Cysteine engineered antibodies and immunoconjugates

Abstract

An antibody engineered with a reactive cysteine residue is provided, and more particularly, antibodies having therapeutic or diagnostic applications are provided. Also provided are immunoconjugates comprising the engineered antibodies. Further provided are drugs or pharmaceutical compositions comprising the antibodies or antibody-drug conjugates, as well as methods and uses of applying the antibodies or immunoconjugates to the treatment of diseases such as tumors.

Description

Cysteine ​​engineered antibodies and immunoconjugates

[0001] The present invention relates to antibodies engineered with reactive cysteine ​​residues, and more specifically, to antibodies having therapeutic or diagnostic applications. The present invention also relates to immunoconjugates comprising the engineered antibodies. The present invention further relates to medicaments or pharmaceutical compositions comprising the antibodies or immunoconjugates, as well as methods and uses of the antibodies or immunoconjugates for treating diseases, such as tumors.

[0002] Background of the Invention

[0003] Although therapeutic antibodies have achieved clinical success, naked antibodies targeting cell surface tumor antigens rarely provide sufficient efficacy alone. In order to increase the low activity of antibodies, new strategies focus on binding them to toxic molecules. Plant and bacterial toxins and small chemotherapeutic molecules can be good candidates because they are very effective and active in very small amounts. Therefore, coupling antibodies to other components to form new immunoconjugates such as antibody-drug conjugates (ADCs) has become a new development strategy for tumor treatment.

[0004] Currently, most ADC conjugation methods still rely on random conjugation, including cysteine ​​and lysine conjugation. This conjugation method can result in a heterogeneous distribution of ADC products. For example, a cysteine-conjugated ADC with an average DAR value of 4 may contain multiple components ranging from DAR0 to DAR8. Even within components with the same DAR value, there are still differences in conjugation at different sites. These components often have different properties. For example, the DAR0 component will competitively bind to the target, while the DAR8 component, due to the high concentration of hydrophobic drugs conjugated, is prone to aggregation and thus more easily cleared from the body. The different pharmacodynamic and pharmacokinetic characteristics corresponding to these components make PK / PD analysis of heterogeneous ADC mixtures more difficult, and require advanced production processes to produce relatively stable ADC products in batches.

[0005] To achieve uniform ADCs, Genentech attempted in 2006 (WO2006034488A2) to mutate a single site in an antibody to cysteine, enabling uniform drug conjugation at this mutated site. The cysteine ​​introduced into the antibodies screened in this patent exhibited high sulfhydryl reactivity, and the cysteine ​​mutation did not affect the antibody's ability to bind to the antigen. Furthermore, the cysteine ​​mutation demonstrated enhanced in vivo efficacy compared to randomly conjugated ADCs. However, this technology still has some limitations.

[0006] In recent years, as more and more studies have found, for some diseases or targets, simply coupling two drugs is far from enough to achieve the expected efficacy, so it is equally important to obtain uniform product technology with higher DAR.

[0007] Existing publicly available strategies for selecting modification sites are limited. To identify sites with higher thiol reactivity and obtain ADCs with higher DAR, existing sites tend to be exposed on the antibody surface and therefore more solvent accessible. However, these strategies fail to consider the negative impacts of these sites, such as ADC stability, hydrophilicity, PK behavior, and in vivo efficacy after drug conjugation.

[0008] Therefore, there is still a need in the art for new cysteine ​​modification technologies for antibodies, which can enable the obtained antibodies to have stronger stability and stronger efficacy when constructing ADCs.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention relates to a modified antibody or antigen-binding fragment thereof, which has a cysteine ​​mutation at a relatively hidden position in the constant region, thereby bringing better stability and / or hydrophilicity to the immunoconjugate containing the same.

[0011] In one embodiment, the cysteine-engineered antibodies of the present invention, after mutating one or two amino acids in their light or heavy chains to cysteine, yield antibody molecules engineered with two or four cysteines due to the dimerization properties of IgG antibodies. Since the thiol groups on cysteine ​​residues can undergo nucleophilic reactions with toxin small molecules with maleimide linkers, site-specific conjugated toxin small molecules can be conjugated to the four cysteines to prepare ADC molecules. The selection of mutation sites is crucial in this invention, as the quality of the site directly determines the properties of the ADC. When a small molecule drug is attached to a site with high solvent accessibility, the drug is relatively exposed, increasing the likelihood of thiol exchange between the small molecule and plasma proteins, leading to reduced efficacy and toxic side effects. Furthermore, relatively exposed small molecules can significantly increase the hydrophobicity of the antibody, potentially affecting its in vivo PK behavior. Therefore, selecting a more secluded site for engineering may result in a more stable and potent ADC molecule.

[0012] The present invention also relates to immunoconjugates comprising the modified antibodies or antigen-binding fragments thereof. The immunoconjugates of the present invention may have a DAR of 4, thereby meeting the needs of more targets or diseases. In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0013] The ADC of the present invention is a site-coupled ADC, which has better physicochemical properties and has one or more of the following advantages compared to the randomly coupled DAR4 ADC:

[0014] (1) More hydrophilic,

[0015] (2) It is more stable in plasma and small molecule toxins are not easily shed;

[0016] (3) It has stronger pharmacodynamics in animals. For example, the ADC of the present invention with a DAR of 4 can have the same pharmacodynamics as the known ADC with a DAR of 8. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows a representative graph used for site-directed coupling DAR value analysis and calculation.

[0018] Figure 2 shows representative graphs used for random coupling DAR value analysis and calculation.

[0019] Figure 3 shows a representative graph of LLC166-HC155-Dxd used for purity analysis.

[0020] Figure 4 shows the hydrophobic interaction profiles of individual DAR2 ADCs.

[0021] Figure 5 shows the hydrophobic interaction profiles of various DAR4 ADCs.

[0022] FIG6A and FIG6B show the cell viability results of the in vitro cell proliferation assay.

[0023] FIG7A and FIG7B show the fluorescence values ​​of each ADC binding to SK-BR-3 in vitro as detected by flow cytometry.

[0024] FIG8A shows a graph showing changes in DAR of each DAR2 ADC in mouse plasma, and FIG8B and FIG8C show graphs showing changes in DAR of each DAR4 ADC in mouse plasma.

[0025] Figures 9A-E show the inhibitory effects of various ADCs on mouse tumors at certain doses (Figures 9A-C and E are for peritoneal administration, and Figure 9D is for intravenous administration).

[0026] Detailed Description of the Invention

[0027] I. Definition

[0028] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0029] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0030] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0031] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0032] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, combinations of the recited elements, integers, or steps are also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0033] The terms "complete antibody", "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules with the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, the full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For conventional four-chain IgG antibodies, the full-length antibody heavy chain is generally composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region are composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0034] The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions. In some embodiments, the heavy chain constant region HC of the antibody of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1. In some preferred embodiments, the heavy chain constant region HC of the antibody of the present invention is

[0035] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3;

[0036] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 3; or

[0037] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 3.

[0038] In some embodiments, the antibody light chain constant region LC of the present invention is a Lambda or Kappa light chain constant region. In some embodiments, the antibody light chain constant region LC of the present invention is a Lambda or Kappa light chain constant region.

[0039] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 5 or 6;

[0040] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 5 or 6; or

[0041] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 5 or 6, or consists of said amino acid sequence.

[0042] The term "CH1 region" refers to the portion of an antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain comprises the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 1).

[0043] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the amino acid residue numbering in the Fc region or heavy chain constant region is numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) in the Fc region may or may not exist. Two Fc regions can achieve dimerization to form a dimeric Fc, such as two different Fc heterodimerizations forming heterodimeric Fc. In this article, the terms "Fc region", "Fc portion" and "dimeric Fc (e.g., heterodimeric Fc)" do not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin and heavy chain constant region CH1 and light chain constant region CL, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases. In one embodiment, a human IgG heavy chain Fc region extends from Asp221, or from Cys226, or from Asp231, to the carboxyl-terminus of the heavy chain.

[0044] In one embodiment, the human IgG1 Fc region polypeptide (including the hinge region) comprises the following amino acid sequence:

[0045] The human IgG4 Fc region polypeptide (including the hinge region) comprises the following amino acid sequence:

[0046] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0047] The term "antigen-binding fragment" of an antibody is a molecule that is different from a full-length antibody and that contains a portion of the full-length antibody, but is capable of binding to the antigen of the full-length antibody or competing with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. In addition, digesting a full-length antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, which is a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab fragment with a hinge region. An Fv fragment consists of the VL and VH domains of a single arm of an antibody. The two domains of the Fv fragment, VL and VH, can be encoded by separate genes, but recombinant methods can also be used to connect the two domains using a synthetic linker peptide so that they are produced as a single protein chain, and in the single protein chain, the VL region and the VH region are paired to form a single-chain Fv (scFv). "Fab fragment" or "Fab" are used interchangeably herein to refer to an immunoglobulin fragment composed of two polypeptide chains, comprising an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, a light chain variable domain VL, and a light chain constant domain CL, wherein one polypeptide chain comprises VH and a constant region selected from CH1 and CL from N-terminus to C-terminus, and the other polypeptide chain comprises VL and another constant region selected from CL and CH1 from N-terminus to C-terminus, wherein the VH domain and VL domain pair to form an antigen binding site. Herein, the Fab polypeptide chain comprising the heavy chain constant region CH1 is also referred to as the "Fab heavy chain"; accordingly, the Fab polypeptide chain comprising the light chain constant region CL is also referred to as the "Fab light chain."

[0048] The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including essentially all proteins or peptides, can serve as an antigen. Additionally, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen that specifically interacts with an antibody molecule.

[0049] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and North's CDR definitions based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Concepts", Journal of Molecular Biology, 406, 228-256 (2011)).

[0050] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0051] As used herein, when referring to amino acid positions in antibody domains other than the variable region (e.g., the constant region, e.g., the Fc region), numbering is according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. When position numbers and / or amino acid residues are assigned to a particular antibody isotype, it is intended to apply to the corresponding positions and / or amino acid residues of any other antibody isotype, as known to those skilled in the art.

[0052] General information on human immunoglobulin light and heavy chains is given in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0053] The term "amino acid substitution" or "amino acid mutation" refers to the replacement of at least one amino acid residue in a predetermined parent amino acid sequence with a different "substituted" amino acid residue. The replacement residue or residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and are selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine ​​(Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val).

[0054] The amino acid position to be mutated to cysteine ​​is generally indicated by "chain type, mutation position". In this article, unless otherwise specified, LLC represents lambda light chain, LC represents kappa light chain, and HC represents heavy chain. Therefore, "LLC160" means that the amino acid at EU position 160 of the lambda light chain is replaced by cysteine ​​(C). "LC165" means that the amino acid at EU position 165 of the kappa light chain is replaced by cysteine ​​(C). When referring to a combination of mutations, the combined mutations are connected with a plus sign (-). "LLC160-LLC166" means that both a cysteine ​​mutation at position 160 of LLC and a cysteine ​​mutation at position 166 of LLC are included.

[0055] When referring to heavy chain amino acid positions herein, unless otherwise specified, these refer to amino acid positions according to the IgG1 heavy chain numbering, i.e., they encompass amino acid positions based on the IgG1 heavy chain numbering, as well as amino acid positions corresponding to these positions on other heavy chains. For example, when referring to HC290, this encompasses amino acid position 290 of the IgG1 heavy chain according to EU numbering.

[0056] "Sequence identity" is defined as the percentage of identical residues in amino acid sequence variants after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0057] "Free cysteine" refers to a cysteine ​​residue that has been engineered into a parent antibody that has a thiol functionality (-SH) (sulfhydryl group) and is not paired with or otherwise part of an intra- or intermolecular disulfide bridge.

[0058] "Parent protein (e.g., parent antibody or parent constant region or parent Fc region)" refers to a protein comprising an amino acid sequence in which one or more amino acid residues are to be replaced with one or more cysteine ​​residues. The parent protein may comprise a native or wild-type sequence. The parent protein may have existing amino acid sequence modifications (such as additions, deletions, and / or substitutions) relative to other native, wild-type, or modified forms of the protein. The parent antibody may be directed against a target antigen of interest, such as a biologically important polypeptide. In some embodiments, the parent antibody is an antibody against HER2, such as trastuzumab.

[0059] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0060] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0061] As used herein, "antibody-drug conjugate (ADC)" refers to a structure obtained by linking an antibody to a drug.

[0062] The term "site-specific conjugation" as used herein refers to conjugation in which a drug / active substance is specifically linked to a specific site of an antibody via a linker.

[0063] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugates of the present invention and is not biologically or otherwise undesirable. The ADC conjugates of the present invention may exist as pharmaceutically acceptable salts thereof, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable, non-toxic acid addition salt refers to a salt formed between the ADC conjugates of the present invention and an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, and the like. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed with organic bases containing an N group.

[0064] The term "solvate" refers to an association formed between one or more solvent molecules and the ADC conjugate of the present invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, and the like.

[0065] As used herein, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably unless there is any contradiction in the context.

