Methods of preparing highly homogeneous antibody-drug conjugates of engineered antibodies

Abstract

Methods for preparing highly homogeneous antibody-drug conjugates (ADCs) of antibodies are provided. In particular, the content of D2, D6, or D2+4 in ADCs prepared by the methods can be achieved at greater than 90%.

Description

[0001] Cross-referencing

[0002] This application claims the benefit of international application PCT / CN2021 / 117009, filed on September 10, 2021, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to a method for preparing antibody-drug conjugates (ADCs). Specifically, this disclosure relates to a bioconjugation method for preparing highly homogeneous antibody-drug conjugates (ADCs) of engineered antibodies, wherein the engineered antibodies are specifically engineered to contain a TCR constant region in a Fab arm. Background Technology

[0004] The specificity of antibodies to specific antigens and molecules on the surface of target cells leads to their widespread use as carriers for a variety of diagnostic and therapeutic agents. For example, antibodies conjugated to markers and reporter groups (such as fluorophores, radioisotopes, and enzymes) can be used for labeling and imaging applications, while conjugation to cytotoxic agents and chemotherapeutic drugs allows for targeted delivery of these agents to specific tissues or structures, such as specific cell types or growth factors, while minimizing the impact on normal healthy tissues and significantly reducing the side effects associated with chemotherapy treatments.

[0005] Bispecific antibodies (bsAbs) are antibodies designed to recognize two different epitopes or antigens and are intended to treat complex, multifaceted diseases by binding two disease targets to a single molecule. They come in various forms. WO2019057122 and WO2020057610 disclose WuXiBody. TM This platform enables the assembly of virtually any monoclonal antibody (mAb) sequence pair into bispecific constructs. Its unique structural flexibility allows for the easy construction of various forms with different valence states. The bispecific antibodies produced by this platform are also stable and exhibit no aggregation issues during production.

[0006] Antibody-drug conjugates (ADCs) are conjugates of antibodies and drugs, with broad potential therapeutic applications in several disease areas, particularly cancer, representing a novel type of targeted therapy. An ADC comprises an antibody for targeting, a linker or conjugate for drug attachment, and a highly efficient payload (e.g., the drug) as the effector. The antibody, through its specificity, directs the drug toward its target for release. Since the FDA approval of Adcetris in 2011 and Kadcyla in 2013, ADC drug development has been widely applied in cancer treatment. Recently, ADCs based on bispecific antibodies (e.g., ZW49) have demonstrated greater effectiveness in cancer therapy.

[0007] One of the challenges of conventional conjugation of ADCs is the heterogeneity of ADC molecules, where the drug moiety attaches to several sites on the antibody, for example, via cysteine ​​chemistry, ranging from 0 to 8 drug moieties per antibody (drug-antibody ratio, DAR). Such mixed ADC molecules not only present difficulties for analysis and characterization but may also exhibit different pharmacokinetic, distribution, toxicity, and efficacy characteristics. Non-specific conjugation also frequently leads to impaired antibody function.

[0008] Strategies to address this problem have been developed. Genetech's THIOMAB and Ambrx's non-natural amino acid incorporation and various enzyme-assisted conjugations aim to introduce drug moieties in a site-directed manner and achieve homogeneity (narrow DAR distribution) in ADCs. However, these methods are all based on antibody engineering, which may lead to side effects in humans. WO2017002776 discloses that by reducing the temperature of the reduction step, DAR4 can be selectively enriched by more than 50%, with most of the drug located on the Fab domain, without altering the IgG sequence.

[0009] However, we still need to develop new bioconjugation methods that can produce ADCs, including bispecific antibodies, with improved homogeneity and simple operation and reduced cost. Invention Overview

[0011] One object of this disclosure is to develop novel conjugation methods that can produce ADCs with improved homogeneity against specific types of bispecific antibodies, and with simple operation and reduced cost. Depending on the specific method applied, the resulting ADCs have high levels of D2, D6, or D2+4 ADCs. ADCs produced by the conjugation methods of this disclosure further exhibit better safety and efficacy.

[0012] This disclosure relates to a combination of metal ion chelation technology and WuXiBody-type bispecific antibodies for developing novel conjugation methods. Compared to conventional conjugation methods, the conjugation method of this disclosure can significantly improve the homogeneity of the antibody-drug conjugate (ADC) products produced. Furthermore, ADC products specifically conjugated to two different drug moieties can be obtained.

[0013] In one aspect, this disclosure provides a method for preparing an antibody-drug conjugate (ADC), wherein the antibody comprises a pair of T cell receptor (TCR) constant regions replacing the CH1 and CL domains in at least one arm, and the pair of TCR constant regions are capable of forming one or more non-natural intrachain disulfide bonds, and wherein the method comprises the following steps:

[0014] (a) Incubate the reducing agent with the antibody in a buffer system;

[0015] (b) Introducing an excess of the linker-drug moiety to react with the reduced thiol group generated in step (a); and

[0016] (c) Recover the obtained antibody-drug conjugate.

[0017] Optionally, the method further includes adding an effective amount of oxidant after step (b) and before recovering the obtained antibody-drug conjugate to reoxidize unreacted thiol groups.

[0018] The inventors have surprisingly discovered that, due to steric hindrance, the non-natural interchain disulfide bonds between the TCR constant regions cannot be approached by reducing agents and therefore cannot be reduced for drug conjugation.

[0019] In some embodiments, the antibody described herein comprises first and second antigen-binding portions, wherein the first antigen-binding portion comprises: a first heavy chain variable domain (VH) operably linked to a first T cell receptor (TCR) constant region (C1), and a first light chain variable domain (VL) operably linked to a second TCR constant region (C2), wherein C1 and C2 are capable of forming non-natural interchain disulfide bonds, and

[0020] The second antigen-binding part is in the form of Fab, scFv, or VHH.

[0021] In some embodiments, the second antigen-binding portion is in the form of Fab and includes a second VH operably linked to the CH1 domain of the antibody heavy chain and a second VL operably linked to the constant (CL) domain of the antibody light chain. In some embodiments, the second antigen-binding portion is in the form of scFv and includes a second VH operably linked to the second VL. In some other embodiments, the second antigen-binding portion is in the form of VHH and includes a single variable domain.

[0022] In some embodiments, the antibody is a bispecific antibody having first and second antigen-binding portions that target different antigens or epitopes. In some other embodiments, the antibody is a monospecific antibody having first and second antigen-binding portions that target the same epitope.

[0023] In some embodiments, the C1 and C2 regions of the antibody disclosed herein comprise engineered T-cell receptor (TCR) constant regions. Specifically, the C1 region may comprise the amino acid sequences of SEQ ID No:2, 7 or variants thereof having at least 90% identity; and the C2 region may comprise the amino acid sequences of SEQ ID No:4, 8, 9 or variants thereof having at least 90% identity. The C1 and C2 regions are capable of forming a dimer, and the non-natural interchain disulfide bond is capable of stabilizing the dimer. In some embodiments, amino acid C58 of SEQ ID No:2 and amino acid C49 of SEQ ID No:4 are capable of forming a non-natural interchain disulfide bond.

[0024] In some embodiments, the antibody comprises an IgG Fc region, such as the Fc region of IgG1, IgG2, IgG3 or IgG4.

[0025] In some implementations, the Fc region also includes a button-in-hole structure. Specifically, the sequence of the hinge region and Fc region in one chain (“button” chain) is shown in SEQ ID No: 5, and the sequence of the hinge region and Fc region in another chain (“hole” chain) is shown in SEQ ID No: 6.

[0026] In some embodiments, the antibody comprises, from the N-terminus to the C-terminus, the following structure (E17): in the first heavy chain, VH1-C1-hinge-Fc; in the second heavy chain, VH2-CH1-hinge-Fc; in the first light chain, VL1-C2; and in the second light chain, VL2-CL, wherein VH1 and VL1 refer to the first VH and VL, respectively, and VH2 and VL2 refer to the second VH and VL, respectively. A "-" indicates an operative link, typically via a peptide linker.

[0027] In some other embodiments, from the N-terminus to the C-terminus, the antibody comprises the following structure: in the first heavy chain, VH1-C1-hinge-Fc-scFv; in the second heavy chain, VH1-CH1-hinge-Fc-scFv; in the first light chain, VL1-C2; and in the second light chain, VL1-CL. scFv constitutes the second antigen-binding moiety and may also be replaced by the VHH form.

[0028] In some embodiments, to obtain a high content of D2 ADC, the incubation in step (a) is carried out in the presence of an effective amount of one or more metal ions, such as divalent metal ions and transition metal ions. The resulting antibody-drug conjugate contains D2 in a content greater than 80 wt%, for example greater than 85 wt%, greater than 90 wt%, or greater than 95 wt%, based on the total weight of D0 and D2.

[0029] In some embodiments, the incubation in step (a) is performed in the presence of effective amounts of metal ions, such as divalent metal ions and transition metal ions, and the method further includes, between steps (b) and (c): removing the transition metal ions or divalent metal ions from the product of step (b), then reintroducing the reducing agent and incubating with an excess of different linker-drug moieties. The resulting antibody-drug conjugate contains D2+4 at a concentration greater than 65 wt%, for example greater than 70 wt%, greater than 80 wt%, or greater than 90 wt%, based on the total weight of the ADC.

[0030] In some embodiments, to obtain a high content of D6 ADC, the incubation in step (a) is not performed in the presence of an effective amount of transition metal ions or divalent metal ions. The resulting antibody-drug conjugate contains D6 in a content greater than 85 wt%, such as greater than 90 wt%, greater than 91 wt%, greater than 92 wt%, or greater than 93 wt%, based on the total weight of D0, D2, D4, and D6.

[0031] In one aspect, this disclosure provides a method for preparing an antibody-drug conjugate (ADC), wherein the antibody comprises first and second antigen-binding moieties.

[0032] The first antigen-binding portion comprises: a first heavy chain variable domain (VH) operably linked to the constant region (C1) of the first T cell receptor (TCR), and a first light chain variable domain (VL) operably linked to the constant region (C2) of the second TCR, and

[0033] The second antigen-binding portion comprises: a second VH operably linked to the CH1 domain of the antibody heavy chain, and a second VL operably linked to the constant (CL) domain of the antibody light chain.

[0034] And the method described therein includes the following steps:

[0035] (a) Incubate a reducing agent and a bispecific antibody in a buffer system in the presence of an effective amount of transition metal ions or divalent metal ions to selectively reduce interchain disulfide bonds within the antibody;

[0036] (b) Introducing an excess of the first linker-drug moiety to react with the reduced thiol group generated in step (a);

[0037] (c) Remove the transition metal ions or divalent metal ions from the product of step (b);

[0038] (d) Reintroduce the reducing agent and incubate with an excess of the second linker-drug fraction; and

[0039] (e) The obtained antibody-drug conjugate is recovered.

[0040] Optionally, the method further includes adding an effective amount of oxidant after step (d) to re-oxidize the unreacted thiol groups.

[0041] In some implementations, the reducing agent added in steps (a) and (d) is different, for example, one is TCEP and the other is TDD. In other implementations, the reducing agent added in steps (a) and (d) is the same, for example, both are TCEP.

[0042] In some implementations, the resulting antibody-drug conjugate contains D2+4 in an amount greater than 65 wt% of the total weight of the ADC, such as greater than 70 wt%, greater than 80 wt%, or greater than 90 wt%.

[0043] In some implementations, the first drug is MMAF and the second drug is DXD.

[0044] In some embodiments, the metal ion in step (a) is selected from divalent ions and transition metal ions, including: Zn2+, Cd2+, Hg2+, Ca2+, Mg2+, or any combination thereof. For example, the metal ion in step (a) is Zn2+. Transition metal ions suitable for use in the coupling method of this disclosure may include, but are not limited to, Zn2+. 2+ Cd 2+ Hg 2+ etc. Among them, due to Zn 2+ Ca 2 + and Mg 2+ Their availability and low cost allow them to be used. For example, suitable transition metal salts or divalent metal ions can be added in step (a), provided they are soluble in the reaction solution, thereby releasing free transition metal ions in the reaction solution. In this regard, ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, zinc formate, and zinc tetrafluoroborate are suitable zinc salts. Similarly, CaCl2, Ca(NO3)2, CaSO4, MgCl2, Mg(NO3)2, and MgSO4 can be used. Likewise, those soluble in the reaction solution and capable of releasing free Cd can be mentioned. 2+ or Hg 2+ Other transition metal salts of ions, including but not limited to CdCl2, Cd(NO3)2, CdSO4, Cd(CH3COO)2, CdI2, CdBr2, cadmium formate and cadmium tetrafluoroborate; HgCl2, Hg(NO3)2, HgSO4, Hg(CH3COO)2, HgBr2, mercuric formate (II) and mercuric tetrafluoroborate (II), etc.

[0045] In some implementations, the buffering system used in step (a) is selected from the group consisting of Hepes, histidine buffers, PBS and MES, and has a pH of about 5.5 to 8.

[0046] In some implementations, the antibody to be conjugated is added in step (a) to a final concentration of approximately 0.01-0.1 mM.

[0047] In some implementations, step (a) is performed at a temperature of about -10°C to 37°C, for example, at a temperature of about 0°C to 20°C.

[0048] In some implementations, the reducing agent in step (a) is TCEP.

[0049] In some implementations, the oxidant is DHAA.

[0050] In some implementations, the linker-drug portion is a maleimide carrying the drug, an organic bromide carrying the drug, or an organic iodide carrying the drug.

