Process for preparing antibody-drug conjugates with improved homogeneity

Abstract

The present invention relates to a bioconjugation process for preparing an antibody-drug conjugate (ADC) composition having improved homogeneity, wherein an antibody-drug conjugate (ADC) with a drug-to-antibody molar ratio of 2 (D2) is present in a high concentration in the composition. In the composition of the ADC produced by the process of this disclosure, the content of D2 is greater than 64 mol%, and the content of D0+D4+D6+D8 is less than 36 mol%, based on the total molar concentrations of D0, D2, D4, D6, and D8.

Description

[Technical Field]

[0001] Priority information This application claims the rights of PCT / CN2023 / 098791, filed on 7 June 2023.

[0002] Field of Invention This disclosure relates to a process for preparing antibody-drug conjugate (ADC) compositions. Specifically, this disclosure relates to a process for preparing antibody-drug conjugate (ADC) compositions in which an antibody-drug conjugate (ADC) with a drug-to-antibody molar ratio of 2 (i.e., DAR2) is present at a high concentration and the homogeneity of the antibody-drug conjugate composition is improved. [Background technology]

[0003] Background of the Invention Antibody-drug conjugates refer to a group of therapeutic molecules in which a therapeutic agent binds to a target-specific antibody, which then guides and releases the therapeutic agent to its target, thereby producing a therapeutic effect. The specificity of antibodies to specific antigens on the surface of target cells and molecules has led to their widespread use as carriers for various diagnostic and therapeutic agents. For example, antibodies conjugated to labeling and reporter groups, such as fluorophores, radioisotopes, and enzymes, have found use in labeling and imaging applications, while antibodies conjugated to cytotoxic and chemotherapeutic agents enable targeted delivery of such drugs to specific tissues or structures, such as specific cell types or growth factors, minimizing the impact on normal, healthy tissue and significantly reducing side effects associated with chemotherapy.

[0004] In the development of antibody-drug conjugates, a therapeutic agent can be coupled to an antibody that specifically targets a particular tumor marker (e.g., a protein found only on tumor cells or on the surface of tumor cells). The antibody tracks these proteins in the body and binds to the surface of cancer cells. The biochemical reaction between the antibody and the target protein (i.e., antigen) triggers a signal within the tumor cell, which then absorbs the antibody together with the therapeutic agent (e.g., a cytotoxic drug) or allows it to move into the tumor cell. After ADCs have moved internally, cytotoxic agents are released, killing tumor cells (Chari, Ravi VJ; Martell, Bridget A.; Gross, Jonathan L.; Cook, Sherrilyn B.; Shah, Sudhir A.; Blattler, Walter A.; McKenzie, Sara J.; Goldmacher, Victor S. (1992). "Immunoconjugates containing novel maytansinoids: promising anticancer drugs." Cancer Research. Vol. 52 (No. 1): pp. 127-131). Thanks to this targeting, ideally, ADCs have fewer side effects than other chemotherapeutic agents and offer a broad therapeutic window.

[0005] For drug binding, functional groups with high reactivity and stability to both the antibody and the linker payload (i.e., the linker drug) are used in the coupling to form a stable covalent bond. Conventional binding methods, i.e., covalent binding of the drug moiety to the antibody via a linker, generally result in a heterogeneous molecular mixture in which the drug moiety is bound to several sites on the antibody. For example, cytotoxic drugs typically conjugate to antibodies via a large number of lysine residues, often resulting in a heterogeneous antibody-drug conjugate mixture.

[0006] For example, antibody-drug conjugates are typically produced by two conventional chemical strategies: lysine-based conjugation and cysteine-based conjugation from the reduction of interchain sulfide bonds. Due to the reaction of primary amine groups on lysine residues, the most widely used connector for linker payloads is the NHS ester (i.e., N-hydroxysuccinimide). However, the application of NHS esters in antibody-drug conjugate production is limited by their inherent properties. For instance, the reaction between NHS esters and primary amines is very slow under acidic conditions, so conjugation must be carried out in buffers with a high pH value (i.e., >7.0), which is sometimes unfavorable for the antibody. Furthermore, NHS is readily hydrolyzed under basic conditions, which complicates the purification and identification of the free drug after conjugation. Additionally, due to the low reactivity of NHS esters to primary amines on antibodies, the reaction must be carried out at high temperatures (i.e., 22°C). Furthermore, due to its low solubility, more organic solvents are required for the linker payload prepared by the NHS ester (i.e., SMCC-DM1) to completely dissolve in the reaction system, which increases the risk of antibody aggregation. Cysteine-based conjugation from reduction of interchain sulfide bonds involves the steps of opening the interchain disulfide bonds in the presence of various reducing agents, e.g., TCEP, DTT, followed by a nucleophilic reaction of the thiol group. In this conjugation process, the antibody-drug conjugate is typically formed by the conjugation of one or more antibody cysteinethiols with one or more linker moieties bound to the drug, thereby forming an antibody-linker-drug complex. Unlike most amines, which are protonated and have low nucleophilicity around pH 7, cysteinethiols are reactive at neutral pH. Because free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins containing cysteine ​​residues often exist in their oxidized form as disulfide-linked oligomers or have internally cross-linked disulfide groups.Antibody cysteinethiol groups are generally more reactive to electrophilic conjugation reagents than antibody amine or hydroxyl groups, i.e., more nucleophilic. Modifying cysteinethiol groups to cysteine ​​amino acids by mutations in various amino acid residues of proteins is potentially problematic, especially in the case of unpaired (free Cys) residues or those that are relatively available for reaction or oxidation. In concentrated protein solutions, whether from E. coli periplasm, culture supernatant, or partially or completely purified protein, unpaired Cys residues on the protein surface can pair and oxidize to form intermolecular disulfides, thus forming protein dimers or polymers. Disulfide dimerization makes the new Cys unresponsive to conjugation to drugs, ligands, or other labels. Furthermore, if the protein oxidatively forms an intramolecular disulfide bond between the newly modified Cys and the existing Cys residue, both Cys groups become unavailable for participation and interaction in the active site. Furthermore, proteins can become inactive or nonspecific due to misfolding or deletion of tertiary structure (Zhang et al. (2002) Anal. Biochem. Vol. 311: pp. 1-9).

[0007] Developing new or ADCs as therapeutic agents is of significant importance. However, conventional conjugation processes always result in heterogeneous molecular mixtures where the drug moiety binds to several sites on the antibody. Depending on the reaction conditions, the heterogeneous mixture typically contains a distribution of antibodies with 0 to about 8 or more attached drug moieties. In addition, within each subgroup of conjugates having a specific integer ratio of drug moiety to a single antibody, there are potentially heterogeneous mixtures where the drug moieties are attached to various sites on the antibody. Analytical and preparative methods are insufficient to separate and characterize the antibody-drug conjugate species molecules within the heterogeneous mixtures produced by the conjugation reaction. The heterogeneous mixtures are very complex and thus difficult and costly to characterize and purify. Each conjugation product in such mixtures potentially has different pharmacokinetic, distribution, toxicity, and efficacy profiles, and non-specific conjugation also frequently results in impairment of antibody function. Antibodies are large, complex, and structurally diverse biomolecules that often have multiple reactive functional groups. Antibody reactivity with linker reagents and drug linker intermediates is influenced by factors such as pH, concentration, salt concentration, and co-solvent.

[0008] Furthermore, the number of drugs coupled to a single antibody molecule is a crucial factor for the efficacy and safety of the resulting ADC. For example, in conjugation processes based on the reduction of undenatured interchain disulfide bonds, interchain disulfide bonds have greater availability to the solvent than other disulfide bonds. Therefore, interchain disulfide bonds can be used as binding sites for coupling drugs (or drug linkers) to antibodies. Generally, a single therapeutic antibody molecule belonging to the IgG1 or IgG4 subclass has four interchain disulfide bonds, each formed by two -SH groups, and thus the number of drugs coupled to a single antibody molecule is 2, 4, 6, or 8. When the number of drugs coupled to a single antibody molecule is 0 (i.e., the drug-to-antibody molar ratio is 0 (i.e., DAR0)), the product is designated as D0 (i.e., representing a single antibody molecule that is not actually conjugated with any drug). Therefore, D2 refers to an ADC molecule in which two drug molecules are coupled to one single antibody molecule (i.e., a drug-to-antibody molar ratio of 2 (i.e., DAR2)), where the two drug molecules can be coupled to -SH groups produced by the reduction of SS bonds between the heavy and light chains, or to -SH groups produced by the reduction of SS bonds between the heavy and heavy chains. D4 refers to an ADC molecule in which four drug molecules are coupled to one single antibody molecule (i.e., a drug-to-antibody molar ratio of 4 (i.e., DAR4)). D6 refers to an ADC molecule in which six drug molecules are coupled to one single antibody molecule (i.e., a drug-to-antibody molar ratio of 6 (i.e., DAR6)). And D8 refers to an ADC molecule in which eight drug molecules are coupled to one single antibody molecule (i.e., a drug-to-antibody molar ratio of 8 (i.e., DAR8)), where all four SS bonds of one antibody molecule are reduced to eight -SH groups, and each -SH group is bound to one drug molecule. Generally, the heterogeneous mixture of ADC molecules produced by conventional conjugation processes is a mixture of D0, D2, D4, D6, and D8.Of these, D0 lacked ADC efficacy, and D6 and D8 are considered to be the reason for in-vivo instability and unpredictable safety and PK profiles due to hydrophobicity induced by the payload (i.e., drug) molecule. Although the in vitro efficacy of antibody-drug conjugates has been shown to depend directly on drug loading (Hamblett KJ et al., Clin Cancer Res. October 15, 2004; Vol. 10 (No. 20): pp. 7063-70), the in vivo antitumor activity of an antibody-drug conjugate with four drug molecules per molecule (D4) was comparable to that of a conjugate with eight drug molecules per molecule (D8) at the same mAb dose, even when the conjugate contained half the amount of drug per mAb. Drug loading also affected plasma clearance, with the D8 conjugate being cleared three times faster than the D4 conjugate and five times faster than the D2 conjugate. Therefore, on the one hand, for payloads with superior efficacy (i.e., highly active payloads), conjugates prepared as D2 can control the safety risks of the drug and improve the circulating stability of the conjugate in vivo, while ensuring efficacy. On the other hand, for specific mechanisms of action and spatial structures such as nucleic acid loading or immunoantagonists, D2 conjugates can minimize the impact of the conjugated high molecular weight payload on antibody function. Generally, when the D2 content in an ADC mixture is high, the ADC is considered to have high homogeneity. Antibody-drug conjugates with improved homogeneity offer therapeutic benefits, such as a higher therapeutic index of the drug, improved efficacy, and reduced toxicity. Homogeneous antibody mixtures also provide more accurate and consistent measurements for diagnostic and imaging applications. Thus, novel preparation processes for ADCs with high homogeneity are highly desirable and have been pursued for a long time.

[0009] Therefore, several methods have been developed to improve the uniformity of antibody-drug conjugates. In this regard, several site-specific labeling techniques have been developed and applied to prepare ADCs for preclinical and clinical research.

[0010] First, the introduction of genetically engineered reactive cysteine residues has become a conventional approach for site-specific conjugation. For example, the ThioMab technology developed by Genentech can be cited. However, the main limitation of the ThioMab technology is that incorrect disulfide bonds may be formed between the two Fabs in the antibody by the thiol group introduction step, which remains an issue to be solved (1).

[0011] Another method of site-specific conjugation is the introduction of non-natural amino acids. The special functional groups of these non-natural amino acids enable site-specific conjugation. However, the production of modified antibodies can be difficult, and antibodies containing non-natural amino acids may induce immunogenicity (2). The hydrophobicity of non-natural amino acids also increases the risk of antibody aggregation (3).

[0012] Ligation using enzymes is also an effective strategy for site-specific conjugation. Through genetic engineering, specific amino acid sequences are artificially induced to be expressed within the antibody, and these sequences are recognized by specific enzymes. Subsequently, specific amino acid residues are modified by the enzymes, enabling site-specific conjugation (4). However, it is worth noting that the modification of amino acid sequences may induce immunogenicity, and the conjugation process of ligation using enzymes becomes more complex.

[0013] Site-specific ADCs can also be generated from glycan remodeling and glycoconjugation. Human IgG molecules have conserved glycosylation sites at each N297 residue of the CH2 domain, and since these glycosylation sites are sufficiently far from the variable region, it is unlikely that conjugation to the bound glycan will affect antigen binding. However, glycosylation is a heterologous posttranslational modification, and the generation of homoglycans for chemical modification is a challenging task. Another complexity associated with glycoengineering approaches is that the produced conjugates may be immunogenic (4).

[0014] Overall, these techniques involve protein engineering, linker payload modification, and / or enzyme catalysis, and therefore have several drawbacks, including reduced antibody expression levels, increased purification complexity, and high costs (5). However, modifying antibody molecules with free cysteine ​​residues still carries the risk of causing rearrangement and recombination reactions with existing cysteine ​​residues within the molecule during antibody folding and assembly, or dimerization through reactions with free cysteine ​​residues in other antibody molecules, potentially leading to impaired antibody function or aggregation.

[0015] Therefore, there is a continued need for the development of novel bioconjugation processes that can generate ADCs without genetic engineering and modification of the linker payload. This disclosure relates to a process for preparing a composition containing an antibody-drug conjugate (ADC), wherein the antibody-drug conjugate (ADC) has a drug-to-antibody molar ratio of 2 (i.e., DAR2) and is present at high concentrations, the antibody-drug conjugate has improved homogeneity, and offers more advantages in terms of safety, in vivo stability, and maintenance of antibody function. The process is simple to perform and low-cost. [Overview of the Initiative] [Problems that the invention aims to solve]

[0016] This disclosure aims to develop a bioconjugation process for preparing compositions containing antibody-drug conjugates (ADCs) with improved homogeneity. In this bioconjugation process, antibody-drug conjugates (ADCs) with a drug-to-antibody molar ratio of 2 (i.e., D2) are present in the composition at high concentrations, and the process is simple and cost-effective. Compared to conventional conjugation processes involving interchain disulfide bond reactions, the content of antibody-drug conjugates (ADCs) with a drug-to-antibody molar ratio of 2 (i.e., D2) exceeds 65 mol% based on the total molar concentration of the produced antibody-drug conjugates (ADCs) (i.e., D0, D2, D4, D6, and D8), thus dramatically improving the homogeneity of antibody-drug conjugates (ADCs) produced from the bioconjugation process of this disclosure. [Means for solving the problem]

[0017] In a first embodiment, the present disclosure relates to a process for preparing an antibody-drug conjugate (ADC) composition, which is described below: (a) Reduction step: Incubate a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP), THPP (tris(3-hydroxypropyl)phosphine), diPPB (2-diphenylphosphanylbenzenesulfonic acid), DTT (dithiothreitol), DPAA (2-(diphenylphosphino)acetic acid), DTE (dithioerythritol), β-mercaptoethanol, LiAlH4, Na2S2O3, KBH4, hydrazine, etc.) and the antibody to be conjugated in a buffer system (e.g., TAPS, bicine, tris, tricine, HEPES, TES, MOPS, PIPES, sodium citrate, histidine buffer, PB (phosphate buffer), PBS, MES, etc.) to reduce the interchain disulfide bonds in the antibody, and optionally purify the reduced antibody; (b) Reoxidation step: Add an oxidizing agent (e.g., hydrogen peroxide (H2O2), nitrate compounds, potassium chlorate (KClO3), peroxydisulfate (H2S2O8), peroxymonosulfate (H2SO5), NaClO, sodium dichromate (Na2Cr2O7), permanganate compounds, sodium perborate, nitrous oxide, sodium bismuthate (NaBiO3), cerium sulfate, NAD, DHAA, DNTB, NADP, 2-aminophenyl disulfide (DDD), etc.) to reoxidize some of the reduced thiol groups, and optionally purify the reoxidized antibody; Step (a) or step (b) involves a transition metal ion (e.g., Zn 2+ Mn 2+ Ni 2+ Fe 2+ Fe 3+ Cu 2+ It is carried out in the presence of (etc.), In step (b), the reduced thiol group not blocked by the transition metal ion is reoxidized; (c) Conjugation step: removing transition metal ions (for example, by adjusting the pH (e.g., by adding NaOH, HCl, etc.), adjusting the temperature, changing the buffer (e.g., by adding NaCl, sodium citrate, etc.), or adding a metal chelating agent (e.g., EDTA, DTPA, ethylenediamine, 2,2'-bipyridine, 1,10-phenanthroline, etc.)), and adding an excess amount of reactive group (e.g., maleimide binder, etc.)-supported payload to react with the reduced thiol groups of the reoxidized antibody; and (d) Recovery step: The step of recovering the obtained antibody-drug conjugate to obtain the antibody-drug conjugate composition, The present invention relates to a process in which an antibody-drug conjugate (D2) having a drug-to-antibody molar ratio of 2 is present in the composition at a high concentration.

[0018] In the context of the present disclosure, the expression "step (a) or step (b) is carried out in the presence of transition metal ions" means that one of step (a) and step (b) is carried out in the presence of transition metal ions. For example, step (a) is carried out in the presence of transition metal ions, or step (b) is carried out in the presence of transition metal ions. However, the present invention does not exclude the possibility that both step (a) and step (b) are carried out in the presence of transition metal ions. For example, the method of the present application can be carried out in a one-pot manner. In this case, if transition metal ions are added in step (a), step (b) will also be carried out in the presence of transition metal ions. From the perspective of economic efficiency, the transition metal ions can be added only once, either in step (a) or step (b).

[0019] In one embodiment, the transition metal ions (e.g., Zn 2+ , Mn 2+ , Ni 2+ , Fe 2+ , Fe 3+ , Cu 2+ etc.) are provided by adding a soluble transition metal salt or a solution of a transition metal salt to step (a) or step (b). Free transition metal ions from the soluble transition metal salt or the solution of the transition metal salt can be released into the reaction solution.

[0020] In one embodiment, a process for preparing a composition of an antibody-drug conjugate (ADC) having improved uniformity comprises the following steps: (a) Reduction step: A reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP), THPP (tris(3-hydroxypropyl)phosphine), diPPB (2-diphenylphosphanylbenzenesulfonic acid), DTT (dithiothreitol), DPAA (2-(diphenylphosphino)acetic acid), DTE (dithioerythritol), β-mercaptoethanol, LiAlH4, Na2S2O3, KBH4, hydrazine, etc.) and an antibody to be conjugated are added to transition metal ions (e.g., Zn 2+ , Mn 2+ , Ni 2+ , Fe2+ Fe 3+ Cu 2+ A step of reducing the interchain disulfide bonds in the antibody by incubating it in a buffer system (e.g., TAPS, bicine, tris, tricine, HEPES, TES, MOPS, PIPES, sodium citrate, histidine buffer, PB (phosphate buffer), PBS, MES, etc.) in the presence of (etc.), and optionally purifying the reduced antibody; (b) Reoxidation step: Add an oxidizing agent (e.g., hydrogen peroxide (H2O2), nitrate compounds, potassium chlorate (KClO3), peroxydisulfate (H2S2O8), peroxymonosulfate (H2SO5), NaClO, sodium dichromate (Na2Cr2O7), permanganate compounds, sodium perborate, nitrous oxide, sodium bismuthate (NaBiO3), cerium sulfate, NAD, DHAA, DNTB, NADP, 2-aminophenyl disulfide (DDD), etc.) to reoxidize the reduced thiol groups that are not blocked by transition metal ions, and optionally purify the reoxidized antibody; (c) Conjugation step: removing transition metal ions (for example, by adjusting the pH (e.g., by adding NaOH, HCl, etc.), adjusting the temperature, changing the buffer (e.g., by adding NaCl, sodium citrate, etc.), or by using a metal chelating agent (e.g., EDTA, DTPA, ethylenediamine, 2,2'-bipyridine, 1,10-phenanthroline, etc.)), and adding an excess amount of reactive group (e.g., maleimide binder, etc.)-supported payload to react with the reduced thiol groups of the reoxidized antibody; and (d) Recovery step: The step of recovering the obtained antibody-drug conjugate to obtain the antibody-drug conjugate composition, The antibody-drug conjugate (D2), having a drug-to-antibody molar ratio of 2, is present in the composition at a high concentration.

[0021] In another embodiment, the process for preparing an antibody-drug conjugate (ADC) composition involves the following steps: (a) Reduction step: Incubate a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP), THPP (tris(3-hydroxypropyl)phosphine), diPPB (2-diphenylphosphanylbenzenesulfonic acid), DTT (dithiothreitol), DPAA (2-(diphenylphosphino)acetic acid), DTE (dithioerythritol), β-mercaptoethanol, LiAlH4, Na2S2O3, KBH4, hydrazine, etc.) and the antibody to be conjugated in a buffer system (e.g., TAPS, bicine, tris, tricine, HEPES, TES, MOPS, PIPES, sodium citrate, histidine buffer, PB (phosphate buffer), PBS, MES, etc.) to reduce the interchain disulfide bonds in the antibody, and optionally purify the reduced antibody; (b) Reoxidation step: Add a soluble transition metal salt or a solution containing a soluble transition metal salt, and add an oxidizing agent (e.g., hydrogen peroxide (H2O2), nitrate compounds, potassium chlorate (KClO3), peroxydisulfate (H2S2O8), peroxymonosulfate (H2SO5), NaClO, sodium dichromate (Na2Cr2O7), permanganate compounds, sodium perborate, nitrous oxide, sodium bismuthate (NaBiO3), cerium sulfate, NAD, DHAA, DNTB, NADP, 2-aminophenyl disulfide (DDD), etc.) to oxidize transition metal ions (e.g., Zn 2+ Mn 2+ Ni 2+ Fe 2+ Fe 3+ Cu 2+ A step of re-oxidizing a reduced thiol group that is not blocked by a transition metal ion in the presence of (etc.), and optionally purifying the re-oxidized antibody; (c) Conjugation step: removing transition metal ions (for example, by adjusting the pH (e.g., by adding NaOH, HCl, etc.), adjusting the temperature, changing the buffer (e.g., by adding NaCl, sodium citrate, etc.), or adding a metal chelating agent (e.g., EDTA, DTPA, ethylenediamine, 2,2'-bipyridine, 1,10-phenanthroline, etc.)), and adding an excess amount of reactive group (e.g., maleimide binder, etc.)-supported payload to react with the reduced thiol groups of the reoxidized antibody; and (d) Recovery step: The step of recovering the obtained antibody-drug conjugate to obtain the antibody-drug conjugate composition, The antibody-drug conjugate (D2), having a drug-to-antibody molar ratio of 2, is present in the composition at a high concentration.

[0022] In the present invention, "a high concentration of antibody-drug conjugates (ADCs) with a drug-to-antibody molar ratio of 2 (D2) is present in the composition" means that, in the ADC composition prepared by the process of the present invention, the content of D2 (i.e., antibody-drug conjugates (ADCs) with a drug-to-antibody molar ratio of 2) exceeds 64 mol%, based on the total molar concentration of the antibody-drug conjugates (ADCs) produced (i.e., D0, D2, D4, D6, and D8).

[0023] In one embodiment, the reducing agent in step (a) is TCEP. In one embodiment, in step (a), the molar ratio of reducing agent to antibody in the reaction solution is in the range of 1 to 20, for example, 2 to 16, preferably 3 to 8. In a particular embodiment, in step (a), the reducing agent is added to the antibody in a molar ratio of 8, i.e., the molar ratio of reducing agent to antibody is 8.

[0024] Transition metal ions suitable for use in the bioconjugation process of this disclosure include Zn 2+ Mn 2+ Ni 2+ Fe 2+ Fe 3+Cu 2+ These may include, but are not limited to, Zn. In particular, due to its easy availability and low cost, 2+ For example, a suitable transition metal salt can be added in step (a) as long as it is soluble in the reaction solution, so that free transition metal ions can be released into the reaction solution. In this regard, suitable zinc salts include ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, Zn(ClO4)2, zinc formate, and zinc tetrafluoroborate. Similarly, soluble and Mn 2+ Ni 2+ Fe 2+ Fe 3+ Cu 2+ Other transition metal salts that can release free transition metal ions into the reaction solution include, but are not limited to, Cu, Ni, Co, Fe, Mn, Cr, V, Ti, Sc, Mg, Sr, Y, Zr, Nb, Mo, Ru, Rh, Pd, Ag, Cd, Ga, and Ge.

[0025] In one embodiment, the transition metal ion in step (a) or step (b) is Zn 2+ In one embodiment, in step (a) or step (b), Zn in the reaction solution 2+ The molar ratio of antibody to transition metal ions is in the range of 0.5 to 50, preferably 1 to 16, and more preferably 2 to 8. In certain embodiments, in step (a) or step (b), transition metal ions are added to the antibody in a molar ratio of 4, i.e., the molar ratio of transition metal ions to antibody is 4.

[0026] Those skilled in the art can select a buffer system suitable for the reaction in step (a) depending on the transition metal ions, which includes, but is not limited to, TAPS, bicine, tris, tricine, HEPES, TES, MOPS, PIPES, sodium citrate, histidine buffer, phosphate buffer (PB), PBS, MES, etc. The pH of the buffer system is about 5 to 8, preferably about 7.0.

[0027] Transition metal ions are removed by appropriate methods known to the art, such as adjusting the pH (e.g., by adding NaOH, HCl, etc.), adjusting the temperature, changing the buffer solution (e.g., by adding NaCl, sodium citrate, etc.), or using chelating reagents such as EDTA, DTPA, ethylenediamine, 2,2'-bipyridine, or 1,10-phenanthroline, and then filtered by subsequent dialysis, ultrafiltration, or gel filtration, if desired.