[0066] The term "drug:antibody ratio" or "DAR" refers to the ratio of the payload moiety (D) coupled to the antibody moiety (A) described herein to the antibody moiety. The DAR can also be calculated as the average DAR of a population of molecules in a product, i.e., the overall ratio of the small molecule drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety in the product as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC), which DAR is referred to herein as the average DAR. For example, some immunoconjugate components in a sample with a payload-to-antibody ratio of 2 may have no or only one payload attached; other immunoconjugate components in the sample will have two, three, four, or even more moieties on individual antibodies. However, the average value in the sample will be 2.

[0067] As an exemplary embodiment, an immunoconjugate with a "DAR of about 2" refers to an immunoconjugate in which the payload to antibody ratio can vary within the range of about 1.4-2.4 payload / antibody, 1.5-2.1 payload / antibody, or 1.5-1.9 payload / antibody. An immunoconjugate with a "DAR of about 4" refers to an immunoconjugate in which the payload to antibody ratio can vary within the range of about 3-4.5, e.g., 3-4 payload / antibody.

[0068] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0069] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0070] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0071] The term "small molecule drug" refers to low molecular weight organic compounds that are capable of regulating biological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.

[0072] As used herein, the term "immunomodulator" refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0073] The term "effective amount" refers to that amount or dosage of an antibody or fragment or composition or combination of the present invention which, after single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.

[0074] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, even more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% relative to an untreated subject.

[0075] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0076] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of the probe or antibody by reacting with another reagent of the direct label.

[0077] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0078] An "isolated" antibody or other molecule (e.g., ADC molecule) is one that has been separated from a component of its natural environment or the environment in which it is expressed. In some embodiments, the antibody or ADC molecule is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0079] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, including but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0080] The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid tumors and hematological tumors.

[0081] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include gastric cancer, pancreatic cancer, or gastroesophageal junction cancer, including metastatic forms of those cancers.

[0082] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0083] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0084] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0085] The term "pharmaceutical combination" or "pharmaceutical combination product" or "combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody or immunoconjugate molecule of the invention, and (ii) other therapeutic agent) are administered to a patient simultaneously, without specific time restrictions, or sequentially at the same or different time intervals, as separate entities, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient's body. In some embodiments, the antibody or immunoconjugate molecule of the invention and other therapeutic agent used in the pharmaceutical combination are administered at levels no greater than when they are used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosages and / or time intervals of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0086] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0087] As used herein, "treat," ...

[0088] As used herein, "prevention" includes the inhibition of the development or progression of a disease or condition, or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0089] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0090] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from a fresh, frozen and / or preserved organ; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0091] II. Cysteine-engineered Antibodies

[0092] In some embodiments, the present invention provides a modified antibody or antigen-binding fragment thereof, wherein the modified antibody or antigen-binding fragment thereof comprises a substitution of one or more amino acids with cysteine ​​at positions 152, 155 or 290 selected from the heavy chain constant region compared to the parent antibody or antigen-binding fragment thereof.

[0093] In some embodiments, the present invention provides a modified antibody or antigen-binding fragment thereof, wherein the modified antibody or antigen-binding fragment thereof comprises a substitution of one or more amino acids with cysteine ​​at positions 138, 160, 165, 166, or 167 of the light chain constant region compared to the parent antibody or antigen-binding fragment thereof. In some embodiments, when the light chain constant region is a kappa light chain constant region, it may further comprise a 178Y mutation at position 178, i.e., the amino acid at position 178 is mutated to tyrosine Y.

[0094] In some embodiments, the present invention provides a modified antibody or antigen-binding fragment thereof, wherein the modified antibody or antigen-binding fragment thereof comprises a substitution of one or more (e.g., 2, 3, or 4) amino acids with cysteine ​​at positions 152, 155, or 290 selected from the heavy chain constant region or at positions 138, 160, 165, 166, or 167 selected from the light chain constant region compared to the parent antibody or antigen-binding fragment thereof.

[0095] In some embodiments, the heavy chain constant region is an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as an IgG1 heavy chain constant region. In some embodiments, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.

[0096] In some embodiments, the present invention provides a modified antibody or antigen-binding fragment thereof, wherein the modified antibody or antigen-binding fragment thereof comprises a cysteine ​​mutation at positions 160 and 166 of the light chain constant region compared to the parent antibody or antigen-binding fragment thereof. In some embodiments, the light chain constant region is a lambda light chain constant region or a kappa light chain constant region, preferably a lambda light chain constant region.

[0097] In some embodiments, the present invention provides a modified antibody or antigen-binding fragment thereof, wherein the modified antibody or antigen-binding fragment thereof comprises a cysteine ​​mutation at positions 160 and 166 of the light chain constant region and a mutation to tyrosine Y at position 178 compared to a parent antibody or antigen-binding fragment thereof. In some embodiments, the light chain constant region is a kappa light chain constant region.

[0098] In some embodiments, the present invention provides a modified antibody or antigen-binding fragment thereof, wherein the modified antibody or antigen-binding fragment thereof comprises a cysteine ​​mutation at position 166 of the light chain constant region and a cysteine ​​mutation at position 152 of the heavy chain constant region compared to the parent antibody or antigen-binding fragment thereof. In some embodiments, the light chain constant region is a lambda light chain constant region or a kappa light chain constant region (preferably a lambda light chain constant region), and / or the heavy chain constant region is an IgG1 heavy chain constant region.

[0099] In some embodiments, the present invention provides a modified antibody or antigen-binding fragment thereof, wherein the modified antibody or antigen-binding fragment thereof comprises a cysteine ​​mutation at position 160 of the light chain constant region and a cysteine ​​mutation at position 155 of the heavy chain constant region compared to the parent antibody or antigen-binding fragment thereof. In some embodiments, the light chain constant region is a lambda light chain constant region or a kappa light chain constant region (preferably a lambda light chain constant region), and / or the heavy chain constant region is an IgG1 heavy chain constant region.

[0100] In some embodiments, the present invention provides a modified antibody or antigen-binding fragment thereof, wherein the modified antibody or antigen-binding fragment thereof comprises a cysteine ​​mutation at position 166 of the light chain constant region and a cysteine ​​mutation at position 155 of the heavy chain constant region compared to the parent antibody or antigen-binding fragment thereof. In some embodiments, the light chain constant region is a lambda light chain constant region or a kappa light chain constant region (preferably a lambda light chain constant region), and / or the heavy chain constant region is an IgG1 heavy chain constant region.

[0101] In some embodiments, the present invention provides a modified antibody or antigen-binding fragment thereof, wherein the modified antibody or antigen-binding fragment thereof comprises a cysteine ​​mutation at position 165 of the light chain constant region and a cysteine ​​mutation at position 152 of the heavy chain constant region compared to the parent antibody or antigen-binding fragment thereof. In some embodiments, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region (preferably a kappa light chain constant region), and / or the heavy chain constant region is an IgG1 heavy chain constant region.

[0102] In some embodiments, the present invention provides a modified antibody or antigen-binding fragment thereof, wherein the modified antibody or antigen-binding fragment thereof comprises a cysteine ​​mutation at position 165 of the light chain constant region and a cysteine ​​mutation at position 155 of the heavy chain constant region compared to the parent antibody or antigen-binding fragment thereof. In some embodiments, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region (preferably a kappa light chain constant region), and / or the heavy chain constant region is an IgG1 heavy chain constant region.

[0103] In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified light chain constant region. In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a light chain comprising a modified light chain constant region.

[0104] In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified heavy chain constant region. In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain constant region comprising a modified CH1. In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain constant region comprising a modified Fc region. In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain constant region comprising a modified CH1 and / or a modified Fc region. In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified CH1. In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified Fc region. In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified CH1 and / or a modified Fc region.

[0105] In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified light chain constant region and a modified heavy chain constant region. In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified light chain constant region and a modified CH1. In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified light chain constant region and a modified Fc region. In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified light chain constant region and a modified Fc region and / or a modified CH1.

[0106] In some embodiments, the parent human Lambda light chain constant region of the present invention comprises

[0107] The amino acid sequence of SEQ ID NO:6 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:6 and not comprising a cysteine ​​mutation.

[0108] In some embodiments, the parent human kappa light chain constant region of the present invention comprises

[0109] The amino acid sequence of SEQ ID NO:5 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:5 and not comprising a cysteine ​​mutation.

[0110] In some embodiments, the parent human IgG1 heavy chain constant region of the present invention comprises

[0111] The amino acid sequence of SEQ ID NO:3 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:3 and not comprising a cysteine ​​mutation.

[0112] In some embodiments, the parent human IgG4 Fc region of the present invention comprises

[0113] The amino acid sequence of SEQ ID NO:4 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:4 and not comprising a cysteine ​​mutation.

[0114] In some embodiments, the parent human IgG1 Fc region of the present invention comprises

[0115] The amino acid sequence of SEQ ID NO:2 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:2 and not comprising a cysteine ​​mutation.

[0116] In some embodiments, a parent human IgG1 CH1 of the invention comprises

[0117] The amino acid sequence of SEQ ID NO: 1 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 and not comprising a cysteine ​​mutation.

[0118] In some embodiments, the modified light chain constant region of the present invention is a Lambda light chain constant region and comprises the amino acid sequence shown in SEQ ID NO: 7, 8 or 28. In some embodiments, the modified light chain constant region of the present invention is a Kappa light chain constant region and comprises the amino acid sequence shown in SEQ ID NO: 9, 12, 14 or 62.

[0119] In some embodiments, the modified light chain constant region of the invention comprises or consists of the following amino acid sequence:

[0120] (1) the amino acid sequence set forth in SEQ ID NO:16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16 and having a cysteine ​​substitution at position 160, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16 and comprising the amino acid sequence set forth in SEQ ID NO:7;

[0121] (2) the amino acid sequence set forth in SEQ ID NO:17, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17 and having a cysteine ​​substitution at position 166, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17 and comprising the amino acid sequence set forth in SEQ ID NO:8;

[0122] (3) the amino acid sequence set forth in SEQ ID NO:18, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18 and having cysteine ​​substitutions at positions 160 and 166, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18 and comprising the amino acid sequence set forth in SEQ ID NO:28;

[0123] (4) the amino acid sequence set forth in SEQ ID NO:19, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:19 and having a cysteine ​​substitution at position 138, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:19 and comprising the amino acid sequence set forth in SEQ ID NO:12;

[0124] (5) the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20 and having a cysteine ​​substitution at position 165, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20 and comprising the amino acid sequence set forth in SEQ ID NO:9;

[0125] (6) the amino acid sequence set forth in SEQ ID NO:21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:21 and having cysteine ​​substituted at position 167, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:21 and comprising the amino acid sequence set forth in SEQ ID NO:14; or

[0126] (7) the amino acid sequence set forth in SEQ ID NO:60, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:60 and having cysteine ​​substitutions at positions 160 and 166 and tyrosine substitutions at position 178, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:60 and comprising the amino acid sequence set forth in SEQ ID NO:62.

[0127] In some embodiments, the modified CH1 of the present invention is the CH1 of IgG1 and comprises the amino acid sequence shown in SEQ ID NO: 10 or 11.

[0128] In some embodiments, the modified CH1 of the present invention comprises or consists of the following amino acid sequence:

[0129] (1) the amino acid sequence set forth in SEQ ID NO:22, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:22 and having a cysteine ​​substitution at position 152, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:22 and comprising the amino acid sequence set forth in SEQ ID NO:10; or

[0130] (2) the amino acid sequence set forth in SEQ ID NO:23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:23 and having cysteine ​​substituted at position 155, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:23 and comprising the amino acid sequence set forth in SEQ ID NO:11.

[0131] In some embodiments, the modified Fc region of the present invention is an IgG1 Fc region, and comprises the amino acid sequence shown in SEQ ID NO: 13, or comprises the amino acid sequences shown in SEQ ID NOs: 67 and 68.

[0132] In some embodiments, the modified Fc region of the present invention comprises or consists of the following amino acid sequence:

[0133] the amino acid sequence set forth in SEQ ID NO:24, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:24 and has a cysteine ​​substitution at position 290, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:24 and comprises the amino acid sequence set forth in SEQ ID NO:13; or

[0134] The amino acid sequence set forth in SEQ ID NO:65, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:65 and has cysteine ​​substitutions at positions 239 and 375, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:65 and comprises the amino acid sequences set forth in SEQ ID NO:67 and SEQ ID NO:68.

[0135] In some embodiments, the modified heavy chain constant region of the present invention is a heavy chain constant region of IgG1, which comprises the amino acid sequence shown in SEQ ID NO: 10, 11 or 13.

