CN117295526A

CN117295526A - Method for preparing highly homogeneous antibody-drug conjugates of engineered antibodies

Info

Publication number: CN117295526A
Application number: CN202280015068.XA
Authority: CN
Inventors: 吉傲; 徐建清; 靳瑾; 徐雷; 王俊; 阴丽
Original assignee: Shanghai Yaoming Helian Biotechnology Co ltd
Current assignee: Shanghai Yaoming Helian Biotechnology Co ltd
Priority date: 2021-09-10
Filing date: 2022-09-06
Publication date: 2023-12-26
Also published as: WO2023036137A1

Abstract

Methods for preparing highly homogeneous antibody-drug conjugates (ADCs) of antibodies are provided. In particular, in the ADC prepared by the method, the content of D2, D6 or d2+4 may reach more than 90%.

Description

Method for preparing highly homogeneous antibody-drug conjugates of engineered antibodies

Cross reference

The present application claims the benefit of International application PCT/CN2021/117009 filed on 9/10 of 2021, the entire contents of which are incorporated herein by reference.

Technical Field

The present disclosure relates to methods of making antibody-drug conjugates (ADCs). In particular, the disclosure relates to bioconjugate methods of making highly homogeneous antibody-drug conjugates (ADCs) of engineered antibodies, particularly antibodies engineered to comprise TCR constant regions in one Fab arm.

Background

The specificity of antibodies for specific antigens and molecules on the surface of target cells has led to their widespread use as carriers for a variety of diagnostic and therapeutic agents. For example, antibodies conjugated to labels and reporter groups (e.g., fluorophores, radioisotopes, and enzymes) may be used in labeling and imaging applications, while conjugation to cytotoxic agents and chemotherapeutic drugs allows targeted delivery of such 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 side effects associated with chemotherapeutic treatments.

Bispecific antibodies (bsAb) refer to an antibody designed to recognize two different epitopes or antigens and are intended to treat a wide variety of complex diseases by binding two disease targets to one molecule. It takes many forms. WO2019057122 and WO2020057610 disclose WuXiBody ^TM Examples of platforms that allow almost any monoclonal antibody (mAb) sequence pair to be assembled into a bispecific construct, the unique structural flexibility of which allows the platform to be conveniently configuredVarious forms with different valence states are built. Bispecific antibodies produced by this platform are also stable and have no aggregation problems during production.

Antibody Drug Conjugates (ADCs) are conjugates of antibodies and drugs with a wide range of potential therapeutic applications in some disease fields, particularly in the cancer field, and are novel targeted drugs for disease treatment. ADCs comprise antibodies for targeting, linkers or linkers for drug attachment, and high potency loads (e.g., drugs) as effectors. Antibodies, by virtue of their specificity, direct drugs to their targets for release. ADC drug development has been widely used for cancer treatment since the us FDA approval was obtained by Adcetris in 2011 and Kadcyla in 2013. Recently, bispecific antibody-based ADCs (e.g., ZW 49) have been demonstrated to be more effective in cancer treatment.

One of the problems faced by conventional conjugation of ADCs is the heterogeneity of ADC molecules, where drug moieties are attached at several sites on the antibody, e.g. by cysteine chemistry, ranging from 0 to 8 drug moieties per antibody (drug-antibody ratio, DAR). The ADC molecules of such mixtures not only present difficulties in analysis and characterization, but may also have different pharmacokinetic, distribution, toxicity and efficacy profiles. Nonspecific coupling also often results in impaired antibody function.

Strategies to solve this problem have been developed. The unnatural amino acid incorporation and various enzyme-assisted couplings of THIOMAB and Ambrx of Genetech are all aimed at introducing drug moieties in a site-directed manner and achieving homogeneity of ADC (narrow DAR distribution). However, these methods are all based on antibody engineering, which may lead to side effects in humans. WO2017002776 discloses that DAR4 can be selectively enriched by more than 50% by lowering the temperature of the reduction step, with most drugs on the Fab domain, without altering the IgG sequence.

However, there is still a need to develop new bioconjugation methods that can produce ADCs including those of bispecific antibodies, with improved homogeneity and with simple handling and reduced costs.

Summary of The Invention

It is an object of the present disclosure to develop a new coupling method that can produce ADCs with improved homogeneity for specific types of bispecific antibodies, with simple handling and reduced costs. Depending on the particular method applied, the resulting ADC has a high content of D2, D6 or d2+4 ADCs. The ADC produced by the coupling methods of the present disclosure further has better safety and efficacy.

The present disclosure relates to a combination of metal ion chelation technology and WuXiBody-type bispecific antibodies for developing new coupling methods. The homogeneity of the antibody-drug conjugate (ADC) product produced by the conjugation methods of the present disclosure can be greatly improved compared to conventional conjugation methods. Furthermore, ADC products can be obtained that are site-specifically coupled to two different drug moieties.

In one aspect, the present disclosure provides a method of making an antibody-drug conjugate (ADC), wherein the antibody comprises a pair of T Cell Receptor (TCR) constant regions in at least one arm in place of CH1 and CL domains, and the pair of TCR constant regions are capable of forming one or more non-native intra-chain disulfide bonds, and wherein the method comprises the steps of:

(a) Incubating a reducing agent with the antibody in a buffer system;

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

(c) Recovering the resulting antibody-drug conjugate.

Optionally, the method further comprises adding an effective amount of an oxidizing agent to reoxidize unreacted thiol groups after step (b) and before recovering the resulting antibody-drug conjugate.

The inventors have surprisingly found that due to steric hindrance, the non-natural interchain disulfide bonds between the pair of TCR constant regions are not accessible to reducing agents and therefore cannot be reduced for drug coupling.

In some embodiments, an 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-native inter-chain disulfide bonds, and

the second antigen binding portion is in the form of a Fab, scFv or VHH.

In some embodiments, the second antigen binding portion is in Fab form and comprises: 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. In some embodiments, the second antigen binding portion is in the form of an scFv and comprises: a second VH operably connected to the second VL. In some other embodiments, the second antigen binding portion is in the form of a VHH and comprises a single variable domain.

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 targeting the same epitope.

In some embodiments, the C1 and C2 regions of the antibodies disclosed herein comprise an engineered T Cell Receptor (TCR) constant region. In particular, the C1 region may comprise the amino acid sequences of SEQ ID Nos. 2, 7 or variants thereof having at least 90% identity; and the C2 region may comprise the amino acid sequences of SEQ ID NOS: 4, 8, 9 or variants thereof having at least 90% identity. The C1 and C2 regions are capable of forming dimers, and the non-natural interchain disulfide bonds are capable of stabilizing the dimers. 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.

In some embodiments, the antibody comprises an IgG Fc region, such as an Fc region in IgG1, igG2, igG3, or IgG 4.

In some embodiments, the Fc region further comprises a knob-in-hole structure. Specifically, the sequences of the hinge region and the Fc region in one strand (the "lock button" strand) are shown in SEQ ID No. 5, and the sequences of the hinge region and the Fc region in the other strand (the "lock hole" strand) are shown in SEQ ID No. 6.

In some embodiments, from N-terminus to C-terminus, the antibody comprises the following structure (E17): in the first heavy chain, VH 1-C1-hinge-Fc; in the second heavy chain, VH2-CH 1-hinge-Fc; in the first light chain, VL1-C2; and in the second light chain, VL2-CL, wherein VH1 and VL1 refer to first VH and VL, respectively, and VH2 and VL2 refer to second VH and VL, respectively. "-" means an operable linkage, typically through a peptide linker.

In some other embodiments, from N-terminus to C-terminus, the antibody comprises the following structure: in the first heavy chain, VH 1-C1-hinge-Fc-scFv; in the second heavy chain, VH1-CH 1-hinge-Fc-scFv; in the first light chain, VL1-C2; and in the second light chain, VL1-CL. The scFv constitutes the second antigen binding portion and may also be replaced by a VHH format.

In some embodiments, to obtain high levels of D2 ADC, the incubation in step (a) is performed 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 comprises D2 in an amount of more than 80wt%, e.g. more than 85wt%, more than 90wt% or more than 95wt%, based on the total weight of D0 and D2.

In some embodiments, the incubation in step (a) is performed in the presence of an effective amount of metal ions, such as divalent metal ions and transition metal ions, and the method further comprises between steps (b) and (c): removing the transition metal ion or divalent metal ion from the product of step (b), and then reintroducing the reducing agent and incubating with an excess of the different linker-drug moiety. The resulting antibody-drug conjugate comprises d2+4 in an amount of more than 65wt%, e.g. more than 70wt%, more than 80wt% or more than 90wt%, based on the total weight of the ADC.

In some embodiments, to obtain high levels 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 comprises D6 in an amount of more than 85wt%, e.g. more than 90wt%, more than 91wt%, more than 92wt% or more than 93wt%, based on the total weight of D0, D2, D4 and D6.

In one aspect, the present disclosure provides a method of making an antibody-drug conjugate (ADC), wherein the antibody comprises first and second antigen binding portions,

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), and

The second antigen binding portion comprises: 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,

and wherein the method comprises the steps of:

(a) Incubating a reducing agent and a bispecific antibody in a buffer system in the presence of an effective amount of a transition metal ion or a divalent metal ion to selectively reduce inter-chain disulfide bonds within the antibody;

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

(c) Removing the transition metal ion or divalent metal ion from the product of step (b);

(d) Reintroducing the reducing agent and incubating with an excess of the second linker-drug moiety; and

(e) Recovering the resulting antibody-drug conjugate.

Optionally, the method further comprises adding an effective amount of an oxidizing agent after step (d) to reoxidize unreacted thiol groups.

In some embodiments, the reducing agent added in steps (a) and (d) is different, e.g., one is TCEP and the other is TDD. In some other embodiments, the reducing agent added in steps (a) and (d) is the same, e.g., both are TCEPs.

In some embodiments, the resulting antibody-drug conjugate comprises d2+4 in an amount of greater than 65wt%, such as greater than 70wt%, greater than 80wt%, or greater than 90wt%, based on the total weight of the ADC.

In some embodiments, the first drug is MMAF and the second drug is DXD.

In some embodiments, the metal ion in step (a) is selected from divalent ions and transition metal ions, comprising: 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 methods of the present disclosure may include, but are not limited to, zn ²⁺ 、Cd ²⁺ 、Hg ²⁺ Etc. Wherein due to Zn ²⁺ 、Ca ² ⁺ And Mg (magnesium) ²⁺ And low cost, and can be used. For example, a suitable transition metal salt or divalent metal ion may be added in step (a) as long as they are soluble in the reaction solution, so that free transition metal ions can be released in the reaction solution. In this respect, znCl ₂ 、Zn(NO ₃ ) ₂ 、ZnSO ₄ 、Zn(CH ₃ COO) ₂ 、ZnI ₂ 、ZnBr ₂ Zinc formate and zinc tetrafluoroborate can be suitable zinc salts. Similarly, caCl may be applied ₂ 、Ca(NO ₃ ) ₂ 、CaSO ₄ 、MgCl ₂ 、Mg(NO ₃ ) ₂ And MgSO ₄ . Likewise, mention may be made of those which are soluble in the reaction solution and which are capable of releasing free Cd ²⁺ Or Hg ²⁺ Other transition metal salts of ions including, but not limited to, cdCl ₂ 、Cd(NO ₃ ) ₂ 、CdSO ₄ 、Cd(CH ₃ COO) ₂ 、CdI ₂ 、CdBr ₂ Cadmium formate and cadmium tetrafluoroborate; hgCl ₂ 、Hg(NO ₃ ) ₂ 、HgSO ₄ 、Hg(CH ₃ COO) ₂ 、HgBr ₂ Mercury (II) formate, and mercury (II) tetrafluoroborate, and the like.

In some embodiments, the buffer system used in step (a) is selected from the group comprising: hepes, histidine buffer, PBS and MES, and a pH of about 5.5 to 8.

