WO2022152308A1

WO2022152308A1 - Engineered anti-trop2 antibody and antibody-drug conjugate thereof

Info

Publication number: WO2022152308A1
Application number: PCT/CN2022/072469
Authority: WO
Inventors: Mingzhi JIN; Qing Zhou; Jia Wang; Li Yin; Jun Wang
Original assignee: Wuxi Biologics (Shanghai) Co., Ltd.; WuXi Biologics Ireland Limited
Priority date: 2021-01-18
Filing date: 2022-01-18
Publication date: 2022-07-21
Also published as: CN116761824B; CN116761824A

Abstract

Provided herein is an engineered dimeric antibody comprising in each monomer a Fab domain specifically binding to a Trop2 antigen operably linked to an engineered hinge region, wherein the engineered hinge region is composed of a portion of truncated IgG1 hinge region and a portion of truncated IgG4 hinge region such that the engineered dimeric antibody comprises at least two inter-chain disulfide bonds in its hinge domain. Also provided is an antibody-drug conjugate comprising the engineered antibody conjugated to one or more drug molecules through a linker, a method of its preparation, a composition comprising same and its use in treatment of cancers.

Description

ENGINEERED ANTI-TROP2 ANTIBODY AND ANTIBODY-DRUG CONJUGATE THEREOF

Field of the Invention

The present invention relates generally to the field of bio-pharmaceutical, and more particularly, engineered antibodies and antibody-drug conjugates.

Background

Antibody-drug conjugate (ADC) is a novel targeted drug comprised by an antibody for targeting, a connector and linker for drug attachment and a high potent payload as effector. Antibody or its relevant forms plays multiple roles in ADC drug. Antibody brings the cytotoxic drugs to antigen-expressing cells by antibody-antigen interaction. At the same time, toxicity of drugs after conjugated to antibody is dramatically reduced. Thus ADC enlarges the therapeutic window by reducing Minimum Effect Dose (MED) and elevating Maximum Tolerance Dose (MTD) .

Cysteine thiols in antibody as strong nucleophiles are ideal reaction groups for conjugation. Since cysteine residues exist as disulfide bonds in native form of antibodies, reduction of disulfide bonds between light-heavy chain and heavy-heavy chain in antibody provides perfect free cysteine thiols for conjugation. Here partial but not full reduction is preferred, since hydrophobicity of drugs and hindrance when all cysteine residues are attached causes the instability of ADC drugs in plasma. As reported, an average of four free thiols after partial reduction for IgG1 type antibodies is preferred as ADCs with drug-antibody ratio (DAR) being 4 exhibit the best therapeutic index in vivo. Since reducibility of the four disulfide bonds in IgG1 antibody cannot be well distinguished, homogeneity of resulted ADC after partial reduction is very poor. ADC species with low drug loading exhibit insufficient therapeutic effect, while ADC species with too high drug loading exhibit instability and toxicity as described above. Improving the homogeneity of ADC can solve this problem. Known approaches are linker-payload design, induction of point mutation, enzyme involved conjugation, conjugation process control

Human Trop2, also referred to as tumor associated calcium signal transducer 2 (TACSTD2) , is a single-pass transmembrane glycoprotein functional as an intracellular calcium single transducer. Trop2 is over-expressed in many cancers, for instance, breast cancer, lung cancer as well as pancreatic cancer. Although Trop2 is expressed in many normal tissues, the over-expression level of Trop2 in cancers is of prognostic significance, thus provides Trop2 as one of the possibility targets for cancer therapy. Human RS7 antibodies targeting Trop2 were described as potential therapeutic antibodies for disease treatment in PCT application No. PCT/GB03/00885 (published as WO03/074566) .

Antibody-drug conjugation is one of the drug formats using Trop2 as therapeutic target. Trodevy (sacituzumab govitecan-hziy) approved by FDA in 2020 is the first anti-Trop2 ADC in market. This ADC used hRS7 as antibody and CL2A-SN38 (see e.g., US 9,102,735 B2; and Oncotarget, 6 (26) : 22496-22512, 01 Sep 2015) as linker-payload for the treatment of Triple-negative Breast Cancer (TNBC) , with a DAR close to 8.0. Other anti-Trop2 antibody-drug conjugates are under developing. Since Trop2 is expressed in many normal tissues, stability of linker-payload on antibody is a main factor for anti-Trop2 ADC design.

Still, there remains a need for ADC products, e.g., anti-Trop2 ADCs, with improved DAR, homogeneity, stability and therapeutic efficacy.

Summary of Invention

The present disclosure provides an engineered anti-Trop2 antibody having an engineered hinge domain. It is surprisingly found that ADCs produced with the engineered anti-Trop2 antibody of the present invention comprise drug molecules mostly linked at the Fab domains, with an average DAR close to or equal to 4. These ADCs are advantageously characterized by high stability and excellent therapeutic efficacy.

In a first aspect, provided herein is an engineered dimeric antibody comprising in each monomer a Fab domain specifically binding to a Trop2 antigen operably linked to an engineered hinge region, wherein the engineered hinge region is composed of a portion of truncated IgG1 hinge region and a portion of truncated IgG4 hinge region such that the engineered dimeric antibody comprises at least two inter-chain disulfide bonds in its hinge domain.

In a further aspect, provided herein is a nucleic acid molecule or a combination of nucleic acid molecules encoding the engineered antibody of the present invention.

In a further aspect, provided herein is an antibody-drug conjugate comprising an engineered antibody of the invention conjugated to one or more drug molecules through a linker.

In a further aspect, provided herein is a composition comprising or consisting of a mixture of antibody-drug conjugates of the invention, wherein at least about 80%of the antibody-drug conjugates have a ratio of drug to antibody being 4.

In a further aspect, provided herein is a pharmaceutical composition comprising an antibody-drug conjugate of the invention and a pharmaceutically acceptable carrier.

In a further aspect, provided herein is a method of preparing the antibody-drug conjugate of the invention, comprising a step of conjugating a partially reduced antibody of the invention with a linker-payload compound bearing a maleimido or haloacetyl moiety via Michael addition reaction.

In a further aspect, provided herein is an antibody-drug conjugate product obtained by the method of the invention, comprising or consisting of a mixture of antibody-drug conjugates of the invention, wherein, at least about 80%of the antibody-drug conjugates have a ratio of drug to antibody being 4.

In a further aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of an antibody-drug conjugate of the invention, wherein the cancer is characterized in over-expression of Trop2.

In a further aspect, provided herein is an antibody-drug conjugate of the invention for use in treatment of cancer in a subject in need thereof, wherein the cancer is characterized in over-expression of Trop2.

The present invention provides various advantages. In particular, for the engineered anti-Trop2 antibody according to the present invention, since native immunoglobulin G hinge sequences are used and swapped at their natural structural positions, without introduction of any de novo amino acid sequence, the obtained engineered antibody will cause less immunogenicity in vivo. And, for the engineered antibodies, comparable protein expression titers can be obtained relative to their IgG1 or IgG4 counterparts. Furthermore, the engineered antibody of the invention allows to obtain highly homogeneous ADC products and to achieve the optimal four linker-payloads attachment under proper conjugation conditions, wherein ADCs with four linker-payloads occupy a high percentage of over 80%in the products.