[0051] In some implementations, the variable regions of the first and second antigen-binding moieties are derived from known, commercially available, or de novo developed antibodies, such as any of the following antibodies: trastuzumab, pertuzumab, sacituzumab, abciximab, adalimumab, alefacept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab (polyethylene glycol). The following are listed: pegol, cetuximab, daclizumab, denosumab, efalizumab, golimumab, inflectra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, secukinumab, and ustekinumab. Preferably, the variable region (or at least the CDR region) of the first and second antigen-binding portions is the same as the variable region (or at least the CDR region) of a known or de novo developed antibody.

[0052] In some embodiments, the first VH and first VL are derived from trastuzumab and the second VH and second VL are derived from pertuzumab, or vice versa. In some other embodiments, the first VH and first VL are derived from trastuzumab and the second VH and second VL are derived from saxizumab, or vice versa.

[0053] In some implementations, the drug to be coupled is selected from the group consisting of: diagnostic agents, therapeutic agents, and labeling agents.

[0054] In some embodiments, metal ions are removed in the purification step by using EDTA as a chelating agent, which is then filtered out in subsequent dialysis, ultrafiltration, or gel filtration.

[0055] In one respect, this disclosure provides antibody-drug conjugates prepared by any of the disclosed methods.

[0056] In one aspect, this disclosure provides pharmaceutical compositions comprising the antibody-drug conjugates disclosed herein and pharmaceutically acceptable carriers or mediators.

[0057] In one aspect, this disclosure provides the use of the antibody-drug conjugates disclosed herein in the preparation of pharmaceutical compositions or kits for treating a condition or ailment of a subject.

[0058] In one aspect, this disclosure provides a method for treating a subject’s condition or ailment, comprising administering to the subject a therapeutically effective amount of the antibody-drug conjugate or pharmaceutical composition disclosed herein.

[0059] In some implementations, the condition or ailment is a tumor, cancer, autoimmune disease, or infectious disease. For example, the cancer is breast cancer. The subject can be a mammal, such as a human.

[0060] The foregoing and other features and advantages of this disclosure will become more apparent from the following detailed description of several embodiments with reference to the accompanying drawings. Attached Figure Description

[0061] Figure 1. Reaction protocol according to some implementation schemes, in which the three pairs of interchain disulfide bonds of the bispecific antibody cAb1 (trastuzumab x pertuzumab) are reduced and specifically coupled to the MC-MMAF site ( Figure 1A ); HIC spectra showing cAb1 coupling of DAR6 abundance ( Figure 1B ); and mass spectra coupled with cAb1 ( Figure 1C ). Figure 1D These are the HIC spectra of two other similarly conjugated bispecific antibodies, cAb3 and cAb4.

[0062] Figure 2. Reaction schemes according to some implementation plans, in which Zn is added. 2+ Furthermore, only one pair of interchain disulfide bonds of the bispecific antibody is reduced and specifically coupled to the MC-MMAF site. Figure 2A );like( Figure 2A The HIC spectra of the four bispecific antibodies cAb1, cAb2, cAb3, and cAb4 conjugated as described above ( Figure 2B ).

[0063] Figure 3. Reaction protocol according to some implementation schemes, wherein the bispecific antibody cAb1 is conjugated with two different drugs (e.g., MMAF and DXD) in two separate steps. Figure 3A The obtained cAb1 ADC ( Figure 3B ), cAb2 ADC ( Figure 3C ), cAb3 ADC ( Figure 3D ) and cAb4 ADC ( Figure 3E MS spectrum of ). Invention Details

[0065] Although this disclosure may be implemented in many different forms, what is disclosed herein are specific embodiments illustrated therein, illustrating the principles of the invention. It should be emphasized that this disclosure is not limited to the specific embodiments illustrated. Furthermore, any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter.

[0066] Generally, the nomenclature and techniques used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6th ed., WB Saunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature and laboratory procedures and techniques used in conjunction with the analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are those well-known and commonly used in the art. All publications mentioned in this specification are incorporated herein by reference in their entirety.

[0067] definition

[0068] To better understand this invention, the following provides definitions and explanations of relevant terms.

[0069] Unless otherwise defined, scientific and technical terms used in conjunction with this disclosure shall have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plurals and plural terms shall include a single term. More specifically, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plurals unless the context explicitly specifies otherwise. Thus, for example, “an antibody” may refer to plurals of antibodies; “a transition metal ion” may refer to plurals of transition metal ions, and so on. In this application, the use of “or” means “and / or” unless otherwise indicated.

[0070] Throughout this disclosure, unless the context otherwise requires, the terms “comprising,” “including,” and “containing” should be understood to imply the inclusion of the stated steps or elements or a group of steps or elements, but do not exclude any other steps or elements or groups of steps or elements. “Constitutes of…” means including and limited to the elements following the phrase “consisting of…”. Therefore, the phrase “consisting of…” indicates that the listed elements are necessary or mandatory, and no other elements may be present. “Substantially constitutes of…” means including any elements listed following the phrase, and is limited to other elements that do not interfere with or promote the activity or effect specified for the listed elements in this invention. Therefore, the phrase “substantially constitutes of…” indicates that the listed elements are necessary or mandatory, but other elements are optional and may be present or absent depending on whether they affect the activity or effect of the listed elements.

[0071] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length by as much as 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In specific implementations, when the terms “about” or “approximately” precede a numerical value, it indicates a range of that value plus or minus 15%, 10%, 5%, or 1%.

[0072] Antibody-drug conjugates (ADCs) are conjugates formed by covalently linking a drug to an antibody, either directly or indirectly via one or more suitable linkers. ADCs are typically in the form of antibody-linker-drug conjugates. Antibody-drug conjugates combine the ideal properties of both antibodies and cytotoxic drugs (or substances with other properties) to enhance their antitumor (or other pharmaceutical) activity by targeting tumor cells (or other cells / organs) expressing antigens with potent cytotoxic (or other) drugs. ADCs are designed to distinguish between healthy cells and diseased tissues, such as tumor cells in a tumor.

[0073] As used herein, the terms "drug" or "load" refer to any cytotoxic molecule that has, for example, antitumor, anti-infective, or anti-inflammatory effects and has at least one substituent group or portion of a structure that allows attachment to a linker structure. Drugs can kill cells (e.g., cancer cells) and / or inhibit the growth, proliferation, or metastasis of cells (e.g., cancer cells), thereby reducing, alleviating, or eliminating one or more symptoms of a disease or condition (e.g., cancer).

[0074] As used in this article, the term "linker" refers to a reactive molecule containing at least two reactive groups, one of which can be covalently bonded to a drug molecule and the other can be covalently coupled to an antibody.

[0075] As used herein, the term "antibody" includes any immunoglobulin that binds to a specific antigen; monoclonal antibodies, polyclonal antibodies, multispecific antibodies, bispecific antibodies, and multivalent or bivalent antibodies. A naturally occurring intact antibody consists of two heavy chains and two light chains. Each heavy chain comprises a variable region ("HCVR") and first, second, and third constant regions (CH1, CH2, and CH3), while each light chain comprises a variable region ("LCVR") and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. Antibodies are typically "Y"-shaped, where the stem of the Y consists of the second and third constant regions of two heavy chains linked together via disulfide bonds. Each arm of the Y comprises the variable region and first constant region of a single heavy chain, which binds to the variable region and constant region 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 typically contain three highly variable loops called complementarity-determining regions (CDRs) (the CDRs for light (L) chains include LCDR1, LCDR2, and LCDR3, and the CDRs for heavy (H) chains include HCDR1, HCDR2, and HCDR3). The CDR boundary of an antibody can be defined or identified by rules such as those of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J Mol Biol. Dec5; 186(3):651-63(1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901(1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83(1989); Kabat EA et al., National Institutes of Health, Bethesda, Md.(1991)). Three CDRs are located between flank segments called framework regions (FRs), which are more conserved than the CDRs and form a scaffold to support the highly variable ring. Each HCVR and LCVR comprises four FRs, and the CDRs and FRs are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are designated into various classes based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively.Several major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain).

[0076] "Antibody fragments" include a portion of a full-length antibody, typically its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; microantibodies (Olafsen et al. (2004) Protein Eng. Design & Sel. 17(4):315-323), fragments generated from Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity-determining regions), and any fragments that bind to epitopes as described herein (which immune-specifically bind to cancer cell antigens, viral antigens, or microbial antigens), single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0077] As used herein, the term "antigen-binding moiety" refers to an antibody fragment formed from a portion of an antibody containing one or more CDRs, or any other antibody fragment that binds an antigen but does not contain the complete structure of the native antibody. Examples of antigen-binding moieties include, but are not limited to, variable domains, variable regions, biantibodies, Fab, Fab', F(ab')2, Fv fragments, scFv, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibodies (ds diabody), multispecific antibodies, camel-derived single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding moiety is capable of binding the same antigen as that bound to the parent antibody. In some embodiments, the antigen-binding moiety may contain one or more CDRs from a specific human antibody that are transposed to a framework region from one or more different human antibodies. For more detailed descriptions of the antigen-binding moiety, see Spiess et al., 2015 (above) and Brinkman et al., mAbs, 9(2), pp.182–212 (2017), the entire contents of which are incorporated herein by reference.

[0078] The term "Fab" in antibody refers to the following portion of the antibody, which consists of a single light chain (both the variable and constant regions) associated with a variable region and a first constant region of a single heavy chain via disulfide bonds. In some embodiments, both the first and second antigen-binding portions of the antibody to be coupled are in Fab form. Further, the constant regions (i.e., CH1 and CL) of the two chains of the Fab are replaced with engineered or modified TCR constant regions.

[0079] The "Fc" in antibody refers to the following portion of the antibody, which contains the second (CH2) and third constant regions (CH3) of the first heavy chain, which bind to the second and third constant regions of the second heavy chain via disulfide bonds and optionally hinge regions. The Fc portion of the antibody is responsible for various effector functions, such as ADCC and CDC, but does not play a role in antigen binding.

[0080] The "hinge region" of an antibody comprises the portion of the heavy chain molecule that connects the CH1 and CH2 domains. This hinge region consists of approximately 25 amino acid residues and is flexible, thereby allowing the two N-terminal antigen-binding regions to move independently.

[0081] As used herein, the term “button-lockhole” refers to the engineering of the CH3 domain of the antibody Fc region to create a “button” or “lockhole” in each heavy chain to facilitate heterodimerization. Typically, a “button” is created by substituting a large residue W for T366 on one heavy chain, while the corresponding “lockhole” is created by a triple mutation of T366S, L368A, and Y407V on another heavy chain. Button-lockhole structures may include other substitutions familiar in the art. When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is generally used (e.g., the EU index reported in Sequences of Proteins of Immunological Interest (5th edition), US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242). Unless otherwise stated herein, references to residue numbers in the constant domain of the Fc region refer to residue numbers obtained through the EU numbering system. In some implementations, the sequence of the hinge region and Fc region in one chain (“button” chain) is shown in SEQ ID No:5, while the sequence of the hinge region and Fc region in another chain (“keyhole” chain) is shown in SEQ ID No:6.

[0082] ASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID No: 5)

[0083] SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID No: 6)

[0084] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting said population are identical except for the possible naturally occurring mutations (which may be present in small amounts). Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike polyclonal antibody formulations which comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring that the population of antibodies be obtained by any particular method. For example, the monoclonal antibodies used according to the present invention can be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or by a recombinant DNA method (see, for example: US 4816567; US5807715). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597.

[0085] As used herein, "WuXiBody" refers to a bispecific antibody, a soluble chimeric protein comprising a variable domain of the antibody and a constant domain of the TCR (e.g., in the first antigen-binding moiety), wherein subunits of the TCR constant domain (e.g., α and β domains) are linked by one or more engineered disulfide bonds. WuXiBody also encompasses WuXiBody 2.0 antibodies, which contain various modified TCR constant domain sequences (see WO2022 / 156687). The TCR constant domain can be engineered to form more than one pair of disulfide bonds, thereby improving stability and / or expression levels. In one type of WuXiBody, the antibody contains a first antigen-binding moiety in one arm and a second antigen-binding moiety in the other arm, both in Fab form and operatively linked at the C-terminus to a single chain of an immunoglobulin Fc region. In a different form of WuXiBody, an Fc region is inserted between the first and second antigen-binding moieties. Specifically, the antibody may contain a first antigen-binding moiety, Fab, at one end of the Fc region and a second antigen-binding moiety, Fab, scFv, or VHH, at the other end of the Fc region. Alternatively, antibodies in the form of WuXiBody may contain a first antigen-binding moiety operatively linked to a second antigen-binding moiety, the second antigen-binding moiety being further operatively linked to the Fc region. Detailed descriptions of the various forms of WuXiBody can be found in WO2019057122, WO201905714, and WO2020057610 (all of which are incorporated herein by reference in their entirety).

[0086] The natural T-cell receptor, or natural TCR, is a heterodimeric T-cell surface protein that associates with the invariant CD3 chain to form a complex capable of mediating signal transduction. The TCR belongs to the immunoglobulin superfamily and is similar to a hapten with a single heavy chain and a single light chain. The natural TCR has extracellular, transmembrane, and intracellular portions. The extracellular domain of the TCR has a proximal constant region and a distal variable region.