In some embodiments, the antibody to be conjugated is added in step (a) to a final concentration of about 0.01-0.1 mM.

In some embodiments, step (a) is performed at a temperature of about-10 ℃ to 37 ℃, for example, about 0 ℃ to 20 ℃.

In some embodiments, the reducing agent in step (a) is TCEP.

In some embodiments, the oxidizing agent is DHAA.

In some embodiments, the linker-drug moiety is a drug-carrying maleimide, a drug-carrying organobromide, or a drug-carrying organoiodide.

In some embodiments, the variable regions of the first and second antigen binding portions are derived from antibodies that are known, on-the-fly, or developed de novo, e.g., any of the following antibodies: trastuzumab (trastuzumab), pertuzumab (pertuzumab), sha Xizhu mab (sacituzumab), acipimab (abciximab), adalimumab (adalimumab), adalimumab (alexaprop), alemtuzumab (alemtuzumab), basiliximab (basiliximab), belimumab (belimumab), bei Luotuo Shu Shan mab (bezlotoxumab), canumab (canakiumab), cetuximab (certolizumab pegol), cetuximab (cetuximab), daclizumab (daclizumab), didanoumab (denosumab), efuzumab (alemtuzumab), golimumab (golimumab), infliximab (infliximab), ipiumab (basilizumab), yizumab (iximab), oxuzumab (bezizumab), oxytuzumab (oxytuzumab) and oxytuzumab (tacuzumab) are assigned to be used as the trastuzumab, the trastuzumab (tacuab) and the oxytuzumab (tacuzumab) is assigned to be the therapeutic antibody. Preferably, the variable regions (or at least CDR regions) of the first and second antigen binding portions are identical to the variable regions (or at least CDR regions) of known or de novo developed antibodies.

In some embodiments, the first VH and the first VL are from trastuzumab and the second VH and the second VL are from pertuzumab, or vice versa. In some other embodiments, the first VH and the first VL are from trastuzumab and the second VH and the second VL are from Sha Xizhu mab, or vice versa.

In some embodiments, the drug to be coupled is selected from the group comprising: diagnostic agents, therapeutic agents, and labeling agents.

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

In one aspect, the present disclosure provides antibody-drug conjugates prepared by any of the methods disclosed.

In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate disclosed herein and a pharmaceutically acceptable carrier or vehicle.

In one aspect, the present disclosure provides the use of an antibody-drug conjugate disclosed herein in the preparation of a pharmaceutical composition or kit for treating a condition or disorder in a subject.

In one aspect, the present disclosure provides a method of treating a condition or disorder in a subject comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate or pharmaceutical composition disclosed herein.

In some embodiments, the condition or disorder is a tumor, cancer, autoimmune disease, or infectious disease. For example, the cancer is breast cancer. The subject may be a mammal, such as a human.

The foregoing and other features and advantages of the disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings.

Drawings

FIG. 1. A reaction scheme according to some embodiments, wherein three pairs of interchain disulfide bonds of bispecific antibody cAb1 (trastuzumab x pertuzumab) are reduced and site-specifically coupled to MC-MMAF (FIG. 1A); a HIC profile of cAb1 coupling showing DAR6 abundance (fig. 1B); mass spectrum coupled to cAb1 (fig. 1C). FIG. 1D is the HIC profile of two other similarly coupled bispecific antibodies cAb3 and cAb 4.

FIG. 2A reaction scheme according to some embodiments wherein Zn is added ²⁺ And only one pair of interchain disulfide bonds of the bispecific antibody is reduced and associated with MC-MMAF site-specific coupling (FIG. 2A); HIC spectra of four bispecific antibodies cAb1, cAb2, cAb3 and cAb4 coupled as described (fig. 2A) (fig. 2B).

FIG. 3. Reaction schemes according to some embodiments wherein bispecific antibody cAb1 is coupled to two different drugs (e.g., MMAF and DXD) in two steps, respectively (FIG. 3A); MS spectra of resulting cAb1 ADC (fig. 3B), cAb2 ADC (fig. 3C), cAb3 ADC (fig. 3D), and cAb4 ADC (fig. 3E).

Detailed Description

While this disclosure may be embodied in many different forms, what is disclosed herein is a specific set forth embodiment thereof, illustrating the principles of the invention. It should be emphasized that this disclosure is not limited to the specific embodiments set forth. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

In general, nomenclature and techniques employed in connection with the 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 employed 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 various general and more specific references cited and discussed throughout the present specification. See, e.g., abbas et al, cellular and Molecular Immunology, 6 th edition, w.b. samanders Company (2010); sambrook J. & Russell d.molecular Cloning: A Laboratory Manual, 3 rd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (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, n.y. (1998); and Coligan et al, short Protocolsin Protein Science, wiley, john & Sons, inc. (2003). The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques, are those well known and commonly employed in the art. All publications mentioned in this specification are herein incorporated in their entirety by reference.

Definition of the definition

For a better understanding of the present invention, definitions and explanations of the relevant terms are provided below.

Unless defined otherwise, scientific and technical terms used in connection with the present disclosure should have meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies; reference to "a transition metal ion" includes mixtures of transition metal ions, and the like. In this application, the use of "or" means "and/or" unless indicated otherwise.

Throughout this disclosure, unless the context requires otherwise, the words "comprise," "comprising," and "include" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. "consisting of …" means including and limited to elements following the phrase "consisting of …". Thus, the phrase "consisting of …" means that the listed elements are required or mandatory and that no other elements may be present. "consisting essentially of …" is intended to include any element listed after the phrase and is limited to other elements that do not interfere with or contribute to the activity or effect specified in the present invention for the listed elements. Thus, the phrase "consisting essentially of …" means that the listed elements are necessary or mandatory, but other elements are optional and may or may not be present, depending on whether they affect the activity or function of the listed elements.

As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In particular embodiments, the term "about" or "approximately" when preceded by a numerical value means that the value is plus or minus a range of 15%, 10%, 5%, or 1%.

An "antibody-drug conjugate" or ADC refers to a conjugate formed by covalently coupling a drug to an antibody, either directly or indirectly via one or more appropriate linkers. ADCs are typically in the form of antibody-linker-drug conjugates. The antibody-drug conjugate combines desirable properties of both antibodies and cytotoxic drugs (or substances with other properties) by targeting potent cytotoxic (or other) drugs to tumor cells (or other cells/organs) expressing the antigen, thereby enhancing their antitumor (or other medical) activity. The design of ADCs is intended to distinguish healthy cells from diseased tissue, such as tumor cells in a tumor.

The term "drug" or "cargo" as used herein refers to any cytotoxic molecule having, for example, an anti-tumor effect, an anti-infective or anti-inflammatory effect and having at least one substituent group or moiety that allows attachment to a linker structure. The agent may kill cells (e.g., cancer cells) and/or inhibit the growth, proliferation, or metastasis of cells (e.g., cancer cells), thereby reducing, alleviating, or eliminating one or more symptoms of a disease or disorder (e.g., cancer).

The term "linker" as used herein refers to a reactive molecule containing at least two reactive groups, one of which may be covalently bonded to a drug molecule and the other of which may be covalently coupled to an antibody.

The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, bispecific antibody, multivalent or bivalent antibody that binds to a particular antigen. A natural intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region ("HCVR") and first, second and third constant regions (CH 1, CH2 and CH 3), while each light chain consists of a variable region ("LCVR") and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. Antibodies typically have a "Y" shape, wherein the stem of Y consists of the second and third constant regions of two heavy chains that are bound together via disulfide bonds. Each arm of Y comprises a variable region and a first constant region of a single heavy chain, in combination with a variable region and a constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region in both chains typically contains three highly variable loops, termed Complementarity Determining Regions (CDRs) (light (L) chain CDRs comprising LCDR1, LCDR2 and LCDR3, and heavy (H) chain CDRs comprising HCDR1, HCDR2 and HCDR 3). The CDR boundaries of antibodies can be defined or identified by the rules of Kabat, chothia or Al-Lazikani et Al (Al-Lazikani, B.; chothia, C.; lesk, A.M., J.Mol.Biol.,273 (4), 927 (1997); chothia, C.; et Al, J Mol biol. Dec5;186 (3): 651-63 (1985); chothia, C. And Lesk, A.M., J.Mol.Biol.,196,901 (1987); chothia, C. Et Al, nature. Dec 21-28;342 (6252): 877-83 (1989); kabat E.A. Et Al, national Institutes of Health, bethesda, md. (1991)). Three CDRs are interposed between flanking segments called Framework Regions (FR) which are more highly conserved than the CDRs and form scaffolds to support highly variable loops. Each HCVR and LCVR includes four FRs, and the CDRs and FRs are arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains are not involved in antigen binding, but rather exhibit various effector functions. Antibodies are assigned to various classes based on the amino acid sequence of the constant region of their heavy chains. The five main classes or isotypes of antibodies are IgA, igD, igE, igG and IgM, which are characterized by the presence of the α, δ, epsilon, 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).

An "antibody fragment" includes a portion of a full-length antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, fab ', F (ab') 2, and Fv fragments; a diabody; a linear antibody; minibodies (Olafsen et al (2004) Protein en. Design & sel.17 (4): 315-323), fragments generated by Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs (complementarity determining regions), and fragments of any of the binding epitopes described herein (which immunospecifically bind to a cancer cell antigen, viral antigen, or microbial antigen), single chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The term "antigen binding portion" as used herein refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds an antigen but does not comprise the complete native antibody structure. Examples of antigen binding moieties include, but are not limited to, variable domains, diabodies, fab ', F (ab ') 2, fv fragments, scFv, disulfide stabilized Fv fragments (dsFv), (dsFv) 2, bispecific dsFv (dsFv-dsFv '), disulfide stabilized diabodies (ds diabodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. The antigen binding portion is capable of binding the same antigen as the parent antibody. In certain embodiments, the antigen binding portion may comprise one or more CDRs from a particular human antibody that are grafted to framework regions from one or more different human antibodies. More and detailed forms of antigen binding moieties are described in Spiess et al, 2015 (supra) and Brinkman et al, mAbs,9 (2), pp.182-212 (2017), the entire contents of which are incorporated herein.

"Fab" with respect to an antibody refers to the portion of an antibody that consists of a single light chain (both variable and constant regions) associated with the variable and first constant regions of a single heavy chain by disulfide bonds. In certain embodiments, both the first and second antigen binding portions of the antibody to be conjugated are in Fab form. Further, the constant regions of both chains of the Fab (i.e., CH1 and CL) are replaced by engineered or modified TCR constant regions.

"Fc" with respect to an antibody refers to the portion of the antibody that comprises the second (CH 2) and third constant regions (CH 3) of the first heavy chain, bound to the second and third constant regions of the second heavy chain via disulfide bonds and optionally hinge regions. The Fc portion of antibodies is responsible for various effector functions, such as ADCC and CDC, but does not play a role in antigen binding.

"hinge region" with respect to antibodies includes the portion of the heavy chain molecule that connects the CH1 domain and the CH2 domain. The hinge region comprises about 25 amino acid residues and is flexible, thereby allowing the two N-terminal antigen binding regions to move independently.

As used herein, the term "lock knob-keyhole" refers to engineering the CH3 domain of an antibody Fc region to create a "lock knob" or "keyhole" in each heavy chain to promote heterodimerization. Typically, a "lock knob" is created by substituting a large residue W for T366 on one heavy chain, while the corresponding "lock hole" is created by triple mutation of T366S, L368A and Y407V on the other heavy chain. The lock knob-keyhole configuration may include other substitutions known in the art. When referring to residues in the immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported in Kabat et al Sequences of Proteins of Immunological Interest (5 th edition), U.S. Dept. Of Health and Human Services, PHS, NIH, NIH publication No. 91-3242). Unless otherwise indicated herein, references to residue numbering in the constant domain of the Fc region refer to residue numbering by the EU numbering system. In some embodiments, the sequences of the hinge region and the Fc region in one strand (the "lock button" strand) are shown in SEQ ID No. 5, while the sequences of the hinge region and the Fc region in the other strand (the "lock hole" strand) are shown in SEQ ID No. 6.

ASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID No:5)

SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID No:6)

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for mutations that may occur naturally (which may be present in minor amounts). Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring that the population of antibodies be obtained by any particular method. For example, monoclonal antibodies for use in accordance with the invention may be prepared by the hybridoma method described first by Kohler et al (1975) Nature 256:495, or may be prepared by recombinant DNA methods (see, e.g., US 4816567;US 5807715). Monoclonal antibodies can also be used, for example, in Clackson et al (1991) Nature,352:624-628; the techniques described in Marks et al (1991) J.mol.biol.,222:581-597, were isolated from phage antibody libraries.

"WuXiBody" as used herein refers to a bispecific antibody comprising a soluble chimeric protein having the variable domain of the antibody and the constant domain of the TCR (e.g., in the first antigen-binding portion), wherein the subunits of the TCR constant domain (e.g., the alpha and beta domains) are linked by one or more engineered disulfide bonds. WuXiBody also encompasses WuXiBody 2.0 antibodies comprising a variety of modified TCR constant domain sequences (see WO 2022/156887). TCR constant domains can be engineered to form more than one pair of disulfide bonds, thereby increasing stability and/or expression levels. In one type of WuXiBody, an antibody comprises a first antigen binding portion in one arm and a second antigen binding portion in the other arm, both in Fab form and operably linked at the C-terminus to one chain of an immunoglobulin Fc region. In a different WuXiBody format, an Fc region is interposed between the first antigen binding portion and the second antigen binding portion. In particular, an antibody may comprise a first antigen binding portion, which is a Fab, at one end of the Fc region, and a second antigen binding portion, which is a Fab, scFv, or VHH, at the other end of the Fc region. Alternatively, the WuXiBody form of the antibody comprises a first antigen binding portion operably linked to a second antigen binding portion, the second antigen binding portion further operably linked to an Fc region. A detailed description of various forms of WuXiBody can be found in WO2019057122, WO201905714 and WO2020057610 (all of which are incorporated herein by reference in their entirety).

A native "T cell receptor" or native "TCR" is a heterodimeric T cell surface protein that associates with a invariant CD3 chain to form a complex capable of mediating signal transduction. TCRs belong to the immunoglobulin superfamily, resembling half antibodies with a single heavy chain and a single light chain. The native TCR has an extracellular portion, a transmembrane portion, and an intracellular portion. The extracellular domain of the TCR has a membrane proximal constant region and a membrane distal variable region.

The terms "trastuzumab x pertuzumab", "pertuzumab x trastuzumab", "trastuzumab x Sha Xizhu mab" and "Sha Xizhu mab x trastuzumab" and similar designations as used herein are named according to the same principle, i.e., refer 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. By "derived from" is meant that the variable region is identical to or has at least 80% homology (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to the variable region in the parent antibody, but still retains the ability to bind to the targeted antigen. For example, the variable regions from a parent antibody may be subjected to humanization, affinity maturation, or glycosylation modification prior to construction into the antibody forms disclosed herein. Methods for modifying variable regions including CDRs and framework regions are familiar to those skilled in the art.

"class" of antibodies refers to the type of constant domain or constant region that the heavy chain has. There are five main classes of antibodies: igA, igD, igE, igG and IgM, and several of these can be further divided into subclasses (isotypes), for example, igG1, igG2, igG3, igG4, igA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively.

An "isolated antibody" refers to an antibody that has been isolated from a component of its natural environment. In some embodiments, the antibodies are purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For reviews of methods for assessing antibody purity, see, e.g., flatman et al, J.chromatogr.B 848:79-87 (2007).

"disulfide" refers to a covalent bond having the structure R-S-S-R'. The amino acid cysteine comprises a thiol group which may form a disulfide bond with a second thiol group, e.g. from another cysteine residue. Disulfide bonds may be formed between sulfhydryl groups of two cysteine residues located on two polypeptide chains, respectively, thereby forming an interchain bridge or interchain bond.

The term "transition metal" as used herein refers to elements of groups 4-11 that are chemically verified by their typical nature, i.e., a broad range of complex ions in various oxidation states, colored complexes, and catalytic properties as elements or as ions (or both). Sc and Y of group 3 are also commonly considered transition metals.

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

The term "DAR" or "drug to antibody ratio" as used herein refers to the average number of drugs conjugated to an antibody, an important attribute of an ADC. DAR values affect the efficacy of drugs because low drug loading can reduce efficacy, while high drug loading can negatively impact Pharmacokinetics (PK) and toxicity. A variety of analytical methods are available for measuring DAR, such as ultraviolet-visible (UV/Vis) spectroscopy, hydrophobic Interaction Chromatography (HIC), reversed-phase high performance liquid chromatography (RP-HPLC), and liquid chromatography coupled electrospray ionization mass spectrometry (LC-ESI-MS). Hydrophobic Interaction Chromatography (HIC) is the leading technique to characterize DAR values and drug load distribution. The conjugate species are isolated based on increased hydrophobicity resulting from increased drug loading. For cysteine-conjugated ADCs, the unconjugated antibody with the lowest hydrophobicity was eluted first, and the most hydrophobic, most drug-conjugated form was eluted last, yielding a quantitative elution profile. The area percent of the peak represents the relative amount of ADC species for each drug load. The payload distribution is derived from the HIC profile, while the average DAR is also calculated from the percentage of peak area. As shown herein, ADCs coupled by the methods disclosed herein are highly homogeneous. Depending on the particular method used, the amount of DAR2, DAR6, or DAR2+4 (i.e., D2, D6, or D2+4) may be up to at least 80wt% of the total ADC. Dar2+4 (d2+4) refers to a dual drug ADC comprising two first drug molecules and four second drug molecules per antibody.

As known in the art, a mixture of antibody-drug conjugates will be produced by conventional conjugation methods. Typically, one therapeutic antibody molecule belonging to the subclass IgG1 or IgG4 has 4 interchain S-S bonds, each formed by two-SH groups. One or more interchain S-S bonds may be partially or fully reduced to form 2n (n is an integer selected from 1, 2, 3 or 4) reactive-SH groups and, thus, the number of drugs coupled to a single antibody molecule is 2, 4, 6 or 8. Different conjugates containing different numbers of drug molecules were designated D0, D2, D4, D6 and D8, depending on the number of drugs conjugated to a single antibody molecule.

If the number of drugs coupled to a single antibody molecule is 0, the product is referred to as D0. Accordingly, D2 refers to an ADC in which two drug molecules are coupled to one single antibody molecule, wherein the two drug molecules may be coupled via a linker to a-SH group generated by reducing an S-S bond between the heavy and light chains, or may be coupled via a linker to a-SH group generated by reducing an S-S bond between the heavy and heavy chains. D4 refers to an ADC in which four drug molecules are coupled to one single antibody molecule, wherein four drug molecules may be coupled via linkers to four-SH groups generated by reducing two S-S bonds between a heavy chain and a light chain or between a heavy chain and a heavy chain, or two drug molecules may be coupled via linkers to two-SH groups generated by reducing one S-S bond between a heavy chain and a light chain and two other drug molecules may be coupled via linkers to two-SH groups generated by reducing one S-S bond between a heavy chain and a heavy chain. D6 refers to an ADC in which six drug molecules are coupled to one single antibody molecule, wherein four drug molecules may be coupled to four-SH groups generated by reducing two S-S bonds between a heavy chain and a light chain via a linker and two drug molecules may be coupled to two-SH groups generated by reducing one S-S bond between a heavy chain and a heavy chain via a linker, or four drug molecules may be coupled to four-SH groups generated by reducing two S-S bonds between a heavy chain and a heavy chain via a linker and two drug molecules may be coupled to two-SH groups generated by reducing one S-S bond between a heavy chain and a light chain via a linker. And D8 refers to an ADC in which eight drug molecules are coupled to one single antibody molecule, i.e. all four S-S bonds in one antibody molecule are reduced to eight-SH groups and each-SH group is attached to one drug molecule. Typically, the heterogeneous mixture of ADC molecules produced by conventional coupling methods or bioconjugate methods of the invention is a mixture of D0, D2, D4, D6 and D8.

And thus the term "homogeneity" of an antibody-drug conjugate is used to describe the property of one particular type of antibody-drug conjugate (preferably one type selected from D2, D4, D6 conjugates) to predominate in a given one of the antibody-drug conjugate mixtures. In the present invention, "homogeneity" of an antibody-drug conjugate means that one particular type of ADC has a high level in the antibody-drug conjugate mixture.

The term "pharmaceutically acceptable" means that the specified carrier, vehicle, diluent, excipient and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

By "pharmaceutically acceptable carrier" is meant an ingredient in a pharmaceutical formulation other than the active ingredient that is biologically active, acceptable and non-toxic to the subject. Pharmaceutically acceptable carriers for use in the pharmaceutical compositions described herein can include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispensing agents, masking or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

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

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

The terms "treat," "treatment" or "treated" as used herein in the context of treating a condition generally refer to treatment or therapy, whether to humans or animals, in which a certain desired therapeutic effect is achieved, such as inhibiting the progression of the condition, and includes a decrease in the rate of progression, a cessation of the rate of progression, a regression of the condition, a remission of the condition, and a cure of the condition. Also included are treatments as a precautionary measure (i.e., prevention, prophylaxis). For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cell, or some combination thereof. For a tumor, "treating" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, inhibiting or delaying tumor progression, or some combination thereof.

Overview of ADC preparation

Antibody-drug conjugates are typically produced by two conventional chemical methods: coupling based on lysine and coupling based on cysteine from interchain sulphur bond reduction. For couplings based on cysteines from interchain reduction, it includes a step of opening interchain disulfide bonds in the presence of various reducing agents (e.g., TCEP, DTT, etc.), followed by nucleophilic reactions of sulfhydryl groups. In this conjugation method, the antibody-drug conjugate is typically formed as follows: one or more antibody cysteine sulfhydryl groups are coupled to one or more drug-binding linker moieties, thereby forming an antibody-linker-drug complex. Since free cysteine thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine often exist in their oxidized form as disulfide-linked oligomers or have internally bridged disulfide groups. Disulfide dimer formation renders Cys unreactive for coupling with drugs, ligands or other labels.

The number of drugs conjugated to a single antibody molecule is an important factor for the efficacy and safety of the resulting ADC. For example, in coupling methods based on reduction of natural interchain disulfide bonds, interchain S-S bonds are more accessible to solvents than other disulfide bonds. Thus, the interchain disulfide bond may serve as a binding site for drug (or drug-linker) coupling to an antibody. Typically, one therapeutic antibody molecule belonging to the IgG1 or IgG4 subtype has 4 interchain S-S bonds, each formed by two-SH groups, so the number of drugs coupled to a single antibody molecule is 2, 4, 6 or 8. If the number of drugs coupled to a single antibody molecule is 0, the product is referred to as D0. Accordingly, D2 refers to an ADC in which two drug molecules are conjugated to one single antibody molecule. D4 refers to an ADC in which four drug molecules are conjugated to one single antibody molecule. D6 refers to ADCs in which six drug molecules are conjugated to a single antibody molecule. And D8 refers to an ADC in which eight drug molecules are coupled to one single antibody molecule, i.e. all four S-S bonds in one antibody molecule are reduced to eight-SH groups and each-SH group is linked to one drug molecule. Typically, the heterogeneous mixture of ADC molecules produced by conventional coupling methods is a mixture of D0, D2, D4, D6 and D8.

It is well known in the art that heterogeneous ADC products generally have lower efficacy and unsatisfactory PK properties. Wherein D0 does not have ADC efficacy and D8 is considered to be a cause of instability in circulation 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 drug loading (Hamblett KJ et al, clin Cancer Res.2004 Oct 15;10 (20): 7063-70), the in vivo antitumor activity of antibody-drug conjugates with four drugs per molecule (D4) is equivalent to conjugates with eight drugs per molecule (D8) at the same mAb dose, even though the conjugates contain half the amount of drug per mAb.