For the anti-Trop2 antibody-drug conjugates according to the present invention, production can be significantly simplified, which can be carried out in a simple (one-pot) conjugation procedure comprising, first, partial reduction using a mild reductant, and then, conjugation in the same buffer. The ADC products according to the present invention advantageously have a high homogeneity, and the percentage of DAR4 species can increases to over 80%. Furthermore, the anti-Trop2 ADCs of the invention exhibit Trop2 expression-relevant cytotoxicity. In particular, the ADCs of the invention exhibit excellent cytotoxicity on cell lines with high Trop2 expression while weak cytotoxicity on cell lines with low or no Trop2 expression. The ADCs according to the present invention have high anti-tumor efficacy. And, the ADCs according to the present invention have excellent in vitro and in vivo stability.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description.

Brief Description of the Figures

The drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1: A schematic diagram of an engineered anti-Trop2 antibody according to the present invention.

Figure 2 shows the structure of antibody 886-36, the HIC-HPLC result of conjugation with MC-vc-PAB-MMAE and characterization of the ADC product.

Figure 3 shows the LC-MS characterization result of 886-36-MMAE. According to the detected abundance of drug molecules attached on the light chain and the detected abundance of drug molecules attached on the heave chain, most of the drug molecules are loaded on the Fab domains.

Figure 4 shows the structure of antibody 886-37, the HIC-HPLC result of conjugation with MC-vc-PAB-MMAE and characterization of the ADC product.

Figure 5 shows the LC-MS characterization result of 886-37-MMAE. According to the detected abundance of drug molecules attached on the light chain and the detected abundance of drug molecules attached on the heave chain, most of the drug molecules are loaded on the Fab domains.

Figure 6 shows the LC-MS characterization result of hRS-SN38 and characterization of the ADC product.

Figure 7 shows HIC-HPLC result of hRS-MMAE and characterization of the ADC product. HIC-HPLC analysis of hRS-MMAE shows normal distribution of conjugates in ADC mixture.

Figure 8 shows the cytotoxicity of different hRS-SN38-conjugated and MMAE-conjugated ADCs on MDA-MB-231 cells, MDA-MB-468 cells, HCC827 cells, BxPC-3 cells and Calu-6 cells. IC ₅₀ values show that MMAE-conjugated ADCs have high cytotoxicity potency in cell lines with high Trop2 expression and weak growth inhibition potency in cell lines with low or no Trop2 expression.

Figure 9 is a summary of the cytotoxicity data in Example 5 and figure 8.

Figure 10 shows cell culture medium stability of different ADCs. DARs of ADCs were determined by LC-MS. According to change in DAR over incubation, 886-36-MMAE and 886-37-MMAE have good stability during incubation.

Figure 11: Light chain and heavy chain sequences of the example antibodies, wherein the engineered hinge region is indicated in italics.

Figure 12 shows rat plasma stability of different ADCs.

Figure 13 shows binding affinity as detected via FACS for different ADCs on MDA-MB-468, HCC827 and BxPC-3.

Figure 14 shows the internalization result of different ADCs on MDA-MB-468, HCC827 and BxPC-3.

Figure 15 shows in vivo efficacy of different ADCs on HCC827.

Figure 16 shows in vivo efficacy of different ADCs on MDA-MB-468.

Figure 17 shows in vivo efficacy of different ADCs on BxPC-3.

Figure 18 shows rat PK results of different ADCs. Concentration of total and conjugated antibodies of each ADCs were determined by ELISA.

Definitions

As used herein, singular forms preceded by “a” , “an” and “the” include plural reference unless the context clearly dictates otherwise. As well, the terms “a” (or “an” ) , “one or more” and “at least one” can be used interchangeably herein.

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%.

The word "exemplary" is used herein to mean "serving as an example, instance, or illustration. " Any aspect described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other aspects.

Herein, the terms “comprise” , “include” , “characterized by (in) ” and “have” , as well as their grammatical variants can be used interchangeably, which should be understood as including the specified step or element without excluding any other steps or elements. Accordingly, they encompasses the exclusive inclusion meant by the close-ended term "consist of" and its grammatical variants, and the semi-closed inclusion meant by the term "consist essentially of" that is only open to qualitatively and/or quantitatively insignificant elements.

One or more features in one embodiment can be combined with any one or more features in another embodiment according to the present disclosure without departing from the spirit and concept of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skills in the fields to which this invention pertains. All publications and patents specifically mentioned herein are incorporated by reference for all purposes. All references cited in this specification are to be taken as indicative of the level of the skill in the art, which should not be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The term “antibody” as used herein encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to one or more specific antigens. Typically, as in the case of a native intact antibody, an antibody comprises two heavy chains and two light chains. Each heavy chain comprises a variable region ( “VH” ) and a first, a second, a third constant regions (CH1, CH2, CH3) and conditionally a fourth constant region (CH4) as in the cases of IgM and IgE antibodies, while each light chain consists of a variable region ( “VL” ) 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 variable regions of the light and the heavy chains are responsible for antigen binding. Each variable region typically contains three highly variable loops called "complementarity determining regions (CDRs) " . CDR boundaries can be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani. 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. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. The five major classes 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) . Accordingly, in context of the present invention, a particular IgG isotype, e.g, "IgG1" or "IgG1 isotype" , refers to IgG isotypes of the defined subclass, and different IgG isotypes refer to IgG isotypes of different subclasses.

The term “variable region” with respect to an antibody as used herein refers to an antibody variable region or a fragment thereof comprising one or more CDRs. Although a variable region may comprise an intact variable region (such as VH or VL) , it is also possible to comprise less than an intact variable region yet still retain the capability of binding to an antigen or forming an antigen-binding site.

The antibody may have a “Y” shape, wherein the two arms are also known as "Antigen-binding Fragments (Fab) " , and the stem portion comprises the hinge domain and the Fc domain of the antibody.

Herein, the terms "Fab" , "Fab domain" and "Fab arm" can be used interchangeably, which refer to the domain consisting of a light chain coupled with a heavy chain along the variable region and first constant region in an immunoglobulin (e.g., an antibody) . Typically, the Fab domain may comprise one or more inter-chain disulfide bonds. In some embodiments, the constant regions of both the light chain and the heavy chain can be replaced with TCR constant regions. The Fab domain is responsible for various antigen binding activities.

As used herein, the term “Fc region” refers to the fragment consisting of the second (CH2) and the subsequent constant regions of a heavy chain, or refers to the fragment consisting of a portion of the hinge region, the second (CH2) and the subsequent constant regions of a heavy chain. And, as used herein, the term "Fc domain" in context of a dimeric antibody refers to the portion of the coupled heavy chains along the Fc region of each. The Fc regions have various effector functions such as ADCC, and CDC.

As used herein, the term "hinge" or “hinge region” of a heavy chain refers to the region that connects the C-terminus of the CH1 to the N-terminus of the CH2 region of the heavy chain. A hinge region may have a length of about 12-62 amino acid residues. In human IgG1, the hinge region spans residues 216 to 230 by EU numbering, and in human IgG4 from residues 219 to 230 by EU numbering. As used herein, the term "hinge domain" in context of a dimeric antibody refers to the portion of the coupled heavy chains along the hinge region of each. Typically, the hinge domain may comprise one, two or more inter-chain disulfide bonds. Hinge regions are flexible, thus allowing the two Fab domains to move independently.