[0087] The terms “trastuzumab x pertuzumab,” “pertuzumab x trastuzumab,” “trastuzumab x sacizumab,” and “sacizumab x trastuzumab,” as well as similar names used herein, are named according to the same principle, namely, referring to a bispecific antibody (preferably WuXiBody BsAb) comprising a first antigen-binding portion derived from a first antibody and a second antigen-binding portion derived from a second antibody. “Derived from” means that the variable region is identical to or has at least 80% homology with the variable region in the parent antibody (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), but still retains the ability to bind to the target antigen. For example, the variable region derived from the parent antibody may be humanized, affinity-matured, or glycosylated prior to constructing the antibody form disclosed herein. Methods for modifying variable regions, including CDRs and frame regions, are well known to those skilled in the art.

[0088] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0089] "Isolated antibody" refers to an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0090] A disulfide bond is a covalent bond with the structure RSS-R'. The amino acid cysteine ​​contains a thiol group, which can form a disulfide bond with a second thiol group, for example, from another cysteine ​​residue. Disulfide bonds can form between the thiol groups of two cysteine ​​residues located on two separate polypeptide chains, thereby forming interchain bridges or interchain bonds.

[0091] As used in this article, the term "transition metals" refers to elements in groups 4–11, as evidenced by their typical chemical characteristics: a wide range of complex ions in various oxidation states, colored complexes, and catalytic properties as elements or as ions (or both). Sc and Y from group 3 are also generally considered transition metals.

[0092] As used herein, the term "effective amount" in relation to metal ions (including transition metal ions and divalent metal ions) refers to the amount of a metal ion sufficient to chelate with disulfide bonds in the antibody hinge region, thereby protecting the disulfide bonds from reduction. "Effective amount" can be considered when using one or more metal ions, and a single metal ion may be considered as given in an effective amount if the desired result is achieved in combination with one or more other metal ions. The effective amount of a metal ion can be determined empirically by those skilled in the art based on the specific composition and conditions used for coupling. In some embodiments, the effective amount of metal ions in the reaction solution in step (a) is about 0.01 mM to 0.2 mM.

[0093] The term “DAR” or “drug-to-antibody ratio” used in this article refers to the average amount of drug conjugated to an antibody, a crucial attribute of ADCs. DAR values ​​affect drug efficacy, as low drug loading reduces potency, while high loading can negatively impact pharmacokinetics (PK) and toxicity. Various analytical methods can be used to measure DAR, such as UV / Vis spectroscopy, hydrophobic interaction chromatography (HIC), reversed-phase high-performance liquid chromatography (RP-HPLC), and liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS). Hydrophobic interaction chromatography (HIC) is a leading technique for characterizing DAR values ​​and drug loading distribution. It separates conjugate species based on the increased hydrophobicity resulting from the increased drug loading. For cysteine-conjugated ADCs, the least hydrophobic unconjugated antibody is eluted first, followed by the most hydrophobic and most drug-conjugated form, producing a quantitative elution profile. The percentage of peak area represents the relative amount of each drug-loaded ADC species. The payload distribution was derived from the HIC distribution plot, and the average DAR was calculated as a percentage of peak area. As shown in this paper, the ADCs conjugated using the methods disclosed herein are highly homogeneous. Depending on the specific method used, the content of DAR2, DAR6, or DAR2+4 (i.e., D2, D6, or D2+4) can reach at least 80 wt% of the total ADC. DAR2+4 (D2+4) refers to a dual-drug ADC containing two first drug molecules and four second drug molecules per antibody.

[0094] As is known in the art, mixtures of antibody-drug conjugates are produced using conventional conjugation methods. Typically, a therapeutic antibody molecule belonging to the IgG1 or IgG4 subclass has four interchain SS bonds, each formed by two -SH groups. One or more interchain SS bonds of the antibody molecule can be partially or completely reduced to form 2n (n is an integer selected from 1, 2, 3, or 4) reactive -SH groups, and thus, the number of drugs conjugated to a single antibody molecule is 2, 4, 6, or 8. Depending on the number of drugs conjugated to a single antibody molecule, different conjugates containing different numbers of drug molecules are named D0, D2, D4, D6, and D8.

[0095] If the number of drugs conjugated to a single antibody molecule is 0, the product is called D0. Correspondingly, D2 refers to an ADC in which two drug molecules are conjugated to a single antibody molecule, wherein the two drug molecules can be conjugated via a linker to -SH groups formed by SS bonds between the reduced heavy and light chains, or via a linker to -SH groups formed by SS bonds between the reduced heavy and light chains. D4 refers to an ADC in which four drug molecules are conjugated to a single antibody molecule, wherein the four drug molecules can be conjugated via a linker to four -SH groups formed by two SS bonds between the reduced heavy and light chains or between the heavy and light chains, or two drug molecules can be conjugated via a linker to two -SH groups formed by one SS bond between the reduced heavy and light chains, and another two drug molecules can be conjugated via a linker to two -SH groups formed by one SS bond between the reduced heavy and light chains. D6 refers to an ADC in which six drug molecules are conjugated to a single antibody molecule. This can be achieved by conjugating four drug molecules via a linker to four -SH groups generated by reducing two SS bonds between the heavy and light chains, and two drug molecules via a linker to two -SH groups generated by reducing one SS bond between the heavy and light chains; or by conjugating four drug molecules via a linker to four -SH groups generated by reducing two SS bonds between the heavy and light chains, and two drug molecules via a linker to two -SH groups generated by reducing one SS bond between the heavy and light chains. D8 refers to an ADC in which eight drug molecules are conjugated to a single antibody molecule, i.e., all four SS bonds in an antibody molecule are reduced to eight -SH groups, and each -SH group is linked to one drug molecule. Typically, heterogeneous mixtures of ADC molecules produced by conventional conjugation methods or the bioconjugation method of this invention are mixtures of D0, D2, D4, D6, and D8.

[0096] Therefore, the term "homogeneity" of antibody-drug conjugates is used to describe the property that a particular type of antibody-drug conjugate (preferably selected from D2, D4, and D6 conjugates) is dominant in a given antibody-drug conjugate mixture. In this invention, "homogeneity" of antibody-drug conjugates refers to a high level of a particular type of ADC in an antibody-drug conjugate mixture.

[0097] The term “pharmaceutical acceptable” means that the specified carrier, medium, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other components constituting the formulation and physiologically compatible with its acceptor.

[0098] "Pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation other than the active ingredient that is biologically acceptable and non-toxic to the subject. Pharmaceutically acceptable carriers used in the pharmaceutical compositions described herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispensing agents, masking or chelating agents, diluents, excipients, or non-toxic adjuvants, other components known in the art, or various combinations thereof.

[0099] The term "subject" includes any person or non-human animal, such as a human.

[0100] As used herein, the term “cancer” refers to any solid tumor or non-solid tumor such as leukemia mediated by the growth, proliferation, or metastasis of malignant cells, and causing a medical condition. “Tumor” includes one or more cancer cells. Examples of cancer include, but are not limited to, epithelial carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer (“NSCLC”), lung adenocarcinoma, and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, anal cancer, penile cancer, and head and neck cancer.

[0101] In the context of treating a condition, the terms “treatment,” “management,” or “treated” as used herein generally refer to a treatment or therapy, whether for humans or animals, in which a desired therapeutic effect is achieved, such as inhibiting the progression of the condition, and including a reduction in the rate of progression, cessation of the rate of progression, regression of the condition, remission of the condition, and cure of the condition. It also includes treatment as a preventative measure (i.e., prevention, avoidance). For cancer, “treatment” can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, “treatment” includes the removal of all or part of the tumor, inhibiting or slowing tumor growth and metastasis, inhibiting or delaying tumor development, or some combination thereof.

[0102] Overview of ADC fabrication

[0103] Antibody-drug conjugates are typically produced using two conventional chemical methods: lysine-based conjugation and cysteine-based conjugation from the reduction of interchain disulfide bonds. For cysteine-based conjugation, it involves opening interchain disulfide bonds in the presence of various reducing agents (such as TCEP, DTT, etc.), followed by a nucleophilic reaction of the thiol group. In this conjugation method, antibody-drug conjugates are typically formed by coupling one or more antibody cysteine ​​thiol groups to one or more drug-binding linker moieties, thereby forming an antibody-linker-drug complex. Because free cysteine ​​thiol (RSH, thiohydryl) groups are relatively reactive, proteins containing cysteine ​​often exist as disulfide-linked oligomers in their oxidized form or with internally bridging disulfide groups. The formation of disulfide dimers renders Cys inactive for conjugation with drugs, ligands, or other labeled substances.

[0104] The number of drugs conjugated to a single antibody molecule is a crucial factor for the efficacy and safety of the resulting ADC. For example, in conjugation methods based on the reduction of natural interchain disulfide bonds, interchain SS bonds are more accessible to the solvent than other disulfide bonds. Therefore, interchain disulfide bonds can serve as binding sites for drug (or drug-linker) conjugation to antibodies. Typically, a therapeutic antibody molecule belonging to the IgG1 or IgG4 subtype has four interchain SS bonds, each formed by two -SH groups, thus the number of drugs conjugated to a single antibody molecule is 2, 4, 6, or 8. If the number of drugs conjugated to a single antibody molecule is 0, the product is called D0. Correspondingly, D2 refers to an ADC in which two drug molecules are conjugated to a single antibody molecule. D4 refers to an ADC in which four drug molecules are conjugated to a single antibody molecule. D6 refers to an ADC in which six drug molecules are conjugated to a single antibody molecule. Furthermore, D8 refers to an ADC in which eight drug molecules are coupled to a single antibody molecule. This means that all four SS bonds in an antibody molecule are reduced to eight -SH groups, with each -SH group linked to one drug molecule. Typically, the heterogeneous mixture of ADC molecules produced by conventional coupling methods is a mixture of D0, D2, D4, D6, and D8.

[0105] It is well known in the art that heterogeneous ADC products often exhibit low efficacy and unsatisfactory PK properties. Among them, D0 lacks ADC efficacy, and D8 is considered to be the cause of instability in cycling due to the hydrophobicity induced by the payload (i.e., drug) molecule. Although the in vitro efficacy of antibody-drug conjugates has been shown to be directly dependent on the drug load (Hamblett KJ et al., Clin Cancer Res. 2004 Oct 15; 10(20):7063-70), the in vivo antitumor activity of an antibody-drug conjugate (D4) with four drugs per molecule is equivalent to that of a conjugate (D8) with eight drugs per molecule at the same mAb dose, even though each mAb contains half the amount of drug.

[0106] Drug loading also affects plasma clearance; D8 conjugates are cleared 3 times faster than D4 conjugates and 5 times faster than D2 conjugates. Antibody-drug conjugates with improved homogeneity offer therapeutic benefits such as a higher therapeutic index, improved efficacy, and reduced drug toxicity. Homogeneous antibody-drug conjugates also provide more accurate and consistent measurements in diagnostic and imaging applications.

[0107] Conventionally, the drug loading of an ADC can be controlled in various ways, for example, by: (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody; (ii) limiting the coupling reaction time or temperature; (iii) partially or limiting the reduction conditions of cysteine ​​thiol modification; and (iv) engineering the amino acid sequence of the antibody by recombinant technology such that the number and position of cysteine ​​residues are modified to control the number and / or position of linker-drug attachments (e.g., thioMab or thioFab and those disclosed in WO2006 / 034488, all of which are incorporated herein by reference).

[0108] Antibodies constructed using the WuxiBody® platform

[0109] In one aspect, this disclosure provides a method or process for preparing a highly homogeneous antibody-drug conjugate (ADC). The antibody comprises engineered Fab whose CH1 and CL domains are replaced by a pair of T-cell receptor (TCR) constant regions, and these TCR constant regions are capable of forming non-natural interchain disulfide bonds. These disulfide bonds stabilize the dimer formed between the pair of TCR constant regions and, as described above, make it more difficult for solvents or reducing agents to approach than natural disulfide bonds.

[0110] In some embodiments, the antibody to be conjugated herein is constructed using the WuXiBody® platform, also referred to as "WuXiBody". "WuXiBody" is typically a bispecific (or multispecific) antibody comprising first and second antigen-binding moieties. The first antigen-binding moiety is an engineered Fab containing a first heavy chain variable domain (VH) operably linked to a first T cell receptor (TCR) constant region (C1) and a first light chain variable domain (VL) operably linked to a second TCR constant region (C2). The second antigen-binding moieties can be in the form of Fab, scFv, VHH, etc. In some embodiments, the second antigen-binding moieties are also in the form of Fab and contain a second VH operably linked to the antibody heavy chain CH1 domain and a second VL operably linked to the antibody light chain constant (CL) domain. In other words, in the first antigen-binding moieties, the normally present CH1 and CL domains are replaced by a pair of TCR constant regions, and thus the native disulfide bond between the CH1 and CL domains is replaced by one or more engineered non-natural disulfide bonds between the TCR C1 and C2 regions. The positions of areas C1 and C2 can also be swapped.

[0111] The antibodies to be conjugated in this article are not limited to bispecific antibodies having first and second antigen-binding moieties targeting different epitopes or antigens. Theoretically, the antibodies to be conjugated in this article can also be monospecific antibodies, wherein the first and second antigen-binding moieties bind to the same antigen or epitope, and the resulting ADC is a highly homogeneous ADC targeting a single antigen or epitope.

[0112] In some embodiments, the antibody comprises the following structure from the N-terminus to the C-terminus: VH1-C1-hinge-Fc in the first heavy chain; VH2-CH1-hinge-Fc in the second heavy chain; VL1-C2 in the first light chain; and VL2-CL in the second light chain, wherein VH1 and VL1 refer to the first VH and VL, respectively, and VH2 and VL2 refer to the second VH and VL, respectively. A "-" indicates an operative link, typically via a peptide linker.