Drug-loading also affected plasma clearance, with D8 conjugate cleared 3-fold faster than D4 conjugate and 5-fold faster than D2 conjugate. Antibody-drug conjugates with improved homogeneity provide therapeutic benefits, such as higher therapeutic index, improved efficacy and reduced drug toxicity. Homogeneous antibody conjugates also provide more accurate and consistent measurements in diagnostic and imaging applications.

Conventionally, the drug load of an ADC may be controlled in different ways, for example, by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the coupling reaction time or temperature, (iii) cysteine thiol-modified moieties or limiting the reducing conditions, (iv) engineering the amino acid sequence of the antibody by recombinant techniques 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, which are incorporated herein by reference in their entirety).

By passing through Platform-constructed antibodies

In one aspect, the present disclosure provides a method or process for preparing a highly homogeneous ADC for an antibody. The antibodies comprise an engineered Fab whose CH1 and CL domains are replaced by a pair of T Cell Receptor (TCR) constant regions, and the pair of TCR constant regions are capable of forming non-native interchain disulfide bonds. Such disulfide bonds stabilize dimers formed between a pair of TCR constant regions and, as noted above, are more difficult to access than natural disulfide bonds to solvents or reducing agents.

In some embodiments, the antibodies to be conjugated herein are prepared byPlatform construction, also known as "WuXiBody". "WuXiBody" is typically a bispecific (or multispecific) antibody comprising first and second antigen-binding portions, the first antigen-binding portion being an engineered Fab comprising 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), while the second antigen-binding portion may be in the form of Fab, scFv, VHH or the like. In certain embodiments, the second antigen-binding portion is also in Fab form and comprises 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. In other words, in the first antigen binding portion, the CH1 domain and CL domain that are normally present are replaced by a pair of TCR constant regions, so the native disulfide bond between the CH1 domain and CL domain is also replaced by one or more engineered non-native disulfide bonds between the C1 and C2 regions of the TCR. The locations of the C1 and C2 regions may also be interchanged.

The antibodies to be conjugated herein are not limited to bispecific antibodies with first and second antigen binding portions targeting different epitopes or antigens. In theory, the antibodies to be conjugated herein may also be monospecific antibodies, wherein the first and second antigen binding portions bind to the same antigen or epitope, and the resulting ADC is a highly homogeneous ADC for a single antigen or epitope.

In some embodiments, from N-terminus to C-terminus, the antibody comprises the following structure: in the first heavy chain, VH 1-C1-hinge-Fc; in the second heavy chain, VH2-CH 1-hinge-Fc; in the first light chain, VL1-C2; and in the second light chain, VL2-CL, wherein VH1 and VL1 refer to first VH and VL, respectively, and VH2 and VL2 refer to second VH and VL, respectively. "-" means an operable linkage, typically through a peptide linker.

WuXiBody can take various other forms. For example, the heavy chain portion of the first antigen binding portion may be operably linked to a second antigen binding portion, which is further operably linked to one chain of the Fc region. Alternatively, the heavy chain portion of the first antigen binding portion is operably linked to one chain of the Fc region, which is further operably linked to one chain of the second antigen binding portion.

In some embodiments, from N-terminus to C-terminus, the antibody comprises the following structure: in the first heavy chain, VH 1-C1-hinge-Fc-scFv; in the second heavy chain, VH1-CH 1-hinge-Fc-scFv; in the first light chain, VL1-C2; and in the second light chain, VL1-CL. The scFv belongs to the second antigen binding portion and may also be replaced by a VHH format.

The first TCR constant region and the second TCR constant region are associated by an unnatural inter-chain disulfide bond. The pair of TCR constant regions in the first antigen-binding portion comprises TCR alpha and beta constant regions (wild-type or preferably engineered) in the light and heavy chains, respectively. The TCR constant regions in bispecific antibodies are capable of forming dimers by associating with each other via unnatural disulfide bonds.

There are two different variants of the human tcrp chain constant region, known as TRBC1 and TRBC2 (IMGT nomenclature). In WuXiBody, the sequence of the TCR β domain is based on the wild-type TCR sequence as follows:

LEDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR (SEQ ID NO: 1), NCBI accession number A0A5B9 (https:// www.uniprot.org/uniprot/A0A5B 9).

In some embodiments, a pair of TCR constant regions comprises an engineered TCR β domain comprising one or more mutation sites, as shown below:

LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR(SEQ ID NO:2)；

LEDLKNVFPPEVAVFEPSECEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR(SEQ ID NO:7)。

The human TCR alpha chain constant region is designated TRAC, NCBI accession number P01848 (https:// www.uniprot.org/uniprot/P01848), the sequence of the wild type TCR alpha domain is:

PDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS(SEQ ID NO:3)。

in some embodiments, a pair of TCR constant regions comprises an engineered TCR alpha chain constant region comprising one or more mutation sites, as shown below:

PDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFPSPESS(SEQ ID NO:4)；

PDIQNPDCVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPECTFFPS(SEQ ID NO:8)；

PDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFCS(SEQ ID NO:9)。

those skilled in the art will readily appreciate that antibodies for use in preparing ADCs as disclosed herein may comprise a variety of C1 and C2 regions, provided that they are capable of stabilizing the first VH and VL regions to form the first antigen binding portion. In particular, an antibody may comprise a TCR C1 region having the amino acid sequence shown in SEQ ID nos. 2 or 7, or a variant thereof at least 90% identical, and a TCR C2 region having the amino acid sequence shown in SEQ ID nos. 4, 8, 9, or a variant thereof at least 90% identical. More than one pair of unnatural disulfide bonds can be formed between the C1 and C2 regions to improve stability and expression levels. In some embodiments, the variant of SEQ ID No. 2 or 7 comprises a substitution, addition and/or deletion of one or more amino acids as compared to SEQ ID No. 2 or 7. Similarly, variants of SEQ ID No. 4, 8 or 9 comprise one or more amino acid substitutions, additions and/or deletions as compared to SEQ ID No. 4, 8 or 9. A detailed description of C1 and C2 variants that can be used to construct the form of the WuXiBody antibody can be found in WO2022/156887, which is incorporated herein by reference in its entirety. Specifically, in some embodiments, the antibodies comprise a combination of C1 and C2 regions having SEQ ID Nos. 2 and 4 (for cAb 1-6), or having SEQ ID Nos. 2 and 8 (for cAb 7), or with SEQ ID Nos. 7 and 9 (for cAb 8), respectively.

For example, the native tcrp chain contains a native cysteine residue at position 76 that is unpaired and thus does not form disulfide bonds in the native α/β TCR. In some bispecific antibodies to WuXiBody, this native cysteine residue at position 76 of the TCR β chain is mutated to an alanine residue. This may help to avoid incorrect intra-or inter-chain pairing. In certain embodiments, certain substitutions increase TCR refolding efficiency in vitro.

Thus, the first and second TCR constant regions of the first antigen-binding portion are capable of forming a dimer comprising at least one non-native interchain disulfide linkage between the TCR constant regions (i.e., CA and CBeta) capable of stabilizing the dimer.

The benefits provided by substituting the CH1 and CL domains with TCR constant regions are significant. In WuXiBody, the first antigen binding portion having at least one unnatural disulfide bond can be recombinantly expressed and assembled into the desired conformation, which stabilizes TCR constant region dimers while providing good antigen binding activity of the antibody variable region. In addition, the first antigen binding portion was found to be better tolerant of conventional antibody engineering, such as modification of glycosylation sites and removal of some native sequences. Furthermore, due to the presence of the TCR constant region in the first antigen-binding portion, bispecific antibodies of this form can be readily expressed and assembled with minimal or substantially no mis-pairing of antigen-binding sequences.

Most importantly, the non-native disulfide bonds in the TCR constant region are less accessible to solvents than native disulfide bonds, thereby providing fewer binding sites (and improved homogeneity) for the conjugated drug (or drug-linker). Furthermore, non-natural disulfide bonds may be less sensitive to reducing agents than natural disulfide bonds.

In some embodiments, the method comprises constructing an antibody based on the parent bispecific antibody or two parent monospecific antibodies and producing the antibody prior to step (a). By "based on" is meant that the first and second VH and VL regions are derived or obtained from the variable region of the parent antibody and assembled with other regions (e.g., TCR constant regions, heavy and light chain constant regions, hinge regions, and Fc regions) into WuxiBody form.

The BsAb for conjugation comprises two antigen binding moieties, which may be Fab, fab', scFv, VHH, and the like. The antigen binding portion may be derived from an antibody (known or newly developed) that targets a particular antigen. In some embodiments, the first antigen binding moiety is derived from trastuzumab and the second antigen binding moiety is derived from pertuzumab, or vice versa. In some other embodiments, the first antigen binding moiety is derived from trastuzumab and the second antigen binding moiety is derived from sabizumab, or vice versa. By "derived from" is meant herein generally that the antigen binding portion comprises the CDR sequences of the parent antibody, and preferably comprises the variable regions of the parent antibody. In some embodiments, the antigen binding portion comprises a variant of a CDR sequence of a parent antibody that retains antigen binding specificity.

Theoretically, parent antibodies from which the antigen binding portion may be derived may include all monoclonal antibodies specific for a particular antigen, e.g., antibodies to 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, sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, trpM4, CRIPTO, CD21, CD79b, fcRH2, NCA, MDP, IL20Ra, brevican, ephB2R, ASLG, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, fcRH1, fcRH5, TENB2, PMEL17, TMEFF1, GDNF-R1, LY6E, TMEM46, LY6G6 95, RET 69, LY6, asd 19, GPR172, GPR 3, and GPR 3; antibodies directed against leukocyte receptors such as MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86 or ligands thereof; CD3 binding antibodies (engager antibodies), NK binding antibodies; an anti-tumor associated antigen that achieves ADCC; monoclonal antibodies directed against TNF, and the like. The antibody may include, but is not limited to, trastuzumab, pertuzumab, sabizumab, acipimab, adalimumab, afaxicon, alemtuzumab, basilizumab, bei Luotuo Shu Shan antibody, cinacalcet, cetuximab polyethylene glycol, cetuximab, daclizumab, denoumab, efalizumab, golimumab, infliximab, ipilimumab, iximab Bei Shan antibody, natalizumab, nivolumab, oxamumab, oxmarizumab, palivizumab, panitumumab, rituximab, toxizumab, threuzumab, and Wu Sinu mab.

Bispecific antibodies for use in the methods provided herein can be antibodies that have binding specificities for a variety of antigens such as Tumor Associated Antigens (TAAs). In certain embodiments, the bispecific antibody to be conjugated has a first specificity for a first TAA antigen and a second specificity for a second TAA antigen. The term "tumor-associated antigen" refers to a target antigen expressed by tumor cells, but may be expressed by the same cell (or healthy cell) prior to transformation into a tumor. In some embodiments, the tumor-associated antigen can only be presented by tumor cells, but not by normal cells, i.e., non-tumor cells. In some other embodiments, the tumor-associated antigen may be expressed on tumor cells only, or may represent a tumor-specific mutation, as compared to non-tumor cells. In some other embodiments, the tumor-associated antigen may be found in both tumor cells and non-tumor cells, but over-expressed on tumor cells compared to non-tumor cells, or antibody binding may occur in tumor cells due to the less compact structure of tumor tissue compared to non-tumor tissue. In some embodiments, the tumor-associated antigen is located on the vasculature of the tumor.