[0028] In one embodiment, the oxidizing agent is selected from, but is not limited to, hydrogen peroxide (H2O2), nitrate compounds, potassium chlorate (KClO3), peroxydisulfate (H2S2O8), peroxymonosulfate (H2SO5), NaClO, sodium dichromate (Na2Cr2O7), permanganate compounds, sodium perborate, nitrous oxide, sodium bismuthate (NaBiO3), cerium sulfate, dehydroascorbic acid (DHAA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), or 2-aminophenyl disulfide (DDD). Those skilled in the art will understand that any other oxidizing agent capable of oxidizing sulfidyl groups can be used in the present invention. In one embodiment, the oxidizing agent added in step (b) is DHAA; in another embodiment, the oxidizing agent added in step (b) is DTNB. In one embodiment, in step (b), the molar ratio of oxidizing agent to antibody in the reaction solution is in the range of 0.5 to 160, for example, 10 to 160 (weak oxidizing agent), 5 to 24 (medium oxidizing agent), or 0.5 to 10 (strong oxidizing agent). In a particular embodiment, in step (b), the oxidizing agent is added to the antibody at a molar ratio of 16, i.e., the oxidizing agent / antibody molar ratio is 16. In one embodiment, DTNB is used as the oxidizing agent, and the oxidizing agent / antibody molar ratio is 1.6 to 7.7. In another embodiment, DHAA is used as the oxidizing agent, and the oxidizing agent / antibody molar ratio is 16 or 40. In yet another embodiment, DDD is used as the oxidizing agent, and the oxidizing agent / antibody molar ratio is 1.82.

[0029] In step (b), in the reoxidized antibody obtained, only one set of reduced interchain disulfide bonds appears to be blocked by the transition metal ion. In some embodiments, the reoxidized antibody is not purified after reoxidation in step (b), and the process for preparing the improved homogeneity antibody-drug conjugate (ADC) composition described herein can be carried out in a one-pot manner. In some embodiments, the reoxidized antibody is purified after reoxidation in step (b).

[0030] The optimal pH for the reaction is typically about 5 to about 8, for example, about 5.5 to about 7.5, or about 6 to about 7.5. The optimal reaction conditions vary depending on the specific reactants used. In specific embodiments, the pH in steps (a) and (b) is about 6 to about 7.5, preferably about 7.0, and the pH in step (c) is about 5 to about 8, preferably about 7.0.

[0031] The incubation time and temperature for each step can be determined by those skilled in the art based on the specific antibody being conjugated. The optimal temperature for the reaction is typically about -10 to 37°C. In a specific embodiment, in step (a) (i.e., the reduction step), the temperature is 0°C to 37°C, preferably 2°C to 25°C, for example 4°C, 12°C, 22°C, or 37°C, and the incubation time is 0.5 hours to 90 hours, for example 0.5 hours to 72 hours, preferably 2 hours to 20 hours, more preferably 16 hours to 20 hours, for example 2 hours, 16 hours, 19 hours, 45 hours, or 92 hours. In step (b) (i.e., the re-oxidation step), the temperature is 0°C to 37°C, preferably 2°C to 25°C or 4 to 12°C, for example 0°C, 12°C, 22°C or 37°C, and the incubation time is 0.5 hours to 170 hours, preferably 0.5 hours to 72 hours, more preferably 0.5 hours to 48 hours, for example 0.5 hours, 1 hour, 2 hours, 4 hours, 24 hours, 47 hours or 170 hours. In step (c) (i.e., the conjugation step), the temperature is 0°C to 37°C, preferably 2°C to 25°C or 4 to 12°C, for example 0°C, 4°C or 12°C, and the incubation time is 0.5 hours to 72 hours, preferably 8 hours to 16 hours, more preferably 1 hour to 4 hours.

[0032] There are no particular limitations on the antibodies that can be conjugated with linker drugs using the bioconjugation process of this disclosure. The choice of antibody depends on the disease or disorder (e.g., cancer) to be treated by the antibody-drug conjugate (ADC). This antibody specifically binds to the corresponding antigen (also called tumor-associated antigen (TAA)), viral antigen, or microbial antigen expressed on cancer cells, possesses antibody-dependent cell-mediated phagocytic (ADCP) activity, and has antitumor, antiviral, or antibacterial activity in vivo. Interchain disulfide bonds of the antibody can function as sites for binding drug linker complexes after reduction.

[0033] In some embodiments, the antibody includes, but is not limited to, monoclonal or polyclonal antibodies. The antibody may also be a monospecific or multispecific antibody, such as a bispecific antibody. Specific examples of the antibody include human antibodies, humanized antibodies, or chimeric antibodies. In certain embodiments, the antibody is a monoclonal antibody, such as a human antibody or humanized antibody. Examples of antibody isotypes of the present invention include IgG (IgG1, IgG2, IgG3, or IgG4). In certain embodiments, the antibody is an IgG1 monoclonal antibody. For example, the three antibodies exemplified in the examples, trastuzumab, rituximab, and cetuximab, are representative IgG1 antibodies. The results of the examples demonstrate that the bioconjugation process of this disclosure is applicable to at least IgG1 antibodies. Furthermore, the bioconjugation process of this disclosure is also applicable to IgG4 antibodies.

[0034] The reactive group-supported payload conjugated to the selected antibody generally takes the form of a drug linker. The drugs and linkers that can be used in the bioconjugation process of this disclosure are not particularly limited, as long as the drug molecule has the desired effect (e.g., cytotoxicity, antitumor, or labeling) and has at least one substituent or substructure that enables connection to the linker structure, and the linker contains at least two reactive groups, one of which can be covalently bonded to the drug molecule and the other can be covalently coupled to the antibody.

[0035] In this technology, a wide variety of known diagnostic agents, therapeutic agents, and labeling agents are conjugated onto antibody molecules. For example, in the broadest sense, conjugated drugs may include diagnostic agents, drug molecules, such as cytotoxic agents, toxins, radionuclides, and fluorescent agents (e.g., amine-derivative fluorescent probes, such as 5-dimethylaminonaphthalene-1-(N-(2-aminoethyl))sulfonamide-dansylethylenediamine, Oregon Green® 488 cadaverine (catalog number O-10465, Molecular Probes), densylcadaverine, N-(2-aminoethyl)-4-amino-3,6-disulfo-1,8-naphthalimide, dipotassium salt (Lucifer Yellow ethylenediamine) or rhodamine B ethylenediamine (catalog number L-2424, Molecular Probes), or thiol-derivative fluorescent probes, such as BODIPY® FLL-cysteine ​​(catalog number B-20340, Molecular Probes)).

[0036] In ADC preparation, the most commonly used reactive group that can be bonded to a thiol group is maleimide. Furthermore, other electron-deficient alkenyl or alkynyl derivatives, mono or di derivatives of disulfides, sulfones, bicyclo[1.1.0]butane derivatives, sulfonyl fluorides, pentafluorophenol esters, palladium oxidative addition complexes, iodoxolones, highly electron-deficient arenes, organobromids, and iodides are also frequently used. Those skilled in the art will understand that any other group that selectively reacts with sulfidyl groups can also be used.

[0037] Depending on the desired drug and the selected linker, those skilled in the art can select an appropriate method for coupling them together. For example, certain conventional coupling methods, such as amine coupling methods, can be used to form a desired drug-linker complex that still contains a reactive group for conjugation to an antibody via covalent linkage. A drug-maleimide complex (i.e., a malamide linker) is an example of a reactive group-supported payload of this disclosure. The drug may include, but is not limited to, cytotoxic reagents, such as chemotherapeutic agents, immunotherapeutic agents, antiviral agents, or antimicrobial agents. The most conventional reactive group that can be bound to a thiol group in ADC preparation is maleimide. In addition, organobromids and iodides are also frequently used. In certain embodiments, the payload comprises a maleimide moiety, a bromide, or an iodide.

[0038] In step (d), a person skilled in the art can select an appropriate purification method to recover the obtained antibody-drug conjugate. Many ADC purification methods are well known in the art. For example, the obtained antibody-drug conjugate may be purified using desalting columns, size exclusion chromatography, etc.

[0039] Using a conjugation process that employs the same steps but without the addition of transition metal ions in step (a) as a negative control (see Example 1 and Figures 1-3), we successfully demonstrated that transition metal ions may be the primary cause of higher levels of D2, as well as lower levels of D0, D0, D6, and D8, in the resulting ADC. This process has been confirmed by several commercially available therapeutic antibodies and has shown remarkable consistency.

[0040] By using the process of this disclosure to produce antibody-drug conjugates, the uniformity of the antibody-drug conjugates is higher than that produced by conventional conjugation processes. Specifically, in ADCs prepared by the process of this disclosure, the D2 content is generally greater than 64 mol%, preferably greater than 75 mol%, and more preferably greater than 80 mol%, whereas in ADCs prepared by conventional conjugation processes, the D2 content is usually greater than 50 mol%. Furthermore, the D0+D4+D6+D8 content in ADCs prepared by the process of this disclosure is less than 36 mol%, preferably less than 25 mol%, more preferably less than 20 mol%, and most preferably less than 15 mol%, compared to the D0+D4+D6+D8 content in ADCs prepared by conventional conjugation processes, which is usually greater than 40 mol%.

[0041] The process of this disclosure avoids any need for protein modification or enzyme catalysis, is based on undenatured interchain disulfide bonds, and requires only transition metal ions. Therefore, compared to conventional processes for preparing ADCs in the absence of transition metal ions, the process of this disclosure is less complex, the homogeneity of the resulting antibody-drug conjugate is dramatically improved, and costs are significantly reduced.

[0042] In a second embodiment, the disclosure relates to a composition of an antibody-drug conjugate (ADC) produced by the process of the first embodiment, wherein the content of D2 is generally greater than 64 mol%, preferably greater than 75 mol%, and more preferably greater than 80 mol%, but the content of D0+D4+D6+D8 in the ADC is less than 36 mol%, preferably less than 25 mol%, more preferably less than 20 mol%, and most preferably less than 15%, based on the total molar concentration of the antibody-drug conjugate (ADC) (i.e., D0, D2, D4, D6, and D8).

[0043] In a third aspect, the disclosure relates to a pharmaceutical composition comprising a composition of an antibody-drug conjugate (ADC) produced by a process of the first aspect and a pharmaceutically acceptable carrier.

[0044] In one embodiment, the pharmaceutical composition is used to treat a condition or disorder of interest, which is selected from, but is not limited to, tumors (e.g., cancer), autoimmune diseases, or infectious diseases.

[0045] In certain embodiments, the infectious disease is a viral or microbial infection.

[0046] In one embodiment, the subject is a mammal, such as a human.

[0047] In a fourth aspect, the disclosure relates to the use of a composition of an antibody-drug conjugate (ADC) produced by a process of the first aspect in the manufacture of a pharmaceutical composition for treating a condition or disorder of interest.

[0048] In a fifth aspect, the Disclosure relates to a method for treating a condition or disorder in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate (ADC) composition produced by a process of the first aspect, or an effective amount of a pharmaceutical composition of the second aspect.

[0049] In certain embodiments, the condition or disorder may be selected from, but is not limited to, tumors (e.g., cancer), autoimmune diseases, or infectious diseases.

[0050] The aforementioned and other features and advantages of this disclosure will become more apparent from the following detailed description of some embodiments, which will proceed with reference to the accompanying drawings. [Brief explanation of the drawing]

[0051] [Figure 1A] Figure 1 shows the HIC (hydrophobic interaction chromatography) results of random DAR2 conjugations of mAb1 (trastuzumab) (A), mAb2 (cetuximab) (B), and mAb3 (rituximab) (C) with MC-VC-PAB-MMAE via a conventional conjugation process. [Figure 1B] Figure 1 shows the HIC (hydrophobic interaction chromatography) results of random DAR2 conjugations of mAb1 (trastuzumab) (A), mAb2 (cetuximab) (B), and mAb3 (rituximab) (C) with MC-VC-PAB-MMAE via a conventional conjugation process. [Figure 1C] Figure 1 shows the HIC (hydrophobic interaction chromatography) results of random DAR2 conjugations of mAb1 (trastuzumab) (A), mAb2 (cetuximab) (B), and mAb3 (rituximab) (C) with MC-VC-PAB-MMAE via a conventional conjugation process.

[0052] [Figure 2A] Figure 2 shows the HIC results of mAb1 (trastuzumab) (A), mAb2 (cetuximab) (B), and mAb3 (rituximab) (C) DAR2 conjugation with MC-VC-PAB-MMAE using the conjugation process of this disclosure. [Figure 2B] Figure 2 shows the HIC results of mAb1 (trastuzumab) (A), mAb2 (cetuximab) (B), and mAb3 (rituximab) (C) DAR2 conjugation with MC-VC-PAB-MMAE using the conjugation process of this disclosure. [Figure 2C] Figure 2 shows the HIC results of mAb1 (trastuzumab) (A), mAb2 (cetuximab) (B), and mAb3 (rituximab) (C) DAR2 conjugation with MC-VC-PAB-MMAE using the conjugation process of this disclosure.

[0053] [Figure 3A] Figure 3 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation with MC-VC-PAB-MMAE (A), MC-MMAF (B), GGFG-Dxd (C), tesirin (PBD) (D), DL1-VC-PAB-MMAE (E), and DM21 (F) using the conjugation process of this disclosure. [Figure 3B]Figure 3 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation with MC-VC-PAB-MMAE (A), MC-MMAF (B), GGFG-Dxd (C), tesirin (PBD) (D), DL1-VC-PAB-MMAE (E), and DM21 (F) using the conjugation process of this disclosure. [Figure 3C] Figure 3 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation with MC-VC-PAB-MMAE (A), MC-MMAF (B), GGFG-Dxd (C), tesirin (PBD) (D), DL1-VC-PAB-MMAE (E), and DM21 (F) using the conjugation process of this disclosure. [Figure 3D] Figure 3 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation with MC-VC-PAB-MMAE (A), MC-MMAF (B), GGFG-Dxd (C), tesirin (PBD) (D), DL1-VC-PAB-MMAE (E), and DM21 (F) using the conjugation process of this disclosure. [Figure 3E] Figure 3 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation with MC-VC-PAB-MMAE (A), MC-MMAF (B), GGFG-Dxd (C), tesirin (PBD) (D), DL1-VC-PAB-MMAE (E), and DM21 (F) using the conjugation process of this disclosure. [Figure 3F] Figure 3 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation with MC-VC-PAB-MMAE (A), MC-MMAF (B), GGFG-Dxd (C), tesirin (PBD) (D), DL1-VC-PAB-MMAE (E), and DM21 (F) using the conjugation process of this disclosure.

[0054] [Figure 4A] Figure 4A shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure under different reducing agents (A: TCEP, B: diPPBS1, C: DPAA). [Figure 4B]Figure 4B shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure under different reducing agents (A: TCEP, B: diPPBS1, C: DPAA). [Figure 4C] Figure 4C shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure under different reducing agents (A: TCEP, B: diPPBS1, C: DPAA).

[0055] [Figure 4D] Figure 4D shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure under different TCEP ratios (D: TCEP, 3.25 equivalents; E: TCEP, 10 equivalents; F: TCEP, 16 equivalents). [Figure 4E] Figure 4E shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure under different TCEP ratios (D: TCEP, 3.25 equivalents; E: TCEP, 10 equivalents; F: TCEP, 16 equivalents). [Figure 4F] Figure 4F shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure under different TCEP ratios (D: TCEP, 3.25 equivalents; E: TCEP, 10 equivalents; F: TCEP, 16 equivalents).

[0056] [Figure 5A] Figure 5 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure in different buffers: (A) HEPES, (B) MES, (C) Tris. [Figure 5B] Figure 5 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure in different buffers: (A) HEPES, (B) MES, (C) Tris. [Figure 5C]Figure 5 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure in different buffers: (A) HEPES, (B) MES, (C) Tris.

[0057] [Figure 6A] Figure 6 shows the HIC results of mAb3 (rituximab) DAR2 conjugation by the conjugation process of this disclosure in different buffers: (A) HEPES, (B) MES, (C) Tris. [Figure 6B] Figure 6 shows the HIC results of mAb3 (rituximab) DAR2 conjugation by the conjugation process of this disclosure in different buffers: (A) HEPES, (B) MES, (C) Tris. [Figure 6C] Figure 6 shows the HIC results of mAb3 (rituximab) DAR2 conjugation by the conjugation process of this disclosure in different buffers: (A) HEPES, (B) MES, (C) Tris.

[0058] [Figure 7A] Figure 7 shows HIC results for mAb3 (rituximab) conjugation by conjugation processes in different buffers: (A) HEPES, (B) MES, (C) Tris(0Zn2+) (transition metal ion-free). [Figure 7B] Figure 7 shows HIC results for mAb3 (rituximab) conjugation by conjugation processes in different buffers: (A) HEPES, (B) MES, (C) Tris(0Zn2+) (transition metal ion-free). [Figure 7C] Figure 7 shows HIC results for mAb3 (rituximab) conjugation by conjugation processes in different buffers: (A) HEPES, (B) MES, (C) Tris(0Zn2+) (transition metal ion-free).

[0059] [Figure 8]Figure 8 shows the HIC results of bispecific antibody (mAb4) DAR2 conjugation by the conjugation process of this disclosure.

[0060] [Figure 9A] Figure 9A shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure using TCEP at different temperatures for reduction: (A) 4°C. [Figure 9B] Figure 9B shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure using TCEP at different temperatures for reduction: (B) 22°C. [Figure 9C] Figure 9C shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure using TCEP at different temperatures for reduction: (C) 37°C.

[0061] [Figure 9D] Figure 9D shows the HIC results for mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure using TCEP at different time intervals for reduction at 4°C: (D) 19 hours. [Figure 9E] Figure 9E shows the HIC results for mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure using TCEP at different time intervals for reduction at 4°C: (E) 45 hours. [Figure 9F] Figure 9F shows the HIC results for mAb1 (trastuzumab) DAR2 conjugation using the conjugation process of this disclosure with TCEP at different time intervals for reduction at 4°C: (F) 92 hours.

[0062] [Figure 10A]Figure 10 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure using various Zn2+-related salts: (A) ZnSO4·7H2O, (B) ZnBr2·2H2O, (C) Zn(ClO4)2·6H2O, (D) Zn(OAc)2·2H2O. [Figure 10B] Figure 10 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure using various Zn2+-related salts: (A) ZnSO4·7H2O, (B) ZnBr2·2H2O, (C) Zn(ClO4)2·6H2O, (D) Zn(OAc)2·2H2O. [Figure 10C] Figure 10 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure using various Zn2+-related salts: (A) ZnSO4·7H2O, (B) ZnBr2·2H2O, (C) Zn(ClO4)2·6H2O, (D) Zn(OAc)2·2H2O. [Figure 10D] Figure 10 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure using various Zn2+-related salts: (A) ZnSO4·7H2O, (B) ZnBr2·2H2O, (C) Zn(ClO4)2·6H2O, (D) Zn(OAc)2·2H2O.

[0063] [Figure 11A] Figure 11 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation using the conjugation process of this disclosure with various amounts of Zn2+ ions: (A) 2 equivalents, (B) 8 equivalents, (C) 16 equivalents. [Figure 11B] Figure 11 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation using the conjugation process of this disclosure with various amounts of Zn2+ ions: (A) 2 equivalents, (B) 8 equivalents, (C) 16 equivalents. [Figure 11C]Figure 11 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation using the conjugation process of this disclosure with various amounts of Zn2+ ions: (A) 2 equivalents, (B) 8 equivalents, (C) 16 equivalents.

[0064] [Figure 12] Figure 12 shows the HIC results for mAb1 (trastuzumab) DAR2 conjugation using the conjugation process described herein, in which TCEP was removed after antibody reduction.

[0065] [Figure 13A] Figure 5 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure under different oxidizing agents: (A) DHAA, (B) DTNB, (C) DDD. [Figure 13B] Figure 5 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure under different oxidizing agents: (A) DHAA, (B) DTNB, (C) DDD. [Figure 13C] Figure 5 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure under different oxidizing agents: (A) DHAA, (B) DTNB, (C) DDD.

[0066] [Figure 14A] Figure 14A shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure at different temperatures for reoxidation: (A) 0°C. [Figure 14B] Figure 14B shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure at different temperatures for reoxidation: (B) 12°C. [Figure 14C] Figure 14C shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure at different temperatures for reoxidation: (C) 22°C. [Figure 14D] Figure 14D shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure at different temperatures for reoxidation: (D) 37°C.

[0067] [Figure 14E] Figure 14E shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure at different time intervals for reoxidation: (E) 1 hour. [Figure 14F] Figure 14F shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure at different time intervals for reoxidation: (F) 2 hours. [Figure 14G] Figure 14G shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure at different time intervals for reoxidation: (G) 4 hours. [Figure 14H] Figure 14H shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by the conjugation process of this disclosure at different time intervals for reoxidation: (H) 24 hours.

[0068] [Figure 15A] Figure 15 shows HIC results of mAb1 (trastuzumab) DAR2 conjugation with linker payload at different temperatures: A: 0°C, B: 12°C. [Figure 15B] Figure 15 shows HIC results of mAb1 (trastuzumab) DAR2 conjugation with linker payload at different temperatures: A: 0°C, B: 12°C.

[0069] [Figure 16] Figure 16 shows the HIC results for mAb1 (trastuzumab) DAR2 conjugation by adding transition metal ions in the reoxidation step and performing reoxidation at 22°C for 47.5 hours.

[0070] [Figure 17A]Figure 17 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by adding transition metal ions in the reoxidation step: (A) reoxidation in PB at 12°C for 170 hours in the presence of transition metal ions, (B) reoxidation in PB at 12°C for 24 hours without transition metal ions (as a negative control), (C) reoxidation in HEPES + PB at 12°C for 170 hours in the presence of transition metal ions, and (D) reoxidation in HEPES at 12°C for 24 hours without transition metal ions (as a negative control). [Figure 17B] Figure 17 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by adding transition metal ions in the reoxidation step: (A) reoxidation in PB at 12°C for 170 hours in the presence of transition metal ions, (B) reoxidation in PB at 12°C for 24 hours without transition metal ions (as a negative control), (C) reoxidation in HEPES + PB at 12°C for 170 hours in the presence of transition metal ions, and (D) reoxidation in HEPES at 12°C for 24 hours without transition metal ions (as a negative control). [Figure 17C] Figure 17 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by adding transition metal ions in the reoxidation step: (A) reoxidation in PB at 12°C for 170 hours in the presence of transition metal ions, (B) reoxidation in PB at 12°C for 24 hours without transition metal ions (as a negative control), (C) reoxidation in HEPES + PB at 12°C for 170 hours in the presence of transition metal ions, and (D) reoxidation in HEPES at 12°C for 24 hours without transition metal ions (as a negative control). [Figure 17D]Figure 17 shows the HIC results of mAb1 (trastuzumab) DAR2 conjugation by adding transition metal ions in the reoxidation step: (A) reoxidation in PB at 12°C for 170 hours in the presence of transition metal ions, (B) reoxidation in PB at 12°C for 24 hours without transition metal ions (as a negative control), (C) reoxidation in HEPES + PB at 12°C for 170 hours in the presence of transition metal ions, and (D) reoxidation in HEPES at 12°C for 24 hours without transition metal ions (as a negative control).

[0071] [Figure 18A] Figure 18 shows the HIC results of mAb3 (rituximab) DAR2 conjugation by adding transition metal ions in the reoxidation step: (A) reoxidation in PB at 12°C for 170 hours in the presence of transition metal ions, and (B) reoxidation in PB at 12°C for 24 hours without transition metal ions (as a negative control). [Figure 18B] Figure 18 shows the HIC results of mAb3 (rituximab) DAR2 conjugation by adding transition metal ions in the reoxidation step: (A) reoxidation in PB at 12°C for 170 hours in the presence of transition metal ions, and (B) reoxidation in PB at 12°C for 24 hours without transition metal ions (as a negative control).

[0072] [Figure 19A] Figure 19 shows HIC results of mAb1 (trastuzumab) DAR2 conjugation with various linker payloads by adding transition metal ions in the reoxidation step: (A) GGFG-Dxd, (B) Tecilin (PBD), (C) MC-VC-PAB-MMAE. [Figure 19B] Figure 19 shows HIC results of mAb1 (trastuzumab) DAR2 conjugation with various linker payloads by adding transition metal ions in the reoxidation step: (A) GGFG-Dxd, (B) Tecilin (PBD), (C) MC-VC-PAB-MMAE. [Figure 19C]Figure 19 shows HIC results of mAb1 (trastuzumab) DAR2 conjugation with various linker payloads by adding transition metal ions in the reoxidation step: (A) GGFG-Dxd, (B) Tecilin (PBD), (C) MC-VC-PAB-MMAE.

[0073] [Figure 20A] Figure 20 shows mAb1 (trastuzumab) DAR2 conjugation under different antibody concentrations: (A) 5 mg / mL Ab, (B) 10 mg / mL Ab. [Figure 20B] Figure 20 shows mAb1 (trastuzumab) DAR2 conjugation under different antibody concentrations: (A) 5 mg / mL Ab, (B) 10 mg / mL Ab.

[0074] [Figure 21A] Figure 21 shows mAb1 (trastuzumab) DAR2 conjugation under different HEPES concentrations: (A) 40 mM HEPES, (B) 20 mM HEPES. [Figure 21B] Figure 21 shows mAb1 (trastuzumab) DAR2 conjugation under different HEPES concentrations: (A) 40 mM HEPES, (B) 20 mM HEPES.

[0075] [Figure 22] Figure 22 is a schematic diagram of the process for preparing the ADC composition of the present invention. (A) and (B) show the procedure for a normal antibody (i.e., a monospecific antibody): (A) the transition metal ion (M2+) is added in the reduction step, and (B) the transition metal ion (M2+) is added in the reoxidation step. (C) shows the procedure for a bispecific antibody in which the transition metal ion (M2+) is added in the reduction step, although the transition metal ion (M2+) can also be added in the reoxidation step (not shown). [Modes for carrying out the invention]

[0076] Detailed description of the invention While this disclosure can be embodied in many different forms, those disclosed herein are specific exemplary embodiments illustrating the principles of this disclosure. It should be emphasized that this disclosure is not limited to the specific embodiments illustrated herein. Furthermore, any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein.

[0077] In general, the nomenclature and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and conventionally used in those techniques. Unless otherwise indicated, the methods and techniques of this disclosure are generally carried out in accordance with conventional methods well known in those techniques and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6th edition, WBSaunders Company (2010); Sambrook J. & Russell D., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature used in relation to analytical chemistry, synthetic organic chemistry, and medicinal chemistry and pharmaceutical chemistry described herein, as well as the methods and experimental techniques for these tests, are well known and conventionally used in the art. Furthermore, any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0078] definition

[0079] To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0080] Unless otherwise specified herein, scientific and technical terms used in connection with this disclosure have meanings that are conventionally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms include plurals and plural terms include singulars. More specifically, when used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple subjects unless otherwise explicitly stated by context. Thus, for example, a reference to “a antibody” includes multiple antibodies, and “a transition metal ion” includes a mixture of transition metal ions. In this application, the use of “or” means “and / or” unless otherwise specified.

