WO2023036137A1

WO2023036137A1 - Process for preparing highly homogenous antibody-drug conjugates for engineered antibodies

Info

Publication number: WO2023036137A1
Application number: PCT/CN2022/117308
Authority: WO
Inventors: Ao JI; Jianqing Xu; Jin Jin; Lei Xu; Jun Wang; Li Yin
Original assignee: Wuxi Biologics (Shanghai) Co. Ltd.; WuXi Biologics Ireland Limited
Priority date: 2021-09-10
Filing date: 2022-09-06
Publication date: 2023-03-16
Also published as: CN117295526A

Abstract

Provided is a process for preparing highly homogenous antibody-drug conjugates(ADCs) for antibodies. Specifically, in the ADCs prepared by the said process, the content of D2, D4 or D2+D4 can reach more than 90%.

Description

Process for Preparing Highly Homogenous Antibody-Drug Conjugates for Engineered Antibodies

CROSS-REFERENCING

This application claims the benefit of International application PCT/CN2021/117709, filed on September 10, 2021, which is incorporated by reference in its entirety.

FIELD

The present disclosure relates to a process for preparing antibody-drug conjugates (ADCs) . Specifically, the present disclosure relates to a bio-conjugation process for preparing highly homogenous antibody-drug conjugates (ADCs) for engineered antibodies, specifically, antibodies that are engineered to comprise TCR constant regions in one Fab arm.

BACKGROUND

The specificity of antibodies for specific antigens on the surface of target cells and molecules has led to their extensive use as carriers of a variety of diagnostic and therapeutic agents. For example, antibodies conjugated to labels and reporter groups such as fluorophores, radioisotopes and enzymes find use in labelling and imaging applications, while conjugation to cytotoxic agents and chemotherapy drugs allows targeted delivery of such agents to specific tissues or structures, for example particular cell types or growth factors, minimizing the impact on normal, healthy tissue and significantly reducing the side effects associated with chemotherapy treatments.

Bispecific antibody (bsAb) refers to a type of antibodies designed to recognize two different epitopes or antigens and aims to treat multifaceted, complex diseases by engaging two disease targets with one molecule. It comes in many formats. WO2019057122 and WO2020057610 disclose examples of WuXiBody ^TM platform that enables almost any monoclonal antibody (mAb) sequence pair to be assembled into a bispecific construct and its unique structural flexibility makes the platform convenient to build various formats with different valency. The bispecific antibody generated by the platform also is stable and has no aggregation issue during production.

Antibody-drug conjugates (ADC) are conjugate of an antibody and a drug and have extensive potential therapeutic applications in several disease areas, particularly in cancer, and become a novel targeted drug for disease treatment. ADC contains an antibody for targeting, a connector or linker for drug attachment and a high potent payload (e.g., a drug) as effector. The antibody, by its specificity, directs the drug to its target to release. Since the approvals of Adcetris in 2011 and Kadcyla in 2013 by US FDA, ADC drug development has widely spread for the treatment of cancer. Recently, it has been demonstrated that ADC based on bispecific antibody (e.g. ZW49) can be more efficacious in cancer treatment.

One of the problems facing the conventional conjugation of ADC is the heterogeneity of the ADC molecules where the drug moieties are attached at several sites on the antibody, for example, ranging from 0 to 8 per antibody (Drug-Antibody Ratio, DAR) by cysteine chemistry. ADC molecules of such a mixture not only bring difficulties in analysis and characterization, but also potentially have different pharmacokinetic, distribution, toxicity and efficacy profiles. And non-specific conjugation also frequently results in impaired antibody function.

Strategies are developed to tackle the problem. Genentech’s THIOMAB and Ambrx’s non-natural amino acid incorporation and various enzyme-assisted conjugations all aim to introduce drug moiety in a site-directed fashion and achieve ADC’s homogeneity (narrow DAR distribution) . These methods however are all based on antibody-engineering which may lead to side effects in human. WO2017002776 discloses that by lowing the temperature of the reduction step, DAR4 can be selectively enriched to more than 50%, with most drug on the Fab domain, without altering IgG sequence.

However, there is a continuing need for developing a novel bio-conjugation process which can generate ADCs, including ADCs of bispecific antibodies, with improved homogeneity, and has simple manipulation and reduced cost.

SUMMARY

The present disclosure has an object to develop a novel conjugation process which can generate ADCs with improved homogeneity for a certain type of bispecific antibody, and has simple manipulation and reduced cost. The resultant ADCs have a high content of D2, D6 or D2+4 ADCs, depending on the specific process applied. The ADCs generated by the conjugation process of the disclosure further have optimized safety and efficacy.

The present disclosure relates to a combination of metal ion chelating technology and a certain type of bispecific antibody, the WuXiBody, for developing new conjugation processes. Compared with conventional conjugation process, the homogeneity of antibody-drug conjugate (ADC) products generated from the conjugation process of the present disclosure can be dramatically improved. Moreover, ADC products site-specifically conjugated with two different drug moieties can be obtained.

In one aspect, the disclosure provides a process for preparing an antibody-drug conjugate (ADC) , wherein the antibody comprises a pair of T cell receptor (TCR) constant regions instead of CH1 and CL domains in at least one arm, and the pair of TCR constant regions is capable of forming a non-native interchain disulfide bond (s) , and wherein the process comprises the following steps:

(a) incubating a reductant and the antibody in a buffer system;

(b) introducing an excess amount of a linker-drug moiety to react with reduced thiol groups resulted from step (a) ; and

(c) recovering the resultant antibody-drug conjugate.

Optionally, the process further comprises adding an effective amount of oxidant to re-oxidize the unreacted thiol groups after step (b) and before recovering the resultant antibody-drug conjugate.

The inventors have surprisingly found that due to steric hindrance, the non-native interchain disulfide bond (s) between the pair of TCR constant regions is/are not accessible to reductants and thus cannot be reduced for drug conjugation.

In some embodiments, the antibody as described herein comprises a first and a second antigen-binding moiety, wherein the first antigen-binding moiety comprises: a first heavy chain variable domain (VH) operably linked to a 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 a non-native interchain disulfide bond, and

the second antigen-binding moiety is in Fab, scFv or VHH format.

In some embodiments, the second antigen-binding moiety is in Fab format 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 other embodiments, the second antigen-binding moiety is in scFv format and comprises: a second VH operably linked to a second VL. In some other embodiments, the second antigen-binding moiety is in VHH format and comprises a single variable domain.

In some embodiments, the antibody is a bispecific antibody with the first and second antigen-binding moieties targeting different antigens or epitopes. In some other embodiments, the antibody is a mono-specific antibody with the first and second antigen-binding moieties targeting the same epitope.

In some embodiments, the C1 region and C2 regions of the antibody as disclosed herein comprises an engineered T cell receptor (TCR) constant regions. Specifically, the C1 region may comprise the amino acid sequence of SEQ ID No: 2, 7 or a variant thereof with at least 90%identity; and the C2 region may comprise the amino acid sequence of SEQ ID No: 4, 8, 9 or a variant thereof with at least 90%identity. The C1 and C2 regions are capable of forming a dimer, and the non-native interchain disulfide bond (s) is/are capable of stabilizing the dimer. In some embodiments, amino acid C58 in SEQ ID No: 2 and amino acid C49 in SEQ ID No: 4 are capable of forming a non-native interchain disulfide bond.

In some embodiments, the antibody comprises an IgG Fc region, such as an IgG1, IgG2, IgG3 or IgG4 isotype.

In some embodiments, the Fc region further comprises a knob into hole structure. Specifically, the sequence of the hinge region and the Fc region in one chain ( “knob” chain) is as shown in SEQ ID No: 5, and the sequence of the hinge region and the Fc region in the other chain ( “hole” chain) is as shown in SEQ ID No: 6.

In some embodiments, from N terminus to C terminus, the antibody comprises the following structure (E17) : in the 1 ^st heavy chain, VH1-C1-hinge-Fc; in the 2 ^nd heavy chain, VH2-CH1-hinge-Fc; in the 1 ^st light chain, VL1-C2; and in the 2 ^nd light chain, VL2-CL, wherein VH1 and VL1 refer to the first VH and VL and VH2 and VL2 refer to the second VH and VL, respectively. “-” represents an operable linkage, generally via a peptide linker.

In some other embodiments, from N terminus to C terminus, the antibody comprises the following structure: in the 1 ^st heavy chain, VH1-C1-hinge-Fc-scFv; in the 2 ^nd heavy chain, VH1- CH1-hinge-Fc-scFv; in the 1 ^st light chain, VL1-C2; and in the 2 ^nd light chain, VL1-CL. The scFv constitutes the second antigen-binding moiety and can also be replaced by a VHH format.

In some embodiments, to obtain a high content of D2 ADCs, the incubation in step (a) is performed in the presence of an effective amount of a metal ion (s) such as a divalent metal ion (s) and a transition metal ion (s) . The resultant antibody-drug conjugate comprises D2 in a content higher than 80 wt%, for example, higher than 85 wt%, higher than 90 wt%, or higher than 95 wt %, on the basis of 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 a divalent metal ion or a transition metal ion and the process further comprises between steps (b) and (c) : removing the transition metal ions or divalent metal ions from the product of step (b) , then introducing a reductant again and incubating with an excess amount of a different linker-drug moiety. The resultant antibody-drug conjugate comprises D2+4 in a content higher than 65 wt%, for example, higher than 70 wt%, higher than 80 wt%, or higher than 90 wt%, on the basis of total weight of ADCs.

In some embodiments, to obtain a high content of D6 ADCs, the incubation in step (a) is not performed in the presence of an effective amount of transition metal ions or divalent metal ions. The resultant antibody-drug conjugate comprises D6 in a content higher than 85 wt%, for example, higher than 90 wt%, higher than 91 wt%, higher than 92 wt%, or higher than 93 wt%, on the basis of total weight of D0, D2, D4, and D6.

In one aspect, the present disclosure provides a process for preparing an antibody-drug conjugate (ADC) , wherein the antibody comprises a first and second antigen-binding moiety,

the first antigen-binding moiety comprises: a first heavy chain variable domain (VH) operably linked to a 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 moiety 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 process comprises the following steps:

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

(b) introducing an excess amount of a first linker-drug moiety to react with reduced thiol groups resulted from step (a) ;

(c) removing the transition metal ions or divalent metal ions from the product of step (b) ;

(d) introducing a reductant again and incubating with an excess amount of a second linker-drug moiety; and

(e) recovering the resultant antibody-drug conjugate.

Optionally, the process further comprises adding an effective amount of oxidant to re-oxidize the unreacted thiol groups after step (d) .

