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WO2025227361A1 - Procédé de préparation d'une composition de conjugués anticorps-médicament (adcs) à haute teneur en d4 - Google Patents

Procédé de préparation d'une composition de conjugués anticorps-médicament (adcs) à haute teneur en d4

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Publication number
WO2025227361A1
WO2025227361A1 PCT/CN2024/090809 CN2024090809W WO2025227361A1 WO 2025227361 A1 WO2025227361 A1 WO 2025227361A1 CN 2024090809 W CN2024090809 W CN 2024090809W WO 2025227361 A1 WO2025227361 A1 WO 2025227361A1
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WO
WIPO (PCT)
Prior art keywords
antibody
drug
composition
adcs
solvent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/090809
Other languages
English (en)
Inventor
Yun He
Ming Chen
Fan Zhang
Hui Shi
Xiaojie YUAN
Mengnan HUANG
Shanshan Dong
Xudong Dai
Shiwen LIN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wuxi Xdc Shanghai Co Ltd
Wuxi Xdc Singapore Private Ltd
Original Assignee
Wuxi Xdc Shanghai Co Ltd
Wuxi Xdc Singapore Private Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wuxi Xdc Shanghai Co Ltd, Wuxi Xdc Singapore Private Ltd filed Critical Wuxi Xdc Shanghai Co Ltd
Priority to PCT/CN2024/090809 priority Critical patent/WO2025227361A1/fr
Publication of WO2025227361A1 publication Critical patent/WO2025227361A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment

Definitions

  • the present disclosure relates to a process for preparing a composition of antibody-drug conjugates (ADCs) with improved homogeneity. Specifically, the present disclosure relates to a bio-conjugation process for preparing a composition of antibody-drug conjugates (ADCs) with high D4 (DAR4) content through simple manipulation.
  • ADCs antibody-drug conjugates
  • DAR4 D4
  • antibodies conjugated to labels and reporter groups such as fluorophores, radioisotopes and enzymes find use in labelling and imaging applications, while conjugation to cytotoxic agents and chemotherapy drugs allows targeted delivery of such agents to specific tissues or structures, for example particular cell types or growth factors, minimizing the impact on normal, healthy tissue and significantly reducing the side effects associated with chemotherapy treatments.
  • ADC antibody-drug conjugates
  • ADC contains an antibody for targeting, a connector or linker for drug attachment and a high potent payload (e.g., a drug) as effector. Since the approvals of Adcetris in 2011 and Kadcyla in 2013 by US FDA, ADC drug development has widely spread for the treatment of cancer.
  • ADCs antibody-drug conjugates
  • ADCs are an important class of highly potent biopharmaceutical drugs designed as a targeted therapy for the treatment of subjects with cancer.
  • ADCs are complex molecules composed of an antibody linked to a biologically active cytotoxic (e.g., anticancer) drug. They combine ideal properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to antigen-expressing cells, thereby enhancing their targeted cytotoxicactivity.
  • antibody-drug conjugates target only cancer cells so that healthy cells are less severely affected (Dijoseph, JF; Armellino, DC; Boghaert, ER; Khandke, K; Dougher, MM; Sridharan, L; Kunz, A; Hamann, PR; Gorovits, B; Udata, C; Moran, JK; Popplewell, AG; Stephens, S; Frost, P; Damle, NK (2004) .
  • a therapeutic agent is coupled to an antibody that specifically targets a certain tumor marker (e.g., a protein that, ideally, is only to be found in or on tumor cells) .
  • a certain tumor marker e.g., a protein that, ideally, is only to be found in or on tumor cells.
  • Antibodies track these proteins down in the body and attach themselves to the surface of cancer cells.
  • the biochemical reaction between the antibody and the target protein i.e., antigen
  • the therapeutic agent e.g., a cytotoxic drug
  • the cytotoxic drug is released and kills the tumor cells (Chari, Ravi V.J.; Martell, Bridget A.; Gross, Jonathan L.; Cook, Sherrilyn B.; Shah, Sudhir A.; Walter A.; McKenzie, Sara J.; Goldmacher, Victor S. (1992) . “Immunoconjugates containing novel maytansinoids: promising anticancer drugs” . Cancer Research. 52 (1) : 127–31. ) . Due to this targeting, ideally the ADC has lower side effects and gives a wider therapeutic window than other chemotherapeutic agents.
  • linker-drug functional groups with high reactivity and stability on both antibody and linker-payload (i.e., linker-drug) were used for the coupling, to form stable covalent bonds.
  • Conventional means of attaching, i.e., covalent bonding of a drug moiety to an antibody via a linker generally leads to a heterogeneous mixture of molecules where the drug moieties are attached at several sites on the antibody.
  • cytotoxic drugs have typically been conjugated to antibodies through the often-numerous lysine residues of an antibody, generating a heterogeneous antibody-drug conjugate mixture.
  • antibody-drug conjugates are usually produced by two conventional chemical strategies, Lysine-based conjugation and conjugation based on Cysteine from the reduction of interchain sulfide bonds.
  • Lysine-based conjugation conjugation based on Cysteine from the reduction of interchain sulfide bonds.
  • Cysteine from the reduction of interchain sulfide bonds.
  • the most widely used connector on linker-payload is the NHS ester (i.e., N-hydroxysuccinimide) .
  • linker-payload prepared by NHS ester i.e., SMCC-DM1
  • linker-payload prepared by NHS ester i.e., SMCC-DM1
  • conjugation based on Cysteine from the reduction of interchain sulfide bonds it comprises a step of opening inter-chain disulfide bonds in the presence of various reductants, such TCEP, DTT and so on, followed by nucleophilic reaction of thiol groups.
  • antibody-drug conjugates are typically formed by conjugating one or more antibody cysteine thiol groups to one or more linker moieties bound to a drug thereby forming an antibody-linker-drug complex.
  • Cysteine thiols are reactive at neutral pH, unlike most amines which are protonated and less nucleophilic near pH 7. Since free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine residues often exist in their oxidized form as disulfide-linked oligomers or have internally bridged disulfide groups. Antibody cysteine thiol groups are generally more reactive, i.e. more nucleophilic, towards electrophilic conjugation reagents than antibody amine or hydroxyl groups.
  • the protein oxidatively forms an intramolecular disulfide bond between the newly engineered Cys and an existing Cys residue, both Cys groups are unavailable for active site participation and interactions. Furthermore, the protein may be rendered inactive or non-specific, by misfolding or loss of tertiary structure (Zhang et al. (2002) Anal. Biochem. 311: 1-9) .
  • the conventional conjugation processes always result in a heterogeneous mixture of molecules where the drug moieties are attached at several sites on the antibody.
  • the heterogeneous mixture typically contains a distribution of antibodies with from 0 to about 8, or more, attached drug moieties.
  • the drug moiety is attached at various sites on the antibody.
  • Analytical and preparative methods are inadequate to separate and characterize the antibody-drug conjugate species molecules within the heterogeneous mixture resulting from a conjugation reaction.
  • the heterogeneous mixture is so complex that it is difficult and expensive to characterize and purify.
  • Each conjugation product in such a mixture potentially has different pharmacokinetic, distribution, toxicity and efficacy profiles, and non-specific conjugation also frequently results in impaired antibody function.
  • Antibodies are large, complex and structurally diverse biomolecules, often with many reactive functional groups. Antibody reactivity with linker reagents and drug-linker intermediates are dependent on factors such as pH, concentration, salt concentration, and co-solvents.
  • the multistep conjugation process may be nonreproducible due to difficulties in controlling the reaction conditions and characterizing reactants and intermediates.
  • the number of drugs coupling to a single antibody molecule is an important factor for the efficacy and safety of the resultant ADC.
  • the inter-chain S-S bonds are more accessible to solvents than other disulfide bonds. Therefore, the inter-chain disulfide bonds can be used as the binding sites for coupling a drug (or a drug-linker) to an antibody.
  • one therapeutic antibody molecule belonging to IgG1 or IgG4 subclass has 4 inter-chain S-S bonds, each of which is formed with two -SH groups, and thus, the number of drugs coupling to a single antibody molecule is 2, 4, 6 or 8.
  • D0 drug to antibody molar ratio is 0 (i.e., DAR0)
  • D2 refers to an ADC molecule in which two drug molecules are coupled to one single antibody molecule (i.e., drug to antibody molar ratio is 2 (i.e., DAR2) )
  • two drug molecules may be coupled to -SH groups generated by reduction of S-S bonds between heavy and light chains, or may be coupled to -SH groups generated by reduction of S-S bonds between heavy and heavy chains.
  • D4 refers to an ADC molecule in which four drug molecules are coupled to one single antibody molecule (i.e., drug to antibody molar ratio is 4 (i.e., DAR4) ) .
  • D6 refers to an ADC molecule in which six drug molecules are coupled to one single antibody molecule (i.e., drug to antibody molar ratio is 6 (i.e., DAR6) ) .
  • D8 refers to an ADC molecule in which eight drug molecules are coupled to one single antibody molecule (i.e., drug to antibody molar ratio is 8 (i.e., DAR8) ) , i.e., all the four S-S bonds in one antibody molecule are reduced to eight -SH groups and each -SH group attaches one drug molecule.
  • the heterogeneous mixture of ADC molecules generated by conventional conjugation processes is a mixture of D0, D2, D4, D6 and D8.It is well known in the art that heterogeneous ADC products are generally instable and have low immunogenicity.
  • D0 has no ADC efficacy, and due to their hydrophobicity induced from payload (i.e., drug) molecules, D6 and D8 is considered to be the reason of instability.
  • payload i.e., drug
  • D6 and D8 is considered to be the reason of instability.
  • antibody-drug conjugate potency in vitro has been shown to be directly dependent on drug loading (Hamblett KJ, et al., Clin Cancer Res. 2004 Oct 15; 10 (20) : 7063-70)
  • in vivo antitumor activity of antibody-drug conjugates with four drugs per molecule (D4) was comparable with conjugates with eight drugs per molecule (D8) at equal mAb doses, even though the conjugates contained half the amount of drug per mAb.
