disorder (e.g., cancer). The drug can be selected from the group consisting of a cytotoxic agent, a toxin, a radionuclide, a fluorescent agent (e.g., an amine derivatized fluorescent probe such as 5-dimethylaminonaphthalene-l-(N-(2-aminoethyl))sulfonamide-dansyl ethylenediamine, Oregon Green® 488cadaverine (catalog no. O-10465, Molecular Probes), dansyl cadaverine, N-(2-aminoethyl)-4-amino-3,6-dithio-l,8-naphthalimide, dipotassium salt (fluorescent yellow ethylenediamine), rhodamine B ethylenediamine (catalog no. L-2424, Molecular Probes), or a thiol derivatized fluorescent probe such as Alexa Fluor® FLL-cysteine (catalog no. B-20340, Molecular Probes)), a chemotherapeutic agent, an immunotherapeutic agent, an antiviral agent, an antimicrobial agent, a molecular degrading agent, an immune agonist, and a nuclear pharmaceutical chelator. In some embodiments, the drug in the linker-drug moiety is selected from the group consisting of an Auristatin class drug, a topoisomerase inhibitor, a Maytansinoid class drug, and a PBD class drug. FLL-cysteine (catalog no. B-20340, Molecular Probes)), a chemotherapeutic agent, an immunotherapeutic agent, an antiviral agent, an antimicrobial agent, a molecular degrading agent, an immune agonist, and a nuclear pharmaceutical chelator. In some embodiments, the drug in the linker-drug moiety is selected from the group consisting of an Auristatin class drug, a topoisomerase inhibitor, a Maytansinoid class drug, and a PBD class drug.

[0040] As used herein, the term "linker" refers to a reactive molecule that contains at least two reactive groups, where one reactive group can covalently bind to the (effective) payload / drug and the other reactive group can covalently conjugate to the antibody.

[0041] As used herein, the term "(effective) payload" refers to the "drug" as defined above that is linked to the "linker" through a chemical bond to form a (effective) payload-linker (complex), which can also be referred to as a drug-linker (complex), a linker-(effective) payload (complex), and a linker-drug (complex).

[0042] As used herein, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains. Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains form the antigen binding site. Bispecific antibody molecules, trispecific antibody molecules, and multispecific antibody molecules are also encompassed within the antibodies according to the application. Chimeric antibodies or humanized antibodies are also encompassed within the antibodies according to the application.

[0043] As used herein, the term "antigen binding fragment" refers to a fragment of an antibody, e.g., a fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion". Antigen binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide stabilized Fv proteins ("dsFv"), single domain antibodies (sdAb, nanobodies), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.

[0044] As used herein, "Fab" of an antibody refers to the following portion of an antibody, which consists of a single light chain (both variable and constant regions) associated with the variable region and the first constant region of a single heavy chain by disulfide bonds. In some embodiments, both the first and second antigen binding portions of the antibody to be conjugated are in Fab format. Further, the constant regions of the two chains of the Fab (i.e., CH1 and CL) are replaced by engineered or modified TCR constant regions.

[0045] As used herein, "Fc" of an antibody refers to the following portion of an antibody, which comprises the second (CH2) and third constant regions (CH3) of the first heavy chain, in conjunction with the second and third constant regions of the second heavy chain via disulfide bonds and optionally the hinge region. The Fc portion of an antibody is responsible for various effector functions, such as ADCC and CDC, but does not play a role in antigen binding.

[0046] As used herein, the term "DAR" also known as Drug to Antibody Ratio, is a unique quality attribute of antibody-drug conjugates. The selection of DAR value depends on the characteristics of the target antigen, the characteristics of the linker-drug / (payload) and needs to be considered in an integrated manner. Various analytical methods can be used to measure DAR, such as ultraviolet-visible (UV / Vis) spectroscopy, hydrophobic interaction chromatography (HIC), reversed-phase high-performance liquid chromatography (RP-HPLC), and liquid chromatography conjugated electrospray ionization mass spectrometry (LC-ESI-MS). Hydrophobic interaction chromatography (HIC) is the leading technology for characterizing DAR values and drug / payload distribution.

[0047] As known in the art, mixtures of antibody-drug conjugates will be produced by conventional conjugation methods. Typically, the antibody molecule can be subjected to partial or complete reduction of one or more interchain S-S bonds to form 2n (n is an integer selected from 1, 2, 3 or 4) reactive -SH groups, and thus, depending on the structure and properties of the linker or linker-(payload) involved in the reaction, the number of drugs conjugated to a single antibody molecule can be 1, 2, 3, 4, 5, 6, 7 or 8. Depending on the number of drugs conjugated to a single antibody molecule, different antibody-drug conjugates containing different numbers of drug molecules can include D0 (DAR value of 0), D1 (DAR value of 1), D2 (DAR value of 2), D3 (DAR value of 3), D4 (DAR value of 4), D5 (DAR value of 5), D6 (DAR value of 6), D7 (DAR value of 7) and D8 (DAR value of 8) antibody-drug conjugates.

[0048] As used herein, the term "homogeneity" or "homogeneity" of antibody-drug conjugates is used to describe the property of a particular type of antibody-drug conjugate (preferably, in this context, one or more types selected from D2, D4, D6 antibody-drug conjugates) to dominate in a given mixture of antibody-drug conjugates. In the present invention, "homogeneity" or "homogeneity" of antibody-drug conjugates means that a particular type of antibody-drug conjugate has a high level in the mixture of antibody-drug conjugates. Antibody-drug conjugates with high homogeneity provide benefits for therapy, such as higher therapeutic index, improved efficacy and reduced drug toxicity.

[0049] As used herein, the term "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," "physiologically acceptable carrier," or "physiologically acceptable excipient" refers to a pharmaceutically-acceptable material, component or medium that is nontoxic to the subject, e.g., a liquid or solid filler, diluent, excipient, solvent or encapsulating material. In some embodiments, each component of the pharmaceutical composition / formulation is "pharmaceutically acceptable" in that it is compatible for use with other ingredients of the formulation and not deleterious to the subject in contact therewith, having no significant toxic, irritating, allergic, immunogenic or other adverse effects with a reasonable benefit / risk ratio.

[0050] As used herein, the term "subject" includes any human or non-human animal, e.g., a human.

[0051] As used herein, the term "cancer" refers to any neoplastic or malignant cell growth, proliferation, or metastasis-mediated solid and non-solid tumors such as leukemias, and that gives rise to a medical condition. A "tumor" includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, penile cancer, thyroid cancer, hepatocellular cancer, anal carcinoma, penile carcinoma, and head and neck cancer.