Hinge region is a flexible linker between the Fab and the Fc of antibody. Length and flexibility of the hinge region varies extensively among the IgG subclasses, IgG1, IgG2, IgG3, and IgG4. Taking IgG1 and IgG4 which are most commonly used as therapeutic biologics for example, the hinge region of IgG1 comprises 15 amino acids (e.g., EPKSCDKTHTCPPCP (SEQ ID NO: 5) ) and is very flexible, while IgG4 has a shorter hinge with only 12 amino acids (Gestur Vidarsson, et al., IgG subclasses and allotypes: from structure to effector functions, Frontiers in Immunology, 20 Oct 2014, 5: 520) . Wild-type IgG1 and IgG4 differ by one amino acid in the core hinge region (226-229 by EU numbering) : Cys-Pro-Pro-Cys in IgG1 and Cys-Pro-Ser-Cys in IgG4. Natural IgG4 presents an equilibrium between inter-and intra-chain cysteine disulfide bonds at the core hinge region, resulting in observable heavy chain arm exchange and the presence of IgG4 half molecules post secretion. S228P mutation for IgG4 (e.g., ESKYGPPCPPCP (SEQ ID NO: 6) ) has been confirmed to markedly stabilize the covalent interaction between IgG4 heavy-chains by preventing natural arm exchange, thus widely applied in IgG4 antibody development and production. The S228P mutation results in a poly-proline helix (PPCPPCP) in the IgG4 hinge, which when combined with the shorter IgG4 hinge length, will further restrict its flexibility compared to the IgG1 hinge. The flexibility difference between different hinges has important implications for antibody bio-conjugation because the cysteine residues located in a flexible hinge fragment are considered more reactive than the ones located in a rigid hinge. There are experiment evidences that both S228P IgG4 inter-heavy-light-chain and inter-heavy-heavy-chain disulfide bond are weakly reactive.

“CH2 domain” as used herein refers to the portion of a heavy chain molecule that extends, e.g., from about amino acid 244 to amino acid 360 of an IgG antibody using conventional numbering schemes (amino acids 244 to 360, Kabat numbering system; and amino acids 231-340, EU numbering system) .

The “CH3 domain” extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 amino acids. Certain immunoglobulin classes, e.g., IgM and IgE, further include a CH4 region.

The terms "Fv" , "Fv fragment" and "Fv domain" can be used interchangeably, which refer to the smallest domain that comprises the complete antigen binding site of an antibody. An Fv domain typically comprises the variable region of the light chain (VL) coupled with the variable region of the heavy chain (VH) .

Percentage (%) of identity between biological sequences, including amino acid sequences and nucleic acid sequences, is defined as the percentage of identical residues between a query sequence and a reference sequence according to an alignment for maximum matching. Sequence identity can be determined using publicly available tools, such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , ClustalW2 (available on the website of European Bioinformatics Institute, and ALIGN or Megalign (DNASTAR) software.

As used herein, the term “specific binding” or “specifically bind” refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen. An engineered antibody provided herein may specifically bind to a Trop2 antigen with a binding affinity (K _D) of ≤ 10 ^-6 M (e.g., ≤ 5x10 ^-7 M, ≤ 2x10 ^-7 M, ≤ 10 ^-7 M, ≤ 5x10 ^-8 M, ≤2x10 ^-8 M, ≤ 10 ^-8 M, ≤ 5x10 ^-9 M, ≤ 2x10 ^-9 M, ≤ 10 ^-9 M, or ≤ 10 ^-10 M) . K _D as used herein refers to the ratio of the dissociation rate to the association rate (k _off/k _on) .

The term “operably link” or “operably linked” refers to a juxtaposition, with or without a spacer or linker, of two or more biological sequences of interest in such a way that they are in a relationship permitting them to function in an intended manner. When used with respect to polypeptides, it is intended to mean that the polypeptide sequences are linked in such a way that permits the linked product to have the intended biological function. For example, an antibody variable region may be operably linked to a constant region so as to provide for a stable product with antigen-binding activity. The term may also be used with respect to polynucleotides. For one instance, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., promoter, enhancer, silencer sequence, etc. ) , it is intended to mean that the polynucleotide sequences are linked in such a way that permits regulated expression of the polypeptide from the polynucleotide.

A sequence less than 100%identical to a reference sequence may comprise mutation at one or more positions, wherein the mutation can be substitution, addition, deletion or a combination thereof. The substitution may be a “conservative substitution” , which refers to replacement with a different amino acid having a side chain of similar physiochemical properties or substitution at a site not critical to the activity or function of the sequence. For example, conservative substitutions can be a replacement between amino acids with a nonpolar side chain (e.g., Met, Ala, Val, Leu, and Ile, Pro, Phe, Trp) , between amino acids with an uncharged polar side chain (e.g., Cys, Ser, Thr, Asn, Gly and Gln) , between amino acids with an acidic side chain (e.g., Asp, Glu) , between amino acids with a basic side chain (e.g., His, Lys, and Arg) , between amino acids with a beta-branched side chain (e.g., Thr, Val and Ile) , between amino acids with a sulfur-containing side chain (e.g., Cys and Met) , or between amino acids with an aromatic side chain (e.g., Trp, Tyr, His and Phe) . Conservative substitution does not cause a significant change in conformational structure, and therefore could retain the biological activity of a protein.

As used herein, the term “subject” refers to a human or a non-human animal subject. Non-human animals may be mammals, such as primates. Examples of non-human animal subjects include but are not limited to domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, and bears. Preferably, the subject is a human. A "subject in need thereof" refers to a subject in need of diagnosis, prognosis, amelioration, prevention and/or treatment of a disease, disorder or condition.

Detailed Description of the Invention

The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety.

In certain embodiments, in the engineered anti-Trop2 antibody according to the invention, by including the engineered hinge domain, reducibility of inter-chain disulfide bonds is made different between the hinge domain and the Fab domains. More specifically, inter-chain disulfide bonds in Fab domains will be opened preferentially during partial reduction by mild reductants, which advantageously leads to a high homogeneity in the ADC products according to the present invention. By optimizing condition of production, the percentage of DAR4 species can increase to over 80%, with most of the drugs loaded on Fab domains. Taking advantages of the simple conjugation procedure, high homogeneity and drug loading position of the final products, CMC (Chemistry, Manufacturing and Control) for the anti-Trop2 ADCs according to the present invention is simple and easy to control. And the anti-Trop2 ADCs according to the present invention have excellent in vitro and in vivo stability, which indicates potential advantages in formulation development and in pharmacokinetic profile. Furthermore, the anti-Trop2 ADCs of the invention exhibit Trop2 expression-relevant cytotoxicity. In particular, the ADCs of the invention exhibit excellent cytotoxicity on cell lines with high Trop2 expression while weak cytotoxicity on cell lines with low or no Trop2 expression. The ADCs according to the present invention have high anti-tumor efficacy.

Engineered Antibody

In one aspect of the disclosure, provided herein is an engineered dimeric antibody comprising in each monomer a Fab domain specifically binding to a Trop2 antigen operably linked to an engineered hinge region, wherein the engineered hinge region is composed of a portion of truncated IgG1 hinge region and a portion of truncated IgG4 hinge region such that the engineered dimeric antibody comprises at least two inter-chain disulfide bonds in its hinge domain.