[0136] In some embodiments, the modified heavy chain constant region of the present invention comprises

[0137] (1) an amino acid sequence as set forth in SEQ ID NO:25, or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:25 and having a cysteine ​​substitution at position 152, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:25 and comprising the amino acid sequence as set forth in SEQ ID NO:10;

[0138] (2) an amino acid sequence as set forth in SEQ ID NO:26 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:26 and having a cysteine ​​substitution at position 155, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:26 and comprising the amino acid sequence as set forth in SEQ ID NO:11;

[0139] (3) an amino acid sequence as set forth in SEQ ID NO:27, or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:27 and having a cysteine ​​substitution at position 290, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:27 and comprising the amino acid sequence as set forth in SEQ ID NO:13; or

[0140] (4) an amino acid sequence as set forth in SEQ ID NO:64 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:64 and in which positions 239 and 375 are substituted with cysteine, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:64 and comprising the amino acid sequences as set forth in SEQ ID NO:67 and SEQ ID NO:68.

[0141] In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified light chain constant region, wherein the light chain constant region

[0142] An amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18 and in which positions 160 and 166 are substituted with cysteine, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18 and comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0143] In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified light chain constant region and a heavy chain constant region, wherein the modified light chain constant region

[0144] an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18 and substituted with cysteine ​​at positions 160 and 166, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18 and comprising the amino acid sequence of SEQ ID NO: 28, and

[0145] The heavy chain constant region comprises a human IgG1 heavy chain constant region, which is, for example,

[0146] The invention also provides a method for preparing an amino acid sequence of SEQ ID NO: 3 or a method for preparing an amino acid sequence of SEQ ID NO: 3. The invention also provides a method for preparing an amino acid sequence of SEQ ID NO: 3 or a method for preparing an amino acid sequence of SEQ ID NO: 3.

[0147] In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified light chain constant region and a heavy chain constant region, wherein the modified light chain constant region

[0148] an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:60, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:60 and substituted with cysteine ​​at positions 160 and 166 and with tyrosine at position 178, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:60 and comprising the amino acid sequence of SEQ ID NO:62, and

[0149] The heavy chain constant region comprises a human IgG1 heavy chain constant region, which is, for example,

[0150] The invention also provides a method for preparing an amino acid sequence of SEQ ID NO: 3 or a method for preparing an amino acid sequence of SEQ ID NO: 3. The invention also provides a method for preparing an amino acid sequence of SEQ ID NO: 3 or a method for preparing an amino acid sequence of SEQ ID NO: 3.

[0151] In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified light chain constant region and a modified heavy chain constant region, wherein

[0152] The modified light chain constant region comprises

[0153] the amino acid sequence of SEQ ID NO:17, or consisting thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:17 and has a cysteine ​​substitution at position 166, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:17 and comprises the amino acid sequence of SEQ ID NO:8;

[0154] And the modified heavy chain constant region comprises

[0155] (1) an amino acid sequence as set forth in SEQ ID NO:25 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:25 and having a cysteine ​​substitution at position 152, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:25 and comprising the amino acid sequence as set forth in SEQ ID NO:10; or

[0156] (2) an amino acid sequence as set forth in SEQ ID NO:26 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:26 and having a cysteine ​​substitution at position 155, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:26 and comprising the amino acid sequence as set forth in SEQ ID NO:11; or

[0157] (3) A modified CH1 comprising

[0158] (i) an amino acid sequence as set forth in SEQ ID NO:22, or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:22, in which position 152 is substituted with cysteine, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:22 and comprising the amino acid sequence as set forth in SEQ ID NO:10; or

[0159] (ii) an amino acid sequence as set forth in SEQ ID NO:23, or consisting thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:23 and has a cysteine ​​substitution at position 155, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:23 and comprises the amino acid sequence as set forth in SEQ ID NO:11.

[0160] In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified light chain constant region and a modified heavy chain constant region, wherein

[0161] The modified light chain constant region comprises

[0162] the amino acid sequence of SEQ ID NO:44, or consisting thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:44 and has a cysteine ​​substitution at position 166, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:44 and comprises the amino acid sequence of SEQ ID NO:66;

[0163] And the modified heavy chain constant region comprises

[0164] (1) an amino acid sequence as set forth in SEQ ID NO:61 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:61 and having a cysteine ​​substitution at position 375, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:61 and comprising the amino acid sequence as set forth in SEQ ID NO:67; or

[0165] (2) A modified Fc region comprising or consisting of the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 63 and substituted with cysteine ​​at position 375, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 63 and comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the modified antibody or antigen-binding fragment thereof of the present invention comprises a modified heavy chain constant region and a light chain constant region, wherein

[0166] The modified heavy chain constant region comprises

[0167] (1) an amino acid sequence as set forth in SEQ ID NO:64, or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:64, in which positions 239 and 375 are substituted with cysteine, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:64, and comprising the amino acid sequences as set forth in SEQ ID NO:67 and SEQ ID NO:68; or

[0168] (2) a modified Fc region comprising or consisting of the amino acid sequence of SEQ ID NO:65, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:65 and a cysteine ​​substitution at position 375, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:65 and comprises the amino acid sequences of SEQ ID NO:67 and SEQ ID NO:68;

[0169] And the light chain constant region comprises a Kappa light chain constant region or a Lambda light chain constant region, which, for example

[0170] The invention also provides a method for preparing an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 5 or 6, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 5 or 6 and having no cysteine ​​substitution.

[0171] In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified light chain constant region and a modified heavy chain constant region, wherein

[0172] The modified light chain constant region comprises

[0173] an amino acid sequence as set forth in SEQ ID NO:20, or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:20 and having a cysteine ​​substitution at position 165, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:20 and comprising the amino acid sequence as set forth in SEQ ID NO:9;

[0174] And the modified heavy chain constant region comprises

[0175] (1) an amino acid sequence as set forth in SEQ ID NO:25 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:25 and having a cysteine ​​substitution at position 152, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:25 and comprising the amino acid sequence as set forth in SEQ ID NO:10; or

[0176] (2) an amino acid sequence as set forth in SEQ ID NO:26 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:26 and having a cysteine ​​substitution at position 155, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:26 and comprising the amino acid sequence as set forth in SEQ ID NO:11; or

[0177] (3) A modified CH1 comprising

[0178] (i) an amino acid sequence as set forth in SEQ ID NO:22, or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:22, in which position 152 is substituted with cysteine, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:22 and comprising the amino acid sequence as set forth in SEQ ID NO:10; or

[0179] (ii) an amino acid sequence as set forth in SEQ ID NO:23, or consisting thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:23 and has a cysteine ​​substitution at position 155, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:23 and comprises the amino acid sequence as set forth in SEQ ID NO:11.

[0180] In some embodiments, the modified antibodies or antigen-binding fragments thereof of the present invention comprise a modified light chain constant region and a modified heavy chain constant region, wherein

[0181] The modified light chain constant region comprises

[0182] the amino acid sequence of SEQ ID NO:16, or consisting thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:16 and has a cysteine ​​substitution at position 160, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:16 and comprises the amino acid sequence of SEQ ID NO:7;

[0183] And the modified heavy chain constant region comprises

[0184] (1) an amino acid sequence as set forth in SEQ ID NO:25 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:25 and having a cysteine ​​substitution at position 152, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:25 and comprising the amino acid sequence as set forth in SEQ ID NO:10; or

[0185] (2) an amino acid sequence as set forth in SEQ ID NO:26 or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:26 and having a cysteine ​​substitution at position 155, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:26 and comprising the amino acid sequence as set forth in SEQ ID NO:11; or

[0186] (3) A modified CH1 comprising

[0187] (i) an amino acid sequence as set forth in SEQ ID NO:22, or consisting thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:22, in which position 152 is substituted with cysteine, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:22 and comprising the amino acid sequence as set forth in SEQ ID NO:10; or

[0188] (ii) an amino acid sequence as set forth in SEQ ID NO:23, or consisting thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:23 and has a cysteine ​​substitution at position 155, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:23 and comprises the amino acid sequence as set forth in SEQ ID NO:11.

[0189] The modified antibodies or antibody binding fragments of the present invention can be in any form known in the art, such as monoclonal, chimeric, humanized, fully human, full-length, bispecific, or multispecific antibodies or antibody fragments thereof.

[0190] In some embodiments, the modified antibody of the present invention specifically binds to an antigen. In some embodiments, the antigen is a tumor-specific antibody (TA) or a tumor-associated antigen (TAA). In some embodiments, the tumor-associated antigen is an immune checkpoint molecule. In some embodiments, the antigen is selected from FAP, CEA, p95 HER2, BCMA, EpCAM, MSLN, MCSP, HER-1, HER-2, CD19, CD20, CD22, CD33, CD38, CD52Flt3, EpCAM, IGF-1R, FOLR1, Trop-2, CA-12-5, HLA-DR, MUC-1 (mucin), GD2, A33-antigen, PSMA, PSCA, transferrin-receptor, TNC (tenascin), CA-IX, CD3, B7H3, Hel, cMET, Axl, GPRC5D, PD-1, PD-L1, CD47, immune activation molecules such as 4-1BB, CD40, OX40, etc. In some embodiments, the antigen is HER2.

[0191] In some embodiments, the parent antibody of the present invention is derived from an antibody that specifically binds to HER2, such as trastuzumab. In some embodiments, the parent antibody of the present invention comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to HER2, such as trastuzumab. In some embodiments, the parent antibody of the present invention comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds to Her2, such as trastuzumab. In some embodiments, the parent antibody of the present invention comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds to HER2, such as trastuzumab. In some embodiments, the parent antibody of the present invention comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known antibody that specifically binds to HER2, such as trastuzumab. In some embodiments, the parent antibody of the present invention comprises the heavy chain variable region and light chain variable region of a known antibody that specifically binds to HER2, such as trastuzumab.

[0192] In some embodiments, the modified antibodies or antigen-binding fragments of the present invention are full-length antibodies comprising a modified heavy chain constant region, or a modified CH1, or a modified Fc region as described herein. In some embodiments, the modified antibodies or antibody-binding fragments of the present invention are full-length antibodies comprising a modified light chain constant region as described herein. In some embodiments, the modified antibodies or antibody-binding fragments of the present invention are full-length antibodies comprising a modified light chain constant region as described herein, and a modified heavy chain constant region as described herein. In some embodiments, the modified antibodies or antigen-binding fragments comprise two heavy chains and two light chains, wherein one or both of the two heavy chains comprise a heavy chain constant region mutation or a combination of mutations as described herein, or one or both of the two light chains comprise a light chain constant region mutation or a combination of mutations as described herein; or both of the two heavy chains and both of the two light chains comprise a heavy chain constant region mutation or a combination of mutations as described herein, and a light chain constant region mutation combination.

[0193] In some embodiments, the modified antibody of the present invention is a bispecific antibody comprising a first antigen-binding region that specifically binds to a first antigen, and a second antigen-binding region that specifically binds to a second antigen.

[0194] First heavy chain: VH Ab1 - first heavy chain constant region;

[0195] First light chain: VL Ab1 - first light chain constant region;

[0196] Second heavy chain: VH Ab2- a second heavy chain constant region; and

[0197] Second light chain: VL Ab2 - second light chain constant region;

[0198] wherein the first light chain constant region and the second light chain constant region are modified light chain constant regions as defined herein; and / or the first heavy chain constant region and the second heavy chain constant region are modified heavy chain constant regions as described herein;

[0199] VH Ab1 and VL Ab1 The heavy chain variable region VH and light chain variable region VL are the antigen binding regions that specifically bind to the first antigen, and VH Ab2 and VL Ab2 These are the heavy chain variable region VH and light chain variable region VL, which are antigen binding regions that specifically bind to a second antigen.

[0200] In some embodiments, the first heavy chain constant region and the second heavy chain constant region are identical, or have the same cysteine ​​mutation.In some embodiments, the first light chain constant region and the second light chain constant region are identical, or have the same cysteine ​​mutation.

[0201] In some embodiments, the first heavy chain constant region and the second heavy chain constant region do not have the cysteine ​​mutations of the present invention, and the first light chain constant region and / or the second light chain constant region have the cysteine ​​mutations of the present invention (and optionally other mutations such as 178Y mutations), preferably the first light chain constant region and the second light chain constant region have the same cysteine ​​mutations of the present invention, more preferably, the first light chain constant region and the second light chain constant region are identical and have the cysteine ​​mutations of the present invention (and optionally other mutations such as 178Y mutations). In some embodiments, the first heavy chain constant region and the second heavy chain constant region are heavy chain constant regions that do not have the cysteine ​​mutations of the present invention, and the first light chain constant region and the second light chain constant region are lambda light chain constant regions that have the cysteine ​​mutations of the present invention at positions 160 and 166, respectively. In some embodiments, the first heavy chain constant region and the second heavy chain constant region are heavy chain constant regions that do not have the cysteine ​​mutations of the present invention, and the first light chain constant region and the second light chain constant region are kappa light chain constant regions that have the cysteine ​​mutations of the present invention at positions 160 and 166, respectively, and a mutation to tyrosine at position 178.

[0202] In some embodiments, the first heavy chain constant region and the second heavy chain constant region each have a cysteine ​​mutation of the present invention, for example, have the same cysteine ​​mutation of the present invention, and the first light chain constant region and the second light chain constant region do not have a cysteine ​​mutation of the present invention.