  • Drug-loading also affected plasma clearance, with the D8 conjugate being cleared 3-fold faster than the D4 conjugate and 5-fold faster than a D2 conjugate. And thus, D6 and D8 may have a shorter therapeutic window, especially when compared with D2 and D4. Generally, due to high DAR (drug to antibody molar ratio) , D6 and D8 may have higher toxicity than D2 and D4. In general, the level of D4 represents the homogeneity of the antibody-drug conjugates. That is, if the content of D4 is high in the mixture of the antibody-drug conjugates (ADCs) , the ADCs are considered to have a high homogeneity.
  • ADCs antibody-drug conjugates
  • Antibody-drug conjugates with improved homogeneity provide benefits in therapy, for example a higher therapeutic index, improving efficacy and reducing toxicity of the drug. Homogeneous antibody conjugates also provide more accurate and consistent measurements in diagnostic and imaging applications. So, novel processes for preparing ADCs with high homogeneity are highly desirable and long-term pursuit.
  • THIOMAB TM improved antibody-drug conjugates
  • THIOMAB TM antibodies can then be conjugated to cytotoxic drugs through the engineered cysteine thiol groups to obtain THIOMAB TM drug conjugates (TDC) with uniform stoichiometry (e.g., up to 2 drugs per antibody in an antibody that has a single engineered cysteine site) .
  • TDC drug conjugates
  • Studies with multiple antibodies against different antigens have shown that TDCs are as efficacious as conventional antibody-drug conjugate in xenograft models and are tolerated at higher doses in relevant preclinical models.
  • THIOMAB TM antibodies have been engineered for drug attachment at different locations of the antibody (e.g., specific amino acid positions (i.e., sites) within the light chain-Fab, heavy chain-Fab and heavy chain-Fc) .
  • THIOMAB TM antibodies provide a unique advantage over conventional antibody-drug conjugates due to their homogeneity and site-specific conjugation to cytotoxic drugs.
  • those technologies involve protein engineering and/or enzyme catalysis, so that those technologies suffer from several drawbacks, such as lower level of antibody expression, complicated purification, and high cost.
  • WO2020164561A1 describes a bio-conjugation process for preparing a mixture of ADCs with high D4 content, said process is performed in presence of transition metal ions such as Zn 2+ and at a low temperature such as 4°C, to produce a mixture of ADCs with D4 content greater than 65wt%, while the mixture of ADCs prepared by conventional conjugation processes in absence of Zn 2+ generally comprises less than 40wt%D4.
  • the bio-conjugation process disclosed in WO2020164561A1 can improve homogeneity of resultant mixture of ADCs. There are some challenges in the temperature control in large scale such as non-GMP or GMP manufacture.
  • the present disclosure has an object to develop a bio-conjugation process which can generate ADCs with improved homogeneity, and has simple manipulation and reduced cost.
  • the ADCs with improved homogeneity generated by the bio-conjugation process of the disclosure further have optimized safety and efficacy.
  • the present disclosure relates to a bio-conjugation process for preparing a composition of antibody-drug conjugates (ADCs) with improved homogeneity.
  • ADCs antibody-drug conjugates
  • the bio-conjugation process of the present disclosure can be performed much simpler and easier, and the composition of antibody-drug conjugates (ADCs) prepared from the bio-conjugation process of the present disclosure has a comparable content of D4.
  • the process for preparing a composition of antibody-drug conjugates (ADCs) with improved homogeneity comprises the following steps:
  • a reductant e.g., Tris (2-carboxyethyl) phosphine (TCEP)
  • TCEP Tris (2-carboxyethyl) phosphine
  • an effective amount of transition metal ions e.g., Zn 2+ , etc.
  • a buffer system e.g., phosphate buffer (PB)
  • PB phosphate buffer
  • step (b) introducing an excess amount of payload bearing reactive groups (e.g., maleimide linking drugs, etc. ) to react with reduced thiol groups resulted from step (a) at room temperature; and
  • payload bearing reactive groups e.g., maleimide linking drugs, etc.
  • oxidant e.g., dehydroascorbic acid (DHAA)
  • ADCs antibody-drug conjugates
  • the process of the present application further comprises: (d) purifying D2 and D4, respectively, from the composition of antibody-drug conjugates (ADCs) , for example, via a resin, preferably via Hydrophobic interaction chromatography (HIC) resin.
  • ADCs antibody-drug conjugates
  • HIC Hydrophobic interaction chromatography
  • the purity of purified D2 and the purity of purified D4 are 90wt%or even higher, preferably higher than 95wt%, more preferably higher than 98wt%, respectively.
  • transition metal ions generate selectivity in disulfide reduction.
  • the two interchain S-S bonds in Fab regions are selectively reduced.
  • four payload-bearing reactive groups i.e., four drug-linker complexes
  • D4 A high content of D4 in the resultant composition of ADCs certainly improve the homogeneity of the ADCs.
  • the reductant may be TCEP.
  • the concentration of the reductant in the reaction solution may be 0.05 mM –0.5 mM.
  • the oxidant to be added in step (c) may be DHAA.
  • the concentration of the oxidant in the reaction solution may be 0.08 mM –0.8 mM.
  • the transition metal ion which is suitable to be used in the bio-conjugation process of the present disclosure may include, but not limited to, Zn 2+ , Cd 2+ , Hg 2+ , and the like.
  • Zn 2+ is used due to its easily availability and low cost.
  • suitable transition metal salts may be added in step (a) as long as they are soluble in the reaction solution so that free transition metal ions can be released in the reaction solution.
  • ZnCl 2 , Zn (NO 3 ) 2 , ZnSO 4 , Zn (CH 3 COO) 2 , ZnI 2 , ZnBr 2 , Zinc Formate, and zinc tetrafluoroborate may be mentioned as suitable zinc salts.
  • transition metal salts which are soluble and can release free Cd 2+ , or Hg 2+ ions in the reaction solution can be mentioned, which include, but not limited to, cadmium formate, and cadmium tetrafluoroborate; and the like.
  • the concentration of the transition metal ions in step (a) is 0.018 mM –0.18 mM.
  • transition metal ions will be removed in purification step by using EDTA as chelating reagent, which will be filtered out in subsequent dialysis, ultrafiltration or gel filtration.
  • Suitable buffer system for the reaction in step (a) including, but not limited to, phosphate buffer (PB) , Hepes, Histidine buffer, PBS, MES, and the like.
  • PB phosphate buffer
  • Hepes Hepes
  • Histidine buffer PBS
  • MES MES
  • the optimum pH for the reaction will typically between about 6.0 and about 7.0, for instance, about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0, or any pH in the range of 6.0 to 7.0.
  • room temperate refers to 16-25°C, for example, 16-20°C.
  • the antibody which can be conjugated with a linker-drug by using the bio-conjugation process of the present disclosure.
  • the selection of the antibody depends on the diseases or disorder (e.g., a cancer) to be treated by the antibody-drug conjugates (ADCs) .
  • the antibody can specifically bind corresponding antigens expressed on cancer cells (also referred to as tumor-associated antigens (TAA) ) , viral antigens, or microbial antigens, have antibody-dependent cell-mediated phagocytosis (ADCP) activity, and have antitumor, antiviral or antimicrobial activity in vivo.
  • TAA tumor-associated antigens
  • ADCP antibody-dependent cell-mediated phagocytosis
  • the interchain S-S bonds in the antibody are the sites for attaching drug-linker complex.
  • the antibody may include, but not limited to, a monoclonal antibody or a polyclonal antibody.
  • the antibody include a human antibody, a humanized antibody or a chimeric antibody.
  • the antibody is a monoclonal antibody, for instance, a human antibody or a humanized antibody.
  • IgG IgG1, IgG2, IgG3, or IgG4
  • the antibody is an IgG1 monoclonal antibody.
  • the antibody is an IgG4 monoclonal antibody.
  • the antibody exemplified in the Example is a representative IgG1 type antibody.
  • Herceptin is a representative IgG1 type antibody.
  • the results of the Example demonstrate that the bio-conjugation process of the present disclosure is at least applicable to IgG1 type antibodies.
  • the bio-conjugation process of the present disclosure may also be applicable to IgG4 type antibodies.
  • the payload bearing reactive group to be conjugated to the selected antibody generally has a format of drug-linker.
  • the drug and linker which can be used in the bio-conjugation process of the present disclosure, as long as the drug molecule has a desired (e.g., cytotoxic, antitumor, or labelling, etc. ) effect and at least one substituted group or a partial structure allowing connection to a linker structure, and the linker contains at least two reactive groups, one of which can covalently bond a drug molecule and the other of which can covalently couple to an antibody.
  • the drug to be conjugated may include a diagnostic agent, a drug molecule, for example a cytotoxic agent, a toxin, a radionuclide, a fluorescent agent (for example an amine derivatized fluorescent probe such as 5-dimethylaminonaphthalene-1- (N- (2-aminoethyl) ) sulfonamide-dansyl ethylenediamine, Oregon 488 cadaverine (catalogue number O-10465, Molecular Probes) , dansyl cadaverine, N- (2-aminoethyl) -4-amino-3, 6-disulfo-l, 8-naphthalimide, dipotassium salt (lucifer yellow ethylenediamine) , or rhodamine B ethylenediamine (catalogue number L-2424, Molecular Probes) ,
  • a fluorescent agent for example an amine derivatized fluorescent probe such as 5-dimethylaminonaphthalene-1- (N
  • a drug-maleimide complex (i.e., maleimide linking drug) is taken as an example of the payload bearing reactive group in the present disclosure.
  • the drug may include, but not limited to, cytotoxic reagents, such as chemo-therapeutic agents, immunotherapeutic agents and the like, antiviral agents or antimicrobial agents. Most common reactive group capable of bonding to thiol group in ADC preparation is maleimide. Additionally, organic bromides, iodides also are frequently used.
  • the payload bearing reactive groups is a maleimide bearing drug, bromide bearing drug, or iodide bearing drug.
  • step (c) those skilled in the art can select proper methods to recover the resultant antibody-drug conjugates.
  • Many ADCs recovery methods are well known in the art.
  • the resultant composition of antibody-drug conjugates may be recovered by using a de-salting column, size exclusion chromatography, and the like.
  • the homogeneity of the antibody-drug conjugates is higher than those produced by conventional conjugation processes.
  • the content of D0+D8 is less than 11wt%and the content of D6 is less than 10wt%.
  • the content of D4 in the composition of ADCs prepared by said process is generally more than 60wt%, and, for example, more than 65wt%, substantively comparable to the content of D4 prepared by process disclosed in WO2020164561A1, while the content D4 is normally less than 40wt%in the composition of ADCs prepared by conventional conjugation processes.
