Ceacam5-antigen-targeting antibody, antibody-drug conjugate thereof and use thereof

Abstract

The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to CEACAM5, a CEACAM5-targeting antibody-drug conjugate (ADC), and a composition containing the antibody or the antigen-binding fragment thereof or the ADC. The present invention also relates to therapeutic and diagnostic uses of the antibody or the ADC.

Description

Antibodies targeting the CEACAM5 antigen, their antibody-drug conjugates, and their uses

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202411620647.4, filed on November 13, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to an antibody that specifically binds to CEACAM5 or an antigen-binding fragment thereof, antibody-drug conjugates (ADCs) targeting CEACAM5, and compositions containing said antibody or its antigen-binding fragment or antibody-drug conjugate. The invention also relates to the therapeutic and diagnostic uses of these antibodies or ADCs.

[0004] Background of the Invention

[0005] CEACAM5, short for Carcinoembryonic antigen-related cell adhesion molecule 5, is a cell surface glycoprotein belonging to the immunoglobulin superfamily of adhesion molecules. Its expression levels are elevated in various cancers, especially gastrointestinal tumors, and therefore it is used clinically as a tumor marker.

[0006] The CEACAM5 protein consists of 642 amino acids and has multiple potential N-linked glycosylation sites, binding to the cell membrane via glycosylphosphatidylinositol (GPI). In normal adult tissues, CEACAM5 is mainly expressed in colonic columnar epithelial cells and goblet cells, while it is highly expressed on the surface of certain cancer cells, including colorectal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, lung adenocarcinoma, small cell lung cancer, breast cancer, bladder cancer, and ovarian cancer.

[0007] In non-small cell lung cancer (NSCLC), CEACAM5 expression is associated with tumor progression and patient clinicopathological features. Studies have shown that CEACAM5 can stimulate NSCLC progression by promoting cell proliferation and migration, possibly through the p38–Smad2 / 3 signaling pathway.

[0008] The potential of CEACAM5 as a therapeutic target is being explored, and several CEACAM5-targeting therapies are currently in clinical trials. These include antibody-drug conjugates (ADCs), bispecific antibodies, and chimeric antigen receptor T-cell therapy (CAR-T). For example, Sanofi's tusamitamab ravtansine (SAR408701) is an ADC targeting CEACAM5. Merck has also developed an ADC targeting CEACAM5 that is currently in a Phase I clinical trial.

[0009] In summary, CEACAM5 is a molecule expressed in a variety of tumors, and its role in tumor development and its potential as a therapeutic target are being actively investigated.

[0010] However, the clinical efficacy of existing CEACAM5 antibodies and ADCs based on them is not ideal. For example, Sanofi's Phase III clinical trial of CEACAM5-ADC has been discontinued, and early data from Merck's CEACAM5-ADC showed low ORR response and high toxicity.

[0011] Therefore, there remains a need in this field to develop new CEACAM5 antibodies and ADCs based on them. Summary of the Invention

[0012] One aspect of the present invention relates to a binding molecule that specifically binds to CEACAM5, such as an anti-CEACAM5 antibody or an antigen-binding fragment thereof, or an immunoconjugate constructed based thereon, such as an antibody-drug conjugate (ADC).

[0013] One aspect of the present invention relates to an anti-CEACAM5 antibody or an antigen-binding fragment thereof.

[0014] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention specifically binds to CEACAM5 (e.g., human CEACAM5 or cynomolgus monkey CEACAM5). In some embodiments, the binding affinity K of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention to CEACAM5 (e.g., human CEACAM5) is [not specified]. D The values ​​are less than or equal to approximately 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or 2 nM, or greater than approximately 0.5 nM or 1 nM, or between these values. In some embodiments, the antibody binding affinity of the present invention is determined by a thin-layer interferometry technique, such as ForteBio Octet.

[0015] In some embodiments, the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment can effectively bind to CEACAM5, such as human CEACAM5.

[0016] In some embodiments, the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment exhibits good endocytic activity on CEACAM5-positive cells, such as CEACAM5-positive tumor cells.

[0017] The present invention also provides an immunoconjugate comprising an antibody targeting CEACAM5 (e.g., the antibody specifically binding to CEACAM5 of the present invention or its antigen-binding fragment) and other payloads. In some aspects, the immunoconjugate is an antibody-drug conjugate (ADC).

[0018] Therefore, the present invention provides antibodies targeting CEACAM5 or antigen-binding fragments thereof and antibody-drug conjugates (ADCs) targeting CEACAM5, such as monotoxin or ditoxin ADC molecules. Attached Figure Description

[0019] Figure 1 shows the RP-HPLC and SEC-HPLC chromatograms of the ADC of the present invention.

[0020] Figure 2 shows the cell survival of the ADCs of the present invention in HPAF-II (A), MKN45 (B), HT55 (C), and HPAC (D) cells.

[0021] Figure 3 shows the cell survival of the ADCs of the present invention in MKN45, HT55 and HPAC cells.

[0022] Figure 4 shows the cell survival of the ADCs of the present invention in MKN45, HT55 and HPAC cells.

[0023] Figures 5A and 5B show the antitumor efficacy and body weight change curves of ADCs in the HT55 tumor-bearing mouse model, respectively.

[0024] Figures 6A and 6B show the antitumor efficacy and body weight change curves of ADCs in the HT55 tumor-bearing mouse model, respectively.

[0025] Figures 7A and 7B show the antitumor efficacy and body weight change curves of ADCs in the HT55 tumor-bearing mouse model, respectively.

[0026] Figures 8A and 8B show the antitumor efficacy and body weight change curves of ADCs in the HT55 tumor-bearing mouse model, respectively.

[0027] Detailed description of the invention:

[0028] Invention Details

[0029] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methodologies, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0030] I. Definition

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0033] When used in conjunction with a numerical value, the term "about" or "approximately" means to cover a range of numerical values ​​having a lower limit of 10% less than the specified numerical value and an upper limit of 10% greater than the specified numerical value, preferably covering a range of numerical values ​​having a lower limit of 5%, 4%, 3%, 2% or 1% less than the specified numerical value and an upper limit of 5%, 4%, 3%, 2% or 1% greater than the specified numerical value.

[0034] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0035] As used herein, the terms “comprising” or “including” mean to include the stated 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, they also cover situations consisting of the stated elements, integers, or steps, unless otherwise specified. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0036] As used herein, the term "CEACAM5" refers to carcinoembryonic antigen-related cell adhesion molecule 5. Human CEACAM5 includes, for example, CEACAM5 having the amino acid sequence shown in UniProt database accession number P06731. In some embodiments, CEACAM5 is human CEACAM5. In some embodiments, CEACAM5 is cynomolgus monkey CEACAM5.

[0037] As used herein, “antibody that binds to CEACAM5” or “anti-CEACAM5 antibody” or “antibody that specifically binds to CEACAM5” means an antibody that can bind to CEACAM5 with appropriate affinity. In some respects, the anti-CEACAM5 antibody described herein also encompasses multispecific antibodies that simultaneously and specifically bind to CEACAM5 and other target antigens, such as bispecific antibodies.

[0038] In some respects, the anti-CEACAM5 antibodies described herein also encompass multispecific antibodies that simultaneously and specifically bind to other target antigens of CEACAM5, such as bispecific antibodies.

[0039] Effector cells include effector T cells (T lymphocytes), such as CD4+ T cells, CD8+ T cells, Th1, Th2, and regulatory T cells (Tregs). Effector cells may also include natural killer cells, macrophages, granulocytes, plasma cells, or B cells (lymphocytes).

[0040] General information about the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0041] For polypeptide sequences, "conservative alteration" includes substitutions, deletions, or additions to the polypeptide sequence that do not substantially change the desired functional activity of the polypeptide sequence. For example, a conserved substitution often results in a particular amino acid being replaced by a chemically similar amino acid. Tables providing conserved substitutions of functionally similar amino acids are well known in the art. The following lists eight groups of amino acids containing mutually conserved substitutions: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conserved sequence alteration" is used to refer to amino acid modifications that do not significantly affect or alter the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, conserved modified variants maintain at least 80%, 85%, 90%, 95%, 98%, 99%, or higher binding affinity to the target antigen relative to the parent antibody or binding protein, such as 100-110% or higher.

[0042] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0043] When it is mentioned that "Ab is derived from antibody", it means that the binding domain of the Ab is or is derived from the binding domain of the antibody's specific binding antigen. For example, the specific binding antigen fragment of the Ab, such as Fab, is or is derived from the corresponding fragment of the antibody, such as Fab. Or the heavy chain variable region and / or light chain variable region of the antigen binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody. Or one, two, three, four, five or six CDRs of the antigen binding region are the CDRs of the antibody.

[0044] In this document, the term "antibody" encompasses any molecule that specifically recognizes and binds to an antigen. This term covers a wide range of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), chimeric or humanized antibodies, full-length antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0045] The terms "full-length antibody" or "complete antibody" are used interchangeably herein and refer to antibody molecules that have the molecular structure of natural immunoglobulins. In the case of a conventional four-chain IgG antibody, a full-length antibody consists of two heavy chains (H) and two light chains (L) linked together by disulfide bonds. In the case of a heavy chain antibody that has only heavy chains and lacks light chains, a full-length antibody consists of two heavy chains (H) linked together by disulfide bonds. For a conventional four-chain IgG antibody, the heavy chain of a full-length antibody typically consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region, wherein the heavy chain constant region contains at least three domains CH1, CH2, and CH3. The light chain of a full-length antibody consists of a light chain variable region (abbreviated as VL in this document) 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 consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region does not directly participate in the binding of antibody to antigen, but exhibits a variety of effector functions. In some embodiments, the antibody heavy chain constant region HC of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, such as the heavy chain constant region of IgG1.

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

[0047] The term "antigen-binding fragment" in antibody refers to a molecule distinct from a full-length antibody. It contains a portion of the full-length antibody but can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using 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, diabody, single-domain antibody (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. Furthermore, digestion of a complete antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, 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 Fab' monomers. Fab' monomers are essentially Fab fragments with hinge regions. Fv fragments consist of the VL and VH domains of the antibody's single arm. The two domains VL and VH of the Fv fragment can be encoded by independent genes, but they can also be produced as a single protein chain by using a recombinant approach, connecting the two domains with a synthetic linker peptide, and pairing the VL and VH regions in the single protein chain to form a single-chain Fv (scFv).