Illustrative examples of tumor-associated antigens are LAG-3, CD10, CD19, CD20, CD22, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, fms-like tyrosine kinase 3 (FLT-3, CD 135), chondroitin sulfate proteoglycan 4 (CSPG 4, melanoma-associated chondroitin sulfate proteoglycan), epidermal Growth Factor Receptor (EGFR), her2neu, her3, IGFR, IL3R, fibroblast Activation Protein (FAP), CDCP1, derlin1, tenascin, frizzled 1-10, vascular antigens VEGFR2 (KDR/FLK 1), VEGFR3 (FLT 4, CD 309), pdgfra (CD 140 a), pdgfra (CD 140 b) Endoglin, CLEC, tem1-8, and Tie2. Further examples may include A33, CAMPATH-1 (CDw 52), carcinoembryonic antigen (CEA), carboxyanhydrase IX (MN/CA IX), de2-7EGFR, EGFRvIII, epCAM, ep-CAM, folate binding protein, G250, fms-like tyrosine kinase 3 (FLT-3, CD 135), c-Kit (CD 117), CSF1R (CD 115), HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (melanoma-associated cell surface chondroitin sulfate proteoglycan), muc-1, prostate Specific Membrane Antigen (PSMA), prostate Stem Cell Antigen (PSCA), prostate Specific Antigen (PSA), and TAG-72.

In certain embodiments, the bispecific antibodies provided herein have a first specificity for a TAA antigen and a second specificity for an infectious disease associated antigen or epitope thereof. Non-limiting examples of infectious disease-associated antigens include, for example, antigens expressed on the surface of viral particles, or antigens preferentially expressed on cells infected with a virus. Wherein the virus is selected from the group consisting of: HIV, hepatitis (type a, type b or type c), herpes viruses (e.g., HSV-1, HSV-2, CMV, HAV-6, VZV, epstein-Barr virus), adenoviruses, influenza viruses, flaviviruses, icoviruses, rhinoviruses, coxsackieviruses, coronaviruses, respiratory syncytial viruses, mumps viruses, rotaviruses, measles viruses, rubella viruses, parvoviruses, vaccinia viruses, HTLV, dengue viruses, papillomaviruses, molluscs, polioviruses, rabies viruses, JC viruses, and arbovirus encephalitis viruses. Alternatively, the target antigen may be an antigen expressed on the surface of a bacterium, or preferentially on a cell infected with a bacterium, wherein the bacterium is selected from the group consisting of: chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci, gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulinum, anthrax, plague, leptospira and lyme disease bacteria. In certain embodiments, the target antigen is an antigen expressed on the surface of a fungus, or preferably an antigen expressed on a cell infected with a fungus, wherein the fungus is selected from the group consisting of: candida (Candida) Candida (albicans), krusei (krusei) Candida, glabra (glabra) Candida, tropical (tropicalis) Candida, etc.), cryptococcus neoformans (Crytococcus neoformans), aspergillus (Aspergillus fumigatus, aspergillus niger, etc.), mucor (Mucorales) trichoderma (mucor, colpitis, rhizopus, etc.), sporotrichia (Sporothrix schenkii), blastodermatitidis (Blastomyces dermatitidis), paracoccidioidosporium brazil (Paracoccidioides brasiliensis), paracoccidiosporium crudus (Coccidioides immitis), and histoplasma capsulatum (Histoplasma capsulatum). In certain embodiments, the target antigen is an antigen expressed on the surface of a parasite, or an antigen preferentially expressed on cells infected with a parasite, wherein the parasite is selected from the group consisting of: endomainamia histolytica (Entamoeba histolytica), balanitidium colonospora (Balanitium coll), grignard (Naegleria fowleri), acanthamoeba (Acanthamoeba) species, giardia lamblia (Giardia), cryptosporidium (Cryptosporidium) species, pneumosporidium californicum (Pneumocystis carinii), plasmodium vivax, babesia microbeta (Babesia microti), trypanosoma brucei (Trypanosoma brucei), trypanosoma cruzi (Trypanosoma cruzi), leishmania donovani (Leishmania donovani), toxoplasma gondii (Toxoplasma gondii), brazilian round nematode (Nippostrongylus brasiliensis), taenia (Taenia crassiceps), and Silk worm (Brugia malayi). Non-limiting examples of specific 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 proteins.

The WuXiBody BsAb to be coupled herein may further comprise a lock knob-lock hole structure in the Fc region. Additional interchain disulfide bonds are created in the knob-keyhole region to increase the stability of the bispecific antibody. In this BsAb there are 5 pairs of interchain disulfide bonds, and it was found that under normal TCEP reduction, only 3 pairs of bonds would be broken to react with maleimide modified drugs, yielding DAR6 (> 90%) rich ADCs. The other 2 pairs of unnatural disulfide bonds (in the TCR and Fc regions) are not structurally exposed to reducing agents and therefore cannot be opened for drug attachment. In the case where the C1 and C2 regions form more than one unnatural disulfide bond, they are also structurally inaccessible to the reducing agent.

In some embodiments, a bispecific antibody to be coupled herein comprises two heavy chains and two light chains, wherein from N-terminus to C-terminus:

the first heavy chain comprises a domain operably linked as in VH 1-C1-hinge-Fc and the first light chain comprises a domain operably linked as in VL 1-C2; the second heavy chain comprises a domain operably linked as in VH2-CH 1-hinge-Fc and the second light chain comprises a domain operably linked as in VL2-CL, wherein VH1 and VL1 refer to the first heavy and light chain variable regions derived from the first parent antibody and VH2 and VL2 refer to the second heavy and light chain variable regions derived from the second parent antibody. VH1, C1, VL1 and C2 form a first antigen-binding portion, and VH2, CH1, VL2 and CL form a second antigen-binding portion.

Coupling method for preparing D6 ADC with high homogeneity

In one aspect, the present disclosure provides a method of preparing an antibody-drug conjugate (ADC) of high homogeneity (50%, 60%, 70%, 80%, 90% or higher of the total ADC produced) of a bispecific antibody as described above, the method comprising the steps of:

(a) Incubating a reducing agent and a bispecific antibody in a buffer system; and

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

(c) Recovering the resulting antibody-drug conjugate.

Optionally, the method further comprises adding an effective amount of an oxidizing agent after step (b) to reoxidize unreacted thiol groups.

As described above, the bispecific antibody to be coupled was constructed as a lock button-keyhole bispecific antibody having a TCR arm and a normal IgG Fab at its variable region. Additional interchain disulfide bonds are created in the knob-keyhole region to increase the stability of the bispecific antibody. For 5 pairs of interchain disulfide bonds, it was found that under normal TCEP reduction, only 3 pairs of bonds would break to react with a drug (e.g., maleimide modified drug) to produce a DAR6 rich (> 80% or even 90%) ADC.

In other words, although the WuXiBody version of the bispecific antibody contains 5 pairs of interchain disulfide bonds, conjugation to the antibody results in a highly homogeneous DAR6 (D6) species with a purity of 85-95%. Two pairs of unnatural disulfide bonds, one pair in the TCR constant region and the other pair in the "lock knob-lock hole" structure, are not reduced by the reducing agent, leaving only 3 pairs of disulfide bonds for reduction and site-specific coupling. Two of which are in the hinge region and the other pair are in the CH1-CL region of the second antigen binding portion.

In the resulting ADC, the content of D6 ADC may reach at least 80wt%, e.g., at least 85wt%, at least 90wt%, at least 91wt%, at least 92wt%, or at least 93wt%, based on the total weight of D0, D2, D4, and D6, as measured by HIC.

In some embodiments, the reducing agent may be TCEP. The concentration of the reducing agent in the reaction solution may be 0.04mM to 0.4mM.

In some embodiments, the oxidizing agent to be added may be DHAA. The concentration of the oxidizing agent in the reaction solution may be 0.08mM to 0.8mM.

The optimum pH for the reaction is typically from about 5.5 to about 8, for example from about 5.5 to 7.5. The optimum reaction conditions will of course depend on the particular reactants used.

In one embodiment, the buffer is PBS, pH 7.

The optimum temperature for the reduction and coupling reactions is typically from about-10 ℃ to 37 ℃, for example from about 4 ℃ to 22 ℃. For example, the reduction reaction is carried out at a temperature of about 22℃for 8-18 hours and the coupling reaction is carried out at a temperature of about 4℃overnight. The temperature and time may vary depending on the amounts of antibody, reducing agent and drug used.

It will be appreciated by those skilled in the art that the period of incubation and temperature in step (a) will depend on the specific antibody to be conjugated. Determination of incubation period and temperature based on specific antibodies is within the ability of one of ordinary skill in the art. For example, the antibody to be conjugated is typically incubated with a reducing agent overnight at 4 ℃.

In some embodiments, the concentration of bispecific antibody in the reaction is 0.01mM to 0.1mM. In a particular embodiment, the concentration of antibody is 0.02mM.

For step (c), the person skilled in the art can select an appropriate purification method to recover the resulting antibody-drug conjugate. Many methods of ADC purification are well known in the art. For example, the resulting antibody-drug conjugate may be purified by using a desalting column, size exclusion chromatography, or the like.

In some embodiments, the resulting antibody-drug conjugate is recovered by any suitable purification method, e.g., using a desalting column, size exclusion chromatography, ultrafiltration, dialysis, UF-DF, or the like.

Coupling method for preparing ADC of D2 with high homogeneity

The transition metal ion and/or divalent metal ion may protect the disulfide bond in the hinge region from being reduced during incubation of the reducing agent and the antibody. The inventors herein combine this metal ion chelating function with WuXiBody antibodies for ADC coupling, resulting in a very narrow DAR2 distribution, which allows for the preparation of low DAR ADCs. Low DAR ADCs are required when the drug (e.g., PBD) has a strong potency and an excessively high DAR leads to toxicity problems.

When a metal ion such as Zn (II) is added to the reaction system, two disulfide bonds in the hinge region are shielded from reduction, and only one pair of disulfide bonds in the CH1-CL region side of IgG is reduced, and only DAR2 species are generated upon drug addition. The PK/PD characteristics of ADCs of this highly homogeneous D2 population are comparable to those produced by antibody engineering. The preparation of such ADCs is also easy. Low DAR ADCs are very important when low DAR is required, for example when the drug is too toxic for high loads.

Accordingly, in one aspect, the present disclosure provides a method of preparing an antibody-drug conjugate (ADC) of a bispecific antibody described above, the method comprising the steps of:

(a) Incubating a reducing agent and a bispecific antibody in a buffer system in the presence of an effective amount of a metal ion, such as a transition metal ion and a divalent metal ion, to selectively reduce interchain disulfide bonds within the antibody;

(c) Recovering the resulting antibody-drug conjugate.

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

In some embodiments, metal ions suitable for use in the bioconjugate methods of the present disclosure are selected from transition metal ions and divalent ions, including, but not limited to: zn2+, cd2+, ca2+, mg2+, hg2+, and the like. Wherein zn2+ may be used due to its simple availability and low cost. For example, suitable transition metal salts may be added in step (a) as long as they are soluble in the reaction solution, so that free transition metal ions may be released in the reaction solution. In this regard, znCl2, zn (NO 3) 2, znSO4, zn (CH 3 COO) 2, znI2, znBr2, zinc formate and zinc tetrafluoroborate may be suitable zinc salts. Likewise, other transition metal salts that are soluble in the reaction solution and capable of releasing free cd2+ or hg2+ ions may be mentioned, including but not limited to CdCl2, cd (NO 3) 2, cdSO4, cd (CH 3 COO) 2, cdI2, cdBr2, cadmium formate and cadmium tetrafluoroborate; hgCl2, hg (NO 3) 2, hgSO4, hg (CH 3 COO) 2, hgBr2, mercury (II) formate, and mercury (II) tetrafluoroborate; etc. Those skilled in the art can select from the transition metal salts and divalent metal salts described above as the source of metal ions.

In one embodiment zn2+ is used in step (a). Water soluble zinc salts may be used. For example, znCl2 is added as a Zn2+ source in step (a).

The concentration of the metal ion in the reaction solution in step (a) may be 0.01mM to 0.2mM.