[0203] In some embodiments, the first heavy chain constant region and the first light chain constant region each have a cysteine ​​mutation of the present invention, e.g., the first heavy chain constant region and the first light chain constant region each have a cysteine ​​mutation described herein, e.g., a modified heavy chain constant region and light chain constant region combination as defined above, respectively, and / or the second heavy chain constant region and the second light chain constant region each have a cysteine ​​mutation of the present invention, e.g., the second heavy chain constant region and the second light chain constant region each have a cysteine ​​mutation described herein, e.g., a modified heavy chain constant region and light chain constant region combination as defined above, respectively. In some embodiments, the first heavy chain constant region and the second heavy chain constant region each have the same cysteine ​​mutation, and the first light chain constant region and the second light chain constant region each have the same cysteine ​​mutation.

[0204] In some embodiments, the first heavy chain constant region and the second heavy chain constant region further comprise a first Fc region and a second Fc region, respectively, and the first Fc region and the second Fc region respectively comprise mutations that are conducive to their heterodimerization. Methods for promoting heterodimerization of the Fc region are known in the art. For example, the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary manner so that each CH3 region (or a heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with other CH3 regions of complementary engineering (so that the CH3 regions of the first and second Fc regions heterodimerize and no homodimer is formed between the two first CH3 regions or the two second CH3 regions). For example, based on the Knob-into-Hole technology, corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region. For this technology, see, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Corresponding mutations can also be introduced into the first CH3 region of the first Fc region and the second CH3 region of the second Fc region based on the Innobody technology. For this technology, see, for example, WO2022143912A1 (the patent is incorporated herein in its entirety).

[0205] In some embodiments, the first antigen and the second antigen are tumor-specific antibodies (TA) or tumor-associated antigens (TAA). In some embodiments, tumor-associated antigens are immune checkpoint molecules. In some embodiments, the antigens are selected from FAP, CEA, p95 HER2, BCMA, EpCAM, MSLN, MCSP, HER-1, HER-2, CD19, CD20, CD22, CD33, CD38, CD52Flt3, EpCAM, IGF-1R, FOLR1, Trop-2, CA-12-5, HLA-DR, MUC-1 (mucin), GD2, A33-antigen, PSMA, PSCA, transferrin-receptor, TNC (tenascin), CA-IX, CD3, B7H3, Hel, cMET, Axl, GPRC5D, PD-1, PD-L1, CD47, immune activation molecules such as 4-1BB, CD40, OX40, etc. In some embodiments, the first antigen and the second antigen are HER3 and EGFR, respectively.

[0206] In some embodiments, the bispecific antibody specifically binding to HER3 and EGFR constructed by the present invention comprising the LLC160-LLC166 mutation site combination exhibits excellent properties in terms of antibody expression yield, one-step purification efficiency, DAR value of the ADC molecule after coupling, and SEC purity (aggregation). Compared with other mutation combinations, the LLC160-LLC166 mutation site combination demonstrates better coupling efficacy.

[0207] In some embodiments, the bispecific antibody ADC molecule specifically binding to HER3 and EGFR constructed based on the present invention comprising the LLC160-LLC166 mutation site combination has one or more of the following properties:

[0208] (1) In the plasma stability test, the bispecific ADC molecule was very stable in plasma, such as mouse plasma or monkey plasma, and no obvious drug shedding was observed;

[0209] (2) In the in vitro cell killing experiment, the ADC constructed with bispecific antibodies had a more significant killing effect on tumor cells with low EGFR expression and high HER3 expression, such as human breast cancer cells MDA-MB-453, than other ADCs constructed with non-mutated bispecific antibodies.

[0210] (3) It has strong affinity for both EGFR and HER3, and the mutation site does not affect the binding activity of the antibody;

[0211] (4) It has anti-tumor effects and / or minor side effects, such as not significantly affecting body weight. For example, it has a significant inhibitory effect on tumors in an H508 tumor-bearing mouse model, a NUGC-4 tumor-bearing mouse model, a SW620 tumor-bearing mouse model, an ASPC1 tumor-bearing mouse model, or an NCI-H1568 tumor-bearing mouse model, and does not significantly affect the weight of the mice.

[0212] III. Preparation, encoding nucleic acid, expression vector and host cells of cysteine-modified antibodies

[0213] In one aspect of the present invention, the present invention relates to a method for preparing a cysteine ​​engineered antibody or antigen-binding fragment thereof, comprising:

[0214] (a) substituting one or more amino acid positions (preferably relatively cryptic sites) in the light chain constant region and / or heavy chain constant region (or CH1 or Fc region) of an antibody or antigen-binding fragment thereof with cysteine;

[0215] (b) introducing nucleic acids encoding each chain of the antibody or antigen-binding fragment thereof obtained in (a) or expression vectors comprising the nucleic acids into host cells;

[0216] b) expressing and assembling the antibody or antigen-binding fragment thereof in a host cell;

[0217] Optionally, the antibody or antigen-binding fragment thereof is purified, for example, by Protein A purification.

[0218] In some embodiments, the more hidden sites are selected by analyzing the protein crystal structure of the antibody. In some embodiments, the selection comprises the following steps:

[0219] To analyze protein crystal structures,

[0220] Select one or more sites within the Fab cavity formed by the CH1 and VH, CL and VL domains of the protein, and the cavity formed by the two CH2 and two CH3 domains of the Fc region;

[0221] selecting a site inside the selected cavity as a candidate cryptic site;

[0222] The properties of antibodies or immunoconjugates prepared therefrom with mutations to cysteine ​​at these cryptic sites are tested.

[0223] In some embodiments, the cysteine ​​engineered antibody or antigen-binding fragment thereof of the present invention has or substantially maintains the expression level and affinity of the parent antibody or antigen-binding fragment thereof before mutation.

[0224] The present invention provides nucleic acids encoding any chain, monomer, or domain of an antibody or antigen-binding fragment thereof of the present invention. Polynucleotide sequences encoding each chain can be generated using methods well known in the art. In addition, the polynucleotides and nucleic acids of the present invention may include a segment encoding a secretory signal peptide, which can be operably linked to a nucleic acid encoding an antibody or antigen-binding fragment thereof of the present invention, thereby directing secretory expression of the antibody or antigen-binding fragment thereof or each chain thereof.

[0225] The present invention also provides vectors comprising the nucleic acids of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector of the present invention is a pCNDA vector, such as the pCNDA3.1 expression vector.

[0226] The present invention also provides host cells containing the nucleic acid or the vector. Host cells suitable for replicating and supporting the expression of the antibodies or antigen-binding fragments thereof or individual chains of the present invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculating large-scale fermenters, thereby obtaining sufficient quantities of multispecific antibodies, such as bispecific antibodies, for clinical use.

[0227] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, preferably mammalian cells such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells or lymphocytes (e.g., Y0, NS0, Sp20 cells) (e.g., CHO cells or 293 cells, such as Expi293 cells, such as Expi293F cells).

[0228] IV. Immunoconjugates

[0229] In one aspect, the present invention also relates to an immunoconjugate comprising a modified antibody or antigen-binding fragment thereof, wherein the modified antibody or antigen-binding fragment thereof is as defined herein.

[0230] The term "immunoconjugate" as used herein refers to a construct in which a payload is linked to an antibody or an antigen-binding fragment thereof via a linker, so that the antibody or the antigen-binding fragment thereof can serve as a carrier for the targeted transport of the payload to a target site.

[0231] The term "payload" refers to an active portion of an antibody or antibody fragment conjugated to the present invention, and may include any portion for attachment to an antibody or antibody fragment. In some embodiments, the payload may be a drug such as a small molecule drug, a radionuclide, DNA, RNA, an enzyme, or a polypeptide.

[0232] In some embodiments, the immunoconjugate encompasses an antibody drug conjugate (ADC), an antibody immunostimulatory drug conjugate (ISAC), an antibody oligonucleotide conjugate (AOC), an antibody polypeptide drug conjugate (APC), an antibody nuclide drug conjugate (RDC), or an antibody degrading drug conjugate (ADeC), among others.

[0233] Suitable payloads or active moieties for attachment to antibodies include, for example, cytotoxic agents, chemotherapeutic agents, innate immune agonists (e.g., Toll-like receptor agonist (TLR) ISAC drugs SBT6050, SBT6290, BDC-1001; STING agonist ISAC drug XMT-2056, Treg cell regulation ISAC drug ADCT-301, etc.), immunomodulators, therapeutic oligonucleotides (siRNA, PMO, etc.) or radionuclides, etc.

[0234] In some embodiments, the immunoconjugates of the present invention are antibody drug conjugates (ADCs). In some embodiments, in the antibody drug conjugate, the drug moiety as the payload is a topoisomerase I inhibitor, such as a camptothecin derivative or a camptothecin compound (e.g., DXd). In some embodiments, the drug moiety can be an anti-tubulin agent, such as auristatins (MMAF, MMAE), taxane analogs (such as epothilone A and B), maytansines (DM1, DM4), tubulysin and its analogs. In some embodiments, the drug moiety can be a DNA-acting drug, including PBD, etc.

[0235] In some embodiments, the immunoconjugates of the invention have a DAR value of 1-6, such as 2, 3, 4, 5, or 6. In preferred embodiments, the DAR value is 2 or 4.

[0236] In some embodiments, the average DAR value of the immunoconjugates of the invention is 1 to 4, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, or 4.4, or a range having two of these values ​​as endpoints.

[0237] In some embodiments, the payload (e.g., drug) of the immunoconjugates of the invention is attached to the modified antibody or antibody fragment via a thiol group of a free cysteine ​​of the antibody or antigen-binding fragment thereof (optionally via a linker). In some embodiments, the payload (e.g., drug) of the immunoconjugates of the invention is linked to the thiol group of the cysteine ​​via a cleavable or non-cleavable linker.

[0238] As used herein, "cleavable" refers to a linker that connects two moieties by covalent attachment, but decomposes under physiological conditions to separate the covalent attachment between the moieties. Cleavage can be enzymatic or non-enzymatic, but generally releases the payload from the antibody without degrading the antibody. As used herein, "non-cleavable" refers to a linker that is insensitive to decomposition under physiological conditions. Although the linker can be physiologically modified, it maintains the payload attached to the antibody until the antibody is substantially degraded, i.e., the antibody degrades in vivo before the linker is cleaved.

[0239] In one embodiment, the linker has a functional group capable of reacting with a sulfhydryl group on a cysteine ​​residue present on the antibody or its antigen-binding fragment to form a covalent bond to the antibody or its antigen-binding fragment. Non-limiting exemplary reactive functional groups of this type are, for example, groups selected from the following: maleimide, iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, haloacetamide, α-haloacetyl, active esters such as succinimidyl ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, anhydride, acyl chloride, sulfonyl chloride, isocyanate and isothiocyanate, preferably maleimide.

[0240] The linker can be composed of one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), hexanoyl-glycine-glycine-phenylalanine-glycine (GGFG), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate ("SMCC"), and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"), ethyleneoxy-CH2CHO- as one or more repeating units ("EO" or "PEO").

[0241] Exemplary linkers include, but are not limited to, maleimido-caproyl-valinealaine (mc-va) linker, maleimidobutanoic acid-valine-citrulline (mb-vc) linker, or maleimido-caproyl-glycine-glycine-phenylalanine-glycine linker (mc-GGFG).

[0242] In some embodiments, the immunoconjugates of the invention comprise thiol-maleimide, haloacetyl, isothiocyanate, and the like.

[0243] In some embodiments, the payload in the immunoconjugates of the present invention may also be present in the form of a pharmaceutically acceptable salt.

[0244] In some embodiments, the payload in the immunoconjugates of the present invention may also be formed with solvent molecules.

[0245] V. Products and Applications

[0246] In some embodiments, the present invention also relates to a composition (eg, a pharmaceutical composition or pharmaceutical preparation) comprising the antibody or antigen-binding fragment thereof of the present invention or the immunoconjugate of the present invention.

[0247] In one embodiment, the composition further comprises a pharmaceutical excipient.In one embodiment, a composition, eg, a pharmaceutical composition, comprises a binding molecule of the invention in combination with one or more other therapeutic agents.

[0248] The compositions of the present invention may also include suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients as known in the art, including buffers. As used herein, "pharmaceutical carriers" include any and all solvents, dispersion media, isotonic agents, or absorption delaying agents that are physiologically compatible. For the use and purposes of pharmaceutical excipients, see also "Handbook of Pharmaceutical Excipients", Eighth Edition, RC Rowe, PJ Eskey and SCO Wen, Pharmaceutical Press, London, Chicago. The compositions of the present invention may be in various forms. These forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. The medicine comprising the antibody or immunoconjugate described herein can be prepared by mixing the binding molecules of the present invention with the desired purity with one or more optional pharmaceutical excipients, for example, in the form of a lyophilized formulation or an aqueous solution.

[0249] In some embodiments, the present invention relates to an antibody or antigen-binding fragment thereof of the present invention or an immunoconjugate of the present invention for use in therapy, such as for treating a tumor such as cancer.