In some embodiments, the reductants added in step (a) and (d) are different, such as one is TCEP and the other is TDD. In some other embodiments, the reductants added in step (a) and (d) are the same, e.g. both are TCEP.

In some embodiments, the resultant antibody-drug conjugate comprises D2+4 in a content higher than 65 wt%, for example, higher than 70 wt%, higher than 80 wt%, or higher than 90 wt%, on the basis of total weight of ADCs.

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

In some embodiments, the metal ions in step (a) are selected from a group of 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+. The transition metal ion which is suitable to be used in the conjugation processes of the present disclosure may include, but not limited to, Zn ²⁺, Cd ²⁺, Hg ²⁺, and the like. Among others, Zn ²⁺, Ca ²⁺ and Mg ²⁺ may be used due to their easily availability and low cost. For example, suitable transition metal salts or divalent metal ions 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 regard, ZnCl ₂, Zn (NO ₃) ₂, ZnSO ₄, Zn (CH ₃COO) ₂, ZnI ₂, ZnBr ₂, Zinc Formate, and zinc tetrafluoroborate may be mentioned as suitable zinc salts. Similarly, CaCl ₂, Ca (NO ₃) ₂, CaSO ₄, MgCl ₂, Mg (NO ₃) ₂, and MgSO ₄ can be applied. Likewise, other transition metal salts which are soluble and can release free Cd ²⁺, or Hg ²⁺ ions in the reaction solution can be mentioned, which include, but not limited to, CdCl ₂, Cd (NO ₃) ₂, CdSO ₄, Cd (CH ₃COO) ₂, CdI ₂, CdBr ₂, cadmium formate, and cadmium tetrafluoroborate; HgCl ₂, Hg (NO ₃) ₂, HgSO ₄, Hg (CH ₃COO) 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 a group comprising Hepes, Histidine buffer, PBS, and MES, and the pH value is about 5.5 to 8.

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

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

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

In some embodiments, the oxidant is DHAA.

In some embodiments, the linker-drug moiety is maleimide bearing a drug, an organic bromide bearing a drug, or an organic iodide bearing a drug.

In some embodiments, the variable regions of the first and second antigen binding moiety are derived from antibodies which are already known, on the market, or developed de novo, such as any of the following antibodies: trastuzumab, pertuzumab, sacituzumab, abciximab, adalimumab, alefacept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, golimumab, inflectra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, trastuzumab, secukinumab, and ustekinumab. Preferably, the variable regions (or at least the CDR regions) of the first and second antigen binding moieties are same as those of the antibodies which are already known or developed de novo.

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 sacituzumab, or vice versa.

In some embodiments, the drug to be conjugated is selected from a group comprising diagnostic agents, therapeutic agents and labelling agents.

In some embodiments, the metal ions will be removed in purification step by using EDTA as chelating reagent, which will be filtered out in subsequent dialysis, ultrafiltration or gel filtration.

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

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

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

In one aspect, the present disclosure provides a method for treating a condition or disorder in a subject, comprising a step of administrating to the subject a therapeutically effective amount of the antibody-drug conjugate or the pharmaceutical composition as 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, for example, 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 figures.

DESCRIPTION OF DRAWINGS

Figure 1. Reaction scheme according to some embodiments wherein three pairs of interchain disulfide bonds of the bispecific antibody cAb1 (Trastuzumab x Pertuzumab) are reduced and site-specifically conjugated with MC-MMAF (Fig. 1A) ; HIC profile of cAb1 conjugation which shows DAR6 abundance (Fig. 1B) ; and Mass Spectrometry profile of cAb1 conjugation (Fig. 1C) . Figure 1D is the HIC profile of two other bispecific antibodies cAb3 and cAb4 which are similarly conjugated.

Figure 2. Reaction scheme according to some embodiments wherein Zn ²⁺ is added and only one pair of interchain disulfide bonds of the bispecific antibody is reduced and site-specifically conjugated with MC-MMAF (Fig. 2A) ; HIC profile of four bispecific antibodies cAb1, cAb2, cAb3 and cAb4 (Fig. 2B) conjugated as described in (Fig. 2A) .

Figure 3. Reaction scheme according to some embodiments wherein the bispecific antibody cAb1 is conjugated in two steps with two different drugs (e.g. MMAF and DXD) , respectively (Fig. 3A) ; MS profiles of resultant cAb1 ADC (Fig. 3B) , cAb2 ADC (Fig. 3C) , cAb3 ADC (Fig. 3D) and cAb4 ADC (Fig. 3E) .

DETAILED DESCRIPTION

While the present disclosure may be embodied in many different forms, disclosed herein are specific illustrative embodiments thereof that exemplify the principles of the disclosure. It should be emphasized that the present disclosure is not limited to the specific embodiments illustrated. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Abbas et al., Cellular and Molecular Immunology, 6th ed., W. B. Saunders Company (2010) ; Sambrook J. &Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., 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 Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1998) ; and Coligan et al., Short Protocols in Protein Science, Wiley, John &Sons, Inc. (2003) . The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. All publications mentioned in this specification are herein incorporated by reference in their entirety.

DEFINITIONS

In order to better understand the disclosure, the definitions and explanations of the relevant terms are provided as follows.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities 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 stated otherwise.

Throughout this disclosure, unless the context requires otherwise, the words “comprise” , “comprises” and “comprising” 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. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of” . Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action 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 as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%.

“Antibody-drug conjugate” or ADC refers to a conjugate formed by covalently coupling a drug to an antibody directly or indirectly via one or more suitable linkers. ADC is generally in a format of antibody-linker-drug conjugate. The Antibody-drug conjugates combine ideal properties of both antibodies and cytotoxic drugs (or those of other properties) by targeting potent cytotoxic (or other) drugs to the antigen-expressing tumor cells (or other cells/organs) , thereby enhancing their anti-tumor (or other medicinal) activity. ADCs are designed with the intention to discriminate between healthy cells and diseased tissue such as tumor cells in a tumor.

The term “drug” or “payload” as used herein refers to any cytotoxic molecule which has for example, an antitumor effect, anti-infection or anti-inflammation effect, and has at least one substituted group or a partial structure allowing connection to a linker structure. The drug may kill cells (e.g. cancer cells) and/or inhibit 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 which contains at least two reactive groups, one of which can covalently bond a drug molecule and the other of which can covalently couple to an antibody.

The term “antibody” as used herein encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multi-specific antibody, bispecific antibody, multivalent or bivalent antibody that binds to a specific antigen. A native intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region ( “HCVR” ) and a first, second, and third constant region (CH1, CH2 and CH3) , while each light chain consists of a variable region (“LCVR” ) and a constant region (CL) . Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. The antibody generally has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light (L) chain CDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs including HCDR1, HCDR2, HCDR3) . CDR boundaries for antibodies may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273 (4) , 927 (1997) ; Chothia, C. et al., J Mol Biol. Dec 5; 186 (3) : 651-63 (1985) ; Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987) ; Chothia, C. et al., Nature. Dec 21-28; 342 (6252) : 877-83 (1989) ; Kabat E.A. et al., National Institutes of Health, Bethesda, Md. (1991) ) , among others. The three CDRs are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. Each HCVR and LCVR comprises four FRs, and the CDRs and FRs are arranged from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain) , IgG2 (γ2 heavy chain) , IgG3 (γ3 heavy chain) , IgG4 (γ4 heavy chain) , IgA1 (α1 heavy chain) , or IgA2 (α2 heavy chain) .

The term “antibody fragment” comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab’, F (ab’) 2, and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al. (2004) Protein Eng. Design &Sel. 17 (4) : 315-323) , fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region) , and epitope -binding fragments of any described herein which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The term “antigen-binding moiety” 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 to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding moiety include, without limitation, a variable domain, a variable region, a diabody, a Fab, a Fab’, a F (ab’) 2, an Fv fragment, a scFv, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv’) , a disulfide stabilized diabody (ds diabody) , a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding moiety is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding moiety may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies. For more and detailed formats of antigen-binding moiety are described in Spiess et al, 2015 (Supra) , and Brinkman et al., mAbs, 9 (2) , pp. 182–212 (2017) , which are incorporated herein by their entirety.

“Fab” with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) associating to the variable region and first constant region of a single heavy chain by a disulfide bond. In certain embodiments, both the first and second antigen-binding moieties of the antibody to be conjugated are in Fab format. Further, the constant regions (i.e. CH1 and CL) of both chains of the Fab are replaced with engineered or modified TCR constant regions.

“Fc” with regard to an antibody refers to that portion of the antibody comprising the second (CH2) and third (CH3) constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonding, and optionally a hinge region. The Fc portion of the antibody is responsible for various effector functions such as ADCC, and CDC, but does not function in antigen binding.

“Hinge region” in terms of an antibody includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 amino acid residues and is flexible, thus allowing the two N-terminus antigen binding regions to move independently.

The term “knob into hole” , as used herein, refers to engineering the CH3 domain of antibody Fc region to create either a “knob” or a “hole” in each heavy chain to promote heterodimerization. Generally, a “knob” is created by replacing T366 with a bulky residue W on one heavy chain, and the corresponding “hole” is made by triple mutations of T366S, L368A and Y407V on the other heavy chain. The knob-into-hole structure may comprise other substitutions, as familiar in the art. The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al. Sequences of Proteins of Immunological Interest (5th Ed. ) , US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242) . Unless stated otherwise herein, references to residue numbers in the constant domain of Fc regions means residue numbering by the EU numbering system. In some embodiments, the sequence of the hinge region and the Fc region in one chain ( “knob” chain) is as shown in SEQ ID No: 5, and the sequence of the hinge region and the Fc region in the other chain ( “hole” chain) is as shown in 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 possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes) , each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see for example: US 4816567; US 5807715) . The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in e.g. Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597.

A “WuXiBody” as used herein refers to a bispecific antibody comprising a soluble chimeric protein with the variable domains of an antibody and the constant domains of a TCR (e.g. in the first antigen-binding moiety) , wherein the subunits (such as alpha and beta domains) of the TCR constant domains are linked by one or more engineered disulfide bonds. The WuXiBody also encompasses WuXiBody 2.0 antibodies which comprise a numerous variety of modified TCR constant domain sequences (see WO2022/156687) . The TCR constant domains may be engineered to form more than one pair of disulfide bonds to improve stability and/or expression level. In one type of the WuXiBody, the antibody comprises a first antigen-binding moiety in one arm and a second antigen-binding moiety in the other arm, both in Fab format and operably linked to one chain of the immunoglobulin Fc region at the C terminus. In a different WuXiBody form, the first antigen-binding moiety and the second antigen-binding moiety is intervened by the Fc region. Specifically, the antibody may comprise a first antigen-binding moiety at one terminal of the Fc region which is a Fab, and a second antigen-binding moiety at the other terminal of the Fc region which is a Fab, scFv or VHH. Alternatively, the antibody in WuXiBody form comprises a first antigen-binding moiety operably linked to the second antigen-binding moiety, which is further operably linked to the Fc region. A detailed description of various formats of WuXiBody can be found in WO2019057122, WO2019057124 and WO2020057610 (all of which incorporated herein by reference in its entirety) .