[0052] As used herein, the terms "treat," "treatment" or "treated" generally refer to a treatment or therapy of a human or animal body, in which some desired therapeutic effect is achieved, such as inhibition of progression of a condition, and includes reduction in the rate of progression, cessation of progression, regression of the condition, remission of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis, prevention) is also included. For cancer, "treatment" can refer to inhibition or reduction of tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. For a tumor, "treatment" includes removal of all or part of the tumor, inhibition or reduction of tumor growth and metastasis, inhibition or delay of tumor development, or some combination thereof.

[0053] Methods of making antibody-drug conjugates

[0054] In one aspect, the present application provides a method for making an antibody-drug conjugate (ADC) with improved homogeneity, comprising the steps of:

[0055] (a) incubating a reducing agent and an antibody to be conjugated in the presence of an effective amount of a first metal ion and a second metal ion, performing a reduction reaction, obtaining a reaction mixture;

[0056] (b) reacting a linker-payload carrying a reactive group with the reaction mixture obtained in step (a); and

[0057] (c) oxidizing the reaction mixture obtained in step (b) with an oxidizing agent, and

[0058] (d) recovering the antibody-drug conjugate obtained.

[0059] The inventors surprisingly found that if an effective amount of a combination of metal ions, in particular Zn 2+ in combination with Ca 2+ or Zn 2+ in combination with Mn 2+ is added in step (a), the homogeneity of the ADC product can be further improved; and a longer reaction plateau can be generated, which is easier to handle for large scale production and leaves more handling time for the work-up.

[0060] In case metal ions are added, a chelating agent is added after the completion of the conjugation to capture the metal ions for easy removal in subsequent steps. In some embodiments, the chelating agent is EDTA, DOTA and DPTA.

[0061] There is no particular limitation on the antibody that can be conjugated with a linker-drug by using the bioconjugation method of the present application. The selection of the antibody depends on the disease or disorder (e.g., cancer) to be treated by the antibody-drug conjugate. In some embodiments, the antibody is selected from the group consisting of a monoclonal antibody and a polyclonal antibody. In some embodiments, the antibody is selected from the group consisting of a human antibody, a humanized antibody, and a chimeric antibody. In some embodiments, the antibody specifically binds to a corresponding antigen (also known as a tumor-associated antigen (TAA)) expressed on a cancer cell, a viral antigen, or a microbial antigen, and has antibody-dependent cell-mediated phagocytosis (ADCP) activity, in vivo anti-tumor, anti-viral, or anti-microbial activity. In some embodiments, the monoclonal antibody is selected from the group consisting of trastuzumab, pertuzumab, sacituzumab, abciximab, adalimumab, alefacept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, golimumab, inflectra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, secukinumab, and ustekinumab. In some embodiments, the monoclonal antibody can also be datopotamab deruxtecan. The interchain S-S bond in the antibody is the site of attachment of the drug-linker moiety.

[0062] The drug in the linker-drug moiety can be any type of drug, as long as the drug molecule has the desired effect. In some embodiments, the drug in the linker-drug moiety can be a diagnostic agent, a therapeutic agent, and a labeling agent. In some embodiments, the drug in the linker-drug moiety can be a cytotoxic agent, a toxin, a radionuclide, a fluorescent agent (e.g., amine-derived fluorescent probes such as 5-dimethylaminonaphthalene-1-(N-(2-aminoethyl))sulfonamide-dansyl ethylenediamine, Oregon 488 cadaverine (Catalog No. O-10465, Molecular Probes), dansyl cadaverine, N-(2-aminoethyl)-4-amino-3,6-dithia-1,8-naphthalimide, dipotassium salt (fluorescein ethylenediamine), rhodamine B ethylenediamine (Catalog No. L-2424, Molecular Probes) or thiol-derivatized fluorescent probes such as FLL-cysteine (Catalog No. B-20340, Molecular Probes)), chemotherapeutic agents, immunotherapeutic agents, antiviral agents, antimicrobial agents, molecular degraders, immune agonists, and radiopharmaceutical chelators. In some embodiments, the drug in the linker-drug moiety can be an Auristatin drug, a topoisomerase inhibitor, a Maytansinoid drug, and a PBD drug.

[0063] The mother liquor of the antibody-drug conjugate obtained by the method according to the present invention can be recovered, post-treated, and purified in any suitable manner to obtain the final product. In some embodiments, the recovery in step (d) includes purification by using a desalting column and / or size exclusion chromatography. In some embodiments, the recovery in step (d) includes first adding N-acetylcysteine to deplete the excess linker-drug moiety and then purification by using a desalting column and / or size exclusion chromatography. In some embodiments, the recovery in step (d) includes adding a chelating agent to capture the transition metal ions and then purification by using a desalting column and / or size exclusion chromatography. In some embodiments, the recovery in step (d) includes first adding N-acetylcysteine to deplete the excess linker-drug moiety, then adding a chelating agent to capture the transition metal ions, and then purification by using a desalting column and / or size exclusion chromatography. The operations before purification are all completed in the same reaction vessel without intermediate separation and purification steps.

[0064] By using the method for preparing an antibody-drug conjugate of the present application, the homogeneity of the antibody-drug conjugate is higher than those prepared by conventional conjugation methods.

[0065] Antibody-drug conjugate

[0066] In another aspect, the application provides antibody-drug conjugates prepared by the methods described herein that are more homogenous than those prepared by conventional conjugation methods.

[0067] In some embodiments, the content of antibody-drug conjugates having a DAR value of 4 in antibody-drug conjugates prepared according to the methods of the application is greater than 60%, preferably greater than 65%, more preferably greater than 70%, for example greater than 71%, 72%, 73% or 74%. In some embodiments where metal ions are added, the sum of the content of antibody-drug conjugates having a DAR value of 0 (DAR0or D0) and a DAR value of 8 (DAR8or D8) in antibody-drug conjugates prepared according to the methods of the application is less than 20%, preferably less than 15%, more preferably less than 10%. In some embodiments, the content of antibody-drug conjugates having a DAR value of 6 (DAR6or D6) in antibody-drug conjugates prepared according to the methods of the application is less than 20%, preferably less than 15%, more preferably less than 10%. In contrast, the content of D4 in antibody-drug conjugates prepared by conventional conjugation methods is typically less than 40%.

[0068] Pharmaceutical compositions

[0069] In one embodiment, the application provides a pharmaceutical composition comprising an antibody-drug conjugate prepared by the methods described herein and a pharmaceutically acceptable carrier or excipient.