[0250] In some embodiments, the present invention relates to a method of treating a disease, such as a tumor, such as cancer, using the antibody or antigen-binding fragment thereof described in the present invention or the immunoconjugate described in the present invention, or a use for such treatment, or a use for preparing a medicament for such treatment.

[0251] In some embodiments, the tumor is a solid tumor or a hematological tumor. In some embodiments, the tumor is a HER2-positive tumor or cancer. In some embodiments, the tumor is breast cancer, such as HER2-positive breast cancer. In some embodiments, the tumor is gastric cancer, such as HER2-positive gastric cancer.

[0252] Depending on its therapeutic use, the antibody or its antigen-binding fragment of the present invention or the immunoconjugate of the present invention can also be combined with other therapeutic agents. For example, when the antibody or its antigen-binding fragment or immunoconjugate is used to treat a tumor, the therapeutic agent is, for example, a various therapeutic agent for treating a tumor, such as a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists).

[0253] In some embodiments, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising an antibody or antigen-binding fragment thereof or immunoconjugate of the present invention, and one or more other therapeutic agents.

[0254] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing regimen or a drug administration interval.

[0255] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0256] - a first container containing a pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or immunoconjugate of the present invention;

[0257] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent. Example

[0258] Materials used in the examples:

[0259] Reagents and columns

[0260] equipment

[0261] Example 1: Protein mutation site selection

[0262] The molecules involved in this invention are cysteine-engineered antibodies. Using trastuzumab as a model antibody, one or two amino acids in its light or heavy chain are mutated to cysteine. Due to the dimerization properties of IgG antibodies, antibody molecules engineered with two or four cysteines are obtained. The sulfhydryl groups on cysteine ​​residues can undergo nucleophilic reactions with small toxin molecules with maleimide linkers, thereby coupling toxin small molecules to the four cysteines to prepare site-specifically conjugated ADC molecules. The selection of mutation sites is crucial in this invention, as the quality of the site directly determines the properties of the ADC. When a small molecule drug is attached to a site with high solvent accessibility, the drug's relatively exposed environment increases the likelihood of sulfhydryl exchange between the small molecule and plasma proteins, resulting in reduced efficacy and toxic side effects. Furthermore, the relatively exposed small molecule can significantly increase the hydrophobicity of the antibody, potentially affecting its in vivo PK behavior. Therefore, selecting a more secluded site for engineering modification may result in a more stable and potent ADC molecule.

[0263] It is generally believed that modifications to the variable region of an antibody may be associated with a higher risk of loss of antigen binding, so in this study, we targeted sites within the antibody constant region. First, we analyzed the protein crystal structure and selected sites within the Fab cavity formed by the CH1 and VH, CL and VL domains, and within the cavity formed by the two CH2 and two CH3 domains in the Fc region. Sites within these cavities, when coupled to hydrophobic small molecules, isolate them from the hydrophilic environment surrounding the antibody, resulting in ADCs that are more hydrophilic than ADCs in which the small molecules are attached to native interchain disulfide bonds.

[0264] The following table lists candidate mutation sites, where LC205 is a site verified by Genentech, LC110 is disclosed in CN101065151A, HC239 is disclosed in CN110352074A, HC118 is disclosed in CN101065151A, HC375 is disclosed in CN101065151B, and HC442 is disclosed in CN102802661B. These mutation sites were used as control sites in this study.

[0265] Example 2: Expression and purification of engineered antibodies

[0266] Expi293F cells (purchased from Thermo Fisher Scientific) were subcultured in Expi293F cell culture medium (purchased from Thermo Fisher Scientific). The cell density was checked one day before transfection and adjusted to 3×10 6 The cell density was adjusted to 3 × 10 cells / mL on the day of transfection. 6 cells / mL.

[0267] Take Opti-MEM medium (purchased from Gibco) with 1 / 10 of the final volume of the transfected Expi293F cells as the transfection buffer, add 10 μg of the corresponding recombinant plasmid (containing Pcdna3.1 plasmids encoding the antibody heavy and light chains, respectively, with a ratio of 1:1 for the light and heavy chain plasmids) per mL of transfection buffer, mix well, add 30 ug of polyethylenimine (PEI) (Polysciences, catalog number: 23966) per mL of transfection buffer, mix well, incubate at room temperature for 20 minutes, then gently pour the PEI / DNA mixture into the Expi293F cell suspension, mix well, and place on a shaker for culture under the conditions of 8% CO2, 36.5°C, and 120 rpm.

[0268] After 16-18 hours of culture, the culture flask was supplemented with 200 g / L FEED (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone) at 1 / 50 the volume of the post-transfection culture, a glucose solution at a final concentration of 5 g / L, and VPA (Gibco, catalog number: 11140-050) at a final concentration of 2.2 mM. The cells were gently mixed and incubated on a shaker at 8% CO₂, 36.5°C, and 120 rpm. Culture was continued for 6 days, and the culture was harvested and centrifuged at 4000 rpm for 30 minutes. The cell supernatant was filtered through a 0.45 μM filter and purified by affinity chromatography and gel chromatography.

[0269] The specific affinity chromatography purification steps are as follows: A MabSelect SuRe LX affinity chromatography column (Cytiva, catalog number: 17547401) was used. Before purification, the column and tubing were detoxified with 0.1M NaOH for 2 hours. Then, the tubing and column were rinsed with distilled water. The column was equilibrated with 5 column volumes of 1× PBS (Gibco, catalog number: 10010023). The collected supernatant was passed through the column, and the column was rinsed with 10 column volumes of 1× PBS to remove nonspecifically bound proteins. The column was rinsed with 5 column volumes of elution buffer (100mM citrate, pH 3.3), and the eluate was collected. The protein pH was then adjusted to 6.0 with 2M Tris for further gel chromatography.

[0270] The specific gel exchange chromatography purification procedure is as follows: a Superdex 200 Increase 10 / 300 GL (GE Healthcare, catalog number: 28-9909-44) gel chromatography column was selected and placed in an AKTA pure system. The AKTA pure system equipped with the Superdex 200 Increase 10 / 300 GL gel chromatography column was detoxified with 0.1 M NaOH for 2 hours. The system and column were then rinsed with distilled water. The column was equilibrated with 2-5 column volumes of 1× PBS until the conductivity and pH stabilized. The protein obtained from the affinity chromatography was loaded and eluted with 1× PBS. Impurities such as aggregates were removed based on the UV absorption peak, and the high-purity sample was collected. The sample was filtered through a 0.22 μm membrane, and the protein concentration was determined and analyzed by SEC.

[0271] SDS-PAGE electrophoresis analysis: Take protein samples, add reducing loading buffer (Yisheng Biotechnology, catalog number: 20315ES05) or non-reducing loading buffer (Beijing Biolabs, catalog number: WE0289), heat at 70 degrees Celsius for 10 minutes, take an appropriate amount of sample and add precast gel 4–20% TGX TM Precast Gel (Bio-rad, catalog number: 4561095) was added, and a protein marker (Biorad, catalog number: 1610375) was added as a control. Electrophoresis was performed at 150 V for 50 minutes, and the gel was stained with a protein stainer (Gensher, L00760C). The gel was then photographed with a gel imager (Shanghai Tianneng, Tanon-1600).

[0272] Example 3: ADC preparation

[0273] I. Preparation of a Single-Site DAR2 ADC: The single-site mutant antibody expressed in Example 2 was conjugated to the linker-drug mc-GGFG-Dxd, resulting in 11 ADC molecules: LLC160-Dxd, LLC166-Dxd, LC165-Dxd, HC152-Dxd, HC155-Dxd, LC138-Dxd, HC290-Dxd, LC167-Dxd, LC205-Dxd, LC160-Dxd, and LC166-Dxd, used as a control. Throughout this document, all numbers follow the Eu numbering scheme, with LLC representing lambda light chain and LC representing kappa light chain. LLC160-Dxd represents an ADC molecule obtained by coupling the lambda light chain at position 160 with cysteine ​​modification and mc-GGFG-Dxd, LLC166-Dxd represents an ADC molecule obtained by coupling the lambda light chain at position 166 with cysteine ​​modification and mc-GGFG-Dxd, LC165-Dxd represents an ADC molecule obtained by coupling the kappa light chain at position 165 with cysteine ​​modification and mc-GGFG-Dxd, HC152-Dxd represents an ADC molecule obtained by coupling the heavy chain at position 152 with cysteine ​​modification and mc-GGFG-Dxd, HC155-Dxd represents an ADC molecule obtained by coupling the heavy chain at position 155 with cysteine ​​modification and mc-GGFG-Dxd, LC138-Dxd represents an ADC molecule obtained by coupling the kappa light chain at position 138 with cysteine ​​modification and mc-GGFG-Dxd. HC290-Dxd represents the ADC molecule obtained by coupling with mc-GGFG-Dxd after cysteine ​​modification at position 290 of the heavy chain, LC167-Dxd represents the ADC molecule obtained by coupling with mc-GGFG-Dxd after cysteine ​​modification at position 167 of the kappa light chain, LC205-Dxd represents the ADC molecule obtained by coupling with mc-GGFG-Dxd after cysteine ​​modification at position 205 of the kappa light chain, LC160-Dxd represents the ADC molecule obtained by coupling with mc-GGFG-Dxd after cysteine ​​modification at position 160 of the kappa light chain, and LC166-Dxd represents the ADC molecule obtained by coupling with mc-GGFG-Dxd after cysteine ​​modification at position 166 of the kappa light chain.

[0274] First, reduce the antibody to release the sulfhydryl group on the cysteine. During the expression process of the engineered antibody, the cysteine ​​will react with cysteine ​​or glutathione in the cell culture medium, becoming blocked and unable to react with small molecule drugs. Therefore, the antibody needs to be reduced to release the cysteine ​​sulfhydryl group. The antibody is replaced with a 20mM histidine solution at pH 6.5 (buffer 1) (for the LC165 site, the coupling solution is a PB solution at pH 7.0 (buffer 2)), and 20mM EDTA and a 20-fold molar excess of the reducing agent TCEP are added to release the free cysteine. The reaction is carried out in a 37°C water bath for 3 hours. After the reaction is completed, the reducing agent and the reduced cysteine ​​or glutathione are removed by overnight dialysis using a dialysis cup.

[0275] Then the interchain disulfide bonds of the antibody were reshaped. In this step, 40 times the molar ratio of the oxidant dehydroascorbic acid (dhAA) was added and oxidized in a water bath at 37°C for 3 hours. The solution was cooled to room temperature, 12 times the molar ratio of mc-GGFG-Dxd was added, and DMSO was added to make the volume ratio of DMSO in the solution 10%, and the reaction was carried out at room temperature for 1 hour. 20 times the molar ratio of N-acetylcysteine ​​was added to the mixture to quench the unreacted small molecule drug. After 15 minutes of reaction, the mixture was treated with Zeba TM The desalting spin column was used to remove unreacted small molecule drugs and other impurities and the ADC was replaced with a 20 mM histidine solution at pH 5.5 for storage.

[0276] 2. Preparation of dual-site DAR4 ADC: The single sites were combined and 13 dual-site mutant antibodies were expressed according to the method of Example 2 for coupling with the linker-drug mc-GGFG-Dxd, resulting in a total of 13 ADC molecules: LLC160-LLC166-Dxd, LLC166-HC152-Dxd, LLC160-HC155-Dxd, LLC166-HC155-Dxd, LC165-HC152-Dxd, LC165-HC155-Dxd, LC 160-LC166-Dxd, LC110-HC239-Dxd, LC110-HC118-Dxd, LC110-HC442-Dxd, HC118-HC239-Dxd, HC118-HC442 -Dxd, HC239-HC442-Dxd, LC160-178-LC166-Dxd, LC166-HC239-Dxd, LC166-HC375-Dxd, HC239-HC375-Dxd.