  • the process of the present disclosure bypasses any need of protein engineering or enzyme catalysis, but is based on native inter-chain disulfide bonds and only needs transition metal ions. Therefore, as compared with conventional processes for preparing ADC, the process of the disclosure is less complicate, the homogeneity of the resultant antibody-drug conjugate is dramatically improved, and the cost will be highly reduced.
  • the process of the present disclosure is performed at or close to room temperature (e.g., 16 °C to 20 °C, e.g., 16 °C or 20 °C) .
  • the scale up and the GMP manufacture can be more easily performed at this temperature, as compared with the technology disclosed in WO2020164561A1, performed at around 4 °C, which is a challenge for scale up and GMP manufacture.
  • the reaction system should be kept at such low temperature and it will be a waste of energy. It will spend long time to decrease the reaction system to the target temperature which will lead to the long process time.
  • the present application relates to a composition of ADCs prepared by the process of the present application.
  • the composition comprises D4 at a content greater than 60wt%, preferably greater than 65wt%, on the basis of the whole weight of D0, D2, D4, D6 and D8.
  • the present application relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the composition of ADCs according to the present application and a pharmaceutically acceptable carrier.
  • the present application relates to a method for treating a disease or disorder in a subject, comprising a step of administrating to the subject a therapeutically effective amount of the composition of ADCs or the pharmaceutical composition according to the present application.
  • the present application relates to use of the composition of ADCs according to the present application in manufacture of a pharmaceutical composition for treating a disease or disorder in a subject.
  • the subject is a mammal, preferably a human.
  • the disease or disorder is a tumor (e.g., cancer) , autoimmune disease, or infectious disease (e.g., viral or microbial infection) .
  • the present application relates to a method for purifying D2 and/or D4 from a composition of antibody-drug conjugates, comprising: performing purification via Hydrophobic interaction chromatography (HIC) , and D2 and D4 are eluted in different fractions.
  • HIC Hydrophobic interaction chromatography
  • the HIC is performed on Butyl sepharose HP resin.
  • Solvent A and Solvent B are used in HIC, wherein Solvent A: 50 mM PB, 2 M NaCl, pH 7.0, Solvent B: 80% (v/v) 50 mM PB, 20% (v/v) ACN, pH 7.0; D4 was eluted with 70% (v/v) Solvent B, and D2 was eluted with 35% (v/v) Solvent B; alternatively, wherein Solvent A: 0.75 M Ammonium sulfate, 25 mM potassium phosphate, pH 7.23, Solvent B: 50 mM potassium phosphate, 15% (v/v) IPA (isopropanol) , pH 7.32; D4 was eluted with 70% (v/v) Solvent B, and D2 was eluted with 35% (v/v) Solvent B.
  • the purity of purified D2 and the purity of purified D4 are 90wt%or even higher, preferably higher than 95wt%, more preferably higher than 98wt%, respectively.
  • D2 and/or D4 purified from a composition of ADCs by the purification method of the present disclosure can be formulated into a pharmaceutical composition, and can be used for treating the diseases or disorders which can be treated with the composition of ADCs or pharmaceutical composition as described in the disclosure.
  • FIG. 1 The HIC (Hydrophobic interaction chromatography) result of a composition of Herceptin-MMAE conjugates prepared by using the process of the disclosure at 20°C and pH 6.8 for reduction of 3 h.
  • FIG. 1 The HIC (Hydrophobic interaction chromatography) result of a composition of Herceptin-MMAE conjugates prepared by using the process of the disclosure at 20°C and pH 6.6 for reduction of 4 h.
  • FIG. 3 The HIC (Hydrophobic interaction chromatography) result of a composition of Herceptin-MMAE conjugates prepared by using the process of the disclosure at 20°C and pH 6.4 for reduction of 5 h.
  • FIG. 4 The HIC (Hydrophobic interaction chromatography) result of a composition of Herceptin-MMAE conjugates prepared by using the process of the disclosure at 20°C and pH 6.2 for reduction of 6 h.
  • FIG. 7 The HIC (Hydrophobic interaction chromatography) result of a composition of Herceptin-MMAE conjugates prepared by using the process of the disclosure at 16°C and pH 6.4 for reduction of 8 h.
  • FIG. 8 The HIC (Hydrophobic interaction chromatography) result of a composition of Herceptin-MMAE conjugates prepared by using the process of the disclosure at 16°C and pH 6.2 for reduction of 8 h.
  • the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%.
  • Antibody-drug conjugate refers to a conjugate formed by covalently coupling a drug to an antibody directly or indirectly via one or more suitable linkers.
  • ADC is generally in a format of antibody-linker-drug conjugate.
  • the Antibody-drug conjugates combine ideal properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to the antigen-expressing tumor cells, thereby enhancing their anti-tumor activity.
  • drug refers to any cytotoxic molecule which has an antitumor effect and at least one substituted group or a partial structure allowing connection to a linker structure.
  • the drug may kill cancer cells and/or inhibit growth, proliferation, or metastasis of cancer cells, thereby reducing, alleviating, or eliminating one or more symptoms of a disease or disorder.
  • linker refers to a reactive molecule which contains at least two reactive groups, one of which can covalently bond a drug molecule and the other of which can covalently couple to an antibody.
  • antibody encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a specific antigen.
  • a native intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region ( “HCVR” ) and a first, second, and third constant region (CH1, CH2 and CH3) , while each light chain consists of a variable region ( “LCVR” ) and a constant region (CL) .
  • HCVR variable region
  • CH1, CH2 and CH3 first, second, and third constant region
  • LCVR variable region
  • Mammalian heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , and mammalian light chains are classified as ⁇ or ⁇ .
  • the antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding.
  • Each arm of the Y includes 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 responsible for antigen binding.
  • the variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light (L) chain CDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs including HCDR1, HCDR2, HCDR3) .
  • CDRs complementarity determining regions
  • CDR boundaries for antibodies may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273 (4) , 927 (1997) ; Chothia, C. et al., J Mol Biol. Dec 5; 186 (3) : 651-63 (1985) ; Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987) ; Chothia, C. et al., Nature. Dec 21-28; 342 (6252) : 877-83 (1989) ; Kabat E.A.
  • Each HCVR and LCVR comprises four FRs, and the CDRs and FRs are arranged from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ heavy chains, respectively.
  • IgG1 ( ⁇ 1 heavy chain) IgG2 ( ⁇ 2 heavy chain)
  • IgG3 ( ⁇ 3 heavy chain) IgG4 ( ⁇ 4 heavy chain)
  • IgA1 ( ⁇ 1 heavy chain) ⁇ 2 heavy chain
  • IgA2 ( ⁇ 2 heavy chain) Several of the major antibody classes are divided into subclasses such as IgG1 ( ⁇ 1 heavy chain) , IgG2 ( ⁇ 2 heavy chain) , IgG3 ( ⁇ 3 heavy chain) , IgG4 ( ⁇ 4 heavy chain) , IgA1 ( ⁇ 1 heavy chain) , or IgA2 ( ⁇ 2 heavy chain) .
  • Antibody fragments comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof.
  • antibody fragments include Fab, Fab’, F (ab’) 2, and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al. (2004) Protein Eng. Design &Sel. 17 (4) : 315-323) , fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region) , and epitope -binding fragments of any described herein which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • variable domain refers to an antibody variable region or a fragment thereof comprising one or more CDRs.
  • a variable domain may comprise an intact variable region (such as HCVR or LCVR) , it is also possible to comprise less than an intact variable region yet still retain the capability of binding to an antigen or forming an antigen-binding site.
  • antigen-binding moiety refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure.
  • antigen-binding moiety include, without limitation, a variable domain, a variable region, a diabody, a Fab, a Fab’, a F (ab’) 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv’) , a disulfide stabilized diabody (ds diabody) , a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody.
  • an antigen-binding moiety is capable of binding to the same antigen to which the parent antibody binds.
  • an antigen-binding moiety may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
  • Fab with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) associating to the variable region and first constant region of a single heavy chain by a disulfide bond.
  • the constant regions of both the light chain and heavy chain are replaced with TCR constant regions.
  • Fab refers to a Fab fragment that includes a portion of the hinge region.
  • F (ab’) 2 refers to a dimer of Fab’.
  • a “fragment difficult (Fd) ” with regard to an antibody refers to the amino-terminal half of the heavy chain fragment that can be combined with the light chain to form Fab.
  • Fc with regard to an antibody refers to that portion of the antibody consisting of the second (CH2) and third (CH3) constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonding.
  • the Fc portion of the antibody is responsible for various effector functions such as ADCC, and CDC, but does not function in antigen binding.
  • “Hinge region” in terms of an antibody includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 amino acid residues and is flexible, thus allowing the two N-terminus antigen binding regions to move independently.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes) , each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see for example: US 4816567; US 5807715) .
  • the monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597; for example.
  • the monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US 4816567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81: 6851-6855) .
  • Chimeric antibodies of interest herein include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc. ) and human constant region sequences.
  • an “intact antibody” herein is one comprising a VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3.
  • the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.
  • the intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc constant region (anative sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC) ; phagocytosis; and down regulation of cell surface receptors such as B cell receptor and BCR.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • immunoglobulin antibodies can be assigned to different "classes” .
  • the heavy-chain constant domains that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • Ig forms include hinge-modifications or hingeless forms (Roux et al. (1998) J. Immunol. 161 : 4083-4090; Lund et al. (2000) Eur. J. Biochem. 267: 7246-7256; US 2005/0048572; US 2004/0229310) .
  • the “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • a “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all or at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • an “isolated antibody” is one which has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95%or 99%purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF) , capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) .
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF) , capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC
  • “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures.
  • native IgG antibodies are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N-to C-terminus, each heavy chain has a variable region (VH) , also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3) . Similarly, from N-to C-terminus, each light chain has a variable region (VL) , also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain.
  • VH variable heavy domain
  • VL variable region
  • the light chain of an antibody may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ) , based on the amino acid sequence of its constant domain.
  • a “cysteine engineered antibody” or “cysteine engineered antibody variant” is an antibody in which one or more residues of an antibody are substituted with cysteine residues.