The dimeric antibody is formed of two paired monomers, wherein each monomer comprises a heavy chain coupled with a light chain. The engineered antibody may have an IgG format. The dimer is formed of the monomers coupled by inter-chain bonding, including inter-chain bonds and/or interactions. Examples of such inter-chain bonding include but are not limited to disulfide bonds, hydrogen bonds, electrostatic interaction, salt bridges, hydrophobic-hydrophilic interaction and Knobs-into-Holes mechanism.

In certain embodiment, the monomer further comprises a Fc region in the heavy chain, and the engineered antibody thus has a dimeric structure as schematically depicted in figure 1. The engineered antibody may be a homodimeric antibody.

The engineered antibody comprises an engineered hinge region of design, which is composed of a portion of truncated IgG1 hinge region and a portion of truncated IgG4 hinge region such that the engineered dimeric antibody comprises at least two inter-chain disulfide bonds in its hinge domain. The engineered hinge region is constituted with natural acids and comprises cysteine residues for forming the at least two inter-chain disulfide bonds between the heavy chains. In context of the engineered dimeric antibody of the invention, the term "hinge domain" refers to the portion of the coupled heavy chains along the hinge region of each.

The wild-type hinge region of IgG1 comprises 15 amino acids (e.g., EPKSCDKTHTCPPCP (SEQ ID NO: 5) ) and is very flexible, while IgG4 has a shorter hinge with only 12 amino acids (supra) . Wild-type IgG1 and IgG4 differ by one amino acid in the core hinge region (226-229 by EU numbering) : Cys-Pro-Pro-Cys in IgG1 and Cys-Pro-Ser-Cys in IgG4, IgG hinge with S228P mutation may be represented by the sequence of ESKYGPPCPPCP (SEQ ID NO: 6) . The S228P mutation results in a poly-proline helix (PPCPPCP) in the IgG4 hinge, which when combined with the shorter IgG4 hinge length, will further restrict its flexibility compared to the IgG1 hinge. The flexibility difference between different hinges has important implications for antibody bio-conjugation because the cysteine residues located in a flexible hinge fragment are considered more reactive than the ones located in a rigid hinge. There are experiment evidences that both S228P IgG4 inter-heavy-light-chain and inter-heavy-heavy-chain disulfide bond are weakly reactive.

The inventors surprisingly find that the engineered hinge region according to the invention offers an improved payload-antibody ratio (PAR, equivalent to DAR) during bio-conjugation, taking advantage of differential accessibility by a reductant among the inter-chain disulfides in the hinge domain and the Fab domains. In particular, as said above, engineered antibodies with the engineered hinge region provide various advantages in ADC production and the ADC product per se, such as improved DAR and DAR homogeneity, simplified production, desirable pharmacokinetic properties and/or pharmcodynamic properties.

In some embodiments, the engineered hinge region may comprise a sequence having the following formula (I) :

EPKx ₁C x ₂ x ₃ x ₄ x ₅ x ₆ x ₇ x ₈ CPPCP (I)

Wherein, x ₁ = null or S, preferably S; x ₂ = null or E or S, preferably null; x ₃ = null or S; x ₄ = null or K or D; x ₅ = Y or K, preferably Y; x ₆ = G or T, preferably G; and/or x ₇x ₈ = PP, PT, HP or HT, preferably PP. In some embodiments, the engineered hinge region comprises a sequence of formula EPKSC x ₂ x ₃ x ₄ x ₅ x ₆ PPCPPCP.

Preferred examples of the engineered hinge region include:

EPKSCESKYGPPCPPCP (SEQ ID NO: 1) ,

EPKSCSKYGPPCPPCP (SEQ ID NO: 2) ,

EPKSCKYGPPCPPCP (SEQ ID NO: 3) ,

EPKSCYGPPCPPCP (SEQ ID NO: 4) ; and

a sequence having an identity of at least 85%, preferably at least 90%and more preferably at least 95%to anyone of the above.

In some embodiments, the engineered hinge region may further comprise an additional hinge segment (e.g., an upper hinge region segment) at either or both sides of the designated region.

In some embodiments, the Fab domain is one of an IgG1 antibody, i.e., one of IgG1 isotype. Preferably, the Fab domain is one of a human IgG1 antibody. According to the invention, the Fab domain specifically binds to a Trop2 antigen, preferably a human Trop2 antigen, and may be a Fab domain of any anti-Trop2 antibodies. Examples of such antibodies include but are not limited to Sacituzumab and Datopotamab.

In some embodiments, the engineered antibody further comprises a Fc domain, preferably a human Fc domain. Preferably, the Fc domain is an IgG Fc domain, i.e., one of IgG class, and more preferably, one of IgG1 or IgG4 isotype. Apparently, the same classification applies to the Fc region of the heavy chain.

In one example, the engineered antibody of the invention comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 8 or a sequence having an identity of at least 85%to SEQ ID NO: 8 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 7 or a sequence having an identity of at least 85%to SEQ ID NO: 7. In another example, the engineered antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 or a sequence having an identity of at least 85%to SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 9 or a sequence having an identity of at least 85%to SEQ ID NO: 9.

In a further aspect of the disclosure, provided here is a nucleic acid molecule or a combination of nucleic acid molecules encoding the engineered antibody of the present invention. In some embodiments, the nucleic acid molecule or each of the combination encodes one or more of the sequences of SEQ ID NOs: 7 to 10 or sequences having an identity of at least 85%to anyone of SEQ ID NOs: 7 to 10. In some embodiments, the nucleic molecule or molecules may be provided in the form of one or more vectors, especially expression vectors. As can be well understood by an ordinarily skilled person in the art, nucleic acids encoding the heavy chain and the light chain of an antibody can be cloned into separate expression vectors and co-transferred into a host to recombinantly produce the antibody, and it is also possible to insert the coding sequences for both chains in one expression vector. Any expression vectors and hosts known in the field are useful for the present invention. Examples include but are not limited to plasmids, viral vectors, synthetic vectors, bacteria hosts, yeasts, insect cells and animal cells, such as CHO cells. In some embodiments, said nucleic acid molecule or a combination of nucleic acid molecules, like vectors, may be provided in the form of a kit, which may optionally comprises instruction on using the nucleic acid molecule or molecules to recombinantly produce the antibody.

Antibody-drug conjugate

In one aspect of the disclosure, provided herein is an antibody-drug conjugate comprising the engineered antibody of the invention conjugated to one or more drug molecules through a linker. The engineered antibody comprises a Fab domain specifically binding to a Trop2 antigen, preferably a human Trop2 antigen, and may be a Fab domain of any anti-Trop2 antibodies. Examples of such antibodies include but are not limited to Sacituzumab and Datopotamab.

The drug (also known as "payload" ) used in the present invention is not particularly limited. Drugs for use in the present invention include cytotoxic drugs, particularly those which are used for cancer therapy. Such drugs include, but are not limited to, DNA damaging agents, DNA binding agents, anti-metabolites, enzyme inhibitors such as thymidylate synthase inhibitors and topoisomerase inhibitors, tubulin inhibitors, and toxins (for example, toxins of a bacterial, fungal, plant or animal origin) . Specific examples include, for example, taxol, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxanes including taxol, taxotere retinoic acid, butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin, esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, hemiasterlins, maytansinoids (including DM1, DM2, DM3, DM4) , auristatins including monomethylauristatin E (MMAE) , monomethylauristatin F (MMAF) and monomethylauristatin D (MMAD) . In some embodiments, auristatins, eg., MMAE, are preferred. Drugs can be linked to the linker via any suitable methods known in the art. In some embodiments, the drug is provided for conjugation in the form of a linker-payload compound as an intermediate, like in the case of "MC-vc-PAB-MMAE" .