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

Based on the metal ion, one 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 a specific embodiment, the buffer system used in step (a) is PBS.

In one embodiment, the buffer is PBS, pH 7.

The optimum temperature for the reduction and coupling reactions is typically from about-10 ℃ to 37 ℃, for example from about 4 ℃ to 22 ℃. For example, the reduction reaction is carried out overnight at a temperature of about 4℃and the coupling reaction is carried out for 2-8 hours at a temperature of about 4 ℃. The temperature and time may vary depending on the amounts of antibody, reducing agent and drug used.

For example, the antibody to be coupled, the metal ion and the reducing agent may be present in the reaction mixture in a molar concentration ratio of 1:2:4. In one embodiment, 0.02mM antibody is incubated with 0.08mM TCEP and 0.04mM ZnCl2 overnight at 4 ℃. Those skilled in the art will appreciate that molar concentrations can also be converted to "equivalents (eq)" compared to antibodies. For example, if 0.02mM antibody is used, "0.04mM ZnCl2" can be converted to "2eq ZnCl2".

In the resulting ADC, the content of D2 ADC may reach at least 80wt%, e.g. at least 85wt%, at least 90wt%, at least 91wt%, at least 92wt%, at least 93wt%, at least 94wt% or at least 95wt%, based on the total weight of D0 and D2, as measured by HIC.

Coupling method for preparing D2+4 dual-drug ADC with high homogeneity

It is often beneficial to combine drugs with different mechanisms of action to treat and may improve the outcome of the treatment. Although targeting methods for ADCs often produce favorable results, (acquired) resistance often occurs, limiting the effectiveness of the ADC. One way to circumvent the problem of ADC-related drug resistance is to combine ADCs with different drugs, or to use ADCs with multiple drug loads. In one aspect, the present disclosure provides a method for producing an ADC having a high degree of homogeneity (greater than 50%, 60%, 70%, 80%, 90% or higher of the total produced ADC) for two different loads.

Based on both of the above approaches, two-step coupling was developed, i.e., coupling the first drug moiety in the presence of an effective amount of a metal ion, and the second drug moiety in the absence of such a metal ion, and resulted in a highly homogeneous ADC of DAR (2+4). Such dual drug ADCs are more resistant to multi-drug resistance from cancer cells of the cancer therapy and are readily produced by this coupling method. The designation "DAR (a+b)" or "D (a+b)" herein refers to a dual drug ADC, wherein the number of first drugs coupled to a single antibody molecule is "a" and the number of second drugs coupled to an antibody molecule is "b".

Accordingly, in one aspect, the present disclosure provides a method of preparing an antibody-drug conjugate (ADC) of a bispecific antibody as described above, wherein the method comprises the steps of:

(c) Removing metal ions from the product of step (b);

(d) Adding the reducing agent again and incubating with an excess of the second linker-drug moiety; and

(e) Recovering the resulting antibody-drug conjugate.

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

The reaction conditions for reduction and coupling may be the same as those disclosed above for preparing D2 ADC. The optimum temperature for the reduction and coupling reactions will generally be between about-10 ℃ and 37 ℃, for example between about 4 ℃ and 22 ℃. The second reduction and coupling of steps (c) and (d) may be slightly different from steps (a) and (b). For example, the reduction reaction of step (c) is carried out at a temperature of about 22℃for 2-8 hours, while the coupling reaction is carried out at about 22℃for 2-8 hours. The temperature and time may vary depending on the amounts of antibody, reducing agent and drug used.

In some embodiments, the first linker-drug moiety is MC-MMAF and the second linker-drug moiety is MC-DXD or DXD with other linkers.

In the resulting ADC, the content of d2+4ADC may reach at least 80wt%, e.g., at least 85wt%, at least 90wt%, at least 91wt%, at least 92wt%, at least 93wt%, at least 94wt%, or at least 95wt%, based on the total weight of D0, D2, d2+2, and D4, as calculated by MS analysis.

Highly homogeneous ADC of bispecific antibodies showed better efficacy

The main problem with conventional conjugation of ADCs is the heterogeneity of ADC molecules, where the drug moiety is attached to several sites on the antibody by e.g. cysteine, e.g. ranging from 0 to 8 per antibody (drug-to-antibody ratio, DAR). Not only does the ADC molecules of such mixtures present difficulties in analysis and characterization, but they may also have different pharmacokinetic, distribution, toxicity and efficacy profiles. Nonspecific coupling also often results in impaired antibody function. Thus, a narrow distribution of DARs is desired to achieve better PK, efficacy, and therapeutic window.

In one aspect, the present disclosure provides highly homogeneous antibody-drug conjugates of bispecific antibodies, wherein the D6 ADC comprises more than 80wt% of the ADC.

In one aspect, the present disclosure provides highly homogeneous antibody-drug conjugates of bispecific antibodies, wherein the D2 ADC comprises more than 80wt% of the ADC.

In one aspect, the present disclosure provides highly homogeneous antibody-drug conjugates of bispecific antibodies, wherein d2+4ADC comprises more than 80wt% of ADC.

In some embodiments, the homogeneity of the antibody-drug conjugate produced by the methods disclosed herein is measured and compared to the homogeneity of a corresponding control antibody-drug conjugate produced by conventional conjugation methods.

Various analytical methods can be used to determine the yield and isomer mixtures of antibody-drug conjugates. For example, in one embodiment, hydrophobic Interaction Chromatography (HIC) is an analytical method for determining yields and isomer mixtures from the resulting antibody-drug conjugates (e.g., for D6 conjugates). This technique enables the isolation of antibodies loaded with different numbers of drugs. Drug loading levels may be determined based on the ratio of absorbance, for example at 250nm and 280 nm. For example, the drug may absorb at 250nm and the antibody at 280 nm. Thus, the ratio of 250/280 increases with increasing drug loading. Using the bioconjugate methods described herein, antibodies with even numbers of drugs are typically observed, as incomplete conjugation results in even DAR.

Linker-drug moiety

Local delivery of cytotoxic or cytostatic agents, i.e. agents that kill or inhibit tumor cells in cancer treatment, using antibody-drug conjugates can target drug moieties to the tumor and accumulate within the cell, whereas systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells as well as tumor cells attempting to eliminate (Thorpe, (1985) ' Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: a Review ', at Monoclonal Antibodies '84:Biological And Clinical Applications,A.Pinchera et al (ed. S), pp. 475-506). Polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al, (1986) Cancer immunol. Immunother., 21:183-87).

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

The drugs and linkers useful in the coupling methods of the present disclosure are not particularly limited as long as the drug molecule has an anti-tumor, anti-viral or anti-microbial effect and contains at least one substituent group or partial structure that allows attachment to the linker structure, and the linker contains at least two reactive groups, one of which can be covalently bound to the drug molecule and the other of which can be covalently coupled to the antibody. Preferably, the linker is susceptible to-SH attack by the antibody and is capable of forming a link with the antibody.

The skilled artisan can select an appropriate method to couple the desired drug and selected linker together, depending on the method. For example, some conventional coupling methods, such as amine coupling methods, may be used to form the desired drug-linker complex, which still contains reactive groups for coupling to the antibody via covalent bonds. In the present disclosure, a drug-maleimide complex (i.e., a maleimide-linked drug) is one example of carrying a load-reactive group.

In one embodiment, the drug may include, but is not limited to, a cytotoxic agent, such as a chemotherapeutic agent, immunotherapeutic agent, etc., an antiviral agent, or an antimicrobial agent. In one embodiment, the drug to be conjugated to the antibody may be selected from, but is not limited to, MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), MMAF (monomethyl auristatin F), and the like.

In ADC preparation, the most common reactive group capable of binding to thiol is maleimide. In addition, organic bromides and iodides are also frequently used.

Enzymatically active toxins and fragments thereof that may be used include diphtheria chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from pseudomonas aeruginosa (Pseudomonas aeruginosa)), ricin a chain, abrin a chain, pristimecin (modeccin) a chain, α -sarcina, aleurites fordii protein, carnation (dianin) protein, pokeberry (Phytolaca americana) protein (PAPI, PAPII and PAP-S), balsam pear (Momordica charantia) inhibitor, jatrophin (curcin), crotin (crotin), soapberry (sapaonaria officinalis) inhibitor, gelonin (gelonin), mitogelonin (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and trichothecene (trichothecene). See, for example, WO 93/21232 published on month 28 of 1993. A variety of radionuclides are useful for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. One or more small molecule toxins, such as calicheamicin (calicheamicin), maytansinoids, dolastatins, auristatins, trichothecenes, and CC1065, and derivatives of these toxins having toxin activity, may be conjugated to an antibody by the methods disclosed herein.

Maytansine compounds suitable for use as the pharmaceutical portion of maytansinoids are well known in the art and may be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinols and maytansinol analogues prepared synthetically according to known methods. Suitable maytansinoids are disclosed, for example, in U.S. Pat. No. 5,208,020. Preferred maytansinoids are maytansinol and maytansinol analogs modified at the aromatic ring or other positions of the maytansinol molecule, such as various maytansinol esters.

Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, cell nuclei and cell division, and have anticancer and antifungal activity (Pettit et al (1998) Antimicrob. Agents chemother. 42:2961-2965). The dolastatin or auristatin drug moiety can be attached to the antibody via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02/088172). Exemplary embodiments including MMAE or MMAF and various linker components are shown below. For example, vcMMAE (Mc-vc-PAB-MMAE) was obtained by using MMAE linked to the lysosomally cleavable dipeptide valine-citrulline (vc) and thiol-reactive maleimide-hexanoyl spacer (Mc) via p-aminobenzyloxycarbonyl ("PAB").

Conjugates of antibodies and cytotoxic agents are prepared using a variety of bifunctional protein coupling agents, for example, N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipic acid HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexamethylenediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene). For example, ricin immunotoxins may be prepared as described in Vitetta et al (1987) Science, 238:1098. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is an exemplary chelator for coupling radionucleotides to antibodies (WO 94/11026).

Additional exemplary embodiments are provided that include MMAE or MMAF and various linker components. For example, vcMMAE (Mc-vc-PAB-MMAE) was obtained by using MMAE linked to the lysosomally cleavable dipeptide valine-citrulline (vc) and thiol-reactive maleimide caproyl spacer (Mc) via p-aminobenzyloxycarbonyl ("PAB").

Pharmaceutical composition

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

The compositions of the present disclosure may be in any form that allows the composition to be administered to an animal. For example, the composition may be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular and intranasal. Parenteral administration includes subcutaneous injections, intravenous injection, intramuscular injection, intrasternal injection or infusion techniques. For example, the composition is administered parenterally. The pharmaceutical compositions of the present invention may be formulated such that the ADCs of the present disclosure are bioavailable when the compositions are administered to animals. The composition may take the form of one or more dosage units, where, for example, a tablet may be a single dosage unit and a container containing an ADC of the present disclosure in aerosol form may hold a plurality of dosage units.

The materials used to prepare the pharmaceutical compositions may be non-toxic in the amounts used. It will be clear to one of ordinary skill in the art that the optimal dosage of the active ingredient in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, but are not limited to, the type of animal (e.g., human), the particular form of ADC, the mode of administration, and the composition used.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavouring agents, thickening agents, colouring agents, emulsifying agents or stabilizing agents, for example sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene and/or propyl gallate. As disclosed herein, inclusion of one or more antioxidants, such as methionine, in the pharmaceutical compositions provided herein reduces oxidation of the polypeptide complex or bispecific polypeptide complex. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving protein stability and maximizing shelf life. Thus, in certain embodiments, compositions are provided comprising a polypeptide complex or bispecific polypeptide complex disclosed herein and one or more antioxidants such as methionine.

To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, ringer's injection, isotonic dextrose injection, sterile water injection or dextrose and lactate ringer's injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, antimicrobial agents of bacteriostatic or fungistatic concentration, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethyl cellulose or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), release or chelating agents such as EDTA (ethylenediamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents useful as carriers can be added to pharmaceutical compositions in multi-dose containers, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoate, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

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 grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like.