[0113] WuXiBody can take various other forms. For example, the heavy chain portion of the first antigen-binding portion can be operatively linked to the second antigen-binding portion, which in turn is operatively linked to a chain in the Fc region. Alternatively, the heavy chain portion of the first antigen-binding portion can be operatively linked to a chain in the Fc region, which in turn is operatively linked to a chain in the second antigen-binding portion.

[0114] In some embodiments, the antibody comprises the following structures from the N-terminus to the C-terminus: in the first heavy chain, VH1-C1-hinge-Fc-scFv; in the second heavy chain, VH1-CH1-hinge-Fc-scFv; in the first light chain, VL1-C2; and in the second light chain, VL1-CL. scFv is part of the second antigen-binding moiety and may also be replaced by the VHH form.

[0115] The first and second TCR constant regions associate via non-natural interchain disulfide bonds. The pair of TCR constant regions in the first antigen-binding moiety comprises the TCRα and β constant regions (wild-type or preferably engineered) in the light and heavy chains, respectively. The TCR constant regions in bispecific antibodies can form dimers by associating with each other via non-natural disulfide bonds.

[0116] The human TCRβ chain constant region has two distinct variants, designated TRBC1 and TRBC2 (IMGT nomenclature). In WuXiBody, the TCRβ domain sequence is based on the following wild-type TCR sequence:

[0117] LEDLKNVFPP K VAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV S TDPQPLKEQPALN DSRY C LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR (SEQ ID NO:1), NCBI accession number A0A5B9 (https: / / www.uniprot.org / uniprot / A0A5B9).

[0118] In some specific implementations, a pair of TCR constant regions contains engineered TCRβ domains, which contain one or more mutation sites, as follows:

[0119] LEDLKNVFPP E VAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV C TDPQPLKEQPAL Q DSRY A LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR (SEQ ID NO: 2);

[0120] LEDLKNVFPP E VAVFEPSE C EISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR (SEQ ID NO: 7).

[0121] The constant region of the human TCRα chain is called TRAC, NCBI accession number P01848 (https: / / www.uniprot.org / uniprot / P01848). The sequence of the wild-type TCRα domain is as follows:

[0122] PDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT N VSQSKDSDVYITDK T VLDMRSMDFKSNSAVAWS N KSDFACANAF N NSIIPEDTFFPSPESS (SEQ ID NO: 3).

[0123] In some specific implementations, a pair of TCR constant regions comprises engineered TCRα chain constant regions, which contain one or more mutation sites, as shown below:

[0124] PDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Q VSQSKDSDVYITDK C VLDMRSMDFKSNSAVAWS Q KSDFACANAF Q NSIIPEDTFFPSPESS(SEQ ID NO: 4);

[0125] PDIQNPD C VYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPE C TFFPS (SEQ ID NO: 8);

[0126] PDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFF CS (SEQ ID NO: 9).

[0127] Those skilled in the art will readily understand that antibodies disclosed herein for preparing ADCs can contain a variety of C1 and C2 regions, provided they are capable of stabilizing the first VH and VL regions to form the first antigen-binding moiety. Specifically, the antibody may contain a TCR C1 region having the amino acid sequence shown in SEQ ID No:2 or 7 or a variant thereof that is at least 90% identical to it, and a TCR C2 region having the amino acid sequences shown in SEQ ID No:4, 8, or 9 or a variant thereof that is at least 90% identical to it. More than one pair of non-natural disulfide bonds may be formed between the C1 and C2 regions to improve stability and expression levels. In some embodiments, variants of SEQ ID No:2 or 7 contain substitutions, additions, and / or deletions of one or more amino acids compared to SEQ ID No:2 or 7. Similarly, variants of SEQ ID No:4, 8, or 9 contain substitutions, additions, and / or deletions of one or more amino acids compared to SEQ ID No:4, 8, or 9. Detailed descriptions of the C1 and C2 variants that can be used to construct WuXiBody antibody forms can be found in WO2022 / 156687, which is incorporated herein by reference in its entirety. Specifically, in some embodiments, the antibody comprises a combination of C1 and C2 regions having SEQ ID No:2 and 4 (for cAb1-6), or having SEQ ID No:2 and 8 (for cAb7), or having SEQ ID No:7 and 9 (for cAb8), respectively.

[0128] For example, the natural TCR β chain contains a natural cysteine ​​residue at position 76, which is unpaired and therefore does not form a disulfide bond in the natural α / β TCR. In some bispecific antibodies from WuXiBody, this natural cysteine ​​residue at position 76 of the TCR β chain is mutated to an alanine residue. This may help avoid incorrect intra- or inter-chain pairing. In some embodiments, certain substitutions can improve the in vitro TCR refolding efficiency.

[0129] Therefore, the first and second TCR constant regions of the first antigen-binding portion can form a dimer containing at least one non-natural interchain disulfide bond between the TCR constant regions (i.e., CAα and CBeta) that can stabilize the dimer.

[0130] The benefits of replacing the CH1 and CL domains with the TCR constant region are significant. In WuXiBody, the first antigen-binding moiety, possessing at least one non-natural disulfide bond, can be recombinantly expressed and assembled into the desired conformation. This stabilizes the TCR constant region dimer while providing good antigen-binding activity to the antibody variable region. Furthermore, the first antigen-binding moiety was found to be well-resistant to conventional antibody engineering, such as modifications to glycosylation sites and the removal of some native sequences. Moreover, due to the presence of the TCR constant region in the first antigen-binding moiety, this form of bispecific antibody can be readily expressed and assembled with minimal or virtually no mispairing of antigen-binding sequences.

[0131] Most importantly, non-natural disulfide bonds in the TCR constant region are less susceptible to solvent access than natural disulfide bonds, thus providing fewer binding sites (and improved homogeneity) for drug coupling (or drug-linkers). Furthermore, non-natural disulfide bonds may be less sensitive to reducing agents than natural disulfide bonds.

[0132] In some embodiments, the method includes, prior to step (a), constructing and generating an antibody based on a parental bispecific antibody or two parental monospecific antibodies. "Based on" means deriving or obtaining first and second VH and VL regions from the variable region of the parental antibody and assembling them with other regions (such as the TCR constant region, heavy and light chain constant regions, hinge region, and Fc region) into a WuxiBody form.

[0133] The BsAb used for conjugation comprises two antigen-binding moieties, which can be Fab, Fab', scFv, VHH, etc. The antigen-binding moieties can be derived from antibodies (known or newly developed) targeting a specific antigen. In some embodiments, the first antigen-binding moieties are derived from trastuzumab, and the second antigen-binding moieties are derived from pertuzumab, or vice versa. In some other embodiments, the first antigen-binding moieties are derived from trastuzumab, and the second antigen-binding moieties are derived from sacizumab, or vice versa. The term "derived from" generally means herein that the antigen-binding moieties include the CDR sequence of the parent antibody, and preferably include the variable region of the parent antibody. In some embodiments, the antigen-binding moieties include a variant of the CDR sequence of the parent antibody that retains antigen-binding specificity.

[0134] Theoretically, parental antibodies that can derive antigen-binding moieties can include all monoclonal antibodies specific to a particular antigen, such as antibodies against tumor-associated antigens or pathways, such as PD-1 / PD-L1, TIM-3, LAG-3, VEGF, HER2, CTLA-4, BMPR1B, E16, STEAP1, MUC16, MPF, Napi2b, Sema5b, 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, TMEFF. 1. GDNF-R1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, Ly6K, GPR19, GPR54, ASPHD1, tyrosinase, TMEM118, GPR172A, CD33, and CLL-1; antibodies against leukocyte receptors such as MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86, or their ligands; CD3-binding antibodies (engager antibodies), NK-binding antibodies; antitumor-associated antigens that enable ADCC; monoclonal antibodies against TNF, etc. Antibodies may include, but are not limited to, trastuzumab, pertuzumab, saxizumab, abcizumab, adalimumab, afaxetine, alenumab, baliximab, belimumab, belotusizumab, canatumab, cetuzumab (pegylated), cetuximab, dazumab, denosumab, efazolin, golimumab, infliximab, ipilimumab, isibetab, natazumab, nivolumab, olamarumab, omalizumab, pallizumab, panitumumab, pembrolizumab, rituximab, tosizumab, secukinumab, and ustekinumab.

[0135] The bispecific antibodies used in the methods provided herein can be antibodies that have binding specificity to multiple antigens, such as tumor-associated antigens (TAAs). In some embodiments, the bispecific antibody to be conjugated has first specificity to a first TAA antigen and second specificity to a second TAA antigen. The term "tumor-associated antigen" refers to a target antigen expressed by tumor cells, however, it may be expressed by similar cells (or healthy cells) before transformation into a tumor. In some embodiments, tumor-associated antigens can only be presented by tumor cells and not by normal cells, i.e., non-tumor cells. In some other embodiments, tumor-associated antigens may be expressed only on tumor cells compared to non-tumor cells, or may represent tumor-specific mutations. In some other embodiments, tumor-associated antigens may be found in both tumor and non-tumor cells, but are overexpressed on tumor cells compared to non-tumor cells, or may bind to the antibody on tumor cells because the structure of tumor tissue is less compact compared to non-tumor tissue. In some embodiments, tumor-associated antigens are located on the vascular system of the tumor.

[0136] Exemplary examples of tumor-associated antigens include LAG-3, CD10, CD19, CD20, CD22, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, Fms-like tyrosine kinase 3 (FLT-3, CD135), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), epidermal growth factor receptor (EGFR), Her2neu, Her3, IGFR, IL3R, fibroblast activation protein (FAP), CDCP1, Derlin1, Tenascin, coil 1-10, vascular antigen VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFRα (CD140a), PDEGFRβ (CD140b), Endoglin, CLEC14, Tem1-8, and Tie2. Further examples may include A33, CAMPATH-1 (CDw52), carcinoembryonic antigen (CEA), carboxylic anhydrase IX (MN / CA IX), de2-7EGFR, EGFRvIII, EpCAM, Ep-CAM, folate-binding protein, G250, Fms-like tyrosine kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (chondroitin sulfate proteoglycan for melanoma-associated cell surface), Muc-1, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate-specific antigen (PSA), and TAG-72.

[0137] In some embodiments, the bispecific antibodies provided herein have primary specificity for TAA antigens and secondary specificity for infectious disease-associated antigens or their epitopes. Non-limiting examples of infectious disease-associated antigens include, for example, antigens expressed on the surface of viral particles or antigens preferentially expressed on virus-infected cells. The viruses are selected from the group consisting of: HIV, hepatitis A, B, or C, herpesviruses (e.g., HSV-1, HSV-2, CMV, HAV-6, VZV, Epstein-Barr virus), adenoviruses, influenza viruses, flaviviruses, echoviruses, rhinoviruses, Coxsackieviruses, coronaviruses, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV, dengue virus, human papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arbovirus encephalitis virus. Alternatively, the target antigen may be an antigen expressed on the surface of the bacteria, or preferably expressed on cells infected by the bacteria, wherein the bacteria are selected from the group consisting of: Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Neisseria meningitidis, Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, Bacillus, Choleraesuis, Clostridium tetani, Clostridium botulinum, Anthrax, Plague, Leptospira, and Lyme disease bacteria. In some embodiments, the target antigen is an antigen expressed on the surface of the fungus, or preferably an antigen expressed on cells infected by the fungus, wherein the fungus is selected from the group consisting of: Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucorales (Mucor, Rhizopus, etc.), Sporothrix chenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.In some embodiments, the target antigen is an antigen expressed on the surface of the parasite or an antigen preferentially expressed on cells infected with the parasite, wherein the parasite is selected from the group consisting of: Entamoeba histolytica, Balantidium coli, Naegleria fowleri, species of Acanthamoeba, Giardia lambia, species of Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus. (Brainia brasiliensis), fat tapeworm (Taenia crassiceps), and Malayan filaria (Brugia malayi). Non-limiting examples of pathogen-associated antigens include, for example, HIV gp120, HIV CD4, hepatitis B glycoprotein L, hepatitis B protein M, hepatitis B glycoprotein S, hepatitis C E1, hepatitis C E2, hepatocyte-specific proteins, herpes simplex virus gB, cytomegalovirus gB, and HTLV envelope protein.

[0138] The WuXiBody BsAb to be coupled in this study can further include a button-lock structure in the Fc region. Additional interchain disulfide bonds are generated in the button-lock region to increase the stability of the bispecific antibody. In this BsAb, there are 5 pairs of interchain disulfide bonds. It was found that under normal TCEP reduction, only 3 pairs of bonds are broken to react with the maleimide-modified drug, resulting in a DAR6-rich (>90%) ADC. The other 2 pairs of non-natural disulfide bonds (in the TCR and Fc regions) are structurally not exposed to the reducing agent and therefore cannot open for drug attachment. In the case where more than one non-natural disulfide bond is formed in the C1 and C2 regions, they are also structurally inaccessible to the reducing agent.

[0139] In some implementations, the bispecific antibody to be conjugated herein comprises two heavy chains and two light chains, wherein from the N-terminus to the C-terminus:

[0140] The first heavy chain includes a domain operatively linked as in VH1-C1-hinge-Fc and the first light chain includes a domain operatively linked as in VL1-C2; the second heavy chain includes a domain operatively linked as in VH2-CH1-hinge-Fc and the second light chain includes a domain operatively linked as in VL2-CL, wherein VH1 and VL1 refer to the variable regions of the first heavy and light chains derived from the first parent antibody, and VH2 and VL2 refer to the variable regions of the second heavy and light chains derived from the second parent antibody. VH1, C1, VL1, and C2 form the first antigen-binding moiety, and VH2, CH1, VL2, and CL form the second antigen-binding moiety.