  • the thiol group (s) of the cysteine engineered antibodies can be conjugated to calicheamicin to form a THIOMAB TM antibody (i.e., a THIOMAB TM drug conjugate (TDC) , wherein in accordance with the present disclosure the drug is a calicheamicin derivative) .
  • the substituted residues occur at accessible sites of the antibody.
  • a THIOMAB TM antibody may be an antibody with a single mutation of a non-cysteine native residue to a cysteine in the light chain (e.g., G64C, K149C or R142C according to Kabat numbering) or in the heavy chain (e.g., D101C or V184C or T205C according to Kabat numbering) .
  • a THIOMAB TM antibody has a single cysteine mutation in either the heavy or light chain such that each full-length antibody (i.e., an antibody with two heavy chains and two light chains) has two engineered cysteine residues.
  • Cysteine engineered antibodies and preparatory methods are disclosed by US 2012/0121615 A1 (incorporated by reference herein in its entirety) .
  • a “disulfide bond” refers to a covalent bond with the structure R-S-S-R’.
  • the amino acid cysteine comprises a thiol group that can form a disulfide bond with a second thiol group, for example from another cysteine residue.
  • the disulfide bond can be formed between the thiol groups of two cysteine residues residing respectively on the two polypeptide chains, thereby forming an interchain bridge or interchain bond.
  • the term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen.
  • the polypeptide complex and the bispecific polypeptide complex provided herein specifically bind an antigen with a binding affinity (K D ) of ⁇ 10 -6 M (e.g., ⁇ 5x10 -7 M, ⁇ 2x10 -7 M, ⁇ 10 -7 M, ⁇ 5x10 -8 M, ⁇ 2x10 -8 M, ⁇ 10 -8 M, ⁇ 5x10 -9 M, ⁇ 2x10 -9 M, ⁇ 10 -9 M, or ⁇ 10 -10 M) .
  • K D refers to the ratio of the dissociation rate to the association rate (koff/kon) , may be determined using surface plasmon resonance methods for example using instrument such as Biacore.
  • transition metal refers to the elements of groups 4-11, justified by their typical chemistry, i.e. a large range of complex ions in various oxidation states, colored complexes, and catalytic properties either as the element or as ions (or both) .
  • Sc and Y in Group 3 are also generally recognized as transition metals.
  • one antibody molecule belonging to IgG1 or IgG4 subclass has 4 inter-chain S-S bonds, each of which is formed with two -SH groups.
  • the antibody molecule can be subjected to partial or complete reduction of one or more interchain S-S bonds to form 2n (n is an integer selected from 1, 2, 3 or 4) reactive -SH groups, and thus, the number of drugs coupling to a single antibody molecule is 2, 4, 6 or 8.
  • D2 refers to the ADC in which two drug molecules are coupled to one single antibody molecule, where two drug molecules may be coupled to -SH groups generated by reduction of S-S bonds between heavy and light chains via linkers, or may be coupled to -SH groups generated by reduction of S-S bonds between heavy and heavy chains via linkers.
  • D4 refers to the ADC in which four drug molecules are coupled to one single antibody molecule, where four drug molecules may be coupled to four -SH groups generated by reduction of two S-S bonds between heavy and light chains via linkers (such ADC is referred to as D4-1) , or four drug molecules may be coupled to four -SH groups generated by reduction of two S-S bonds between heavy and heavy chains via linkers (such ADC is referred to as D4-2) , or two drug molecules may be coupled to two -SH groups generated by reduction of one S-S bond between heavy and light chains via linkers and the other two drug molecules may be coupled to two -SH groups generated by reduction of one S-S bond between heavy and heavy chains vis linkers (such ADC is referred to as D4-3) .
  • D6 refers to the ADC in which six drug molecules are coupled to one single antibody molecule, where four drug molecules may be coupled to four -SH groups generated by reduction of two S-S bonds between heavy and light chains via linkers and two drug molecules may be coupled to two -SH groups generated by reduction of one S-S bonds between heavy and heavy chains via linkers (such ADC is referred to as D6-1) , or four drug molecules may be coupled to four -SH groups generated by reduction of two S-S bonds between heavy and heavy chains via linkers and two drug molecules may be coupled to two -SH groups generated by reduction of one S-S bonds between heavy and light chains via linkers (such ADC is referred to as D6-2) .
  • D8 refers to the ADC in which eight drug molecules are coupled to one single antibody molecule, i.e., all the four S-S bonds in one antibody molecule are reduced to eight -SH groups and each -SH group attaches one drug molecule.
  • the heterogeneous mixture of ADC molecules generated by conventional conjugation processes or the bio-conjugation process of the present disclosure is a mixture of D0, D2, D4, D6 and D8.
  • the “homogeneity” of antibody-drug conjugates is used to describe the property of dominance of one specific type of antibody-drug conjugate (i.e., one type selected from D0, D2, D4, D6 and D8 conjugates) in one given mixture of antibody-drug conjugates.
  • the ADCs are considered to have a high homogeneity.
  • the “homogeneity” of antibody-drug conjugates refers to a high level of D4 in the mixture of antibody-drug conjugates.
  • “improved homogeneity” of ADCs refers to a higher level of D4 in the mixture of antibody-drug conjugates generated by the process of the present disclosure as compared with the mixture of ADCs generated by conventional conjugation processes.
  • the content of D4 is generally more than 60wt%, for example, more than 65wt%, while the content of D4 is normally less than 40wt%in the ADCs prepared by conventional conjugation processes.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g. Natural Killer (NK) cells, neutrophils, and macrophages
  • NK cells Natural Killer cells
  • neutrophils neutrophils
  • macrophages cytotoxic cells
  • the antibodies “arm” the cytotoxic cells and are absolutely required for such killing.
  • the primary cells for mediating ADCC, NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991) .
  • an in vitro ADCC assay such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998) .
  • complement dependent cytotoxicity refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen.
  • C1q first component of the complement system
  • a CDC assay e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) , may be performed.
  • pharmaceutically acceptable indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is bioactivity acceptable and nontoxic to a subject.
  • Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
  • subject includes any human or nonhuman animal, for example, humans.
  • cancer refers to any or a tumor or a malignant cell growth, proliferation or metastasis-mediated, solid tumors and non-solid tumors such as leukemia and initiate a medical condition.
  • a “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastema, sarcoma, and leukemia or lymphoid malignancies.
  • cancers include squamous cell cancer (e.g., epithelial squamous cell cancer) , lung cancer including small-cell lung cancer, non-small cell lung cancer ( “NSCLC” ) , adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • squamous cell cancer e.g., epithelial squamous cell cancer
  • lung cancer including small-cell lung cancer, non-small cell lung cancer ( “NSCLC”
  • treatment refers generally to treatment and therapy, whether of a human or an animal, in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition.
  • Treatment as a prophylactic measure i.e., prophylaxis, prevention
  • treating may refer to dampen or slow the tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof.
  • treatment includes removal of all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.
  • the antibody is against tumor-associated antigens (TAA) , an antigen of a cell that is responsible for producing autoimmune antibodies, a viral or a microbial antigen.
  • TAA tumor-associated antigens
  • Tumor-associated antigens are known in the art, and can be prepared for use in generating antibodies using methods and information which are well known in the art.
  • TAA tumor-associated antigens
  • researchers have sought to identify transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type (s) of cancer cell as compared to on one or more normal non-cancerous cell (s) .
  • s normal non-cancerous cell
  • tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells.
  • the identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody -based therapies.
  • TAA tumor-associated antigens
  • TAA tumor-associated antigens
  • NBI National Center for Biotechnology Information
  • Tumor-associated antigens targeted by antibodies include all amino acid sequence variants and isoforms possessing at least about 70%, 80%, 85%, 90%, or 95%sequence identity relative to the sequences identified in the cited references, or which exhibit substantially the same biological properties or characteristics as a TAA having a sequence found in the cited references.
  • a TAA having a variant sequence generally is able to bind specifically to an antibody that binds specifically to the TAA with the corresponding sequence listed.
  • TAA TUMOR-ASSOCIATED ANTIGENS
  • BMPR1B bone morphogenetic protein receptor-type 1B, Genbank accession no. NM_001203
  • BMPR1B bone morphogenetic protein receptor-type 1B, Genbank accession no. NM_001203
  • WO2004063362 (Claim 2)
  • WO2003042661 (Claim 12)
  • US2003134790-A1 (Page 38-39)
  • WO2002102235 (Claim 13; Page 296)
  • WO2003055443 Page 91-92
  • WO200299122 Example 2; Page 528-530
  • WO2003029421 (Claim 6)
  • WO2003024392 Claim 2; Fig 112)
  • WO200298358 (Claim 1; Page 183)
  • WO200254940 (Page 100-101)
  • WO200259377 (Page 349- 350)
  • WO200254940 (Page 100-101)
  • WO200259377 (Page 349- 350)
  • WO2004065577 (Claim 6) ; WO2004027049 (Fig 1L) ; EP1394274 (Example 11) ; WO2004016225 (Claim 2) ; WO2003042661 (Claim 12) ; US2003157089 (Example 5) ; US2003185830 (Example 5) ; US2003064397 (Fig 2) ; WO200289747 (Example 5; Page 618-619) ; WO2003022995 (Example 9; Fig 13A, Example 53; Page 173, Example 2; Fig 2A) ; NP_036581 six transmembrane epithelial antigen of the prostate Cross-references: MIM: 604415; NP_036581.1; NM_012449_1.
  • MPF MPF
  • MSLN MSLN
  • SMR megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823
  • Yamaguchi N., et al. Biol. Chem. 269 (2) , 805-808 (1994) , Proc. Natl. Acad. Sci. U.S.A. 96 (20) : 11531-11536 (1999) , Proc. Natl. Acad. Sci. U.S.A. 93 (1) : 136-140 (1996) , J. Biol. Chem.
  • Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate) , member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424) J. Biol. Chem. 277 (22) : 19665-19672 (2002) , Genomics 62 (2) : 281-284 (1999) , J.A., et al. (1999) Biochem. Biophys. Res. Commun.
  • Sema 5b (FLJ10372, KIAA1445, Mm. 42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like) , transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878) Nagase T., et al. (2000) DNA Res.
  • PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628) ; Ross et al. (2002) Cancer Res.