[0277] In this article, all numbers are based on the Eu numbering principle. LLC160-LLC166-Dxd represents the ADC molecules obtained by coupling the lambda light chain 160 and 166 with cysteine ​​modification and mc-GGFG-Dxd. LLC166-HC152-Dxd represents the ADC molecules obtained by coupling the lambda light chain 166 and heavy chain 152 with cysteine ​​modification and mc-GGFG-Dxd. LLC160-HC155-Dxd represents the ADC molecules obtained by coupling the lambda light chain 166 and heavy chain 152 with cysteine ​​modification and mc-GGFG-Dxd. The ADC molecule is obtained by coupling lambda light chain 160 and heavy chain 155 with mc-GGFG-Dxd after cysteine ​​modification. LLC166-HC155-Dxd represents the ADC molecule obtained by coupling lambda light chain 166 and heavy chain 155 with mc-GGFG-Dxd after cysteine ​​modification. LC165-HC152-Dxd represents the ADC molecule obtained by coupling kappa light chain 165 and heavy chain 152 with mc-GGFG-Dxd after cysteine ​​modification. ADC molecules, LC165-HC155-Dxd represents the ADC molecule obtained by coupling the kappa light chain 165 and heavy chain 155 with cysteine ​​modification and mc-GGFG-Dxd, LC160-LC166-Dxd represents the ADC molecule obtained by coupling the kappa light chain 160 and 166 with cysteine ​​modification and mc-GGFG-Dxd, LC110-HC239-Dxd represents the ADC molecule obtained by coupling the kappa light chain 110 and heavy chain 239 with cysteine ​​modification and mc-GGFG-Dxd. The ADC molecules obtained by coupling with mc-GGFG-Dxd after modification are LC110-HC118-Dxd, which represents the ADC molecules obtained by coupling with mc-GGFG-Dxd after modification of cysteine ​​at positions 110 and 118 of the kappa light chain. LC110-HC442-Dxd represents the ADC molecules obtained by coupling with mc-GGFG-Dxd after modification of cysteine ​​at positions 110 and 442 of the kappa light chain. HC118-HC239-Dxd represents an ADC molecule obtained by coupling mc-GGFG-Dxd after cysteine ​​modification at positions 118 and 239 of the heavy chain, HC118-HC442-Dxd represents an ADC molecule obtained by coupling mc-GGFG-Dxd after cysteine ​​modification at positions 118 and 442 of the heavy chain, HC239-HC442-Dxd represents an ADC molecule obtained by coupling mc-GGFG-Dxd after cysteine ​​modification at positions 239 and 442 of the heavy chain, LC160-178-LC166-Dxd represents an ADC molecule obtained by coupling mc-GGFG-Dxd after cysteine ​​modification at positions 160 and 166 of the kappa light chain and mutation of amino acid T to Y at position 178,LC166-HC239-Dxd represents an ADC molecule obtained by coupling mc-GGFG-Dxd with cysteine ​​residues at positions 166 and 239 of the kappa light chain. LC166-HC375-Dxd represents an ADC molecule obtained by coupling mc-GGFG-Dxd with cysteine ​​residues at positions 166 and 375 of the kappa light chain. HC239-HC375-Dxd represents an ADC molecule obtained by coupling mc-GGFG-Dxd with cysteine ​​residues at positions 239 and 375 of the heavy chain.

[0278] Among them, the LC160-178-LC166 antibody not only mutated cysteine ​​at sites 160 and 166, but also mutated the T at site 178 near 160 to a Y near the LLC160 site. By changing the amino acid environment near the LC160 site, the properties of the LC160 site were made closer to LLC160.

[0279] Preparation of LLC160-LLC166-Dxd:

[0280] First, reduce the antibody to release the sulfhydryl group on the cysteine. During the expression process of the engineered antibody, the cysteine ​​will react with cysteine ​​or glutathione in the cell culture medium, thus being blocked and unable to react with small molecule drugs. Therefore, the antibody needs to be reduced to release the sulfhydryl group of the cysteine. The LLC160-LLC166 antibody was replaced with a 20mM histidine solution at pH 6.5 (buffer 1), and 20mM EDTA and a 20-fold molar excess of the reducing agent TCEP were added to release the free cysteine. The reaction was carried out in a 37°C water bath for 3 hours. After the reaction, the reducing agent and the reduced cysteine ​​or glutathione were removed by overnight dialysis using a dialysis cup.

[0281] Then the interchain disulfide bonds of the antibody were reshaped. In this step, 40 times the molar ratio of the oxidant dehydroascorbic acid (dhAA) was added and oxidized in a water bath at 37°C for 3 hours. The solution was cooled to room temperature, 12 times the molar ratio of mc-GGFG-Dxd was added, and DMSO was added to make the volume ratio of DMSO in the solution 10%, and the reaction was carried out at room temperature for 1 hour. 20 times the molar ratio of N-acetylcysteine ​​was added to the mixture to quench the unreacted small molecule drug. After 15 minutes of reaction, the mixture was treated with Zeba TM The desalting spin column was used to remove unreacted small molecule drugs and other impurities and the ADC was replaced with a 20 mM histidine solution at pH 5.5 for storage.

[0282] LC160-LC166-Dxd, LC110-HC239-Dxd, LC110-HC118-Dxd, LC110-HC442-Dxd, HC118-HC239-Dxd, HC118-HC442-Dxd, HC239-HC442-Dxd, LC160-178-LC166-Dxd, LC166-HC239-Dxd, LC166-HC375-Dxd, HC239-HC375-Dxd were prepared similarly.

[0283] Preparation of LLC166-HC152-Dxd:

[0284] LLC166-HC152 antibody was replaced with a 20 mM histidine solution (pH 7.0) (Buffer 2). 20 mM EDTA and a 20-fold molar excess of the reducing agent TCEP were added to release free cysteine. The reaction was carried out in a 37°C water bath for 3 hours. After the reaction, the reducing agent and reduced cysteine ​​or glutathione were removed by overnight dialysis using a dialysis cup.

[0285] A 40-fold molar ratio of oxidant dhAA was added and oxidized in a 37°C water bath for 3 hours to reshape the interchain disulfide bonds of the antibody. The solution was cooled to room temperature, and a 12-fold molar ratio of mc-GGFG-Dxd was added. DMSO was added to make the volume ratio of DMSO in the solution 10% and the reaction was continued at room temperature for 1 hour. A 20-fold molar ratio of N-acetylcysteine ​​was added to the mixture to quench the unreacted small molecule drug. After 15 minutes of reaction, the mixture was treated with Zeba TM The desalting spin column was used to remove unreacted small molecule drugs and other impurities and the ADC was replaced with a 20 mM histidine solution at pH 5.5 for storage.

[0286] Preparation of LLC160-HC155-Dxd:

[0287] The LLC160-HC155 antibody was replaced with a 20mM histidine solution (buffer 2) at pH 7.0, and 20mM EDTA and a 20-fold molar excess of the reducing agent TCEP were added to release free cysteine. The reaction was carried out in a 37°C water bath for 3 hours. After the reaction, the reducing agent and the reduced cysteine ​​or glutathione were dialyzed overnight using a dialysis cup. A 40-fold molar ratio of the oxidizing agent dhAA was added and oxidized in a 37°C water bath for 3 hours. The solution was cooled to room temperature, a 12-fold molar ratio of mc-GGFG-Dxd was added, and DMSO was supplemented to make the volume ratio of DMSO in the solution 10%, and the reaction was carried out at room temperature for 1 hour. A 20-fold molar ratio of N-acetylcysteine ​​was added to the mixture to quench the unreacted small molecule drug. After 15 minutes of reaction, the mixture was treated with Zeba TMThe desalting spin column was used to remove unreacted small molecule drugs and other impurities and the ADC was replaced with a 20 mM histidine solution at pH 5.5 for storage.

[0288] Preparation of LLC166-HC155-Dxd:

[0289] The LLC166-HC155 antibody was replaced with a 20mM histidine solution (buffer 2) at pH 7.0, and 20mM EDTA and a 20-fold molar excess of the reducing agent TCEP were added to release free cysteine. The reaction was carried out in a 37°C water bath for 3 hours. After the reaction was completed, the reducing agent and the reduced cysteine ​​or glutathione were dialyzed overnight using a dialysis cup. A 40-fold molar ratio of the oxidant dhAA was added and oxidized in a 37°C water bath for 3 hours. The solution was cooled to room temperature, a 12-fold molar ratio of mc-GGFG-Dxd was added, and DMSO was supplemented to make the volume ratio of DMSO in the solution 10%, and the reaction was carried out at room temperature for 1 hour. A 20-fold molar ratio of N-acetylcysteine ​​was added to the mixture to quench the unreacted small molecule drug reaction for 15 minutes, and then the mixture was treated with Zeba TM The desalting spin column was used to remove unreacted small molecule drugs and other impurities and the ADC was replaced with a 20 mM histidine solution at pH 5.5 for storage.

[0290] Preparation of LC165-HC152-Dxd:

[0291] The LC165-HC152 antibody was replaced with a 20mM histidine solution (buffer 2) at pH 7.0, and 20mM EDTA and a 20-fold molar excess of the reducing agent TCEP were added to release free cysteine. The reaction was carried out in a 37°C water bath for 3 hours. After the reaction was completed, the reducing agent and the reduced cysteine ​​or glutathione were dialyzed overnight using a dialysis cup. A 40-fold molar ratio of the oxidant dhAA was added and oxidized in a 37°C water bath for 3 hours. The solution was cooled to room temperature, a 12-fold molar ratio of mc-GGFG-Dxd was added, and DMSO was supplemented to make the volume ratio of DMSO in the solution 10%, and the reaction was carried out at room temperature for 1 hour. A 20-fold molar ratio of N-acetylcysteine ​​was added to the mixture to quench the unreacted small molecule drug reaction for 15 minutes, and then the mixture was treated with Zeba TM The desalting spin column was used to remove unreacted small molecule drugs and other impurities and the ADC was replaced with a 20 mM histidine solution at pH 5.5 for storage.

[0292] Preparation of LC165-HC155-Dxd:

[0293] The LC165-HC155 antibody was replaced with a 20mM histidine solution (buffer 2) at pH 7.0, and 20mM EDTA and a 20-fold molar excess of the reducing agent TCEP were added to release free cysteine. The reaction was carried out in a 37°C water bath for 3 hours. After the reaction, the reducing agent and the reduced cysteine ​​or glutathione were dialyzed overnight using a dialysis cup. A 40-fold molar ratio of the oxidant dhAA was added and oxidized in a 37°C water bath for 3 hours. The solution was cooled to room temperature, a 12-fold molar ratio of mc-GGFG-Dxd was added, and DMSO was supplemented to make the volume ratio of DMSO in the solution 10%, and the reaction was carried out at room temperature for 1 hour. A 20-fold molar ratio of N-acetylcysteine ​​was added to the mixture to quench the unreacted small molecule drug reaction for 15 minutes, and then the mixture was treated with Zeba TM The desalting spin column was used to remove unreacted small molecule drugs and other impurities and the ADC was replaced with a 20 mM histidine solution at pH 5.5 for storage.

[0294] Preparation of TZB-Dxd-DAR4 / TZB-Dxd-DAR2:

[0295] Randomly conjugated DAR4 ADC and DAR2 ADC were prepared as control groups. First, the ratio of the reducing agent TCEP was tested. Different ratios of TCEP were added to the trastuzumab antibody solution, and after reacting at 25°C for 2 hours, a 10-fold molar ratio of mc-GGFG-Dxd was added, and DMSO was added to make the final system contain 10% by volume. The reaction was continued at 25°C for 1 hour. A 20-fold molar ratio of N-acetylcysteine ​​was added to quench the unreacted small molecule drug. After 15 minutes of reaction, the reaction was carried out with Zeba TM A desalting spin column was used to remove unreacted small molecule drug and other impurities, and the ADC was then exchanged into a 20 mM histidine solution at pH 5.5 for storage. The DAR was calculated by RP-HPLC, and linear regression was performed for different TCEP ratios to determine the TCEP ratio required to achieve a DAR of 4 or 2. Based on the obtained TCEP ratios, ADC molecules with an average DAR of 4 or 2 were prepared according to the experimental method described above.

[0296] Preparation of TZB-Dxd-DAR8:

[0297] DAR8 ADC was prepared as a control group. First, 20-fold molar ratio of TCEP was added to the Trastuzumab antibody solution, and the reaction was carried out at 25°C for 2 hours. Then 12-fold molar ratio of mc-GGFG-Dxd was added, and DMSO was added to make the final system contain 10% by volume. The reaction was continued at 25°C for 1 hour. 20-fold molar ratio of N-acetylcysteine ​​was added to quench the unreacted small molecule drug. After 15 minutes of reaction, the reaction was carried out with Zeba TMThe desalting spin column was used to remove unreacted small molecule drugs and other impurities and the ADC was replaced with a 20 mM histidine solution at pH 5.5 for storage.

[0298] Preparation of negative control IgG-Dxd-DAR4

[0299] IgG-Dxd-DAR4 ADC was prepared as a control group. First, the proportion of the reducing agent TCEP was tested. Different proportions of TCEP were added to the IgG-anti Hen Egg Lysozyme (Hel) antibody (heavy chain: SEQ ID NO: 38; light chain: SEQ ID NO: 39) solution, and after reacting at 25°C for 2 hours, a 10-fold molar ratio of mc-GGFG-Dxd was added, and DMSO was added to make the final system contain a volume ratio of 10%, and the reaction was continued at 25°C for 1 hour. After adding 20-fold molar ratio of N-acetylcysteine ​​to quench the unreacted small molecule drug, the reaction was continued for 15 minutes with Zeba TM A desalting spin column was used to remove unreacted small molecule drug and other impurities, and the ADC was then exchanged into a 20 mM histidine solution at pH 5.5 for storage. The DAR was calculated using RP-HPLC, and linear regression was performed for different TCEP ratios to determine the TCEP ratio required to achieve a DAR of 4. Based on the obtained TCEP ratios, ADC molecules with an average DAR of 4 were prepared according to the experimental method described above.