[0141] Coupling method for preparing D6 ADCs with high homogeneity

[0142] In one aspect, this disclosure provides a method for preparing a highly homogeneous antibody-drug conjugate (ADC) (accounting for 50%, 60%, 70%, 80%, 90% or more of the total generated ADC) of bispecific antibodies as described above, the method comprising the following steps:

[0143] (a) Incubating the reducing agent and bispecific antibody in a buffer system; and

[0144] (b) Introducing an excess of the linker-drug moiety (e.g., MC-MMAF) to react with the reduced thiol group generated in step (a); and

[0145] (c) Recover the obtained antibody-drug conjugate.

[0146] Optionally, the method further includes adding an effective amount of oxidant after step (b) to re-oxidize the unreacted thiol groups.

[0147] As described above, the bispecific antibody to be conjugated is constructed as a button-and-key bispecific antibody with a TCR arm and normal IgGFab in its variable region. Additional interchain disulfide bonds are generated in the button-and-key region to increase the stability of the bispecific antibody. Of the five pairs of interchain disulfide bonds, it was found that under normal TCEP reduction, only three pairs cleave to react with the drug (e.g., maleimide-modified drugs), producing an ADC rich in DAR6 (>80% or even 90%).

[0148] In other words, although the bispecific antibody in WuXiBody form contains 5 pairs of interchain disulfide bonds, conjugation with the antibody produces highly homogeneous DAR6 (D6) strains with a purity of 85-95%. Two pairs of non-natural disulfide bonds, one in the TCR constant region and the other in a "button-lock" structure, are not reduced by reducing agents, leaving only 3 pairs of disulfide bonds for reduction and site-specific conjugation. Two of these pairs are in the hinge region, and the other pair is in the CH1-CL region of the second antigen-binding site.

[0149] In the obtained ADC, the content of D6 ADC can reach at least 80 wt% based on the total weight of D0, D2, D4 and D6, for example, at least 85 wt%, at least 90 wt%, at least 91 wt%, at least 92 wt% or at least 93 wt%, as measured by HIC.

[0150] In some implementations, the reducing agent may be TCEP. The concentration of the reducing agent in the reaction solution may be from 0.04 mM to 0.4 mM.

[0151] In some embodiments, the oxidant to be added may be DHAA. The concentration of the oxidant in the reaction solution may be from 0.08 mM to 0.8 mM.

[0152] The optimal pH for the reaction is typically between about 5.5 and about 8, for example, about 5.5 to 7.5. Optimal reaction conditions, of course, depend on the specific reactants used.

[0153] In one implementation, the buffer solution is PBS with a pH of 7.

[0154] The optimal temperatures for reduction and coupling reactions are typically between approximately -10°C and 37°C, for example, approximately 4°C to 22°C. For instance, reduction reactions are carried out at approximately 22°C for 8–18 hours, and coupling reactions are carried out overnight at approximately 4°C. Temperature and time may vary depending on the amount of antibody, reducing agent, and drug used.

[0155] Those skilled in the art will understand that the incubation period and temperature in step (a) depend on the specific antibody to be conjugated. Determining the incubation period and temperature based on a specific antibody is within the capabilities of those skilled in the art. For example, the antibody to be conjugated is typically incubated overnight with a reducing agent at 4°C.

[0156] In some embodiments, the concentration of the bispecific antibody in the reaction is from 0.01 mM to 0.1 mM. In one particular embodiment, the antibody concentration is 0.02 mM.

[0157] For step (c), those skilled in the art can choose an appropriate purification method to recover the obtained antibody-drug conjugate. Many ADC purification methods are well known in the art. For example, the obtained antibody-drug conjugate can be purified using a desalting column, size exclusion chromatography, etc.

[0158] In some implementations, the obtained antibody-drug conjugate is recovered by any suitable purification method, such as using a desalting column, size exclusion chromatography, ultrafiltration, dialysis, UF-DF, etc.

[0159] Coupling method for preparing ADCs with highly homogeneous D2

[0160] Transition metal ions and / or divalent metal ions can protect disulfide bonds in the hinge region from reduction during incubation with reducing agents and antibodies. The inventors of this paper combined this metal ion chelating function with WuXiBody antibodies for ADC conjugation, resulting in a very narrow DAR2 distribution, which allows for the preparation of low-DAR ADCs. Low-DAR ADCs are needed when drugs (such as PBD) are highly potent and excessively high DAR leads to toxicity issues.

[0161] When metal ions such as Zn(II) are added to the reaction system, two disulfide bonds in the hinge region are shielded from reduction, and only one pair of disulfide bonds on the CH1-CL side of IgG is reduced, resulting in only the DAR2 class upon drug addition. The PK / PD properties of this highly homogeneous D2 population ADC are comparable to those produced by antibody engineering. The preparation of this ADC is also easy. Low-DAR ADCs are crucial when low DAR is required, such as when the drug is too toxic for high loads.

[0162] Therefore, in one aspect, this disclosure provides a method for preparing an antibody-drug conjugate (ADC) of the bispecific antibody described above, the method comprising the following steps:

[0163] (a) In a buffer system, in the presence of effective amounts of metal ions such as transition metal ions and divalent metal ions, a reducing agent and a bispecific antibody are incubated to selectively reduce interchain disulfide bonds within the antibody.

[0164] (b) Introducing an excess of the linker-drug moiety (e.g., MC-MMAF) to react with the reduced thiol group generated in step (a); and

[0165] (c) Recover the obtained antibody-drug conjugate.

[0166] Optionally, the method further includes adding an effective amount of oxidant after step (b) to re-oxidize the unreacted thiol groups.

[0167] In some embodiments, the reducing agent may be TCEP. The concentration of the reducing agent in the reaction solution may be from 0.04 mM to 0.4 mM. The oxidizing agent to be added after step (b) may be DHAA. The concentration of the oxidizing agent in the reaction solution may be from 0.08 mM to 0.8 mM.

[0168] In some embodiments, the metal ions suitable for use in the bioconjugation method of this disclosure are selected from transition metal ions and divalent ions, including but not limited to: Zn2+, Cd2+, Ca2+, Mg2+, and Hg2+. Zn2+ is particularly suitable because it is readily available and inexpensive. For example, suitable transition metal salts can be added in step (a), provided they are soluble in the reaction solution, thereby releasing free transition metal ions in the reaction solution. In this regard, ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, zinc formate, and zinc tetrafluoroborate are suitable zinc salts. Similarly, other transition metal salts that are soluble in the reaction solution and can release free Cd²⁺ or Hg²⁺ ions can be mentioned, including but not limited to CdCl₂, Cd(NO₃)₂, CdSO₄, Cd(CH₃COO)₂, CdI₂, CdBr₂, cadmium formate and cadmium tetrafluoroborate; HgCl₂, Hg(NO₃)₂, HgSO₄, Hg(CH₃COO)₂, HgBr₂, mercuric formate(II) and mercuric tetrafluoroborate(II); etc. Those skilled in the art can select from the above transition metal salts and divalent metal salts as sources of metal ions.

[0169] In one implementation, Zn2+ is used in step (a). A water-soluble zinc salt can be used. For example, ZnCl2 is added in step (a) as a source of Zn2+.

[0170] In step (a), the concentration of metal ions in the reaction solution can be from 0.01 mM to 0.2 mM.

[0171] EDTA will be used as a chelating agent to remove metal ions in the purification step, and the EDTA will be filtered out in subsequent dialysis, ultrafiltration or gel filtration.

[0172] Based on the metal ion, those skilled in the art can select a suitable buffer system for the reaction in step (a), including but not limited to Hepes, histidine buffer, PBS, MES, etc. In one specific embodiment, the buffer system used in step (a) is PBS.

[0173] The optimal pH for the reaction is typically between about 5.5 and about 8, for example, about 5.5 to 7.5. Optimal reaction conditions, of course, depend on the specific reactants used.

[0174] In one implementation, the buffer is PBS, pH 7.

[0175] The optimal temperatures for reduction and coupling reactions are typically between approximately -10°C and 37°C, for example, approximately 4°C to 22°C. For instance, reduction reactions are carried out overnight at approximately 4°C, and coupling reactions are carried out for 2–8 hours at approximately 4°C. Temperature and time may vary depending on the amount of antibody, reducing agent, and drug used.

[0176] For example, the antibody, metal ions, and reducing agent to be coupled can be present in the reaction mixture at a molar ratio of 1:2:4. In one embodiment, 0.02 mM antibody is incubated overnight at 4°C with 0.08 mM TCEP and 0.04 mM ZnCl2. Those skilled in the art will understand that molar concentration can also be converted to “equivalents (eq)” relative to the antibody. For example, if 0.02 mM antibody is used, “0.04 mM ZnCl2” can be converted to “2 eq ZnCl2”.

[0177] In the obtained ADC, the content of D2 ADC can reach at least 80 wt% based on the total weight of D0 and D2, such as at least 85 wt%, at least 90 wt%, at least 91 wt%, at least 92 wt%, at least 93 wt%, at least 94 wt%, or at least 95 wt%, as measured by HIC.

[0178] Conjugation method for preparing highly homogeneous D2+4 dual-drug ADCs

[0179] Combining drugs with different mechanisms of action for treatment is generally beneficial and can improve treatment outcomes. While targeted approaches to ADCs typically yield favorable results, (acquired) resistance frequently occurs, limiting the effectiveness of ADCs. One way to circumvent ADC-related resistance is to combine ADCs with different drugs or to use ADCs with multiple drug loads. In one aspect, this disclosure provides a method for producing ADCs with highly homogeneous (more than 50%, 60%, 70%, 80%, 90%, or higher of the total produced ADCs) two different loads.

[0180] Based on the two methods described above, a two-step conjugation method was developed: conjugation of the first drug component in the presence of an effective amount of metal ions, and conjugation of the second drug component in the absence of such metal ions, resulting in a highly homogeneous DAR(2+4) ADC. This dual-drug ADC exhibits stronger resistance to multidrug resistance in cancer cells from cancer therapies and is readily generated using this conjugation method. In this paper, the designations "DAR(a+b)" or "D(a+b)" refer to dual-drug ADCs, where the number of the first drug conjugated to a single antibody molecule is "a," and the number of the second drug conjugated to the antibody molecule is "b."

[0181] Therefore, in one aspect, this disclosure provides a method for preparing an antibody-drug conjugate (ADC) of the bispecific antibody as described above, wherein the method includes the following steps:

[0182] (a) In a buffer system, in the presence of an effective amount of metal ions, such as transition metal ions and divalent metal ions, a reducing agent and a bispecific antibody are incubated to selectively reduce interchain disulfide bonds within the antibody.

[0183] (b) Introducing an excess of the first linker-drug moiety to react with the reduced thiol group generated in step (a);

[0184] (c) Remove metal ions from the product of step (b);

[0185] (d) Add the reducing agent again and incubate with an excess of the second linker-drug fraction; and

[0186] (e) The obtained antibody-drug conjugate is recovered.

[0187] Optionally, the method further includes adding an effective amount of oxidant after step (b) to re-oxidize the unreacted thiol groups.

[0188] The reducing agent, metal ions, buffer system, and oxidizing agent can be the same as those disclosed above for the preparation of D2 ADCs. For step (c), the metal ions can be removed in the purification step by using EDTA as a chelating agent, which will be filtered out in subsequent dialysis, ultrafiltration, or gel filtration.

[0189] The reaction conditions for reduction and coupling can be the same as those disclosed above for the preparation of D2 ADC. The optimal temperatures for the reduction and coupling reactions are typically between approximately -10°C and 37°C, for example, approximately 4°C to 22°C. The second reduction and coupling in steps (c) and (d) can differ slightly from steps (a) and (b). For example, the reduction reaction in step (c) is carried out at approximately 22°C for 2–8 h, while the coupling reaction is carried out at approximately 22°C for 2–8 h. The temperature and time can vary depending on the amount of antibody, reducing agent, and drug used.

[0190] In some implementations, the first linker-drug portion is MC-MMAF, and the second linker-drug portion is MC-DXD or DXD with other linkers.

[0191] In the obtained ADC, the content of D2+4 ADC can reach at least 80 wt% based on the total weight of D0, D2, D2+2 and D4, for example, at least 85 wt%, at least 90 wt%, at least 91 wt%, at least 92 wt%, at least 93 wt%, at least 94 wt% or at least 95 wt%, as calculated by MS analysis.

[0192] Highly homogeneous ADCs of bispecific antibodies show better efficacy.

[0193] A major problem with conventional conjugation of ADCs is the heterogeneity of ADC molecules, where the drug portion is chemically attached to several sites on the antibody via, for example, cysteine ​​residues, ranging from 0 to 8 per antibody (drug-to-antibody ratio, DAR). Such a mixture of ADC molecules not only presents challenges for analysis and characterization but may also exhibit different pharmacokinetic, distribution, toxicity, and efficacy characteristics. Non-specific conjugation also frequently leads to impaired antibody function. Therefore, a narrow distribution of DAR is desired to achieve better pharmacokinetic, efficacy, and therapeutic window.

[0194] In one aspect, this disclosure provides highly homogeneous antibody-drug conjugates of bispecific antibodies, wherein D6ADC accounts for more than 80 wt% of the ADC.