[0048] The term "Fab fragment" or "Fab" is used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains containing 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). One polypeptide chain contains VH and a constant region selected from CH1 and CL from its N-terminus to its C-terminus, while the other polypeptide chain contains VL and another constant region selected from CL and CH1 from its N-terminus to its C-terminus. The VH and VL domains pair to form an antigen-binding site. In this document, the Fab polypeptide chain containing the heavy chain constant region CH1 is also referred to as the "Fab heavy chain," and correspondingly, the Fab polypeptide chain containing the light chain constant region CL is also referred to as the "Fab light chain."

[0049] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those 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 many known antibody CDR assignment schemes, including, for example: Chothia (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)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (available at imgt.cines.fr / on the World Wide Web), and the North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing manner. A CDR may also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention).

[0050] In one embodiment, HCDR1 in the anti-CEACAM5 antibody of the present invention is determined according to the union of the Kabat and Chothia schemes (denoted as Kabat & Chothia), and HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to Kabat rules:

[0051] The following is a calculation of sequence identity between sequences.

[0052] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0053] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody while exhibiting low immunogenicity when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the remaining portion of the antibody with its corresponding human portion (i.e., replacing the non-binding portion of the variable region with the corresponding portion of the human antibody).

[0054] As used herein, the terms “anti,” “binding,” or “specific binding” mean that the binding interaction is selective for the target or antigen and can be distinguished from unwanted or nonspecific interactions. The ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as radioimmunoassay (RIA), thin-layer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0055] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can usually be determined by the dissociation constant (K). D This indicates that the dissociation constant is the dissociation rate constant and the association rate constant (Kdissociation and Kassociation, respectively). dis and K onThe ratio of affinity to kinetic binding affinity. Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay described in this paper.

[0056] As used herein, the term "multispecific antibody" refers to an antibody having at least two antigen-binding regions, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. The term "bispecific antibody" refers to an antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to one antigen or epitope and the second antigen-binding region binds to another antigen or another epitope.

[0057] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, an immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, amino acid residues in the Fc region or heavy chain constant region are numbered 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 Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Two Fc regions can dimerize to form a dimer Fc, and two distinct Fc regions can heterodimerize to form a heterodimeric Fc. In this document, 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 immunoglobulins, nor the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region. In one embodiment, the human IgG heavy chain Fc region extends from Asp221, Cys226, or Asp231 to the carboxyl terminus of the heavy chain. In one embodiment, the Fc region is derived from a human Fc region. In one embodiment, the Fc region comprises all or part of the human constant region. The antibody Fc region directly participates in complement activation, C1q binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region belongs to the human IgG4 subclass. In one embodiment, the Fc region belongs to the human IgG1 subclass. In one embodiment, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4.

[0058] The term "CH1 region" refers to the portion of the antibody heavy chain polypeptide that extends from EU position 118 to EU position 220 (EU numbering system).

[0059] As used herein, when referring to amino acid positions in domains other than the variable region of an antibody (e.g., constant regions, such as the Fc region), the numbering follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. When a position number and / or amino acid residue is assigned to a particular antibody isotype, it is intended to be applicable to the corresponding position and / or amino acid residue in any other antibody isotype, as is known to those skilled in the art.

[0060] General information about the light and heavy chains of human immunoglobulins is given in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0061] The term "amino acid substitution" or "amino acid mutation" refers to replacing at least one amino acid residue in a predetermined parental amino acid sequence with a different "substituted" amino acid residue. This substituted residue or residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and are selected from: 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).

[0062] The position of the amino acid to be mutated to cysteine ​​is generally indicated by "chain type, mutation position". In this article, unless otherwise specified, LLC represents the lambda light chain, LC represents the kappa light chain, and HC represents the heavy chain. Therefore, "LLC160C" means that the amino acid at position 160 of EU in the lambda light chain is replaced by cysteine ​​(C).

[0063] When referring to the position of an amino acid in the heavy chain in this invention, unless otherwise specified, it refers to the amino acid position based on the IgG1 heavy chain number. That is, it encompasses the amino acid position based on the IgG1 heavy chain number, as well as the corresponding amino acid positions on other heavy chains. For example, when referring to HC239, it encompasses the 239th amino acid of the IgG1 heavy chain under the EU number, and also the position of the 239th amino acid of that IgG1 in other IgG isoforms.

[0064] "Sequence identity" is defined as the percentage of identical residues in an amino acid sequence variant after aligning sequences and, where necessary, introducing gaps to achieve maximum percentage sequence identity.

[0065] "Parental protein (e.g., parental antibody, parental constant region, or parental Fc region)" refers to a protein containing an amino acid sequence in which one or more amino acid residues are to be replaced by one or more other amino acid residues, such as cysteine ​​residues. Parental proteins can contain natural or wild-type sequences. Parental proteins may have existing amino acid sequence modifications (such as additions, deletions, and / or substitutions) relative to other natural, wild-type, or modified forms of proteins. Parental antibodies can target antigens of interest, such as biologically important peptides.

[0066] A hyphen ("-") not between two letters or symbols indicates the linking site of a substituent. For example, -OR4 indicates that the group is linked to the rest of the molecule via an oxygen atom. The "-" may be omitted when the linking site of the substituent is obvious to those skilled in the art (e.g., for halogens, CN, OH, etc.).

[0067] The term "halogen" or "halogenated" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I), preferably fluorine, chlorine or bromine.

[0068] The term "alkyl" alone or as part of other groups refers to a fully saturated straight-chain or branched hydrocarbon group consisting of carbon and hydrogen atoms. Preferably, the alkyl group has 1-6 carbon atoms (C6H ... 1-6 Alkyl groups, 1-4 carbon atoms (C 1-4 Alkyl group or 1-3 carbon atoms (C 1-3 Alkyl groups. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (Pr) (including n-propyl and isopropyl), butyl (Bu) (including n-butyl, isobutyl, sec-butyl and tert-butyl), pentyl (including n-pentyl, isopentyl, neopentyl, etc.), hexyl, heptyl, octyl, etc.

[0069] The term "alkylene" refers to an alkyl group as defined above, but which is divalent, i.e. has two single bonds attached to two other groups. Non-limiting examples of alkylene include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH(-CH2CH3)-, or -CH2CH(-CH3)-.

[0070] The term "alkenyl" alone or as part of other groups refers to a straight-chain or branched hydrocarbon group consisting of carbon and hydrogen atoms and containing at least one double bond. Preferably, the alkenyl group has 2-6 carbon atoms (C6H ... 2-6 alkenyl), 2-4 carbon atoms (C 2-4 alkenyl) or 2-3 carbon atoms (C 2-3 Alkenyl). Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, allyl, butenyl, isobutenyl, pentenyl, isopentenyl, and hexenyl.

[0071] The term "alkynyl" alone or as part of other groups refers to a straight-chain or branched hydrocarbon group consisting of carbon and hydrogen atoms and containing at least one triple bond. Preferably, the alkynyl group has 2-6 carbon atoms (C6H ... 2-6 acetylinyl group), 2-4 carbon atoms (C 2-4 (alkynyl group) or 2-3 carbon atoms (C 2-3 (Alynyl group). Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, propynyl, butynyl, isobutynyl, penynyl, isopentenynyl, and hexynyl.

[0072] The terms "alkoxy" and "alkyl-O-" are used interchangeably to refer to an alkyl group as defined above, linked by an oxygen atom. Preferably, the alkoxy group has 1-6 carbon atoms (C... 1-6 alkoxy group), 1-4 carbon atoms (C 1-4 alkoxy group or 1-3 carbon atoms (C 1-3 Alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, etc.), pentoxy (including n-pentoxy, isopentoxy, neopentoxy, etc.), hexoxy, heptoxy, octoxy, etc.

[0073] The term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) halogens. It will be understood that when there is more than one halogen substituent, the halogen substituents may be the same or different, and may be located on the same or different carbon atoms. Preferably, the haloalkyl group is C10. 1-6 Haloalkyl, C 1-4 Halogenated alkyl or C 1-3Haloalkyl groups. Examples of haloalkyl groups include, but are not limited to, fluoromethyl, chloromethyl, difluoromethyl, dichloromethyl, fluorochloromethyl, trifluoromethyl, trichloromethyl, dichlorofluoromethyl, difluoroethyl, trifluoroethyl, trichloroethyl, difluorochloroethyl, difluoropropyl, and trifluoropropyl.

[0074] The term “haloalkenyl” refers to an alkenyl group as defined herein, which is substituted with one or more halogen groups as defined herein. The term “haloynyl” refers to an ynyl group as defined herein, which is substituted with one or more halogen groups as defined herein. The meaning of “halogenated” as defined for “haloalkyl” may apply to both “haloalkenyl” and “haloynyl”.

[0075] The term "heteroaryl" refers to a monocyclic or bicyclic group having one or more, preferably 1, 2, 3, 4, 5, or 6 heteroatoms independently selected from N, O, or S, with the remaining ring members being carbon, wherein at least one ring is aromatic, and the other rings (if present) may be aromatic or non-aromatic. Any N and S heteroatoms of the heteroaryl group may optionally be oxidized (e.g., in NO, SO, SO2) and said any N heteroatomium may optionally be quaternized (e.g., in [NR]). + Cl - [NR] + OH - (In Chinese). The heteroaryl group is preferably a 5-8 membered heteroaryl group (e.g., monocyclic, nitrogen-containing). Representative examples of heteroaryl groups include, but are not limited to: pyrrole, furanyl, thiophene, pyrazolyl, imidazolyl, oxazolyl, isothiazolyl, thiazolyl, isothiazolyl, triazolyl, pyridinyl, pyrazinyl, pyranyl, thiaranyl, oxazinyl, oxadiazinyl, indole, isoyindole, azaindole (e.g., 7-azaindole, 6-azaindole, 5-azaindole, 4-azaindole), etc. The heteroaryl group can be attached to the rest of the compound via a carbon atom or a heteroatom, provided it is chemically feasible.

[0076] The term "subunit" or "sub...unit" refers to a divalent group derived by removing two hydrogen atoms from a molecule.

[0077] The term "OH" represents a hydroxyl group.

[0078] The term "SH" stands for thiol group.

[0079] The term "NH2" represents amino.

[0080] The term "-CO-" or "-C(=O)-" represents a carbonyl group.

[0081] The term "-SO-" represents a sulfinyl group.

[0082] The term "-SO2-" represents a sulfonyl group.

[0083] The term "-COOH" represents a carboxyl group.

[0084] The term "NO2" stands for nitro.

[0085] The term "leaving group" refers to an atom or functional group that readily detaches from a molecule during a chemical reaction. Examples include, but are not limited to: halogens; such as F, Cl, or Br; sulfonyl groups, such as methanesulfonyl or p-toluenesulfonyl; sulfonyloxy groups, such as alkylsulfonyloxy (e.g., methanesulfonyloxy), trifluoromethylsulfonyloxy, or arylsulfonyloxy (e.g., p-toluenesulfonyloxy); tertiary amine groups (e.g., Me3N). + or Et3N + ); or diazonium salts.