The drug used in the present invention can be bound to an antibody via a linker. Various linkers for ADCs are known in the art. Linkers useful in the present invention are not particularly limited, as long as it includes a moiety capable reacting with a thoil group rendered by an antibody and thereby linking to the antibody. Particularly useful in the present invention are amleimido or haloactyl functionalized linkers. Examples include, but are not limited to -MC-vc-PAB- ("MC" : Maleimide-caproyl; "-vc-" : the dipeptide of -Val-Cit-; "PAB" : para-aminobenzyl) , -MC-GGFG- ("-GGFG-" : the tetrapeptide of –Gly-Gly-Phe-Gly-) , -MC-vc-, -MC-and -SMCC- (succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate) . In some embodiments, the linker is -MC-vc-PAB-.

In some embodiments, the linker-payloads are connected to the cysteine residues provided by selected inter-chain disulfide bonds opened by reduction. In some embodiments, the conjugate of the invention may have a ratio of drug to antibody (DAR) ranging from about 2 to about 8, preferably about 2 to about 6, and more preferably about 4. The ratio may refer to an average ratio in a population, such as an average of DAR4 for a population of ADCs. And, in a preferred embodiment, the conjugate comprises the drug molecules mostly attached at Fab domains, and in some cases, comprises all the four drug molecules attached at Fab domains.

In a further aspect, provided herein is a composition comprising or consisting of a mixture of antibody-drug conjugates according to the invention, wherein at least about 80%, at least about 85%, at least about 90%, or at least about 95%of the mixture have a DAR of 4. In some cases, the conjugates having a DAR of 4 have the four drug molecules all linked at the Fab domains.

In a further aspect, provided herein is a pharmaceutical composition comprising the antibody-drug conjugate or the mixture thereof as described above and a pharmaceutically acceptable carrier.

Preparation of antibody-drug conjugate

The ADCs of the invention can be prepared using any suitable methods known in this field. According to the invention, linker-payloads are conjugated at cysteine residues freed from disulfide bonds via reduction using a mild reductant. Particularly, the engineered hinge domain according to the invention alters reducibility of the disulfide bonds in the hinge domain, which leads to selective reduction of the disulfide bonds in the Fab domains, when the antibody is partially reduced using a mild reductant. This advantageously provides a highly homogeneous product comprising predominantly conjugates with four linker-payloads mostly attached at the Fab domains.

Herein is provided a method of preparing the antibody-drug conjugate of the invention. In brief, the method may involve partial reduction of the antibody and conjugation between the partially reduced antibody and a linker-payload. Preferably, the conjugation is conducted in a reduction buffer with an organic solvent as additive to help dissolve the linker-payload. Specifically, the method may comprise a step of conjugating a partially reduced antibody of the invention with a linker-payload compound bearing a maleimido or haloacetyl moiety via Michael addition reaction. The partially reduced antibody may be produced by partially reducing an engineered antibody of the invention using a mild reductant. In some embodiments, the method may comprise:

partially reducing an engineered antibody of the invention using a mild reductant, and

conjugating the partially reduced antibody with a linker-payload compound bearing a maleimido or haloacetyl moiety via Michael addition reaction.

In some embodiments, the mild reductant is TCEP or DTT. In some embodiments, the reductant/antibody ratio is about 1 to 20, preferably about 3 to 10, about 3 to 8, about 3 to 6 or about 3 to 5. In some embodiments, the partial reduction is conducted at pH of about 4.0 to 8.0, preferably about 5 to 6. In some embodiments, the partial reduction is conducted for a period of about 0.5 to 24 hours (hr) , preferably about 1 to 20 hours, about 2 to 16 hours or about 3 to 5 hours. In some embodiments, the partial reduction is conducted at a temperature of about 4 to 37 ℃, preferably about 4 to 15 ℃ or about 4 to 10 ℃.

In some embodiments, the conjugation is carried out in a buffer of pH ranging from about 4.0 to 8.0, optionally in presence of an organic additive (e.g., organic solvent or organic co-solvent) at an amount of about 0.0%to 20.0%by weight, preferably about 5.0%to 15.0%or about 10.0%to 15.0%. In some embodiments, the drug/antibody ratio may be about 7 to 20, preferably about 7 to 10; the reaction temperature may be about 4 to 37 ℃, preferably about 4 to 20 ℃ or about 4 to 10 ℃; and/or the time of reaction may be about 0.5 to 4 hours, preferably about 1 to 3 hours.

In some embodiments, the method of the invention provides a product comprising or consisting of a mixture of antibody-drug conjugates, wherein at least about 80%, at least about 85%, at least about 90%, or at least about 95%of the mixture have a DAR of 4. In some cases, the conjugates having a DAR of 4 have the four drug molecules all linked at the Fab domains.

Treatment

The antibody-drug conjugate of the invention can be used for treating cancers that are characterized in over-expression of Trop2. Accordingly, also provided herein is a method of treating cancer in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of the antibody-drug conjugate of the invention, wherein the cancer is characterized in over-expression of Trop2. Also provided herein is an antibody-drug conjugate according to the present invention for use in treatment of cancer in a subject in need thereof, wherein the cancer is characterized in over-expression of Trop2. In particular, the cancer can be selected from the group consisting to breast cancer, pancreatic cancer and lung cancer.

Abbreviation

ADC: Antibody-drug conjugate

CH: Constant region of heavy chain

CMC: Chemistry, Manufacturing and Controls

DAR: Drug-antibody ratio

DMA: N, N’-Dimethylacetamide

DTT: 1, 4-Dithiothreitol

EGFR: Epidermal growth factor receptor

Fab: Antigen-binding fragment

Fc: Crystallizable fragment,

FDA: Food and Drug Administration

FGE: Formylglycine-generating enzyme

HIC: Hydrophobic interaction chromatography

HPLC: High performance liquid chromatography

IC50: The half maximal inhibitory concentration

IgG: Immunoglobulin G

MC: Maleimide-caproyl

MED: Minimum Effect Dose

MMAE: Monomethyl auristatin E

MTD: Maximum Tolerance Dose

MWCO: Molecular weight cut-off

NaCl: Sodium Chloride

NNAA: Non-natural amino acid

mTG: Microbial transglutaminase

PAB: para-aminobenzyl

PAR: Payload-antibody ratio

RP: Reverse phase

SEC: Size exclusion chromatography

TCEP: Tris (2-carboxyethyl) phosphine

VH: Variable region of heavy chain

eq: Molar ratio of reductant/antibody

Examples

General Method

Antibody Preparation

All antibody molecules described in this document were codon optimized for Cricetulus griseus, synthesized, cloned into production vectors, and then maxi-prepared from TOP10 E. coli cells following standard molecular biology procedures.

CHO K1 host cells were seeded at 2-4E5 cells/mL in CD CHO medium 72 hours before transfection. The host cells were counted for cell density using Vi-CELL, centrifuged at 290 g for 7 min and then resuspended in pre-warmed fresh CD CHO medium prior to transfection. The re-suspended host cells were incubated in a Kuhner shaker (36.5℃, 75%humidity, 6%CO ₂, 120 rpm) before use.