[0070] In some embodiments, the antibody-drug conjugates provided herein are provided as part of a pharmaceutical composition comprising the antibody-drug conjugate in combination with an appropriate pharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science of Pharmacy, 20thEdition; Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7thEdition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rdEdition, Pharmaceutical Press (2000)). Such carriers can be selected from pharmaceutically acceptable carriers and excipients. The excipients include, but are not limited to, solubilizing agents, surfactants, emulsifying agents, and suspending agents.

[0071] Examples of solubilizers suitable for pharmaceutical compositions include, but are not limited to, alcohols and polyols such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butylene glycol and its isomers, glycerol, pentaerythritol, sorbitol, mannitol, diethylene glycol monoethyl ether, isosorbide dimethyl ether, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrins and cyclodextrin derivatives, 2-pyrrolidone, N-alkyl pyrrolidones, polyvinyl pyrrolidone, ethyl propionate, tributyl citrate, triethyl citrate, ethyl oleate, ethyl caprylate, ethyl butyrate, glyceryl triacetate, and propylene glycol monoacetate.

[0072] Examples of surfactants suitable for pharmaceutical compositions include, but are not limited to, sodium lauryl sulfate (SDS), gelatin, casein, sodium docusate, benzalkonium chloride, calcium stearate, polyethylene glycol, phosphates, polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 80, polysorbate 20), gum acacia, cholesterol, tragacanth, polyoxyethylene 20 stearyl ether, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, macrogolglycerol hydroxystearate, fatty acid sorbitan esters, vitamin E or tocopherol derivatives, tocopherol esters, lecithin, phospholipids and derivatives thereof, poloxamer, stearic acid, oleic acid, oleyl alcohol, cetyl alcohol, monoglycerides and diglycerides, propylene glycol fatty acid esters, glyceryl fatty acid esters, ethylene glycol palmitostearate, polyoxyglycerides, propylene glycol monocaprylate, propylene glycol monolaurate, alkyl aryl polyether alcohols, and polyglyceryl oleate.

[0073] Examples of emulsifiers suitable for pharmaceutical compositions include, but are not limited to, acacia, tragacanth gum gelatin, natural phospholipids (e.g., soy lecithin), fatty acid sorbitan, polysorbate, and poloxamer.

[0074] Examples of suspending agents suitable for pharmaceutical compositions include, but are not limited to, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth.

[0075] In some embodiments, the provided pharmaceutical compositions can be prepared in the form of a liquid suspension or solution using liquids such as oils, water, alcohols, and combinations thereof.

[0076] In some embodiments, the provided pharmaceutical compositions can be prepared in the form of a sterile injectable preparation, which can be either aqueous or oleaginous suspensions. These suspensions can be prepared according to known techniques.

[0077] In some embodiments, kits comprising an antibody-drug conjugate provided herein (or a composition comprising an antibody-drug conjugate provided herein) are also provided, which are provided in a manner convenient for practicing the methods described herein. In some embodiments, the kit includes an antibody-drug conjugate provided herein (or a composition comprising an antibody-drug conjugate provided herein) packaged in a container, such as a sealed bottle or vessel, and a label affixed to the container or included in the kit describing the use of the antibody-drug conjugate or composition to practice the methods provided herein. In some embodiments, the antibody-drug conjugate or composition is packaged in unit dose form. In some embodiments, the kit further includes an apparatus suitable for administering the antibody-drug conjugate or composition according to a predetermined route of administration. In some embodiments, the kit includes an antibody-drug conjugate provided herein and instructions for administering the antibody-drug conjugate to a cancer patient.

[0078] Uses

[0079] In some embodiments, the present application provides uses of an antibody-drug conjugate prepared by the methods described herein or a pharmaceutical composition comprising the antibody-drug conjugate in the manufacture of a medicament for treating cancer, autoimmune diseases, inflammation, and / or metabolic diseases.

[0080] In some embodiments, an antibody-drug conjugate prepared by the methods described herein or a pharmaceutical composition comprising the antibody-drug conjugate can be used in any patient that can benefit from the compounds provided herein. In some embodiments, the patients can be mammals, such as humans and companion animals. In some embodiments, the patient is a human.

[0081] A therapeutically effective amount of an ADC provided herein will depend on various factors known in the art, such as body weight, age, past medical history, current medications, the health of the subject, and the likelihood of cross-reactions, allergies, sensitivities, and adverse side effects, as well as the route of administration and the extent of disease progression. The dosage can be proportionally reduced or increased as indicated by these and other conditions or requirements. In some embodiments, an ADC or pharmaceutical composition provided herein can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In certain of these embodiments, an ADC or pharmaceutical composition provided herein is administered at a dose of about 50 mg / kg or less, and in certain of these embodiments, the dose is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less.

[0082] In some embodiments, the dose administered can vary over the course of treatment. For example, in some embodiments, the initial dose administered can be higher than the subsequent doses administered. In some embodiments, the dose administered can vary over the course of treatment depending on the subject’s response.

[0083] In some embodiments, the disease treated by the antibody-drug conjugate described herein is selected from the group consisting of cancer, autoimmune disease, inflammation, and metabolic disease.

[0084] In some embodiments, the present application provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate described herein or a pharmaceutical composition described herein, the disease is selected from the group consisting of cancer, autoimmune disease, inflammation, and metabolic disease.

[0085] In some embodiments, the present application provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate described herein or a pharmaceutical composition described herein and another therapeutic agent for treating cancer, autoimmune disease, inflammation, and metabolic disease.

[0086] Advantages of the present invention

[0087] The present invention uses two metal ions (e.g., Zn 2+ and Ca 2+ , Zn 2+ and Mn 2+ , etc.) together for the ADC preparation method at room temperature, compared to the preparation method disclosed in WO2020164561A1 using only Zn 2+ , the reaction temperature and reduction reaction time of the method according to the present invention are more suitable for scale-up and production processes, thereby simplifying the equipment and operation, and improving the homogeneity of the product. BRIEF DESCRIPTION OF DRAWINGS

[0088] FIGS. 1-7 are, in order, hydrophobic interaction chromatography (HIC-HPLC) graphs of Herceptin-MC-VC-PAB-MMAE conjugates prepared in the experiments of Example 1 Nos. 1-7, respectively.

[0089] FIGS. 8-19 are, in order, hydrophobic interaction chromatography (HIC-HPLC) graphs of Herceptin-MC-VC-PAB-MMAE conjugates prepared in the experiments of Example 2 Nos. 8-19, respectively.