[0086] The term "polyol group" refers to an alkyl group as defined above containing a plurality of (e.g., 2-10, e.g., 3, 4, 5, 6, 7, or 8) hydroxyl groups, optionally containing one or more (e.g., 2, 3, or 4) other groups (e.g., amino, carbonyl). Non-limiting examples of "polyol group" include, for example... Preferred

[0087] The term "amino acid" refers to naturally occurring and synthetic amino acids. Amino acids can be L or D isomers. The common amino acids discussed in this article are written according to their usual usage. See, for example, Immunology-A Synthesis (2 nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this disclosure, amino acids are generally represented by single-letter and three-letter abbreviations well known in the art. For example, amino acids may be selected from phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), and glutamine (Gln).

[0088] The term "pentose," also known as a five-carbon sugar, refers to a monosaccharide containing five carbon atoms. Pentoses include D- and / or L-pentoses. Examples of pentoses include, but are not limited to, xylose, arabinose, ribose, and deoxyribose.

[0089] The term "hexose," also known as a six-carbon sugar, refers to a monosaccharide containing six carbon atoms. Hexoses include D- and / or L-pentoses. Examples of hexoses include, but are not limited to, glucose, galactose, mannose, and fructose.

[0090] The term "penturonic acid" refers to compounds formed by oxidizing the primary hydroxyl group of a pentose sugar as defined above to a carboxyl group. Examples of penturonic acids include, but are not limited to, xyuronic acid and arabinuronic acid.

[0091] The term "hexuronic acid" refers to compounds formed by oxidizing the primary hydroxyl group of a hexose as defined above to a carboxyl group. Examples of hexuronic acids include, but are not limited to, glucuronic acid, galacturonic acid, and mannuronic acid.

[0092] The term "disaccharide" refers to a disaccharide formed by two molecules of monosaccharides (such as pentoses and / or hexoses as defined above) through a glycosidic bond. Examples of disaccharides include, but are not limited to, sucrose, lactose, and maltose.

[0093] The term "trisaccharide" refers to a sugar formed by three monosaccharide molecules (such as pentoses and / or hexoses as defined above) through glycosidic bonds.

[0094] The term "glycosidic bond" refers to the chemical bond that connects the sugar and the other hydroxyl-containing compound (such as an alcohol, phenol, or another sugar) to form an acetal derivative through dehydration condensation of the hydroxyl group on the hemiacetal structure of a sugar.

[0095] It should be understood that when referring to pentose, hexose, penturonic acid, hexuronic acid, disaccharide, and trisaccharide as a group in a structure, it refers to a group formed by the dehydration condensation reaction between the hydroxyl group (e.g., at position 1, 2, 3, 4, 5, or 6, preferably position 1) of the corresponding sugar or uronic acid and the hydroxyl group or other group of another molecule. It can also be referred to as pentose, hexose, etc. Preferably, the sugar or uronic acid is covalently linked to other structures or partially via a glycosidic bond.

[0096] The expressions “optional,” “optional,” or “optionally” mean that the event described below may or may not occur, and the expression includes both the occurrence and non-occurrence of the event. For example, “optionally substituted by…” includes both the case where the event is not substituted and the case where it is substituted. “Optional substituent” indicates that the substituent may or may not be present.

[0097] When any variable appears more than once in a structural formula, it is defined independently each time it appears. For example, the expression "optionally substituted with one or more substituents independently selected from..." means substitution by one or more independently selected substituents, which can be the same or different. Combinations of substituents and / or variables are permitted, as long as a stable compound is produced.

[0098] The term "substance of the invention" includes the antibody or antigen-binding fragment thereof or its immunoconjugate such as antibody-drug conjugate or its salt or ester, solvate (e.g., hydrate), tautomer, stereoisomer, prodrug, metabolite, or isotope label (e.g., deuterated or tritated). In some embodiments, "substance of the invention" specifically refers to the antibody or antigen-binding fragment thereof or its immunoconjugate such as antibody-drug conjugate, or its salt or ester, solvate (e.g., hydrate), tautomer, stereoisomer, prodrug, metabolite, or isotope label (e.g., deuterated or tritated).

[0099] In this document, the designation of a general formula includes the general formula itself, any subformulation thereof, and any embodiment falling within the scope of the general formula. For example, the designation of formula (II) includes formulas (IIa), (II-1) to (II-11), and any embodiment falling within the scope of formula (II).

[0100] The term "medicinal" refers to a substance or composition that, when administered to animals such as humans, does not produce adverse reactions, allergic reactions, or other unwanted reactions.

[0101] The term "medicinal salt" refers to those salts that retain the biological effectiveness of the substances of the present invention and are not biologically or otherwise undesirable. The substances of the present invention can be in the form of salts, preferably pharmaceutically acceptable salts, including acid addition salts and base addition salts. Acid addition salts can be formed from inorganic or organic acids. Inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, and perchloric acid. Organic acids include formic acid, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, glutaric acid, adipic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, dihydroxynaphthaleneic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, ethanedisulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, xylenesulfonic acid, hydroxyethanesulfonic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, salicylic acid, oleic acid, nicotinic acid, palmitic acid, stearic acid, furoic acid, hippuric acid, orotic acid, and pyruvic acid. Base addition salts can be formed from 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 by reacting with an organic base containing an N-group. Salts can be synthesized from parent compounds using conventional methods.

[0102] Pharmaceutical salts are preferred. However, other salts may also be useful, for example, in separation or purification steps, and can be used during preparation, and are therefore included within the scope of this invention.

[0103] As used herein, the term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules may also exist as geometric isomers (cis / trans).

[0104] The term "tautomer" refers to structural isomers with different energies that can interconvert through low energy barriers. For example, proton tautomers (also known as proton shift tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via the rearrangement of some bonding electrons.

[0105] In this invention, solid lines, solid wedges, or imaginary wedges can be used to depict the valence bonds in the substances of this invention. Solid lines connecting to asymmetric carbon atoms are intended to indicate all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) at that carbon atom. Solid or imaginary wedges connecting to asymmetric carbon atoms are intended to indicate the presence of the indicated stereoisomers. In racemic mixtures, solid and imaginary wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise specified, the substances of this invention can exist as stereoisomers, including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-blocking isomers, and mixtures thereof. The substances of this invention can exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0106] This invention also includes prodrugs of the substances of the invention. The term "prodrug" refers to a chemically modified active or inactive compound that, upon administration to an individual, undergoes physiological processes in vivo (e.g., hydrolysis, metabolism, etc.) to be converted into the substances of the invention. Therefore, in this invention, the term "administration" includes administering a prodrug of the substances of the invention to treat various diseases or conditions, wherein the prodrug is converted into the substances of the invention in vivo. Techniques for manufacturing and using prodrugs are well known to those skilled in the art.

[0107] This invention also includes all pharmaceutically acceptable isotopic labels that are identical to the substances of this invention, but in which one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number than the dominant atomic mass or mass number found in nature. Examples of isotopes suitable for incorporation into the substances of this invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H); carbon isotopes (e.g., H); 11 C 13 C and14 C); isotopes of chlorine (e.g. 36 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); isotopes of phosphorus (e.g., O); phosphorus isotopes ... 32 P); and isotopes of sulfur (e.g. 35 S). Designations of compounds of the present invention include their isotopic labels. Similarly, designations of elements or groups also include their isotopes or isotopic labels. For example, the designation "H" or "hydrogen" includes H. 1 H 2 and H 3 .

[0108] This invention also includes metabolites of the substances of this invention, i.e., substances formed in the body after administration of the substances of this invention. These can be produced by, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, etc. Therefore, this invention includes metabolites of the substances of this invention, including compounds obtained by methods that expose the substances of this invention to mammals for a time sufficient to produce their metabolites.

[0109] Some of the substances of this invention can exist in non-solventized and solvated forms, including hydrates. The term "solvent" refers to a complex of solvent molecules with a substance of this invention. Examples of solvents that form solvates include water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" refers to a complex in which the solvent molecules are water. Methods of solvation are well known in the art.

[0110] As used herein, the term "linker" refers to a linker that can be used to covalently link a small molecule toxin or payload to an antibody or its antigen-binding fragment (Ab), and in this invention may be represented as L. a1 -L a2 -L a3 -L a4 -Partial, L b Parts, etc.

[0111] The term "joint unit" refers to a component of a connector (L). a1 It has reactive groups that can form covalent bonds with functional groups of antibodies or antibody fragments. The role of the linker unit is to attach the linker, or the linker as part of a drug linker conjugate, to the antibody or its antigen-binding fragment.

[0112] As used herein, the term "bridging spacer" refers to one or more linker components covalently attached together to form a divalent moiety, wherein the divalent moiety comprises a divalent amino acid or peptide spacer, etc. (e.g., L...). a3 It is attached to a reactive group or a coupling group.

[0113] The term "cleavable linker" refers to a linker that can utilize the differences in conditions between the bloodstream and tumor cells (e.g., pH, protease hydrolysis, or glutathione concentration) or the specific action of antibodies within lysosomes (e.g., lysosomal proteolytic enzymes) to cleave and release the payload, thereby exerting drug activity. Cleavable linkers include pH-sensitive linkers (e.g., hydrazone linkers), glutathione-sensitive linkers (e.g., disulfide linkers), and enzyme-cleavable linkers (e.g., short peptide linkers, β-glucuronic acid linkers, β-galacturonic acid linkers), etc.

[0114] The term "suicide spacer" refers to a spacer that is embedded between a cleavable linker and a small molecule drug, or is itself part of a cleavable linker. It is capable of spontaneous structural rearrangement, releasing the small molecule drug. Examples of suicide spacers include, for example, the p-aminobenzyloxycarbonyl (PABC) or m-aminobenzyloxycarbonyl classes.

[0115] As used herein, the term "antibody-drug conjugate" or "ADC" refers to a substance obtained by conjugating a small molecule drug payload to an antibody or its antigen-binding fragment (Ab, responsible for targeting function and sometimes also having biological activity) via a linker, including its pharmaceutically usable salts or esters, solvates, isotope labels, stereoisomers, tautomers, etc., unless otherwise indicated or the context is clearly contradictory.

[0116] The term “drug:antibody ratio” or “DAR” refers to the ratio of the drug portion (D1 or D2) to the Ab portion conjugated herein.