In certain embodiments, the pharmaceutical composition is formulated as an injectable composition. The injectable pharmaceutical composition may be prepared in any conventional form, for example, as a liquid solution, suspension, emulsion or solid form suitable for producing a liquid solution, suspension or emulsion. The injectable preparation may include sterile and/or pyrogen-free solutions ready for injection, sterile dry soluble products such as lyophilized powders ready for combination with solvents prior to use, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products ready for combination with vehicles prior to use, and sterile and/or pyrogen-free emulsions. The solution may be aqueous or non-aqueous.

In certain embodiments, the unit dose parenteral preparation is packaged in an ampoule, vial or syringe with needle. All formulations for parenteral administration should be sterile and pyrogen-free, as known and practiced in the art.

In certain embodiments, sterile lyophilized powders are prepared by dissolving the ADCs disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those skilled in the art. In one embodiment, the solution is about neutral pH. The solution is then sterile filtered and then freeze-dried under standard conditions known to those skilled in the art to provide the desired formulation. In one embodiment, the resulting solution will be dispensed into vials for lyophilization. Each vial may contain a single dose or multiple doses of an ADC provided herein or a composition thereof. To facilitate accurate sample extraction and accurate administration, an overfilled vial may be received in an amount slightly (e.g., about 10%) greater than that required for a dose or group of doses. The lyophilized powder may be stored under suitable conditions, for example, at a temperature of about 4 ℃ to room temperature.

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

In addition, the antibody-drug conjugate or pharmaceutical composition may be formulated into a kit including inserts indicating application information such as indication, amount of use, route of administration, and the like.

Use of ADC with improved homogeneity

In one aspect, the present disclosure relates to the use of an antibody-drug conjugate with improved homogeneity prepared by the foregoing method for the preparation of a pharmaceutical composition or kit for treating a condition or disorder in a subject.

The subject may be a mammal, such as a human.

The condition or disorder to be treated may be a tumor, cancer, autoimmune disease or infectious disease. In particular embodiments, the infectious disease may be a viral or microbial infection.

In one aspect, the present disclosure also relates to a method for treating a subject having a condition or disorder, comprising: administering to a subject in need thereof a therapeutically effective amount of an ADC having improved homogeneity prepared by a method disclosed herein or a therapeutically effective amount of a pharmaceutical composition comprising an ADC having improved homogeneity prepared by a method disclosed herein, thereby treating or preventing the condition or disorder.

In certain embodiments, the subject has been identified as having a condition or disorder that is likely to respond to an ADC provided herein.

The subject may be a mammal, such as a human.

The therapeutically effective amount of the ADCs provided herein will depend on various factors known in the art, such as body weight, age, history of previous disease, current medication, health status of the subject, and the likelihood of cross-reactivity, allergy, sensitivity and adverse side effects, as well as the route of administration and the extent of disease progression. The dosage may be reduced or increased proportionally by one of ordinary skill in the art (e.g., a doctor or veterinarian), as indicated by these and other circumstances or requirements.

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

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

The ADCs or pharmaceutical compositions provided herein may be administered by any route known in the art, such as parenterally (e.g., subcutaneously, intraperitoneally, intravenously, including intravenous infusion, intramuscular, or intradermal injection) or parenterally (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical administration).

In certain embodiments, the condition or disorder treated by the ADCs or pharmaceutical compositions provided herein is cancer or a cancerous condition, an autoimmune disease, or an infectious disease.

The cancer may be an antigen-positive cancer, including lung cancer, breast cancer, colon cancer, ovarian cancer, and pancreatic cancer, such as cancer associated with a tumor-associated antigen.

Other specific types of cancers that may be treated with the ADCs or pharmaceutical compositions provided herein include, but are not limited to, solid tumors, including, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphoendothelioma, synovial carcinoma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, renal 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, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary gland carcinoma, cyst adenocarcinoma, medullary carcinoma, bronchi carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryo carcinoma, wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial cancer, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniofoam, ependymoma, pineal tumor, angioma, glioblastoma, oligodendroglioma, skin cell carcinoma, melanoma, retinoblastoma, meningioma; cancers of blood origin, 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 megakaryoblastic leukemia, acute granulomonocytic lymphoma, acute nonlymphoblastic leukemia, acute undifferentiated leukemia, chronic Myelogenous Leukemia (CML), chronic Lymphoblastic Leukemia (CLL), hairy cell leukemia, multiple myeloma; lymphoma: b-cell lymphoma, optionally hodgkin's lymphoma or non-hodgkin's lymphoma, wherein the non-hodgkin's lymphoma comprises: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphohistiolymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL) or Mantle Cell Lymphoma (MCL), acute Lymphoblastic Leukemia (ALL) or Waldenstrom Macroglobulinemia (WM).

Autoimmune diseases may include, but are not limited to, active chronic hepatitis, addison's disease, allergic alveolitis, allergic reactions, allergic rhinitis, alport syndrome, allergy (anaplaxis), ankylosing spondylitis, antiphospholipid syndrome, arthritis, ascariasis, aspergillosis, atopic allergies, atrophic dermatitis, atrophic rhinitis, behcet's disease, bird-fed lung, bronchial asthma, capelan syndrome, cardiomyopathy, celiac disease, chagas's disease, chronic glomerulonephritis, ke Genshi syndrome, condensed collectin disease, congenital rubella infection, CREST syndrome, crohn's disease, cryoglobulinemia, cushin syndrome, dermatomyositis, lupus, dressler syndrome, eaton-Lambert syndrome, ehrymal virus infection, encephalomyelitis, endocrinopathy, epstein-Barr virus infection, equine heavies (Equipment heavies), erythema, evan syndrome, feltey syndrome, fibromyalgia, fuch's ciliary inflammation, gastric atrophy, gastrointestinal allergies, 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 fibrinolysis, igA nephropathy, inflammatory bowel disease, insulin dependent diabetes mellitus, juvenile arthritis, juvenile diabetes mellitus (type I), lambert-Eaton syndrome, petechiae, lichen planus, lupus hepatitis, lupus, lymphopenia, meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyadenopathy, early dementia, primary agarop globulinemia, primary liver cirrhosis, psoriasis, raynaud's disease, recurrent abortion, reiter syndrome, rheumatic fever, rheumatoid arthritis, samter syndrome, schistosomiasis, schmidt syndrome, scleroderma, shulman syndrome, sjorgen syndrome, stiff-Man syndrome, sympathoophthalmia, systemic lupus erythematosus, takayasu arteritis, temporal arteritis, thyroiditis, thrombocytopenia, thyrotoxicities, toxic epidermolysis, insulin resistance type B, diabetes type I, ulcerative colitis, uveitis, vitiligo, waldenstrom macroglobulinemia, wegener granulomatosis.

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

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

In some embodiments, the therapeutic agent is an anti-autoimmune disease agent. Suitable anti-autoimmune disease agents include, but are not limited to, cyclosporine a, mycophenolate mofetil (mycophenylate mofetil), sirolimus, tacrolimus, etanercept, prednisone, azathioprine, methotrexate-cyclophosphamide, prednisone, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorobenzoyl hydrazine, DHEA, danazol, bromocriptine, meloxicam, and infliximab.

In some embodiments, the therapeutic agent is an anti-infective agent. In some embodiments, the anti-infective agent is, but is not limited to, an antibacterial agent: [ beta ] -lactam antibiotics: penicillin G, penicillin V, cloxacillin, dicloxacillin, methicillin, nevuxillin, oxacillin, ampicillin, amoxicillin, baoxacillin, azlocillin, carbenicillin, mezlocillin, piperacillin, ticarcillin; aminoglycosides: amikacin, gentamicin, kanamycin, neomycin, natamycin, streptomycin, tobramycin; macrolides: azithromycin, clarithromycin, erythromycin, lincomycin, clindamycin; tetracyclines: the combination of minocycline, doxycycline, minocycline, oxytetracycline, and tetracycline; quinolones: xin Meisu nalidixic acid; fluoroquinolones: ciprofloxacin, enoxacin, glafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin, and terofloxacin; polypeptide: bacitracin, colistin, polymyxin B; sulfonamide: sulfoisoazole, sulfamethoxazole, sulfadiazine, sulfamethoxazole, and sulfaacetamide; hybrid antibacterial agents: trimethoprim, sulfamethoxazole, chloramphenicol, vancomycin, metronidazole, quinapril, megafostine, rifampin, spectinomycin, nitrofurantoin; antiviral agents: general antiviral agents: iodoside (idoxuridine), vidarabine, trifluoperazine, acyclovir, famciclovir, pan Xihuan, valacyclovir, gan Xihuan, foscarnet, ribavirin, amantadine, rimantadine, cidovir, antisense oligonucleotides, immunoglobulins, interferons; drugs for HIV infection: zidovudine, didanosine, zalcitabine, stavudine (Stavudine), lamivudine, nevirapine, delavirdine, saquinavir, ritonavir, indinavir, nelafenavir.

The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials, and methods described below fall within the scope of the invention, in whole or in part. These particular compositions, materials, and methods are not intended to limit the invention but are merely illustrative of specific embodiments that fall within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials and methods without departing from the scope of the invention. It should be understood that many variations may be made in the process described herein while still remaining within the scope of the present invention. It is the intention of the inventors to include such variations within the scope of the invention.

Examples

The present disclosure will now be described in detail with reference to the following examples. However, it will be understood by those skilled in the art that the following examples are provided for illustration only and are not intended to limit the present disclosure in any way.

Example 1 production of wuxibody bispecific antibodies

1.1 Construction of a bispecific antibody in the form of WuXiBody E17

TABLE 1

Antibodies to	Parent antibodies
		cAb1	Trastuzumab x pertuzumab
cAb2	Pertuzumab x trastuzumab
		cAb3	Trastuzumab x Sha Xizhu mab
cAb4	Sha Xizhu mab x trastuzumab
		cAb5	Sha Xizhu monoclonal antibody x cetuximab
cAb6	Cetuximab x Sha Xizhu mab
		cAb7	Trastuzumab x pertuzumab
cAb8	Trastuzumab x pertuzumab

8 bispecific antibodies were constructed (Table 1). The VL, VH genes (obtained from the parent antibodies in table 1) were amplified by PCR from existing plasmid templates. TCR C.alpha.and C.beta.genes (except for encoding SEQ ID Nos: 2 and 4, cAb7 and cAb 8) were synthesized by Genewiz Inc (Suzhou, china) using the different C1 and C2 region sequences shown in SEQ ID Nos: 7-9. VL (VL) ₁ -Cα and VL ₂ The DNA fragment of CL was inserted into a linearization vector containing the CMV promoter and the human light chain signal peptide, respectively. VH (VH) ₁ The DNA fragment of Cβ was inserted into a linearized vector containing the human IgG1 constant region CH2-CH3 with a "lock knob" mutation. VH (VH) ₂ Insertion of a DNA fragment of-CH 1 containing a mutation with a "keyholeIs a linearized vector of the human IgG1 constant region CH2-CH 3. The vector comprises a CMV promoter and a human antibody heavy chain signal peptide. An exemplary structure of cAb1 is shown in the reaction scheme of fig. 1A.

1.2 Expression and purification of WuXiBody bispecific antibodies

The heavy and light chain expression plasmids were co-transfected into an Expi293 cell (Invitrogen-A14527) using the expression system kit (Invitrogen-A14524) according to the manufacturer's instructions. 5 days after transfection, the supernatant was collected and used for protein purification using protein A chromatography (GE Healthcare-17543802). Further purification was performed using size exclusion chromatography (GE Healthcare-17104301), if desired. Antibody concentration was measured by Nano Drop. The purity of the protein was evaluated by SDS-PAGE and HPLC-SEC.