  • ETBR Endothelin type B receptor, Genbank accession no. AY275463
  • Nakamuta M. et al. Biochem. Biophys. Res. Commun. 177, 34-39, 1991
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  • MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. NM_017763) ; WO2003104275 (Claim 1) ; WO2004046342 (Example 2) ; WO2003042661 (Claim 12) ; WO2003083074 (Claim 14; Page 61) ; WO2003018621 (Claim 1) ; WO2003024392 (Claim 2; Fig 93) ; WO200166689 (Example 6) ; Cross-references: Locus ID: 54894; NP_060233.2; NM_017763_1.
  • STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138) ; Lab. Invest.
  • TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636) Xu, X.Z., et al. Proc. Natl. Acad. Sci. U.S.A. 98 (19) : 10692-10697 (2001) , Cell 109 (3) : 397-407 (2002) , J. Biol. Chem.
  • CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212) Ciccodicola, A., et al. EMBO J. 8 (7) : 1987-1991 (1989) , Am. J. Hum. Genet.
  • CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs. 73792 Genbank accession no. M26004) Fujisaku et al. (1989) J. Biol. Chem. 264 (4) : 2118-2125) ; Weis J.J., et al. J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al. Proc. Natl. Acad. Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al. Mol. Immunol. 35, 1025-1031, 1998; Weis J.J., et al. Proc. Natl.
  • CD79b (CD79B, CD79 ⁇ , IGb (immunoglobulin-associated beta) , B29, Genbank accession no. NM_000626 or 11038674) Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7) : 4126-4131, Blood (2002) 100 (9) : 3068-3076, Muller et al. (1992) Eur. J. Immunol.
  • FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a) , SPAP1B, SPAP1C, Genbank accession no. NM_030764, AY358130) Genome Res. 13 (10) : 2265-2270 (2003) , Immunogenetics 54 (2) : 87-95 (2002) , Blood 99 (8) : 2662-2669 (2002) , Proc. Natl. Acad. Sci. U.S.A. 98 (17) : 9772-9777 (2001) , Xu, M. J., et al. (2001) Biochem. Biophys. Res. Commun.
  • HER2 ErbB2, Genbank accession no. M11730
  • NCA CEACAM6, Genbank accession no. M18728
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  • Tawaragi Y. et al. Biochem. Biophys. Res. Commun. 150, 89-96, 1988
  • Strausberg R.L. et al. Proc. Natl. Acad. Sci. U.S.A.
  • MDP DPEP1, Genbank accession no. BC017023
  • WO2003016475 (Claim 1)
  • WO200264798 (Claim 33; Page 85-87)
  • JP05003790 (Fig 6-8)
  • W09946284 (Fig 9)
  • Cross-references MIM: 179780; AAH17023.1; BC017023_1.
  • IL20R ⁇ (IL20R ⁇ , ZCYTOR7, Genbank accession no. AF184971) ; Clark H.F., et al. Genome Res. 13, 2265-2270, 2003; Mungall A.J., et al. Nature 425, 805-811, 2003; Blumberg H., et al. Cell 104, 9-19, 2001; Dumoutier L., et al. J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J., et al. J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al.
  • EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no. NM_004442) ; Chan, J. and Watt, V.M., Oncogene 6 (6) , 1057-1061 (1991) Oncogene 10 (5) : 897-905 (1995) , Annu. Rev. Neurosci. 21: 309-345 (1998) , Int. Rev. Cytol.
  • PSCA Prostate stem cell antigen precursor, Genbank accession no. AJ297436
  • Reiter R.E. et al. Proc. Natl. Acad. Sci. U.S.A. 95, 1735-1740, 1998
  • Gu Z. et al. Oncogene 19, 1288-1296, 2000
  • BAFF-R B cell-activating factor receptor, BLyS receptor 3, BR3, Genbank accession No. AF116456
  • BAFF receptor /pid NP_443177.1 -Homo sapiens Thompson, J.S., et al.
  • CD22 B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession No. AK026467
  • CD22 B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession No. AK026467
  • CD79a (CD79A, CD79 ⁇ , immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation) , pI: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19ql3.2, Genbank accession No.
  • CXCR5 (Burkitt’s lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia) ; 372 aa, pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accession No.
  • NP_001707.1 WO2004040000; WO2004015426; US2003105292 (Example 2) ; US6555339 (Example 2) ; WO200261087 (Fig 1) ; WO200157188 (Claim 20, page 269) ; WO200172830 (pages 12-13) ; WO200022129 (Example 1, pages 152-153, Example 2, pages 254-256) ; W09928468 (claim 1, page 38) ; US5440021 (Example 2, col 49-52) ; W09428931 (pages 56-58) ; W09217497 (claim 7, Fig 5) ; Dobner et al. (1992) Eur. J. Immunol. 22: 2795-2799; Barella et al. (1995) Biochem. J. 309: 773-779.
  • HLA-DOB Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+ T lymphocytes
  • P2X5 Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability
  • Gene Chromosome: 17p13.3, Genbank accession No. NP_002552.2) Le et al. (1997) FEBS Lett. 418 (1-2) : 195-199; WO2004047749; WO2003072035 (claim 10) ; Touchman et al. (2000) Genome Res. 10: 165-173; WO200222660 (claim 20) ; WO2003093444 (claim 1) ; WO2003087768 (claim 1) ; WO2003029277 (page 82) .
  • CD72 B-cell differentiation antigen CD72, Lyb-2, 359 aa, pI: 8.66, MW: 40225 TM: 1 [P] Gene Chromosome: 9p13.3, Genbank accession No. NP_001773.1) ; WO2004042346 (claim 65) ; WO2003026493 (pages 51-52, 57-58) ; WO200075655 (pages 105-106) ; Von Hoegen et al. (1990) J. Immunol. 144 (12) : 4870-4877; Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99: 16899-16903.
  • LY64 Lymphocyte antigen 64 (RP105) , type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis) ; 661 aa, pI: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12, Genbank accession No. NP_005573.1) ; US2002193567; WO9707198 (claim 11, pages 39-42) ; Miura et al. (1996) Genomics 38 (3) : 299-304; Miura et al. (1998) Blood 92: 2815-2822; WO2003083047; W09744452 (claim 8, pages 57-61) ; WO200012130 (pages 24-26) .
  • RP105 type I membrane protein of the leucine rich repeat
  • FcRH1 Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation
  • IRTA2 Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies
  • 977 aa, pI: 6.88 MW: 106468 TM 1 [P] Gene Chromosome: 1q21, Genbank accession No.
  • TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin) ; 374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbank accession No.
  • PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL) ; ME20; gp100) BC001414; BT007202; M32295; M77348; NM_006928; McGlinchey, R.P. et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106 (33) , 13731-13736; Kummer, M. P. et al. (2009) J. Biol. Chem. 284 (4) , 2296-2306.
  • TMEFF1 transmembrane protein with EGF-like and two follistatin-like domains 1; Tomoregulin-1) ; H7365; C9orf2; C90RF2; U19878; X83961; NM_080655; NM_003692; Harms, P.W. (2003) Genes Dev. 17 (21) , 2624-2629; Gery, S. et al. (2003) Oncogene 22 (18) : 2723-2727.
  • GDNF-Ra1 GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1) ; U95847; BC014962; NM 145793 NM_005264; Kim, M. H. et al. (2009) Mol. Cell. Biol. 29 (8) , 2264-2277; Treanor, J.J. et al. (1996) Nature 382 (6586) : 80-83.
  • Ly6E lymphocyte antigen 6 complex, 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 103 (6) , 768-774; Zammit, D. J. et al. (2002) Mol. Cell. Biol. 22 (3) : 946-952.
  • TMEM46 shisa homolog 2 (Xenopus laevis) ; SHISA2) ; NP_001007539.1; NM_001007538.1; Furushima, K. et al. (2007) Dev. Biol. 306 (2) , 480-492; Clark, H.F. et al. (2003) Genome Res. 13 (10) : 2265-2270.
  • Ly6G6D lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1 ; NP_067079.2; NM_021246.2; Mallya, M. et al. (2002) Genomics 80 (1) : 113-123; Ribas, G. et al. (1999) J. Immunol. 163 (1) : 278-287.
  • 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. 170 (5) : 537-545; Yamamoto, Y. et al. (2003) Hepatology 37 (3) : 528-533.
  • 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. 100 (10) : 1895-1901; Narita, N. et al. (2009) Oncogene 28 (34) : 3058-3068.
  • LY6K lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226) ; NP_059997.3; NM_017527.3; Ishikawa, N. et al. (2007) Cancer Res. 67 (24) : 11601-11611; de Nooij-van Dalen, A G. et al. (2003) Int. J. Cancer 103 (6) : 768-774.
  • GPR19 G protein-coupled receptor 19; Mm. 4787
  • NP_006134.1 G protein-coupled receptor 19; Mm. 4787
  • NM_006143.2 Montpetit, A. and Colltt, D. (1999) Hum. Genet. 105 (1-2) : 162-164; O’Dowd, B.F. et al. (1996) FEBS Lett. 394 (3) : 325-329.
  • GPR54 KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12
  • NP_115940.2 NM_032551.4
  • ASPHD1 aspartate beta-hydroxylase domain containing 1; LOC253982
  • NP_859069.2 NM_181718.3
  • Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3) ; NP_000363.1; NM_000372.4; Bishop, D.T. et al. (2009) Nat. Genet. 41 (8) : 920-925; Nan, H. et al. (2009) Int. J. Cancer 125 (4) : 909-917.
  • TMEM118 ring finger protein, transmembrane 2; RNFT2; FLJ14627
  • NP_001103373.1 NM_001109903.1
  • GPR172A G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e) ; NP_078807.1; NM_024531.3; Ericsson, T.A. et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100 (l l) : 6759-6764; Takeda, S. et al. (2002) FEBS Lett. 520 (1-3) : 97-101.
  • CD33 a member of the sialic acid binding, immunoglobulin-like lectin family, is a 67-kDa glycosylated transmembrane protein. CD33 is expressed on most myeloid and monocytic leukemia cells in addition to committed myelomonocytic and erythroid progenitor cells. It is not seen on the earliest pluripotent stem cells, mature granulocytes, lymphoid cells, or nonhematopoietic cells (Sabbath et al., (1985) J. Clin. Invest. 75: 756-56; Andrews et al., (1986) Blood 68: 1030-5) . CD33 contains two tyrosine residues on its cytoplasmic tail, each of which is followed by hydrophobic residues similar to the immunoreceptor tyrosine-based inhibitory motif (ITIM) seen in many inhibitory receptors.