[0117] In some embodiments described herein, the DAR value of the ADC molecule is determined by m or n in formula (II) or formula (III). For example, DAR can be an integer from 1 to 20, such as 2-20, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0118] In other embodiments, the DAR value is calculated as the average DAR value of a molecular population in the product, i.e., the overall proportion of the drug portion (D1 or D2) coupled to the Ab portion described herein to the Ab portion in the product, as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC). This DAR is referred to herein as the average DAR or the measured DAR. For example, the average DAR can be any value between 0.5 and 20.0. It should be understood that when referring to the average DAR value, the ADC of the present invention refers to a population of ADC molecules or a mixture of ADC molecules containing ADC molecules having the same and / or different DARs. In some embodiments, the ADC of the present invention is in the form of an antibody-drug conjugate comprising one or more of formulas (II) and / or (III) or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled composition thereof.

[0119] The term "individual" or "patient" refers to an animal, preferably a mammal. Examples of individuals include, but are not limited to, primates (e.g., humans and non-human primates such as monkeys), horses, cattle, sheep, cats, dogs, rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual is a person, including children, adolescents, or adults.

[0120] The term "treatment" means (i) treating or preventing a particular disease, symptom, or disorder; (ii) reducing, improving, or eliminating one or more symptoms of a particular disease, symptom, or disorder; and optionally (iii) preventing or delaying the onset of one or more symptoms of a particular disease, symptom, or disorder described herein. In some embodiments, "treatment" means improving at least one bodily parameter, which may not be perceptible to the patient. In other embodiments, "treatment" means regulating a disease or symptom from a physical (e.g., stabilizing perceptible symptoms) or physiological (e.g., stabilizing bodily parameters) or both.

[0121] The term "prevention" refers to the administration of one or more pharmaceutical substances, particularly the compounds of the present invention and / or their pharmaceutically acceptable salts, to an individual with a predisposition to the disease or condition, in order to prevent the individual from contracting the disease.

[0122] The terms “inhibition” and “reduction” refer to the reduction or inhibition of a specific patient, symptom, condition, or disease, or a significant reduction in the baseline activity of a biological activity or process.

[0123] The term "effective dose" refers to the amount of medication required to achieve a desired therapeutic or preventative effect at the necessary dosage and for the required duration. It can be determined by the physician or veterinary practitioner involved and will vary depending on factors such as the compound, the state of the disease being treated, the severity of the disease, the individual's age and relevant health conditions, the route and form of administration, and the judgment of the attending physician or veterinary practitioner. Generally, the "preventive effective dose" will be less than the "therapeutic effective dose."

[0124] The term "preventive effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because the prophylactic dose is administered to the subject before or at an early stage of the disease, the preventive effective dose will be less than the therapeutic effective dose.

[0125] The term "therapeutic effective amount" refers to the amount that, at the required dose and sustained for the required period of time, effectively achieves the desired therapeutic outcome. A therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody or antibody fragment or ADC or composition or combination are less than the beneficial therapeutic effect. Relative to an untreated subject, the "therapeutic effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, and more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0126] The terms "formulation" or "pharmaceutical composition" refer to a composition suitable for administration to animals, preferably mammals (including humans), comprising at least one active ingredient and at least one inactive ingredient, such as a pharmaceutically acceptable excipient. The formulations of this invention can be any formulation applicable in the art, such as tablets, capsules, liquid formulations, lyophilized powder formulations, etc.

[0127] The term "pharmaceutical excipient" refers to components in a pharmaceutical preparation other than the active ingredient, including physiologically compatible fillers, solvents, dispersion media, isotonic agents, and absorption delay agents. Examples of pharmaceutically acceptable excipients include, but are not limited to, binders, disintegrants, lubricants, solvents, dispersion media, buffers, excipients, antioxidants, preservatives, or flavoring agents. For more information on pharmaceutical excipients, please refer to "Handbook of Pharmaceutical Excipients," 8th edition, R.C. Rowe, P.J. Seskey, and S.O. Wen, Pharmaceutical Press, London, Chicago.

[0128] The term "drug combination" refers to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that active ingredients (e.g., (i) antibodies of the present invention or their immunoconjugates such as ADC molecules, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. In some embodiments, the antibodies of the present invention or their immunoconjugates such as ADC molecules and other therapeutic agents used in the drug combination are administered at levels not exceeding those achieved when 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. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in separate formulations, and their formulations may be the same or different.

[0129] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0130] When referring to chemical reactions, “processing,” “contacting,” and “reaction” mean the addition or mixing of two or more reagents under appropriate conditions to produce the shown and / or desired product. It should be understood that the reaction producing the shown and / or desired product may not necessarily originate directly from the combination of the two initially added reagents; that is, one or more intermediates may be present in the mixture that ultimately lead to the formation of the shown and / or desired product.

[0131] 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). In some embodiments, the therapeutic agent is an antitumor compound.

[0132] The term "cytotoxic agent" in this invention refers to a substance that inhibits or prevents cell function and / or causes cell death or damage.

[0133] The terms “toxin,” “drug component,” “payload,” and “antibody-drug conjugate” used herein are used interchangeably to refer to the component of an antibody-drug conjugate or drug-linker conjugate responsible for killing tumor cells. After administration, the ADC undergoes lysis between or within target cells, releasing the toxin or its derivatives, thereby exerting its biological activity. To avoid ambiguity, it should be noted that “drug” does not only refer to “medicines” approved by pharmaceutical regulatory authorities, but should also include any compound with potential biological activity in clinical practice or in research and development and academic studies.

[0134] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which foreign nucleic acids have been introduced, including the progeny of such cells.

[0135] As used herein, the term "label" 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 conjugated or fused reagent. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of probes or antibodies by conjugation (i.e., physical linking) to a detectable substance and indirect labeling of probes or antibodies by reaction with another directly labeled reagent.

[0136] "Isolated" antibodies or other molecules (e.g., ADC molecules) are antibodies or molecules that have been separated from components of their natural environment or the environment in which they are expressed. In some embodiments, the antibody or ADC molecule is purified to a purity of more than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0137] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0138] "Subject / Patient / Individual Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds that are naturally occurring and do not mix with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0139] The term “antitumor” or “antitumor effect” refers to biological effects that can be demonstrated through a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0140] The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth. Cancer can be in its early, middle, or late stages, or it can be metastatic.

[0141] The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. “Tumor” encompasses solid tumors and hematologic malignancies as well as metastatic lesions. The terms “cancer,” “cancerous,” and “tumor” are not mutually exclusive when used in this article.

[0142] II. Anti-CEACAM5 antibody, its encoding nucleic acid, and its preparation

[0143] II-1 anti-CEACAM5 antibody or its antigen-binding fragment

[0144] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (LCDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region and three complementarity-determining regions (LCDRs) from the light chain variable region.

[0145] In some aspects, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH). In some aspects, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises a light chain variable region (VL). In some aspects, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises both a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises three complementarity-determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity-determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0146] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention further comprises an antibody heavy chain constant region. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention further comprises an antibody light chain constant region. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention further comprises both a heavy chain constant region and a light chain constant region.

[0147] In some embodiments, the heavy chain variable region of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention includes:

[0148] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:4; or

[0149] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:4; or

[0150] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any of (i)-(ii), preferably, the amino acid alterations do not occur in the CDR region.

[0151] In some embodiments, the light chain variable region of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention...

[0152] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from SEQ ID NO:8; or

[0153] (ii) Contains or consists of an amino acid sequence selected from or composed of the amino acid sequence shown in SEQ ID NO:8;

[0154] or

[0155] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from any of (i)-(ii), preferably, the amino acid alterations do not occur in the CDR region.

[0156] In some embodiments, the three complementarity-determining regions (HCDRs) of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention, HCDR1, HCDR2, and HCDR3, derived from the heavy chain variable region, are selected from...

[0157] (i) the three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:4; or

[0158] (i) A sequence that, relative to any one of (i), contains at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions.

[0159] The HCDR can be determined according to any scheme for determining the CDR, such as the schemes of Kabat, AbM, Chothia, Contact, or IMGT or combinations thereof.

[0160] For example, HCDR1 is determined by the union of the Kabat and Chothia schemes, while HCDR2 and HCDR3 are determined by the Kabat scheme.

[0161] In some embodiments, the three complementarity-determining regions (LCDRs) of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention, LCDR1, LCDR2, and LCDR3, derived from the light chain variable region, are selected from...

[0162] (i) the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:8, or

[0163] (i) A sequence that, relative to any one of (i), contains at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions.

[0164] The LCDR can be determined according to any scheme for determining the CDR, such as Kabat, AbM, Chothia, Contact, or IMGT or a combination thereof.

[0165] For example, LCDR1, LCDR2 and LCDR3 are determined according to the Kabat scheme.

[0166] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises:

[0167] The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:4, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:8;

[0168] The HCDR and LCDR can be determined according to any scheme for determining the CDR, such as the schemes of Kabat, AbM, Chothia, Contact, or IMGT or combinations thereof.

[0169] For example, HCDR1 is determined by the union of the Kabat and Chothia schemes, HCDR2 and HCDR3 are determined by the Kabat scheme, and LCDR1, LCDR2 and LCDR3 are determined by the Kabat scheme.

[0170] In some embodiments, HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:1, or HCDR1 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:1.

[0171] In some embodiments, HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:2, or HCDR2 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:2.

[0172] In some embodiments, HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:3, or comprises an amino acid sequence having one, two, or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:3.

[0173] In some embodiments, LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:5, or LCDR1 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:5.

[0174] In some embodiments, LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:6, or LCDR2 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:6.

[0175] In some embodiments, LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:7, or LCDR3 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:7.

[0176] In some specific embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), a third heavy chain complementarity-determining region (HCDR3), and a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3), wherein...

[0177] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or are composed of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7.

[0178] In some embodiments, the VH of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein;

[0179] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or are composed of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7.

[0180] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises:

[0181] VH comprising the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence.

[0182] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:8.

[0183] In one embodiment of the invention, the amino acid alterations described herein include substitutions, insertions, or deletions of amino acids. In some embodiments, the amino acid alterations described herein are conserved amino acid alterations. Preferably, the amino acid alterations described herein are amino acid substitutions, preferably conserved substitutions. In a preferred embodiment, the amino acid alterations described herein occur in regions outside the CDR (e.g., in the FR). More preferably, the amino acid alterations described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region.