[0081] Throughout this disclosure, unless required by context, the words “comprise,” “comprises,” and “comprising” are understood to mean including the steps or elements, or groups of steps or elements, described, but not to mean excluding any other steps or elements, or groups of steps or elements. “Consisting of” means including and being limited to everything that follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the enumerated elements are required or essential, and that other elements cannot be present. “Consisting essentially of” means including any elements enumerated after this phrase, and being limited to other elements that do not interfere with, or contribute to, the activity or action of the enumerated elements as specified in this disclosure. Thus, the phrase “consisting essentially of” indicates that the enumerated elements are required or essential, but other elements are optional and may or may not be present, depending on whether they affect the activity or action of the enumerated elements.

[0082] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by approximately 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a baseline quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In certain embodiments, the terms “about” or “approximately” refer to a value that is plus or minus a range of 15%, 10%, 5%, or 1%, if followed by a number.

[0083] Throughout this disclosure, any reference to “one embodiment,” “embodiment,” “specific embodiment,” “related embodiment,” “a particular embodiment,” “additional embodiment,” or “further embodiment,” or any combination thereof, means that any particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of this disclosure. Therefore, occurrences of the aforementioned terms in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0084] An "antibody-drug conjugate" or ADC refers to a conjugate formed by covalently coupling a drug to an antibody, either directly or indirectly via one or more suitable linkers. ADCs are generally a format of antibody-linker-drug conjugates. Antibody-drug conjugates combine the ideal properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs toward antigen-expressing cells, thereby enhancing their antitumor activity.

[0085] The term “drug” as used herein refers to any cytotoxic molecule having an antitumor effect and having at least one substituent or substructure that enables connection to a linker structure. A drug can kill cancer cells and / or inhibit the growth, proliferation or metastasis of cancer cells, thereby reducing, alleviating or eliminating one or more symptoms of a disease or disorder.

[0086] As used herein, the term "linker" refers to a reactive molecule containing at least two reactive groups, one of which can be covalently bonded to a drug molecule and the other can be covalently coupled to an antibody.

[0087] The term “antibody,” as used herein, encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a particular antigen. An intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region ("HCVR") and first, second, and third constant regions (CH1, CH2, and CH3), and each light chain consists of a variable region ("LCVR") and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. The antibody has a “Y” shape, and the axis of the Y consists of the second and third constant regions of two heavy chains bound together via disulfide bonds. Each arm of the Y contains the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are involved in antigen binding. The variable regions of both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (the light (L) chain CDRs include LCDR1, LCDR2, and LCDR3, while the heavy (H) chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundary of an antibody can be defined or identified by the Kabat, Chothia, or Al-Lazikani conferences (Al-Lazikani, B., Chothia, C., Lesk, AM, J.Mol.Biol., Vol. 273 (No. 4), p. 927 (1997); Chothia, C. et al., J Mol Biol. December 5; Vol. 186 (No. 3): pp. 651-63 (1985); Chothia, C. and Lesk, AM, J.Mol.Biol., Vol. 196, pp. 196, 901 (1987); Chothia, C. et al., Nature. December 21-28; Vol. 342 (No. 6252): pp. 877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs intersect between the flanking stretches known as framework regions (FRs), which are more conserved than the CDRs and form a scaffold supporting high-frequency variable loops.Each HCVR and LCVR contains four FRs, and the CDRs and FRs are arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The constant regions of the heavy and light chains do not participate in antigen binding and exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as IGg1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IGg3 (γ3 heavy chain), IGg4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0088] An "antibody fragment" contains a portion of a full-length antibody, generally including its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al., (2004) Protein Eng. Design & Sel. Vol. 17 (No. 4): pp. 315-323); fragments produced by Fab expression libraries; anti-idiotype (anti-Id) antibodies; CDRs (complementarity-determining regions); and epitope-binding fragments described herein that bind immunospecifically to cancer cell antigens, viral antigens, or microbial antigens; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0089] The term "variable domain," as used herein from the perspective of antibodies, refers to an antibody variable region or fragment thereof comprising one or more CDRs. A variable domain may include an intact variable region (e.g., HCVR or LCVR), but may still retain, to a lesser extent than, the ability to bind to an antigen or form an antigen-binding site compared to an intact variable region.

[0090] The term “antigen-binding moiety,” as used herein, refers to an antibody fragment formed from a portion of an antibody containing one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain an intact, undenatured antibody structure. Examples of antigen-binding moieties include, but are not limited to, variable domains, variable regions, bispecific antibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bispecific antibodies (ds-bispecific antibodies), multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding moiety can bind to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding moiety may include one or more CDRs from a particular human antibody grafted onto a framework region from one or more different human antibodies. More detailed formats of the antigen-binding moiety are described in Spiess et al., 2015 (see above) and Brinkman et al., mAbs, Vol. 9 (No. 2), pp. 182–212 (2017), which are incorporated herein by reference in their entirety.

[0091] "Fab" refers to the antibody portion consisting of a single light chain (both variable and constant regions) associated with a single heavy chain's variable region and a first constant region via disulfide bonds, from an antibody perspective. In certain embodiments, both the light and heavy chain constant regions are replaced by a TCR constant region.

[0092] "Fc" refers to the portion of the antibody consisting of the second (CH2) and third (CH3) constant regions of the first heavy chain, which are bound to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc portion of an antibody is involved in various effector functions, such as ADCC and CDC, but does not function in antigen binding.

[0093] The "hinge region," from an antibody perspective, contains the heavy chain molecule portion that binds the CH1 domain to the CH2 domain. This hinge region consists of approximately 25 amino acid residues and is mobile, thus allowing the two N-terminal antigen-binding regions to move independently.

[0094] When used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies in the population are identical except for the possibility of naturally occurring mutations that may exist in small amounts. Monoclonal antibodies are highly specific and directed to a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which contain different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to these specificities, monoclonal antibodies have the advantage of being able to be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used in the present invention can be produced by the hybridoma method first described in Kohler et al. (1975) Nature, Vol. 256: p. 495, or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567; U.S. Patent No. 5,807,715). Monoclonal antibodies can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al. (1991) Nature, Vol. 352: p. 624-628; Marks et al. (1991) J. Mol. Biol., Vol. 222: p. 581-597.

[0095] The monoclonal antibodies described herein include, specifically, “chimeric” antibodies in which, to the extent that they exhibit the desired biological activity, portions of the heavy chain and / or light chain are identical or homologous to a corresponding sequence in an antibody derived from a particular species or in an antibody belonging to a particular class or subclass of antibody, and the remaining chain is identical or homologous to a corresponding sequence in an antibody derived from another species or in an antibody belonging to another class or subclass of antibody, as well as in fragments of such antibodies (U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci USA, Vol. 81: pp. 6851-6855 (1984)). The chimeric antibodies of interest herein include “primatized” antibodies that contain variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0096] As used herein, “intact antibody” includes a VL domain and a VH domain, as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domain may be an undenatured sequence constant domain (e.g., a human undenatured sequence constant domain) or an amino acid sequence variant thereof. An intact antibody may have one or more “effector functions,” where the effector function refers to their biological activity that contributes to the antibody’s Fc constant region (undenatured sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include Clq binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors, such as B cell receptors and BCRs.

[0097] Intact antibodies can be assigned to different "classes" depending on the amino acid sequence of the heavy chain constant domain. The five main classes of intact immunoglobulin antibodies are IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional arrangements of different classes of immunoglobulins are well known. The Ig form includes the hinged or non-hingeed form (Roux et al. (1998) J.Immunol. 161: pp. 4083-4090; Lund et al. (2000) Eur.J.Biochem. 267: pp. 7246-7256; U.S. Patent Application Publication No. 2005 / 0048572; U.S. Patent Application Publication No. 2004 / 0229310).

[0098] The "class" of an antibody refers to the type of constant domain or constant region in its heavy chain. The five main classes of antibodies are IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0099] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining heavy chain and / or light chain is derived from a different source or species.

[0100] A "human antibody" is an antibody produced from a human or human cell, or an antibody that has an amino acid sequence corresponding to the amino acid sequence of an antibody derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0101] A “humanized” antibody refers to a chimeric antibody containing amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody comprises substantially all, or at least one, typically two, variable domains, where all, or substantially all, HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all, or substantially all, FRs correspond to the FRs of the human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0102] "Isolated antibodies" are those separated from components of the natural environment. In some embodiments, antibodies are purified to a purity of 95% or more than 99%, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B848: pp. 79-87 (2007).

[0103] "Undenatured antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, an undenatured IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chains of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0104] A "cysteine-modified antibody" or "cysteine-modified antibody variant" is an antibody in which one or more residues of the antibody are substituted with cysteine ​​residues. According to this disclosure, the thiol group of a cysteine-modified antibody can be conjugated to calicheamicin to form a THIOMAB® antibody (i.e., a THIOMAB® drug conjugate (TDC), where the drug is a calicheamicin derivative according to this disclosure). In certain embodiments, the substituted residues occur at the usable site of the antibody. By substituting these residues with cysteine, a reactive thiol group can be positioned at the usable site of the antibody and used to conjugate the antibody to the drug portion to create an immunoconjugate, as further described herein. For example, a THIOMAB® antibody may have a single mutation from an undenatured cysteine ​​residue to cysteine ​​in either the light chain (e.g., G64C, K149C, or R142C, according to Kabat numbering) or the heavy chain (D101C, V184C, or T205C, according to Kabat numbering). In certain examples, a THIOMAB® antibody has a single cysteine ​​mutation in either the heavy chain or the light chain such that each full-length antibody (i.e., an antibody having two heavy chains and two light chains) has two modified cysteine ​​residues. Cysteine-modified antibodies and methods for their preparation are disclosed in U.S. Patent Application Publication No. 2012 / 0121615(A1), which is incorporated herein by reference in its entirety.

[0105] A "disulfide bond" refers to a covalent bond having the structure RSS-R'. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond with, for example, the second thiol group of another cysteine ​​residue. Disulfide bonds are formed between the thiol groups of two cysteine ​​residues present in each of two polypeptide chains, thereby forming interchain bridges or interchain bonds.

[0106] The term “specific binding” or “specifically binding” as used herein refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen. In certain embodiments, the polypeptide complexes and bispecific polypeptide complexes provided herein are ≤10 -6 M (for example, ≤ 5 × 10) -7 M, ≤ 2 × 10 -7 M, ≤10 -7 M, ≤ 5 × 10 -8 M, ≤ 2 × 10 -8 M, ≤10 -8 M, ≤ 5 × 10 -9 M, ≤ 2 × 10 -9 M, ≤10 -9 M, or ≤10 -10 Binding affinity (K) of M D ) specifically binds to the antigen. D When used herein, koff refers to the ratio of the dissociation rate to the association rate (koff / kon), which can be determined using surface plasmon resonance spectroscopy and instruments such as Biacore.

[0107] The term "transition metal," as used herein, refers to elements of groups 4-11, justified by typical chemistry, i.e., by a wide range of complex ions with various oxidation states, colored complexes, and catalytic properties, either as elements or ions (or both). Sc and Y of group 3 are also commonly recognized as transition metals.

[0108] As discussed above, antibody-drug conjugate mixtures are produced by conventional conjugation processes or the bioconjugation processes of this disclosure. Generally, a single antibody molecule belonging to the IgG1 or IgG4 subclass has four interchain disulfide bonds, each formed by two -SH groups. An antibody molecule can undergo partial or complete reduction of one or more interchain disulfide bonds to form 2n (where n is an integer selected from 1, 2, 3, or 4) reactive -SH groups, and therefore the number of drugs coupled to a single antibody molecule is 2, 4, 6, or 8. Different conjugates containing different numbers of drug molecules are named D0, D2, D4, D6, and D8 depending on the number of drugs coupled to the single antibody molecule. When the number of drugs coupled to the single antibody molecule is 0 (i.e., the drug-to-antibody molar ratio is (i.e., DAR0)), the product is designated D0 (i.e., representing a single antibody molecule that is not actually conjugated with any drug). Therefore, D2 refers to an ADC molecule in which two drug molecules are coupled to one single antibody molecule (i.e., a drug-to-antibody molar ratio of 2 (i.e., DAR2)), where the two drug molecules can be coupled to an -SH group produced by the reduction of SS bonds between heavy and light chains via a linker, or to an -SH group produced by the reduction of SS bonds between heavy chains via a linker.D4 refers to an ADC molecule in which four drug molecules are coupled to one single antibody molecule (i.e., a drug-to-antibody molar ratio of 4 (i.e., DAR4)), where the four drug molecules may be coupled to four -SH groups produced by the reduction of two SS bonds between the heavy and light chains via a linker (such an ADC is designated D4-1), or the four drug molecules may be coupled to four -SH groups produced by the reduction of two SS bonds between the heavy and heavy chains via a linker (such an ADC is designated D4-2), or two drug molecules may be coupled to two -SH groups produced by the reduction of one SS bond between the heavy and light chains via a linker, and the other two drug molecules may be coupled to two -SH groups produced by the reduction of one SS bond between the heavy and heavy chains via a linker (such an ADC is designated D4-3). D6 refers to an ADC molecule in which six drug molecules are coupled to one single antibody molecule (i.e., a drug-to-antibody molar ratio of 6 (i.e., DAR6)), where four drug molecules may be coupled to four -SH groups produced by the reduction of two SS bonds between the heavy and light chains via a linker, and two drug molecules may be coupled to two -SH groups produced by the reduction of one SS bond between the heavy and light chains via a linker (such an ADC is designated D6-1), or four drug molecules may be coupled to four -SH groups produced by the reduction of two SS bonds between the heavy and light chains via a linker, and two drug molecules may be coupled to two -SH groups produced by the reduction of one SS bond between the heavy and light chains via a linker (such an ADC is designated D6-2). D8 refers to an ADC molecule in which eight drug molecules are coupled to one single antibody molecule (i.e., a drug-to-antibody molar ratio of 8 (i.e., DAR8)), meaning that all four disulfide bonds of one antibody molecule are reduced to eight -SH groups, and each -SH group is bound to one drug molecule.Generally, the heterogeneous mixture of ADC molecules produced by conventional conjugation processes, or the bioconjugation processes of this disclosure, is a mixture of D0, D2, D4, D6, and D8 (also referred to as the “composition of ADCs”). Thus, the “homogeneity” of an antibody-drug conjugate is used to describe the superiority of one particular type of antibody-drug conjugate (i.e., one type selected from the D0, D2, D4, D6, and D8 conjugates) in a given mixture of antibody-drug conjugates. While the in vitro efficacy of antibody-drug conjugates has been shown to directly depend on drug loading (Hamblett KJ et al., Clin Cancer Res., October 15, 2004; Vol. 10 (No. 20): pp. 7063-70), the in vivo therapeutic (antitumor) activity of an antibody-drug conjugate with four drug molecules per molecule (D4) is comparable to that of a conjugate with eight drug molecules per molecule (D8) at the same mAb dose, even if the conjugate contains half the amount of drug per mAb. Drug loading also affects plasma clearance; D8 conjugates are cleared three times faster than D4 conjugates and five times faster than D2 conjugates. In cases where the conjugated molecule is highly toxic (i.e., a highly toxic payload), D2 may be preferable. In practice, for payloads with superior efficacy, conjugates prepared as D2 can control drug safety risks and improve the circulating stability of the conjugate in vivo, while ensuring efficacy. On the other hand, for specific mechanisms of action and spatial structures such as nucleic acid loading or immunoantagonists, D2 conjugates can minimize the impact of the conjugated high molecular weight payload on antibody function. Generally, when the D2 content in the mixture is high, the ADC is considered to have high homogeneity. In this disclosure, "homogeneity" of an antibody-drug conjugate refers to a high level of D2 in the mixture of antibody-drug conjugates.

[0109] Therefore, the “improved homogeneity” of the ADC, as used herein, refers to a higher level of D2 in the mixture of antibody-drug conjugates produced by the process of this disclosure compared to the mixture of ADCs produced by conventional conjugation processes. In the ADCs prepared by the process of this disclosure, the D2 content is generally 65 mol% or more, for example 70 mol% or more, preferably 75 mol% or more, or 80 mol% or more, based on the total molar concentration of the antibody-drug conjugates (ADCs) produced (i.e., D0, D2, D4, D6, and D8), whereas in ADCs prepared by conventional conjugation processes, the D2 content is usually less than 50 mol%. Furthermore, the content of D0+D4+D6+D8 in the ADC prepared by the process of this disclosure is less than 35 mol%, preferably less than 25 mol%, more preferably less than 20 mol%, and most preferably less than 15 mol%, whereas the content of D0+D4+D6+D8 in the ADC prepared by conventional conjugation processes is usually more than 40 mol%.

[0110] The term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient, and / or salt are generally chemically and / or physically compatible with the other components of the formulation and are physiologically compatible with the recipient of the formulation.

[0111] "Medically acceptable carriers" refer to components in a pharmaceutical formulation other than the active ingredient, which are acceptable to the target and have non-toxic biological activity. Medically acceptable carriers used in the pharmaceutical compositions disclosed herein may include, for example, medicamentally acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / diffusing agents, metal ion sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0112] The term "subject" includes any human or non-human animal, such as humans.

[0113] As used herein, the term “cancer” refers to any malignant tumor, such as a solid tumor or a non-solid tumor, such as a leukemia, caused by malignant proliferation, reproduction, or metastasis. “Cancer” 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 specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung cancer including adenocarcinoma and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric or abdominal cancer including gastrointestinal cancer, pancreatic cancer, gliablastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0114] The terms “treatment,” “treating,” or “treated,” as used herein, generally relate to a treatment or therapy, whether in humans or animals, in the context of treating a condition, in which some desired therapeutic effect is achieved, such as inhibiting the progression of a condition, reducing the rate of progression, stopping the rate of progression, regression of the condition, remission of the condition, and cure of the condition. Treatment as a preventive measure (i.e., prevention, avoidance) is also included. In cancer, “treating” may mean the attenuation or slowing of the growth, proliferation, or metastasis of tumor or malignant cells, or some combination thereof. In tumors, “treatment” includes the removal of all or part of the tumor, inhibition or slowing of tumor growth and metastasis, prevention or delay of tumor development, or some combination thereof.

[0115] Antibodies are against tumor-associated antigens (TAAs), cellular antigens that cause the production of autoimmune antibodies, viral antigens, or microbial antigens. Tumor-associated antigens are known in the art and can be prepared for use in antibody production using methods and information known in the art. In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane, or otherwise tumor-associated, polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells compared to one or more normal non-cancerous cells. Often, such tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells compared to the surface of non-cancerous cells. Identifying such tumor-associated cell surface antigen polypeptides gives rise to the ability to specifically target cancer cells for destruction via antibody-based therapy.

[0116] Examples of tumor-associated antigens (TAAs) include, but are not limited to, TAA(1)–(53) listed herein. For convenience, information relating to these antigens is listed herein, all of which is known to the art and includes names, alternative names, GenBank accession numbers, and primary standards in accordance with the National Center for Biotechnology Information (NCBI) Nucleic Acid and Protein Sequence Validation Conference (see International Publication No. 2017 / 068511(A1) brochure, which is incorporated herein by reference in its entirety). Nucleic acid and protein sequences corresponding to TAA(1)–(53) are available in public databases such as GenBank. Tumor-associated antigens targeted by antibodies include all amino acid sequence variants and isoforms that have at least approximately 70%, 80%, 85%, 90%, or 95% sequence identity with the sequence identified in the cited standard, or that exhibit substantially the same biological properties or characteristics as a TAA having the sequence found in the cited standard. For example, a TAA having a variant sequence can generally bind specifically to an antibody that specifically binds to a TAA having the corresponding sequence listed. Standard sequences and disclosures specifically cited herein are explicitly incorporated by reference.

[0117] Tumor-associated antigens (TAAs)

[0118] (1) BMPR1B (Bone morphogenetic protein receptor - type 1B, GenBank accession number NM_001203) ten Dijke, P. et al., Science Vol. 264 (No. 5155): pp. 101-104 (1994), Oncogene Volume 14 (Issue 11): pp. 1377-1382 (1997); International Publication No. 2004 / 063362 (Claim 2); International Publication No. 2003 / 042661 (Claim 12); US Patent Application Publication No. 2003134790(A1) Specification (pp. 38-39); International Publication No. 2002 / 102235 (Claim 13; pp. 296); International Publication No. 2003 / 055443 (pp. 91-92); International Publication No. 2002 / 99122 (Example 2; pp. 528-530); International Publication No. 2003 / 029421 (Claim 6); International Publication No. 2003 Brochure No. / 024392 (Claim 2; Figure 112); International Publication No. 2002 / 98358 (Claim 1; page 183); International Publication No. 2002 / 54940 (pages 100-101); International Publication No. 2002 / 59377 (pages 349-350); International Publication No. 2002 / 30268 (Claim 27; page 376); International Publication No. 2001 / 48204 (Examples; Figure 4) NP_001194 Osteogenesis Imperative Receptor, Type IB / pid=NP_001194.1 - Cross-reference: MIM:603248;NP_001194.1;AY065994.

[0119] (2) E16 (LAT1, SLC7A5, GenBank accession number NM_003486) Biochem. Biophys. Res. Commun. Vol. 255 (No. 2), pp. 283-288 (1999), Nature Vol. 395 (No. 6699): pp. 288-291 (1998), Gaugitsch, HW et al., (1992) J. Biol. Chem. Vol. 267 (No. 16): pp. 11267-11273); International Publication No. 2004 / 048938 (Example 2); International Publication No. 2004 / 032842 (Example IV); International Publication No. 2003 / 042661 (Claim 12); International Publication No. 2003 / 016475 (Claim 1); International Publication No. 2002 / 78524 (Example 2); International Publication No. 2002 / 99074 (Claim 19; pp. 127-129); International Publication No. 2002 / 864 Brochure No. 43 (Claim 27; pp. 222, 393); International Publication No. 2003 / 003906 (Claim 10; pp. 293); International Publication No. 2002 / 64798 (Claim 33; pp. 93-95); International Publication No. 2000 / 14228 (Claim 5; pp. 133-136); US Patent Application Publication No. 2003 / 224454 (Figure 3); International Publication No. 2003 / 025138 (Claim 12; pp. 150); NP_003477 Solute Carrier Family 7 (Cationic Amino Acid Transporter, y+ System), Member 5 / pid=NP_003477.3-Homo sapiens (Hopm sapiens) Cross reference: MIM:600182;NP_003477.3;NM_015923;NM_003486_1.

[0120] (3) STEAP1 (six transmembrane epithelial antigen of prostate, GenBank accession number NM_012449), Cancer Res. Vol. 61 (No. 15), pp. 5857-5860 (2001), Hubert, RS et al., (1999) Proc. Natl. Acad. Sci. USA Vol. 96 (No. 25): pp. 14523-14528); International Publication No. 2004 / 065577 (Claim 6); International Publication No. 2004 / 027049 (Figure 1L); European Patent No. 1394274 (Example 11); International Publication No. 2004 / 016225 (Claim 2); International Publication No. 2003 / 042661 Pamphlet No. (Claim 12); U.S. Patent Application Publication No. 2003 / 157089 (Example 5); U.S. Patent Application Publication No. 2003 / 185830 (Example 5); U.S. Patent Application Publication No. 2003 / 064397 (Figure 2); International Publication No. 2002 / 89747 Pamphlet (Example 5; pp. 618-619); International Publication No. 2003 / 022995 Pamphlet (Example 9; Figure 13A, Example 53; p. 173, Example 2; Figure 2A); NP_036581 Prostatic 6-Transmembrane Epithelial Antigen Cross-reference: MIM:604415;NP_036581.1;NM_012449_1.

[0121] (4)0772P (CA125, MUC16, GenBank accession number AF361486) J. Biol. Chem. Vol. 276 (No. 29): pp. 27371-27375 (2001)); International Publication No. 2004 / 045553 (Claim 14); International Publication No. 2002 / 92836 (Claim 6; Figure 12); International Publication No. 2002 / 83866 (Claim 15; pp. 116-121); U.S. Patent Application Publication No. 2003 / 124140 (Example 16); U.S. Patent No. 798959. Cross-reference: GI:34501467; AAK74120.3; AF361486_1.

[0122] (5) MPF (MPF, MSLN, SMR, megakaryocyte-enhancing factor mesothelin, GenBank accession number NM_005823), Yamaguchi, N. et al., Biol. Chem. Vol. 269 (No. 2), pp. 805-808 (1994), Proc. Natl. Acad. Sci. USA Vol. 96 (No. 20): pp. 11531-11536 (1999), Proc. Natl. Acad. Sci. USA Vol. 93 (No. 1): pp. 136-140 (1996), J. Biol. Chem. Vol. 270 (No. 37): pp. 21984-2 Page 1990 (1995); International Publication No. 2003 / 101283 (Claim 14); International Publication No. 2002 / 102235 (Claim 13; pages 287-288); International Publication No. 2002 / 101075 (Claim 4; pages 308-309); International Publication No. 2002 / 71928 (pages 320-321); International Publication No. 9410312 (pages 52-57); Cross-reference: MIM:601051; NP_005814.2; NM_005823_1.

[0123] (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate) member 2, type II sodium-dependent phosphate transporter 3b, GenBank accession number NM_006424) J. Biol. Chem. Vol. 277 (No. 22): pp. 19665-19672 (2002), Genomics Vol. 62 (No. 2): pp. 281-284 (1999), JA et al., (1999) Biochem. Biophys. Res. Commun. Vol. 258 (No. 3): pp. 578-582); International Publication No. 2004 / 022778 (Claim 2); European Patent No. 1394274 (Example 11); International Publication No. 2002 / 102235 (Claim 13; p. 326); European Japanese Patent No. 875569 (Claim 1; pp. 17-19); International Publication No. 2001 / 57188 ​​(Claim 20; pp. 329); International Publication No. 2004 / 032842 (Example IV); International Publication No. 2001 / 75177 (Claim 24; pp. 139-140); Cross-reference: MIM:604217;NP_006415.1;NM_006424_1.