A native “T cell receptor” or a native “TCR” is a heterodimeric T cell surface protein which is associated with invariant CD3 chains to form a complex capable of mediating signal transduction. TCR belongs to the immunoglobulin superfamily, and is similar to a half antibody with a single heavy chain and a single light chain. a native TCR has an extracellular portion, a transmembrane portion, and an intracellular portion. The extracellular domain of a TCR has a membrane-proximal constant region and a membrane-distal variable region.

The terms “Trastuzumab x Pertuzumab” , “Pertuzumab x Trastuzumab” , “Trastuzumab x Sacituzumab” and “Sacituzumab x Trastuzumab” and similar designations, as used herein, are named following the same principle, i.e. refer to bispecific antibodies (preferably WuXiBody BsAbs) that comprise a first antigen binding moiety derived from the first antibody, and a second antigen-binding moiety derived from the second antibody. By “derived from” it is meant that the variable regions are same as those in the parent antibody or have at least 80%homology (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) yet still retain the binding ability to the targeted antigen. For example, the variable regions from parental antibodies may be humanized, affinity matured, or glycosylation modified before being constructed into the antibody format as disclosed herein. The methods for modification of the variable regions, including CDRs and framework regions, are familiar to a person in the art.

The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes) , e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

An “isolated antibody” is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95%or 99%purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF) , capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) . For review of methods for assessment of antibody purity, see, e.g., Flatman et al, J. Chromatogr. B 848: 79-87 (2007) .

A “disulfide bond” refers to a covalent bond with the structure R-S-S-R’. The amino acid cysteine comprises a thiol group that can form a disulfide bond with a second thiol group, for example from another cysteine residue. The disulfide bond can be formed between the thiol groups of two cysteine residues residing respectively on the two polypeptide chains, thereby forming an interchain bridge or interchain bond.

The term “transition metal” , as used herein, refers to the elements of groups 4-12, justified by their typical chemistry, i.e. a large range of complex ions in various oxidation states, colored complexes, and catalytic properties either as the element or as ions (or both) . Sc and Y in Group 3 are also generally recognized as transition metals.

The term “effective amount” used herein in relation to the metal ions (including transition metal ions and divalent metal ions) , refer to an amount of the metal ions that is sufficient to chelate with the disulfide bonds in the hinge region of the antibody and thus protect the disulfide bonds from reduction. The “effective amount” may be considered in the context of utilizing one or more metal ions, and a single metal ion may be considered as given in an effective amount if, in conjunction with one or more other metal ions, the desirable result is achieved. The effective amount of the metal ions can be empirically determined by a person skilled in the art based on the specific compositions and conditions used for conjugation. In some embodiments, the effective amount of metal ions in the reaction solution in step (a) is about 0.01 mM to 0.2 mM.

The term “DAR” or “Drug-to-Antibody Ratio” , as used herein, refers to the average number of drugs conjugated to an antibody, which is an important attribute of ADCs. The DAR value affects the efficacy of the drug, as low drug loading reduces the potency, while high drug loading can negatively affect pharmacokinetics (PK) and toxicity. Various analytical methods can be used to measure DAR, such as Ultraviolet-Visible (UV/Vis) spectroscopy, Hydrophobic interaction chromatography (HIC) , Reversed phase high-performance liquid chromatography (RP-HPLC) and Liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS) . Hydrophobic interaction chromatography (HIC) is a leading technique for the characterization of DAR values and drug loading distribution. The conjugated species are separated based on an increased hydrophobicity caused by the increased drug-load. In terms of cysteine-conjugated ADCs, the unconjugated antibody with the least hydrophobicity is eluted first while the most hydrophobic, most drug conjugated form elutes last, generating a quantitative elution profile. The area percentage of a peak represents the relative amount of each drug-loaded ADC species. The payload distribution is derived from the HIC profile while the average DAR is also calculated from the percentage peak area. As demonstrated herein, the ADCs conjugated by the process as disclosed herein are highly homogeneous. The content of DAR2, DAR6 or DAR2+4 (i.e. D2, D6, or D2+4) , depending on the specific processes used, can reach at least 80 wt%of total ADCs. DAR2+4 (D2+4) refers to a bi-drug ADC that comprises two molecules of the first drug and four molecules of the second drug per antibody.

As is known in the art, a mixture of antibody-drug conjugates will be generated by the conventional conjugation processes. In general, one antibody molecule belonging to IgG1 or IgG4 subclass has 4 inter-chain S-Sbonds, each of which is formed with two -SH groups. The antibody molecule can be subjected to partial or complete reduction of one or more interchain S-Sbonds to form 2n (n is an integer selected from 1, 2, 3 or 4) reactive -SH groups, and thus, the number of drugs coupling to a single antibody molecule is 2, 4, 6 or 8. In accordance with the number of drugs coupling to a single antibody molecule, the different conjugates containing different number of drug molecules are denominated as D0, D2, D4, D6 and D8.

If the number of drugs coupling to a single antibody molecule is 0, the product is referred to as D0. Accordingly, D2 refers to the ADC in which two drug molecules are coupled to one single antibody molecule, where two drug molecules may be coupled to -SH groups generated by reduction of S-Sbonds between heavy and light chains via linkers, or may be coupled to -SH groups generated by reduction of S-Sbonds between heavy and heavy chains via linkers. D4 refers to the ADC in which four drug molecules are coupled to one single antibody molecule, where four drug molecules may be coupled to four -SH groups generated by reduction of two S-Sbonds between heavy and light chains, or between heavy and heavy chains via linkers, or two drug molecules may be coupled to two -SH groups generated by reduction of one S-Sbond between heavy and light chains via linkers and the other two drug molecules may be coupled to two -SH groups generated by reduction of one S-Sbond between heavy and heavy chains vis linkers. D6 refers to the ADC in which six drug molecules are coupled to one single antibody molecule, where four drug molecules may be coupled to four -SH groups generated by reduction of two S-Sbonds between heavy and light chains via linkers and two drug molecules may be coupled to two -SH groups generated by reduction of one S-Sbonds between heavy and heavy chains via linkers, or four drug molecules may be coupled to four -SH groups generated by reduction of two S-Sbonds between heavy and heavy chains via linkers and two drug molecules may be coupled to two -SH groups generated by reduction of one S-Sbonds between heavy and light chains via linkers. And D8 refers to the ADC in which eight drug molecules are coupled to one single antibody molecule, i.e., all the four S-Sbonds in one antibody molecule are reduced to eight -SH groups and each -SH group attaches one drug molecule. In general, the heterogeneous mixture of ADC molecules generated by conventional conjugation processes or the bio-conjugation process of the present disclosure is a mixture of D0, D2, D4, D6 and D8.

And thus, the term “homogeneity” of antibody-drug conjugates is used to describe the property of dominance of one specific type of antibody-drug conjugate (preferably, one type selected from D2, D4, D6 conjugates) in one given mixture of antibody-drug conjugates. In the present disclosure, the “homogeneity” of antibody-drug conjugates refers to a high level of one specific type of ADC in the mixture of antibody-drug conjugates.

The term “pharmaceutically acceptable” indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is bioactivity acceptable and nontoxic to a subject. Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

The term “subject” includes any human or nonhuman animal, for example, humans.

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

The term “treatment” , “treating” or “treated” , as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal, in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis, prevention) is also included. For cancer, “treating” may refer to dampen or slow the tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. For tumors, “treatment” includes removal of all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

ADC Preparation in General

Antibody-drug conjugates are usually produced by two conventional chemical strategies, Lysine based conjugation and Cysteine from the reduction of interchain sulfide bonds based conjugation. For conjugation based on Cysteine from the reduction of interchain sulfide bonds, it comprises a step of opening inter-chain disulfide bonds in the presence of various reductants, such as TCEP, DTT and so on, followed by nucleophilic reaction of thiol groups. In this conjugation process, antibody-drug conjugates are typically formed by conjugating one or more antibody cysteine thiol groups to one or more linker moieties bound to a drug thereby forming an antibody-linker-drug complex. Since free cysteine thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine residues often exist in their oxidized form as disulfide-linked oligomers or have internally bridged disulfide groups. Disulfide dimer formation renders the Cys unreactive for conjugation to a drug, ligand, or other label.

The number of drugs coupling to a single antibody molecule is an important factor for the efficacy and safety of the resultant ADC. For example, in the conjugation process based on native inter-chain disulfide bond reduction, the inter-chain S-Sbonds are more accessible to solvents than other disulfide bonds. Therefore, the inter-chain disulfide bonds can be used as the binding sites for coupling a drug (or a drug-linker) to an antibody. In general, one therapeutic antibody molecule belonging to IgG1 or IgG4 subclass has 4 inter-chain S-Sbonds, each of which is formed with two -SH groups, and thus, the number of drugs coupling to a single antibody molecule is 2, 4, 6 or 8. If the number of drugs coupling to a single antibody molecule is 0, the product is referred to as D0. Accordingly, D2 refers to the ADC in which two drug molecules are coupled to one single antibody molecule. D4 refers to the ADC in which four drug molecules are coupled to one single antibody molecule. D6 refers to the ADC in which six drug molecules are coupled to one single antibody molecule. And D8 refers to the ADC in which eight drug molecules are coupled to one single antibody molecule, i.e., all the four S-Sbonds in one antibody molecule are reduced to eight -SH groups and each -SH group attaches one drug molecule. In general, the heterogeneous mixture of ADC molecules generated by conventional conjugation processes is a mixture of D0, D2, D4, D6 and D8.

It is well known in the art that heterogeneous ADC products are generally of lower efficacy and unsatisfactory PK properties. Among them, D0 has no ADC efficacy, and due to their hydrophobicity induced from payload (i.e., drug) molecules, D8 is considered to be the reason of instability in the circulation. Although antibody-drug conjugate potency in vitro has been shown to be directly dependent on drug loading (Hamblett KJ, et al., Clin Cancer Res. 2004 Oct 15; 10 (20) : 7063-70) , in-vivo antitumor activity of antibody-drug conjugates with four drugs per molecule (D4) was comparable with conjugates with eight drugs per molecule (D8) at equal mAb doses, even though the conjugates contained half the amount of drugs per mAb.