[0090] FIGS. 20 and 21 are, in order, hydrophobic interaction chromatography (HIC-HPLC) graphs of Herceptin-MC-VC-PAB-MMAE conjugates prepared in the experiments of Example 3 Nos. 20 and 21, respectively.

[0091] FIGS. 22 and 23 are, in order, hydrophobic interaction chromatography (HIC-HPLC) graphs of Herceptin-MC-GGFG-DXD conjugates prepared in the experiments of Example 4 Nos. 22 and 23, respectively.

[0092] FIGS. 24 and 25 are, in order, hydrophobic interaction chromatography (HIC-HPLC) graphs of Herceptin-MC-GGFG-DXD conjugates prepared in the experiments of Example 5 Nos. 24 and 25, respectively.

[0093] FIGS. 26 and 27 are, in order, hydrophobic interaction chromatography (HIC-HPLC) graphs of Cetuximab-MC-VC-PAB-MMAE conjugates prepared in the experiments of Example 6 Nos. 26 and 27, respectively.

[0094] FIGS. 28 and 29 are, in order, hydrophobic interaction chromatography (HIC-HPLC) graphs of Datopotamab-MC-VC-PAB-MMAE conjugates prepared in the experiments of Example 7 Nos. 28 and 29, respectively.

[0095] EXAMPLES

[0096] The application will now be elucidated with reference to the following examples. However, the skilled person will understand that the following examples are provided for illustrative purposes only and are not intended to limit the application in any way.

[0097] Example 1. Preparation of Herceptin-MC-VC-PAB-MMAE conjugate by using the method according to the present application and homogeneity of the conjugate

[0098] Herceptin-MC-VC-PAB-MMAE conjugate was prepared in a one-pot reaction (the following ratios refer to molar ratios):

[0099] (1) ZnCl2(ZnCl2 / antibody ratio see Table 2), CaCl2(CaCl2 / antibody ratio see Table 2), and TCEP (tris(2-carboxyethyl)phosphine), TCEP / antibody ratio 3.25) were added to a solution of the antibody (Herceptin was dissolved in 40 mM phosphate buffer, pH 7.0 at a concentration of 5.56 mg / mL), and the reaction mixture was allowed to react at a reaction temperature of 12°C for 20 hours in a 1.5 mL reaction vessel at a total reaction scale of 5 mg (reaction volume 0.9 mL);

[0100] (2) MC-VC-PAB-MMAE (CAS: 646502-53-6, commercially available from Lenena, Biopharma, MC-VC-PAB-MMAE / antibody ratio 10.0, solution volume 0.1 mL) in DMA (dimethylacetamide, commercially available from General) was introduced to the reaction mixture in (1), and the reaction was continued at 12°C for 2 hours; the reaction concentration of the antibody after this step was 5 mg / mL, the reaction volume was 1 mL; the DMA (containing MC-VC-PAB-MMAE) solution accounted for 10% of the total volume.

[0101] (3) NAC (N-acetyl-L-cysteine, NAC / antibody ratio 12.0) was added to the reaction mixture in (2) to deplete excess MC-VC-PAB-MMAE;

[0102] (4) EDTA (ethylenediaminetetraacetic acid, EDTA / antibody ratio 30.0) was added to the reaction mixture in (3) to capture metal ions, and DHAA (dehydroascorbic acid, commercially available from BIOSYNTH, DHAA / antibody ratio 8.0) was added to oxidize excess thiol, to obtain the Herceptin-MC-VC-PAB-MMAE conjugate.

[0103] Finally, the drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. The analysis was performed by hydrophobic interaction chromatography (HIC) on a HIC column (Protein-Pak TM The detection of the DAR was performed by hydrophobic interaction chromatography (HIC) on a Hi Res HIC 2.5 μm 4.6 x 35 mm Column at ambient temperature with a flow rate of 0.5 mL / min. The mobile phase A was 1.5 M (NH4)2SO4 and 50 mM potassium phosphate dibasic, pH 7.0; the mobile phase B was 75% v / v 50 mM potassium phosphate and 25% v / v acetonitrile, pH 7.0.

[0104] The elution gradient is shown in Table 1.

[0105] Table 1 HIC column elution gradient

[0106] The results of the ZnCl2 / antibody ratio and CaCl2 / antibody ratio in the above reaction and the drug / antibody ratio (DAR) and product distribution are shown in Table 2.

[0107] Table 2

[0108] As shown in Table 2, only with Zn 2+ D4% was about 66.2% in the case of the transition metal ion only with Ca 2+ The D4% of the sample could not be directly improved, but in the presence of Zn 2+ CaCl2was added in the presence of Zn 2+ The D4% of the sample increased significantly after the addition of Ca 2+ The D4% could be improved to 70.8% when Ca 2+ was 8 equivalents of the antibody.

[0109] Example 2. Preparation of Herceptin-MC-VC-PAB-MMAE conjugate by using the method according to the present application and homogeneity of the conjugate

[0110] The Herceptin-MC-VC-PAB-MMAE conjugate was prepared in a one-pot reaction (the ratios below refer to molar ratios):

[0111] (1) ZnCl2(ZnCl2 / antibody ratio 4.0), CaCl2(CaCl2 / antibody ratio see Table 4), and TCEP (TCEP / antibody ratio 3.25) were added to a solution of antibody (Herceptin was dissolved in 40 mM phosphate buffer, pH 7.0 at a concentration of 5.56 mg / mL) and the reaction mixture was allowed to react at a reaction temperature of 12°C in a 50 mL reaction vessel at a total reaction scale of 50 mg (reaction volume 9 mL), the reduction reaction time is shown in Table 4;

[0112] (2) MC-VC-PAB-MMAE (MC-VC-PAB-MMAE / antibody ratio 10.0, solution volume 1 mL) in DMA (dimethylacetamide, commercially available from General) was introduced to the reaction mixture in (1) and the reaction was continued at 12°C for 2 hours; the reaction concentration of antibody after the addition of DMA in this step was 5 mg / mL, the reaction volume was 10 mL; the DMA (containing MC-VC-PAB-MMAE) solution accounted for 10% of the total volume.

[0113] (3) NAC (NAC / antibody ratio 12.0) was added to the reaction mixture in (2) to deplete excess MC-VC-PAB-MMAE;

[0114] (4) EDTA (EDTA / antibody ratio 30.0) was added to the reaction mixture in (3) to capture metal ions and DHAA (commercially available from BIOSYNTH, DHAA / antibody ratio 8.0) was added to oxidize excess thiol to obtain Herceptin-MC-VC-PAB-MMAE conjugate.