[0124] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospongin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, GenBank accession number AB040878) Nagase T. et al., (2000) DNA Res. Vol. 7 (No. 2): pp. 143-150); International Publication No. 2004 / 000997 pamphlet (Claim 1); International Publication No. 2003 / 003984 pamphlet (Claim 1); International Publication No. 2002 / 06339 pamphlet (Claim 1; pp. 50); International Publication No. 2001 / 88133 pamphlet (Claim 1; pp. 41-43, 48-58); International Publication No. 2003 / 054152 pamphlet (Claim 20); International Publication No. 2003 / 101400 pamphlet (Claim 11); Commissioned by: Q9P283; EMBL; AB040878; BAA95969.1.Genew; HGNC:10737.

[0125] (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, GenBank accession number AY358628); Ross et al. (2002) Cancer Res. 62: pp. 2546-2553; U.S. Patent Application Publication No. 2003 / 129192 (Claim 2); U.S. Patent Application Publication No. 2004 / 044180 (Claim 12); US2004044179 (Claim 11); U.S. Patent Application Publication No. 2003 / 096961 (Claim 11); U.S. Patent Application Publication No. 2003 / 232056 (Example 5); International Publication No. 2003 / 105758 (Claim 12); U.S. Patent Application Publication No. 2003 / 206918 (Example 5); European Patent No. 1347046 (Claim 1); International Publication No. 2003 / 025148 (Claim 20); Cross-reference: GE37182378; AAQ88991.1; AY358628_1.

[0126] (9) ETBR (endothelin type B receptor, GenBank accession number AY275463); Nakamuta M. et al., Biochem. Biophys. Res. Commun. Vol. 177, pp. 34-39, 1991; Ogawa Y. et al., Biochem. Biophys. Res. Commun. Vol. 178, pp. 248-255, 1991; Arai H. et al., Jpn. Circ. J. Vol. 56, pp. 1303-1307, 1992; Arai H. et al., J. Biol. Chem. Vol. 268, pp. 3463-3470, 1993; Sakamoto A., Yanagisawa M. et al., Biochem. Biophys. Res. Commun. Vol. 178, pp. 656-663, 1991; Elshourbagy NA et al., J. Biol. Chem. Vol. 268, pp. 3873-3879, 1993; Haendler B. et al., J. Cardiovasc. Pharmacol. Vol. 20, s1-S4, 1992; Tsutsumi M. et al., Gene Vol. 228, pp. 43-49, 1999; Strausberg RL et al., Proc. Natl. Acad. Sci. USA Vol. 99, pp. 16899-16903, 2002; Bourgeois C. et al., J. Clin. Endocrinol. Metab. Vol. 82, pp. 3116-3123, 1997; Okamoto Y. et al., Biol. Chem. Vol. 272, pp. 21589-21596, 1997; Verheij JB et al., Am.J.Med.Genet. Vol. 108, pp. 223-225, 2002; Hofstra RMW et al., Eur.J.Hum.Genet. Vol. 5, pp. 180-185, 1997; Puffenberger EG et al., Cell Vol. 79, pp. 1257-1266, 1994; Attie T. et al., Hum.Mol.Genet. Vol. 4, pp. 2407-2409, 1995; Auricchio A. et al., Hum.Mol.Genet. Vol. 5, pp. 351-354, 1996; Amiel J. et al., Hum.Mol.Genet. Vol. 5, pp. 355-357, 1996; Hofstra RMW et al., Nat.Genet. Vol. 12, pp. 445-447, 1996; Svensson PJ et al., Hum.Genet.Volume 103, pp. 145-148, 1998; Fuchs S. et al., Mol. Med. Vol. 7, pp. 115-124, 2001; Pingault V. et al., (2002) Hum. Genet. Vol. 1, pp. 198-206; International Publication No. 2004 / 045516 (Claim 1); International Publication No. 2004 / 048938 (Example 2); International Publication No. 2004 / 040000 (Claim 151); International Publication No. 2003 / 087768 (Claim 1); International Publication No. 2003 / 016475 (Claim 1); International Publication No. 2003 / 016475 (Claim 1); International Publication No. 2002 / 61087 (Figure 1); International Publication No. 2003 / 016 Brochure No. 494 (Figure 6); International Publication No. 2003 / 025138 (Claim 12; page 144); International Publication No. 2001 / 98351 (Claim 1; pages 124-125); European Patent No. 522868 (Claim 8; Figure 2); International Publication No. 2001 / 77172 (Claim 1; pages 297-299); US Patent Application Publication No. 2003 / 109676; US Patent No. 6518404 (Figure 3); US Patent No. 5773223 (Claim 1a; columns 31-34); International Publication No. 2004 / 001004.

[0127] (10) MSG783 (RNF124, virtual protein FLJ20315, GenBank accession number NM_017763); International Publication No. 2003 / 104275 (Claim 1); International Publication No. 2004 / 046342 (Example 2); International Publication No. 2003 / 042661 (Claim 12); International Publication No. 2003 / 083074 (Claim 14; page 61); International Publication No. 2003 / 018621 (Claim 1); International Publication No. 2003 / 024392 (Claim 2; Figure 93); International Publication No. 2001 / 66689 (Example 6); Cross-reference: Locus ID: 54894; NP_060233.2; NM_017763_1.

[0128] (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-related gene 1, prostate cancer-related protein 1, 6-transmembrane epithelial antigen of prostate 2, 6-transmembrane prostate protein, GenBank accession number AF455138); Lab.Invest. 82(11):1573-1582 (2002); International Publication No. 2003 / 087306 brochure; U.S. Patent Application Publication No. 2003 / 064397 specification (Claim 1; Figure 1); International Publication No. 2002 / 72596 brochure (Claim 13; pages 54-55); International Publication No. 2001 / 72962 pamphlet Fret (Claim 1; Figure 4B); International Publication No. 2003 / 104270 (Claim 11); International Publication No. 2003 / 104270 (Claim 16); U.S. Patent Application Publication No. 2004 / 005598 (Claim 22); International Publication No. 2003 / 042661 (Claim 12); U.S. Patent Application Publication No. 2003 / 060612 (Claim 12; Figure 10); International Publication No. 2002 / 26822 (Claim 23; Figure 2); International Publication No. 2002 / 16429 (Claim 12; Figure 10); Cross-reference: GE22655488; AAN04080.1; AF455138_1.

[0129] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, GenBank accession number NM_017636) Xu, XZ et al., Proc. Natl. Acad. Sci. USA Vol. 98 (No. 19): pp. 10692-10697 (2001), Cell Volume 109 (Issue 3): pp. 397-407 (2002), J. Biol. Chem. Volume 278 (Issue 33): pp. 30813-30820 (2003); U.S. Patent Application Publication No. 2003 / 143557 (Claim 4); International Publication No. 2000 / 40614 (Claim 14; pp. 100-103); International Publication No. 2002 / 10382 (Claim 1; Figure 9A); International Publication No. 2003 / 042661 (Claim 12); International Publication No. 2002 / 30268 (Claim 27; page 391); U.S. Patent Application Publication No. 2003 / 219806 (Claim 4); International Publication No. 2001 / 62794 (Claim 14; Figures 1A-D); Cross-reference: MIM:606936;NP_060106.2;NM_017636_1.

[0130] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-inducing growth factor, GenBank accession number NP_003203 or NM_003212) Ciccodicola, A. et al., EMBO J. Vol. 8 (No. 7): pp. 1987-1991 (1989), Am. J. Hum. Genet. Vol. 49 (No. 3): pp. 555-565 (1991)); US Patent Application Publication No. 2003 / 224411 (Claim 1); International Publication No. 2003 / 083041 (Example 1); International Publication No. 2003 / 034984 (Claim 12); International Publication No. 2002 / 88170 (Claim 2; pp. 52-53); Country International Publication No. 2003 / 024392 (Claim 2; Figure 58); International Publication No. 2002 / 16413 (Claim 1; pp. 94-95, 105); International Publication No. 2002 / 22808 (Claim 2; Figure 1); U.S. Patent No. 5854399 (Example 2; columns 17-18); U.S. Patent No. 5792616 (Figure 2); Cross-reference: MIM:187395; NP_003203.1; NM_003212_1.

[0131] (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barrwill receptor) or Hs.73792 GenBank accession number M26004) Fujisaku et al., (1989) J. Biol. Chem. Vol. 264 (No. 4): pp. 2118-2125; Weis JJ et al., Exp. Med. Vol. 167, pp. 1047-1066, 1988; Moore M. et al., Proc. Natl. Acad. Sci. USA Vol. 84, pp. 9194-9198, 1987; Barel M. et al., Mol. Immunol. Vol. 35, pp. 1025-1031, 1998; Weis JJ et al., Proc. Natl. Acad. Sci. USA Vol. 83, pp. 5639-5643, 1986; Sinha SK et al., (1993) J.Immunol. Vol. 150, pp. 5311-5320; International Publication No. 2004 / 045520 (Example 4); U.S. Patent Application Publication No. 2004 / 005538 (Example 1); International Publication No. 2003 / 062401 (Claim 9); International Publication No. 2004 / 045520 (Example 4); International Publication No. 91 / 02536 (Figures 9.1-9.9); International Publication No. 2004 / 020595 (Claim 1); Commissioned: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

[0132] (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29, GenBank accession number NM_000626 or 11038674) Proc. Natl. Acad. Sci. USA (2003) Vol. 100 (No. 7): pp. 4126-4131, Blood (2002) Vol. 100 (No. 9): pp. 3068-3076, Muller et al., (1992) Eur. J. Immunol. Vol. 22 (No. 6): pp. 1621-1625); International Publication No. 2004 / 016225 (Claim 2, Figure 140); International Publication No. 2003 / 087768, US Patent Application Publication No. 2004 / 1 Patent No. 01874 (Claim 1, p. 102); International Publication No. 2003 / 062401 (Claim 9); International Publication No. 2002 / 78524 (Example 2); U.S. Patent Application Publication No. 2002 / 150573 (Claim 5, p. 15); U.S. Patent No. 5644033; International Publication No. 2003 / 048202 (Claim 1, pp. 306 and 309); International Publication No. 99 / 558658, U.S. Patent No. 6534482 (Claim 13, Figure 17A / B); International Publication No. 2000 / 55351 (Claim 11, pp. 1145-1146); Cross-reference: MIM: 147245; NP_000617.1; NM_000626_1.

[0133] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, GenBank accession number NM_030764, AY358130) Genome Res. Vol. 13 (No. 1): pp. 2265-2270 (2003), Immunogenetics Vol. 54 (No. 2): pp. 87-95 (2002), Blood Vol. 99 (No. 8): pp. 2662-2669 (2002), Proc. Natl. Acad. Sci. USA Vol. 98 (No. 17): pp. 9772-9777 (2001), Xu, MJ et al., (2001) Biochem. Biophys. Res. Commun. Vol. 280 (No. 3): pp. 768-775; International Publication No. 2004 / 016225 pamphlet (Claim 2); International Publication No. 2003 / 077836; International Publication No. 20013 / 8490 (Claim 5; Figures 18D-1 to 18D-2); International Publication No. 2003 / 097803 (Claim 12); International Publication No. 2003 / 089624 (Claim 25); Cross-reference: MIM:606509; NP_110391.2; NM_030764_1.

[0134] (17) HER2 (ErbB2, GenBank accession number M11730) Coussens L. et al., Science (1985) Vol. 230 (No. 4730): pp. 1132-1139; Yamamoto T. et al., Nature Vol. 319, pp. 230-234, 1986; Semba K. et al., Proc. Natl. Acad. Sci. USA Vol. 82, pp. 6497-6501, 1985; Swiercz JM et al., J. Cell Biol. Vol. 165, pp. 869-880, 2004; Kuhns JJ et al., J. Biol. Chem. Vol. 274, pp. 36422-36427, 1999; Cho H.-S. et al., Nature Vol. 421, pp. 756-760, 2003; Ehsani A.et al., (1993) Genomics Vol. 15, pp. 426-429; International Publication No. 2004 / 048938 (Example 2); International Publication No. 2004 / 027049 (Figure 11); International Publication No. 2004 / 009622; International Publication No. 2003 / 081210; International Publication No. 2003 / 089904 (Claim 9); International Publication No. 2003 / 016475 (Claim 1); U.S. Patent Application Publication No. 2003 / 11859 Specification No. 2; International Publication No. 2003 / 008537 (Claim 1); International Publication No. 2003 / 055439 (Claim 29; Figures 1A-B); International Publication No. 2003 / 025228 (Claim 37; Figure 5C); International Publication No. 2002 / 22636 (Example 13; pages 95-107); International Publication No. 2002 / 12341 (Claim 68; Figure 7); International Publication No. 2002 / 13847 (pages 71-74); International Publication No. 2002 / 14503 (pages 114-117); International Publication No. 2001 / 53463 (Claim 2; pages 41-46); International Publication No. 2001 / 41787 (page 15); International Publication No. 2000 / 44899 (Claim 52; Figure 7); International Publication No. 2000 / 20579 (Claim 3; Figure 2); U.S. Patent No. 5869445 (Claim 3) Columns 31-38); International Publication No. 9630514 (Claim 2; pages 56-61); EP1439393 (Claim 7); International Publication No. 2004 / 043361 (Claim 7); International Publication No. 2004 / 022709; International Publication No. 2001 / 00244 (Example 3; Figure 4); Commissioned: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1.

[0135] (18) NCA (CEACAM6, GenBank accession number M18728); Barnett T. et al., Genomics Vol. 3, pp. 59-66, 1988; Tawaragi Y. et al., Biochem. Biophys. Res. Commun. Vol. 150, pp. 89-96, 1988; Strausberg RL et al., Proc. Natl. Acad. Sci. USA Vol. 99: pp. 16899-16903, 2002; International Publication No. 2004 / 063709; EP1439393 (Claim 7); International Publication No. 2004 / 044178 (Example 4); International Publication No. 2004 / 031238; International Publication No. 2003 / 042661 (Claim 12); International Publication No. 2002 / 78524 (Example 2); International Publication No. 2002 / 86443 (Claim 27; p. 427); International Publication No. 2002 / 60317 (Claim 2); Commissioned: P40199; Q14920; EMBL; M29541; AAA59915.1.EMBL; M18728.

[0136] (19) MDP (DPEP1, GenBank accession number BC017023) Proc. Natl. Acad. Sci. USA Vol. 99 (No. 26): pp. 16899-16903 (2002)); International Publication No. 2003 / 016475 (Claim 1); International Publication No. 2002 / 64798 (Claim 33; pp. 85-87); Patent No. 5003790 Specification (Figures 6-8); International Publication No. 99 / 46284 (Figure 9); Cross-reference: MIM:179780;AAH17023.1;BC017023_1.

[0137] (20) IL20Rα (IL20Rα, ZCYTOR7, GenBank accession number AF184971); Clark HF et al., Genome Res. Vol. 13, pp. 2265-2270, 2003; Mungall AJ et al., Nature Vol. 425, pp. 805-811, 2003; Blumberg H. et al., Cell Vol. 104, pp. 9-19, 2001; Dumoutier L. et al., J. Immunol. Vol. 167, pp. 3545-3549, 2001; Parrish-Novak J. et al., J. Biol. Chem. Vol. 277, pp. 47517-47523, 2002; Pletnev S. et al., (2003) Biochemistry Vol. 42: pp. 12617-12624; Sheikh F. et al., (2004) J.Immunol. Vol. 172, pp. 2006-2010; EP1394274 (Example 11); U.S. Patent Application Publication No. 2004 / 005320 (Example 5); International Publication No. 2003 / 029262 (pp. 74-75); International Publication No. 2003 / 002717 (Claim 2; p. 63); International Publication No. 2002 / 22153 (pp. 45-47) ); US Patent Application Publication No. 2002 / 042366 Specification (pages 20-21); International Publication No. 2001 / 46261 Brochure (pages 57-59); International Publication No. 2001 / 46232 Brochure (pages 63-65); International Publication No. 09 / 837193 Brochure (Claim 1; pages 55-59) Commissioned: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

[0138] (21) Brevican (BCAN, BEHAB, GenBank accession number AF229053), Gary SC et al., Gene Vol. 256, pp. 139-147, 2000; Clark HF et al., Genome Res. Vol. 13, pp. 2265-2270, 2003; Strausberg RL et al., Proc. Natl. Acad. Sci. USA, Vol. 99, pp. 16899-16903, 2002; US2003186372 (Claim 11); U.S. Patent Application Publication No. 2003 / 186373 (Claim 11); U.S. Patent Application Publication No. 2003 / 119131 (Claim 1; Figure 52); U.S. Patent Application Publication No. 2003 / 119122 (Claim 1; Figure 52); U.S. Patent Application Publication No. 2003 / 119126 (Claim 1); U.S. Patent Application Publication No. U.S. Patent Application Publication No. 2003 / 119121 (Claim 1; Figure 52); U.S. Patent Application Publication No. 2003 / 119129 (Claim 1); U.S. Patent Application Publication No. 2003 / 119130 (Claim 1); U.S. Patent Application Publication No. 2003 / 119128 (Claim 1; Figure 52); U.S. Patent Application Publication No. 2003 / 119125 (Claim 1); International Publication Brochure No. 2003 / 016475 (Claim 1); International Publication Brochure No. 2002 / 02634 (Claim 1).

[0139] (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, GenBank accession number NM_004442) Chan, J. and Watt, VM, Oncogene Vol. 6 (No. 6), pp. 1057-1061 (1991) Oncogene Vol. 10 (No. 5): pp. 897-905 (1995), Annu. Rev. Neurosci. Vol. 21: pp. 309-345 (1998), Int. Rev. Cytol. Vol. 196: pp. 177-244 (2000)); International Publication No. 2003 / 042661 (Claim 12); International Publication No. 2000 / 53216 (Claim 1; p. 41); International Publication No. 2004 / 065576 (Claim 1); International Publication No. 2004 / 020583 (Claim 9); International Publication No. 2003 / 004529 (pages 128-132); International Publication No. 2000 / 53216 (Claim 1; page 42); Cross-reference: MIM:600997; NP_004433.2; NM_004442_1.

[0140] (23) ASLG659 (B7h, GenBank accession number AX092328); U.S. Patent Application Publication No. 2004 / 0101899 (Claim 2); International Publication No. 2003 / 104399 (Claim 11); International Publication No. 2004 / 000221 (Figure 3); U.S. Patent Application Publication No. 2003 / 165504 (Claim 1); U.S. Patent Application Publication No. 2003 / 124140 (Example 2); U.S. National Patent Application Publication No. 2003 / 065143 (Figure 60); International Publication No. 2002 / 102235 (Claim 13; page 299); US Patent Application Publication No. 2003 / 091580 (Example 2); International Publication No. 2002 / 10187 (Claim 6; Figure 10); International Publication No. 2001 / 94641 (Claim 12; Figure 7b); International Publication No. 2002 / 02624 (Claim 1) 3; Figures 1A-1B); US Patent Application Publication No. 2002 / 034749 Specification (Claim 54; pp. 45-46); International Publication No. 2002 / 06317 Brochure (Example 2; pp. 320-321, Claim 34; pp. 321-322); International Publication No. 2002 / 71928 Brochure (pp. 468-469); International Publication No. 2002 / 02587 Brochure (Example 1; Figure 1); International Publication No. 2001 / 40269 Brochure (Example 3; pages 190-192); International Publication No. 2000 / 36107 Brochure (Example 2; pages 205-207); International Publication No. 2004 / 053079 Brochure (Claim 12); International Publication No. 2003 / 004989 Brochure (Claim 1); International Publication No. 2002 / 71928 Brochure (pages 233-234, 452-453); International Publication No. 01 / 16318 Brochure.

[0141] (24) PSCA (Prostate stem cell antigen precursor, GenBank accession number AJ297436) Reiter RE et al., Proc. Natl. Acad. Sci. USA Vol. 95, pp. 1735-1740, 1998; Gu Z. et al., Oncogene Volume 19, pp. 1288-1296, 2000; Biochem. Biophys. Res. Commun. (2000) Volume 275 (No. 3): pp. 783-788; International Publication No. 2004 / 022709 pamphlet; European Patent No. 1394274 specification (Example 11); US Patent Application Publication No. 2004 / 018553 specification (Claim 17); International Publication No. 2003 / 008537 pamphlet (Claim 1); International Publication No. 2002 / 81646 pamphlet (Claim 1; p. 164); International Publication No. 2003 / 003906 pamphlet (Claim 1) Item 10; page 288); International Publication No. 2001 / 40309 (Example 1; Figure 17); U.S. Patent Application Publication No. 2001 / 055751 (Example 1; Figure 1b); International Publication No. 2000 / 32752 (Claim 18; Figure 1); International Publication No. 98 / 51805 (Claim 17; page 97); International Publication No. 98 / 51824 (Claim 10; page 94); International Publication No. 98 / 40403 (Claim 2; Figure 1B); Commissioned: 043653; EMBL; AF043498; AAC39607.1.

[0142] (25)GEDA (GenBank accession number AY260763); AAP14954 lipoma HMGIC fusion partner-like protein / pid=AAP14954.1-homo sapiens species: Homo sapiens (human) International Publication No. 2003 / 054152 (Claim 20); International Publication No. 2003 / 000842 (Claim 1); International Publication No. 2003 / 023013 (Example 3, Claim 20); U.S. Patent Application Publication No. 2003 / 194704 (Claim 45); Cross-reference: GI:30102449;AAP14954.1;AY260763_1.

[0143] (26) BAFF-R (B-cell activator receptor, BlyS receptor 3, BR3, GenBank accession number AF116456); BAFF receptor / pid=NP_443177.1-Homo sapiens Thompson, JS et al., Science Vol. 293 (No. 5537), pp. 2108-2111 (2001); International Publication No. 2004 / 058309 pamphlet; International Publication No. 2004 / 011611 pamphlet; International Publication No. 2003 / 045422 pamphlet (Examples; pp. 32-33); International Publication No. 2003 / 014294 pamphlet (Claim 35; Figure 6B); International Publication No. 2003 / 035846 pamphlet Lett (Claim 70; pp. 615-616); International Publication No. 2002 / 94852 (columns 136-137); International Publication No. 2002 / 38766 (Claim 3; p. 133); International Publication No. 2002 / 24909 (Example 3; Figure 3); Cross-reference: MIM:606269; NP_443177.1; NM_052945_1; AF132600.

[0144] (27) CD22 (B cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, GenBank accession number AK026467); Wilson et al., (1991) J. Exp. Med. 173: pp. 137-146; International Publication No. 2003 / 072036 (Claim 1; Figure 1); Cross-reference: MIM:107266; ​​NP_001762.1; NM_001771_1.

[0145] (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) to form a complex with IgM molecules on its surface, and transmits signals involved in B cell differentiation), pI: 4.84, MW: 25028 TM: 2 [P]Gene Chromosome:19ql3.2, GenBank accession number NP_001774.10); International Publication No. 2003 / 088808 pamphlet, US Patent Application Publication No. 2003 / 0228319 specification; International Publication No. 2003 / 062401 pamphlet (Claim 9); US Patent Application Publication No. 2002 / 150573 specification (Claim 4, pp. 13-14); W09958658 (Claim 13, Figure 16); International Publication No. 92 / 07574 pamphlet (Figure 1); US Patent No. 5644033 specification; Ha et al. (1992) J.Immunol. Vol. 148 (No. 5): pp. 1526-1531; Mueller et al. (1992) Eur. J.Biochem. Vol. 22: pp. 1621-1625; Hashimoto et al. (1994) Immunogenetics Volume 40 (Issue 4): pp. 287-295; Preud'homme et al. (1992) Clin. Exp. Immunol. Volume 90 (Issue 1): pp. 141-146; Yu et al. (1992) J. Immunol. Volume 148 (Issue 2): pp. 633-637; Sakaguchi et al. (1988) EMBO J. Volume 7 (Issue 11): pp. 3457-3464.

[0146] (29) CXCR5 (Burkitt lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection, and possibly plays a role in the development of AIDS, lymphoma, melanoma, and leukemia); 372 aa, pI:8.54 MW:41959 TM:7 [P]Gene Chromosome:11q23.3, GenBank accession number NP_001707.1); International Publication No. 2004 / 040000; International Publication No. 2004 / 015426; U.S. Patent Application Publication No. 2003 / 105292 (Example 2); U.S. Patent No. 6555339 (Example 2); International Publication No. 2002 / 61087 (Figure 1); International Publication No. 2001 / 57188 ​​(Claim 20, page 269); International Publication No. 2001 / 72830 (12-1 Page 3); International Publication No. 2000 / 22129 pamphlet (Example 1, pages 152-153; Example 2, pages 254-256); W09928468 (Claim 1, page 38); U.S. Patent No. 5440021 (Example 2, columns 49-52); W09428931 (pages 56-58); W09217497 (Claim 7, Figure 5); Dobner et al. (1992) Eur. J. Immunol. Vol. 22: pages 2795-2799; Barella et al. (1995) Biochem. J. Vol. 309: pages 773-779.

[0147] (30) HLA-DOB (beta subunit of MHC class II molecules (Ia antigens) that bind to peptides and present them to CD4+ T lymphocytes); 273 aa, pI:6.56 MW:30820 TM:1 [P]Gene Chromosome:6p21.3, GenBank accession number NP_002111.1) Tonnelle et al. (1985) EMBO J. Vol. 4 (No. 11): pp. 2839-2847; Jonsson et al. (1989) Immunogenetics Vol. 29 (No. 6): pp. 411-413; Beck et al. (1992) J.Mol.Biol. Vol. 228: pp. 433-441; Strausberg et al. (2002) Proc.Natl.Acad.Sci USA Volume 99: pp. 16899-16903; Servenius et al. (1987) J. Biol. Chem. Volume 262: pp. 8759-8766; Beck et al. (1996) J. Mol. Biol. Volume 255: pp. 1-13; Naruse et al. (2002) Tissue Antigens Volume 59: pp. 512-519; W09958658 (Claim 13, Figure 15); U.S. Patent No. 6,153408 (columns 35-38); U.S. Patent No. 5,976551 (columns 168-170); U.S. 6011146 (columns 145-146); Kasahara et al. (1989) Immunogenetics Volume 30 (Issue 1): pp. 66-68; Larhammar et al. (1985) J. Biol. Chem. Volume 260 (Issue 26): pp. 14111-14119.