Drug-loading also affected plasma clearance, with D8 conjugate being cleared 3-fold faster than D4 conjugate and 5-fold faster than a D2 conjugate. Antibody-drug conjugates with improved homogeneity provide benefits in therapy, for example a higher therapeutic index, improving efficacy and reducing toxicity of the drug. Homogeneous antibody conjugates also provide more accurate and consistent measurements in diagnostic and imaging applications.

Conventionally, the drug-loading of an ADC may be controlled in different ways, e.g., by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reductive conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number and/or position of linker-drug attachments (such as thioMab or thioFab and those disclosed in WO2006/034488, herein incorporated by reference in its entirety) .

Antibodies Constructed via

Platform

In one aspect, the present disclosure provides a method or process for preparing highly homogeneous ADCs for antibodies. 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 is capable of forming a non-native interchain disulfide bond. This disulfide bond is capable of stabilizing the dimer formed between the pair of TCR constant regions, and as described above, is less accessible to solvents or reductants than native disulfide bonds.

In some embodiments, the antibodies to be conjugated herein are constructed via

Platform and also named as “WuXiBody” . A “WuXiBody” is generally a bispecific (or multispecific) antibody that comprises a first and second antigen-binding moiety, the first antigen-binding moiety is 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 moiety may be in Fab, scFv, VHH format, among others. In certain embodiments, the second antigen-binding moiety is also in Fab format 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 moiety, the commonly present CH1 domain and CL domain are replaced by a pair of TCR constant regions, thus the native disulfide bond between CH1 domain and CL domain is also replaced by one or more engineered non-native disulfide bond (s) between the TCR C1 and C2 regions. The positions of C1 and C2 regions can also be exchanged.

The antibodies to be conjugated herein are not limited to bispecific antibodies that have the first and second antigen-binding moieties targeting different epitopes or antigens. Theoretically, the antibodies to be conjugated herein could also be monospecific antibodies wherein the first and second antigen-binding moieties bind to a same antigen or epitope, and the resultant ADCs are highly homogenous ADCs against a single antigen or epitope.

In some embodiments, from N terminus to C terminus, the antibody comprises the following structure: in the 1 ^st heavy chain, VH1-C1-hinge-Fc; in the 2 ^nd heavy chain, VH2-CH1-hinge-Fc; in the 1 ^st light chain, VL1-C2; and in the 2 ^nd light chain, VL2-CL, wherein VH1 and VL1 refer to the first VH and VL and VH2 and VL2 refer to the second VH and VL, respectively. “-” represents an operable linkage, generally via a peptide linker.

The WuXiBody may adopt various other formats. For example, the heavy chain portion of the first antigen binding moiety may be operably linked to the second antigen binding moiety, and the latter one is further operably linked to one chain of the Fc region. Alternatively, the heavy chain portion of the first antigen binding moiety is operably linked to one chain of the Fc region, and the latter one is further operably linked to one chain of the second antigen binding moiety.

In some embodiments, from N terminus to C terminus, the antibody comprises the following structure: in the 1 ^st heavy chain, VH1-C1-hinge-Fc-scFv; in the 2 ^nd heavy chain, VH1-CH1-hinge-Fc-scFv; in the 1 ^st light chain, VL1-C2; and in the 2 ^nd light chain, VL1-CL. The scFv belongs to the second antigen-binding moiety and can also be replaced by a VHH format.

The first TCR constant region and the second TCR constant region are associated via a non-native interchain disulfide bond. The pair of TCR constant regions in the first antigen-binding moiety includes TCR alpha and beta constant regions (wild-type or preferably engineered) in the light chain and heavy chain respectively. The TCR constant regions in the bispecific antibodies are capable of associating with each other to form a dimer through a non-native disulfide bond.

Human TCR beta chain constant region has two different variants, known as TRBC1 and TRBC2 (IMGT nomenclature) . In WuXiBody, the sequence of TCR beta domain is based on wild type TCR sequence as:

LEDLKNVFPP KVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV STDPQPLKEQPAL NDSRY CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR (SEQ ID NO: 1) , with the NCBI accession number of A0A5B9 (https: //www. uniprot. org/uniprot/A0A5B9) .

In some specific embodiments, the pair of TCR constant regions comprises an engineered TCR beta domain comprising one or more mutated sites, as shown below:

Human TCR alpha chain constant region is known as TRAC, with the NCBI accession number of P01848 (https: //www. uniprot. org/uniprot/P01848) , the sequence of wild type TCR alpha domain is:

In some specific embodiments, the pair of TCR constant regions comprises an engineered TCR alpha chain constant region comprising one or more mutated sites, as shown below:

A person in the art would easily appreciate that, the antibody for preparing ADCs as disclosed herein may comprise a variety of C1 and C2 regions, as long as they can stabilize the first VH and VL regions to form the first antigen-binding moiety. Specifically, the antibodies may comprise a TCR C1 region with the amino acid sequence as shown in SEQ ID No: 2 or 7 or a variant thereof with at least 90%identity, and a TCR C2 region with the amino acid sequence as shown in SEQ ID No: 4, 8, 9 or a variant thereof with at least 90%identity. More than one pair of non-native disulfide bonds can be formed between the C1 and C2 regions to improve stability and expression level. In some embodiments, the variants of SEQ ID No: 2 or 7 comprise substitutions, additions and/or deletions of one or more amino acids compared to SEQ ID No: 2 or 7. Similarly, the variants of SEQ ID No: 4, 8, or 9 comprise substitutions, additions and/or deletions of one or more amino acids compared to SEQ ID No: 4, 8, or 9. A detailed description of C1 and C2 variants that can be utilized for constructing WuXiBody antibody format can be found in WO2022/156687, which is entirely incorporated herein by reference. Specifically, in some embodiments, the antibodies comprise a combination of C1 and C2 regions with SEQ ID Nos: 2 and 4 (for cAb1-6) , or with SEQ ID Nos: 2 and 8 (for cAb7) , or with SEQ ID Nos: 7 and 9 (for cAb8) , respectively.

For example, the native TCR beta chain contains a native cysteine residue at position 76, which is unpaired and therefore does not form a disulfide bond in a native alpha/beta TCR. In some bispecific antibodies of WuXiBody, this native cysteine residue at position 76 of TCR beta chain is mutated to an alanine residue. This may be useful to avoid incorrect intrachain or interchain pairing. In certain embodiments, the substitution in certain embodiments can improve the TCR refolding efficiencies in vitro.

Therefore, the first and the second TCR constant regions of the first antigen-binding moiety are capable of forming a dimer comprising, between the TCR constant regions (i.e., CAlpha and CBeta) , at least one non-native interchain disulfide bond that is capable of stabilizing the dimer.

The benefits provided by replacing CH1 and CL domains with TCR constant regions is significant. In the WuXiBody, the first antigen-binding moiety with at least one non-native disulfide bond can be recombinantly expressed and assembled into the desired conformation, which stabilizes the TCR constant region dimer while providing for good antigen-binding activity of the antibody variable regions. Moreover, the first antigen-binding moiety is found to well tolerate routine antibody engineering, for example, modification of glycosylation sites, and removal of some natural sequences. Furthermore, the bispecific antibodies in such format can be readily expressed and assembled with minimal or substantially no mispairing of the antigen-binding sequences due to the presence of the TCR constant regions in the first antigen-binding moiety.

Most importantly, the non-native disulfide bond in the TCR constant regions is less accessible to solvents than native disulfide bonds, thereby providing fewer binding sites (and improved homogeneity) for coupling a drug (or a drug-linker) . Besides, the non-native disulfide bonds are possibly less sensitive to reductants than native disulfide bonds.

In some embodiments, the process comprises constructing the antibody based on a parental bispecific antibody or two parental monospecific antibodies and generation of the antibody, prior to step (a) . By “based on” it is meant to derive or obtain the first and second VH and VL regions from the variable regions of the parental antibodies and assemble into the WuxiBody format with other regions e.g. TCR constant regions, heavy and light chain constant regions, hinge regions and Fc regions.

The BsAbs to be used for conjugation comprise two antigen-binding moieties which may be a Fab, Fab’, scFv, VHH etc. The antigen-binding moiety may be derived from an antibody (already known or developed de novo) targeted to a certain 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 Sacituzumab, or vice versa. By “derived from” , it is generally meant herein that the antigen-binding moiety comprises the CDR sequences of the parent antibody, and preferably, comprises the variable regions of the parent antibody. In some embodiments, the antigen-binding moiety comprises the variants of the CDR sequences of the parent antibody which retain the antigen-binding specificity.

Theoretically, the parent antibodies that can derive the antigen-binding moieties can include all monoclonal antibodies that have specificity for a certain antigen, such as antibodies against tumor related antigens or pathways, e.g. 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, HER2, NCA, MDP, IL20Ra, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, FcRH5, TENB2, PMEL17, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, Ly6K, GPR19, GPR54, ASPHD1, Tyrosinase, TMEM118, GPR172A, CD33 and CLL-1; antibodies against leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86 or their ligands; CD3 engager antibodies, NK engager antibodies; ADCC enabling anti-Tumor associated antigens; monoclonal antibodies to TNF, among others. The antibodies may include but not limited to, trastuzumab, pertuzumab, sacituzumab, abciximab, adalimumab, alefacept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, golimumab, inflectra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, trastuzumab, secukinumab, and ustekinumab.

The bispecific antibody used in the method provided herein may be antibodies that have binding specificity for a numerous variety of antigens, such as tumor associated antigens (TAA) . In certain embodiments, the bispecific antibodies to be conjugated have 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, however may be expressed by the cognate cell (or healthy cells) prior to transforming into a tumor. In some embodiments, the tumor associated antigens can be presented only by tumor cells and not by normal, i.e. non-tumor cells. In some other embodiments, the tumor associated antigens can be exclusively expressed on tumor cells or may represent a tumor specific mutation compared to non-tumor cells. In some other embodiments, the tumor associated antigens can be found in both tumor cells and non-tumor cells, but is overexpressed on tumor cells when compared to non-tumor cells or are accessible for antibody binding in tumor cells due to the less compact structure of the tumor tissue compared to non-tumor tissue. In some embodiments, the tumor associated antigen is located on the vasculature of a tumor.