[0300] Example 4: Analysis of ADC physical and chemical properties

[0301] RP-HPLC was used to analyze the number of small molecules conjugated to each engineered antibody. First, a small amount of the ADC solution prepared in Example 3 was taken, the pH was adjusted to 7.5 with Tris buffer, and the reducing agent DTT was added to fully reduce the ADC in a 37°C water bath. HPLC analysis was then performed, and the DAR value was calculated based on the peak area of ​​each peak at UV 280 nm. The analysis conditions are as follows.

[0302] The results are shown in Figures 1 and 2.

[0303] Figure 1 shows a representative graph used for site-specific conjugation DAR analysis and calculation. LC represents the light chain component without a drug attached, LC+1 represents the light chain conjugated with one drug, HC represents the heavy chain component without a drug attached, and HC+1 represents the heavy chain conjugated with one drug.

[0304] Figure 2 shows a representative plot for random conjugation DAR analysis and calculation. LC represents a light chain component without a drug attached, LC+1 represents a light chain conjugated with one drug, HC represents a heavy chain component without a drug attached, HC+1 represents a heavy chain conjugated with one drug, HC+2 represents a heavy chain conjugated with two drugs, and HC+3 represents a heavy chain conjugated with three drugs.

[0305] The DAR calculation results of each ADC are shown in Tables 1 and 2.

[0306] Table 1: Average DAR of DAR2 ADC:

[0307] Table 2: Average DAR of DAR4 ADCs:

[0308] ADC purity analysis

[0309] The purity of the ADC was analyzed using size exclusion chromatography (SEC) according to the conditions in the table below. The purity of the ADC was determined based on the peak area ratio of monomer, aggregate, and oligomer peaks at UV 280 nm.

[0310] The purity of each ADC is shown in Tables 3 and 4 below.

[0311] Table 3: Purity of DAR2 ADC:

[0312] Table 4: Purity of DAR4 ADC:

[0313] Figure 3 is a representative graph used for purity analysis (LLC166-HC155-Dxd).

[0314] From the above, we can see that the DAR of ADC is around 3.5 and the purity is above 95%.

[0315] Following the above analytical method, the DAR values ​​and purity of the ADCs were re-measured. The results are shown in the table below. As can be seen, the DAR values ​​of the ADCs in each group were approximately 3.5, with minimal differences between groups. Furthermore, the purity was consistently above 97%, allowing for subsequent in vitro and in vivo comparisons.

[0316] Table 5. Analysis of ADC physical and chemical properties

[0317] Hydrophobicity analysis

[0318] The hydrophobicity of the ADC was analyzed using hydrophobic interaction chromatography (HIC). The analysis conditions are shown in the table below. The hydrophilicity of different ADCs was compared based on the peak elution time at UV 280 nm.

[0319] The peak times of various ADCs are shown in Tables 6 and 7 below.

[0320] Table 6: DAR2 ADC retention time:

[0321] Figure 4 shows the hydrophobic interaction profiles of various DAR2 ADCs.

[0322] Table 7: Retention Time of DAR4 ADC:

[0323] FIG5 is a hydrophobic interaction spectrum of each DAR4 ADC, showing the peak time of the ADC on the hydrophobic interaction chromatography.

[0324] Hydrophilicity is generally considered to predict the in vivo PK behavior of ADCs. More hydrophobic ADC molecules are more susceptible to aggregation and clearance, thus reducing efficacy. The earlier the peak elution time on the HIC spectrum, the greater the hydrophilicity. The later the peak elution position, the less hydrophilicity. As shown in Figure 5 and Table 6, the DAR4 ADCs in Groups 1-6 are more hydrophilic than the randomly conjugated TZB-Dxd-DAR4 ADC. The LLC160-LLC166-Dxd peaked at 11.611 minutes, while the other control site combinations all had later peak times. HC118-HC442-Dxd peaked at 14.809 minutes, while LC110-HC118-Dxd, which had the earliest peak, peaked at 12.346 minutes. This demonstrates the significant hydrophilicity advantage of LLC160-LLC166-Dxd. This superior hydrophilicity predicts excellent pharmacokinetic behavior in the body, leading to greater efficacy.

[0325] Example 5: In vitro cell proliferation assay

[0326] In vitro cell proliferation assays can be used to evaluate the in vitro activity of ADC molecules. Human breast adenocarcinoma SK-BR-3 cells (from ATCC) were plated at a density of 2,000 cells per well in 96-well plates. The 96-well plates were incubated overnight at 37°C in a CO2 incubator.

[0327] ADC samples were diluted to different concentrations and added to the well plate in sequence, with two replicate wells for each sample. Culture was continued in the incubator for 6 days. 100 μL was added to each well. The assay reagent was shaken evenly for 2 minutes and then allowed to stand at room temperature for 10 minutes to stabilize the luminescence signal. The absorbance at 450 nm was measured using a spectrophotometer. The results were fitted using GraphPad Prism software, and the EC50 values ​​were calculated. Figures 6A and 6B show the cell viability results. Tables 8-1 and 8-2 show the EC50 values ​​for cell killing.

[0328] Table 8-1: EC50 of each sample.

[0329] Table 8-2

[0330] In in vitro cell cytotoxicity assays, ZB-Dxd-DAR8 demonstrated the strongest cytotoxicity, while the other ADC groups showed no significant differences in cytotoxicity, with similar EC50 values. This is because the DAR values ​​of the ADCs in each group were similar, differing only in the conjugation site. The relatively simple in vitro cell culture environment prevented significant differences between the groups. However, in the complex physiological environment of the body, differences in the conjugation sites become apparent.

[0331] Example 6: In vitro binding

[0332] Resuspend SK-BR-3 cells in 2 mL FACS buffer and adjust the cell density to 1.5-2.0 × 10 6 100 μL / mL, add 50 μL / well to a 96-well plate and place in a 4°C refrigerator until ready. Dilute the test antibody or ADC to 200 nM in FACS buffer, and then serially dilute from this concentration to 200 nM. Mix the diluted antibody with cells in a 1:1 ratio and incubate at 4°C for 30 minutes. After incubation, centrifuge and discard the supernatant, then wash the cells with FACS buffer. Then, add the diluted fluorescent secondary antibody, Goat Anti-Human IgG-PE, to the cells, resuspend, and incubate at 4°C in the dark for 30 minutes. After centrifugation and washing, measure fluorescence using a flow cytometer. Data were processed using FlowJo V10 and GraphPad Prism 8 software. The results are shown in Figures 7A and 7B and Table 9.

[0333] Table 9-1: EC50 of each sample binding to cells in vitro.

[0334] Table 9-2: EC50 of each sample binding to cells in vitro.

[0335] As can be seen from Figure 7A and Table 9-1, each group has a similar EC50 value and is almost indistinguishable from the wild-type Trastuzumab antibody, indicating that the mutant antibody does not affect its ability to bind to the antigen after being coupled to ADC.

[0336] As shown in Figure 7B and Table 9-2, each group had similar EC50 values, with little difference compared to wild-type trastuzumab, indicating that neither the antibody mutation nor the conjugated ADC affected antigen binding. There was no significant difference in cell binding between the LLC160-LLC166-Dxd group and the LC160-LC160-Dxd group.

[0337] Example 7: Evaluation of stability in plasma

[0338] ADCs were diluted to 0.2 mg / mL in mouse plasma and incubated in a 37°C CO2 incubator for 0, 2, 7, and 14 days. ADCs were then extracted from the plasma using affinity chromatography for further analysis. The extraction process was as follows. First, a gel resin (anti-human IgG-agarose goat antibody, Sigma, A3316) that specifically binds to the Fc region of human IgG was incubated with the sample for 30 minutes at room temperature. The plasma was removed by centrifugation, and unbound proteins were washed with PBS. Finally, the resin was mixed with a pH 3.0 citric acid solution and incubated for 10 minutes. The ADC solution was collected by centrifugation and the pH was adjusted to 7.5 with Tris. The ADCs were fully reduced by adding the reducing agent DTT in a 37°C water bath. The DAR values ​​at each time point were then analyzed by HPLC. The DAR values ​​at each time point are shown in Tables 9 and 10. The reduction ratio of DAR over 14 days is calculated as (DAR at day 14 - initial DAR) / DAR at day 14.

[0339] The DAR profiles of various ADCs in mouse plasma are shown in Figures 8A (DAR2 ADC), 8B, and 8C (DAR4 ADC). The DAR2 stability results show that all site-specifically conjugated DAR2 ADCs exhibited greater stability than the randomly conjugated DAR2 ADC (TZB-Dxd-DAR2). Among them, LLC160-Dxd was more stable than LC205-Dxd. LC166-Dxd exhibited only a 4% decrease in DAR over 14 days, demonstrating relatively good stability. LC160-Dxd had an initial DAR of 1.72. Once in plasma, the drug rapidly shed, with the DAR decreasing to 1.56 at time T0. After 14 days of incubation in plasma, the DAR had decreased to 1.05, indicating relatively poor stability. Based on the comparison of single-site ADC plasma stability results, it can be inferred that the stability of the dual-site combination of LC160-Dxd and LC166-Dxd is generally inferior to that of the dual-site combination of LLC160-Dxd and LLC166-Dxd. This means that LLC160-LLC166-Dxd is more stable in plasma than LC160-LC166-Dxd, thereby resulting in better efficacy.

[0340] From the DAR4 stability results (Table 11), it can be seen that TZB-Dxd-DAR4 reduced DAR by 44.5% within 14 days, while LLC166-LLC160-Dxd only reduced DAR by 5.5%, which has certain advantages.

[0341] As shown in Table 12, LLC160-LLC166-Dxd showed the most stable DAR, with a decrease of only 6.46% over 14 days. In contrast, the DAR of the LC160-LC166-Dxd ADC decreased more rapidly, decreasing by 24.73% over 14 days. After modification at position LC178, the stability of LC160-178-LC166-Dxd was significantly increased, indicating that the amino acid environment surrounding position 160 is crucial for ADC stability. This demonstrates that LLC160-LLC166-Dxd is more stable than LC160-LC166-Dxd in mouse plasma.

[0342] Table 10: DAR drop in mouse plasma for various DAR2 ADCs.

[0343] Table 11: DAR drop in mouse plasma for various DAR4 ADCs.

[0344] Table 12: DAR drop in mouse plasma for various DAR4 ADCs.

[0345] Example 8: In vivo pharmacodynamic evaluation

[0346] Transgenic mice that highly express Her2 were used to determine the in vivo efficacy of the ADC. Human gastric cancer cells NCI-N87, purchased from ATCC, were subcutaneously transplanted into CB17-SCID mice (6-8 weeks old, Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) at a seeding density of 2×10 6 Tumor size was monitored using vernier calipers and the average tumor volume was calculated.

[0347] On day 12, mice were randomly divided into groups (n=5) and dosed once. The doses are shown in Figure 9 (Figures 9A-9C and 9E are for peritoneal administration, and Figure 9D is for intravenous injection). Tumor volumes in each treatment group were measured every three days. The results are shown in Figure 9.

[0348] The efficacy results of the DAR2 ADCs showed that TZB-Dxd-DAR8 exhibited the strongest tumor suppression activity. TZB-Dxd-DAR4 (traditional conjugate) had the weakest efficacy among all experimental groups. The LLC160-Dxd and LLC166-Dxd groups showed the best efficacy, outperforming the control group, LC205-Dxd. The efficacy results of DAR4 ADCs showed that the antitumor activity of all site-specifically conjugated DAR4 ADCs was between that of TZB-Dxd-DAR8 and TZB-Dxd-DAR4. All groups of ADCs had better efficacy than TZB-Dxd-DAR4. Among them, LLC166-LLC160-Dxd and LC160-LC166-Dxd had similar pharmacodynamic activities (no significant differences) and the best efficacy, which was better than other controls LC110-HC239-Dxd, LC110-HC118-Dxd, LC110-HC442-Dxd, HC118-HC239-Dxd, HC118-HC442-Dxd, and HC239-HC442-Dxd, and was similar to the efficacy of TZB-Dxd-DAR8 ADC. The in vivo efficacy superiority of LLC160-LLC166-Dxd was demonstrated.