A total 4 mg of plasmids encoding the antibody of interest were added into the re-suspended host cells, followed by 12 mg polyetherimide. The transfected cultures were incubated in a Kuhner shaker at 36.5℃, 75%humidity, 6%CO ₂, 120 rpm for 4 hours. After proprietary supplements were added, the transfected cultures were then incubated in a Kuhner shaker at 31 ℃, 75%humidity, 6%CO ₂, 120 rpm for 9-10 days.

On the harvest day, transfected cultures were clarified by primary centrifugation at 1,000 g for 10 min, and secondary centrifugation at 10,000 g for 40 min, followed by sterile filtration through 0.22 μm filter. The supernatants were measured for titers and purified by ProA chromatography. The ProA eluate were neutralized by adding 1-2%neutralization buffer (1 M Tris-HCl, pH 9.0) and formulated in 20 mM Histidine-Acetate buffer, pH 5.5.

All proteins were subjected to quality control tests before conjugation, including reducing and non-reducing SDS-PAGE, SEC-HPLC, endotoxin level detection by LAL gel clot assay and molecular identification by mass spectrometry.

Preparation of ADC

To an antibody solution concentration 1mg/ml to 20mg/ml in a buffer with pH 4.0-8.0, such as Histidine-acetate, 1 to 20 eq (e.g., in some embodiments, 3-10eq) of reducing reagent such as TCEP or DTT was added. The reduction was performed at 4-37 ℃ for 0.5hr to 24hr with gentle shaking or stirring. Without purification, organic co-solvent such as DMA was added to the partial reduced antibody to a concentration of 0%to 20%, with 7-20eq of Maleimido or Haloacetyl functionalized linker-payload. The conjugation was performed at 4-37 ℃ for 0.5hr to 4hr with gentle shaking or stirring. Final conjugated product was characterized with UV-vis for concentration, HIC-HPLC for conjugate distribution and DAR, RP-HPLC for drug loading on light chain and heave chain as well as free drug residue, SEC-HPLC for aggregation and purity, and kinetic turbidimetric for Endotoxin level.

HIC-HPLC

SEC-HPLC

RP-HPLC for drug loading

Procedure: mix 20μl of ADC sample with 75μl 8M Guanidine-HCl and 5μl Tris-HCl, pH 8.0. Then add 1μl 0.5M TCEP solution into the mixture. The reaction was performed at 37 ℃ for 30min and then tested with RP-HPLC for drug loading on antibody.

RP-HLPC for free drug determination

Procedure: 85μl ADC solution was mixed with 15μl DMA and then protein was precipitated with 100μl precipitation buffer (37.5%v/v of Methanol in Acetonitrile, saturated with NaCl) and vortex at 1400rpm for 10min at 22 ℃.

Sample was centrifuged at 16000rpf for 10min. The supernatant was taken for RP-HPLC assay together with standard sample for free drug determination.

LC-MS for DAR determination

Procedure: 85μl ADC solution was mixed with 15μl 50mM TCEP and then incubated for 30min at 22 ℃. Samples were subjected to LC-MS for DAR determination.

The following Examples are provided merely for illustration, with no intends to limit scope of the invention.

Example 1

Anti-Trop2 antibody 886-36, also referred to as "WBP886-36" in FIG. 11, was constructed using the engineered hinge domain comprising the sequence of SEQ ID NO: 2. It has the light chain (LC) sequence of SEQ ID NO: 7 and the heavy chain (HC) sequence of SEQ ID NO: 8. The variable regions VH and VL have the sequences as disclosed in PCT/GB03/00885 for an RS7 antibody. The antibody was recombinantly produced as described in the part of general method.

The antibody was dissolved in 20mM Histidine-acetate pH 5.5 to a concentration of 7.2mg/ml. 5eq of TCEP was added to the antibody solution and the mixture was incubated at 4 ℃ for 2hr. Then DMA was added to the reduced antibody to a concentration of 10%, followed by 10eq of MC-vc-PAB-MMAE (Levena Biopharma, SET0201) . Conjugation reaction was performed at 4 ℃ for 1hr. The conjugated product was purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. Final product was characterized with HIC-HPLC for DAR and drug distribution determination, SEC-HPLC for purity and aggregation level test, LC-MS for drug loading test, RP-HPLC for free drug residue and kinetic turbidimetric for Endotoxin level (Fig. 2 and Fig. 3) . As seen, the product was highly homogeneous, predominantly comprising the DAR4 species with a percentage as high as 82.3%. Further, according to the LC-MS results, the drug molecules were mostly loaded on the Fab domains. And, product characterization shows that the ADC designated as "886-36-MMAE" can be used for in vitro and in vivo study.

HIC-HPLC result of DAR and drug distribution:

ADC	TCEP ratio/T	D0	D2	D4	D6	D8	DAR
886-36-MMAE	5.0/4℃	1.2	5.7	82.3	8.7	2.1	4.1

Example 2

Anti-Trop2 antibody 886-37, also referred to as "WBP886-37" in FIG. 11, was constructed using the engineered hinge domain comprising the sequence of SEQ ID NO: 2. It has the light chain (LC) sequence of SEQ ID NO: 9 and the heavy chain (HC) sequence of SEQ ID NO: 10. The variable regions VH and VL have the sequences as disclosed in PCT/GB03/00885 for another RS7 antibody. The antibody was recombinantly produced as described in the part of general method.

The antibody was dissolved in 20mM Histidine-acetate pH 5.5 to a concentration of 6.9mg/ml. 3.5eq of TCEP was added to the antibody solution and the mixture was incubated at 4 ℃ for 2hr. Then DMA was added to the reduced antibody to a concentration of 10%, followed by 8eq of MC-vc-PAB-MMAE. Conjugation reaction was performed at 4℃for 1hr. The conjugated product was purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. Final product was characterized with HIC-HPLC for DAR and drug distribution determination, SEC-HPLC for purity and aggregation level test, LC-MS for drug loading test, RP-HPLC for free drug residue and kinetic turbidimetric for Endotoxin level (Fig. 4 and Fig. 5) . As seen, the product was highly homogeneous, predominantly comprising the DAR4 species with a percentage as high as 82.0%. Further, according to the LC-MS results, the drug molecules were mostly loaded on the Fab domains. And, product characterization shows that the ADC designated as "886-37-MMAE" can be used for in vitro and in vivo study.

HIC-HPLC result of DAR and drug distribution:

ADC

TCEP ratio/T

D0

D2

D4

D6

D8

DAR

886-37-MMAE

3.5/4℃

1.6

7.7

82.0

7.8

0.9

4.0

Example 3

Anti-Trop2 antibody Sacituzumab (hRS7) was dissolved in 20mM Histidine-acetate pH 5.5 to a concentration of 5.0mg/ml. 8.0eq of TCEP was added to the antibody solution and the mixture was incubated at 37℃ for 3hr. Then DMA was added to the reduced antibody to a concentration of 10%, followed by 14eq of CL2A-SN38 (Levena Biopharma, SET0217) . Conjugation reaction was performed at 4 ℃ for 1hr. The conjugated product was purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. The final product "hRS7-SN38" was characterized with LC-MS for DAR determination, SEC-HPLC for purity and aggregation level test, RP-HPLC for free drug residue and kinetic turbidimetric for Endotoxin level (Fig. 6) .