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • CLL-1 (CLEC12A, MICL, and DCAL2) , encodes a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily.
  • CTL/CTLD C-type lectin/C-type lectin-like domain
  • Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-cell signaling, glycoprotein turnover, and roles in inflammation and immune response.
  • the protein encoded by this gene is a negative regulator of granulocyte and monocyte function.
  • Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined.
  • This gene is closely linked to other CTL/CTLD superfamily members in the natural killer gene complex region on chromosome 12p13 (Drickamer K (1999) Curr. Opin. Struct. Biol. 9 (5) : 585-90; van Rhenen A, et al, (2007) Blood 110 (7) : 2659-66; Chen CH, et al. (2006) Blood 107 (4) : 1459-67; Marshall AS, et al. (2006) Eur. J. Immunol. 36 (8) : 2159-69; Bakker AB, et al. (2005) Cancer Res. 64 (22) : 8443-50; Marshall AS, et al. (2004) J. Biol. Chem. 279 (15) : 14792-802) .
  • CLL-1 has been shown to be a type II transmembrane receptor comprising a single C-type lectin-like domain (which is not predicted to bind either calcium or sugar) , a stalk region, a transmembrane domain and a short cytoplasmic tail containing an ITIM motif.
  • the present disclosure relates to a process for preparing a composition of antibody-drug conjugates (ADCs) with improved homogeneity, comprising the following steps:
  • step (b) introducing an excess amount of payload bearing reactive groups to react with reduced thiol groups resulted from step (a) at room temperature;
  • the process of the present application further comprises: (d) purifying D2 and D4, respectively, from the composition of antibody-drug conjugates (ADCs) , for example, via a resin, preferably via Hydrophobic interaction chromatography (HIC) , and the purity of purified D2 and the purity of purified D4 are 90wt%or even higher, preferably higher than 95wt%, more preferably higher than 98wt%, respectively.
  • ADCs antibody-drug conjugates
  • HIC Hydrophobic interaction chromatography
  • Transition metal ions generate selectivity in disulfide reduction. In the presence of transition metal ions, the two interchain S-S bonds in Fab regions are selectively reduced. And thus, four payload bearing reactive groups (i.e., four drug-linker complexes) are attached to one antibody to form D4. A high content of D4 in the resultant ADCs certainly improve the homogeneity of the ADCs.
  • the reductant may be TCEP.
  • the concentration of the reductant in the reaction solution may be 0.05 mM to 0.5 mM.
  • the oxidant to be added in step (c) may be DHAA.
  • the concentration of the oxidant in the reaction solution may be 0.08 mM to 0.8 mM.
  • the transition metal ion which is suitable to be used in the bio-conjugation process of the present disclosure may include, but not limited to, Zn 2+ , Cd 2+ , and Hg 2+ , and the like.
  • Zn 2+ is used due to its easily availability and low cost.
  • suitable transition metal salts may be added in step (a) as long as they are soluble in the reaction solution so that free transition metal ions can be released in the reaction solution.
  • ZnCl 2 , Zn (NO 3 ) 2 , ZnSO 4 , Zn (CH 3 COO) 2 , ZnI 2 , ZnBr 2 , Zinc Formate, and zinc tetrafluoroborate may be mentioned as suitable zinc salts.
  • transition metal salts which are soluble and can release free Cd 2+ or Hg 2+ ions in the reaction solution can be mentioned, which include, but not limited to, CdCl 2 , Cd (NO 3 ) 2 , CdSO 4 , Cd (CH 3 COO) 2 , CdI 2 , CdBr 2 , cadmium formate, and cadmium tetrafluoroborate; HgCl 2 , Hg (NO 3 ) 2 , HgSO 4 , Hg (CH 3 COO) 2 , HgBr 2 , Mercury (II) formate, and Mercury (II) tetrafluoroborate; and the like.
  • Those skilled in the art can make a selection from the above transition metal salts as the source of transition metal ions.
  • Zn 2+ is used in step (a) .
  • Zinc salts which are water soluble are available.
  • ZnCl 2 may be added in step (a) as the Zn 2+ source.
  • the concentration of the transition metal ions in the reaction solution in step (a) is 0.018 mM to 0.18 mM.
  • transition metal ions will be removed in purification step by using EDTA as chelating reagent, which will be filtered out in subsequent dialysis, ultrafiltration or gel filtration.
  • buffer system for the reaction in step (a) , including, but not limited to, phosphate buffer (PB) , Hepes, Histidine buffer, PBS, MES, and the like.
  • PB phosphate buffer
  • the buffer system used in step (a) is PBS.
  • the optimum pH for the reaction will typically between about 6.0 and about 7.0, for instance, about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0, or any pH in the range of 6.0 to 7.0.
  • the buffer is PB buffer, pH 6.0-7.0, e.g., pH 6.8, 6.6, 6.4, or 6.2.
  • room temperate refers to 16-25°C, for example, 16-20°C.
  • the concentration of the antibody in the reaction is from 0.01 mM to 0.1 mM. In a specific embodiment, the concentration of the antibody is 0.02 mM.
  • the antibody to be conjugated, the transitional metal ions and the reductant may be present in the reaction mixture in a molar ratio of 1: 1.8: 5.
  • 0.02 mM antibody is incubated with 0.10 mM TCEP and 0.036 mM ZnCl 2 at room temperature (e.g., 16-25°C, e.g., 16°C or 20°C) for several hours (e.g., about 3-8 hours) .
  • the antibody is an antibody which binds to one or more tumor-associated antigens or cell-surface receptors as described elsewhere herein.
  • the antibody may include, but not limited to, a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a multispecific antibody, or an antibody derivate.
  • 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., a Fv, Fab, Fab’, scFv, diabody, or F (ab’) 2 fragment. In another embodiment, the antibody is a substantially full-length antibody, e.g., an IgG1 antibody, IgG4 antibody or other antibody class or isotype as defined herein. In a specific embodiment, the antibody is an IgG1 antibody.
  • the antibody is selected from Herceptin (trastuzumab) , Rituxan (rituximab) , Erbitux (Cetuximab) , or antibodies against any one of the following antigens: BMPR1B, E16, STEAP1, MUC16, MPF, Napi2b, Sema 5b, PSCA 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
  • Useful Polyclonal antibody is a heterogeneous population of antibody molecules derived from the sera of immunized animals.
  • Various procedures well known in the art may be used for the production of polyclonal antibodies to an antigen-of-interest.
  • various host animals can be immunized by injection with an antigen of interest or derivative thereof, including but not limited to rabbits, mice, rats, and guinea pigs.
  • Useful monoclonal antibody is a homogeneous population of antibodies to a particular antigen (e.g., a cancer cell antigen, a viral antigen, a microbial antigen covalently linked to a second molecule) .
  • a monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any subclass thereof.
  • the hybridoma producing the mAbs of use in this invention may be cultivated in vitro or in vivo.
  • Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies.
  • Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16) .
  • a human antibody may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge.
  • an immunogen for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005) .
  • Human antibodies can also be made by hybridoma-based methods.
  • Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (See, e.g., Kozbor J. Immunol, 133: 3001 (1984) ; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987) ; and Boerner et al., J. Immunol., 147: 86 (1991) ) .
  • Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci.
  • Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are well known in the art.
  • chimeric and humanized monoclonal antibodies comprising both human and non-human portions, which can be made using standard recombinant DNA techniques
  • Ligands are useful Ligands.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein by reference in their entirety.
  • Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Publication No. WO 87/02671; European Patent Publication No. 184,187; European Patent Publication No. 171, 496; European Patent Publication No. 173,494; International Publication No. WO 86/01533; U.S. Pat. No.
  • antibodies for the treatment or prevention of cancer are used in the invention.
  • Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art, such as, e.g., chemical synthesis or recombinant expression techniques.
  • the nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g. from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
  • HERCEPTIN Trastuzumab; Genentech, CA
  • Herceptin trastuzumab
  • RITUXAN rituximab; Genentech
  • Erbitux Cetuximab; Merck
  • OvaRex AltaRex Corporation, MA
  • Panorex Gaxo Wellcome, NC
  • BEC2 ImClone Systems Inc., NY
  • IMC-C225 Imclone Systems Inc., NY
  • Vitaxin MedImmune, Inc., MD
  • antibodies useful in the treatment of cancer include, but are not limited to, antibodies against the following antigens: CA125 (ovarian) , CA15-3 (carcinomas) , CA19-9 (carcinomas) , L6 (carcinomas) , Lewis Y (carcinomas) , Lewis X (carcinomas) , alpha fetoprotein (carcinomas) , CA 242 (colorectal) , placental alkaline phosphatase (carcinomas) , prostate specific antigen (prostate) , prostatic acid phosphatase (prostate) , epidermal growth factor (carcinomas) , MAGE-1 (carcinomas) , MAGE-2 (carcinomas) , MAGE-3 (carcinomas) , MAGE 4 (carcinomas) , anti-transferrin receptor (carcinomas) , p97 (melanoma) , MUC1-KLH (breast cancer) , CEA (colorectal) , gp100 (mel
  • BR96 mAb Trail, P.A., Willner, D., Lasch, S.J., Henderson, A.J., Hofstead, S.J., Casazza, A.M., Firestone, R.A., I., K.E., “Cure of Xenografted Human Carcinomas by BR96-Doxorubicin Immunoconjugates” Science 1993, 261, 212-215) , BR64 (Trail, Pa., Willner, D, Knipe, J., Henderson, A.J., Lasch, S.J., Zoeckler, M.E., Trailsmith, M.D., Doyle, T.W., King, H.D., Casazza, A.M., Braslawsky, G.R., Brown, J.P., Hofstead, S.J., Greenfield, Ill.
  • BR96 mAb Trail, P.A., Willner, D., Lasch, S.J., Henderson, A.J., Hof
  • antibodies for the treatment or prevention of an autoimmune disease are used in accordance with the process of the disclosure.
  • Antibodies immunospecific for an antigen of a cell that is responsible for producing autoimmune antibodies can be obtained from any organization (e.g., a university scientist or a company such as Genentech) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques.