[0195] In one aspect, this disclosure provides highly homogeneous antibody-drug conjugates of bispecific antibodies, wherein D2ADC accounts for more than 80 wt% of the ADC.

[0196] In one aspect, this disclosure provides highly homogeneous antibody-drug conjugates of bispecific antibodies, wherein the D2+4 ADC accounts for more than 80 wt% of the ADC.

[0197] In some implementations, the homogeneity of the antibody-drug conjugate produced by the methods disclosed herein is measured and compared with the homogeneity of a corresponding control antibody-drug conjugate produced by conventional conjugation methods.

[0198] Various analytical methods can be used to determine the yield and isomer mixture of antibody-drug conjugates. For example, in one embodiment, hydrophobic interaction chromatography (HIC) is an analytical method for determining the yield and isomer mixture from the resulting antibody-drug conjugate (e.g., for the D6 conjugate). This technique is capable of separating antibodies loaded with different numbers of drug. The drug loading level can be determined based on the ratio of absorbance, for example, at 250 nm and 280 nm. For example, the drug may absorb at 250 nm while the antibody absorbs at 280 nm. Therefore, the 250 / 280 ratio increases with increasing drug loading. Using the bioconjugation methods described herein, antibodies with an even number of drug units are typically observed because incomplete conjugation results in an even number of DARs.

[0199] Linker-drug component

[0200] Using antibody-drug conjugates for local delivery of cytotoxic agents or cell inhibitors—drugs that kill or inhibit tumor cells in cancer treatment—allows for partial targeted delivery of the drug to the tumor, where it accumulates intracellularly. Systemic administration of unconjugated drugs can result in unacceptable levels of toxicity to both normal cells and the tumor cells being eliminated (Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506). Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87).

[0201] Drugs that can be used in ADCs include chemotherapeutic agents such as daunorubicin, doxorubicin, methotrexate, and vindesine; toxins such as bacterial toxins like diphtheria toxin, plant toxins like ricin, small molecule toxins such as geldanamycin, maytansinoids, and calicheamicin; and auristatin peptides, auristatin E (AE) and monomethyl auristatin (MMAE), which are synthetic analogs of dolastatin. MMAE is a synthetic derivative of dolastatin 10, a natural cell-inhibiting pseudopeptide. Toxins can exert their cytotoxic and cell-inhibitory effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to become inactive or less active when conjugated to large antibodies or protein receptor ligands.

[0202] There are no specific limitations on the drugs and linkers that can be used in the conjugation methods of this disclosure, as long as the drug molecule has antitumor, antiviral, or antimicrobial activity and contains at least one substituent or partial structure that allows it to be linked to the linker structure, and the linker contains at least two reactive groups, one of which can covalently bind to the drug molecule and the other can be covalently coupled to the antibody. Preferably, the linker is susceptible to -SH attack by the antibody and is capable of forming a linker with the antibody.

[0203] Depending on the desired drug and the selected linker, those skilled in the art can choose a suitable method to couple them together. For example, some conventional coupling methods, such as amine coupling methods, can be used to form the desired drug-linker complex, which still contains reactive groups for covalent coupling with the antibody. In this disclosure, the drug-maleimide complex (i.e., the maleimide-linked drug) is an example of carrying a loaded reactive group.

[0204] In one embodiment, the drug may include, but is not limited to, cytotoxic agents, such as chemotherapeutic agents, immunotherapeutic agents, antiviral agents, or antimicrobial agents. In one embodiment, the drug to be conjugated to the antibody may be selected from, but is not limited to, MMAE (monomethylaurestatin E), MMAD (monomethylaurestatin D), MMAF (monomethylaurestatin F), etc.

[0205] In ADC preparation, the most common reactive group that can bind to a thiol group is maleimide. In addition, organic bromides and iodides are also frequently used.

[0206] Available enzymatically active toxins and their fragments include diphtheria A chain, non-bound active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, absinthecin A chain, modeccin A chain, α-Dacococcus, Aleutites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, jatropha curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecene. See, for example, WO 93 / 21232 published on October 28, 1993. Various radionuclides can be used to produce radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. One or more small molecule toxins, such as calicheamicin, maytansine compounds, dolastatin, etc., can be conjugated to antibodies using the methods disclosed herein. Trichothecene, CC1065, and their toxic derivatives.

[0207] Maytansine compounds suitable for use as pharmaceutical fractions in maytansine derivatives are well known in the art and can be isolated from natural sources by known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or synthesized by known methods, including maytanol and maytanol analogues. Suitable maytansine compounds are disclosed, for example, in U.S. Patent 5,208,020. Preferred maytansine compounds are maytanol and maytanol analogues modified at the aromatic ring or other positions of the maytanol molecule, such as various maytanol esters.

[0208] Dolasstatin and Dolatasstatin has been shown to interfere with microtubule dynamics, GTP hydrolysis, cell nucleus and cell division, and possesses anticancer and antifungal activities (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). Dolatasstatin or... The drug is attached to the antibody (WO 02 / 088172). Exemplary embodiments including MMAE or MMAF and various linker components are shown below. For example, VcMMAE (Mc-vc-PAB-MMAE) is obtained by using an MMAE linked via a p-aminobenzyloxycarbonyl group (“PAB”) to a lysosomal cleavable dipeptide valine-citrulline (vc) and a thiol-reactive maleimide-hexanoyl spacer (Mc).

[0209] Antibody-cytotoxic agent conjugates are prepared using a variety of bifunctional protein conjugates, such as N-succinimide-3-(2-pyridyldithiool)propionate (SPDP), iminothiones (IT), bifunctional derivatives of imide esters (e.g., dimethyl adipic acid HCl), active esters (e.g., disuccinimide-amino octanoate), aldehydes (e.g., glutaraldehyde), diazid compounds (e.g., bis(p-azidobenzoyl)hexamethylenediamine), diazido derivatives (e.g., bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and difluorinated compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described by Vitetta et al. (1987) Science, 238:1098. Carbon-14 labeled 1-isothiocyanobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies (WO94 / 11026).

[0210] Further exemplary embodiments including MMAE or MMAF and various linker components are provided. For example, VcMMAE (Mc-vc-PAB-MMAE) is obtained by using an MMAE connected via a p-aminobenzyloxycarbonyl group (“PAB”) to a lysosomal cleavable dipeptide valine-citrulline (vc) and a thiol-reactive maleimide hexanoyl spacer (Mc).

[0211] Pharmaceutical Composition

[0212] In one aspect, this disclosure relates to a pharmaceutical composition comprising an effective amount of an ADC with improved homogeneity prepared by the methods disclosed herein and a pharmaceutically acceptable carrier or mediator. The composition is suitable for veterinary or human administration.

[0213] The compositions disclosed herein can be in any form that allows for administration to animals. For example, the compositions can be in solid, liquid, or gaseous (aerosol) form. Typical routes of administration include, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, and intranasal administration. Parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection, intrasternal injection, or infusion techniques. For example, the composition may be administered parenterally. The pharmaceutical compositions of the present invention can be formulated such that the ADC of the present disclosure is bioavailable when administered to animals. The compositions can be in the form of one or more dose units, wherein, for example, tablets may be single dose units, and containers containing the ADC of the present disclosure in aerosol form may contain multiple dose units.

[0214] The materials used to prepare the pharmaceutical composition may be non-toxic in the amount used. It will be apparent to those skilled in the art that the optimal dosage of the active ingredient in a pharmaceutical composition will depend on a variety of factors. These factors include, but are not limited to, the type of animal (e.g., human), the specific form of the ADC, the method of administration, and the composition used.

[0215] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, mercaptoacetic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants such as methionine in the pharmaceutical compositions provided herein reduces the oxidation of peptide complexes or bispecific peptide complexes. This reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving protein stability and maximizing shelf life. Therefore, in some embodiments, compositions comprising peptide complexes or bispecific peptide complexes disclosed herein and one or more antioxidants such as methionine are provided.

[0216] To further illustrate, acceptable carriers for drugs may include, for example, aqueous media such as sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, or glucose and lactated Ringer's injection; non-aqueous media such as plant-derived fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at antibacterial or antifungal concentrations; isotonic agents such as sodium chloride or glucose; 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 carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); isolating or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multi-dose containers, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzoyl chloramine. Suitable excipients may include, for example, water, saline, glucose, glycerol, or ethanol. Suitable non-toxic adjuvants may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0217] The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained-release formulation, or powder. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0218] In some embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solutions, suspensions, emulsions, or solid forms suitable for producing liquid solutions, suspensions, or emulsions. Injectable preparations may include sterile and / or pyrogen-free solutions prepared for injection, sterile dry-solubilized products prepared for use with a solvent, such as lyophilized powders, including subcutaneous tablets, sterile suspensions prepared for injection, sterile dry-insoluble products prepared for use with a carrier, and sterile and / or pyrogen-free emulsions. Solutions may be aqueous or non-aqueous.

[0219] In some embodiments, unit doses of the parenteral preparation are packaged in ampoules, vials, or syringes with needles. All preparations intended for parenteral administration should be sterile and pyrogen-free, as is known and practiced in the art.

[0220] In some embodiments, a sterile lyophilized powder is prepared by dissolving the ADC disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability of the powder or a reconstituted solution prepared from the powder, or other pharmacological components. Excipients that may be used include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable reagents. The solvent may contain buffers, such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art. In one embodiment, the solution is approximately neutral pH. The solution is then sterilely filtered and then lyophilized under standard conditions known to those skilled in the art to provide the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single dose or multiple doses of the ADC or combinations thereof provided herein. For the purpose of accurate sample extraction and accurate dosing, overfilling of vials in amounts slightly greater than required for one dose or a group of doses (e.g., about 10%) is acceptable. The lyophilized powder may be stored under suitable conditions, such as at temperatures from about 4°C to room temperature.

[0221] Reconstitution of lyophilized powder with water for injection provides a formulation for parenteral administration. In one embodiment, for reconstitution, sterile and / or pyrogen-free water or other suitable liquid carrier is added to the lyophilized powder. The precise amount depends on the chosen therapy and can be determined empirically.

[0222] In addition, antibody-drug conjugates or drug compositions can be formulated into kits that include inserts indicating application information such as indications, dosage, and route of administration.

[0223] Applications of ADCs with improved homogeneity

[0224] In one aspect, this disclosure relates to the use of antibody-drug conjugates with improved homogeneity prepared by the foregoing method in the preparation of pharmaceutical compositions or kits for treating a subject’s condition or ailment.

[0225] Subjects can be mammals, such as humans.

[0226] The condition or ailment to be treated can be a tumor, cancer, autoimmune disease, or infectious disease. In a specific implementation, an infectious disease can be a viral or microbial infection.

[0227] In one aspect, this disclosure also relates to a method for treating a subject suffering from a condition or symptom, comprising: administering to the subject in need a therapeutically effective amount of an ADC having improved homogeneity prepared by the method disclosed herein, or a therapeutically effective amount of a pharmaceutical composition comprising an ADC having improved homogeneity prepared by the method disclosed herein, thereby treating or preventing said condition or symptom.

[0228] In some implementations, the subject has been identified as having a condition or ailment that may respond to the ADC provided herein.

[0229] Subjects can be mammals, such as humans.

[0230] The condition or ailment to be treated can be a tumor, cancer, autoimmune disease, or infectious disease. In a specific implementation, an infectious disease can be a viral or microbial infection.

[0231] The therapeutically effective dose of the ADCs described herein will depend on various factors known in the art, such as weight, age, medical history, current medications, the subject's health status, and the likelihood of cross-reactions, allergies, sensitivities, and adverse side effects, as well as the route of administration and the extent of disease development. The dosage may be proportionally reduced or increased by a person skilled in the art (e.g., a physician or veterinarian), as indicated by these and other circumstances or requirements.

[0232] In some embodiments, the ADC or pharmaceutical composition provided herein may be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In some of these embodiments, the ADC or pharmaceutical composition provided herein is administered at a dose of about 50 mg / kg or less, and in some of these embodiments, the dose is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In some embodiments, the administered dose may be varied during treatment. For example, in some embodiments, the initial administered dose may be higher than subsequent administered doses. In some embodiments, the administered dose may be varied during treatment based on the subject's response.

[0233] Dosing regimens can be adjusted to provide the best expected response (e.g., therapeutic response). For example, a single dose can be administered, or several separate doses can be administered over time.

[0234] The ADCs or pharmaceutical compositions provided herein can be administered via any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-parenteral routes (e.g., oral, intranasal, intraocular, sublingual, rectal or topical administration).

[0235] In some implementations, the condition or ailment treated by the ADC or pharmaceutical composition provided herein is cancer or cancerous condition, autoimmune disease or infectious disease.

[0236] Cancers can be antigen-positive cancers, including lung cancer, breast cancer, colon cancer, ovarian cancer, and pancreatic cancer, such as cancers associated with tumor-associated antigens.

[0237] Other specific types of cancer that can be treated with the ADCs or pharmaceutical compositions provided in this article include, but are not limited to, solid tumors, including but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphoangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, laryngeal cancer, squamous cell carcinoma, basal cell carcinoma. Cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary gland cancer, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, retinoblastoma Tumors; blood-borne cancers, including but not limited to: acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia (AML), acute promyelocytic leukemia (APL), acute monocytic leukemia, acute erythrocytic leukemia, acute megakaryocytic leukemia, acute myelomonocytic lymphoma, acute non-lymphocytic leukemia, acute undifferentiated leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy Cellular leukemia, multiple myeloma; lymphoma: B-cell lymphoma, optionally Hodgkin lymphoma or non-Hodgkin lymphoma, of which non-Hodgkin lymphoma includes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL) or mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL) or Waldenstrom macroglobulinemia (WM).