[0148] (31) P2X5 (purinergic receptor P2X ligand-opening ion channel 5, an ion channel opened by extracellular ATP, potentially involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability); 422 aa, pI:7.63, MW:47206 TM:1 [P]Gene Chromosome:17p13.3, GenBank accession number NP_002552.2) Le et al. (1997) FEBS Lett. Vol. 418 (Nos. 1-2): pp. 195-199; International Publication No. 2004047749; International Publication No. 2003 / 072035 pamphlet (Claim 10); Touchman et al. (2000) Genome Res. Vol. 10: pp. 165-173; International Publication No. 2002 / 22660 pamphlet (Claim 20); International Publication No. 2003 / 093444 pamphlet (Claim 1); International Publication No. 2003 / 087768 pamphlet (Claim 1); International Publication No. 2003 / 029277 pamphlet (p. 82).

[0149] (32) CD72 (B cell differentiation antigen CD72, Lyb-2, 359 aa, pI:8.66, MW:40225 TM:1 [P]Gene Chromosome:9p13.3, GenBank accession number NP_001773.1) International Publication No. 2004 / 042346 (Claim 65); International Publication No. 2003 / 026493 (pp. 51-52, 57-58); International Publication No. 2000 / 75655 (pp. 105-106); Von Hoegen et al. (1990) J.Immunol. Vol. 144 (No. 12): pp. 4870-4877; Strausberg et al. (2002) Proc.Natl.Acad.Sci USA Vol. 99: pp. 16899-16903.

[0150] (33)LY64 (Lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family, modulates B cell activation and apoptosis, and loss of function is associated with increased disease activity in patients with systemic lupus erythematosus); 661 aa, pI:6.20, MW:74147 TM:1 [P]Gene Chromosome:5q12, GenBank accession number NP_005573.1); U.S. Patent Application Publication No. 2002 / 193567; International Publication No. 97 / 07198 (Claim 11, pp. 39-42); Miura et al. (1996) Genomics Vol. 38 (No. 3): pp. 299-304; Miura et al. (1998) Blood Volume 92: pp. 2815-2822; Pamphlet International Publication No. 2003 / 083047; W09744452 (Claim 8, pp. 57-61); Pamphlet International Publication No. 2000 / 12130 (pp. 24-26).

[0151] (34) FcRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain containing C2 type Ig-like and ITAM domains, possibly playing a role in B lymphocyte differentiation); 429 aa, pI: 5.28, MW: 46925 TM: 1 [P]Gene Chromosome: 1q21~1q22, GenBank accession number NP_443170.1); International Publication No. 2003 / 077836; International Publication No. 2001 / 38490 (Claim 6, Figures 18E-1~18E-2); Davis et al. (2001) Proc. Natl. Acad. Sci USA Volume 98 (Issue 17): Pages 9772-9777; International Publication No. 2003 / 089624 (Claim 8); European Patent No. 1347046 (Claim 1); International Publication No. 2003 / 089624 (Claim 7).

[0152] (35) IRTA2 (Immunoglobulin superfamily receptor translocation-associated 2, a putative immune receptor that may play a role in B cell development and lymphoma formation; deregulation of the gene due to translocation occurs in some B cell malignancies); 977 aa, pI:6.88 MW:106468 TM:1 [P]Gene Chromosome:1q21, GenBank accession number Humans: AF343662, AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187, AY358085; Mouse: AK089756, AY158090, AY506558; NP_112571.1, International Publication No. 2003 / 024392 (Claim 2, Figure 97); Nakayama et al. (2000) Biochem. Biophys. Res. Commun. Vol. 277 (No. 1): pp. 124-127; International Publication No. 2003 / 077836; International Publication No. 2001 / 38490 (Claim 3, Figures 18B-1~18B-2).

[0153] (36)TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, associated with the growth factors EGF / heregulin family and follistatin); 374 aa, NCBI accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; GenBank accession number AF179274; AY358907, CAF85723, CQ782436 International Publication No. 2004 / 074320 (SEQ ID NO: 810); Patent Publication No. 2004 / 113151 (SEQ ID NOs: 2, 4, 8); International Publication No. 2003 / 042661 (SEQ ID NO: 580); International Publication No. 2003 / 009814 (SEQ ID NO: 411); European Patent No. 1295944 (pages 69-70); International Publication No. 2002 / 30268 (page 329); International Publication No. 2001 / 90304 Fret (Sequence ID 2706); U.S. Patent Application Publication No. 2004 / 249130; U.S. Patent Application Publication No. 2004 / 022727; International Publication No. 2004 / 063355; U.S. Patent Application Publication No. 2004 / 197325; U.S. Patent Application Publication No. 2003 / 232350; U.S. Patent Application Publication No. 2004 / 005563; U.S. Patent Application Publication No. 2003 / 124579; Horie et al. (2000) Genomics Volume 67: pp. 146-152; Uchida et al. (1999) Biochem. Biophys. Res. Commun. Volume 266: pp. 593-602; Liang et al. (2000) Cancer Res. Volume 60: pp. 4907-4912; Glynne-Jones et al. (2001) Int J Cancer. October 15; Volume 94 (No. 2): pp. 178-1784.

[0154] (37) PMEL17 (silver homologue; SILV; D12S53E; PMEL17; SI; SIL); ME20; gp100) BC001414; BT007202; M32295; M77348; NM_006928; McGlinchey, RP et al. (2009) Proc. Natl. Acad. Sci. USA Vol. 106 (No. 33), pp. 13731-13736; Kummer, MP et al. (2009) J. Biol. Chem. Vol. 284 (No. 4), pp. 2296-2306.

[0155] (38) TMEFF1 (a transmembrane protein having EGF-like and two follistatin-like domains 1; tomolegrin 1); H7365; C9orf2; C90RF2; U19878; X83961; NM_080655; NM_003692; Harms, PW (2003) Genes Dev. Vol. 17 (No. 21), pp. 2624-2629; Gery, S. et al. (2003) Oncogene Vol. 22 (No. 18): pp. 2723-2727.

[0156] (39) GDNF-Ra1 (GDNF family receptor alpha-1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha-1; GFR-ALPHA-1); U95847; BC014962; NM 145793 NM_005264; Kim, MH et al. (2009) Mol. Cell. Biol. Vol. 29 (No. 8), pp. 2264-2277; Treenor, JJ et al. (1996) Nature Vol. 382 (No. 6586): pp. 80-83.

[0157] (40) Ly6E (Lymphocyte antigen 6 complex, gene locus E; Ly67, RIG-E, SCA-2, TSA-1); NP_002337.1; NM_002346.2; de Nooij-van Dalen, A G. et al. (2003) Int. J. Cancer Vol. 103 (No. 6), pp. 768-774; Zammit, DJ et al. (2002) Mol. Cell. Biol. Vol. 22 (No. 3): pp. 946-952.

[0158] (41) TMEM46 (shisa homologue 2 (Xenopus laevis); SHISA2); NP_001007539.1; NM_001007538.1; Furushima, K. et al. (2007) Dev. Biol. Vol. 306 (No. 2), pp. 480-492; Clark, HF et al. (2003) Genome Res. Vol. 13 (No. 10): pp. 2265-2270.

[0159] (42) Ly6G6D (Lymphocyte antigen 6 complex, gene locus G6D; Ly6-D, MEGT1); NP_067079.2; NM_021246.2; Mallya, M et al. (2002) Genomics Vol. 80 (No. 1): pp. 113-123; Ribas, G. et al. (1999) J. Immunol. Vol. 163 (No. 1): pp. 278-287.

[0160] (43) LGR5 (Leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67); NP_003658.1; NM_003667.2; Salanti, G. et al. (2009) Am. J. Epidemiol. Vol. 170 (No. 5): pp. 537-545; Yamamoto, Y. et al. (2003) Hepatology Vol. 37 (No. 3): pp. 528-533.

[0161] (44)RET(ret proto-oncogene;MEN2A;HSCR1;MEN2B;MTC1;PTC;CDHF12;Hs.168114;RET51;RET-ELE1);NP_066124.1;NM_020975.4;Tsukamoto, H. et al. (2009) Cancer Sci. Vol. 100 (No. 10): pp. 1895-1901;Narita, N. et al. (2009) Oncogene Vol. 28 (No. 34): pp. 3058-3068 Hs.168114

[0162] (45)LY6K (lymphocyte antigen 6 complex, gene locus K;LY6K;HSJ001348;FLJ35226);NP_059997.3;NM_017527.3;Ishikawa, N. et al. (2007) Cancer Res. Vol. 67 (No. 24): pp. 11601-11611;de Nooij-van Dalen, A G. et al. (2003) Int. J. Cancer Vol. 103 (No. 6): pp. 768-774.

[0163] (46) GPR19 (G protein-coupled receptor 19; Mm.4787); NP_006134.1; NM_006143.2; Montpetit, A. and Sinnett, D. (1999) Hum. Genet. Vol. 105 (Nos. 1-2): pp. 162-164; O'Dowd, BF et al. (1996) FEBS Lett. Vol. 394 (No. 3): pp. 325-329.

[0164] (47) GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12); NP_115940.2; NM_032551.4; Navenot, JM et al. (2009) Mol. Pharmacol. Vol. 75 (No. 6): pp. 1300-1306; Hata, K. et al. (2009) Anticancer Res. Vol. 29 (No. 2): pp. 617-623.

[0165] (48) ASPHD1 (containing aspartate beta-hydroxylase domain 1; LOC253982); NP_859069.2; NM_181718.3; Gerhard, DS et al. (2004) Genome Res. Vol. 14 (No. 10B): pp. 2121-2127.

[0166] (49) Tyrosinase (TYR;OCAIA;OCA1A;Tyrosinase;SHEP3);NP_000363.1;NM_000372.4;Bishop, DT et al. (2009) Nat. Genet. Vol. 41 (No. 8): pp. 920-925;Nan, H. et al. (2009) Int. J. Cancer Vol. 125 (No. 4): pp. 909-917.

[0167] (50) TMEM118 (ring finger protein, transmembrane 2; RNFT2; FLJ14627); NP_001103373.1; NM_001109903.1; Clark, HF et al. (2003) Genome Res. Vol. 13 (No. 10): pp. 2265-2270; Scherer, SE et al. (2006) Nature Vol. 440 (No. 7082): pp. 346-351.

[0168] (51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e); NP_078807.1; NM_024531.3; Ericsson, TA et al. (2003) Proc. Natl. Acad. Sci. USA Vol. 100 (No. 11): pp. 6759-6764; Takeda, S. et al. (2002) FEBS Lett. Vol. 520 (Nos. 1-3): pp. 97-101.

[0169] (52) CD33 is a member of the sialic acid-binding immunoglobulin-like lectin family. CD33 is a 67 kDa glycosylated transmembrane protein. CD33 is expressed in most myeloid cells and monocytic leukemia cells, in addition to the myelomonocytic progenitor cells and erythroid progenitor cells in which it is involved. It is not found in the earliest pluripotent stem cells, mature granulocytes, lymphoid cells, or nonhematopoietic cells (Sabbath et al., (1985) J. Clin. Invest. Vol. 75: pp. 756-756; Andrews et al., (1986) Blood Vol. 68: pp. 1030-1035). CD33 contains two tyrosine residues in its cytoplasmic tail, each followed by a hydrophobic residue similar to the immunoreceptor tyrosine-based inhibitory motif (ITIM) found in many inhibitory receptors.

[0170] (53) CLL-1 (CLEC12A, MICl, and DCAL2) encodes a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as roles in cell adhesion, intercellular signaling, glycoprotein metabolism, and inflammation and immune responses. The protein encoded by this gene acts as a negative regulator of granulocyte and monocyte function. Several alternatively spliced ​​transcriptional variants of this gene have been described, but the full length of some of these variants has not been determined. This gene is closely related to other CTL / CTLD superfamily members in the natural killer gene complex region of chromosome 12p13 (Drickamer K (1999) Curr. Opin. Struct. Biol. Vol. 9 (No. 5): pp. 585-590; van Rhenen A et al., (2007) Blood Vol. 110 (No. 7): pp. 2659-2666; Chen CH et al., (2006) Blood Vol. 107 (No. 4): pp. 1459-1467; Marshall AS et al., (2006) Eur. J. Immunol. Vol. 36 (No. 8): pp. 2159-2169; Bakker AB et al., (2005) Cancer Res. Vol. 64 (No. 22): pp. 8443-8450; Marshall AS et al., (2004) J. Biol. Chem. Vol. 279 (No. 15): pp. 14792-802. CLL-1 has been shown to be a type II transmembrane receptor containing a single C-type lectin-like domain (not presumed to bind to either calcium or sugar), a stalk region, a transmembrane domain, and a short cytoplasmic tail containing an ITIM motif.

[0171] The Conjugation Process of the Present Invention

[0172] In one embodiment, the present disclosure relates to a process for preparing an antibody-drug conjugate (ADC) composition, comprising the following steps: (a) Reduction step: Incubating the reducing agent and antibody in a buffer system to reduce the interchain disulfide bonds in the antibody, and optionally purifying the reduced antibody; (b) Reoxidation step: A step in which an oxidizing agent is added to reoxidize a portion of the reduced thiol groups, and optionally the reoxidized antibody is purified; Step (a) or step (b) is carried out in the presence of a transition metal ion. In step (b), the reduced thiol group not blocked by the transition metal ion is reoxidized; (c) Conjugation step: a step of removing transition metal ions and adding an excess amount of reactive group-supported payload to react with the reduced thiol groups of the reoxidized antibody; and (d) Recovery step: The process comprises the step of recovering the obtained antibody-drug conjugate to obtain an antibody-drug conjugate (ADC) composition.

[0173] In the context of this disclosure, the expression "step (a) or step (b) is carried out in the presence of a transition metal ion" means that either step (a) or step (b) is carried out in the presence of a transition metal ion, for example, either step (a) is carried out in the presence of a transition metal ion, or step (b) is carried out in the presence of a transition metal ion. However, the present invention does not exclude the possibility that both step (a) and step (b) are carried out in the presence of a transition metal ion. For example, the method of this application can be carried out in a one-pot manner, in which case if a transition metal ion is added in step (a), step (b) is also carried out in the presence of a transition metal ion. From the standpoint of economic efficiency, the transition metal ion may be added only once, either in step (a) or step (b).

[0174] In one embodiment, the process for preparing an antibody-drug conjugate (ADC) composition with improved homogeneity involves the following steps: (a) Reduction step: Incubate the reducing agent and the antibody to be conjugated in a buffer system in the presence of transition metal ions to reduce the interchain disulfide bonds in the antibody, and optionally purify the reduced antibody; (b) Reoxidation step: Add an oxidizing agent to reoxidize the reduced thiol groups that are not blocked by the transition metal ions, and optionally purify the reoxidized antibody; (c) Conjugation step: a step of removing transition metal ions and adding an excess amount of reactive group-supported payload (e.g., maleimide conjugate) to react with the reduced thiol groups of the reoxidized antibody; and (d) Recovery step: The step of recovering the obtained antibody-drug conjugate to obtain the antibody-drug conjugate composition, The antibody-drug conjugate (ADC)(D2), having a drug-to-antibody molar ratio of 2, is present in the composition at a high concentration.

[0175] In another embodiment, the process for preparing an antibody-drug conjugate (ADC) composition involves the following steps: (a) Reduction step: Incubating the reducing agent and the conjugated antibody in a buffer system to reduce the interchain disulfide bonds in the antibody, and optionally purifying the reduced antibody; (b) Reoxidation step: Add a soluble transition metal salt or a solution containing a soluble transition metal salt, add an oxidizing agent to reoxidize the reduced thiol groups that are not blocked by the transition metal ions in the presence of the transition metal ions, and purify the reoxidized antibody as necessary; (c) Conjugation step: a step of removing transition metal ions and adding an excess amount of reactive group-supported payload to react with the reduced thiol groups of the reoxidized antibody; and (d) Recovery step: The step of recovering the obtained antibody-drug conjugate to obtain the antibody-drug conjugate composition, The antibody-drug conjugate (D2), having a drug-to-antibody molar ratio of 2, is present in the composition at a high concentration.

[0176] In the present invention, "a high concentration of antibody-drug conjugate (ADC) (D2) with a drug-to-antibody molar ratio of 2 is present in the composition" means that in the ADC prepared by the process of the present invention, the content of D2 (i.e., antibody-drug conjugate (ADC) with a drug-to-antibody molar ratio of 2) exceeds 64 mol%, preferably exceeds 75 mol%, and more preferably exceeds 80 mol%, based on the total molar concentration of the antibody-drug conjugates (ADCs) produced (i.e., D0, D2, D4, D6, and D8).

[0177] In one embodiment, the reducing agent is selected from, but is not limited to, tris(2-carboxyethyl)phosphine (TCEP), THPP (tris(3-hydroxypropyl)phosphine), diPPB (2-diphenylphosphanylbenzenesulfonic acid), DTT (dithiothreitol), DPAA (2-(diphenylphosphino)acetic acid), DTE (dithioerythritol), β-mercaptoethanol, LiAlH4, Na2S2O3, KBH4, or hydrazine. In one embodiment, the reducing agent in step (a) is TCEP. In one embodiment, in step (a), the molar ratio of reducing agent / antibody in the reaction solution is in the range of 1 to 20, for example, 2 to 16, preferably 3 to 8. In a particular embodiment, in step (a), the reducing agent is added to the antibody in a molar ratio of 8, i.e., the molar ratio of reducing agent / antibody is 8.

[0178] In one embodiment, the transition metal ion in step (a) or step (b) is Zn 2+ In one embodiment, in step (a) or step (b), Zn in the reaction solution 2+ The molar ratio of the antibody to the transition metal ion is in the range of 0.5 to 50, preferably 1 to 16, and more preferably 2 to 8. In certain embodiments, in step (a) or step (b), the transition metal ion is added to the antibody in a molar ratio of 4, i.e., the transition metal ion / antibody molar ratio is 4. Water-soluble zinc salts are suitable for the process of this disclosure. For example, in step (a), Zn 2+ZnCl2 can be added as a source.

[0179] Those skilled in the art can select a buffer system suitable for the reaction in step (a) depending on the transition metal ions, which includes, but is not limited to, TAPS, bicine, tris, tricine, HEPES, TES, MOPS, PIPES, sodium citrate, histidine buffer, PB (phosphate buffer), PBS, MES, and the like. In a particular embodiment, the buffer system used in step (a) is PB buffer, and the pH of the buffer system is about 5 to 8, preferably about 7.0.

[0180] Transition metal ions are removed by appropriate methods known to the art, such as adjusting the pH (e.g., by adding NaOH, HCl, etc.), adjusting the temperature, changing the buffer solution (e.g., by adding NaCl, sodium citrate, etc.), or using chelating reagents such as EDTA, DTPA, ethylenediamine, 2,2'-bipyridine, or 1,10-phenanthroline, and then filtered by subsequent dialysis, ultrafiltration, or gel filtration, if desired.

[0181] In one embodiment, the oxidizing agent is selected from, but is not limited to, hydrogen peroxide (H2O2), nitrate compounds, potassium chlorate (KClO3), peroxydisulfate (H2S2O8), peroxymonosulfate (H2SO5), NaClO, sodium dichromate (Na2Cr2O7), permanganate compounds, sodium perborate, nitrous oxide, sodium bismuthate (NaBiO3), cerium sulfate, dehydroascorbic acid (DHAA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), or 2-aminophenyl disulfide (DDD). Those skilled in the art will understand that any other oxidizing agent capable of oxidizing sulfidyl groups can be used in the present invention. In one embodiment, the oxidizing agent added in step (b) is DHAA; in another embodiment, the oxidizing agent added in step (b) is DTNB. In one embodiment, in step (b), the molar ratio of oxidizing agent to antibody in the reaction solution is in the range of 0.5 to 160, for example, 10 to 160 (weak oxidizing agent), 5 to 24 (medium oxidizing agent), or 0.5 to 10 (strong oxidizing agent). In a particular embodiment, in step (b), the oxidizing agent is added to the antibody at a molar ratio of 16, i.e., the oxidizing agent / antibody molar ratio is 16. In one embodiment, DTNB is used as the oxidizing agent, and the oxidizing agent / antibody molar ratio is 1.6 to 7.7. In another embodiment, DHAA is used as the oxidizing agent, and the oxidizing agent / antibody molar ratio is 16 or 40. In yet another embodiment, DDD is used as the oxidizing agent, and the oxidizing agent / antibody molar ratio is 1.82.

[0182] The optimal pH for the reaction is typically about 5 to about 8, for example, about 5.5 to about 7.5, or about 6 to about 7.5. The optimal reaction conditions vary depending on the specific reactants used. In specific embodiments, the pH in steps (a) and (b) is about 6 to about 7.5, preferably about 7.0, and the pH in step (c) is about 5 to about 8, preferably about 6.5 to 7.0.

[0183] In one embodiment, the buffer solution is PB (phosphate buffer) with a pH of 6.90.

[0184] The incubation time and temperature for each step can be determined by those skilled in the art based on the specific antibody being conjugated. The optimal temperature for the reaction is typically around -10 to 37°C. The reaction can take place, for example, at a temperature of about 0 to 20°C overnight.

[0185] Those skilled in the art should understand that the incubation time and temperature in step (a) may depend on the specific antibody being conjugated. Determining the incubation time and temperature based on a specific antibody is within the capabilities of those skilled in the art. For example, the antibody to be conjugated is typically incubated with a reducing agent in the presence of transition metal ions at 4°C overnight or at 12°C for 20 hours. In specific embodiments, in step (a) (i.e., the reduction step), the temperature is 0°C to 37°C, preferably 2°C to 25°C, e.g., 4°C, 12°C, 22°C, or 37°C, and the incubation time is 0.5 hours to 90 hours, e.g., 0.5 hours to 72 hours, preferably 2 hours to 20 hours, more preferably 16 hours to 20 hours, e.g., 2 hours, 16 hours, 20 hours, 45 hours, or 90 hours. In step (b) (i.e., the re-oxidation step), the temperature is 0°C to 37°C, preferably 2°C to 25°C or 4 to 12°C, for example 0°C, 12°C, 22°C or 37°C, and the incubation time is 0.5 hours to 170 hours, preferably 0.5 hours to 72 hours, more preferably 0.5 hours to 48 hours, for example 0.5 hours, 1 hour, 2 hours, 4 hours, 24 hours, 47 hours or 170 hours. In step (c) (i.e., the conjugation step), the temperature is 0°C to 37°C, preferably 2°C to 25°C or 4 to 12°C, for example 0°C, 4°C or 12°C, and the incubation time is 0.5 hours to 72 hours, preferably 8 hours to 16 hours, more preferably 1 hour to 4 hours.

[0186] In some embodiments, the conjugated antibody, transition metal ions, and reducing agent may be present in the reaction mixture in a molar ratio of 1:2:4, but are not limited to this ratio. In one embodiment, 0.02 mM antibody is incubated overnight at 4°C with 0.08 mM TCEP and 0.04 mM ZnCl2.

[0187] There are no specific limitations on the antibodies that are conjugated. Those skilled in the art can select suitable antibodies useful for the bioconjugation process of this disclosure, depending on the antigen associated with the disease or disorder (e.g., a specific tumor-associated antigen, viral antigen, or microbial antigen). In some embodiments, the antibody is an antibody that binds to one or more tumor-associated antigens or cell surface receptors as described elsewhere in this specification. Antibodies may include, but are not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, or antibody derivatives.

[0188] In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In a further embodiment, the antibody is an antibody fragment, e.g., Fv, Fab, Fab', scFv, a bispecific antibody, or an F(ab')2 fragment. In yet another embodiment, the antibody is a substantially full-length antibody, e.g., an IgG1 antibody, an IgG4 antibody, or another antibody class or isotype as defined herein. In a particular embodiment, the antibody is an IgG1 antibody.

[0189] In some other embodiments, the antibody may be Trastuzumab, Rituxan, Cetuximab, or one of the following antigens: BMPR1B, E16, STEAP1, MUC16, MPF, Napi2b, Sema 5b, PSCA The antibody is selected from one of the following: hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, HER2, NCA, MDP, IL20Ra, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, FcRH5, TENB2, PMEL17, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, Ly6K, GPR19, GPR54, ASPHD1, tyrosinase, TMEM118, GPR172A, CD33, and CLL-1.

[0190] Useful polyclonal antibodies are a heterogeneous population of antibody molecules derived from the serum of immunized animals. Various procedures known in this technique can be used to produce polyclonal antibodies against an antigen of interest. For example, a variety of host animals, including but not limited to rabbits, mice, rats, and guinea pigs, can be immunized by injection with the antigen of interest or a derivative thereof to produce polyclonal antibodies.

[0191] A useful monoclonal antibody is a homogeneous population of antibodies against a specific antigen (e.g., cancer cell antigen, viral antigen, microbial antigen covalently linked to a second molecule). A monoclonal antibody (mAb) against an antigen of interest can be prepared using any technique known to that technique, for example, by producing antibody molecules by a serial cell system in culture. Such techniques include, but are not limited to, the hybridoma technique first described by Kohler and Milstein (1975, Nature vol. 256, pp. 495-497), the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today vol. 4: p. 72), and the EBV hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. The mAb-producing hybridomas used in this invention can be cultured in vitro or in vivo.

[0192] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies can be produced by any of the many techniques known to the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 7308-7312; Kozbor et al., 1983, Immunology Today, Vol. 4, pp. 72-79; and Olsson et al., 1982, Meth. Enzymol., Vol. 92, pp. 3-16).