  • useful ligand antibodies that are immunospecific for the treatment of autoimmune diseases include, but are not limited to, Anti-Nuclear Antibody; Anti dsDNA; Anti ssDNA, Anti Cardiolipin Antibody IgM, IgG; Anti Phospholipid Antibody IgK, IgG; Anti SM Antibody; Anti Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody, Thyroglobulin Antibody, Anti SCL-70; Anti-Jo; Anti-U1RNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti GBM Antibody.
  • useful antibodies that are immunospecific for a viral or a microbial antigen are monoclonal antibodies.
  • antibodies that are immunospecific for a viral antigen or microbial antigen may be humanized or human monoclonal antibodies.
  • the term “viral antigen” includes, but is not limited to, any viral peptide, polypeptide protein (e.g. HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g.
  • microbial antigen includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptide including, e.g., LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune response.
  • Antibodies immunospecific for a viral or microbial antigen can be obtained commercially, for example, from Genentech (San Francisco, Calif. ) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques.
  • the nucleotide sequence encoding antibodies that are immunospecific for a viral or microbial antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
  • useful antibodies are those that are useful for the treatment or prevention of viral or microbial infection.
  • examples of antibodies available useful for the treatment of viral infection or microbial infection include, but are not limited to, SYNAGIS (MedImmune, Inc., MD) which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody useful for the treatment of patients with RSV infection; PRO542 (Progenics) which is a CD4 fusion antibody useful for the treatment of HIV infection; OSTAVIR (Protein Design Labs, Inc., CA) which is a human antibody useful for the treatment of hepatitis B virus; PROTVIR (Protein Design Labs, Inc., CA) which is a humanized IgG1 antibody useful for the treatment of cytomegalovirus (CMV) ; and anti-LPS antibodies.
  • SYNAGIS MedImmune, Inc., MD
  • RSV humanized anti-respiratory syncytial virus
  • PRO542 Progenics
  • antibodies useful in the treatment of infectious diseases include, but are not limited to, antibodies against the antigens from pathogenic strains of 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,
  • 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, Tryoanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria) ; or Helminiths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloid
  • antibodies useful in this invention for treatment of viral disease include, but are not limited to, antibodies against antigens of pathogenic viruses, including as examples and not by limitation: Poxyiridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, 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 virus, Rhinoviridae, Coronaviridae, Rotoviridae, and Human Immunodeficiency Virus.
  • Poxyiridae Herpesviridae, Herpes Simple
  • the antibodies suitable for use in the bio-conjugation process provided herein can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and, for example, are produced by recombinant expression techniques.
  • the payload bearing reactive group to be conjugated to the selected antibody generally has a format of drug-linker.
  • the drug and linker which can be used in the bio-conjugation process of the present disclosure, as long as the drug molecule has an antitumor, antiviral or antimicrobial effect and contains at least one substituted group or a partial structure allowing connection to a linker structure, and the linker contains at least two reactive groups, one of which can covalently bond a drug molecule and the other of which can covalently couple to an antibody.
  • a drug-maleimide complex i.e., maleimide linking drug
  • a drug-maleimide complex is taken as an example of the payload bearing reactive group in the present disclosure.
  • the drug may include, but not limited to, cytotoxic reagents, such as chemo-therapeutic agents, immunotherapeutic agents and the like, antiviral agents or antimicrobial agents.
  • the drug to be conjugated with an antibody may be selected from, but not limited to, MMAE (monomethyl auristatin E) , MMAD (monomethyl auristatin D) , MMAF (monomethyl auristatin F) , and the like.
  • Drug loading is represented by the number of drug moieties per antibody in a molecule of ADC.
  • the drug loading may be limited by the number of attachment sites on the antibody.
  • the drug loading may range from 0 to 8 drug moieties per antibody.
  • higher drug loading e.g. p ⁇ 5
  • the average drug loading for an antibody-drug conjugate ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5.
  • the optimal ratio of drug moieties per antibody may be 4 (see, e.g., WO2013190292) .
  • the resulting product is a mixture of antibody-drug conjugate compounds with a distribution of one or more drug moieties attached to an antibody.
  • the average number of drugs per antibody may be calculated from the mixture by a dual ELISA antibody assay, which is specific for antibody and specific for the drug.
  • Individual antibody-drug conjugate molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography (see, e.g., McDonagh et al. (2006) Prot. Engr. Design &Selection 19 (7) : 299-307; Hamblett et al. (2004) Clin. Cancer Res.
  • a homogeneous antibody-drug conjugate with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.
  • the homogeneity of the antibody-drug conjugates is higher than those produced by conventional conjugation processes.
  • the content of D0+D8 is less than 11wt%and the content of D6 is less than 10wt%.
  • the content of D4 is generally more than 60wt%, and, for example, more than 65wt%, while the content D4 is normally less than 40wt%in the composition of ADCs prepared by conventional conjugation processes.
  • the resultant antibody-drug conjugates are recovered by any suitable purification method, such as using a de-salting column, size exclusion chromatography, ultrafiltration, dialysis, UF-DF, and the like.
  • the present disclosure relates to a composition of antibody-drug conjugates (ADCs) with improved homogeneity prepared by the process of the first aspect.
  • ADCs antibody-drug conjugates
  • the improved homogeneity of the composition of ADC is represented by high level of D4 in the resultant composition of ADCs.
  • the homogeneity of the composition of antibody-drug conjugates generated by the process of the first aspect is measured, and compared with the homogeneity of corresponding control composition of antibody-drug conjugates generated by conventional conjugation processes.
  • HIC is the analytical method used to determine yields and isomeric mixtures from resultant antibody-drug conjugates (e.g., for D4 conjugates) .
  • This technique is able to separate antibodies loaded with various numbers of drugs.
  • the drug loading level can be determined based on the ratio of absorbances, e.g., at 250 nm and 280 nm. For example, if a drug can absorb at 250 nm while the antibody absorbs at 280 nm. The 250/280 ratio therefore increases with drug loading.
  • Using the bio-conjugation process described herein generally antibodies with even numbers of drugs were observed to be conjugated to the antibody since reduction of disulfides yields even numbers of free cysteine thiols.
  • the composition of antibody-drug conjugates generated by the process of the first aspect have improved homogeneity, which is represented by the increased content of D4 in the resultant composition of ADCs.
  • the content of D4 in the composition of antibody-drug conjugates generated by the process of the present disclosure is higher than 60wt%, for example, higher than 65wt%, comparable with the content of D4 in the composition of antibody-drug conjugates (ADCs) generated by conventional conjugation processes is usually less than 40wt%, as described in WO2020164561A1.
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the composition of ADCs with improved homogeneity prepared by the process of the first aspect and a pharmaceutically acceptable carrier or vehicle.
  • the compositions are suitable for veterinary or human administration.
  • compositions of the present invention can be in any form that allows for the composition to be administered to an animal.
  • the composition can be in the form of a solid, liquid or gas (aerosol) .
  • routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, and intranasal.
  • Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • the compositions are administered parenterally.
  • Pharmaceutical compositions of the invention can be formulated so as to allow ADCs of the invention to be bioavailable upon administration of the composition to an animal.
  • Compositions can take the form of one or more dosage units, where for example, a tablet can be a single dosage unit, and a container of ADCs of the invention in aerosol form can hold a plurality of dosage units.
  • compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient (s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of animal (e.g., human) , the particular form of the ADCs of the Invention, the manner of administration, and the composition employed.
  • Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, 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 hydroxanisol, butylated hydroxytoluene, and/or propyl gallate.
  • compositions that comprise the composition of ADCs disclosed herein and one or more antioxidants such as methionine.
  • pharmaceutical acceptable carriers may 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 fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA
  • Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol.
  • Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
  • compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the pharmaceutical compositions are formulated into an injectable composition.
  • the injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion.
  • Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions.
  • the solutions may be either aqueous or non-aqueous.
  • unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.
  • a sterile, lyophilized powder is prepared by dissolving the composition of ADCs as disclosed herein in a suitable solvent.
  • the solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents.
  • the solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH.
  • the resulting solution will be apportioned into vials for lyophilization.
  • Each vial can contain a single dosage or multiple dosages of the composition of ADCs provided herein or pharmaceutical composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing.
  • the lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.
  • Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration.
  • the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.
  • composition of antibody-drug conjugates or the pharmaceutical composition may be manufactured into a kit, including an insert which indicates the information for the application, such as the indications, the amount in use, the route to be administrated, and the like.
  • the present disclosure relates to the use of the composition of antibody-drug conjugates with improved homogeneity prepared by the process of the first aspect in the manufacture of a pharmaceutical composition or a kit for treating a disease or disorder in a subject.
  • the subject may be a mammal, for example, a human.
  • the disease or disorder to be treated may be a tumor, cancer, autoimmune disease, or infectious disease.
  • the infectious disease may be viral or microbial infection.
  • the present disclosure also relates to a method for treating a subject having a disease or disorder, comprising: administrating a therapeutically effective amount of the composition of ADCs with improved homogeneity prepared by the process of the first aspect or a therapeutically effective amount of the pharmaceutical composition comprising the composition of ADCs with improved homogeneity prepared by the process of the first aspect to a subject in need thereof, thereby treating or preventing the disease or disorder.
  • the subject has been identified as having a disease or disorder likely to respond to the composition of ADCs provided herein.
  • the subject may be a mammal, for example, a human.
  • the disease or disorder to be treated may be a tumor, cancer, autoimmune disease, or infectious disease.
  • the infectious disease may be viral or microbial infection.
  • composition of ADCs provided herein will depend on various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.
  • the composition of ADCs or pharmaceutical composition provided herein may be administered at a therapeutically effective dosage of about 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg) .
  • the composition of ADCs or pharmaceutical composition provided herein are administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage 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.
  • the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) .
  • a single dose may be administered, or several divided doses may be administered over time.
  • composition of ADCs or pharmaceutical composition provided herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.
  • parenteral e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection
  • non-parenteral e.g., oral, intranasal, intraocular, sublingual, rectal, or topical routes.
  • the disease or disorder treated by the composition of ADCs or pharmaceutical composition provided herein is cancer or a cancerous condition, autoimmune disease or infectious disease.