[0184] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises an antibody heavy chain. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises an antibody light chain. In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises a heavy chain and a light chain. In some embodiments, the heavy chain of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises, or is composed of, the heavy chain variable region and the heavy chain constant region described herein. In some embodiments, the light chain of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention comprises, or is composed of, the light chain variable region and the light chain constant region described herein. In some embodiments, the antibody of the present invention comprises two of the described heavy chains and two of the described light chains, or is composed of, two of the described heavy chains and two of the described light chains. In some embodiments, the antibody of the present invention comprises, or is composed of, two identical described heavy chains and two identical described light chains.

[0185] In some embodiments, the heavy chain constant region of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention is or is derived from the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, such as the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the heavy chain constant region of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention is the (human) IgG heavy chain constant region, such as the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the heavy chain constant region is the human IgG1 heavy chain constant region, for example, comprising the amino acid sequence shown in SEQ ID NO:20 or SEQ ID NO:21, or having 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:20 or SEQ ID NO:21, and not containing an amino acid sequence with a cysteine ​​mutation.

[0186] In some embodiments, the light chain constant region of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention is or is derived from the lambda or Kappa light chain constant region, preferably the Kappa light chain constant region, such as the human lambda or Kappa light chain constant region. In some embodiments, the light chain constant region of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention is the (human) Lambda light chain constant region. In some embodiments, the Lambda light chain constant region comprises the amino acid sequence shown in SEQ ID NO:28 or 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:28 and does not contain an amino acid sequence with a cysteine ​​mutation. In some embodiments, the light chain constant region of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention is the (human) Kappa light chain constant region. In some embodiments, the Kappa light chain constant region comprises the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having 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:30.

[0187] In one aspect, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention is modified with cysteine ​​to mutate one or more amino acids in its heavy or light chain to cysteine. Based on the fact that the thiol group on the cysteine ​​can undergo a nucleophilic reaction with a toxin small molecule having a maleimide linker, a site-specifically coupled ADC molecule is prepared by coupling a toxin small molecule to the cysteine.

[0188] In one aspect, the antibodies or antigen-binding fragments of the present invention have cysteine ​​mutations at relatively hidden sites in the constant region, thereby providing better stability and / or hydrophilicity to the ADCs containing them. As defined herein, a "cysteine ​​mutation" refers to the substitution of a non-cysteine ​​amino acid for a cysteine ​​in a protein.

[0189] In some embodiments, the antibody or antigen-binding fragment of the present invention is an antibody or antigen-binding fragment having a cysteine ​​mutation in a light chain constant region, wherein the antibody or antigen-binding fragment comprises one or two Lambda light chain constant regions and has a cysteine ​​substitution (LLC160C or LLC160) at position 160 (EU number) of the Lambda light chain constant region.

[0190] In some embodiments, the antibody or antigen-binding fragment of the present invention is an antibody or antigen-binding fragment having a cysteine ​​mutation in the heavy chain constant region, wherein the antibody or antigen-binding fragment comprises one or two heavy chain constant regions and has a cysteine ​​substitution (HC239C) at position 239 (EU number) of the heavy chain constant region.

[0191] In some embodiments, the light chain constant region of the antibody or antigen-binding fragment of the present invention has a cysteine ​​mutation at position 160 (Eu number) relative to the wild-type Lambda light chain, such as the amino acid sequence shown in SEQ ID NO:28.

[0192] In some embodiments, the constant region of the Lamda light chain with a cysteine ​​mutation at position 160 contains the amino acid sequence shown in SEQ ID NO:29 or 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:29 and contains the amino acid sequence VKAGVCTTTPS (SEQ ID NO:34).

[0193] In some embodiments, the heavy chain constant region of the antibody or its antigen-binding fragment of the present invention has a cysteine ​​mutation at position 239 (Eu number) relative to the wild-type human IgG1 heavy chain constant region, such as the amino acid sequence shown in SEQ ID NO:20 or SEQ ID NO:21.

[0194] In some embodiments, the heavy chain constant region with a cysteine ​​mutation at position 239 contains the amino acid sequence shown in SEQ ID NO:22 or 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:22 and contains the amino acid sequence GGPCVFLFP (SEQ ID NO:35).

[0195] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises...

[0196] (i) One or both of the Lamda light chain constant regions having a cysteine ​​mutation at position 160; or

[0197] (ii) One or two heavy chain constant regions with a cysteine ​​mutation at position 239.

[0198] In some specific embodiments, the antibody or its antigen-binding fragment comprises two heavy chain constant regions and two light chain constant regions, wherein

[0199] (i) One heavy chain constant region contains a cysteine ​​mutation at position 239, and another heavy chain constant region does not contain a cysteine ​​mutation at position 239 (or another heavy chain constant region does not contain a cysteine ​​mutation), and two light chain constant regions do not contain a cysteine ​​mutation (e.g., the light chain constant region is a Kappa light chain constant region).

[0200] (ii) The two light chain constant regions are both Lambda light chains, and one light chain constant region contains a cysteine ​​mutation at position 160, while the other light chain constant region does not contain a cysteine ​​mutation at position 160; or

[0201] (iii) The two light chain constant regions are Lambda light chains, and each of the two light chain constant regions contains a cysteine ​​mutation at position 160.

[0202] In some embodiments, in an ADC molecule containing the antibody or antigen-binding fragment of the present invention, the cysteine ​​mutation causes the antibody or antigen-binding fragment to be coupled to a linker via the mutated cysteine, for example, to achieve site-directed coupling of a toxin.

[0203] In some embodiments, the heavy chain constant region of the antibody or its antigen-binding fragment of the present invention comprises or is composed of CH1 and Fc regions.

[0204] In some embodiments, CH1 is or is derived from IgG1, for example, comprising the amino acid sequence shown in SEQ ID NO:23 or having 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:23 and not containing an amino acid sequence with a cysteine ​​mutation.

[0205] In some embodiments, the Fc region is or is derived from (human) IgG Fc, such as IgG1, IgG2, IgG3, or IgG4 Fc. In some embodiments, the Fc region is the Fc region of human IgG1, for example, containing the amino acid sequence shown in SEQ ID NO:24 or SEQ ID NO:25, or having 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:24 or SEQ ID NO:25, and not containing an amino acid sequence with a cysteine ​​mutation. In some embodiments, the Fc region is an Fc region with a cysteine ​​mutation at position 239. In some embodiments, the Fc region with a cysteine ​​mutation at position 239 contains the amino acid sequence shown in SEQ ID NO:26 or 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:26 and contains the amino acid sequence GGPCVFLFP (SEQ ID NO:35).

[0206] In some embodiments, the antibody or antigen-binding fragment of the present invention may also contain modifications in the heavy chain constant region or the Fc region contained therein that alter the binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region contains a mutation that reduces binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in the present invention has a mutation that reduces binding to the Fcγ receptor, such as the L234A / L235A mutation. In still some preferred embodiments, the Fc fragment may have a mutation that results in an increased serum half-life, such as a mutation that improves the binding of the Fc fragment to FcRn. In some embodiments, the Fc region having a cysteine ​​mutation at position 239 and a mutation that reduces binding to the Fcγ receptor comprises the amino acid sequence shown in SEQ ID NO:27 or 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:27 and comprises the amino acid sequence GGPCVFLFP (SEQ ID NO:35) and the L234A / L235A mutation.

[0207] In some embodiments, when the antibody or its antigen-binding fragment of the present invention comprises two different light chains, or when the antibody or its antigen-binding fragment of the present invention comprises two different heavy chains, the Fc region may contain mutations that facilitate heterodimerization between the first Fc region and the second Fc region. In one embodiment, mutations are introduced into the CH3 regions of the two Fc regions. Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 regions of the first Fc region and the CH3 regions of the second Fc region are engineered in a complementary manner such that each CH3 region (or the heavy chain containing it) can no longer homodimerize with itself but is forced to heterodimerize with other complementary engineered CH3 regions (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). Preferably, based on Knob-in-Hole technology, corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region. This technique is illustrated in, 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). In one particular embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knob mutation); while in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (numbered according to EU index). In some embodiments, the knob mutation and hole mutation in the Fc region may also include substitution with a cysteine ​​residue, thereby obtaining a non-natural disulfide bond connection. In some implementations, the serine residue at position 354 in one Fc region is replaced with a cysteine ​​residue (S354C), and in another Fc region, the tyrosine residue at position 349 is replaced with a cysteine ​​residue (Y349C) (numbering in accordance with EU index).Therefore, in yet another embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine ​​residue (S354C) or the glutamate residue at position 356 is replaced with a cysteine ​​residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine ​​residue) (knob mutation); while in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the EU index), optionally the tyrosine residue at position 349 is replaced with a cysteine ​​residue (Y349C) (numbering according to the EU index) (hole mutation). In one specific implementation, one Fc region contains the amino acid substitution T366W (knob mutation), and the other Fc region contains the amino acid substitutions T366S, L368A, and Y407V (numbered according to the EU index) (hole mutation). In another specific implementation, one Fc region contains the amino acid substitutions S354C and T366W (knob mutation), and the other Fc region contains the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbered according to the EU index) (hole mutation).

[0208] Therefore, in one specific embodiment, the antibody or its antigen-binding fragment of the present invention comprises two heterodimerized Fc regions, one Fc region polypeptide comprising the knob mutations S354C and T366W (EU number), and the other Fc region polypeptide comprising the hole mutations Y349C, T366S, L368A, and Y407V (EU number). Optionally, one or both of the Fc regions further comprise a 239C substitution. Optionally, one or both of the Fc regions further comprise mutations that reduce binding to the Fcγ receptor, such as L234A / L235A (EU number).

[0209] Therefore, in a specific embodiment, the antibody or antigen-binding fragment of the present invention, for example when the two heavy chains are different or the two light chains are different, comprises two Fc region heterodimerizations, one of which is an Fc region polypeptide containing or consisting of the amino acid sequence shown in SEQ ID NO:31 or 33, and the other is an Fc region polypeptide containing or consisting of the amino acid sequence shown in SEQ ID NO:32.

[0210] Therefore, in one specific embodiment, the antibody or antigen-binding fragment of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:31 or 33, and the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:32.

[0211] Therefore, in some specific embodiments, the antibody or antigen-binding fragment of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:31 and comprises the mutations S354C and T366W, and optionally the L234A / L235A mutation, and the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:32 and comprises the mutations Y349C, T366S, L368A, and Y407V, and optionally the L234A / L235A mutation.

[0212] Therefore, in some specific embodiments, the antibody or antigen-binding fragment of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:33 and comprises mutations 239C, S354C, and T366W, and optionally L234A / L235A mutations, and the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:32 and comprises mutations Y349C, T366S, L368A, and Y407V, and optionally L234A / L235A mutations.