[0115] (5) The crude ADC sample obtained in (4) was subjected to liquid exchange and purification (liquid exchange to 20 mM histidine / histidine hydrochloride buffer, pH 6.0) using a centrifugal ultrafiltration tube.

[0116] Finally, the drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. The detection of DAR was performed by hydrophobic interaction chromatography (HIC) on a HIC column (Protein-Pak TM Hi Res HIC 2.5 μm 4.6 x 35 mm Column) at a flow rate of 0.5 mL / min at ambient temperature. Mobile phase A was 1.5 M (NH4)2SO4 and 50 mM potassium phosphate dibasic, pH 7.0; mobile phase B was 75% v / v 50 mM potassium phosphate and 25% v / v acetonitrile, pH 7.0.

[0117] The elution gradient is shown in Table 3.

[0118] Table 3 HIC column eluent gradient

[0119] The results of the CaCl2 / antibody ratio and drug / antibody ratio (DAR) and product distribution in the above reaction are shown in Table 4.

[0120] Table 4

[0121] As shown in Table 4, in the presence of Zn 2+ , the D4 content was significantly improved (more than 6%) after the addition of Ca 2+ , and the reduction reaction time reached a plateau at 12 h (74.0%) for the D4 content, while the sample without Ca 2+ reached a plateau at 20 h for the D4 content, only 67.2%. 2+

[0122] Example 3. Preparation of Herceptin-MC-VC-PAB-MMAE conjugate by using the method according to the present application with the addition of MnCl2and homogeneity of the conjugate

[0123] The Herceptin-MC-VC-PAB-MMAE conjugate was prepared in a one-pot reaction (the following ratios refer to molar ratios):

[0124] (1) ZnCl2(ZnCl2 / antibody ratio of 4.0), MnCl2(MnCl2 / antibody ratio as shown in Table 6), and TCEP(tris(2-carboxyethyl)phosphine) (TCEP / antibody ratio of 3.5) were added to a solution of the antibody (Herceptin was dissolved in 40 mM phosphate buffer at a concentration of 5.56 mg / mL, pH 7.0), and the reaction mixture was allowed to react at a reaction temperature of 12°C for 20 hours in a 1.5 mL reaction vessel at a total reaction scale of 1 mg (reaction volume 0.18 mL);

[0125] (2) MC-VC-PAB-MMAE (MC-VC-PAB-MMAE / antibody ratio of 10.0) in DMA (dimethylacetamide, commercially available from General) was introduced to the reaction mixture in (1) at a solution volume of 0.02 mL, and the reaction was continued at 12°C for 2 hours; the reaction concentration of the antibody after the addition of DMA (containing MC-VC-PAB-MMAE) in this step was 5 mg / mL, and the reaction volume was 0.2 mL; the DMA (containing MC-VC-PAB-MMAE) solution accounted for 10% of the total volume.

[0126] ​(3) To the reaction mixture in (2), NAC (N-acetyl-L-cysteine, NAC / antibody ratio of 12.0) was added to deplete excess MC-VC-PAB-MMAE;

[0127] (4) To the reaction mixture in (3), EDTA (ethylenediaminetetraacetic acid, EDTA / antibody ratio of 30.0) was added to capture metal ions and DHAA (dehydroascorbic acid, commercially available from BIOSYNTH, DHAA / antibody ratio of 8.0) was added to oxidize excess thiol, to obtain Herceptin-MC-VC-PAB-MMAE conjugate.

[0128] Finally, the drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. The detection of DAR was performed by hydrophobic interaction chromatography (HIC) on a HIC-HPLC column (Tosoh Bioscience Butyl-NPR Column, 4.6 mm x 3.5 cm, 2.5 μm) at a flow rate of 1 mL / min at ambient temperature. The mobile phase A was 1.5 M (NH4)2SO4 and 50 mM potassium phosphate dibasic, pH 7.0; the mobile phase B was 50 mM potassium phosphate dibasic and 25% v / v isopropanol, pH 7.0.

[0129] The elution gradient is shown in Table 5.

[0130] Table 5 HIC column elution gradient

[0131] The results of drug / antibody ratio (DAR) and product distribution in the above reaction are shown in Table 6.

[0132] Table 6

[0133] As shown in Table 6, the content of D4 was significantly increased (more than 3%) after the addition of Mn 2+ compared with the sample without the addition of Mn 2+ . 2+

[0134] Example 4. Preparation of Herceptin-MC-GGFG-DXD conjugate by using the method according to the present application and homogeneity of the conjugate

[0135] The Herceptin-MC-GGFG-DXD conjugate was prepared in a one-pot reaction (the ratios below refer to molar ratios):

[0136] ​​(1) ZnCl2(ZnCl2 / antibody ratio of 4.0), CaCl2(CaCl2 / antibody ratio as shown in Table 8), and TCEP (TCEP / antibody ratio of 3.5) were added to a solution of the antibody (Herceptin was dissolved in 40 mM phosphate buffer, pH 7.0 at a concentration of 5.56 mg / mL) and the reaction mixture was allowed to react at a reaction temperature of 12°C in a 1.5 mL reaction vessel at a total reaction scale of 1 mg (reaction volume 0.18 mL), and the reduction reaction time was 16 h;

[0137] (2) MC-GGFG-DXD (CAS: 1599440-33-1, MC-GGFG-DXD / antibody ratio of 10.0, solution volume 0.02 mL) in DMSO (dimethyl sulfoxide, commercially available from AppliChem) was introduced to the reaction mixture in (1), and the reaction was continued at 12°C for 2 hours; the reaction concentration of the antibody after the addition of DMSO in this step was 5 mg / mL, and the reaction volume was 0.2 mL; the DMSO (containing MC-GGFG-DXD) solution accounted for 10% of the total volume.

[0138] (3) NAC (NAC / antibody ratio of 12.0) was added to the reaction mixture in (2) to deplete excess MC-GGFG-DXD;

[0139] (4) EDTA (EDTA / antibody ratio of 30.0) was added to the reaction mixture in (3) to capture metal ions, and DHAA (commercially available from BIOSYNTH, DHAA / antibody ratio of 8.0) was added to oxidize excess thiol, to obtain a Herceptin-MC-GGFG-DXD conjugate.