  • the cancer may be antigen positive carcinomas including those of the lung, breast, colon, ovaries, and pancreas, for example, the cancers associated with tumor-associated antigens listed in (1) - (53) under the heading of “TUMOR-ASSOCIATED ANTIGENS (TAA) ” .
  • cancers that can be treated with the composition of ADCs or pharmaceutical composition provided herein include, but are not limited to, solid tumors, including but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophogeal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillar
  • the autoimmune disease may include, but not limited to, Active Chronic Hepatitis, Addison’s Disease, Allergic Alveolitis, Allergic Reaction, Allergic Rhinitis, Alport’s Syndrome, Anaphlaxis, Ankylosing Spondylitis, Anti-phosholipid Syndrome, Arthritis, Ascariasis, Aspergillosis, Atopic Allergy, Atropic Dermatitis, Atropic Rhinitis, Behcet’s Disease, Bird- Fancier’s Lung, Bronchial Asthma, Caplan’s Syndrome, Cardiomyopathy, Celiac Disease, Chagas’ Disease, Chronic Glomerulonephritis, Cogan’s Syndrome, Cold Agglutinin Disease, Congenital Rubella Infection, CREST Syndrome, Crohn’s Disease, Cryoglobulinemia, Cushing’s Syndrome, Dermatomyositi, Discoid Lupus, Dressler’s Syndrome, Eaton-Lambert Syndrome, Echovirus Infection, Encephalomye
  • Bacterial Diseases Diptheria, Pertussis, Occult Bacteremia, Urinary Tract Infection, Gastroenteritis, Cellulitis, Epiglottitis, Tracheitis, Adenoid Hypertrophy, Retropharyngeal Abcess, Impetigo, Ecthyma, Pneumonia, Endocarditis, Septic Arthritis, Pneumococcal, Peritonitis, Bactermia, Meningitis, Acute Purulent Meningitis, Urethritis, Cervicitis, Proctitis, Pharyngitis, Salpingitis, Epididymitis, Gonorrhea, Syphilis, Listeriosis, Anthrax, Nocardiosis, Salmonella, Typhoid Fever, Dysentery, Conjuntivitis, Sinusitis, Brucellosis, Tullaremia
  • the present disclosure includes a method for treating disease or disorder in a subject, comprising administering to the subject an effective amount of the composition of ADCs or pharmaceutical composition provided herein and another therapeutic agent.
  • said another therapeutic agent is an anti-cancer agent.
  • Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel.
  • said another therapeutic agent is an anti-autoimmune disease agent.
  • Suitable anti-autoimmune disease agents include, but are not limited to, cyclosporine, cyclosporine A, mycophenylate mofetil, Sirolimus, tacrolimus, etanercept, prednisone, azathioprine, methotrexate cyclophosphamide, prednisone, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, DHEA, danazol, bromocriptine, meloxicam, and infliximab.
  • said another therapeutic agent is anti-infectious disease agent.
  • the anti-infectious disease agent is, but not limited to, antibacterial agents: [beta] -Lactam Antibiotics: Penicillin G, Penicillin V, Cloxacilliin, Dicloxacillin, Methicillin, Nafcillin, Oxacillin, Ampicillin, Amoxicillin, Bacampicillin, Azlocillin, Carbenicillin, Mezlocillin, Piperacillin, Ticarcillin; Aminoglycosides: Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin; Macrolides: Azithromycin, Clarithromycin, Erythromycin, Lincomycin, Clindamycin; Tetracyclines: Demeclocycline, Doxycycline, Minocycline, Oxytetracyclinem, Tetracycline; Quinolones: Cinoxacin, Nalidixic Acid; Fluoroquino
  • the present disclosure relates to a method for purifying D2 and/or D4 from a composition of antibody-drug conjugates, comprising: performing purification via Hydrophobic interaction chromatography (HIC) , and D2 and D4 are eluted in different fractions.
  • HIC Hydrophobic interaction chromatography
  • the HIC is performed on Butyl sepharose HP resin.
  • Solvent A and Solvent B are used in HIC, wherein Solvent A: 50 mM PB, 2 M NaCl, pH 7.0, Solvent B: 80% (v/v) 50 mM PB, 20% (v/v) ACN, pH 7.0; D4 was eluted with 70% (v/v) Solvent B, and D2 was eluted with 35% (v/v) Solvent B; alternatively, Solvent A: 0.75 M Ammonium sulfate, 25 mM potassium phosphate, pH 7.23, Solvent B: 50 mM potassium phosphate, 15% (v/v) IPA (isopropanol) , pH 7.32; D4 was eluted with 70% (v/v) Solvent B, and D2 was eluted with 35% (v/v) Solvent B.
  • the purity of purified D2 and the purity of purified D4 are 90wt%or even higher, preferably higher than 95wt%, more preferably higher than 98wt%, respectively.
  • D2 and/or D4 purified from a composition of ADCs by the purification method of the present disclosure can be formulated into a pharmaceutical composition, and can be used for treating the diseases or disorders which can be treated with the composition of ADCs or pharmaceutical composition as described in the disclosure.
  • Example 1 A composition of Herceptin-MC-VC-PAB-MMAE conjugates is prepared in a one-pot reaction (scale: 5 mg)
  • composition of Herceptin-MC-VC-PAB-MMAE conjugates is prepared in a one-pot reaction (scale: 5 mg) :
  • reaction mixture was subjected to recovery using the de-salting column (type: 40K, 0.5 ml, Prod#87766, Lot#UI285695, Thermo Zeba TM Spin Desalting Column) ;
  • HIC resin type: Butyl sepharose HP resin
  • Method 1 Solvent A: 50 mM PB, 2 M NaCl, pH 7.0, Solvent B: 80% (v/v) 50 mM PB, 20% (v/v) ACN, pH 7.0; D4 was eluted with 70% (v/v) Solvent B, and D2 was eluted with 35% (v/v) Solvent B; or
  • Solvent B was 80% (v/v) 50 mM sodium phosphate pH 7 and 20%(v/v) acetonitrile (ACN) .
  • the column was previously equilibrated with 5 column volumes of solvent A.
  • the composition of ADCs was mixed with 0.67 volume of 5 M NaCl (2.0 M final) and applied to the column. D0 was not retained by the column.
  • the different drug loaded species were eluted by sequential step gradients: D2 was eluted with 35% (v/v) Solvent B, D4 was eluted with 70%(v/v) Solvent B, D6 was eluted with 95% (v/v) Solvent B, and D8 was eluted with 100% (v/v) Solvent B.
  • Figures 1-8 show HIC results of the composition of Herceptin-MMAE conjugates prepared by using the process of the present disclosure at close to R.T (20°C or 16°C) and different pH (e.g., 6.8, 6.6, 6.4 or 6.2) .
  • the content of D4 is 73.5wt%in the presence of Zn 2+ and 38.3wt%in absence of Zn 2+ )
  • the content of D4 produced by the process of the present disclosure in which reaction temperature was close to room temperature e.g., 20°C or 16°C
  • the content of D6 is less than 10wt%
  • the content of D4 is generally more than 60wt%, preferably more than 65wt%.
  • the advantage that the reaction temperature was increased to close to room temperature of the present disclosure has great benefit on large manufacture, such as easy operation and reduce cost.
  • D2 and D4 after isolation and purification via HIC resin, D2 and D4 could be eluted in different fractions, so that D2 and D4 could be purified, respectively; the purity of D4 could be as high as 99.49%or 99.53%, and the purity of D2 could be 98.50%or 98.80%.

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Abstract

L'invention concerne un procédé de bio-conjugaison pour préparer une composition de conjugués anticorps-médicament (ADC) avec une homogénéité améliorée, c'est-à-dire une composition de conjugués anticorps-médicament (ADC) à haute teneur en D4 (DAR4) par une manipulation simple, le procédé étant mis en œuvre à température ambiante. La composition résultante d'ADC comprend D4 dans une teneur supérieure à 60 % en poids, de préférence supérieure à 65 % en poids, sur la base du poids total de D0, D2, D4, D6 et D8.
PCT/CN2024/090809 2024-04-30 2024-04-30 Procédé de préparation d'une composition de conjugués anticorps-médicament (adcs) à haute teneur en d4 Pending WO2025227361A1 (fr)

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CN111556876A (zh) * 2017-10-27 2020-08-18 辉瑞公司 对cd123具有特异性的抗体和抗体-药物缀合物及其用途
CN113423730A (zh) * 2019-02-15 2021-09-21 上海药明生物技术有限公司 用于制备具有改善的同质性的抗体-药物缀合物的方法
US20210379193A1 (en) * 2019-02-15 2021-12-09 WuXi Biologics Ireland Limited Process for preparing antibody-drug conjugates with improved homogeneity
WO2024041544A1 (fr) * 2022-08-22 2024-02-29 Suzhou Bioreinno Biotechnology Limited Company Procédé de préparation d'un anticorps ayant des modifications spécifiques à un site
WO2024041542A1 (fr) * 2022-08-22 2024-02-29 Suzhou Bioreinno Biotechnology Limited Company Procédé de gestion programmatique d'une modification spécifique d'un site de liaison disulfure d'anticorps
CN117838879A (zh) * 2023-12-21 2024-04-09 上海药明合联生物技术有限公司 一种提高抗体-药物缀合物同质性的制备方法及其应用
CN118416247A (zh) * 2024-04-30 2024-08-02 上海药明合联生物技术有限公司 用于制备具有高d4含量的抗体-药物缀合物（adc）组合物的方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111556876A (zh) * 2017-10-27 2020-08-18 辉瑞公司 对cd123具有特异性的抗体和抗体-药物缀合物及其用途
CN113423730A (zh) * 2019-02-15 2021-09-21 上海药明生物技术有限公司 用于制备具有改善的同质性的抗体-药物缀合物的方法
US20210379193A1 (en) * 2019-02-15 2021-12-09 WuXi Biologics Ireland Limited Process for preparing antibody-drug conjugates with improved homogeneity
WO2024041544A1 (fr) * 2022-08-22 2024-02-29 Suzhou Bioreinno Biotechnology Limited Company Procédé de préparation d'un anticorps ayant des modifications spécifiques à un site
WO2024041542A1 (fr) * 2022-08-22 2024-02-29 Suzhou Bioreinno Biotechnology Limited Company Procédé de gestion programmatique d'une modification spécifique d'un site de liaison disulfure d'anticorps
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