[0238] Autoimmune diseases may include, but are not limited to, active chronic hepatitis, Addison's disease, allergic alveolitis, allergic reactions, allergic rhinitis, Alport syndrome, anaphylaxis, ankylosing spondylitis, antiphospholipid syndrome, arthritis, ascariasis, aspergillosis, atopic dermatitis, atrophic rhinitis, Behcet's disease, bird pulmonary disease, bronchial asthma, Kaplan syndrome, cardiomyopathy, Celiac disease, Chagas disease, chronic glomerulonephritis, Korghan's syndrome, cold agglutinin disease, congenital rubella infection, CREST syndrome, Crohn's disease, cryoglobulinemia, Cushing's syndrome, dermatomyositis, discoid lupus, Dressler's syndrome, Eaton-Lambert syndrome, Echovirus infection, encephalomyelitis, endocrine ophthalmopathy, Epstein-Barr virus infection, and equine weight (Equine virus infection). Heaves disease, erythema, Evan syndrome, Felty syndrome, fibromyalgia, Fuch's cyclitis, gastric atrophy, gastrointestinal allergy, giant cell arteritis, glomerulonephritis, Goodpaste syndrome, graft-versus-host disease, Graves' disease, Guillain-Barre disease, Hashimoto's thyroiditis, hemolytic anemia, allergic purpura, idiopathic adrenal atrophy, idiopathic pulmonary fibrillitis, IgA nephropathy, inflammatory bowel disease, insulin-dependent diabetes mellitus, juvenile arthritis, juvenile diabetes mellitus (type I), Lambert-Eaton syndrome, laminitis, lichen planus, lupus hepatitis, lupus, lymphopenia, Meniere's disease, mixed connective tissue disease, multiple sclerosis, severe illness Myasthenia gravis, pernicious anemia, polyadenomas, early-onset dementia, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, recurrent miscarriage, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, Sampter's syndrome, schistosomiasis, Schmidt's syndrome, scleroderma, Shulman's syndrome, Sjorgen's syndrome, Stiff-Man's syndrome, sympathetic ophthalmia, systemic lupus erythematosus, hyperarteritis, temporal arteritis, thyroiditis, thrombocytopenia, thyroid toxicity, toxic epidermal necrolysis, insulin resistance type B, type I diabetes, ulcerative colitis, uveitis, vitiligo, Waldenstrom's macroglobulinemia, Wegener's granulomatosis.

[0239] In one embodiment, this disclosure includes a method of treating a subject's disease or condition, comprising administering to the subject an effective amount of the ADC or pharmaceutical composition provided herein and another therapeutic agent.

[0240] In some embodiments, the therapeutic agent is an anticancer agent. Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cyclophosphamide, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, edararubicin, daunorubicin, spectinomycin, procainoxine, mitoxantrone, asparaginase, vincristine, vinorelbine, paclitaxel, and docetaxel.

[0241] In some embodiments, the 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, prednisolone, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorobenzoyl hydrazine, DHEA, danazol, bromocriptine, meloxicam, and infliximab.

[0242] In some embodiments, the therapeutic agent is an anti-infective agent. In some embodiments, the anti-infective agent is, but is not limited to, antibacterial agents: [β]-lactam antibiotics: penicillin G, penicillin V, cloxacillin, dicloxacillin, methicillin, nefocillin, oxacillin, ampicillin, amoxicillin, bamocillin, azlocillin, carbenicillin, mezlocillin, piperacillin, ticarcillin; aminoglycosides: amikacin, gentamicin, kanamycin, neomycin, natamycin. Tobramycin, streptomycin, tobramycin; macrolides: azithromycin, clarithromycin, erythromycin, lincomycin, clindamycin; tetracyclines: norchlorotetracycline, doxycycline, minocycline, oxytetracycline, tetracycline; quinolones: styromycin, nalidixic acid; fluoroquinolones: ciprofloxacin, enoxacin, gapfloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin, terofol; polypeptides Bacitracin, colistin, polymyxin B; Sulfonamides: sulfamethoxazole, sulfamethoxazole, sulfadiazine, sulfamethoxazole, sulfaacetamide; Mixed antibacterial agents: trimethoprim, sulfamethoxazole, chloramphenicol, vancomycin, metronidazole, quinapril, darafosine, rifampin, spectinomycin, nitrofurantoin; Antiviral agents: General antiviral agents: idoxuridine, vidarabin, trifluralin, Acyclovir, Panxicyclovir, Panxicyclovir, Valacyclovir, Ganxicyclovir, Foscarboxylic Acid, Ribavirin, Amantadine, Remantadine, Siddovir, Antisense Oligonucleotides, Immunoglobulins, Interferon; Drugs used for HIV infection: Zidovudine, Didanoxin, Zacitabine, Stavudine, Lamivudine, Nevirapine, Delaviridine, Saquinavir, Ritonavir, Indinavir, Nelfenavir.

[0243] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below fall in whole or in part within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention but are merely illustrative of specific embodiments falling within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials, and methods without exercising inventive capabilities and without departing from the scope of the invention. It should be understood that many variations can be made in the processes described herein while still remaining within the scope of the invention. The inventors intend that such variations be included within the scope of the invention. Example

[0244] This disclosure will now be described in detail with reference to the following examples. However, those skilled in the art will understand that the following examples are provided for illustrative purposes only and are not intended to limit this disclosure in any way.

[0245] Example 1. Generation of WuXiBody bispecific antibodies

[0246] 1.1 Construction of WuXiBody E17 bispecific antibody

[0247] Table 1

[0248] Antibody Parental antibodies cAb1 Trastuzumab x Pertuzumab cAb2 Pertuzumab x Trastuzumab cAb3 Trastuzumab x Saxizumab cAb4 Saxizumab x Trastuzumab cAb5 Saxizumab x Cetuximab cAb6 Cetuximab x Saxizumab cAb7 Trastuzumab x Pertuzumab cAb8 Trastuzumab x Pertuzumab

[0249] Eight bispecific antibodies were constructed (Table 1). The VL and VH genes (obtained from the parental antibodies in Table 1) were amplified from existing plasmid templates by PCR. The TCR Cα and Cβ genes (encoding SEQ ID Nos: 2 and 4, excluding cAb7 and cAb8, which used different C1 and C2 region sequences shown in SEQ ID Nos: 7-9) were synthesized by Genewiz Inc (Suzhou, China). DNA fragments of VL1-Cα and VL2-CL were inserted into linearized vectors containing a CMV promoter and a human light chain signal peptide, respectively. The DNA fragment of VH1-Cβ was inserted into a linearized vector containing the human IgG1 constant region CH2-CH3 with a "lockhole" mutation. The DNA fragment of VH2-CH1 was inserted into a linearized vector containing the human IgG1 constant region CH2-CH3 with a "keyhole" mutation. The vectors contained a CMV promoter and a human antibody heavy chain signal peptide. An exemplary structure of cAb1 is shown below. Figure 1A The reaction scheme is shown below.

[0250] 1.2 Expression and purification of WuXiBody bispecific antibody

[0251] Following the manufacturer's instructions, heavy and light chain expression plasmids were co-transfected into Expi293 cells (Invitrogen-A14527) using an expression system kit (Invitrogen-A14524). Five days post-transfection, the supernatant was collected and purified using protein A chromatography (GE Healthcare-17543802). Further purification was performed using size exclusion chromatography (GE Healthcare-17104301) if necessary. Antibody concentration was measured using Nano Drop. Protein purity was evaluated by SDS-PAGE and HPLC-SEC.

[0252] Example 2. Coupling to form D6 species

[0253] conduct Figure 1A The reaction protocol shown is illustrated (using cAb1 as an example). 10 mg / mL mAb in PBS buffer was reduced with 7.5 eq of TCEP, and the reaction vial was incubated at 22°C for 8 hours. The reduced antibody solution was then directly used for the next conjugation step without removing the TCEP.

[0254] DMA and linker-loading solution (10 mg / mL stock solution of DMA, relative to 18.0 eq. of antibody) were added to the reduced antibody solution. The reactants were mixed appropriately, and the reaction vials were incubated at 4°C for 18 hours. The crude product was buffer-exchanged to its storage buffer (1X PBS buffer, pH 7.4, Gibco) using a rotary desalting column (40 kD). The resulting ADC was characterized.

[0255] Homogeneity assay. Drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. D0, D2, D4, and D6 were purified by hydrophobic interaction chromatography (HIC) on a Toyopearl phenyl 650M HIC column (Tosoh Biosciences, Montgomeryville, PA) at a flow rate of 10 mL / min at ambient temperature. The loading volume was 7.5 mg ADC per 1 mL column volume. Solvent A consisted of 2.0 M NaCl and 50 mM sodium phosphate at pH 7. Solvent B consisted of 80% v / v 50 mM sodium phosphate at pH 7 and 20% v / v acetonitrile. The column was pre-equilibrated with 5 column volumes of solvent A. ADC was mixed with 0.67 volumes of 5 M NaCl (final 2.0 M) and applied to the column. D0 was not retained by the column. Different drug loads were eluted in a sequential gradient: D2 was eluted with 35% solvent B, D4 with 70% solvent B, and D6 with 95% solvent B.

[0256] For MS analysis, 75.0 μl of 8.0 mol / L Gdn-HCl, 5.0 μl of 1.0 mol / L Tris-HCl, and 2.0 μl of 1 mol / L DTT were added to 20.0 μl of ADC sample solution. The new solution was thoroughly mixed and then incubated at 10–30°C for 10–30 min. The drug / antibody ratio (DAR) and product distribution were then analyzed using LC-MS.

[0257] As shown in Table 2 and Figure 1B-1D As shown, cAb1, cAb3, and cAb4 antibodies yielded ADCs with approximately 90% or higher homogeneity of D6 class. Similar results were obtained for the other five antibodies.

[0258] Table 2: HPLC results of ADC homogeneity

[0259] Lot. mAb Connector-Load D6 % cAb1-MMAF cAb 1 MC-MMAF 91 cAb3-MMAF cAb 3 MC-MMAF 90 cAb4-MMAF cAb 4 MC-MMAF 94

[0260] Example 3. Coupling to form D2 species

[0261] Add 3.0 eq TCEP and 1.0 eq ZnCl2 to the mAb. Incubate the reaction vial at 4°C for 18 hours. Add DMA and linker-loading solution (10 mg / mL stock solution of DMA) to the reduced antibody solution. Mix the reactants appropriately and incubate the reaction vial at 4°C for 2 hours.

[0262] Add cysteine ​​to the mixture. Then incubate the new mixture at 4°C for 15 minutes. After adding 3.0 eq EDTA and 8.0 eq DHAA, incubate the mixture at 22°C for 2 hours.

[0263] The crude product was buffer-exchanged into its storage buffer (1X PBS buffer, pH 7.4, Gibco) using a rotary desalting column (40 kD). The resulting ADC ( Figure 2A ) is characterized.

[0264] As shown in Table 3 and Figure 2B As shown, cAb1, cAb2, cAb3, and cAb4 antibodies yielded ADCs with over 80% homogeneity of D2 class. Similar results were obtained for the other four antibodies.

[0265] Table 3

[0266] Lot. mAb Final mAb concentration (mg / ml) Connector-payload / mAb ratio Cysteine / mAb ratio Connector - Payload D2 % cAb1-MMAF-D2 cAb 1 9.7 4 8 MC-MMAF 91 cAb2-MMAF-D2 cAb 2 7.5 10 14 MC-MMAF 96 cAb3-MMAF-D2 cAb 3 7.9 5 9 MC-MMAF 90 cAb4-MMAF-D2 cAb 4 5.1 10 14 MC-MMAF 81

[0267] Example 4. Coupling to form D(2+4) species

[0268] use Figure 3A Following the reaction protocol shown in Table 4 (using cAb1 as an example), reduce the D2 ADC (mAb coupled to linker-loador 1) in TCEP-reduced buffer (1X PBS buffer, pH 7.4, Gibco) and incubate the reaction vial at 22°C for 2 hours. Proceed directly to the next coupling step with the reduced antibody solution without removing the TCEP.

[0269] DMA and linker-loador 2 solution (10 mg / mL stock solution of DMA) were added to the reduced antibody solution. The reactants were mixed appropriately, and the reaction vials were incubated at 22°C for 2 hours.

[0270] The crude product was buffer-exchanged into its storage buffer (1X PBS buffer, pH 7.4, Gibco) using a rotary desalting column (40 kD). The resulting ADC was characterized. Figure 3B-3E ).

[0271] from Figure 3B The MS results show that the content of D2+4 ADC reaches more than 90% (taking cAb1 as an example).

[0272] Table 4

[0273] Lot. mAb Reduced mAb concentration (mg / ml) TCEP / mAb ratio Connector-payload / mAb ratio Connector - Payload 1 Connector - Payload 2 cAb1-MMAF+DXD cAb 1 6.6 7.5 15 MC-MMAF Deruxtecan cAb2-MMAF+DXD cAb 2 4.3 11.5 18 MC-MMAF Deruxtecan cAb3-MMAF+DXD cAb 3 5.4 9.1 15 MC-MMAF Deruxtecan cAb4-MMAF+DXD cAb 4 4.2 10.0 16 MC-MMAF Deruxtecan

[0274] Those skilled in the art will further understand that the invention can be practiced in other specific forms without departing from the spirit or central attributes of the invention. Since the foregoing description of the invention discloses only exemplary embodiments thereunder, it should be understood that other variations are also within the scope of the invention. Therefore, the invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims as an indication of the scope and content of the invention.