[0193] In addition, combination antibodies, such as chimeric and humanized monoclonal antibodies that contain both human and non-human portions and can be produced using standard combination DNA technology, are useful ligands. Chimeric antibodies are molecules in which different portions originate from different animal species, for example, a variable region derived from a mouse monoclonal molecule and a human immunoglobulin constant region. (See, for example, Cabilly et al., U.S. Patent No. 4,816,567, and Boss et al., U.S. Patent No. 4,816,397, both of which are incorporated herein by reference). Humanized antibodies are non-human antibody molecules in which one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule. (See, for example, Queen, U.S. Patent No. 5,585,089, both of which are incorporated herein by reference).Such chimeric and humanized monoclonal antibodies are incorporated herein by reference, for example, by recombinant DNA technology known to the art, in whole, as described in International Publication No. 87 / 02671; European Patent No. 184,187; European Patent No. 171,496; European Patent No. 173,494; International Publication No. 86 / 01533; U.S. Patent No. 4,816,567; European Patent No. 125,023; Berter et al., 1988, Science Vol. 240: pp. 1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA Volume 84: pp. 3439-3443; Liu et al., 1987, J. Immunol. Volume 139: pp. 3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA Volume 84: pp. 214-218; Nishimura et al., 1987, Canc. Res. Volume 47: pp. 999-1005; Wood et al., 1985, Nature Volume 314: pp. 446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. Volume 80: pp. 1553-1559; Morrison, 1985, Science Volume 229: pp. 1202-1207; Oi et al., 1986, BioTechniques It can be produced using the methods described in Volume 4: 214 pages; U.S. Patent No. 5,225,539; Jones et al., 1986, Nature Vol. 321: pp. 552-525; Verhoeyan et al. (1988), Science Vol. 239: pp. 1534; and Beidler et al., 1988, J. Immunol. Vol. 141: pp. 4053-4060.

[0194] In certain embodiments, antibodies known for the treatment or prevention of cancer are used in the present invention. Antibodies immune to cancer cell antigens can be commercially available or produced by any method known to those skilled in the art, such as chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immune to cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, published literature, or by routine cloning and sequencing. Examples of antibodies available for cancer treatment include HERCEPTIN (trastuzumab; Genentech, CA), a humanized anti-HER2 monoclonal antibody used to treat patients with metastatic breast cancer (Stebbing J., Copson, E., and O'Reilly, S. "Herceptin (trastuzamab) in advanced breast cancer," Cancer Treat Rev. Vol. 26, pp. 287-289, 2000); RITUXAN (rituximab; Genentech), a chimeric anti-CD20 monoclonal antibody used to treat patients with non-Hodgkin lymphoma; Erbitux (cetuximab; Merck), an IgG1 monoclonal antibody against the EGF receptor used to treat colorectal cancer; OvaRex (AltaRex Corporation, MA), a mouse antibody used to treat ovarian cancer; and Panorex (Glaxo), a mouse IgG2a antibody used to treat colorectal cancer. Wellcome (NC); BEC2 (ImClone Systems Inc., NY), a mouse IgG antibody that treats lung cancer; IMC-C225 (Imclone Systems Inc., NY), a chimeric IgG antibody that treats head and neck cancer; Vitaxin (MedImmune, Inc., MD), a humanized antibody that treats sarcoma; Campath I / H (Leukosite, MA), a humanized IgG1 antibody that treats chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., CA), a humanized IgG antibody that treats acute myeloid leukemia (AML); LymphoCide (Immunomedics, Inc., CA), a humanized IgG antibody that treats non-Hodgkin lymphoma.This includes, but is not limited to, Smart ID10 (Protein Design Labs, Inc., CA), a humanized antibody for treating non-Hodgkin lymphoma; Oncolym (Techniclone, Inc., CA), a mouse antibody for treating non-Hodgkin lymphoma; Allomune (BioTransplant, CA), a humanized anti-CD2 mAb for treating Hodgkin lymphoma or non-Hodgkin lymphoma; anti-VEGF (Genentech, Inc., CA), a humanized antibody for treating lung cancer and colorectal cancer; CEAcide (Immunomedics, NJ), a humanized anti-CEA antibody for treating colorectal cancer; IMC-1C11 (ImClone Systems, NJ), an anti-KDR chimeric antibody for treating colorectal cancer, lung cancer, and melanoma; and cetuximab (ImClone, NJ), an anti-EGFR chimeric antibody for treating epidermal growth factor-positive cancer. .

[0195] Other antibodies useful in cancer treatment include the following antigens: CA125 (ovary), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (carcinoma), Lewis Y (carcinoma), Lewis X (carcinoma), alpha-fetoprotein (carcinoma), CA242 (colorectal), placental alkaline phosphatase (carcinoma), prostate-specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinoma), MAGE-1 (carcinoma), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE Antibodies against 4 (carcinoma), anti-transferrin receptor (carcinoma), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colorectal cancer), gp100 (melanoma), MART1 (melanoma), PSA (prostate cancer), IL-2 receptor (T-cell leukemia and lymphoma), CD20 (non-Hodgkin lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinoma), P21 (carcinoma), MPG (melanoma), and Neu oncogene product (carcinoma) are included, but are not limited to, these. Some specific useful antibodies include BR96 mAb (Trail, PA, Willner, D., Lasch, SJ, Henderson, AJ, Hofstead, SJ, Casazza, AM, Firestone, RA, Hellstrom, I., Hellstrom, KE), "Cure of Xenografted Human Carcinomas by BR96-Doxorubicin Immunoconjugates" Science. 1993, vol. 261, pp. 212-215), BR64 (Trail, Pa., Willner, D., Knipe, J., Henderson, A. J., Lasch, S. J., Zoeckler, ME., Trailsmith, MD., Doyle, TW., King, HD, Casazza, AM, Braslawsky, GR, Brown, JP, Hofstead, SJ, Greenfield, Ill. S., Firestone, RA, Mosure, K., Kadow, DF, Yang, MB, Hellstrom, K. E., and Hellstrom, I."Effect of Linker Variation on the Stability, Potency, and Efficacy of Carcinoma-reactive BR64-Doxorubicin Immunoconjugates" Cancer Research 1997, Vol. 57, pp. 100-105), mAbs against the CD40 antigen, e.g., S2C6 mAb (Francisco, JA, Donaldson, KL, Chace, D., Siegall, CB, and Wahl, AF, "Agonistic properties and in vivo antitumor activity of the anti-CD-40 antibody, SGN-14" Cancer Res. 2000, Vol. 60, pp. 3225-3231), mAbs against the CD70 antigen, e.g., 1F6 mAb, and mAbs against the CD30 antigen, e.g., AC10 (Bowen, MA, Olsen, KJ, Cheng, L., Avila, D., and Podack, ER, "Functional effects of CD30 on a large This includes, but is not limited to, the granular lymphoma cell line YT (J. Immunol., Vol. 151, pp. 5896-5906, 1993). Many other internally distributed antibodies that bind to tumor-associated antigens can be used in the present invention and have been summarized (Franke, AE, Sievers, EL, and Scheinberg, DA, "Cell surface receptor-targeted therapy of acute myeloid leukemia: a review," Cancer Biother Radiopharm. 2000, Vol. 15, pp. 15, 459-476; Murray, JL, "Monoclonal antibody treatment of solid tumors: a coming of age," Semin Oncol. 2000, Vol. 27, pp. 64-70; Breitling, F., and Dubel, S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).

[0196] In another specific embodiment, antibodies known for the treatment or prevention of autoimmune diseases are used in accordance with the process of this disclosure. Antibodies that are immune-specific to the antigens of cells involved in the production of autoimmune antibodies can be obtained from any entity (e.g., university scientists, or companies such as Genentech) or produced by any method known to those skilled in the art, such as chemical synthesis or recombinant expression techniques. In another embodiment, useful ligand antibodies that are immune-specific for the treatment of autoimmune diseases include, but are not limited to, antinuclear antibodies; anti-dsDNA; anti-ssDNA; anti-cardiolipin antibodies IgM, IgG; antiphospholipid antibodies IgK, IgG; anti-SM antibodies; anti-mitochondrial antibodies; thyroid antibodies; microsomal antibodies; thyroglobulin antibodies; anti-SCL-70; anti-Jo; anti-U1RNP; anti-La / SSB; anti-SSA; anti-SSB; anti-parietal cell antibodies; anti-histones; anti-RNP; C-ANCA; P-ANCA; anti-centromere; anti-fibrillarin; and anti-GBM antibodies.

[0197] In another specific embodiment, a useful antibody that is immune-specific to a viral or microbial antigen is a monoclonal antibody. For example, an antibody immune-specific to a viral or microbial antigen may be a humanized or human monoclonal antibody. As used herein, the term “viral antigen” includes, but is not limited to, any viral peptides, polypeptide proteins (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoproteins (e.g., gB, gC, gD, and gE), and hepatitis B surface antigen) that can induce an immune response. As used herein, the term “microbial antigen” includes, but is not limited to, any microbial peptides, polypeptides, proteins, sugars, polysaccharides, or lipid molecules (e.g., bacteria, fungi, pathogenic protozoa, or yeast polypeptides containing, for example, LPS and capsular polysaccharides 5 / 8) that can induce an immune response.

[0198] Antibodies immune to viral or microbial antigens can be obtained commercially, for example, from Genentech (San Francisco, Calif.), or produced by any method known to those skilled in the art, such as chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immune to viral or microbial antigens can be obtained, for example, from the GenBank database or similar databases, published literature, or by routine cloning and sequencing.

[0199] In certain embodiments, a useful antibody is one that is useful for the treatment or prevention of a viral or microbial infection. Examples of antibodies available that are useful for the treatment of viral or microbial infections include, but are not limited to, SYNAGIS (MedImmune, Inc., MD), a humanized anti-RSV monoclonal antibody useful for the treatment of patients with respiratory syncytial virus (RSV) infection; PRO542 (Progenics), a CD4 fusion antibody useful for the treatment of HIV infection; OSTAVIR (Protein Design Labs, Inc., CA), a human antibody useful for the treatment of hepatitis B virus; PROTVIR (Protein Design Labs, Inc., CA), a humanized IgG1 antibody useful for the treatment of cytomegalovirus (CMV); and anti-LPS antibodies.

[0200] Other antibodies useful for the treatment of infectious diseases include bacteria (Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio colerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma species, Rickettsia prowazeki, Rickettsia tsutsugumushi, Clamydia species);Pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); Protozoa (Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Tryanosoma) gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or helminths ((Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella) This includes, but is not limited to, antibodies against antigens from pathogenic strains of *Scistosoma spiralis*, *Strongyloides stercoralis*, *Schistosoma japonicum*, *Schistosoma mansoni*, *Schistosoma haematobium*, and hookworms.

[0201] Other antibodies useful in the present invention for the treatment of viral diseases include, but are not limited to, antibodies against antigens of pathogenic viruses, including, as examples, voxviridae, herpesviridae (herpes simplex virus 1, herpes simplex virus 2), adenoviridae, papovaviridae, enteroviridae, picornaviridae, parvoviridae, reoviridae, retroviridae, influenza virus, parainfluenza virus, mumps, measles, respiratory syncytial virus, rubella, arboviridae, rhabdoviridae, arenaviridae, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, non-A / non-B hepatitis viruses, rhinoviridae, coronavirusidae, rotaviridae (and human immunodeficiency virus).

[0202] Antibodies suitable for use in bioconjugation processes provided herein can be produced by any method known to the art for antibody synthesis, in particular by chemical synthesis or recombinant expression, for example, by recombinant expression technology.

[0203] The reactive group-supported payload conjugated to the selected antibody generally has the format of a drug linker. The drugs and linkers that can be used in the bioconjugation process of this disclosure are not particularly limited, as long as the drug molecule has antitumor, antiviral or antimicrobial effect and contains at least one substituent or substructure that enables connection to the linker structure, and the linker contains at least two reactive groups, one of which can be covalently bonded to the drug molecule and the other can be covalently bonded to the antibody.

[0204] Depending on the desired drug and selected linker, those skilled in the art can select an appropriate method for coupling them together. For example, certain conventional coupling methods, such as amine coupling methods, can be used to form a desired drug-linker complex that still contains a reactive group for conjugation to an antibody via covalent linkage. A drug-maleimide complex (i.e., a maleimide linker) is an example of a reactive group-supported payload of this disclosure.

[0205] In some embodiments, the drug may include, but is not limited to, cytotoxic reagents, such as chemotherapeutic agents, immunotherapeutic agents, antiviral agents, or antimicrobial agents. In some embodiments, the drug conjugated to the antibody may be selected from, but is not limited to, MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), MMAF (monomethyl auristatin F), and the like.

[0206] In ADC preparation, the most commonly used reactive group that can be bonded to a thiol group is maleimide. In addition, organobromids and iodides are also frequently used.

[0207] The drug load is expressed by the number of drug moieties per antibody in the ADC molecule. In some antibody-drug conjugates, the drug load may be limited by the number of antibody binding sites. For example, if the binding is cysteinethiol, as in certain exemplary embodiments described herein, the drug load may range from 0 to 8 drug moieties per antibody. In certain embodiments, a larger drug load, e.g., p ≥ 5, may cause aggregation, insolubility, toxicity, or loss of cell permeability in certain antibody-drug conjugates. In certain embodiments, the average drug load of an antibody-drug conjugate is in the range of 1 to about 8, about 2 to about 6, or about 2 to about 5.

[0208] When more than one nucleophile reacts with a drug, it should be understood that the resulting product is a mixture of antibody-drug conjugate compounds having a distribution of one or more drug moieties bound to the antibody. The average number of drugs per antibody can be calculated from the mixture by a dual ELISA antibody assay, which is antibody-specific and drug-specific. Individual antibody-drug conjugate molecules can be identified in the mixture by mass spectrometry and separated by HPLC, for example, by hydrophobic interaction chromatography (e.g., McDonagh et al. (2006) Prot.Engr. Design & Selection Vol. 19 (No. 7): pp. 299-307; Hamblet et al. (2004) Clin. Cancer Res. Vol. 10: pp. 7063-7070; Hamblet, KJ et al., "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate," Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004; Alley, SC et al., "Controlling the location of drug attachment in antibody-drug conjugates," Abstract No. 627, American Association for (See Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004). In certain embodiments, a homogeneous antibody-drug conjugate having a single loading value can be isolated from the conjugation mixture by electrophoresis or chromatography.

[0209] To improve the uniformity of ADCs, the conjugation process requires the isolation of ADCs with a specific drug load or the selective binding of the drug moiety to the antibody. However, isolating ADCs with a specific drug load involves complex operations and high costs. Using a conjugation process that employs the same steps but without the addition of transition metal ions as a negative control (see U.S. Patent No. 7,659,241(B2)), the inventors have successfully demonstrated that transition metal ions were the primary cause of higher concentrations of D2, as well as lower concentrations of D0, D4, D6, and D8, in the resulting ADCs. By using the process of this disclosure to produce antibody-drug conjugates, the uniformity of the antibody-drug conjugates is higher than that produced by conventional conjugation processes. Specifically, in the ADC composition prepared by the process of this disclosure, the D2 content is generally greater than 64 mol%, preferably greater than 75 mol%, and more preferably greater than 80 mol%, whereas in ADC prepared by conventional conjugation processes that do not use transition metal ions, the D2 content is usually greater than 50 mol%. Furthermore, the D0+D4+D6+D8 content in the ADC prepared by the process of this disclosure is less than 36 mol%, preferably less than 25 mol%, more preferably less than 20 mol%, and most preferably less than 15 mol%, whereas the D0+D4+D6+D8 content in ADC prepared by conventional conjugation processes is usually greater than 40 mol%.

[0210] In one embodiment, the obtained antibody-drug conjugate is recovered or further purified by any suitable method, such as desalting column chromatography, size exclusion chromatography, ultrafiltration, dialysis, or UF-DF.

[0211] Antibody-drug conjugates with improved homogeneity

[0212] In one embodiment, the present disclosure relates to a composition of an antibody-drug conjugate (ADC) produced by the method of the present invention, wherein the D2 content is generally greater than 64 mol%, preferably greater than 75 mol%, and more preferably greater than 80 mol%, but the D0+D4+D6+D8 content in the ADC is less than 36 mol%, preferably less than 25 mol%, more preferably less than 20 mol%, and most preferably less than 15%, based on the total molar concentrations of D0, D2, D4, D6, and D8.

[0213] In one embodiment, the uniformity of the antibody-drug conjugate produced by the process of the first embodiment is measured and compared with the uniformity of the corresponding control antibody-drug conjugate produced by a conventional conjugation process.

[0214] Various analytical methods can be used to determine the yield and isomer mixture of antibody-drug conjugates. For example, in one embodiment, HIC is an analytical method used to determine the yield and isomer mixture of the obtained antibody-drug conjugate (e.g., D2 conjugate). This technique can separate antibodies loaded with varying numbers of drugs. The drug loading levels can be determined based on the absorbance ratio, for example, at 250 nm and 280 nm. For example, if the drug can absorb at 250 nm, the antibody will absorb at 280 nm. Therefore, the 250 / 280 ratio increases with the amount of drug loaded. Using the bioconjugation process described herein, it has been observed that antibodies with an even number of drugs are generally conjugated, since the reduction of disulfide produces an even number of free cysteine ​​thiols.

[0215] Compared to the corresponding control antibody-drug conjugates generated by conventional conjugation processes, the antibody-drug conjugates generated by the process of the present invention have improved uniformity, which is represented by an increase in the D2 content in the resulting ADC. In certain embodiments, as shown in Tables 2-3, the content of D2 in the antibody-drug conjugates produced by the process of the present disclosure is greater than 75 mol%, preferably greater than 80 mol%, while the content of D2 in the antibody-drug conjugates produced by conventional conjugation processes is typically less than 50 mol% (Table 1).

[0216] Pharmaceutical composition

[0217] In a further aspect, the present disclosure relates to a pharmaceutical composition comprising a composition of an antibody-drug conjugate (ADC) produced by the process of the present invention and a pharmaceutically acceptable carrier. This pharmaceutical composition is suitable for veterinary or human administration. In the pharmaceutical composition, a composition of an antibody-drug conjugate (ADC) produced by the process of the present invention is present in an effective amount, for example, an amount effective to treat a condition or disorder of a subject.

[0218] The pharmaceutical composition of the present invention can be in any form that enables the pharmaceutical composition to be administered to an animal. For example, the pharmaceutical composition can be in solid, liquid, or gaseous (aerosol) form. Typical routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, intravaginal, intraocular, and intranasal. Parenteral administration includes subcutaneous injection, intravenous, intramuscular, intracardiac injection, or infusion techniques. For example, the composition is administered parenterally. The pharmaceutical composition of the present invention can be formulated such that the ADC of the present invention is bioavailable upon administration of the pharmaceutical composition to an animal. The pharmaceutical composition can be in the form of one or more dosage units. For example, a tablet can be a single dosage unit, and a container of the ADC of the present invention in aerosol form can hold multiple dosage units.

[0219] The substances used to prepare the pharmaceutical composition may be nontoxic in the amount used. It will be apparent to those skilled in the art that the optimal dose of the active ingredient in the pharmaceutical composition is determined by various factors. These factors include, but are not limited to, the type of animal (e.g., human), the specific form of the ADC of the present invention, the mode of administration, and the composition used.

[0220] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole (hydroxanisol), butylated hydroxytoluene, and / or propyl gallate. Including one or more antioxidants, such as methionine, in the pharmaceutical compositions provided herein, as disclosed herein, reduces oxidation of polypeptide complexes or bispecific polypeptide complexes. Reduced oxidation prevents or reduces loss of binding affinity, thereby improving protein stability and maximizing shelf life. Accordingly, in certain embodiments, compositions are provided that include a polypeptide complex or bispecific polypeptide complex disclosed herein, and one or more antioxidants, such as methionine.

[0221] To further illustrate, pharmaceutically acceptable carriers include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as plant-derived fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at bacteriostatic or fungiostatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffer; antioxidants such as sodium bisulfate; and topical anesthetics. The following may be included: anesthetics, such as procaine hydrochloride; suspending and dispersing agents, such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers, such as polysorbate 80 (TWEEN®-80); metal ion sequestering or chelating agents, such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers, including phenol or cresol, mercury compounds, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride, and benzethonium chloride, may be added to the pharmaceutical composition in multi-dose containers. Suitable excipients may include, for example, water, saline solution, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or pharmaceuticals such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0222] Pharmaceutical compositions may be liquids, suspensions, emulsions, pills, capsules, tablets, sustained-release formulations, or powders. Oral formulations may include standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, and magnesium carbonate.

[0223] In certain embodiments, a pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition may be prepared in any conventional form suitable for producing a liquid, suspension, or emulsion, such as an aqueous solution, suspension, emulsion, or solid. Injectable preparations may include sterile and / or nonpyrogenic solutions prepared for injection, tablets for subcutaneous injection, sterile dry soluble products such as lyophilized powders prepared for combination with a solvent immediately before use, sterile suspensions prepared for injection, sterile insoluble products prepared for combination with a vehicle immediately before use, and sterile and / or nonpyrogenic emulsions. The solutions may be aqueous or non-aqueous.

[0224] In certain embodiments, a unit dose parenteral preparation is packed in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration should be sterile and not pyrogenic, as is known and practiced in the art.

[0225] In certain embodiments, a sterile lyophilized powder is prepared by dissolving the ADC disclosed herein in a suitable solvent. The solvent may contain other pharmacological components of the powder or excipients that improve the stability of the reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitan, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer, e.g., citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art, at approximately a neutral pH in one embodiment. The desired formulation is provided by sterile filtration of the subsequent solution, followed by lyophilization under standard conditions known to those skilled in the art. In one embodiment, the resulting liquid formulation is distributed into vials for lyophilization. Each vial may contain a single dose or multiple dose of the ADC or its composition provided herein. Overfilling the vials with a small amount (e.g., about 10%) more than the amount required for the dose or dose set is permissible to facilitate accurate sampling and accurate dosing. The freeze-dried powder can be stored under appropriate conditions, for example, from about 4°C to room temperature.

[0226] Reconstitution of lyophilized powder with sterile water for injection provides formulations for parenteral administration. In one embodiment, sterile and / or non-pyrogenic water, or other liquid suitable as a carrier, is added to the lyophilized powder for reconstitution. The exact amount may be determined empirically, depending on the selected therapy provided.

[0227] In addition, the antibody-drug conjugate or pharmaceutical composition of the present invention may be manufactured into a kit that includes a package insert containing information about its application, such as indications, dosage, and route of administration.

[0228] Use of antibody-drug conjugates with improved homogeneity

[0229] In a further embodiment, the disclosure relates to the use of a composition of an antibody-drug conjugate having improved homogeneity, prepared by the process of the present invention, in the manufacture of a pharmaceutical composition or kit for treating a condition or disorder of interest.

[0230] The target could be a mammal, such as a human.

[0231] The condition or disorder being treated may be a tumor (e.g., cancer), an autoimmune disease, or an infectious disease. In certain embodiments, the infectious disease may be a viral infection or a microbial infection.

[0232] In a further embodiment, the present disclosure relates to a method for treating a subject having a condition or disorder, comprising the step of administering to a subject in need of the improved homogeneity of a therapeutically effective amount of an ADC composition or a therapeutically effective amount of a pharmaceutical composition of the present invention, prepared by the process of the present invention, to the subject having the condition or disorder, thereby treating or preventing the condition or disorder.

[0233] In certain embodiments, the subject is identified to have a condition or fault that may respond to the ADC provided herein.

[0234] The target could be a mammal, such as a human.

[0235] The condition or disorder being treated may be a tumor (e.g., cancer), an autoimmune disease, or an infectious disease. In certain embodiments, the infectious disease may be a viral infection or a microbial infection.

[0236] The therapeutically effective dose of the ADC compositions provided herein depends on various factors known to the art, such as the subject's weight, age, medical history, current drug therapy, health status and potential for cross-reactivity, allergies, sensitivity and adverse side effects, as well as the route of administration and the severity of the disease. The dose may be reduced or increased by those skilled in the art (e.g., physicians or veterinarians) in proportion to these and other circumstances or requirements.

[0237] In certain embodiments, the ADC composition or pharmaceutical composition provided herein may be administered in therapeutically effective doses of about 0.01 mg / kg to about 10 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 embodiments, the ADC composition or pharmaceutical composition provided herein is administered in doses of about 50 mg / kg or less, and in certain 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. In certain embodiments, the dose may change during the course of treatment. For example, in certain embodiments, the initial dose may be greater than subsequent doses. In certain embodiments, the dose may change during the course of treatment depending on the subject's response.

[0238] The dosage regimen may be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[0239] The ADCs or pharmaceutical compositions provided herein can be administered by any route known in the art, e.g., parenterally (e.g., intravenous, intramuscular, or intradermal injection including subcutaneous, intraperitoneal, intravenous injection), or non-parenterally (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes and the like.

[0240] In certain embodiments, the conditions or disorders treated by the ADCs or pharmaceutical compositions provided herein are cancer, or cancerous conditions, autoimmune diseases, or infectious diseases.

[0241] The cancer may be an antigen-positive carcinoma, including carcinomas of the lung, breast, colon, ovary, and pancreas, e.g., cancers associated with the tumor-associated antigens listed in (1)-(53) under the heading "Tumor-Associated Antigen (TAA)".

[0242] Other specific types of cancer that can be treated with ADCs or pharmaceutical compositions provided herein include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, and bronchial carcinoma. Solid tumors include, but are not limited to, uterine lung cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminocarcinoma, embryonic carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymocyte tumor, pineal gland tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinoblastoma; acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia (AML), acute promyelocytic leukemia (APL), acute monoblastic leukemia, and acute erythroleukemia (acute Hematological cancers include, but are not limited to, erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocytic leukemia, acute anaplastic leukemia, chronic myeloid leukemia "CML", chronic lymphocytic leukemia "CLL", pilocytic cell leukemia, and multiple myeloma; Lymphoma: B-cell lymphoma, optionally Hodgkin lymphoma or non-Hodgkin lymphoma (non-Hodgkin lymphoma includes diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), and mucosa-associated lymphatic tissue lymphoma). This includes, but is not limited to, lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), or mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), or Waldenström hypergammaglobulinemia (WM).