Conjugation result:

ADC	TCEP ratio/T	LC	LC+1	LC+2	HC+1	HC+2	HC+3	DAR
hRS7-SN38	8.0/37℃	3.1	96.0	0.8	0.1	2.1	97.7	7.9

Example 4

Anti-Trop2 antibody Sacituzumab (hRS7) was dissolved in 20mM Histidine-acetate pH 5.5 to a concentration of 8.0mg/ml. 2.1eq of TCEP was added to the antibody solution and the mixture was incubated at 37 ℃ for 3hr. Then DMA was added to the reduced antibody to a concentration of 10%, followed by 7eq of MC-vc-PAB-MMAE. Conjugation reaction was performed at 4 ℃ for 1hr. The conjugated product was purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. The final product "hRS7-MMAE" was characterized with HIC-HPLC for DAR and drug distribution determination, SEC-HPLC for purity and aggregation level test, LC-MS for drug loading test, RP-HPLC for free drug residue and kinetic turbidimetric for Endotoxin level (Fig. 7) .

HIC-HPLC result of DAR and drug distribution:

ADC	TCEP ratio/T	D0	D2	D4	D6	D8	DAR
hRS7-MMAE	2.1/37℃	5.3	23.8	44.1	22.4	4.4	3.9

Example 5

In vitro cytotoxicity experiment: The antibody-drug conjugates targeting Trop2 were tested for cytotoxicity on human breast cancer cell lines MDA-MB-231 (Trop2 low expression) and MDA-MB-468 (Trop2 high expression) , human non-small cell lung cancer cell line HCC827 (Trop2 high expression) , pancreatic carcinoma cell line BxPC-3 (Trop2 high expression) and human pulmonary adenocarcinoma cell line Calu-6 (Trop2 low expression) , respectively.

All five cell lines were cultured in RPMI-1640 medium supplemented with 10%fetal bovine serum. All five cell lines were plated in 96-well-plate at 3000 cells/well and treated with ADCs 24hr after cells were plated. Viability of cells was analyzed after 5 days treatment with ADCs at 37 ℃. Percentage of inhibition and maximum Percentage of inhibition were calculated (Fig. 8) . The data were further summarized in Fig. 9.

As seen, the ADCs of the invention have high cytotoxicity potency in cell lines with high Trop2 expression, and weak growth inhibition potency in cell lines with low Trop2 expression.

Example 6

hRS7-SN38, hRS7-MMAE, 886-36-MMAE and 886-37-MMAE were incubated with RPMI-1640 medium at 0.5mg/mL at 37 ℃. Samples were taken at 0 day, 1 day, 3 days and 5 days, respectively. DAR of samples at each time point was determined with deconvolution result of LC-MS (Fig. 10) .

According to change in DAR over incubation, 886-36-MMAE and 886-37-MMAE have good stability during incubation.

Example 7

hRS7-SN38, hRS7-MMAE, 886-36-MMAE and 886-37-MMAE were incubated with rat plasma at 0.5mg/mL at 37 ℃. Samples were taken at 6 hours, 24 hours, 72 hours, 120 hours and 240 hours, respectively. Percentage (%) of drug remaining in samples at each time point was determined with deconvolution result of DARs of ADCs determined by LC-MS compared to their original ones (Fig. 12) .

According to change in %of drug remaining over incubation, 886-36-MMAE and 886-37-MMAE have good rat plasma stability during incubation.

Example 8

FACS was used to detect the binding of ADCs to human Trop2. On the assay day, tumor cells (1×10 ⁵ cells/well) were incubated with serially-diluted ADCs and antibody for 1-2 hours at 4℃. Human IgG isotype antibodies were used as negative controls. Negative control 1 was the isotype control for 886-36 and negative control 2 the isotype control for 886-37. The negative controls shared the same Fc region with the test antibodies respectively, but do not bind to any antigen. After washing the cells with FACS staining buffer, the secondary antibody, Alexa647-conjugated goat anti-human IgG Fc was diluted in FACS staining buffer and then added to cells. The plates were incubated at 4℃ for 20-60 minutes in the dark. The fluorescence intensity of the cells was measured by the flow cytometer and analyzed by FlowJo software. The EC50 values were calculated using GraphPad Prism software (Fig. 13) .

According to EC50, 886-36 and 886-37, hRS7-SN38, hRS7-MMAE, 886-36-MMAE and 886-37-MMAE have similar binding affinity to hRS7 antibody.

Example 9

Fab-ZAP assay was used to detect antibody internalization. Tumor cell lines, MDA-MB-468, HCC827 and BxPC-3 cells, were routinely cultured in RPMI1640 medium. One day before the assay day, tumor cells were seeded into 96-well plates in cell culture medium at appropriate cell densities. Next day, antibody was mixed with Fab-ZAP serially diluted in cell culture medium and then added into the 96-well plates pre-plated with tumor cells. Human IgG isotype antibodies were used as negative controls. Negative control 1 was the isotype control for 886-36 and negative control 2 the isotype control for 886-37. The negative controls shared the same Fc region with the test antibodies respectively, but do not bind to any antigen. The plates were kept in an incubator set to 37℃, 5%CO ₂ for 3-6 days. After incubation, cell viability was test using CellTiter-Glo. The IC50 values were calculated using GraphPad Prism software (Fig. 14) .

According to IC50, 886-36 and 886-37 have similar internalization efficiency to hRS7 antibody.

Example 10

The tumor cells (HCC827) were maintained in vitro and was routinely subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Balb/c nude mice, female, 6-8 weeks, weighing approximately 18-22 g, were inoculated subcutaneously at the right flank with MDA-MB-468 tumor cells (10 x 10 ⁶) in 0.2 ml of PBS and Matrigel (1: 1) for tumor development. When the tumor volumes reache approximately 150-200 mm ³ on average by visual check, all animals were weighed and measured for individual tumor volumes. Then all mice were randomly assigned into groups and injected with 3 mg/kg of hRS7-MMAE, 886-36-MMAE or 886-37-MMAE, respectively. Body weight and tumor size were recorded every 3-4 days (Fig. 15) .

As seen, the ADCs of the invention have high tumor inhibition efficacy in HCC827 CDX model.

Example 11

The tumor cells (MDA-MB-468) were maintained in vitro and was routinely sub-cultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Balb/c nude mice, female, 6-8 weeks, weighing approximately 18-22 g, were inoculated subcutaneously at the right flank with HCC827 tumor cells (1 x 10 ⁶) in 0.2 ml of PBS and Matrigel (1: 1) for tumor development. When the tumor volumes reache approximately 150-200 mm ³ on average by visual check, all animals were weighed and measured for individual tumor volumes. Then all mice were randomly assigned into groups and injected with 3 mg/kg of hRS7-MMAE, 886-36-MMAE or 886-37-MMAE, respectively. Body weight and tumor size were recorded every 3-4 days (Fig. 16) .

As seen, the ADCs of the invention have high tumor inhibition efficacy in MDA-MB-468 CDX model.