[0213] The antibodies or antigen-binding fragments thereof of the present invention may be any form of antibody or antigen-binding fragment known in the art, such as monoclonal, chimeric, humanized, fully human, bispecific, or multispecific antibodies or antibody fragments thereof.

[0214] In some embodiments, the heavy chain of the antibody of the present invention comprises

[0215] The amino acid sequence comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 9, 10, 13, or 15;

[0216] Contains or consists of an amino acid sequence selected from or composed of said amino acid sequence; or

[0217] The amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 9, 10, 13, or 15.

[0218] In some embodiments, the light chain of the antibody of the present invention comprises

[0219] The amino acid sequence comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 11, 12, or 14;

[0220] Contains or consists of an amino acid sequence selected from or composed of said amino acid sequence; or

[0221] The amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 11, 12, or 14.

[0222] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises heavy chain 1, heavy chain 2, light chain 1, and light chain 2, wherein...

[0223] Heavy chain 1 includes the VH described herein or HCDR1, HCDR2 and HCDR3 described herein, and the Fc region and CH1 containing the Knob mutations S354C and T366W and L234AL235A described herein, or the heavy chain constant region containing the Knob mutations S354C and T366W and L234AL235A described herein;

[0224] Heavy chain 2 includes the VH described herein or the HCDR1, HCDR2 and HCDR3 described herein, and the Fc region and CH1 containing the Hole mutations Y349C, T366S, L368A and Y407V and L234AL235A described herein, or the heavy chain constant region containing the Hole mutations Y349C, T366S, L368A and Y407V and L234AL235A described herein;

[0225] Light chain 1 includes the VL described herein or LCDR1, LCDR2, and LCDR3 described herein, and the Lambda light chain constant region containing 160C described herein; and / or

[0226] Light chain 2 includes the VL described herein or LCDR1, LCDR2 and LCDR3 described herein, and the Lambda light chain constant region described herein.

[0227] In one specific embodiment, the antibody or its antigen-binding fragment of the present invention comprises heavy chain 1, heavy chain 2, light chain 1, and light chain 2, wherein...

[0228] The heavy chain 1 comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:9.

[0229] The heavy chain 2 comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 10.

[0230] The light chain 1 comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11; and / or

[0231] The light chain 2 comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:12.

[0232] In a more specific embodiment, the antibody or antigen-binding fragment of the present invention comprises heavy chain 1, heavy chain 2, light chain 1, and light chain 2, wherein...

[0233] The heavy chain 1 contains or is composed of the amino acid sequence shown in SEQ ID NO:9.

[0234] The heavy chain 2 contains or is composed of the amino acid sequence shown in SEQ ID NO:10.

[0235] The light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:11; and / or

[0236] The light chain 2 contains or consists of the amino acid sequence shown in SEQ ID NO:12.

[0237] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0238] The heavy chain includes the VH described herein or HCDR1, HCDR2, and HCDR3 described herein, and the Fc region and CH1 containing L234AL235A described herein, or the heavy chain constant region containing L234AL235A described herein; and / or

[0239] The light chain includes the VL described herein or LCDR1, LCDR2 and LCDR3 described herein, and the Kappa light chain constant region described herein.

[0240] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0241] The heavy chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:13, and / or

[0242] The light chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:14.

[0243] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0244] The heavy chain contains or consists of the amino acid sequence shown in SEQ ID NO:13, and / or

[0245] The light chain contains or consists of the amino acid sequence shown in SEQ ID NO:14.

[0246] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0247] The heavy chain includes the VH described herein or HCDR1, HCDR2 and HCDR3 described herein, and the Fc region and CH1 containing L234AL235A described herein, or the heavy chain constant region containing L234AL235A described herein.

[0248] The light chain includes the VL described herein or LCDR1, LCDR2 and LCDR3 described herein, and the Lambda light chain constant region containing 160C described herein.

[0249] In one specific embodiment, the antibody or its antigen-binding fragment of the present invention comprises two identical heavy chains and two identical light chains, wherein...

[0250] The heavy chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:13, and / or

[0251] The light chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:11.

[0252] In a more specific embodiment, the antibody or antigen-binding fragment of the present invention comprises two identical heavy chains and two identical light chains, wherein...

[0253] The heavy chain contains or consists of the amino acid sequence shown in SEQ ID NO:13, and / or

[0254] The light chain contains or consists of the amino acid sequence shown in SEQ ID NO:11.

[0255] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises heavy chain 1, heavy chain 2, and two identical light chains, wherein

[0256] Heavy chain 1 includes the VH described herein or HCDR1, HCDR2 and HCDR3 described herein, and the Fc region and CH1 containing Knob mutations S354C and T366W and L234AL235A and 239C described herein, or the heavy chain constant region containing Knob mutations S354C and T366W and L234AL235A and 239C described herein;

[0257] Heavy chain 2 comprises the VH described herein or HCDR1, HCDR2, and HCDR3 described herein, and the Fc region and CH1 containing Hole mutations Y349C, T366S, L368A, Y407V, and L234AL235A described herein, or the heavy chain constant region containing Hole mutations Y349C, T366S, L368A, Y407V, and L234AL235A described herein; and / or

[0258] The light chain includes the VL described herein or LCDR1, LCDR2 and LCDR3 described herein, and the Kappa light chain constant region described herein.

[0259] In one specific embodiment, the antibody or its antigen-binding fragment of the present invention comprises heavy chain 1, heavy chain 2, and two identical light chains, wherein...

[0260] The heavy chain 1 comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:15.

[0261] The heavy chain 2 comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:10, and / or

[0262] The light chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:14.

[0263] In a more specific embodiment, the antibody or antigen-binding fragment of the present invention comprises heavy chain 1, heavy chain 2, and two identical light chains, wherein...

[0264] The heavy chain 1 contains or is composed of the amino acid sequence shown in SEQ ID NO:15.

[0265] The heavy chain 2 contains or is composed of the amino acid sequence shown in SEQ ID NO:10, and / or

[0266] The light chain contains or consists of the amino acid sequence shown in SEQ ID NO:14.

[0267] In some embodiments, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites on the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. When the antibody contains an Fc region, the sugars attached to it can be altered. In some applications, modifications to remove unwanted glycosylation sites can be useful, for example, removing the fucosylation motif to enhance antibody-dependent cytotoxicity (ADCC) function. In other applications, galactosylation modifications can be performed to modify complement-dependent cytotoxicity (CDC).

[0268] In some embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0269] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention has one or more of the following characteristics:

[0270] (i) Shows the same or similar binding affinity and / or specificity to CEACAM5 as mAb7, mAb8, mAb9 or mAb10;

[0271] (ii) Inhibit (e.g., competitively inhibit) the binding of mAb7, mAb8, mAb9 or mAb10 to CEACAM5;

[0272] (iii) Epitopes that bind to the same or overlapping mAb7, mAb8, mAb9 or mAb10;

[0273] (iv) Competes with mAb7, mAb8, mAb9 or mAb10 to bind to CEACAM5;

[0274] (v) Having one or more of the biological characteristics of mAb7, mAb8, mAb9 or mAb10.

[0275] In some implementations, the anti-CEACAM5 antibody is a monoclonal antibody.

[0276] In some implementations, the anti-CEACAM5 antibody is humanized.

[0277] In some implementations, the anti-CEACAM5 antibody is a chimeric antibody.

[0278] In some embodiments, the anti-CEACAM5 antibody of the present invention is a full-length antibody.

[0279] In one embodiment, the anti-CEACAM5 antibody of the present invention also encompasses its antibody fragments (e.g., antigen-binding fragments), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), bivalent antibodies, or linear antibodies.

[0280] In one embodiment, the anti-CEACAM5 antibody of the present invention also encompasses bispecific or multispecific antibodies that specifically bind to CEACAM5.

[0281] II-2. Polynucleotides, carriers, and host cells

[0282] In one aspect, the present invention provides a nucleic acid encoding any strand or any monomer or domain of any of the above-mentioned anti-CEACAM5 antibodies or their antigen-binding fragments.

[0283] As will be apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0284] In some embodiments, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO:4, 8, and 9-15, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO:4, 8, and 9-15.

[0285] The nucleic acid sequence encoding the molecule of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification.

[0286] In one aspect, the present invention also provides a vector comprising the nucleic acid of the present invention. In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector. The vector includes, but is not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC). In a preferred embodiment, the expression vector is pCDNA, such as pCDNA3.1.

[0287] In one aspect, the invention also provides a host cell comprising the nucleic acid or the vector. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells, such as HEK 293 or 293F cells or 293FT cells or Expi293 cells). In yet another embodiment, the host cell is prokaryotic.

[0288] Suitable host cells include prokaryotic microorganisms such as *Escherichia coli*, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can also be used. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells or 293FT cells or Expi293 cells), young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (WI-38), human liver cancer cells (Hep G2), CHO cells, NSO cells, and myeloma cell lines such as YB2 / 0, NSO, P3X63, and Sp2 / 0. Mammalian host cell lines suitable for antibody production are known in the art. In a preferred embodiment, the host cell is a CHO, HEK293, 293F, 293FT, or Expi293 cell.

[0289] II-3. Production and purification of antibody molecules

[0290] In another aspect, the present invention provides a method for producing the anti-CEACAM5 antibody or an antigen-binding fragment thereof, the method comprising: culturing a host cell containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the molecule; optionally further comprising assembling the polypeptide chain to produce the anti-CEACAM5 antibody or an antigen-binding fragment thereof under conditions suitable for the polypeptide chain to assemble into the molecule.

[0291] To facilitate production and purification, anti-CEACAM5 antibodies or their antigen-binding fragments can be fused to a secretory signal peptide at the N-terminus or C-terminus (e.g., C-terminus), and / or a tagged peptide that facilitates purification, such as a hexahistine tag or biotin label.

[0292] For recombinant production, a polynucleotide encoding a polypeptide chain of the present invention's anti-CEACAM5 antibody or its antigen-binding fragment can be inserted into one or more vectors for further cloning and / or expression in host cells. Expression vectors can be constructed using methods well known to those skilled in the art.

[0293] In one embodiment, each polynucleotide encoding the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment can be transfected into a different vector, optionally the vector containing a heavy chain constant region or a light chain constant region (e.g., nucleotides encoding a heavy chain variable region are transfected into a vector containing a heavy chain constant region, or nucleotides containing a light chain variable region are transfected into a vector containing a light chain constant region, so that the vector can express the full-length heavy chain or the full-length light chain; or nucleotides encoding a heavy chain are transfected into a vector to express the full-length heavy chain, and nucleotides encoding a light chain are transfected into a vector to express the full-length light chain, so that the vector can express the full-length heavy chain or the full-length light chain).