[0349] Sequence Listing

Claims

1. A cysteine-modified antibody or antigen-binding fragment thereof, wherein (i) the antibody or antigen-binding fragment thereof comprises a modified lambda light chain constant region, wherein the amino acid at position 160 of the modified lambda light chain constant region is mutated to cysteine ​​compared to a parent lambda constant region; (ii) the antibody or antigen-binding fragment thereof comprises a modified lambda light chain constant region, wherein the amino acids at positions 160 and 166 of the modified lambda light chain constant region are mutated to cysteine ​​compared to a parent lambda constant region; (iii) the antibody or antigen-binding fragment thereof comprises a modified lambda light chain constant region and a modified heavy chain constant region, wherein the modified lambda light chain constant region is mutated to cysteine ​​at amino acid position 160 compared to the parent lambda constant region, and the modified heavy chain constant region is mutated to cysteine ​​at amino acid position 152 or 155 compared to the parent heavy chain constant region; (iv) the antibody or antigen-binding fragment thereof comprises a modified lambda light chain constant region and a modified heavy chain constant region, wherein the modified lambda light chain constant region is mutated to cysteine ​​at amino acid position 166 compared to the parent lambda constant region, and the modified heavy chain constant region is mutated to cysteine ​​at amino acid position 152 or 155 compared to the parent heavy chain constant region; (v) the antibody or antigen-binding fragment thereof comprises a modified Kappa light chain constant region and a modified heavy chain constant region, wherein the amino acid at position 165 of the modified Kappa light chain constant region is mutated to cysteine ​​compared to the parent Kappa light chain constant region, and the amino acid at position 152 or 155 of the modified heavy chain constant region is mutated to cysteine ​​compared to the parent heavy chain constant region; (vi) the antibody or antigen-binding fragment thereof comprises a modified Kappa light chain constant region, wherein the amino acids at positions 160 and 166 of the modified Kappa light chain constant region are mutated to cysteine, and the amino acid at position 178 is mutated to tyrosine compared to the parent Kappa constant region; (vii) the antibody or antigen-binding fragment thereof comprises a modified kappa light chain constant region and a modified heavy chain constant region, wherein the amino acid at position 166 of the modified kappa light chain constant region is mutated to cysteine ​​compared to the parent kappa light chain constant region, and the amino acid at position 375 of the modified heavy chain constant region is mutated to cysteine ​​compared to the parent heavy chain constant region; or (viii) the antibody or antigen-binding fragment thereof comprises a modified heavy chain constant region, wherein the amino acids at positions 239 and 375 of the modified heavy chain constant region are mutated to cysteine ​​compared to the parent heavy chain constant region.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the parent heavy chain constant region is the heavy chain constant region of human IgG1, for example, it comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:3 and does not comprise a cysteine ​​mutation.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the parent lambda light chain constant region is a human lambda light chain constant region, e.g., comprising the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 6 and not comprising a cysteine ​​mutation; or wherein the parent kappa light chain constant region is a human kappa light chain constant region, for example, it comprises the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO:5 and does not comprise a cysteine ​​mutation.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein (i) wherein the modified lambda light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 7; (ii) wherein the modified lambda light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 28; (iii) wherein the modified lambda light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 7, and the modified heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 10 or 11; (iv) wherein the modified lambda light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 8, and the modified heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 10 or 11; (v) wherein the modified kappa light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 9, and the modified heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 10 or 11; (vi) wherein the modified kappa light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 62; (vii) wherein the modified kappa light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 66, and the modified heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 67; or The modified heavy chain constant region described in (viii) comprises the amino acid sequence shown in SEQ ID NO:67 and the amino acid sequence shown in SEQ ID NO:

68.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The modified light chain constant region in (i) comprises or consists of the following amino acid sequence: the amino acid sequence of SEQ ID NO:16, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:16 and has cysteine ​​substituted at position 160, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:16 and comprises the amino acid sequence of SEQ ID NO:7; or (ii) the modified light chain constant region comprises or consists of the following amino acid sequence: the amino acid sequence of SEQ ID NO:18, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:18 and wherein positions 160 and 166 are substituted with cysteine, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:18 and comprises the amino acid sequence of SEQ ID NO:28; or (iii) the modified light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO:16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16 and substituted with cysteine ​​at position 160, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16 and comprising the amino acid sequence set forth in SEQ ID NO:7, and The modified heavy chain constant region comprises or consists of the following amino acid sequence: the amino acid sequence of SEQ ID NO:25, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:25 and has cysteine ​​substituted at position 152, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:25 and comprises the amino acid sequence of SEQ ID NO:10; or the amino acid sequence of SEQ ID NO:26, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:26 and has cysteine ​​substituted at position 155, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:26 and comprises the amino acid sequence of SEQ ID NO:11; or The modified heavy chain constant region comprises a modified CH1, and the modified CH1 comprises or consists of the following amino acid sequence: The amino acid sequence of SEQ ID NO:22, or a sequence that is at least 90%, 91%, 92%, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:22 and comprising the amino acid sequence set forth in SEQ ID NO:10; or the amino acid sequence set forth in SEQ ID NO:23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:23 and having cysteine ​​substituted at position 155, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:23 and comprising the amino acid sequence set forth in SEQ ID NO:11; (iv) the modified light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO:17, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17 and substituted with cysteine ​​at position 166, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17 and comprising the amino acid sequence set forth in SEQ ID NO:8; and The modified heavy chain constant region comprises or consists of the following amino acid sequence: the amino acid sequence of SEQ ID NO:25, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:25 and has cysteine ​​substituted at position 152, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:25 and comprises the amino acid sequence of SEQ ID NO:10; or the amino acid sequence of SEQ ID NO:26, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:26 and has cysteine ​​substituted at position 155, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:26 and comprises the amino acid sequence of SEQ ID NO:11; or The modified heavy chain constant region comprises a modified CH1, and the modified CH1 comprises or consists of the following amino acid sequence: the amino acid sequence of SEQ ID NO:22, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:22 and has cysteine ​​substituted at position 152, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:22 and comprises the amino acid sequence of SEQ ID NO:10; or the amino acid sequence of SEQ ID NO:23, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:23 and has cysteine ​​substituted at position 155, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:23 and comprises the amino acid sequence of SEQ ID NO:11; or (v) the modified light chain constant region comprises or consists of the following amino acid sequence: the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20 and substituted with cysteine ​​at position 165, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20 and comprising the amino acid sequence set forth in SEQ ID NO:9; and The modified heavy chain constant region comprises or consists of the following amino acid sequence: The amino acid sequence set forth in SEQ ID NO:25, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:25 and having cysteine ​​substituted at position 152, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:25 98% or 99% sequence identity and comprises the amino acid sequence shown in SEQ ID NO: 10; or the amino acid sequence of SEQ ID NO:26, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:26 and has cysteine ​​substituted at position 155, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:26 and comprises the amino acid sequence of SEQ ID NO:11; or The modified heavy chain constant region comprises a modified CH1, and the modified CH1 comprises or consists of the following amino acid sequence: the amino acid sequence of SEQ ID NO:22, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:22 and has cysteine ​​substituted at position 152, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:22 and comprises the amino acid sequence of SEQ ID NO:10; or the amino acid sequence set forth in SEQ ID NO:23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:23 and having cysteine ​​substituted at position 155, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:23 and comprising the amino acid sequence set forth in SEQ ID NO:11; (vi) the modified light chain constant region comprises or consists of the following amino acid sequence: the amino acid sequence of SEQ ID NO:60, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:60 and substituted with cysteine ​​at positions 160 and 166 and substituted with tyrosine at position 178, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:60 and comprising the amino acid sequence of SEQ ID NO:62; (vii) the modified light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO:44, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:44 and substituted with cysteine ​​at position 166, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:44 and comprising the amino acid sequence set forth in SEQ ID NO:66; and the modified heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:61, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:61 and has cysteine ​​substituted at position 375, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:61 and comprises the amino acid sequence of SEQ ID NO:67; or the modified heavy chain constant region comprises a modified Fc region that comprises or consists of the amino acid sequence of SEQ ID NO:63, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:63 and has cysteine ​​substituted at position 375 an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:63 and having cysteine ​​substituted at position 375, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:63 and comprising the amino acid sequence set forth in SEQ ID NO:67; (viii) the modified heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:64, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:64 and has cysteine ​​substituted at positions 239 and 375, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:64 and comprises the amino acid sequences of SEQ ID NO:67 and SEQ ID NO:68; or the modified heavy chain constant region comprises a modified Fc region that comprises or consists of the amino acid sequence of SEQ ID NO:65, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:65 and has cysteine ​​substituted at positions 239 and 375. An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:65 and substituted with cysteine ​​at position 375, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:65 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and comprises the amino acid sequences shown in SEQ ID NO:67 and SEQ ID NO:

68.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2 or diabody.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof specifically binds to an antigen, such as a tumor-associated antigen, such as an immune checkpoint molecule, such as HER2.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the antibody or antigen-binding fragment thereof that specifically binds to HER2 comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known antibody that specifically binds to HER2, such as trastuzumab; or comprises a heavy chain variable region and a light chain variable region of a known antibody that specifically binds to HER2, such as trastuzumab.

9. The antibody or antigen-binding fragment thereof according to claim 7, wherein the parent antibody or antigen-binding fragment thereof is trastuzumab or an antigen-binding fragment thereof.

10. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-9.

11. An expression vector comprising the nucleic acid molecule of claim 10.

12. A host cell comprising the isolated nucleic acid molecule of claim 10 or the expression vector of claim 11.

13. A method for preparing a cysteine ​​engineered antibody or an antigen-binding fragment thereof, comprising: Introducing a nucleic acid encoding each chain of the antibody or antigen-binding fragment thereof of any one of claims 1 to 9 or an expression vector comprising the nucleic acid into a host cell; and expressing and assembling the antibody or antigen-binding fragment thereof in a host cell; Optionally, the antibody or antigen-binding fragment thereof is purified, for example, by Protein A purification.

14. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a payload.

15. The immunoconjugate of claim 14, wherein the payload is selected from a drug such as a small molecule drug, a radionuclide, DNA, RNA, an enzyme or a polypeptide; for example, a cytotoxic agent, a chemotherapeutic agent, an innate immune agonist (e.g., Toll-like receptor agonist (TLR) ISAC drug SBT6050, SBT6290, BDC-1001; STING agonist ISAC drug XMT-2056, Treg cell regulation ISAC drug ADCT-301, etc.), an immunomodulator, a therapeutic oligonucleotide (siRNA, PMO, etc.) or a radionuclide, etc.

16. The immunoconjugate of claim 14, wherein the immunoconjugate is selected from an antibody drug conjugate (ADC), an antibody immunostimulatory drug conjugate (ISAC), an antibody oligonucleotide conjugate (AOC), an antibody polypeptide drug conjugate (APC), an antibody nuclide drug conjugate (RDC) or an antibody degradation drug conjugate (ADeC).

17. The immunoconjugate of claim 16, wherein the immunoconjugate is an antibody drug conjugate (ADC), and wherein the payload is a drug moiety selected from the group consisting of: topoisomerase I inhibitors, such as camptothecin derivatives or camptothecin compounds (e.g., DXd); anti-tubulin agents, such as auristatins (MMAF, MMAE), taxane analogs (such as epothilone A and B), maytansines (DM1, DM4), tubulysin and analogs thereof; DNA-acting drugs, such as PBD, etc.

18. The immunoconjugate of claim 16 or 17, wherein the immunoconjugate is an antibody drug conjugate, and wherein the average DAR value of the ADC is between 1 and 4.

19. The immunoconjugate of any one of claims 14-18, wherein the antibody or antigen-binding fragment thereof is linked to a payload via a cysteine ​​introduced by mutation, e.g., via or without a linker.

20. The immunoconjugate of claim 19, wherein the linker comprises a functional group capable of reacting with a thiol group on a cysteine ​​residue present on the antibody or antigen-binding fragment thereof to form a covalent bond, for example a group selected from the group consisting of maleimide, iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, haloacetamide, α-haloacetyl, active esters such as succinimidyl ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, anhydride, acyl chloride, sulfonyl chloride, isocyanate and isothiocyanate, preferably maleimide.

21. The immunoconjugate of claim 19, wherein the linker is selected from a maleimido-caproyl-valinealaine (mc-va) linker, a maleimidobutanoic acid-valine-citrulline (mb-vc) linker, or a maleimido-caproyl-glycine-glycine-phenylalanine-glycine linker (mc-GGFG).

22. The immunoconjugate of any one of claims 17 to 21, wherein the immunoconjugate comprises the antibody or antigen-binding fragment thereof as defined in any one of claims 7 to 9.

23. The immunoconjugate of claim 22, wherein the drug moiety of the immunoconjugate is a topoisomerase I inhibitor, such as a camptothecin compound (eg, DX-8951 derivative DXd).

24. The immunoconjugate of claim 22 or 23, wherein the linker of the immunoconjugate is maleimido-hexanoyl-glycine-glycine-phenylalanine-glycine linker (mc-GGFG).

25. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9 or the immunoconjugate of any one of claims 14-24.

26. A pharmaceutical combination comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9 or the immunoconjugate of any one of claims 14-24, and one or more other therapeutic agents.

27. The antibody or antigen-binding fragment thereof of any one of claims 1-9 or the immunoconjugate of any one of claims 14-24 for use in therapy.

28. The antibody or antigen-binding fragment thereof of any one of claims 1 to 9 or the immunoconjugate of any one of claims 14 to 24 for use in treating a tumor such as cancer.

29. A method for treating a disease tumor such as cancer, wherein the method comprises administering the antibody or antigen-binding fragment thereof of any one of claims 1 to 9, the immunoconjugate of any one of claims 14 to 24, or the pharmaceutical composition of claim 25 to an individual in need thereof.

30. The method of claim 29, wherein the method further comprises administering the antibody or antigen-binding fragment thereof of any one of claims 1-9, the immunoconjugate of any one of claims 14-24, or the pharmaceutical composition of claim 25 in combination with one or more other therapeutic agents, for example, the other therapeutic agents are various therapeutic agents for treating tumors.

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