Example 12

The tumor cells (BxPC-3) were maintained in vitro and was routinely subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Balb/c nude mice, female, 6-8 weeks, weighing approximately 18-22 g, were inoculated subcutaneously at the right flank with BxPC-3 tumor cells (5 x 10 ⁶) in 0.1 ml of PBS for tumor development. When the tumor volumes reache approximately 150-200 mm ³ on average by visual check, all animals were weighed and measured for individual tumor volumes. Then all mice were randomly assigned into groups and injected with 3 mg/kg of hRS7-MMAE, 886-36-MMAE or 886-37-MMAE, respectively. Body weight and tumor size were recorded every 3-4 days (Fig. 17) .

As seen, the ADCs of the invention have high tumor inhibition efficacy in BxPC-3 CDX model.

Example 13

Juvenile male Sprague-Dawley rats, 7-10 weeks, were randomly assigned into groups and administered with a single bolus intravenous injection of 10 mg/kg of hRS7-MMAE, 886-36-MMAE or 886-37-MMAE, respectively. Blood samples, about 0.25 mL per time point, were taken at 0.083, 6, 24, 48, 72 and 144 hours, respectively, and transferred into pre-chilled EDTA-K2 tubes and placed on wet ice until centrifugation. Plasma samples were collected after centrifugation and quick frozen over dry ice until ELISA analysis.

Total Antibody ELISA. Nunc MaxiSorp 96-well plates were coated with human Trop2 protein and incubated overnight at 4 ℃, and washed 5 times with 0.05%Tween-20 in PBS buffer (pH 7.4) . Diluted standards and the ADC plasma samples were added to the wells and incubated for 1 h at 37℃. The plates were washed 6 times and a detection antibody, goat anti-human Kappa antibody conjugated to horseradish peroxidase, was added to the wells and incubated for 1 h at 37℃. The plates were washed 6 times and developed using TMB peroxidase substrate. The assay range for ADC was 0.32768-1250 μg/mL with a minimum dilution of 1: 2.5.

Conjugated Antibody ELISA. Nunc MaxiSorp 96-well plates were coated with anti-MMAE antibody and incubated overnight at 4℃. The plates were washed 5 times with 0.05%Tween-20 in PBS buffer (pH 7.4) . Diluted standards and the ADC plasma samples were added to the wells and incubated for 1 h at 37℃. The plates were washed 6 times and a detection antibody, goat anti-human Kappa antibody conjugated to horseradish peroxidase, was added for 1 h at 37℃. All plates were developed using TMB peroxidase substrate. The assay range for ADC was 0.3-600 μg/mL with a minimum dilution of 1: 1.5 (Fig. 18) .

As seen, the ADCs of the invention have similar PK stability in the perspective of total antibody, and obviously better stability in the perspective of conjugated antibodies, as compared with hRS7-MMAE.

Claims

An engineered dimeric antibody comprising in each monomer a Fab domain specifically binding to a Trop2 antigen operably linked to an engineered hinge region, wherein the engineered hinge region is composed of a portion of truncated IgG1 hinge region and a portion of truncated IgG4 hinge region such that the engineered dimeric antibody comprises at least two inter-chain disulfide bonds in its hinge domain.
The engineered antibody of claim 1, wherein, the engineered hinge region comprises a sequence having the following formula:

EPKx ₁C x ₂ x ₃ x ₄ x ₅ x ₆ x ₇ x ₈ CPPCP (I)

Wherein, x ₁ = null or S; x ₂ = null or E or S, preferably null; x ₃ = null or S; x ₄ = null or K or D; x ₅ = Y or K; x ₆ = G or T; and/or x ₇x ₈ = PP, PT, HP or HT.
The engineered antibody of claim 1, wherein, the engineered hinge region comprises (a) a sequence of EPKSCESKYGPPCPPCP (SEQ ID NO: 1) , EPKSCSKYGPPCPPCP (SEQ ID NO: 2) , EPKSCKYGPPCPPCP (SEQ ID NO: 3) , or EPKSCYGPPCPPCP (SEQ ID NO: 4) ; or (b) a sequence having an identity of at least 85%to (a) .
The engineered antibody of claim 1, wherein, the Fab domain is of IgG1 isotype, preferably human IgG1 isotype.
The engineered antibody of claim 1, wherein, each monomer further comprises a Fc region of IgG class and preferably of IgG1 or IgG4 isotype; and, the Fc region is preferably a human Fc region.
The engineered antibody of claim 1, wherein, each monomer comprises,

(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 8 or a sequence having an identity of at least 85%to SEQ ID NO: 8 and a light chain comprising the amino acid sequence of SEQ ID NO: 7 or a sequence having an identity of at least 85%to SEQ ID NO: 7; or

(b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 or a sequence having an identity of at least 85%to SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 9 or a sequence having an identity of at least 85%to SEQ ID NO: 9.
A nucleic acid molecule or a combination of nucleic acid molecules encoding the engineered antibody of claim 1.
The nucleic acid molecule or combination of nucleic acid molecules of claim 7, wherein, the nucleic acid molecule or each nucleic acid molecule of the combination encodes one or more of the sequences of SEQ ID NOs: 7 to 10 or sequences having an identity of at least 85%to anyone of SEQ ID NOs: 7 to 10.
The nucleic acid molecule or combination of nucleic acid molecules of claim 7, wherein the nucleic acid molecule or each nucleic acid molecule of the combination is a vector.
A kit comprising a nucleic acid molecule or a combination of nucleic acid molecules according to claim 7.
An antibody-drug conjugate comprising an engineered antibody according to claim 1 conjugated to one or more drug molecules through a linker.
The antibody-drug conjugate of claim 11, characterized in one or more of the followings:

wherein, the linker is -MC-vc-PAB-;

wherein, the drug is MMAE;

wherein, the conjugate has a ratio of drug to antibody being 2 to 8, preferably 4; and/or

wherein, the conjugate has all four drug molecules linked at Fab domains of the antibody.
A composition comprising or consisting of a mixture of antibody-drug conjugates according to claim 11, wherein, at least 80%of the antibody-drug conjugates have a ratio of drug to antibody being 4.
A pharmaceutical composition comprising an antibody-drug conjugate according to claim 11 and a pharmaceutically acceptable carrier.
A method of preparing the antibody-drug conjugate of claim 11, comprising a step of conjugating a partially reduced antibody of claim 1 with a linker-payload compound bearing a maleimido or haloacetyl moiety via Michael addition reaction.
The method of claim 15, further comprising a step of partially reducing an engineered antibody of claim 1 using a mild reductant to provide the partially reduced antibody.
The method of claim 16, characterized in one or more the followings:

wherein, the mild reductant is TCEP or DTT;

wherein, the reductant/antibody ratio is 1 to 20, preferably 3 to 10;

wherein, the step of partially reducing is conducted at pH of 4.0 to 8.0, preferably 5 to 6;

wherein, the step of partially reducing is conducted for a period of 0.5 to 24 hours, preferably 1 to 20 hours; and/or

wherein, the step of partially reducing is conducted at a temperature of 4 to 37 ℃, preferably 4 to 15 ℃.
An antibody-drug conjugate product obtained by the method of claim 15, comprising or consisting of a mixture of antibody-drug conjugates according to claim 11, wherein, at least 80%of the antibody-drug conjugates have a ratio of drug to antibody being 4.
A method of treating cancer in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of an antibody-drug conjugate according to claim 11, wherein the cancer is characterized in over-expression of Trop2.
The method of claim 19, wherein the cancer is selected from the group consisting to breast cancer, pancreatic cancer and lung cancer.