[0140] Finally, HIC-HPLC was used to analyze the drug / antibody ratio (DAR) and product distribution. The detection of DAR was performed by hydrophobic interaction chromatography (HIC) on a HIC column (Phenyl-5PW Column, 7.7 mm x 7.5 cm, 10 μm) at a flow rate of 0.5 mL / min at 2-8°C. The mobile phase A was 1 M (NH4)2SO4 and 50 mM phosphate, pH 7.0; the mobile phase B was 50 mM phosphate and 20% v / v isopropanol, pH 7.0.

[0141] The elution gradient is shown in Table 7.

[0142] Table 7 HIC column elution gradient

[0143] ​The results of the TCEP / antibody ratio in the above reaction and the drug / antibody ratio (DAR) and product distribution are shown in Table 8.

[0144] Table 8

[0145] As shown in Table 8, in the presence of Zn 2+ CaCl2significantly increased the D4 content (~5%) compared to the sample without Ca 2+ Cl2. 2+

[0146] Example 5. Preparation of Herceptin-MC-MMAF conjugate by using the method according to the present application and homogeneity of the conjugate

[0147] The Herceptin-MC-MMAF conjugate was prepared in a one-pot reaction (the ratios below refer to molar ratios):

[0148] (1) ZnCl2(ZnCl2 / antibody ratio of 4.0), CaCl2(CaCl2 / antibody ratio as shown in Table 10), and TCEP (TCEP / antibody ratio of 3.5) were added to a solution of the antibody (Herceptin was dissolved in 40 mM phosphate buffer, pH 7.0, at a concentration of 5.56 mg / mL) and the reaction mixture was allowed to react at a reaction temperature of 12°C in a 1.5 mL reaction vessel at a total reaction scale of 1 mg (reaction volume 0.18 mL), the reduction reaction time being 16 h;

[0149] (2) MC-MMAF (CAS: 863971-19-1, MC-MMAF / antibody ratio of 10.0) in DMA (dimethylacetamide, commercially available from General) (solution volume 0.02 mL) was introduced to the reaction mixture in (1) and the reaction was continued at 12°C for 2 hours; the reaction concentration of the antibody after the addition of DMA (containing MC-MMAF) in this step was 5 mg / mL, the reaction volume was 0.2 mL; the DMA (containing MC-MMAF) solution accounted for 10% of the total volume.

[0150] (3) NAC (NAC / antibody ratio of 12.0) was added to the reaction mixture in (2) to consume excess MC-MMAF;

[0151] (4) EDTA (EDTA / antibody ratio of 30.0) was added to the reaction mixture in (3) to capture metal ions and DHAA (commercially available from BIOSYNTH, DHAA / antibody ratio of 8.0) was added to oxidize excess thiol to obtain the Herceptin-MC-MMAF conjugate.

[0152] ​Finally, the drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. The detection of DAR was performed by hydrophobic interaction chromatography (HIC) at a flow rate of 1 mL / min on a HIC column ( Butyl-NPR Column, 4.6mm x 3.5cm, 2.5μm) at ambient temperature. Mobile phase A was 1.5M (NH4)2SO4 and 50mM dipotassium hydrogen phosphate, pH 7.0; mobile phase B was 50mM dipotassium hydrogen phosphate and 25% v / v isopropanol, pH 7.0.

[0153] The elution gradient is shown in Table 9.

[0154] Table 9 HIC column eluent gradient

[0155] The results of the TCEP / antibody ratio, drug / antibody ratio (DAR), and product distribution in the above reaction are shown in Table 10.

[0156] Table 10

[0157] As shown in Table 10, in the presence of Zn 2+ compared with the sample without Ca 2+ after adding Ca 2+ the content of D4 increased significantly (more than 6%).

[0158] Example 6. Preparation of Cetuximab-MC-VC-PAB-MMAE conjugate by using the method according to the present invention and the homogeneity of the conjugate

[0159] The Cetuximab-MC-VC-PAB-MMAE conjugate was prepared by a one-pot reaction (the following ratios refer to molar ratios):

[0160] (1) ZnCl2 (ZnCl2 / antibody ratio of 4.0), CaCl2 (CaCl2 / antibody ratio as shown in Table 12), and TCEP (TCEP / antibody ratio of 3.5) were added to a solution of Cetuximab (https: / / go.drugbank.com / drugs / DB00002) (Cetuximab was dissolved in 40mM phosphate buffer at a concentration of 5.56mg / mL, pH 7.0) and the reaction mixture was allowed to react in a 1.5mL reaction vessel at a reaction temperature of 12°C with a total reaction scale of 1mg (reaction volume 0.18mL) for 16h for the reduction reaction;

[0161] (2) To the reaction mixture in (1), MC-VC-PAB-MMAE in DMA (dimethylacetamide, commercially available from General) was introduced (MC-VC-PAB-MMAE / antibody ratio was 10.0, solution volume 0.02 mL) and the reaction was continued for 2 hours at 12°C; the reaction concentration of antibody after this step was 5 mg / mL and the reaction volume was 0.2 mL; the DMA (containing MC-VC-PAB-MMAE) solution accounted for 10% of the total volume.

[0162] (3) To the reaction mixture in (2), NAC (NAC / antibody ratio was 12.0) was added to deplete excess MC-VC-PAB-MMAE;

[0163] (4) To the reaction mixture in (3), EDTA (EDTA / antibody ratio was 30.0) was added to capture metal ions and DHAA (commercially available from BIOSYNTH, DHAA / antibody ratio was 8.0) was added to oxidize excess thiol to obtain Cetuximab-MC-VC-PAB-MMAE conjugate.

[0164] Finally, HIC-HPLC was used to analyze the drug / antibody ratio (DAR) and product distribution. The detection of DAR was performed by hydrophobic interaction chromatography (HIC) on a HIC column (Tosoh Butyl-NPR Column, 4.6 mm x 3.5 cm, 2.5 μm) at ambient temperature with a flow rate of 1 mL / min. Mobile phase A was 1.5 M (NH4)2SO4 and 50 mM potassium phosphate dibasic, pH 7.0; mobile phase B was 50 mM potassium phosphate dibasic and 25% v / v isopropanol, pH 7.0. Butyl-NPR Column,4.6mm x3.5cm,2.5μm) on a HIC column (Tosoh Butyl-NPR Column, 4.6 mm x 3.5 cm, 2.5 μm) at ambient temperature with a flow rate of 1 mL / min. Mobile phase A was 1.5 M (NH4)2SO4 and 50 mM potassium phosphate dibasic, pH 7.0; mobile phase B was 50 mM potassium phosphate dibasic and 25% v / v isopropanol, pH 7.0.