Illustrative examples of a tumor associated antigen are LAG-3, CD10, CD19, CD20, CD22, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, Fms-like tyrosine kinase 3 (FLT-3, CD135) , chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan) , Epidermal growth factor receptor (EGFR) , Her2neu, Her3, IGFR, IL3R, fibroblast activating protein (FAP) , CDCP1, Derlin1, Tenascin, frizzled 1-10, the vascular antigens VEGFR2 (KDR/FLK1) , VEGFR3 (FLT4, CD309) , PDGFR-alpha (CD140a) , PDGFR-beta (CD140b) Endoglin, CLEC14, Tem1-8, and Tie2. Further examples may include A33, CAMPATH-1 (CDw52) , Carcinoembryonic antigen (CEA) , Carboanhydrase IX (MN/CA IX) , de2-7 EGFR, EGFRvIII, EpCAM, Ep-CAM, Folate-binding protein, G250, Fms-like tyrosine kinase 3 (FLT-3, CD135) , c-Kit (CD117) , CSF1R (CD115) , HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (Melanoma-associated cell surface chondroitin sulphate proteoglycane) , 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 an epitope thereof. Non-limiting examples of infectious disease-associated antigens include, e.g., an antigen that is expressed on the surface of a virus particle, or preferentially expressed on a cell that is infected with a virus, wherein the virus is selected from the group consisting of HIV, hepatitis (A, B or C) , herpes virus (e.g., HSV-1 , HSV-2, CMV, HAV-6, VZV, Epstein Barr virus) , adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus. Alternatively, the target antigen can be an antigen that is expressed on the surface of a bacterium, or preferentially expressed on a cell that is infected with a bacterium, wherein the bacterium is selected from the group consisting of chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci, gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospira, and Lyme disease bacteria. In certain embodiments, the target antigen is an antigen that is expressed on the surface of a fungus, or preferentially expressed on a cell that is infected with a fungus, wherein the fungus is selected from the group consisting of Candida (albicans, krusei, glabrata, tropicalis, etc. ) , Crytococcus neoformans, Aspergillus (fumigatus, niger, etc. ) , Mucorales (mucor, absidia, rhizopus, etc. ) , Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum. In certain embodiments, the target antigen is an antigen that is expressed on the surface of a parasite, or preferentially expressed on a cell that is infected with a parasite, wherein the parasite is selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis, Taenia crassiceps, and Brugia malayi. Non-limiting examples of specific pathogen-associated antigens include, e.g., HIV gp120, HIV CD4, hepatitis B glucoprotein L, hepatitis B glucoprotein M, hepatitis B glucoprotein S, hepatitis C E1, hepatitis C E2, hepatocyte-specific protein, herpes simplex virus gB, cytomegalovirus gB, and HTLV envelope protein.

The WuXiBody BsAb to be conjugated herein may further comprise a knob-into-hole structure in the Fc region. An extra inter-chain disulfide bond is created in the knob-hole region to increase the stability of the bispecific antibody. With 5 pairs of inter-chain disulfide bonds in such BsAbs, it is found that under normal TCEP reduction, only 3 pairs of the bonds will be broken to react with maleimide-modified drug to give DAR6 enriched (>90%) ADC. The other 2 pairs of non-native disulfide bonds (in the TCR region and Fc region) are structurally not exposed to reducing agent and thus cannot be opened for drug attachment. In the circumstances where C1 and C2 regions form more than one non-native disulfide bonds, they are also structurally inaccessible to reducing agents.

In some embodiments, the bispecific antibodies to be conjugated herein comprise two heavy chains and two light chains, wherein from N terminal to C terminal:

the first heavy chain comprises domains operably linked as in VH1-C1-hinge-Fc and the first light chain comprises domains operably linked as in VL1-C2; the second heavy chain comprises domains operably linked as in VH2-CH1-hinge-Fc and the second light chain comprises domains operably linked as in VL2-CL, wherein VH1 and VL1 refer to the first heavy chain and light chain variable regions derived from a first parental antibody, while VH2 and VL2 refer to the second heavy chain and light chain variable regions derived from a first parental antibody. VH1, C1, VL1 and C2 constitute the first antigen binding moiety, and VH2, CH1, VL2 and CL constitute the second antigen binding moiety.

Conjugation process for preparing ADCs with high homogeneity of D6

In one aspect, the disclosure provides a process for preparing a highly homogeneous (occupies more than 50%, 60%, 70%, 80%, 90%or higher of total produced ADCs) antibody-drug conjugate (ADC) for the bispecific antibody as described above, the process comprises the following steps:

(a) incubating a reductant and the bispecific antibody in a buffer system; and

(b) introducing an excess amount of a linker-drug moiety (e.g. MC-MMAF) to react with reduced thiol groups resulted from step (a) ; and

(c) recovering the resultant antibody-drug conjugate.

Optionally, the process further comprises adding an effective amount of oxidant to re-oxidize the unreacted thiol groups after step (b) .

As described above, the bispecific antibody to be conjugated is constructed as a knob-into-hole bispecific antibody with a TCR arm and a normal IgG Fab at its variable regions. An extra inter-chain disulfide bond is created in the knob-hole region to increase the stability of the bispecific antibody. With 5 pairs of inter-chain disulfide bonds, it is found that under normal TCEP reduction, only 3 pairs of the bonds will be broken to react with drugs (e.g. maleimide-modified drug) , giving a DAR6 enriched (>80%, or even 90%) ADC.

In other words, conjugation with bispecific antibodies in WuXiBody form generate highly homogeneous DAR6 (D6) species with 85～95%purity, although the antibody contains 5 pairs of inter-chain disulfide bonds. Two pairs of non-native disulfide bonds, one in the TCR constant regions and the other in the “knob-hole” structure, are not reduced by the reductant, which leaves only 3 pairs of disulfide bonds for reduction and site-specific conjugation. Among which, two pairs are in the hinge region and the other pair is in the CH1-CL region of the second antigen-binding moiety.

In the resultant ADCs, the content of D6 ADCs can reach at least 80 wt%, for example, at least 85 wt%, at least 90 wt%, at least 91 wt%, at least 92 wt%or at least 93 wt%, on the basis of total weight of D0, D2, D4 and D6, as measured by HIC.

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

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

The optimum pH for the reaction will typically be between about 5.5 and about 8, for instance, about 5.5 to 7.5. The optimal reaction conditions will of course depend upon the specific reactants employed.

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

The optimum temperature for the reduction reaction and conjugation reaction will typically be between about -10℃ and 37℃, e.g. about 4℃-22℃. For instance, the reduction reaction occurs at a temperature of about 22℃ for 8-18 h, and the conjugation reaction occurs at a temperature of about 4℃ overnight. The temperature and time may vary depending on the amounts of antibody, reductant and drugs used.

Those skilled in the art should understand that the incubation time period and temperature in step (a) depend on specific antibodies to be conjugated. The determination of the incubation time period and temperature based on specific antibodies is within the abilities of ordinary skilled persons in the art. For example, the antibody to be conjugated is typically incubated with the reductant at 4℃ overnight.

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

As for step (c) , those skilled in the art can select proper purification methods to recover the resultant antibody-drug conjugates. Many ADC purification methods are well known in the art. For example, the resultant antibody-drug conjugates may be purified by using a de-salting column, size exclusion chromatography, and the like.

In some embodiments, the resultant antibody-drug conjugates are recovered by any suitable purification method, such as using a de-salting column, size exclusion chromatography, ultrafiltration, dialysis, UF-DF, and the like.

Conjugation process for preparing ADCs with high homogeneity of D2

Transitional metal ions and/or divalent metal ions could protect the disulfide bonds in the hinge region from being reduced during the incubation of the reductant and the antibody. The inventors herein combined such metal ion chelating function with WuXiBody antibody for ADC conjugation, giving very narrow DAR2 distribution which allows the preparation of ADCs of low DAR. When drugs (such as PBD) are highly potent and too high DARs lead to toxicity issues, ADCs of low DAR are desired.

When a metal ion such as Zn (II) is added into the reaction system, two disulfide bonds in the hinge region are shielded from the reducing agent and only one pair of the disulfide bond in the CH1-CL region of the IgG side is reduced, and only DAR2 species is generated upon drug addition. The PK/PD properties of ADC of such highly homogeneous D2 population rivals that generated by antibody engineering. The manufacture of such ADC is also easy. The low-DAR ADC is of great importance when low DAR is required, e.g. when the drug is too toxic for high loading.

Thus, in one aspect, the disclosure provides a process for preparing an antibody-drug conjugate (ADC) for the bispecific antibody as described above, wherein the process comprises the following steps:

(a) incubating a reductant and the bispecific antibody in the presence of an effective amount of metal ions such as transition metal ions and divalent metal ions in a buffer system to selectively reduce inter-chain disulfide bonds within the antibody;

(c) recovering the resultant antibody-drug conjugate.

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

In some embodiments, the metal ion which is suitable to be used in the bio-conjugation process of the present disclosure is selected from transition metal ions and divalent ions, including but not limited to, Zn2+, Cd2+, Ca2+, Mg2+ and Hg2+, and the like. Among others, Zn2+ is used due to its easy 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 can be released in the reaction solution. In this regard, ZnCl2, Zn (NO3) 2, ZnSO4, Zn (CH3COO) 2, ZnI2, ZnBr2, Zinc Formate, and zinc tetrafluoroborate may be mentioned as suitable zinc salts. Likewise, other transition metal salts which are soluble and can release free Cd2+ or Hg2+ ions in the reaction solution can be mentioned, which include, but not limited to, CdCl2, Cd (NO3) 2, CdSO4, Cd (CH3COO) 2, CdI2, CdBr2, cadmium formate, and cadmium tetrafluoroborate; HgCl2, Hg(NO3) 2, HgSO4, Hg (CH3COO) 2, HgBr2, Mercury (II) formate, and Mercury (II) tetrafluoroborate; and the like. Those skilled in the art can make a selection from the above transition metal salts and divalent metal salts as the source of metal ions.

In an embodiment, Zn2+ is used in step (a) . Zinc salts which are water soluble are available. For example, ZnCl2 may be added in step (a) as the Zn2+ source.

The concentration of the metal ions in the reaction solution in step (a) is 0.01 mM to 0.2 mM.

The metal ions will be removed in purification step by using EDTA as chelating reagent, which will be filtered out in subsequent dialysis, ultrafiltration or gel filtration.

Depending on the metal ions, those skilled in the art can select suitable buffer system for the reaction in step (a) , including, but not limited to, Hepes, Histidine buffer, PBS, MES, and the like. In a specific embodiment, the buffer system used in step (a) is PBS.

The optimum pH for the reaction will typically between about 5.5 and about 8, for instance, about 5.5 to 7.5. The optimal reaction conditions will of course depend upon the specific reactants employed.

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

The optimum temperature for the reduction reaction and conjugation reaction will typically be between about -10℃ and 37℃, e.g. about 4℃-22℃. For instance, the reduction reaction occurs at a temperature of about 4℃ overnight, and the conjugation reaction occurs at a temperature of about 4℃ for 2-8 h. The temperature and time may vary depending on the amounts of antibody, reductant and drugs used.