[0294] Once an expression vector containing one or more polynucleotides of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. In one embodiment, one or more vectors encoding multiple chains of an antibody are transfected into the same host cell, such that the antibody chains assemble into a complete antibody within the host cell.

[0295] A variety of techniques can be used to achieve this goal, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

[0296] The molecules prepared as described herein can be purified using known existing techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A affinity chromatography), size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art.

[0297] The purity of the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and high-performance liquid chromatography. The physical / chemical properties and / or biological activity of the antibody molecules provided herein can be identified, screened, or characterized by a variety of assays known in the art.

[0298] II-4. Determination Method

[0299] The anti-CEACAM5 antibodies or their antigen-binding fragments or ADC molecules provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. Examples illustrate methods for determining the properties of the anti-CEACAM5 antibodies or their antigen-binding fragments or ADC molecules of the present invention, such as thin-layer interferometry techniques like ForteBio Octet.

[0300] III. Immunoconjugates

[0301] In one aspect, the present invention provides an immunoconjugate comprising the anti-CEACAM5 antibody of the present invention or an antigen-binding fragment thereof and one or more other active ingredients (e.g., active ingredients derived from a medicament or therapeutic agent for treating the disease of the present invention, such as small molecules or antitumor compounds that enhance the molecular therapeutic effect of the present invention).

[0302] In some embodiments, the immunoconjugates of the present invention are antibody-drug conjugates (ADCs).

[0303] Antibody-drug conjugates (ADCs)

[0304] In some embodiments, antibody-drug conjugates are provided comprising the anti-CEACAM5 antibody of the present invention conjugated with one or more identical or different linker toxins, or an antigen-binding fragment thereof.

[0305] In some embodiments, an antibody-drug conjugate of formula (II) or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof is provided:

[0306] in,

[0307] Ab is the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention;

[0308] D1 has the structure of formula (Ia):

[0309] in,

[0310] R d1 It is H, OH, C 1-6 Alkyl, C 1-6 Alkoxy, Pentoose, penturonic acid, hexose, hexuronic acid, of which R dm R dn and R dp Each is independently H or C 1-6 Alkyl; or R d1 Represents the valence bond, and is related to L. a2 L a3 and L a4 Any one of them is connected to form a loop, preferably with L. a4 Connect them into a ring;

[0311] R d2 and R d3 Each is independently H, OH, C 1-6 Alkyl, C 1-6Alkyl groups, pentoses, penturonic acid, hexoses, hexuronic acid;

[0312] The wavy line represents L. a4 connect;

[0313] L a1 It is a connector unit;

[0314] L a2 There is no or a bridging spacer;

[0315] L a3 Yes: (i) a short chain containing 1-10 amino acid residues, wherein said amino acid residues are optionally selected from C10 by one or more (e.g., 1, 2, 3 or 4) of C10. 1-6 Substitution of alkyl and polyol groups; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3 or 4). 1-6 Substituents of alkyl and polyol groups; or (iii) none; wherein L a3 When present, N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1;

[0316] L a4 There are no non-existent, or the connectors are either cuttable or self-destructive; and

[0317] m can be 1, 2, 3, 4, 5, or 6.

[0318] It should be understood that m refers to the -L atom linked to Ab in antibody-drug conjugate molecules of formula (II). a1 -L a2 -L a3 -L a4 The number of -D1 molecules can also be referred to as the DAR value of ADC molecules.

[0319] It should also be understood that, in this document, the expression "connected to B" includes the case of connecting to B itself, the case of connecting to B's adjacent variables when B does not exist, and the case of connecting to the next adjacent variable when neither B nor the adjacent variable exists, and so on. For example, the expression for D1, "the wavy line represents the connection to L," is used to describe the connection between B and B. a4 "Connection" includes L a4 The case of self-connection, when L a4 When it does not exist, it is related to the adjacent variable L. a3 Connection scenarios, when L a4 and L a3When neither exists, the next adjacent variable L a2 Connection scenarios, and when L a4 L a3 and L a2 When neither exists, the next adjacent variable L a1 The connection situation.

[0320] In some embodiments, the antibody-drug conjugate of formula (II) of the present invention has the structure of formula (IIa):

[0321] The variables are defined as described in this paper, for example, as defined in equation (II).

[0322] In some implementation schemes, R d1 It is H, OH, Where R dm R dn and R dp Each is independently H or C 1-6 Alkyl groups such as C 1-4 Alkyl group, preferably H.

[0323] In some implementation schemes, R d1 It is H, OH, (For example )or (For example ), where R dm R dn and R dp Each is independently H or C 1-6 Alkyl groups such as C 1-4 Alkyl group, preferably H.

[0324] In some implementation schemes, R d1 It is H, OH, (For example ), where R dm R dn and R dp Each is independently H or C 1-6 Alkyl groups such as C 1-4 Alkyl group, preferably H.

[0325] In some implementation schemes, R d1 It is H, OH,

[0326] In some implementation schemes, R d1 It is H, OH,

[0327] In some implementation schemes, R dmR dn and R dp Each is independently H or C 1-4 Alkyl group, preferably H.

[0328] In some implementation schemes, R d2 and R d3 Independently speaking, they are: H and C 1-6 Alkyl, C 1-6 Alkoxy,

[0329] In some implementation schemes, R d2 and R d3 Each is C independently 1-6 Alkoxy, (For example )or (For example ).

[0330] In some implementation schemes, R d2 and R d3 Each is C independently 1-6 Alkoxy groups, such as C 1-4 Alkyl groups, preferably methoxy, ethoxy, propoxy, or butoxy. In some embodiments, R... d2 and R d3 Each is an methoxy group independently.

[0331] In some implementations, L a1 yes:

[0332] One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0333] In some implementations, L a1 yes: One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0334] In some implementations, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0335] In some implementations, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0336] It is understandable that the expression L a1 The two "with L" a2 "Connectivity" covers La2 The case of self-connection, when L a2 When it does not exist and L a3 Connection scenarios, when L a2 and L a3 When neither exists and L a4 Connection scenarios, and when L a2 L a3 and L a4 The case where neither exists when connected to D1. Other similar statements should be understood accordingly.

[0337] In some implementations, L a2 The bridging spacer either does not exist or is selected from the following: *-(CR p R q ) s1 -(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -L M -(CH2CH2O) t -(CR m R n ) s2 -CO-**; and *-(CR) p R q ) s1 -(OCH2CH2) t -(CR m R n ) s2 -CO-**;

[0338] Among them, * end and L a1 Connect, and the **end is connected to L a3 Connect, and where R p R q R m and R n Each is independently H or C 1-6 Alkyl groups, preferably H or C 1-4 Alkyl, L M It is -NH-CO-, -CO-NH-, -CO-, -NH- or -O, and s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0339] In some implementations, L a2 The bridging spacer either does not exist or is selected from the following: *-(CR p R q )s1 -(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -NH-CO-(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -CO-NH-(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -CO-(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -NH-(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -O-(CH2CH2O) t -(CR m R n ) s2 -CO-**; and *-(CR) p R q ) s1 -(OCH2CH2) t -CO-**;

[0340] Among them, * end and L a1 Connect, and the **end is connected to L a3 Connect, and where R p R q R m and R n Each is independently H or C 1-6 Alkyl groups, preferably H or C 1-4Alkyl groups, s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0341] In some implementations, L a2 The bridging spacer either does not exist or is selected from the following: *-(CH2) s1 -(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -NH-CO-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -CO-NH-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -CO-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -NH-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -O-(CH2CH2O) t -(CH2) s2 -CO-**; and *-(CH2) s1 -(OCH2CH2) t -CO-**;

[0342] Among them, * end and L a1 Connect, and the **end is connected to L a3 Connect , where s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0343] In some implementations, L a2 The bridging spacer either does not exist or is selected from the following: *-(CR p R q ) s1 -CO-**;*-(CH2CH2O) t -(CR m R n ) s2 -CO-**; and *-(CR) p R q ) s1 -(OCH2CH2) t -CO-**; where the * terminator is related to the L terminator. a1 Connect, and the **end is connected to La3 Connect, and where R p R q R m and R n Each is independently H or C 1-6 Alkyl groups, preferably H or C 1-4 Alkyl groups, s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0344] In some implementations, L a2 The bridging spacer either does not exist or is selected from the following: *-(CH2) s1 -CO-**;*-(CH2CH2O) t -(CH2) s2 -CO-**; and *-(CH2) s1 -(OCH2CH2) t -CO-**; where the * terminator is related to the L terminator. a1 Connect, and the **end is connected to L a3 Connect , where s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0345] In some implementations, L a2 It does not exist or is the formula *-(CR) p R q ) s1 -CO-** bridging spacer, where the * end is connected to L a1 Connect, and the **end is connected to L a3 Connection, R p and R q Each is independently H or C 1-6 Alkyl, preferably H or C 1-4 Alkyl groups, and s1 is 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0346] In some implementations, L a2 It does not exist or is expressed as *-(CH2). s1 -CO-** bridging spacer, where the * end is connected to L a1 Connect, and the **end is connected to L a3 The connection, and s1 are 0, 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, 6 or 7, more preferably 5 or 6.

[0347] In some implementations, L a2The following are possible values: -CO-, *-(CH2)-CO-**, *-(CH2)2-CO-**, *-(CH2)3-CO-**, *-(CH2)4-CO-**, *-(CH2)5-CO-**, *-(CH2)6-CO-**, *-(CH2)7-CO-**, or *-(CH2)8-CO-**, preferably *-(CH2)5-CO-**, wherein the * terminus is associated with the L terminus. a1 Connect, and the **end is connected to L a3 connect.

[0348] In some implementations, -L a1 -L a2 -yes Wherein s1 and s2 are each independently 1, 2, 3, 4, 5, 6, 7 or 8, preferably 4, 5 or 6, and t is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, 6, 7 or 8, and the left side of the group is connected to Ab, and the right side is connected to L. a3 connect.

[0349] In some implementations, -L a1 -L a2 -yes Where s1 is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, and the left side of the group is connected to Ab, and the right side is connected to L. a3 connect.

[0350] Understandable, for L a2 or -L a1 -L a2 - Regarding the statement "right side and L" a3 "Connectivity" covers L a3 The case of self-connection, when L a3 When it does not exist and L a4 Connection scenarios, and when L a3 and L a4 The case where neither exists when connected to D1. Other similar statements should be understood accordingly.