[0243] Autoimmune diseases include active chronic hepatitis, Addison's disease, allergic alveolitis, allergic reactions, allergic rhinitis, Alport syndrome, anaphylaxis, ankylosing spondylitis, antiphospholipid syndrome, arthritis, roundworm infection, aspergillosis, atopic allergy, atopic dermatitis, atopic rhinitis, Behçet's disease, avian lung, bronchial asthma, Kaplan syndrome, cardiomyopathy, celiac disease, Chagas disease, chronic glomerulonephritis, Cogan syndrome, cold agglutinin disease, congenital rubella infection, CREST syndrome, Crohn's disease, cryoglobulinemia, Cushing's syndrome, dermatomyositis, and lupus discoid. Lupus, Dressler syndrome, Eaton-Lambert syndrome, echovirus infection, encephalomyelitis, endocrine eye disorders, Epstein-Barr virus infection, equine chronic emphysema, lupus erythematosus, Evans syndrome, Felty syndrome, fibromyalgia, Fuch's cyclitis, gastric atrophy Atrophy, gastrointestinal allergy, giant cell arteritis, glomerulonephritis, Goodpasture syndrome, graft-versus-host disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, idiopathic adrenal atrophy, idiopathic pulmonary fibrosis, IgA nephropathy, inflammatory bowel disease, insulin-dependent diabetes mellitus, juvenile arthritis, juvenile diabetes mellitus (Type 1), Lambert-Eaton syndrome, laminitis, lichen planus, lupoid hepatitis, lupus, lymphopenia, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyglandular syndrome, presenile dementia, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, recurrent miscarriage, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, Sampter's syndrome This may include, but is not limited to, the following: syndrome, schistosomiasis, Schmidt syndrome, scleroderma, Schulman syndrome, Sjögren's syndrome, Stiffman syndrome, sympathetic ophthalmitis, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyroiditis, thrombocytopenia, thyrotoxicosis, toxic epidermal necrolysis, type B insulin resistance, type I diabetes, ulcerative colitis, uveitis, vitiligo, Waldenström hypergammaglobulinemia, and Wegener's granulomatosis.

[0244] Certain types of infectious diseases that can be treated with the ADC or pharmaceutical composition of this disclosure include: bacterial diseases: diphtheria, pertussis, occult bacteremia, urinary tract infections, gastroenteritis, cellulitis, epiglottitis, tracheitis, adenoid hypertrophy, retropharyngeal abscess, impetigo, pustules, pneumonia, endocarditis, suppurative arthritis, pneumococcal infections, peritonitis, bacteremia, meningitis, acute suppurative meningitis, urethritis, cervicitis, proctitis, pharyngitis, salpingitis, epididymitis, gonorrhea, syphilis, listeriosis, anthrax, nocardiosis, salmonella, typhoid fever, dysentery, conjunctivitis, sinusitis, brucellosis, tularemia, cholera, bubonic plague, tetanus, necrotizing enterocolitis, actinomycosis, and mixed types. Anaerobic bacterial infections: syphilis, relapsing fever, leptospirosis, Lyme disease, rat-bite fever, tuberculosis, lymphadenitis, leprosy, chlamydia, chlamydial pneumonia, trachoma, inclusion conjunctivitis; systemic mycoses: histoplasmosis, coccidioidomycosis, blastomycosis, sporotrichumosis, cryptococcosis, systemic candidiasis, aspergillosis, mucormycosis, mycomas, chromomycosis; rickettsial diseases: typhus, Rocky Mountain spotted fever, ehrlichiosis, scrub rickettsia (Eastern Tick-Borne Rickettsioses, Rickettsial pox, Q fever, Bartonellosis; Parasitic diseases: Malaria, Babesiosis, African sleeping sickness, Chagas disease, Leishmaniasis, Dumb-Dum fever, Toxoplasmosis, Meningoencephalitis, Keratitis, Amebiasis, Giardiasis, Cryptosporidiosis, Isosporasis, Cyclosporiasis, Microsporiasis, Ascariasis, Whipworm infection, Hookworm infection, Pinworm infection, Ocular larval migrans, Trichinella, Dracunculosis, Lymphangiofilariasis, Loa filariasis, River blindness, Canine Heartworm Infection, Schistosomiasis, Swamp dermatitis, Oriental Lung Fluke, Oriental Liver Fluke fluke), hepatic fluke, hypertrophic paragonimiasis, opisthorchiasis, taeniasis, hydatidosis, polyhydatidosis;Viral diseases: Measles, subacute sclerosing panencephalitis, common cold, mumps, rubella, roseola, varicella, varicella, respiratory syncytial virus infection, croup, bronchiolitis, infectious mononucleosis, poliomyelitis, herpangina, hand-foot-and-mouth disease, bornholm's disease, genital herpes, genital warts, aseptic meningitis, myocarditis, pericarditis, gastroenteritis, acquired immunodeficiency syndrome (AIDS), Reye's syndrome, Kawasaki syndrome, influenza, bronchitis, viral "walking" pneumonia, acute febrile respiratory illness This list includes, but is not limited to, diseases such as acute pharyngoconjunctival fever, epidemic keratoconjunctivitis, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), herpes zoster, giant cell inclusion body disease, rabies, progressive multifocal leukoencephalopathy, kuru disease, fatal familial insomnia, Creutzfeldt-Jakob disease, Gerstmann-Streussler-Scheinker disease, tropical spastic paraplegia, Western equine encephalitis, California encephalitis, St. Louis encephalitis, yellow fever, dengue fever, lymphocytic choriomeningitis, Lassa fever, hemorrhagic fever, hantavirus pulmonary syndrome, Marburg virus infection, Ebola virus infection, and smallpox.

[0245] In one embodiment, the present disclosure includes a method for treating a disease or disorder in question, comprising the step of administering an effective amount of an ADC or pharmaceutical composition provided herein and another therapeutic agent to the subject.

[0246] In some embodiments, the therapeutic agent is an anticancer agent. Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cytoxane, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel.

[0247] In some embodiments, the therapeutic agent is an autoimmune disease agent. Suitable autoimmune disease agents include, but are not limited to, cyclosporine, cyclosporine A, mycophenolate mofetil, sirolimus, tacrolimus, etanercept, prednisone, azathioprine, methotrexate + cyclophosphamide, prednisone, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, DHEA, danazol, bromocriptine, meloxicam, and infliximab.

[0248] In some embodiments, the therapeutic agent is an anti-infective disease agent. In one embodiment, the anti-infective disease agent is an antibacterial agent: [beta]lactam antibiotics: penicillin G, penicillin V, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, bacampicillin, azurocillin, carbenicillin, mezlocillin, piperacillin, ticalcillin; aminoglycoside drugs: amikacin, gentamicin, kanamycin, neomycin, netylmycin, streptomycin, tobramycin; macrolides Drugs: Azithromycin, clarithromycin, erythromycin, lincomycin, clindamycin; Tetracycline drugs: Demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline; Quinolone drugs: Synoxacin, nalidixic acid; Fluoroquinolone drugs: Ciprofloxacin, enoxacin, glepafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin Polypeptide-based drugs: bacitracin, colistin, polymyxin B; Sulfonamide-based drugs: sulfisoxazole, sulfamethoxazole, sulfadiazine, sulfamethizol, sulfacetamide; Other antibacterial agents: trimethoprim, sulfamethoxazole, chloramphenicol, vancomycin, metronidazole, quinupristin, dalfopristin, rifampin, spectinomycin, nitrofurantoin; Antiviral agents: General antiviral agents: idoxuridiol Drugs for HIV infection include, but are not limited to, vidarabine, trifluridine, acyclovir, famciclovir, penciclovir, valacyclovir, ganciclovir, foscarnet, ribavirin, amantadine, rimantadine, cidofovir, antisense oligonucleotides, immunoglobulins, and interferon; and drugs for HIV infection such as zidovudine, didanosine, zalcitabine, stabudine, lamivudine, nevirapine, delavirdine, saquinavir, ritonavir, indinavir, and nelfinavir.

[0249] In specific embodiments, to demonstrate the process of this disclosure, three antibodies were selected: trastuzumab, rituximab, and cetuximab (each produced by WuXi Biologics using a standard method for preparing monoclonal antibodies according to the publicly available corresponding protein sequences) and conjugated with MC-VC-PAB-MMAE (HY-15575, MedChemExpress), MC-MMAF (HY-15579, MedChemExpress), GGFG-Dxd (HY-13631E, MedChemExpress), or Tesirine (C210323012-FP, WuXi Biologics).

[0250] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, substances, and methods described below are, in whole or in part, within the scope of the invention. These specific compositions, substances, and methods are not intended to limit the invention, but merely to illustrate specific embodiments that fall within the scope of the invention. Those skilled in the art can develop equivalent compositions, substances, and methods without practicing the capabilities of the invention and without departing from the scope of the invention. It will be understood that many modifications to the procedures described herein may still remain within the boundaries of the invention. It is the inventors' intention that such modifications fall within the scope of the invention. [Examples]

[0251] The present disclosure is illustrated in detail by reference to the following embodiments. However, those skilled in the art should understand that the following embodiments are provided for illustrative purposes only and are not intended to limit the present disclosure in any way.

[0252] Example 1. Conjugate Mc-VC-PAB-MMAE into mAb1, mAb2, and mAb3 using a conventional conjugation process.

[0253] mAb1 (trastuzumab, 838-1W220125, commercially available from Genentech, but in this invention, manufactured by WuXi Biologics according to standard molecular biological methods, its sequence information is shown in Table A), mAb2 (cetuximab, 120SD151029H13X01, commercially available from Merck, but in this invention, manufactured by WuXi Biologics according to standard molecular biological methods, its sequence information is shown in Table A), and mAb3 (rituximab, 254SD131223D01X01E01, commercially available from Genentech, but in this invention, manufactured by WuXi PB buffer (40 mM, pH 6.90; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)) prepared by Biologics according to standard molecular biological methods (sequence information shown in Table A) was individually reduced with TCEP (C4706-2G, Sigma-Aldrich) (TCEP / mAb molar ratio approximately 1.3, e.g., TCEP / mAb1-3 were 1.3 / 1.5 / 1.3 respectively) at 4°C for approximately 20 hours (e.g., 20 / 23 / 23 hours). A DMA (dimethylacetamide, ARK2190-1L, SAFC) solution containing linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress, abbreviated MC-VC-MMAE) (10 mg / mL in DMA, linker payload / mAb molar ratio 10.0) was added to the elution solution and mixed to form a conjugation reaction solution. The conjugation reaction was maintained at 4°C for 1-2 hours (e.g., 2 / 1 / 2 hours) to obtain the intermediate mAb conjugate.

[0254] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich) (10 mM stock in ddH2O, NAC / mAb molar ratio 16) was added to the conjugation reaction solution, and the conjugation was quenched at 4°C for 0.5 hours.

[0255] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0256] Each recovered ADC was characterized. The DAR values ​​are shown in Table 1 and Figure 1 (A-C).

[0257] [Table 1] remarks: a. "Random DAR2 conjugation" means that two payload molecules are randomly conjugated to a pair of reducing thiol groups without the addition and reoxidation reactions of transition metals. b. ADCs having DAR0, DAR2, DAR4, DAR6, and DAR8 are also referred to as D0, D2, D4, D6, and D8, respectively. c. The proportion of ADCs having DAR0, DAR2, DAR4, DAR6, and DAR8 is expressed in mole percent (mol%).

[0258] Example 2. Different antibodies (e.g., mAb1, mAb2, and mAb3) are conjugated with a linker payload (MC-VC-PAB-MMAE) in phosphate buffer (PB) in the presence of transition metal ions to form high-ratio DAR2 ADCs.

[0259] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics), mAb2 (cetuximab, 120SD151029H13X01, WuXi Biologics), and mAb3 (rituximab, 254SD131223D01X01E01, WuXi Biologics) were individually reduced in PB buffer (40 mM, pH 6.90; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4) using TCEP (C4706-2G, Sigma-Aldrich, TCEP / mAb molar ratio 3) at 12°C for 20 hours.

[0260] A DHAA (261565-1G, Sigma-Aldrich) solution (DHAA / mAb molar ratio 12) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 3.5 / 3 / 3 hours.

[0261] This intermediate product was purified using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher) with PB buffer (40 mM, pH 7.09, molar ratio).

[0262] A DMA (dimethylacetamide, ARK2190-1L, SAFC) solution of EDTA (EDTA·2Na, 10009719, SCR) (50 mM stock in ddH2O) with a final maximum concentration of 5 mM and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) (10 mg / mL in DMA, linker payload / mAb molar ratio 10.0) was added to the elution solution and mixed to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb with two linker payloads conjugated.

[0263] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0264] Each recovered ADC was characterized. The DAR values ​​are shown in Table 2 and Figure 2.

[0265] [Table 2]

[0266] The results in Table 2 show that, in the ADC compositions produced by the conjugation process of this application using three different antibodies, DAR 2% (mol percent) was the highest, DAR 0% and DAR 4% were low, and DAR6 ADC and DAR8 ADC were not produced or were produced at very low percentages.

[0267] Example 3. Various linker payloads are conjugated with mAb1 in phosphate buffer (PB) in the presence of transition metal ions to form high-ratio DAR2 ADCs.

[0268] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics) in PB buffer (40 mM, pH 6.90; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)) was reduced with TCEP (C4706-2G, Sigma-Aldrich, TCEP / mAb molar ratio 3 / 3 / 3 / 3 / 8 / 8) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4) at 12°C for 20 hours. The reduced antibody solution was directly subjected to the next step of reoxidation without removal of TCEP.

[0269] A solution of DHAA (261565-1G, Sigma-Aldrich) (10 mM stock in ddH2O, DHAA / mAb molar ratio 12) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vials and the conjugation groups of MC-VC-PAB-MMAE (HY-15575, MedChemExpress), MC-MMAF (HY-15579, MedChemExpress), Tesirine (PBD) (C210323012-FP, WuXiBiologics), and GGFG-Dxd (HY-13631E, MedChemExpress) were left to stand at 12°C for 3 / 3.5 / 4 / 3 hours, respectively.

[0270] DTNB (D8130-500MG, Sigma-Aldrich, DTNB / mAb molar ratio 7.6) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vials containing DM21 or DL1-VC-PAB-MMAE groups were left at 12°C for 0.5 hours.

[0271] This intermediate product was purified using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher) with PB buffer (40 mM, pH 6.90; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)).

[0272] EDTA (EDTA-2Na, 10009719, SCR; 50 mM stock in ddH2O, final concentration up to 5 mM) and linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress; MC-MMAF, HY-15579, MedChemExpress; Tesirine (PBD), C210323012-FP, WuXiBiologics; DM21, HY-13631E, MedChemExpress; or DL1-VC-PAB) A conjugation reaction solution was formed by adding a solution of DMA (dimethylacetamide, ARK2190-1L, SAFC) containing MMAE (WuXi Biologics) (10 mg / mL in DMA, linker payload / mAb molar ratio 10.0 / 10.0 / 7.0 / 5.0 / 15.0) or a solution of DMSO containing the linker payload (GGFG-Dxd, HY-13631E, MedChemExpress) (dimethyl sulfoxide, D4540-1L, Sigma-Aldrich; 10 mg / mL in DMSO, linker payload / mAb molar ratio 6.0) to the elution solution and mixing. The conjugation reaction was maintained at 12°C for 1 hour or 23 hours to obtain an intermediate mAb conjugated with two linker payloads / mAbs.

[0273] The molecular structure of DL1-VC-PAB-MMAE is represented by formula (I): [ka]

[0274] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich) (10 mM stock in ddH2O, NAC / mAb molar ratio 16) was added to the conjugation reaction solution, and the conjugation was quenched at 4°C for 0.5 hours.

[0275] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0276] Each recovered ADC was characterized. The DAR values ​​are shown in Table 3 and Figure 3 (A-F).

[0277] [Table 3]

[0278] The results in Table 3 show that, among the ADC compositions produced by the conjugation process of this application using six different linker payloads, DAR2% (mol percent) was the highest (over 75 mol%), DAR0% and DAR4% were low, and DAR6 ADC and DAR8 ADC were not produced or were produced at very low percentages.

[0279] It is known in the art that the six linker payloads used in this embodiment may represent those conventionally used in the field of bioconjugation. Therefore, the results in Table 3 may also demonstrate that the conjugation process of this application can be widely used with a variety of antibodies and a variety of linker payloads.

[0280] Example 4-1. Production of high-ratio DAR2 ADC using mAb1 and various reducing agents

[0281] mAb1 (trastuzumab, 838-1W220125, Genentech) is commercially available at 5 mg / mL, but in this invention, WuXi is used according to standard molecular biological methods. PB buffer (40 mM, pH 6.90; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)) or HEPES buffer (40 mM, pH 6.99), manufactured by Biologics (sequence information shown in Table A), were individually reduced at 12°C for approximately 20 hours with diPPBS1 (2-diphenylphosphanylbenzenesulfonic acid, diPPBS1 / mAb molar ratio 35), TCEP (C4706-2G, Sigma-Aldrich, TCEP / mAb molar ratio 3.25), or DPAA (DPAA / mAb molar ratio 10) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4).

[0282] A solution of DHAA (261565-1G, Sigma-Aldrich) (DHAA / mAb molar ratio 12) or DTNB (D8130-500MG, Sigma-Aldrich, DTNB / mAb molar ratio 2.2) was added to the antibody-reducing solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 3 hours.

[0283] This intermediate product was purified using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher) with PB buffer (40 mM, pH 6.90, molar ratio).

[0284] A DMA (dimethylacetamide, ARK2190-1L, SAFC) solution of EDTA (EDTA·2Na, 10009719, SCR) (50 mM stock in ddH2O) with a final maximum concentration of 5 mM and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) (10 mg / mL in DMA, linker payload / mAb molar ratio 10.0) was added to the elution solution and mixed to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb with two linker payloads conjugated.

[0285] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0286] The recovered ADCs were characterized. The DAR values ​​are shown in Table 4-1 and Figure 4 (A-C).

[0287] [Table 4-1]

[0288] The results in Table 4-1 show that compositions of ADCs produced by the conjugation process of this application under different reducing agents (Figure 4(A~C): A: TCEP, B: diPPBS1, C: DPAA) yielded significantly higher DAR2 ratios.

[0289] Example 4-2. Manufacturing a high-ratio DAR2 ADC using mAb1 and TCEP in different ratios.

[0290] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, commercially available from Genentech, but in this invention, it was manufactured by WuXi Biologics according to standard molecular biological methods, and its sequence information is shown in Table A) in HEPES buffer (40 mM, pH 6.99) was individually reduced with TCEP (C4706-2G, Sigma-Aldrich) (TCEP / mAb molar ratio 3.25 / 10 / 16) (TCEP / mAb molar ratio 3.25 / 10 / 16) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4) at 12°C for approximately 20 hours. The antibody reduction solution was either directly subjected to the next step in reoxidation without removal of TCEP, or purified to remove TCEP for the next step in reoxidation.

[0291] DTNB (D8130-500MG, Sigma-Aldrich, DTNB / mAb molar ratio 2.2) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 2 / 26 / 26 hours.

[0292] This intermediate product was either left unpurified or purified using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher) with PB buffer (40 mM, pH 6.90, molar ratio).

[0293] A DMA (dimethylacetamide, ARK2190-1L, SAFC) solution of EDTA (EDTA·2Na, 10009719, SCR; 50 mM stock in ddH2O, final concentration maximal 5 mM) and linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) (10 mg / mL in DMA, linker payload / mAb molar ratio 10.0) was added to the elution solution and mixed to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads were conjugated.

[0294] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich) (NAC / mAb molar ratio 18) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0295] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0296] The recovered ADCs were characterized. The DAR values ​​are shown in Table 4-2 and Figure 4 (D-F).

[0297] [Table 4-2]

[0298] The results in Table 4-2 show that compositions of ADCs produced by the conjugation process of this application with different ratios of TCEP (Figure 4(DF): (D)TCEP, 3.25 equivalents, (E)TCEP, 10 equivalents, (F)TCEP, 16 equivalents) were able to obtain significantly higher DAR2 ratios, indicating that a high TCEP / mAb molar ratio is not necessary, and that 3.25 equivalents of TCEP is desirable.

[0299] Example 5. Manufacturing a high-ratio DAR2 ADC using mAb1 with various buffers.

[0300] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics) in HEPES buffer (40 mM, pH 6.99; H3375-500G, Sigma-Aldrich) / MES buffer (40 mM, pH 6.51, M3671-250G, Sigma-Aldrich) / Tris buffer (40 mM, pH 7.01, T1503-1KG, Sigma-Aldrich) was reduced with TCEP (C4706-2G, Sigma-Aldrich) (TCEP / mAb molar ratio 3.25) at 12°C for 18 hours in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4). The reduced antibody solution was directly subjected to the next step of reoxidation without removal of TCEP.

[0301] DTNB (D8130-500MG, Sigma-Aldrich, DTNB / mAb molar ratio 2.2) was added to the antibody-reducing solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 2 hours.

[0302] A solution of EDTA (EDTA-2Na, 10009719, SCR, final concentration up to 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0303] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich, NAC / mAb molar ratio 16) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0304] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0305] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 5 and Figure 5.

[0306] [Table 5]

[0307] The results in Table 5 show that compositions of ADCs produced by the conjugation process of this application in different buffers (Figure 5: (A) HEPES, (B) MES, (C) Tris) yielded significantly higher DAR2 ratios, demonstrating that conventional buffers can be used in the conjugation process of this application.

[0308] Example 6. Manufacturing a high-ratio DAR2 ADC using mAb3 with various buffers.

[0309] 5 mg / mL of mAb3 (rituximab, 254SD131223D01X01E01, WuXi Biologics) was reduced in HEPES buffer (40 mM, pH 6.99; H3375-500G, Sigma-Aldrich) / MES buffer (40 mM, pH 6.51, M3671-250G, Sigma-Aldrich) / Tris buffer (40 mM, pH 7.01, T1503-1KG, Sigma-Aldrich) with TCEP (C4706-2G, Sigma-Aldrich) (TCEP / mAb molar ratio 3.25) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4) for 20 hours at 12°C. The reduced antibody solution was directly subjected to the next step of reoxidation without removal of TCEP.

[0310] A DTNB (D8130-500MG, Sigma-Aldrich) solution (DTNB / mAb molar ratio 2.2) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 2 hours.

[0311] A solution of EDTA (EDTA-2Na, 10009719, SCR, final concentration up to 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0312] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich) (NAC / mAb molar ratio 18) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0313] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0314] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 6 and Figure 6.

[0315] [Table 6]

[0316] The results in Table 6 show that compositions of ADCs produced by the conjugation process of this application in different buffers (Figure 6: (A) HEPES, (B) MES, (C) Tris) yielded significantly higher DAR2 ratios, demonstrating that conventional buffers can be used in the conjugation process of this application.

[0317] Example 7. Production of ADCs using mAb3 in various buffer solutions that do not contain transition metal ions.

[0318] 5 mg / mL of mAb3 (rituximab, 254SD131223D01X01E01, WuXi Biologics) in HEPES buffer (40 mM, pH 6.99; H3375-500G, Sigma-Aldrich) / MES buffer (40 mM, pH 6.51; M3671-250G, Sigma-Aldrich) / Tris buffer (40 mM, pH 7.01; T1503-1KG, Sigma-Aldrich) was reduced with TCEP (C4706-2G, Sigma-Aldrich, TCEP / mAb molar ratio 3.25) at 12°C for 20 hours. The reduced antibody solution was directly subjected to the next step of reoxidation without removal of TCEP.

[0319] DTNB (D8130-500MG, Sigma-Aldrich) solution was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 2 hours.

[0320] A solution of EDTA (EDTA-2Na, 10009719, SCR, final concentration maximum 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour.

[0321] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich, NAC / mAb molar ratio 18) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0322] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0323] The purified ADCs were characterized. The DAR values ​​are shown in Table 7 and Figure 7.

[0324] [Table 7]

[0325] The results in Table 7 show that high DAR2 ratios were not obtained in ADC compositions produced by conjugation processes using different buffers that did not contain transition metal ions (Figure 7(A)HEPES, (B)MES, (C)Tris). 2+ This demonstrates that ) is important to the conjugation process of this application.

[0326] Example 8. Production of high-ratio DAR2 ADC using bispecific antibody (mAb4) in phosphate buffer (PB).

[0327] 9.53 mg / mL of mAb4 (W329001-U5T5.E17R-57.uIgG1, WuXi Biologics, sequence information shown in Table A) in PB buffer (40 mM, pH 7.12; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)) was reduced with TCEP (C4706-2G, Sigma-Aldrich) (TCEP / mAb molar ratio 10) at 4°C for 20 hours in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4). The reduced antibody solution was directly subjected to the next step of reoxidation without removal of TCEP.

[0328] DTNB (261565-1G, Sigma-Aldrich) solution (DTNB / mAb molar ratio 11) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 0.5 hours.

[0329] A DMA (dimethylacetamide, ARK2190-1L, SAFC) solution of EDTA (EDTA·2Na, 10009719, SCR; 50 mM stock in ddH2O, final maximum concentration 5 mM) and linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) (10 mg / mL in DMA, linker payload / mAb molar ratio 10.0) was added to the elution solution and mixed to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 0.5 hours to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0330] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich) (10 mM stock in ddH2O, NAC / mAb molar ratio 40) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0331] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher).

[0332] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 8 and Figure 8.

[0333] [Table 8]

[0334] The results in Table 8 demonstrate that, in addition to monospecific antibodies, the conjugation process of this application is also applicable to bispecific antibodies, and can produce ADC compositions with a remarkably high DAR2 ratio.

[0335] Example 9. Reduction temperature and reduction time

[0336] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics) in HEPES buffer (40 mM, pH 6.99; H3375-500G, Sigma-Aldrich) was reduced with TCEP (C4706-2G, Sigma-Aldrich) (TCEP / mAb molar ratio 3.25 at 4°C / 19 hours) / (TCEP / mAb molar ratio 3.0 at 22°C / 19 hours) / (TCEP / mAb molar ratio 2.8 at 37°C / 2 hours) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4) and the antibody-reduced solution was directly subjected to the next reoxidation step without removing TCEP.

[0337] DTNB (D8130-500MG, Sigma-Aldrich, DTNB / mAb molar ratio 2.2) was added to the antibody-reducing solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 2 hours.

[0338] A solution of EDTA (EDTA-2Na, 10009719, SCR, final concentration up to 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0339] AC (acetylcysteine, A9165-100G, Sigma-Aldrich) (NAC / mAb molar ratio 18) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0340] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0341] The recovered ADCs were characterized. The DAR values ​​are shown in Table 9-1 and Figure 9(A-C).