EXAMPLE 2 coupling to form D6 species

The reaction scheme shown in FIG. 1A (cAb 1 as an example) was performed. 10mg/mL mAb in PBS buffer was reduced by 7.5eq TCEP and the reaction vials were left at 22℃for 8 hours. The reduced antibody solution was directly subjected to the next coupling without removal of TCEP.

DMA and linker-loading solution (10 mg/mL stock in DMA, relative to antibody 18.0 eq.) were added to the reduced antibody solution. The reactants were mixed appropriately and the reaction vials were left at 4 ℃ for 18 hours. The crude product was buffer exchanged into its storage buffer (1 XPBS buffer, pH 7.4, gibco) using a spin-desalting column (40 kD). The resulting ADC was characterized.

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 10mL/min at ambient temperature. The loading was 7.5mg of ADC per 1mL column volume. Solvent A was 2.0M NaCl and 50mM sodium phosphate, pH 7. Solvent B was 80% v/v 50mM sodium phosphate pH 7 and 20% v/v acetonitrile. The column was equilibrated beforehand with 5 column volumes of solvent a. The ADC was mixed with 0.67 volumes of 5M NaCl (final 2.0M) and applied to the column. D0 is not retained by the column. Different drug loading species were eluted by sequential step gradients: d2 eluted with 35% solvent B, D4 eluted with 70% solvent B, and D6 eluted with 95% solvent B.

For MS analysis, 75.0. Mu.l of 8.0mol/L Gdn-HCl, 5.0. Mu.l of 1.0mol/L Tris-HCl and 2.0. Mu.l of 1mol/L DTT were added to 20.0. Mu.l of the ADC sample solution. The new solution is fully mixed and then incubated for 10-30 min at 10-30 ℃. The drug/antibody ratio (DAR) and product distribution were then analyzed using LC-MS.

As shown in table 2 and fig. 1B-1D, the cAb1, cAb3, and cAb4 antibodies obtained ADCs with about 90% or higher homogeneity of the D6 species. Similar results were obtained for the other 5 antibodies.

Table 2: HPLC results of ADC homogeneity

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

EXAMPLE 3 coupling to form D2 species

3.0eq TCEP and 1.0eq ZnCl2 were added to the mAb. The reaction vials were left at 4℃for 18 hours. DMA and linker-loading solution (10 mg/mL stock in DMA) were added to the reduced antibody solution. The reactants were mixed appropriately and the reaction vials were left at 4 ℃ for 2 hours.

Cysteine was added to the mixture. The new mixture was then left at 4℃for 15 minutes. After addition of 3.0eq EDTA and 8.0eq DHAA, the mixture was left at 22℃for 2 hours.

The crude product was buffer exchanged into its storage buffer (1 XPBS buffer, pH 7.4, gibco) using a spin-desalting column (40 kD). The resulting ADC (fig. 2A) was characterized.

As shown in table 3 and fig. 2B, the cAb1, cAb2, cAb3, and cAb4 antibodies obtained ADCs with more than 80% homogeneity of the D2 species. Similar results were obtained with the other 4 antibodies.

TABLE 3 Table 3

EXAMPLE 4 coupling to form the D (2+4) species

Using the reaction scheme shown in FIG. 3A and Table 4 (in the case of cAb 1), D2 ADC (mAb conjugated to linker-payload 1) in TCEP reduction buffer (1 XPBS buffer, pH 7.4, gibco) was used and the reaction vials were left at 22℃for 2 hours. The reduced antibody solution was directly subjected to the next coupling without removal of TCEP.

DMA and linker-payload 2 solution (10 mg/mL stock in DMA) were added to the reduced antibody solution. The reactants were mixed appropriately and the reaction vials were left at 22 ℃ for 2 hours.

The crude product was buffer exchanged into its storage buffer (1 XPBS buffer, pH 7.4, gibco) using a spin-desalting column (40 kD). The resulting ADC was characterized (fig. 3B-3E).

From the MS results of fig. 3B, it can be calculated that the d2+4adc content reaches more than 90% (in the case of cAb 1).

TABLE 4 Table 4

Those skilled in the art will further appreciate that the invention may be embodied in other specific forms without departing from the spirit or central attributes thereof. Since the foregoing description of the invention discloses only exemplary embodiments thereof, it should be understood that other variations are also within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims as indicative of the scope and content of the invention.

Claims

1. A method for preparing an antibody-drug conjugate (ADC), wherein the antibody comprises a first antigen binding portion and a second antigen binding portion,

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 one or more non-natural inter-chain disulfide bonds, and

the second antigen binding portion comprises a Fab, scFv or VHH,

and wherein the method comprises the steps of:

(a) Incubating a reducing agent and the antibody in a buffer system;

(c) Recovering the resulting antibody-drug conjugate.

2. The method of claim 1, wherein the second antigen-binding portion comprises 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.

3. The method of claim 1 or 2, wherein the incubating in step (a) is performed in the presence of an effective amount of one or more transition metal ions and/or divalent metal ions.

4. The method according to claim 1 or 2, wherein the incubation in step (a) is not performed in the presence of an effective amount of transition metal ions or divalent metal ions.

5. A method according to claim 3, wherein the method further comprises, between steps (b) and (c): removing the metal ions from the product of step (b) and then reintroducing the reducing agent and incubating with an excess of the different linker-drug moiety.

6. A method for preparing an antibody-drug conjugate (ADC), wherein the antibody comprises a first antigen binding portion and a second antigen binding portion,

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-native inter-chain disulfide bonds, and

and wherein the method comprises the steps of:

(a) Incubating a 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 inter-chain disulfide bonds within the antibody;

(c) Removing the metal ions from the product of step (b);

(d) Incubating again with a reducing agent (optionally the same as the reducing agent in step (a)) and introducing an excess of a second linker-drug moiety; and

(e) Recovering the resulting antibody-drug conjugate.

7. The method of any one of the preceding claims, wherein the method further comprises adding an effective amount of an oxidizing agent (e.g., DHAA) to reoxidize unreacted thiol groups prior to recovering the resulting antibody-drug conjugate.

8. The method according to any of the preceding claims, wherein the C1 region comprises the amino acid sequence of SEQ ID nos. 2, 7 or variants thereof having at least 90% identity and the C2 region comprises the amino acid sequence of SEQ ID nos. 4, 8, 9 or variants thereof having at least 90% identity, or

The C1 region comprises the amino acid sequences of SEQ ID Nos. 4, 8, 9 or variants thereof having at least 90% identity, and the C2 region comprises the amino acid sequences of SEQ ID Nos. 2, 7 or variants thereof having at least 90% identity.

9. The method according to any of the preceding claims, wherein the antibody comprises an IgG Fc region, e.g. of the IgG1 or IgG4 isotype, preferably the Fc region comprises a knob-keyhole (knob-in-hole) structure.

10. The method according to any one of claims 3-9, wherein the transition metal ion and/or divalent metal ion in step (a) is selected from the group consisting of: zn (zinc) ²⁺ 、Cd ²⁺ 、Hg ²⁺ 、Ca ²⁺ 、Mg ²⁺ And any combination thereof.

11. The method of claim 10, wherein the metal ion is selected from Zn ²⁺ 、Ca ²⁺ And Mg (magnesium) ²⁺ 。

12. The method according to any of the preceding claims, wherein the buffer system used in step (a) is selected from the group comprising: hepes, histidine buffer, PBS and MES, and a pH of about 5.5 to 8.

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

14. The method according to any of the preceding claims, wherein step (a) is performed at a temperature of about-10 ℃ to 37 ℃, such as about 0 ℃ to 22 ℃.

15. The method according to any of the preceding claims, wherein the reducing agent in step (a) is TCEP.

16. The method of any one of the preceding claims, wherein the linker-drug moiety is a drug-carrying maleimide, a drug-carrying organobromide, or a drug-carrying organoiodide.

17. The method according to any of the preceding claims, wherein the drug to be coupled is selected from the group comprising: diagnostic agents, therapeutic agents, and labeling agents.

18. The method according to any one of the preceding claims, wherein the drug is selected from maytansinoids such as DM1, DM3, DM4, dolastatins, dolastatin peptide analogues and derivatives such as australistatins, calicheamicin, trichothecene and CC1065, optionally MMAE, DM1 or MMAF.

19. The method of any one of the preceding claims, wherein the method comprises producing the antibody prior to step (a), wherein the variable region of the first antigen binding portion is derived from a first parent antibody and the variable region of the second antigen binding portion is derived from a second parent antibody.

20. The method of claim 19, wherein the first parent antibody and the second parent antibody are the same.

21. The method of claim 20, wherein the parent antibody is a monospecific, bispecific or multispecific antibody.

22. The method according to any one of the preceding claims, wherein the variable regions of the first and second antigen binding portions are derived from any of the following parent antibodies: trastuzumab (trastuzumab), pertuzumab (pertuzumab), sha Xizhu mab (sacituzumab), acipimab (abciximab), adalimumab (adalimumab), adalimumab (alexaprop), alemtuzumab (alemtuzumab), basiliximab (basiliximab), belimumab (belimumab), bei Luotuo Shu Shan mab (bezlotoxumab), canumab (canakiumab), cetuximab (certolizumab pegol), cetuximab (cetuximab), daclizumab (daclizumab), didanoumab (denosumab), efuzumab (alemtuzumab), golimumab (golimumab), infliximab (infliximab), ipiumab (basilizumab), yizumab (iximab), oxuzumab (bezizumab), oxytuzumab (oxytuzumab) and oxytuzumab (tacuzumab) are assigned to be used as the trastuzumab, the trastuzumab (tacuab) and the oxytuzumab (tacuzumab) is assigned to be the therapeutic antibody.

23. The method of claim 22, wherein the first VH and first VL are from trastuzumab and the second VH and second VL are from pertuzumab, or vice versa.

24. The method of claim 22, wherein the first VH and first VL are from trastuzumab and the second VH and second VL are from Sha Xizhu mab, or vice versa.

25. A method according to claim 3, wherein the resulting antibody-drug conjugate comprises D2 in an amount of more than 80wt%, such as more than 85wt%, more than 90wt% or more than 95wt%, based on the total weight of D0 and D2.

26. The method according to claim 4, wherein the resulting antibody-drug conjugate comprises D6 in an amount of more than 85wt%, such as more than 90wt%, more than 91wt%, more than 92wt% or more than 93wt%, based on the total weight of D0, D2, D4 and D6.

27. The method according to claim 5 or 6, wherein the resulting antibody-drug conjugate comprises d2+4 in an amount of more than 65wt%, such as more than 70wt%, more than 80wt% or more than 90wt%, based on the total weight of the ADC.

28. An antibody-drug conjugate prepared by the method of any one of the preceding claims.

29. A pharmaceutical composition comprising an effective amount of the antibody-drug conjugate of claim 28 and a pharmaceutically acceptable carrier or vehicle.

30. Use of an antibody-drug conjugate according to claim 28 in the manufacture of a pharmaceutical composition or kit for treating a condition or disorder in a subject.

31. Use of an antibody form in the preparation of a highly homogeneous ADC for a selected antigen, wherein the antibody form comprises a first antigen binding portion and a second antigen binding portion,

the first antigen binding portion specifically binds a first selected antigen and 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 one or more non-natural inter-chain disulfide bonds,

the second antigen-binding portion specifically binds a second selected antigen and comprises 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, an

The one or more non-natural interchain disulfide bonds are not accessible to the reducing agent and are not coupled to the linker-drug moiety.

32. The use of claim 31, wherein the highly homogeneous ADC comprises a high content of D6 ADC, D2 ADC, or dual drug d2+4ADC.

33. A method of treating a condition or disorder in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate of claim 28 or a pharmaceutical composition of claim 29.

34. The method of claim 33, wherein the condition or disorder is cancer, an autoimmune disease, or an infectious disease.

35. The method of any one of claims 33-34, wherein the subject is a mammal, such as a human.

36. A kit comprising one or more containers comprising the antibody-drug conjugate of claim 28 or the pharmaceutical composition of claim 29.