For instance, the antibody to be conjugated, the metal ions and the reductant may be present in the reaction mixture in a ratio of 1: 2: 4 in molar concentration. In one embodiment, 0.02 mM antibody is incubated with 0.08 mM TCEP and 0.04 mM ZnCl2 at 4℃ overnight. It will be understood by a person skilled in the art that a molar concentration may also be converted into “eq” compared to antibody. For instance, “0.04 mM ZnCl2” may be converted into “2 eq ZnCl2” if antibody is used at 0.02 mM.

In the resultant ADCs, the content of D2 ADCs can reach at least 80 wt%, for example, at least 85 wt%, at least 90 wt%, at least 91 wt%, at least 92 wt%, at least 93 wt%, at least 94 wt%, or at least 95 wt%on the basis of total weight of D0 and D2, as measured by HIC.

Conjugation process for preparing bi-drug ADCs with high homogeneity of D2+4

Combining drugs with different mechanisms of action for treatment is often beneficial and may improve treatment outcomes. Although the targeted approach of an ADC typically yields favorable results, (acquired) drug resistance often emerges, limiting the effectiveness of ADCs. One way to circumvent the problem of ADC-associated drug resistance is combining ADCs with different drugs or using ADCs with multiple-drug payloads. In one aspect, the present disclosure provides a process for producing highly homogeneous (more than 50%, 60%, 70%, 80%, 90%or higher of total produced ADCs) ADCs with two different payloads.

Based on the above two processes, a two-step conjugation –with the presence of effective amount of metal ions for conjugation of the first drug moiety and without such metal ions for conjugation of the second drug moiety -is developed and produces a highly homogeneous ADC of DAR (2+4) . Such bi-drug ADCs are more resistant to multi-drug resistance of cancer cells rising from cancer therapies and can be easily produced by this conjugation process. The designation of “DAR (a+b) ” or “D (a+b) ” herein refers to a bi-drug ADC in which the number of the first drug conjugated to a single antibody molecule is “a” and the number of the second drug conjugated to the antibody molecule is “b” .

Thus, in one aspect, the disclosure provides a process for preparing an antibody-drug conjugate (ADC) of the bispecific antibodies as described above, wherein the process comprises the following steps:

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

(d) adding a reductant again and incubating with an excess amount of a second linker-drug moiety; and

(e) recovering the resultant antibody-drug conjugate.

The reductant, metal ions, buffer systems and oxidants may be the same as disclosed above for preparing D2 ADC. For step (c) , the metal ions can be removed in purification step by using EDTA as chelating reagent, which will be filtered out in subsequent dialysis, ultrafiltration or gel filtration.

The reaction conditions for reducing and conjugation may be the same as disclosed above for preparing D2 ADC. The optimum temperature for the reduction reaction and conjugation reaction will typically be between about -10℃ and 37℃, e.g. about 4℃-22℃. The second reduction and conjugation of steps (c) and (d) may have slight differences from steps (a) and (b) . For instance, the reduction reaction for step (c) occurs at a temperature of about 22℃ for 2-8h, and the conjugation reaction occurs at about 22℃ for 2-8 h. The temperature and time may vary depending on the amounts of antibody, reductant and drugs 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 resultant ADCs, the content of D2+4 ADCs can reach at least 80 wt%, for example, at least 85 wt%, at least 90 wt%, at least 91 wt%, at least 92 wt%, at least 93 wt%, at least 94 wt%, or at least 95 wt%on the basis of total weight of D0, D2, D2+2 and D4, as calculated from MS analysis.

Highly homogenous ADC of Bispecific Antibody Shows Better Efficacy

The main problem of conventional conjugation of ADC is the heterogeneity of the ADC molecules where the drug moieties are attached at several sites on the antibody, for example, ranging from 0 to 8 per antibody (Drug-Antibody Ratio, DAR) by cysteine chemistry. ADC molecules of such a mixture not only brings difficulties in analysis and characterization, but also potentially has different pharmacokinetic, distribution, toxicity and efficacy profiles. And non-specific conjugation also frequently results in impaired antibody function. Therefore, narrow distribution of DAR is desired for better PK, efficacy and therapeutic window.

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

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

In one aspect, the present disclosure provides a highly homogeneous antibody-drug conjugates of bispecific antibodies, wherein the D2+4 ADC constitute more than 80 wt%of the ADCs.

In some embodiments, the homogeneity of the antibody-drug conjugates generated by the process as disclosed herein is measured and compared with the homogeneity of corresponding control antibody-drug conjugates generated by conventional conjugation processes.

Various analytical methods can be used to determine the yields and isomeric mixtures of the antibody-drug conjugates. For example, in one embodiment, HIC is the analytical method used to determine yields and isomeric mixtures from resultant antibody-drug conjugates (e.g., for D6 conjugates) . This technique is able to separate antibodies loaded with various numbers of drugs. The drug loading level can be determined based on the ratio of absorbances, e.g., at 250 nm and 280 nm. For example, if a drug can absorb at 250 nm while the antibody absorbs at 280 nm. The 250/280 ratio therefore increases with drug loading. Using the bio-conjugation process described herein, generally antibodies with even numbers of drugs were observed because conjugation incompletion leads to even number of DAR.

Linker-Drug moiety

The use of antibody-drug conjugates for local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, " in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (ed. s) , pp. 475-506) . Both polyclonal antibodies and monoclonal antibodies have been reported as 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 daunomycin, doxorubicin, methotrexate, and vindesine; toxins, for example bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin, maytansinoids, and calicheamicin; auristatin peptides, auristatin E (AE) and monomethylauristatin (MMAE) , which are synthetic analogs of dolastatin. MMAE is a synthetic derivative of dolastatin 10, a natural cytostatic pseudo peptide. The toxins may achieve their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

There are no specific limitations to the drug and linker which can be used in the conjugation process of the present disclosure, as long as the drug molecule has an antitumor, antiviral or antimicrobial effect and contains at least one substituted group or a partial structure allowing connection to a linker structure, and the linker contains at least two reactive groups, one of which can covalently bond a drug molecule and the other of which can covalently couple to an antibody. Preferably, the linker is susceptible to –SH attack from the antibody and capable of forming a linkage with the antibody.

Depending on the desired drug and selected linker, those skilled in the art can select suitable method for coupling them together. 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 conjugating to the antibodies through covalent linkage. A drug-maleimide complex (i.e., maleimide linking drug) is taken as an example of the payload bearing reactive group in the present disclosure.

In an embodiment, the drug may include, but not limited to, cytotoxic reagents, such as chemo-therapeutic agents, immunotherapeutic agents and the like, antiviral agents or antimicrobial agents. In an embodiment, the drug to be conjugated with an antibody may be selected from, but not limited to, MMAE (monomethyl auristatin E) , MMAD (monomethyl auristatin D) , MMAF (monomethyl auristatin F) , and the like.

Most common reactive group capable of bonding to thiol group in ADC preparation is maleimide. Additionally, organic bromides, iodides also are frequently used.

Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa) , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S) , momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. See, e.g., WO 93/21232 published October 28, 1993. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. One or more small molecule toxins, such as a calicheamicin, maytansinoids, dolastatins, auristatins, a trichothecene, and CC1065, and the derivatives of these toxins that have toxin activity, can be conjugated with the antibody by the process as disclosed herein.

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

Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division and have anticancer and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother. 42: 2961-2965) . The dolastatin or auristatin drug moiety may be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172) . Exemplary embodiments comprising MMAE or MMAF and various linker components are shown below. For example, VcMMAE (Mc-vc-PAB-MMAE) is obtained by using MMAE linked via p-aminobenzyloxycarbonyl ( “PAB” ) to the lysosomally cleavable dipeptide valine-citrulline (vc) and a thiolreactive maleimidocaproyl spacer (MC) .

Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP) , iminothiolane (IT) , bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl) , active esters (such as disuccinimidyl suberate) , aldehydes (such as glutaraldehyde) , bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine) , 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, a ricin immunotoxin can be prepared as described in Vitetta et al (1987) Science, 238: 1098. Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody (WO94/11026) .

Additional exemplary embodiments comprising MMAE or MMAF and various linker components. For example, VcMMAE (Mc-vc-PAB-MMAE) is obtained by using MMAE linked via p-aminobenzyloxycarbonyl ( “PAB” ) to the lysosomally cleavable dipeptide valine-citrulline (vc) and a thiolreactive maleimidocaproyl spacer (MC) .

Pharmaceutical Composition

In one aspect, the present disclosure relates to a pharmaceutical composition comprising an effective amount of the ADCs with improved homogeneity prepared by the process as disclosed herein and a pharmaceutically acceptable carrier or vehicle. The compositions are suitable for veterinary or human administration.

The compositions of the present disclosure can be in any form that allows for the composition to be administered to an animal. For example, the composition can be in the form of a solid, liquid or gas (aerosol) . Typical routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, and intranasal. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. For example, the compositions are administered parenterally. Pharmaceutical compositions of the invention can be formulated so as to allow ADCs of the disclosure to be bioavailable upon administration of the composition to an animal. Compositions can take the form of one or more dosage units, where for example, a tablet can be a single dosage unit, and a container of ADCs of the disclosure in aerosol form can hold a plurality of dosage units.

Materials used in preparing the pharmaceutical compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient (s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of animal (e.g., human) , the particular form of the ADCs, the manner of administration, and the composition employed.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, inclusion of one or more antioxidants such as methionine in a pharmaceutical composition provided herein decreases oxidation of the polypeptide complex or the bispecific polypeptide complex. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving protein stability and maximizing shelf-life. Therefore, in certain embodiments, compositions are provided that comprise the polypeptide complex or the bispecific polypeptide complex disclosed herein and one or more antioxidants such as methionine.

To further illustrate, pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer’s injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer’s injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid) , ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

The pharmaceutical compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.

In certain embodiments, the pharmaceutical compositions are formulated into an injectable composition. The injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion. Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions. The solutions may be either aqueous or non-aqueous.

In certain embodiments, unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.

In certain embodiments, a sterile, lyophilized powder is prepared by dissolving the ADCs as disclosed herein in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides a desirable formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial can contain a single dosage or multiple dosages of the ADCs provided herein or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4 ℃ to room temperature.

Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given and can be empirically determined.

Additionally, the antibody-drug conjugates or the pharmaceutical composition may be manufactured into a kit, including an insert which indicates the information for the application, such as the indications, the amount in use, the route to be administrated, and the like.