Claims

1. A method for preparing a highly homogeneous antibody-drug conjugate (ADC) with a drug-to-antibody ratio of 6 (D6), wherein the antibody comprises a first antigen-binding moiety, a second antigen-binding moiety, a hinge region, and an Fc region, wherein both the first and second antigen-binding moieties are in Fab form. The first antigen-binding portion includes a first heavy chain variable domain (VH) operably linked to a first T cell receptor (TCR) constant region (C1) and a first light chain variable domain (VL) operably linked to a second TCR constant region (C2); the second antigen-binding portion includes a second VH operably linked to an antibody heavy chain CH1 domain and a second VL operably linked to an antibody light chain constant (CL) domain. A non-natural interchain disulfide bond is formed between C1 and C2, a natural interchain disulfide bond exists between CH1 and CL, and two natural interchain disulfide bonds exist between the hinge regions. C1 and C2 are selected from the following group: (1) The C1 region consisting of the amino acid sequence of SEQ ID No: 2, and the C2 region consisting of the amino acid sequence of SEQ ID No: 4 or 8, or (2) The C1 region consisting of the amino acid sequence of SEQ ID No: 7, and the C2 region consisting of the amino acid sequence of SEQ ID No: 9; And the method described therein includes the following steps: (a) Incubate the reducing agent and the antibody in a buffer system; (b) Introducing an excess of the linker-drug moiety to react with the reduced thiol group generated in step (a); and (c) Recover the obtained antibody-drug conjugate.

2. A method for preparing a highly homogeneous antibody-drug conjugate (ADC) with a drug-to-antibody ratio of 2 (D2), wherein the antibody comprises a first antigen-binding moiety, a second antigen-binding moiety, a hinge region, and an Fc region, wherein both the first and second antigen-binding moieties are in Fab form. The first antigen-binding portion includes a first heavy chain variable domain (VH) operably linked to a first T cell receptor (TCR) constant region (C1) and a first light chain variable domain (VL) operably linked to a second TCR constant region (C2); the second antigen-binding portion includes a second VH operably linked to an antibody heavy chain CH1 domain and a second VL operably linked to an antibody light chain constant (CL) domain. A non-natural interchain disulfide bond is formed between C1 and C2, a natural interchain disulfide bond exists between CH1 and CL, and two natural interchain disulfide bonds exist between the hinge regions. C1 and C2 are selected from the following group: (1) The C1 region consisting of the amino acid sequence of SEQ ID No: 2, and the C2 region consisting of the amino acid sequence of SEQ ID No: 4 or 8, or (2) The C1 region consisting of the amino acid sequence of SEQ ID No: 7, and the C2 region consisting of the amino acid sequence of SEQ ID No: 9; And the method described therein includes the following steps: (a) Incubate the reducing agent and the antibody in a buffer system in the presence of an effective amount of one or more transition metal ions and / or divalent metal ions; (b) Introducing an excess of the linker-drug moiety to react with the reduced thiol group generated in step (a); and (c) Recover the obtained antibody-drug conjugate.

3. A method for preparing highly homogeneous antibody-drug conjugates (ADCs), wherein the ratio of a first drug to an antibody in the ADC is 2 and the ratio of a second drug to an antibody is 4 (D2+4), wherein the antibody comprises a first antigen-binding moiety, a second antigen-binding moiety, a hinge region, and an Fc region, and both the first and second antigen-binding moieties are in Fab form. The first antigen-binding portion includes a first heavy chain variable domain (VH) operably linked to a first T cell receptor (TCR) constant region (C1) and a first light chain variable domain (VL) operably linked to a second TCR constant region (C2); the second antigen-binding portion includes a second VH operably linked to an antibody heavy chain CH1 domain and a second VL operably linked to an antibody light chain constant (CL) domain. C1 and C2 are selected from the following group: (1) The C1 region consisting of the amino acid sequence of SEQ ID No: 2, and the C2 region consisting of the amino acid sequence of SEQ ID No: 4 or 8, or (2) The C1 region consisting of the amino acid sequence of SEQ ID No: 7, and the C2 region consisting of the amino acid sequence of SEQ ID No: 9; A non-natural interchain disulfide bond forms between C1 and C2, a natural interchain disulfide bond exists between CH1 and CL, and two natural interchain disulfide bonds exist between the hinge regions. And the method described therein includes the following steps: (a) Incubating the reducing agent and the antibody in a buffer system in the presence of an effective amount of one or more transition metal ions and / or divalent metal ions to selectively reduce interchain disulfide bonds within the antibody; (b) Introducing an excess of the first linker-first drug moiety to react with the reduced thiol group generated in step (a); (c) Remove the metal ions from the product of step (b); (d) Incubate again with the reducing agent and introduce an excess of the second linker-second drug portion, wherein the reducing agent is the same as or different from the reducing agent in step (a); and (e) The obtained antibody-drug conjugate is recovered.

4. The method according to any one of the preceding claims, wherein the method further comprises adding an effective amount of oxidant to reoxidize unreacted thiol groups before recovering the obtained antibody-drug conjugate.

5. The method according to claim 4, wherein the oxidant is DHAA.

6. The method according to any one of claims 1-3, wherein the antibody is an IgG1 or IgG4 isotype.

7. The method of claim 6, wherein the Fc region comprises a button-in-hole structure.

8. The method according to any one of claims 2-3, wherein the transition metal ion and / or divalent metal ion in step (a) is selected from the group consisting of Zn 2+ Cd 2+ Hg 2+ Ca 2+ Mg 2+ and any combination thereof.

9. The method according to claim 8, wherein the metal ion is selected from Zn. 2+ Ca 2+ and Mg 2+ .

10. The method according to any one of claims 1-3, wherein the buffering system used in step (a) is selected from the group consisting of Hepes, histidine buffers, PBS and MES, and has a pH of 5.5 to 8.

11. The method according to any one of claims 1-3, wherein the antibody in step (a) is added at a final concentration of 0.01-0.1 mM.

12. The method according to any one of claims 1-3, wherein step (a) is performed at a temperature of -10°C to 37°C.

13. The method according to claim 12, wherein step (a) is performed at a temperature of 0°C to 22°C.

14. The method according to any one of claims 1-3, wherein the reducing agent in step (a) is TCEP.

15. The method according to any one of claims 1-3, wherein the linker-drug portion is a maleimide carrying a drug, an organic bromide carrying a drug, or an organic iodide carrying a drug.

16. The method according to any one of claims 1-3, wherein the drug to be coupled is selected from the group consisting of diagnostic agents, therapeutic agents, and labeling agents.

17. The method according to any one of claims 1-3, wherein the drug is selected from maytansine compounds, dolalastatins, aurestatins, calciferine, trichothecene, and CC1065.

18. The method of claim 17, wherein the maytansine compound is selected from DM1, DM3 and DM4.

19. The method of claim 17, wherein the drug is selected from MMAE, DM1, and MMAF.

20. The method according to any one of claims 1-3, wherein the method comprises generating the antibody prior to step (a), wherein a first antigen-binding portion and a second antigen-binding portion of the antibody specifically bind to a selected antigen.

21. The method according to any one of claims 1-3, wherein the variable region of the first antigen-binding portion originates from the first parent antibody and the variable region of the second antigen-binding portion originates from the second parent antibody.

22. The method of claim 21, wherein the first parent antibody and the second parent antibody are identical.

23. The method of claim 22, wherein the parent antibody is a monospecific or multispecific antibody.

24. The method of claim 22, wherein the parent antibody is a bispecific antibody.

25. The method according to any one of claims 1-3, wherein the variable regions of the first and second antigen-binding portions are derived from any of the following parental antibodies: trastuzumab, pertuzumab, sacituzumab, abciximab, adalimumab, alefacept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab (polyethylene glycol). The following are listed: pegol, cetuximab, daclizumab, denosumab, efalizumab, golimumab, inflectra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, secukinumab, and ustekinumab.

26. The method of claim 25, wherein the first VH and the first VL are derived from trastuzumab and the second VH and the second VL are derived from pertuzumab, or the first VH and the first VL are derived from pertuzumab and the second VH and the second VL are derived from trastuzumab.

27. The method of claim 25, wherein the first VH and the first VL are derived from trastuzumab and the second VH and the second VL are derived from sacizumab, or the first VH and the first VL are derived from sacizumab and the second VH and the second VL are derived from trastuzumab.

28. The method of claim 2, wherein the content of D2 ADC in the resulting antibody-drug conjugate is greater than 80 wt% based on the total weight of D0 and D2 ADC.

29. The method of claim 1, wherein the content of D6 ADC in the resulting antibody-drug conjugate is greater than 85 wt% based on the total weight of D0, D2, D4 and D6 ADC.

30. The method of claim 3, wherein the content of D2+4 ADC in the resulting antibody-drug conjugate is greater than 65 wt% based on the total weight of the ADC.

31. An antibody-drug conjugate prepared by any one of the methods of the preceding claims.

32. An antibody-drug conjugate, wherein the antibody comprises a first antigen-binding moiety, a second antigen-binding moiety, a hinge region, and an Fc region, wherein both the first antigen-binding moiety and the second antigen-binding moiety are in Fab form. The first antigen-binding portion includes a first heavy chain variable domain (VH) operably linked to a first T cell receptor (TCR) constant region (C1) and a first light chain variable domain (VL) operably linked to a second TCR constant region (C2); the second antigen-binding portion includes a second VH operably linked to an antibody heavy chain CH1 domain and a second VL operably linked to an antibody light chain constant (CL) domain. A non-natural interchain disulfide bond is formed between C1 and C2, a natural interchain disulfide bond exists between CH1 and CL, and two natural interchain disulfide bonds exist between the hinge regions. C1 and C2 are selected from the following group: (1) The C1 region consisting of the amino acid sequence of SEQ ID No: 2, and the C2 region consisting of the amino acid sequence of SEQ ID No: 4 or 8, or (2) The C1 region consisting of the amino acid sequence of SEQ ID No: 7, and the C2 region consisting of the amino acid sequence of SEQ ID No: 9; In the antibody-drug conjugate mentioned above: (a) The drug-to-antibody ratio is 6, and the drug is coupled to the cysteine ​​site of the reduced natural disulfide bond in the second antigen-binding moiety and the cysteine ​​site of the reduced natural disulfide bond in the hinge region. (b) The drug-to-antibody ratio is 2, and the drug is coupled to the cysteine ​​residue of the reduced natural disulfide bond in the second antigen-binding moiety; or (c) The ratio of the first drug to the antibody is 2 and the ratio of the second drug to the antibody is 4, and the first drug is coupled to the cysteine ​​site of the reduced natural disulfide bond in the second antigen-binding region, and the second drug is coupled to the cysteine ​​site of the reduced natural disulfide bond in the hinge region.

33. A pharmaceutical composition comprising an effective amount of an antibody-drug conjugate according to any one of claims 31-32 and a pharmaceutically acceptable carrier or mediator.

34. Use of the antibody-drug conjugate according to any one of claims 31-32 in the preparation of a pharmaceutical composition for treating a condition or symptom in a subject, wherein the condition or symptom is related to an antigen targeted by the antibody.

35. The use according to claim 34, wherein the condition or ailment is cancer, an autoimmune disease, or an infectious disease.

36. The use according to claim 34 or 35, wherein the subject is a mammal.

37. The use according to claim 36, wherein the subject is a human.

38. Use of an antibody having the following form in the preparation of a highly homogeneous D6, D2, or dual-drug D2+4 ADC against a selected antigen, wherein the antibody form comprises a first antigen-binding moiety, a second antigen-binding moiety, a hinge region, and an Fc region, wherein both the first and second antigen-binding moieties are in Fab form. The first antigen-binding portion includes a first heavy chain variable domain (VH) operably linked to a first T cell receptor (TCR) constant region (C1) and a first light chain variable domain (VL) operably linked to a second TCR constant region (C2); the second antigen-binding portion includes a second VH operably linked to an antibody heavy chain CH1 domain and a second VL operably linked to an antibody light chain constant (CL) domain. A non-natural interchain disulfide bond is formed between C1 and C2, a natural interchain disulfide bond exists between CH1 and CL, and two natural interchain disulfide bonds exist between the hinge regions. C1 and C2 are selected from the following group: (1) The C1 region consisting of the amino acid sequence of SEQ ID No: 2, and the C2 region consisting of the amino acid sequence of SEQ ID No: 4 or 8, or (2) The C1 region consisting of the amino acid sequence of SEQ ID No: 7, and the C2 region consisting of the amino acid sequence of SEQ ID No: 9; in, (a) For D6 ADC, the drug is coupled to the cysteine ​​site of the reduced natural disulfide bond in the second antigen-binding moiety and the cysteine ​​site of the reduced natural disulfide bond in the hinge region. (b) For D2 ADC, the drug is coupled to the cysteine ​​site of the reduced native disulfide bond in the second antigen-binding moiety. (c) For a dual-drug D2+4 ADC, the ratio of the first drug to the antibody is 2 and the ratio of the second drug to the antibody is 4. The first drug is coupled to the cysteine ​​site of the reduced natural disulfide bond in the second antigen-binding region, and the second drug is coupled to the cysteine ​​site of the reduced natural disulfide bond in the hinge region.

39. A kit comprising one or more containers, said containers containing an antibody-drug conjugate according to any one of claims 31-32 or a pharmaceutical composition according to claim 33.

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