[0342] [Table 9-1]

[0343] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics) in HEPES buffer (40 mM, pH 6.99; H3375-500G, Sigma-Aldrich) was reduced with TCEP (C4706-2G, Sigma-Aldrich, TCEP / mAb molar ratio 3.25) at 4°C for 19 hours / 45 hours / 92 hours in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4). The reduced antibody solution was then subjected directly to the next reoxidation step without removing TCEP.

[0344] DTNB (D8130-500MG, Sigma-Aldrich, DTNB / mAb molar ratio 2.2) was added to the antibody-reducing solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 2 hours.

[0345] A solution of EDTA (EDTA-2Na, 10009719, SCR, final concentration up to 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0346] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich, NAC / mAb molar ratio 18) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0347] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0348] The recovered ADCs were characterized. The DAR values ​​are shown in Table 9-2 and Figure 9 (D-F).

[0349] [Table 9-2]

[0350] The results in Tables 9-1 and 9-2 indicate that reduction is preferable at approximately 4°C, room temperature, or 37°C for approximately 2 to 45 hours.

[0351] Example 10. Various Zn 2+ Related salts

[0352] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi) in HEPES buffer (40 mM, pH 6.99; H3375-500G, Sigma-Aldrich). Biologics (Biologics) was reduced with TCEP (C4706-2G, Sigma-Aldrich) (TCEP / mAb molar ratio 3.25) at 12°C for 20 hours in the presence of either ZnSO4·7H2O (Z0251-100G, Sigma-Aldrich), ZnBr2·2H2O (546739-100G, Sigma-Aldrich), Zn(ClO4)2·6H2O (401439-100G, Sigma-Aldrich), or Zn(OAc)2·2H2O (96459-250G, Zn(II) / mAb molar ratio 4), respectively. The reduced antibody solution was then directly subjected to the next step of reoxidation without removing TCEP.

[0353] DTNB (D8130-500MG, Sigma-Aldrich, DTNB / mAb molar ratio 2.2) was added to the antibody-reducing solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 2 hours.

[0354] A solution of EDTA (EDTA-2Na, 10009719, SCR, final concentration up to 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0355] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich) (NAC / mAb molar ratio 16) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0356] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher).

[0357] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 10 and Figure 10.

[0358] [Table 10]

[0359] The results in Table 10 show free Zn 2+ As long as ions are released into the reaction solution, Zn 2+ This demonstrated that there are no restrictions on the types of related salts.

[0360] Example 11. Zn 2+ Different molar ratios of ions

[0361] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics) in PB buffer (40 mM, pH 6.90; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)) or HEPES buffer (40 mM, pH 6.99; H3375-500G, Sigma-Aldrich) was reduced with TCEP (C4706-2G, Sigma-Aldrich) (TCEP / mAb molar ratio 8 / 8 / 3.25) in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 2 / 8 / 16) at 4°C / 17 hours or 12°C / 20 hours. The reduced antibody solution was directly subjected to the next step of reoxidation without removal of TCEP.

[0362] DTNB (D8130-500MG, Sigma-Aldrich, DTNB / mAb molar ratio 7.6 / 7.6 / 2.2) was added to the antibody-reducing solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 1 or 2 hours.

[0363] A solution of EDTA (EDTA-2Na, 10009719, SCR, final concentration up to 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0364] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich) (NAC / mAb molar ratio 16) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0365] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher).

[0366] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 11 and Figure 11.

[0367] [Table 11]

[0368] The results in Table 11 show that for 2 to 16 equivalents of Zn 2+ Ions (i.e., Zn) 2+ It was shown that using an ion / mAb molar ratio of 2:16 is preferable.

[0369] Example 12. The reduced antibody solution was subjected to a reoxidation step after TCEP removal.

[0370] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics) in HEPES buffer (40 mM, pH 6.99; H3375-500G, Sigma-Aldrich) was reduced with TCEP (C4706-2G, Sigma-Aldrich) (TCEP / mAb molar ratio 8) at 4°C for 17 hours in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4). The reduced antibody solution was subjected to a reoxidation step after TCEP removal.

[0371] To remove TCEP, this intermediate product was purified using PB buffer (40 mM, pH 6.90; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)) with a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0372] A DHAA (261565-1G, Sigma-Aldrich) solution (DHAA / mAb molar ratio 8) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 2 hours.

[0373] This intermediate product was purified using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher) with PB buffer (40 mM, pH 6.90; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)).

[0374] A solution of EDTA (EDTA-2Na, 10009719, SCR, final concentration maximal 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) was added to the elution solution and mixed to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0375] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich) (NAC / mAb molar ratio 16) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0376] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher).

[0377] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 12 and Figure 12.

[0378] [Table 12]

[0379] The data in Table 12, compared to the data in Table 3, showed that removing excess TCEP after antibody reduction had no effect.

[0380] Example 13. Production of high-concentration DAR2 ADC using different oxidizing agents.

[0381] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics) in PB buffer (40 mM, pH 6.90; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)) or HEPES buffer (40 mM, pH 6.99; H3375-500G, Sigma-Aldrich) was individually reduced with TCEP (C4706-2G, Sigma-Aldrich, TCEP / mAb molar ratio approximately 3.25) at 12°C for 20 hours in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4).

[0382] To the antibody-reducing solution, DHAA (261565-1G, Sigma-Aldrich) solution (DHAA / mAb molar ratio 12), DTNB solution (D8130-500MG, Sigma-Aldrich, DTNB / mAb molar ratio 2.2), and DDD solution (2-aminophenyl disulfide, Sigma-Aldrich, DDD / mAb molar ratio 1.82) were added. The reaction mixture was properly mixed, and the reaction vial was left at 22°C for 2 hours.

[0383] A conjugation reaction solution was formed by adding a DMA (dimethylacetamide, ARK2190-1L, SAFC) solution of EDTA (EDTA·2Na, 10009719, SCR; 50 mM stock in ddH2O, final concentration maximum 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) (10 mg / mL in DMA, linker payload / mAb molar ratio 10.0) to the aforementioned solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads were conjugated.

[0384] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich) (10 mM stock in ddH2O, NAC / mAb molar ratio 18) was added to the conjugation reaction solution, and the conjugation was quenched at 4°C for 0.5 hours.

[0385] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0386] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 13 and Figure 13.

[0387] [Table 13]

[0388] The results in Table 13 show that many oxidizing agents conventionally used for antibody oxidation (e.g., oxidation of free thiol groups in antibodies) can be used in the conjugation process of this disclosure.

[0389] Example 14. Temperature and duration of re-oxidation

[0390] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics) in PB buffer (40 mM, pH 6.90; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)) or HEPES buffer (40 mM, pH 6.99; H3375-500G, Sigma-Aldrich) was reduced with TCEP (C4706-2G, Sigma-Aldrich, TCEP / mAb molar ratio approximately 3.0) at 12°C / 20 hours in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4). The reduced antibody solution was directly subjected to the next step of reoxidation without removal of TCEP.

[0391] DTNB (D8130-500MG, Sigma-Aldrich, DTNB / mAb molar ratio approximately 2.0) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left to stand at 4°C / 12°C / 22°C / 37°C for 2 hours.

[0392] A solution of EDTA (EDTA-2Na, 10009719, SCR, final concentration up to 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0393] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich) (NAC / mAb molar ratio 18) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0394] The product was buffer-replaced using a desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher) and then buffer-replaced with storage buffer (20 mM histidine-acetone buffer, pH 5.5).

[0395] The recovered ADCs were characterized. The DAR values ​​are shown in Table 14-1 and Figure 14 (A-D).

[0396] [Table 14-1]

[0397] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics) in HEPES buffer (40 mM, pH 6.99; H3375-500G, Sigma-Aldrich) was reduced with TCEP (C4706-2G, Sigma-Aldrich, TCEP / mAb molar ratio 3.25) at 12°C / 20 hours in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4). The reduced antibody solution was directly subjected to the next step of reoxidation without removal of TCEP.

[0398] DTNB solution (D8130-500MG, Sigma-Aldrich, DTNB / mAb molar ratio 2.2) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 0.5 hours, 2 hours, 4 hours, or 24 hours.

[0399] A solution of EDTA (EDTA-2Na, 10009719, SCR, final concentration up to 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0400] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich) (NAC / mAb molar ratio 18) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0401] The product was subjected to buffer exchange with storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0402] The recovered ADCs were characterized. The DAR values ​​are shown in Table 14-2 and Figure 14(E~H).

[0403] [Table 14-2]

[0404] The results in Tables 14-1 and 14-2 indicate that reoxidation should ideally be carried out at room temperature for approximately 1 to 24 hours.

[0405] Example 15. Conjugation temperature

[0406] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics) in PB buffer (40 mM, pH 6.90; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)) was reduced with TCEP (C4706-2G, Sigma-Aldrich, TCEP / mAb molar ratio 3) at 12°C for 20 hours in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4). The reduced antibody solution was directly subjected to the next step of reoxidation without removal of TCEP.

[0407] DNTB (22582-5G, Sigma-Aldrich) solution (DNTB / mAb molar ratio 1.8) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 3 hours. The reoxidized antibody solution was directly used in the next step to conjugate the reactive group-supported payload without purifying the reoxidized antibody.

[0408] A DMA (dimethylacetamide, ARK2190-1L, SAFC) solution of EDTA (EDTA-2Na, 10009719, SCR; 50 mM stock in ddH2O, final concentration maximum 5 mM) and linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) (10 mg / mL in DMA, linker payload / mAb molar ratio 10.0) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C or 0°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0409] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher).

[0410] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 15 and Figure 15.

[0411] [Table 15]

[0412] The results in Table 15 indicate that conjugation is preferable to be performed at 0°C or 12°C.

[0413] Example 16. In the reoxidation step, transition metal ions are added, and the reoxidation is carried out at 22°C for 47.5 hours.

[0414] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics) in PB buffer (40 mM, pH 6.90; Na2HPO8 (24.4 mM), NaH2PO4 (15.6 mM)) was reduced with TCEP (C4706-2G, Sigma-Aldrich; TCEP / mAb molar ratio 8) at 12°C for 18 hours. The reduced antibody solution was purified to remove TCEP for the subsequent reoxidation step.

[0415] To remove TCEP, this intermediate product was purified using PB buffer (40 mM, pH 6.90; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)) with a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0416] DTNB (D8130-500MG, Sigma-Aldrich) solution (5 mM stock in 40 mM PB buffer, DTNB / mAb molar ratio 1) and ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4) were added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 22°C for 47.5 hours.

[0417] A solution of EDTA (EDTA-2Na, 10009719, SCR, 50 mM stock in ddH2O, final concentration up to 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0418] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher).

[0419] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 16 and Figure 16.

[0420] [Table 16]

[0421] The results in Table 16 show that adding transition metal ions in the reoxidation step can produce an ADC composition with a high proportion of DAR2.

[0422] Example 17. In the reoxidation step, transition metal ions are added, and the reoxidation is carried out at 12°C for 170 hours.

[0423] HEPES buffer (40mM, pH 6.99; H3375-500G, Sigma-Aldrich) / PB buffer (40mM, pH 6.90; Na2HPO4 (24.4mM), NaH2PO4 (15.6mM)) containing 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi The antibody (manufactured by Biologics) was reduced by TCEP (C4706-2G, Sigma-Aldrich, TCEP / mAb molar ratio 8) at 12°C for 18 hours. The reducing antibody solution was purified to remove TCEP for the next reoxidation step. To remove TCEP, this intermediate product was purified using PB buffer (40mM, pH 6.90; Na2HPO4 (24.4mM), NaH2PO4 (15.6mM)) with a spin desalting column (40kDa, 0.5mL, REF:87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0424] ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4) was added to the reducing antibody solution and reacted at 12°C for 0.5 hours. DTNB (D8130-500MG, Sigma-Aldrich) solution (DTNB / mAb molar ratio 2.04 / 2.12) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 170 hours.

[0425] A solution of EDTA (EDTA-2Na, 10009719, SCR, 50 mM stock in ddH2O, final concentration up to 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0426] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich) (10 mM stock in ddH2O, NAC / mAb molar ratio 18) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0427] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher).

[0428] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 17 and Figure 17.

[0429] [Table 17]

[0430] The results in Table 17 show that, in the case of mAb1, if transition metal ions are not added in the conjugation process of this application (i.e., the reduction step or the reoxidation step), no ADC is produced.

[0431] Example 18. In the reoxidation step, transition metal ions are added, and reoxidation is carried out at 12°C for 170 hours (mAb3).

[0432] 5 mg / mL of mAb3 (rituximab, 254SD131223D01X01E01, WuXi Biologics) in PB buffer (40 mM, pH 6.90; Na2HPO8 (24.4 mM), NaH2PO4 (15.6 mM)) was reduced by TCEP (C4706-2G, Sigma-Aldrich; TCEP / mAb molar ratio 8) at 12°C for 18 hours. The reduced antibody solution was purified to remove TCEP for the next reoxidation step. To remove TCEP, this intermediate product was purified using a spin desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher) in PB buffer (40 mM, pH 6.90; Na2HPO4 (24.4 mM), NaH2PO4 (15.6 mM)).

[0433] ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4) was added to the reducing antibody solution and reacted at 12°C for 0.5 hours. DTNB (D8130-500MG, Sigma-Aldrich) solution (5 mM stock in 40 mM PB buffer, DTNB / mAb molar ratio 2.50) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 170 hours.

[0434] A solution of EDTA (EDTA-2Na, 10009719, SCR, 50 mM stock in ddH2O, final concentration up to 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0435] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich) (10 mM stock in ddH2O, NAC / mAb molar ratio 18) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0436] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher).

[0437] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 18 and Figure 18.

[0438] [Table 18]

[0439] The results in Table 18 show that, in the case of mAb3, if no transition metal ions are added in the conjugation process of this application (i.e., the reduction step or the reoxidation step), only a small amount of D2 is produced.

[0440] Example 19. In the reoxidation step, transition metal ions are added, and reoxidation is carried out at 12°C for 170 hours (mAb1 + various linker payloads).

[0441] HEPES buffer (40mM, pH 6.99; H3375-500G, Sigma-Aldrich) / PB buffer (40mM, pH 6.90; Na2HPO4 (24.4mM), NaH2PO4 (15.6mM)) containing 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi The antibody (manufactured by Biologics) was reduced by TCEP (C4706-2G, Sigma-Aldrich, TCEP / mAb molar ratio 8) at 12°C for 18 hours. The reducing antibody solution was purified to remove TCEP for the next reoxidation step. To remove TCEP, this intermediate product was purified using PB buffer (40mM, pH 6.90; Na2HPO4 (24.4mM), NaH2PO4 (15.6mM)) with a spin desalting column (40kDa, 0.5mL, REF:87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0442] ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4) was added to the reducing antibody solution and reacted at 12°C for 0.5 hours. DTNB (D8130-500MG, Sigma-Aldrich) solution (DTNB / mAb molar ratio 2.12) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 170 hours.

[0443] A solution of EDTA (EDTA-2Na, 10009719, SCR; 50 mM stock in ddH2O, final concentration up to 5 mM) and a linker payload (MC-VC-PAB-MMAE, HY-15575; GGFG-Dxd, HY-13631E, MedChemExpress; or Tesirine (PBD), C210323012-FP, WuXiBiologics) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0444] NAC (acetylcysteine, A9165-100G, Sigma-Aldrich; 10 mM stock in ddH2O, NAC / mAb molar ratio 18) was added to the conjugation reaction solution, and the conjugation was quenched at 12°C for 0.5 hours.

[0445] The product was subjected to buffer exchange to storage buffer (20 mM histidine-acetone buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher).

[0446] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 19 and Figure 19.

[0447] [Table 19]

[0448] The results in Table 19 show that compositions with a high DAR2 ratio can be produced by adding transition metal ions in the reoxidation step.

[0449] Example 20. Antibody concentration

[0450] 5 mg / mL and 10 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics) in HEPES buffer (40 mM, pH 6.99; H3375-500G, Sigma-Aldrich) were reduced with TCEP (C4706-2G, Sigma-Aldrich, TCEP / mAb molar ratio 3.25) at 12°C / 20 hours in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4). The reduced antibody solution was directly subjected to the next step of reoxidation without removal of TCEP.

[0451] DNTB (22582-5G, Sigma-Aldrich) solution (DNTB / mAb molar ratio 2.2) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 2 hours. The reoxidized antibody solution was directly used in the next step to conjugate the reactive group-supported payload without purifying the reoxidized antibody.

[0452] A DMA (dimethylacetamide, ARK2190-1L, SAFC) solution of EDTA (EDTA-2Na, 10009719, SCR; 50 mM stock in ddH2O, final maximum concentration 5 mM) and linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) (10 mg / mL in DMA, linker payload / mAb molar ratio 10.0) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0453] The product was subjected to buffer exchange with storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0454] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 20 and Figure 20.

[0455] [Table 20]

[0456] The results in Table 20 show that compositions with a high ratio of DAR2 can be produced at different antibody concentrations.

[0457] Example 21. Concentration of HEPES buffer

[0458] 5 mg / mL of mAb1 (trastuzumab, 838-1W220125, WuXi Biologics) in HEPES buffer (20 mM and 40 mM, pH 6.99; H3375-500G, Sigma-Aldrich) was reduced with TCEP (C4706-2G, Sigma-Aldrich, TCEP / mAb molar ratio 3.25) at 12°C / 20 hours in the presence of ZnCl2 (14422-500G, Sigma-Aldrich, Zn(II) / mAb molar ratio 4). The reduced antibody solution was directly subjected to the next step of reoxidation without removal of TCEP.

[0459] DNTB (22582-5G, Sigma-Aldrich) solution (DNTB / mAb molar ratio 2.2) was added to the reducing antibody solution. The reaction mixture was properly mixed, and the reaction vial was left at 12°C for 2 hours. The reoxidized antibody solution was directly used in the next step to conjugate the reactive group-supported payload without purifying the reoxidized antibody.

[0460] A DMA (dimethylacetamide, ARK2190-1L, SAFC) solution of EDTA (EDTA-2Na, 10009719, SCR; 50 mM stock in ddH2O, final maximum concentration 5 mM) and linker payload (MC-VC-PAB-MMAE, HY-15575, MedChemExpress) (10 mg / mL in DMA, linker payload / mAb molar ratio 10.0) was added to the aforementioned solution to form a conjugation reaction solution. The conjugation reaction was maintained at 12°C for 1 hour to obtain an intermediate mAb in which two linker payloads / mAbs were conjugated.

[0461] The product was subjected to buffer exchange with storage buffer (20 mM histidine-acetone buffer, pH 5.5) using a desalting column (40 kDa, 0.5 mL, REF: 87766, lot number SJ251704, manufacturer: Thermo Fisher).

[0462] The recovered ADCs were feature-defined. The DAR values ​​are shown in Table 21 and Figure 21.

[0463] [Table 21]

[0464] The results in Table 21 show that changes in HEPES concentration have a slight effect on the ratio of DAR2 in the composition obtained as a result of ADC.

[0465] [Table A-1] [Table A-2]

[0466] Those skilled in the art will further understand that the present invention can be embodied in other specific forms without departing from its spirit or central attributes. It should be understood that other modifications are considered within the scope of the invention, given that the foregoing description of the invention discloses only exemplary embodiments. Therefore, the invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims indicating the scope and content of the invention.

[0467] References (1) Junutula Jagath R, Raab Helga,Clark Suzanna et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. [J] .Nat Biotechnol, 2008, 26: 925-32. (2) Hallam Trevor J, Smider Vaughn V,Unnatural amino acids in novel antibody conjugates. [J] .Future Med Chem, 2014, 6: 1309-24. (3) Hallam Trevor J, Wold Erik, Wahl Alan et al. Antibody conjugates with unnatural amino acids. [J] .Mol Pharm, 2015, 12: 1848-62. (4) Agarwal Paresh, Bertozzi Carolyn R, Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. [J] .Bioconjug Chem, 2015, 26: 176-92. (5) Sarrett Samantha M, Rodriguez Cindy, Rymarczyk Grzegorz et al. Lysine-Directed Site-Selective Bioconjugation for the Creation of Radioimmunoconjugates. [J]. Bioconjug Chem, 2022, 33: 1750-1760.

Claims

1. A process for preparing an antibody-drug conjugate (ADC) composition, the following: (a) Reduction step: Incubating the reducing agent and antibody in a buffer system to reduce the interchain disulfide bonds in the antibody, and optionally purifying the reduced antibody; (b) Reoxidation step: A step in which an oxidizing agent is added to reoxidize a portion of the reduced thiol groups, and optionally the reoxidized antibody is purified; Step (a) or step (b) is carried out in the presence of a transition metal ion. In step (b), the reduced thiol group not blocked by the transition metal ion is reoxidized; (c) Conjugation step: a step of removing transition metal ions and adding an excess amount of reactive group-supported payload to react with the reduced thiol groups of the reoxidized antibody; and (d) Recovery step: A step of recovering the obtained antibody-drug conjugate to obtain an antibody-drug conjugate (ADC) composition, A process that includes this.

2. The transition metal ion is Zn ２＋ Mn ２＋ Ni ２＋ Fe ２＋ Fe ３＋ ,Cd ２＋ , or a combination thereof, preferably the transition metal ion is Zn ２＋ The process according to claim 1.

3. The process according to claim 1, wherein the buffer system used in step (a) is selected from TAPS, bicine, tris, tricine, HEPES, TES, MOPS, PIPES, sodium citrate, histidine buffer, PB (phosphate buffer), PBS, or MES, and the pH value of the buffer system is about 5 to 8, preferably about 7.

0.

4. The process according to claim 1, wherein in step (a), the reducing agent is added in a molar ratio of 1 to 20 relative to the antibody.

5. The process according to claim 1, wherein the transition metal ion is added to the antibody in a molar ratio of 0.5 to 50.

6. The reducing agent in step (a) is tris(2-carboxyethyl)phosphine (TCEP), THPP (tris(3-hydroxypropyl)phosphine), diPPB (2-diphenylphosphanylbenzenesulfonic acid), DTT (dithiothreitol), DPAA (2-(diphenylphosphino)acetic acid), DTE (dithioerythritol), β-mercaptoethanol, LiAlH ４ , Na ２ S ２ O ３ , KBH ４ , or hydrazine, the process according to claim 1.

7. The oxidizing agent in step (b) is hydrogen peroxide (H ２ O ２ ), nitrate compounds, potassium chlorate (KClO ３ ), peroxydisulfate (H ２ S ２ O ８ ), peroxymonosulfate (H ２ SO ５ ), NaClO, sodium dichromate (Na ２ Cr ２ O ７ ), permanganate compounds, sodium perborate, nitrous oxide, sodium bismuthate (NaBiO ３ The process according to claim 1, wherein the substance is selected from cerium sulfate, dehydroascorbic acid (DHAA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), or 2-aminophenyl disulfide (DDD), preferably DHAA or DTNB.

8. The process according to claim 1, wherein in step (b), the oxidizing agent is added in a molar ratio of 0.5 to 160 relative to the antibody.

9. The process according to claim 1, wherein in step (c), the transition metal ions are removed by adjusting the pH, adjusting the temperature, changing the buffer solution, or by adding a metal chelating agent, the metal chelating agent being EDTA, DTPA, ethylenediamine, 2,2'-bipyridine, or 1,10-phenanthroline.

10. The process according to claim 1, wherein the payload comprises a maleimide moiety, a bromide, an iodide, an electron-deficient alkenyl or alkynyl, a mono or di-derivative of a disulfide, a sulfone, a bicyclo[1.1.0]butane derivative, a sulfonyl fluoride, a pentafluorophenol ester, a palladium oxidative addition complex, an iodoxolone, or a highly electron-deficient arene.

11. The process according to any one of claims 1 to 10, wherein the antibody is a monoclonal antibody or a polyclonal antibody, or the antibody is a single-specificity antibody or a multispecificity antibody, for example, a bispecificity antibody.

12. The process according to claim 11, wherein the antibody is a human antibody, a humanized antibody, a chimeric antibody, or an antigen-binding portion thereof.

13. The process according to any one of claims 1 to 12, wherein the payload comprises a diagnostic agent, a therapeutic agent, or a labeling agent.

14. The composition contains D2 in an amount exceeding 64 mol%, preferably exceeding 75 mol%, or exceeding 80 mol%, based on the total molar concentration of D0, D2, D4, D6, and D8. D0 represents an antibody molecule that is not conjugated with any drug. D2 represents an ADC molecule in which two drug molecules are coupled to one single antibody molecule. D4 represents an ADC molecule in which four drug molecules are coupled to one single antibody molecule. D6 represents an ADC molecule in which six drug molecules are coupled to one single antibody molecule, and The process according to any one of claims 1 to 13, wherein D8 represents an ADC molecule in which eight drug molecules are coupled to one single antibody molecule.

15. The process according to claim 14, wherein the composition contains less than 36 mol%, preferably less than 25 mol%, more preferably less than 20 mol%, and most preferably less than 15%, of D0, D2, D4, D6, and D8 in total content, based on the total molar concentration of D0, D2, D4, D6, and D8.

16. A composition of an antibody-drug conjugate (ADC) prepared by a process according to any one of claims 1 to 15, comprising D2 in an amount exceeding 64 mol%, preferably exceeding 75 mol%, or even exceeding 80 mol%, based on the total molar concentration of D0, D2, D4, D6, and D8.

17. The composition according to claim 16, wherein the composition contains less than 36 mol%, preferably less than 25 mol%, more preferably less than 20 mol%, and most preferably less than 15% of D0, D2, D4, D6, and D8 in total content, based on the total molar concentration of D0, D2, D4, D6, and D8.

18. A pharmaceutical composition comprising an effective amount of the composition according to claim 16 or 17 and a pharmaceutically acceptable carrier.

19. A method for treating a condition or disorder in a subject, comprising the step of administering to the subject a therapeutically effective amount of the composition according to claim 16 or 17 or the pharmaceutical composition according to claim 18.

20. The method according to claim 19, wherein the condition or disorder is a tumor (e.g., cancer), an autoimmune disease, or an infectious disease (e.g., a viral or microbial infection).

21. The method according to claim 20, wherein the subject is a mammal, preferably a human.

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