[0165] The elution gradient is shown in Table 11.

[0166] Table 11 HIC column elution gradient

[0167] The TCEP / antibody ratio in the above reaction and the results of drug / antibody ratio (DAR) and product distribution are shown in Table 4.

[0168] Table 12

[0169] As shown in Table 12, in the presence of Zn 2+ , the D4 content was significantly improved (more than 6%) after the addition of Ca 2+ . 2+

[0170] ​Example 7. Preparation of Datopotamab-MC-VC-PAB-MMAE conjugate by using the method according to the present application and homogeneity of the conjugate

[0171] Datopotamab-MC-VC-PAB-MMAE conjugate was prepared in one-pot reaction (the following ratios refer to molar ratios):

[0172] (1) ZnCl2(ZnCl2 / antibody ratio of 4.0), CaCl2(CaCl2 / antibody ratio as shown in Table 14), and TCEP (TCEP / antibody ratio of 3.5) were added to a solution of Datopotamab (https: / / go.drugbank.com / drugs / DB16410) dissolved in 40 mM phosphate buffer, pH 7.0 at a concentration of 5.56 mg / mL and the reaction mixture was allowed to react at a reaction temperature of 12°C in a 1.5 mL reaction vessel at a total reaction scale of 1 mg (reaction volume 0.18 mL), the reduction reaction time being 16 h;

[0173] (2) MC-VC-PAB-MMAE (MC-VC-PAB-MMAE / antibody ratio of 10.0) in DMA (dimethylacetamide, commercially available from General) was introduced to the reaction mixture in (1) and the reaction was continued at 12°C for 2 hours; the reaction concentration of the antibody after the addition of DMA in this step was 5 mg / mL and the reaction volume was 0.2 mL; the DMA (containing MC-VC-PAB-MMAE) solution accounted for 10% of the total volume.

[0174] (3) NAC (NAC / antibody ratio of 12.0) was added to the reaction mixture in (2) to deplete excess MC-VC-PAB-MMAE;

[0175] (4) EDTA (EDTA / antibody ratio of 30.0) was added to the reaction mixture in (3) to capture metal ions and DHAA (commercially available from BIOSYNTH, DHAA / antibody ratio of 8.0) was added to oxidize excess thiol to obtain Datopotamab-MC-VC-PAB-MMAE conjugate.

[0176] Finally, the drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. The HIC column (Toyopearl Butyl-650S, 21.5 x 250 mm, Tosoh Bioscience) was equilibrated with 50 mM Na2SO4, 50 mM NaH2PO4, 0.02% NaN3, pH 7.0 (Buffer A) and the sample was eluted with a linear gradient of Buffer A to Buffer B (50 mM Na2SO4, 50 mM NaH2PO4, 0.02% NaN3, pH 7.0) at a flow rate of 0.5 mL / min. DAR was detected by hydrophobic interaction chromatography (HIC) on a Butyl-NPR Column, 4.6 mm x 3.5 cm, 2.5 μιη at ambient temperature with a flow rate of 1 mL / min. Mobile phase A was 1.5 M (NH4)2S04and 50 mM potassium phosphate dibasic, pH 7.0; mobile phase B was 50 mM potassium phosphate dibasic and 25% v / v isopropanol, pH 7.0.

[0177] The elution gradient is shown in Table 13.

[0178] Table 13 HIC column elution gradient

[0179] The results of the TCEP / antibody ratio and drug / antibody ratio (DAR) and product profile in the above reaction are shown in Table 14.

[0180] Table 14

[0181] As shown in Table 14, in the presence of Zn 2+ , the D4 content was significantly increased (~8%) after the addition of Ca 2+ compared to the sample without Ca 2+ .

Claims

1. A method for preparing an antibody-drug conjugate (ADC) with improved homogeneity, comprising the following steps: (a) incubating a reducing agent and an antibody to be conjugated in the presence of an effective amount of a first metal ion and a second metal ion, performing a reduction reaction, to obtain a reaction mixture.

2. The method of claim 1, wherein, The method further comprises the following step: (b) reacting a linker-payload carrying a reactive group with the reaction mixture obtained in step (a).

3. The method of claim 1 or 2, wherein, The method further comprises the following step: (c) oxidizing the reaction mixture obtained in step (b) with an oxidizing agent.

4. The method of any one of claims 1 to 3, wherein, The method further comprises the following step: (d) recovering the antibody-drug conjugate obtained in step (c).

5. The method of any one of claims 1 to 4, wherein, The reducing agent of step (a) is selected from one or more of tris(2-carboxyethyl)phosphine (TCEP) and its hydrochloride salt, diphenylphosphinoacetic acid, 2-[2-(diphenylphosphino)ethyl]pyridine, 3-(diphenylphosphino)benzenesulfonic acid, 4-(diphenylphosphino)benzoic acid, 2-(diphenylphosphino)ethylamine, 3-(diphenylphosphino)propylamine, 3-(diphenylphosphino)propanoic acid, 2-(diisopropylphosphino)ethylamine, 2-(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphine, 1,3,5-triaza-7-phosphanorbornane, n-butyldi(1-adamantyl)phosphine, tris(3-hydroxypropyl)phosphine (THPP), 2-(diphenylphosphino)benzenesulfonic acid (diPPBs), dithiothreitol (DTT), 2-(diphenylphosphino)acetic acid (DPAA), dithioerythritol (DTE), β-mercaptoethanol, LiAlH4, Na2S2O3, KBH4, or hydrazine.

6. The method of any one of claims 1 to 5, wherein, The pH value of the buffer system in step (a) is about 5.5 to 9; preferably, the pH value of the buffer system used in step (a) is about 5.5 to 8, preferably, the pH value of the buffer system used in step (a) is 7.

7. The method of any one of claims 1 to 6, wherein, The buffer system of step (a) comprises one or more of HEPES, PBS, MES, Tris, TAPS, Bicine, Tricine, TES, MOPS, PIPES, sodium citrate, or histidine buffer.