Use of the ADCs with Improved Homogeneity

In one aspect, the present disclosure relates to the use of the antibody-drug conjugates with improved homogeneity prepared by the process of the first aspect in the manufacture of a pharmaceutical composition or a kit for treating a condition or disorder in a subject.

The subject may be a mammal, for example, a human.

The condition or disorder to be treated may be a tumor, cancer, autoimmune disease, or infectious disease. In specific embodiments, the infectious disease may be 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: administrating a therapeutically effective amount of the ADCs with improved homogeneity prepared by the process as disclosed herein or a therapeutically effective amount of the pharmaceutical composition comprising the ADCs with improved homogeneity prepared by the process as disclosed herein to a subject in need thereof, thereby treating or preventing the condition or disorder.

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

The subject may be a mammal, for example, a human.

The therapeutically effective amount of the ADCs provided herein will depend on various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.

In certain embodiments, the ADCs or pharmaceutical composition provided herein may be administered at a therapeutically effective dosage of about 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg) . In certain of these embodiments, the ADCs or pharmaceutical composition provided herein are administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage is 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less. In certain embodiments, the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) . For example, a single dose may be administered, or several divided doses may be administered over time.

The ADCs or pharmaceutical composition provided herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

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

The cancer may be antigen positive carcinomas including those of the lung, breast, colon, ovaries, and pancreas, for example, the cancers associated with tumor-associated antigens.

Other particular types of cancers that can be treated with the ADCs or pharmaceutical composition provided herein include, but are not limited to, solid tumors, including but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophogeal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung carcinoma, bladder carcinoma, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, retinoblastoma; blood-borne cancers, including but not limited to: acute lymphoblastic leukemia “ALL” , acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia “AML” , acute promyelocytic leukemia “APL" , acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia “CML” , chronic lymphocytic leukemia “CLL” , hairy cell leukemia, multiple myeloma; Lymphomas: B cell lymphoma, optionally Hodgkin lymphoma or non-Hodgkin lymphoma, wherein the non-Hodgkin lymphoma comprises: Diffuse large B-cell lymphoma (DLBCL) , Follicular lymphoma, Marginal zone B-cell lymphoma (MZL) , Mucosa-Associated Lymphatic Tissue lymphoma (MALT) , Small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL) , or Mantle cell lymphoma (MCL) , Acute Lymphoblastic Leukemia (ALL) , or Waldenstrom’s Macroglobulinemia (WM) .

The autoimmune disease may include, but not limited to, Active Chronic Hepatitis, Addison’s Disease, Allergic Alveolitis, Allergic Reaction, Allergic Rhinitis, Alport’s Syndrome, Anaphlaxis, Ankylosing Spondylitis, Anti-phosholipid Syndrome, Arthritis, Ascariasis, Aspergillosis, Atopic Allergy, Atropic Dermatitis, Atropic Rhinitis, Behcet’s Disease, Bird-Fancier’s Lung, Bronchial Asthma, Caplan’s Syndrome, Cardiomyopathy, Celiac Disease, Chagas’ Disease, Chronic Glomerulonephritis, Cogan’s Syndrome, Cold Agglutinin Disease, Congenital Rubella Infection, CREST Syndrome, Crohn’s Disease, Cryoglobulinemia, Cushing’s Syndrome, Dermatomyositi, Discoid Lupus, Dressler’s Syndrome, Eaton-Lambert Syndrome, Echovirus Infection, Encephalomyelitis, Endocrine opthalmopathy, Epstein-Barr Virus Infection, Equine Heaves, Erythematosis, Evan’s Syndrome, Felty’s Syndrome, Fibromyalgia, Fuch’s Cyclitis, Gastric Atrophy, Gastrointestinal Allergy, Giant Cell Arteritis, Glomerulonephritis, Goodpasture’s Syndrome, Graft v. Host Disease, Graves’ Disease, Guillain-Barre Disease, Hashimoto’s Thyroiditis, Hemolytic Anemia, Henoch-Schonlein Purpura, Idiopathic Adrenal Atrophy, Idiopathic Pulmonary Fibritis, IgA Nephropathy, Inflammatory Bowel Diseases, Insulin-dependent Diabetes Mellitus, Juvenile Arthritis, Juvenile Diabetes Mellitus (Type I) , Lambert-Eaton Syndrome, Laminitis, Lichen Planus, Lupoid Hepatitis, Lupus, Lymphopenia, Meniere’s Disease, Mixed Connective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis, Pernicious Anemia, Polyglandular Syndromes, Presenile Dementia, Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, Psoriatic Arthritis, Raynauds Phenomenon, Recurrent Abortion, Reiter’s Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Sampter’s Syndrome, Schistosomiasis, Schmidt’s Syndrome, Scleroderma, Shulman’s Syndrome, Sjorgen’s Syndrome, Stiff-Man Syndrome, Sympathetic Ophthalmia, Systemic Lupus Erythematosis, Takayasu’s Arteritis, Temporal Arteritis, Thyroiditis, Thrombocytopenia, Thyrotoxicosis, Toxic Epidermal Necrolysis, Type B Insulin Resistance, Type I Diabetes Mellitus, Ulcerative Colitis, Uveitis, Vitiligo, Waldenstrom’s Macroglobulemia, Wegener’s Granulomatosis.

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

In some embodiments, the therapeutic agent is an anti-cancer agent. Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel.

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

In some embodiments, the therapeutic agent is anti-infectious disease agent. In some embodiments, the anti-infectious disease agent is, but not limited to, antibacterial agents: [beta] -Lactam Antibiotics: Penicillin G, Penicillin V, Cloxacilliin, Dicloxacillin, Methicillin, Nafcillin, Oxacillin, Ampicillin, Amoxicillin, Bacampicillin, Azlocillin, Carbenicillin, Mezlocillin, Piperacillin, Ticarcillin; Aminoglycosides: Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin; Macrolides: Azithromycin, Clarithromycin, Erythromycin, Lincomycin, Clindamycin; Tetracyclines: Demeclocycline, Doxycycline, Minocycline, Oxytetracyclinem, Tetracycline; Quinolones: Cinoxacin, Nalidixic Acid; Fluoroquinolones: Ciprofloxacin, Enoxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxicin; Polypeptides: Bacitracin, Colistin, Polymyxin B; Sulfonamides: Sulfisoxazole, Sulfamethoxazole, Sulfadiazine, Sulfamethizole, Sulfacetamide; Miscellaneous Antibacterial Agents: Trimethoprim, Sulfamethazole, Chloramphenicol, Vancomycin, Metronidazole, Quinupristin, Dalfopristin, Rifampin, Spectinomycin, Nitrofurantoin; Antiviral Agents: General Antiviral Agents: Idoxuradine, Vidarabine, Trifluridine, Acyclovir, Famcicyclovir, Pencicyclovir, Valacyclovir, Gancicyclovir, Foscarnet, Ribavirin, Amantadine, Rimantadine, Cidofovir, Antisense Oligonucleotides, Immunoglobulins, Inteferons; Drugs for HIV infection: Zidovudine, Didanosine, Zalcitabine, Stavudine, Lamivudine, Nevirapine, Delavirdine, Saquinavir, Ritonavir, Indinavir, Nelfinavir.

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.

EXAMPLES

Now the present disclosure will be illustrated in detail with reference to the following examples. However, those skilled in the art should understand that, the following examples are only provided for illustration, but not intended to limit the present disclosure in any way.

Example 1. Generation of WuXiBody Bispecific antibodies

1.1 Construction of WuXiBody E17 format bispecific antibodies

Table 1

Antibody	Parental antibodies
cAb1	Trastuzumab x Pertuzumab
cAb2	Pertuzumab x Trastuzumab
cAb3	Trastuzumab x Sacituzumab
cAb4	Sacituzumab x Trastuzumab
cAb5	Sacituzumab x Cetuximab
cAb6	Cetuximab x Sacituzumab
cAb7	Trastuzumab x Pertuzumab
cAb8	Trastuzumab x Pertuzumab

8 bispecific antibodies were constructed (Table 1) . The VL, VH genes (obtained from the parental antibodies in Table 1) were amplified by PCR from existing plasmid template. The TCR Cα and Cβ genes (encoding SEQ ID Nos: 2 and 4, except cAb7 and cAb8 which use different C1 and C2 region sequences shown in SEQ ID Nos: 7-9) were synthesized by Genewiz Inc (Suzhou, China) . The DNA fragments of VL ₁-Cα and VL ₂-CL were inserted into a linearized vector respectively, which contains a CMV promoter and a human light chain signal peptide. The DNA fragments of VH ₁-Cβ were inserted into a linearized vector containing human IgG1 constant region CH2-CH3 with “knob” mutations. The DNA fragments of VH ₂-CH1 were inserted into a linearized vector containing human IgG1 constant region CH2-CH3 with “hole” mutations. The vectors contain a CMV promoter and a human antibody heavy chain signal peptide. An exemplary structure of cAb1 is shown in the reaction scheme of Figure 1A.

1.2 Expression and purification of WuXiBody bispecific antibodies

Heavy chain and light chain expression plasmids were co-transfected into Expi293 cells (Invitrogen-A14527) using expression system kit (Invitrogen-A14524) according to the manufacturer’s instructions. 5 days after transfection, the supernatants were collected and used for protein purification using protein A chromatography (GE Healthcare-17543802) . If needed, further purifications were conducted using size exclusion chromatography (GE Healthcare-17104301) . Antibody concentration was measured by Nano Drop. The purity of proteins was evaluated by SDS-PAGE and HPLC-SEC.

Example 2. Conjugation to form D6 species

The reaction scheme (exemplified by cAb1) shown in Figure 1A was performed. 10 mg/mL mAb in PBS buffer was reduced by 7.5 eq TCEP and the reaction vial was stood at 22 ℃ for 8 hours. The reduced antibody solution was directly submitted to next step conjugation without TCEP removal.

DMA and Linker-payload solution (10 mg/mL stock in DMA, 18.0 eq. to antibody) was added into the reduced antibody solution. The reactions were mixed properly and the reaction vials were stood at 4 ℃ for 18 hours. The crude product was submitted to a buffer exchange into its storage buffer (1X PBS buffer, pH 7.4, Gibco) using spin desalting column (40 kD) . The resulting ADCs were submitted to do the characterizations.

Homogeneity assays. The drug/antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification of D0, D2, D4, and D6 by hydrophobic interaction chromatography (HIC) was performed on a Toyopearl phenyl 650M HIC column (Tosoh Biosciences, Montgomeryville, PA) at a flow rate of 10 mL/min at ambient temperature. The column size was 1 mL per 7.5 mg of ADCs. Solvent A was 2.0 M NaCl and 50 mM sodium phosphate pH 7. Solvent B was 80%v/v 50 mM sodium phosphate pH 7 and 20%v/v acetonitrile. The column was previously equilibrated with 5 column volumes of solvent A. the ADCs were mixed with 0.67 volume of 5 M NaCl (2.0 M final) and applied to the column. D0 was not retained by the column. The different drug loaded species were eluted by sequential step gradients: D2 was eluted with 35%solvent B, D4 was eluted with 70%solvent B, D6 was eluted with 95%solvent B.