8. The method of any one of claims 1 to 7, wherein, The first metal ion and the second metal ion in step (a) are independently selected from the group consisting of: Zn 2+ , Cd 2+ , Hg 2+ , Mg 2+ , Sr 2+ , Ba 2+ , Ti 2+ , Ti 3+ , Zr 2+ , Cr 2+ , Cr 3+ , Mo 2+ , Mn 2+ , Mn 3+ , Fe 2+ , Fe 3+ , Co 2+ , Co 3+ , Ni 2+ , Pd 2+ , Pt 2+ , Cu + , Cu 2+ , Ag + and / or Ca 2+ , preferably from the group consisting of Zn 2+ and Ca 2+ or Mn 2+ , preferably the first metal ion is Zn 2+ and the second metal ion is Ca 2+ , preferably the first metal ion is Zn 2+ and the second metal ion is Mn 2+ ; more preferably the first metal ion and the second metal ion are selected from the group consisting of: Zn 2+ and Ca 2+ , Zn 2+ and Mn 2+ , Zn 2+ and Ni 2+ , Zn 2+ and Fe 2+ , Zn 2+ and Fe 3+ , Zn 2+ and Cd 2+ , Zn 2+ and Cu 2+ , Mn 2+ and Ni 2+ , Mn 2+ and Fe 2+ , Mn 2+ and Fe 3+ , Mn 2+ and Cu 2+ , Mn 2+ and Ca 2+ , Ni 2+ and Fe 2+ , Ni 2+ and Fe 3+ , Ni 2+ and Cu 2+ , Ni 2+ and Ca 2+ , Fe 2+ and Fe 3+ , Fe 2+ and Cu 2+ , Fe 2+ and Ca 2+ , Fe 3+ and Cu 2+ , Fe 3+ and Ca 2+ , Cu 2+ and Ca 2+ ; wherein the ratio of the first ion to the antibody in step (a) is in the range of about 1:1 to 30:1, preferably in the range of about 1:1.5 to 20:1, preferably in the range of about 2:1 to 16:1, preferably in the range of about 4:1 to 8:1, by molar ratio; wherein the ratio of the second ion to the antibody in step (a) is above 0:1, preferably in the range of about 1:1 to 30:1, preferably in the range of about 1.5:1 to 20:1, preferably in the range of about 2:1 to 16:1, preferably in the range of about 3:1 to 11:1, preferably in the range of about 4:1 to 8:1, by molar ratio; wherein the ratio of the sum of the first ion and the second ion to the antibody in step (a) is in the range of about 1:1 to 30:1, preferably in the range of about 2:1 to 20:1, preferably in the range of about 4:1 to 16:1, more preferably in the range of about 8:1 to 12:1, by molar ratio; or wherein the ratio of the first ion to the second ion in step (a) is in the range of about 1 :0.2 to 1 :15, preferably in the range of about 1 :0.5 to 1 :8, preferably in the range of about 1 :1 to 1 :4, more preferably in the range of about 1 :2 to 1 :4 by molar ratio.

9. The method of any one of claims 1 to 8, wherein, The incubation temperature in step (a) is about 0 °C to room temperature (about 20-25 °C); preferably, the incubation temperature in step (a) is about 12 °C to 15 °C; and / or, the incubation time in step (a) is about 2-24 hours, preferably 4-20 hours, more preferably 8-20 hours, still more preferably 12-20 hours, most preferably about 12 hours; most preferably, the reaction conditions in step (a) are a reaction at a temperature of about 12 °C to 20 °C for 12-24 hours.

10. The method of any one of claims 1 to 9, wherein, The antibody in step (a) is selected from the group consisting of a monoclonal antibody and a polyclonal antibody, an antibody fusion protein; or, the antibody is selected from the group consisting of a human antibody, a humanized antibody, and a chimeric antibody; preferably, the monoclonal antibody is selected from the group consisting of trastuzumab, pertuzumab, sibrotuzumab, abciximab, adalimumab, alfaferone, alemtuzumab, basiliximab, belimumab, belotufosumab, canakinumab, pexidartinib, cetuximab, daclizumab, denosumab, efalizumab, guselkumab, infliximab, ipilimumab, isibizumab, natalizumab, nivolumab, olaratumab, ocrelizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, sucralfate, and ustekinumab.

11. The method of any one of claims 1 to 10, wherein, The linker comprises a reactive group or moiety selected from the group consisting of maleimide, bromo, iodo, sulfone, mono or di derivatives of disulfide, bicyclo[l.l.0]butane, sulfonyl fluoride, pentafluorophenol ester, palladium oxidative addition complex, iodoxolones, highly electron-deficient arenes, alkenes or alkynes bearing electron-deficient groups.

12. The method of any one of claims 1 to 11, wherein, the drug in the linker-drug moiety is selected from the group consisting of diagnostic agents, therapeutic agents, and labeling agents; or, the drug in the linker-drug moiety is selected from the group consisting of cytotoxic agents, toxins, radionuclides, fluorescent agents (e.g., amine derivatized fluorescent probes such as 5-dimethylaminonaphthalene-l-(N-(2-aminoethyl))sulfonamide-dansyl ethylenediamine, Oregon Green® 488cadaverine, dansylcadaverine, N-(2-aminoethyl)-4-amino-3,6-dithio-l,8-naphthalimide, dipotassium salt (fluorophor Yellow ethylenediamine), rhodamine B ethylenediamine, or thiol derivatized fluorescent probes such as FLL-cysteine, chemotherapeutic agents, immunotherapeutic agents, antiviral agents, antimicrobial agents, molecular degrading agents, immune agonists, and nuclear pharmaceutical chelators; or, the drug in the linker-drug moiety is selected from the group consisting of Auristatin drugs, topoisomerase inhibitors, Maytansinoid drugs, and PBD drugs.

13. An antibody-drug conjugate prepared by the method of any one of claims 1-12, wherein the content of antibody-drug conjugates having a DAR value of 4 is greater than 60%, preferably greater than 65%, more preferably greater than 70%, for example greater than 71%, 72%, 73% or 74%; or wherein the sum of the content of antibody-drug conjugates having a DAR value of 0 (DAR0 or D0) and a DAR value of 8 (DAR8 or D8) is less than 20%, preferably less than 15%, more preferably less than 10%; or wherein the content of antibody-drug conjugates having a DAR value of 6 (DAR6 or D6) is less than 20%, preferably less than 15%, more preferably less than 10%.

14. A pharmaceutical composition comprising an antibody-drug conjugate prepared by the method of any one of claims 1-12 and a pharmaceutically acceptable carrier or excipient.

15. Use of an antibody-drug conjugate prepared by the method of any one of claims 1-12 or a pharmaceutical composition according to claim 14 for the manufacture of a medicament for the treatment of a disease selected from the group consisting of cancer, autoimmune diseases, inflammation and metabolic diseases.

16. A method of treating a disease selected from the group consisting of cancer, autoimmune disease, inflammation and metabolic disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate prepared by the method of any one of claims 1-12 or a pharmaceutical composition of claim 14.

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