For MS analysis, 20.0 μl ADC sample solution was added with 75.0 μL 8.0 mol/L Gdn-HCl, 5.0 μL 1.0 mol/L Tris-HCl and 2.0 μL 1 mol/L DTT. The new solution was mixed well and then incubated at 10～30℃ for 10～30 min. Then the drug/antibody ratio (DAR) and product distribution were analyzed using LC-MS.

As shown in Table 2 and Figures 1B-1D, cAb1, cAb3 and cAb4 antibodies obtained ADCs with about 90%or higher homogeneity of D6 species. The other 5 antibodies achieve similar results.

Table 2: HPLC result of the homogeneity of ADCs

Lot.	mAb	Linker-payload	D6 %
cAb1-MMAF	cAb	1	MC-MMAF	91
cAb3-MMAF	cAb	3	MC-MMAF	90
cAb4-MMAF	cAb	4	MC-MMAF	94

Example 3. Conjugation to form D2 species

3.0 eq TCEP and 1.0 eq ZnCl2 was added into mAb. The reaction vial was stood at 4 ℃ for 18 hours. DMA and Linker-payload solution (10 mg/mL stock in DMA) was added into the reduced antibody solution. The reactions were mixed properly and the reaction vials were stood at 4 ℃ for 2 hours.

Cysteine was added into the mixture. Then the new mixture was stood at 4 ℃ for 15 minutes. After 3.0 eq EDTA and 8.0 eq DHAA were added, the mixture was stood at 22 ℃ for 2 hours.

The crude product was submitted to a buffer exchange into its storage buffer (1X PBS buffer, pH 7.4, Gibco) using spin desalting column (40 kD) . The resulting ADCs (Figure 2A) were submitted to do the characterizations.

As shown in Table 3 and Figure 2B, cAb1, cAb2, cAb3 and cAb4 antibodies obtained ADCs with more than 80%homogeneity of D2 species. The other 4 antibodies achieve similar results.

Table 3

Example 4. Conjugation to form D (2+4) species

Using the reaction scheme (exemplified by cAb1) shown in Figure 3A and Table 4, the D2 ADC (mAb conjugated with Linker-payload 1) in buffer (1X PBS buffer, pH 7.4, gibco) was reduced by TCEP and the reaction vial was stood at 22 ℃ for 2 hours. The reduced antibody solution was directly submitted to next step conjugation without TCEP removal.

DMA and Linker-payload 2 solution (10 mg/mL stock in DMA) was added into the reduced antibody solution. The reactions were mixed properly and the reaction vials were stood at 22 ℃ for 2 hours.

The crude product was submitted to a buffer exchange into its storage buffer (1X PBS buffer, pH 7.4, gibco) using spin desalting column (40 kD) . The resulting ADCs were submitted to do the characterizations (Figures 3B-3E) .

As can be calculated from the MS result of Figure 3B, the content of D2+4 ADCs reach more than 90% (exemplified by cAb1) .

Table 4

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

Claims

A process for preparing antibody-drug conjugates (ADC) , wherein the antibody comprises a first and a second antigen-binding moiety,

the first antigen-binding moiety 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-native interchain disulfide bonds, and

the second antigen-binding moiety comprises a Fab, scFv or VHH,

and wherein the process comprises the following steps:

(a) incubating a reductant and the antibody in a buffer system;

(b) introducing an excess amount of a linker-drug moiety to react with reduced thiol groups resulted from step (a) ; and

(c) recovering the resultant antibody-drug conjugates.
The process according to claim 1, wherein the second antigen-binding moiety 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.
The process according to claim 1 or 2, wherein the incubation in step (a) is performed in the presence of an effective amount of a transition metal ion (s) and/or divalent metal ion (s) .
The process according to claim 1 or 2, wherein the incubation in step (a) is not performed in the presence of an effective amount of a transition metal ion (s) or divalent metal ion (s) .
The process according to claim 3, wherein the process further comprises between steps (b) and (c) : removing the metal ions from the product of step (b) , then introducing a reductant again and incubating with an excess amount of a different linker-drug moiety.
A process for preparing antibody-drug conjugates (ADC) , wherein the antibody comprises a first and a second antigen-binding moiety,

the first antigen-binding moiety 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 a non-native interchain disulfide bond (s) , and

the second antigen-binding moiety 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 process comprises the following steps:

(a) incubating a reductant and the antibody in the presence of an effective amount of a transition metal ion (s) and/or divalent metal ion (s) in a buffer system to selectively reduce inter-chain disulfide bonds within the antibody;

(b) introducing an excess amount of a first linker-drug moiety to react with reduced thiol groups resulted from step (a) ;

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

(d) incubating with a reductant (optionally same as the reductant in step (a) ) again and introducing an excess amount of a second linker-drug moiety; and

(e) recovering the resultant antibody-drug conjugates.
The process according to any of the preceding claims, wherein the process further comprises adding an effective amount of oxidant (such as DHAA) to re-oxidize the unreacted thiol groups before recovering the resultant antibody-drug conjugate.
The process according to any of the preceding claims, wherein the C1 region comprises the amino acid sequence of SEQ ID No: 2, 7 or a variant thereof with at least 90%identity and the C2 region comprises the amino acid sequence of SEQ ID No: 4, 8, 9 or a variant thereof with at least 90%identity, or the C1 region comprises the amino acid sequence of SEQ ID No: 4, 8, 9 or a variant thereof with at least 90%identity and the C2 region comprises the amino acid sequence of SEQ ID No: 2, 7 or a variant thereof with at least 90%identity.
The process according to any of the preceding claims, wherein the antibody comprises an IgG Fc region, such as in IgG1 or IgG4 isotype, preferably the Fc region comprises a knob into hole structure.
The process according to any of claims 3-9, wherein the transition metal ions and/or divalent metal ions in step (a) are selected from a group comprising Zn ²⁺, Cd ²⁺, Hg ²⁺, Ca ²⁺, Mg ²⁺and any combination thereof.
The process according to claim 10, wherein the metal ion (s) are selected from Zn ²⁺, Ca ²⁺and Mg ²⁺.
The process according to any of the preceding claims, wherein the buffer system used in step (a) is selected from a group comprising Hepes, Histidine buffer, PBS, and MES, and the pH value is about 5.5 to 8.
The process according to any of the preceding claims, wherein the antibody in step (a) is added in a final concentration of about 0.01 to 0.1 mM.
The process according to any of the preceding claims, wherein step (a) is performed at a temperature of about -10℃ to 37℃, for example, at about 0℃ to 22℃.
The process according to any of the preceding claims, wherein the reductant in step (a) is TCEP.
The process according to any of the preceding claims, wherein the linker-drug moiety is maleimide bearing a drug, an organic bromide bearing a drug, or an organic iodide bearing a drug.
The process according to any of the preceding claims, wherein the drug to be conjugated is selected from a group comprising diagnostic agents, therapeutic agents and labelling agents.
The process according to any of the preceding claims, wherein the drug is selected from maytansinoids such as DM1, DM3, DM4, dolastatins, dolostatin peptidic analogs and derivatives such as auristatins, calicheamicin, trichothecene, and CC1065, optionally is MMAE, DM1 or MMAF.
The process according to any of the preceding claims, wherein the process comprises generating the antibody prior to step (a) , wherein the variable regions of the first antigen-binding moiety are derived from a first parental antibody and the variable regions of the second antigen-binding moiety are derived from a second parental antibody.
The process according to claim 19, wherein the first and second parental antibody is the same.
The process according to claim 20, wherein the parental antibody is a monospecific, bispecific or multi-specific antibody.
The process according to any of the preceding claims, wherein the variable regions of the first and second antigen binding moiety are derived from any of the following parental antibodies: trastuzumab, pertuzumab, sacituzumab, abciximab, adalimumab, alefacept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, golimumab, inflectra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, secukinumab, and ustekinumab.
The process according to claim 22, wherein the first VH and the first VL are from Trastuzumab and the second VH and the second VL are from pertuzumab, or vice versa.
The process according to claim 22, wherein the first VH and the first VL are from Trastuzumab and the second VH and the second VL are from sacituzumab, or vice versa.
The process according to claim 3, wherein the resultant antibody-drug conjugate comprises D2 in a content higher than 80 wt%, for example, higher than 85 wt%, higher than 90 wt%, or higher than 95 wt%, on the basis of total weight of D0 and D2.
The process according to claim 4, wherein the resultant antibody-drug conjugate comprises D6 in a content higher than 85 wt%, for example, higher than 90 wt%, higher than 91 wt%, higher than 92 wt%, or higher than 93 wt%, on the basis of total weight of D0, D2, D4, and D6.
The process according to claim 5 or 6, wherein the resultant antibody-drug conjugate comprises D2+4 in a content higher than 65 wt%, for example, higher than 70 wt%, higher than 80 wt%, or higher than 90 wt%, on the basis of total weight of ADCs.
Antibody-drug conjugates prepared by the process of any one of preceding claims.
A pharmaceutical composition comprising an effective amount of the antibody-drug conjugates according to claim 28 and a pharmaceutically acceptable carrier or vehicle.
Use of the antibody-drug conjugates according to claim 28 in the manufacture of a pharmaceutical composition or a kit for treating a condition or disorder in a subject.
Use of an antibody format in preparing highly homogenous ADCs against selected antigens, wherein the antibody format comprises a first and second antigen-binding moiety,

the first antigen-binding moiety specifically binds to 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-native interchain disulfide bond (s) ,

the second antigen-binding moiety specifically binds to 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, and

said one or more non-native interchain disulfide bond (s) are not accessible to reductants and cannot be conjugated with a linker-drug moiety.
The use of claim 31, wherein the highly homogenous ADCs comprise a high content of D6 ADCs, D2 ADCs or bi-drug D2+4 ADCs.
A method for treating a condition or disorder in a subject, comprising administrating to the subject a therapeutically effective amount of the antibody-drug conjugates according to claim 28 or the pharmaceutical composition according to claim 29.
The method of claim 33, wherein the condition or disorder is cancer, autoimmune disease, or infectious disease.
The method of any of claims 33-34, wherein the subject is a mammal, for example, a human.
A kit, comprising one or more containers comprising the antibody-drug conjugates according to claim 28 or the pharmaceutical composition according to claim 29.