[0351] In some implementations, L a3 Yes: (i) a short chain containing 1-8 amino acid residues, such as a single amino acid residue or a peptide containing 2, 3, 4, 5, 6, 7 or 8 amino acids, preferably 2, 3 or 4 amino acids, wherein said amino acid is optionally bounded by one or more (e.g. 1, 2, 3 or 4, preferably 1 or 2) C 1-6 Alkyl substitution; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3 or 4, preferably 1 or 2). 1-6Alkyl substituents; or (iii) none; wherein L a3 When present, N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connection (i.e., with L) a1 -L a2 -Partial connection), C-end and L a4 Connection or when L a4 If it does not exist, connect it to D1 (i.e., connect it to -L). a4 -D1 partial connection).

[0352] In some embodiments, each of the amino acids is independently selected from valine (Val), alanine (Ala), glycine (Gly), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), serine (Ser), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), methionine (Met), tryptophan (Trp), cysteine ​​(Cys), histidine (His), and threonine (Thr), wherein the amino acid is optionally converted by one or more C 1-6 Alkyl substitution.

[0353] In some embodiments, the amino acid is selected from valine, alanine, glycine, lysine, citrulline, glutamine, glutamic acid, phenylalanine, and leucine, wherein the amino acid is optionally converted by one or more C... 1-6 Alkyl substitution.

[0354] In some embodiments, the amino acid is selected from valine, alanine, glycine, lysine, citrulline, and glutamine, wherein the amino acid (e.g., lysine) is optionally oxidized by one or more C14 groups. 1-6 Alkyl substitution.

[0355] In some embodiments, the amino acid is selected from valine, alanine, glycine, and lysine, wherein the lysine is optionally oxidized by one or more C2O groups. 1-6 Alkyl substitution, preferably N-substitution.

[0356] In some implementations, L a3Selected from: Val, Ala, Gly, Lys, Cit, Gln, Glu, Phe, Leu; Val-Val, Val-Ala, Val-Gly, Val-Lys, Val-Cit, Val-Gln, Val-Glu, Val-Phe, Val-Leu; Ala-Ala, Ala-Gly, Ala-Lys, Ala-Cit, Ala-Gln, Ala-Glu, Ala-Phe, Ala-Leu; Gly-Gly, Gly-Lys, Gly-Cit, Gly-Gln, Gly-Glu, Gly-Phe, Gly-Leu; Lys-Lys, Lys-Cit, Lys-Gln, Lys-Glu, Lys-Phe, Lys-Leu; Cit-Cit, Cit-Gln, Cit-Glu , Cit-Phe, Cit-Leu; Gln-Gln, Gln-Glu, Gln-Phe, Gln-Leu; Glu-Glu, Glu-Phe, Glu-Leu; Ph e-Phe, Phe-Leu, Leu-Leu; Ala-Ala-Ala, Ala-Ala-Asn, Val-Lys-Gly, Gln-Val-Ala, Phe-Ph -NH-(CH2) 2-6 -CO- or absent, wherein the amino acid (preferably lysine) is optionally surrounded by one or more C-. 1-6 Alkyl substitution, preferably N-substitution, and wherein L a3 When L exists a3 The N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connect, and C end is connected to L a4 Connection or when L a4 If it does not exist, it is connected to D1. For example, for Val-Ala, it includes the N end of Val connected to L. a2 Connected and Ala's C terminal is connected to L a4 The connection details, and the relationship between the N-terminal of Ala and the L-terminal. a2 Connected and Val's C end to L a4 Connection status.

[0357] In some implementations, L a3Is Gly, Val-Ala, Val-Cit, Phe-Lys, Val-Lys, Leu-Cit, Val-Lys-Gly, Val-(N6,N6-C 1-6 Alkyl group (-Lys)-Gly, -NH-(CH2) 2-6 -CO- or absent, L is preferred. a3 It is Gly, Val-Ala, Val-Lys-Gly, Val-(N6,N6-diisopropyl-Lys)-Gly, or -NH-(CH2). 2-6 -CO-, and L is preferred a3 It is Gly, Val-Ala, Val-Lys-Gly, Val-(N6,N6-diisopropyl-Lys)-Gly or -NH-(CH2)2-CO-; where L a3 The N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connect, and C end is connected to L a4 Connection or when L a4 If it does not exist, connect it to D1.

[0358] In some implementations, L a3 It is -NH-(CH2) 2-4 -CO-, such as -NH-(CH2)2-CO-, -NH-(CH2)3-CO- and -NH-(CH2)4-CO-, preferably -NH-(CH2)2-CO-, wherein L a3 The N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connect, and C end is connected to L a4 Connection or when L a4 If it does not exist, connect it to D1.

[0359] In some implementations, L a3 yes (Gly) Or -NH-(CH2)2-CO-, where L a3 The N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1.

[0360] In some implementations, L a4 It does not exist or is -NH-CH2-. The right side of the group is connected to D1, and the left side is connected to L. a3 connect.

[0361] In some implementations, L a4 Does not exist or The right side of the group is connected to D1, and the left side is connected to L. a3 connect.

[0362] In some implementation schemes, R L Selected from H, C 1-6 Alkyl, C 1-6 Alkoxy, pentose, penturonic acid, hexose, hexuronic acid, disaccharide, trisaccharide, and (optionally selected by one or more of C) 1-6 5-8 membered heteroaryl groups substituted with alkyl and nitro groups)-(C 1- 4-alkylene)-O-.

[0363] In some implementation schemes, R L It is H.

[0364] In some implementation schemes, R L It is H. (like ), (like ),or

[0365] In some implementation schemes, R L It is H. (like ),or

[0366] In some implementations, L a4 Does not exist or Terminal C is connected to D1, and terminal N is connected to L. a3 connect.

[0367] In some implementations, L a4 Does not exist or Terminal C is connected to D1, and terminal N is connected to L. a3 connect.

[0368] In some implementations, L a4 yes For example Terminal C is connected to D1, and terminal N is connected to L. a3 connect.

[0369] In some implementation schemes, R d1 Indicates valence bond, L a4 yes R L yes Where R L 1st bit and R d1 Connect and 2 bits with L a4 Connect, and where L a4 The C terminal is connected to D1, and the N terminal is connected to L. a3 connect.

[0370] In some implementation schemes, R d1 Indicates valence bond, L a4 express For example Where L a4 Bit 1 is connected to D1, and bit 2 is connected to R. d1 Connect in a ring, and 3 bits with L a3 connect.

[0371] In some embodiments, a compound of formula (II) or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof is provided:

[0372] in,

[0373] Ab is the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention;

[0374] D1 has the structure of formula (Ib):

[0375] in,

[0376] R d1 It is H, OH, (For example );

[0377] R d2 and R d3 Each is C independently 1-4 Alkyl groups, preferably methoxy groups;

[0378] The wavy line represents L. a4 connect;

[0379] -L a1 -L a2 -yes Where s1 is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, and the left side of the group is connected to Ab, and the right side is connected to L. a3 connect;

[0380] L a3yes (Gly) Or -NH-(CH2)2-CO-, where the N-terminus is associated with the L-terminus. a2 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1;

[0381] L a4 Does not exist or (For example ), (For example ),or Preferred is For example Where L a4 When present, terminal C is connected to D1, and terminal N is connected to L. a3 connect;

[0382] Or, R d1 Indicates valence bond, L a4 express For example Where L a4 Bit 1 is connected to D1, and bit 2 is connected to R. d1 Connect in a ring, and 3 bits with L a3 Connection; and

[0383] m can be 1, 2, 3, 4, 5, or 6.

[0384] In some implementations, -L a1 -L a2 -L a3 -L a4 -D1 is selected from:

[0385] The wavy line indicates that the valence key is connected to Ab.

[0386] In some implementations, -L a1 -L a2 -L a3 -L a4 -D1 is selected from:

[0387] The wavy line indicates that the valence key is connected to Ab.

[0388] In some implementations, the antibody-drug conjugate of formula (II) has the following subform:

[0389] Wherein, Ab is the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment, such as mAb7, mAb8, mAb9, or mAb10; and

[0390] m is 1, 2, 3, 4, 5 or 6; preferably, m is 1; and even more preferably, m is 2.

[0391] In some implementations, the antibody-drug conjugate of formula (II) has the following subform:

[0392] Wherein, Ab is the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment, such as mAb7, mAb8, mAb9, or mAb10; and

[0393] m is 1, 2, 3, 4, 5 or 6; preferably, m is 1; and even more preferably, m is 2.

[0394] In some implementations, m is 1, 2, 3, or 4. In some implementations, m is 1. In some implementations, m is 2.

[0395] In some embodiments, the antibody-drug conjugates of formula (II) or its sub-forms (including formulas IIa and II-1 to II-11) of the present invention are in the form of a composition comprising one or more of the antibody-drug conjugates or their pharmaceutically acceptable salts or esters, solvates or isotopic labels, and the composition has an average DAR of 0.6-6, preferably 0.8-4.0, more preferably 0.8-2.2. In some embodiments, the antibody-drug conjugates of formula (II) or its sub-forms of the present invention, or the composition comprising one or more of the antibody-drug conjugates, have an average DAR selected from any value within the range of 1 ± 0.4, 2 ± 0.4, 3 ± 0.4 or 4 ± 0.4. Preferably, the antibody-drug conjugates of formula (II) or its sub-forms of the present invention, or the composition comprising one or more of the antibody-drug conjugates, have an average DAR of about 1.0. Further preferably, the antibody-drug conjugates of formula (II) of the present invention, or the composition comprising one or more of the antibody-drug conjugates, have an average DAR of about 2.0.

[0396] In some embodiments, the antibody-drug conjugate of formula (II) of the present invention or a composition comprising one or more of the antibody-drug conjugates are those prepared in the examples.

[0397] In another aspect, antibody-drug conjugates of formula (III) are provided:

[0398] in:

[0399] Ab is the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention;

[0400] D1 has the structure of formula (Ia):

[0401] in,

[0402] R d1 It is H, OH, C 1-6 Alkyl, C 1-6 Alkoxy, Pentoose, penturonic acid, hexose or hexuronic acid, wherein R dm R dn and R dp Each is independently H or C 1-6 Alkyl; or R d1 Represents the valence bond, and is related to L. a2 L a3 and L a4 Any one of them is connected to form a loop, preferably with L. a4 Connect them into a ring;

[0403] R d2 and R d3 Each is independently H, OH, C 1-6 Alkyl, C 1-6 Alkoxy, pentose, penturonic acid, hexose or hexuronic acid;

[0404] The wavy line represents L. a4 connect;

[0405] L a1 It is a connector unit;

[0406] L a2 There is no or a bridging spacer;

[0407] L a3 Yes: (i) a short chain containing 1-10 amino acid residues, wherein said amino acid residues are optionally selected from C10 by one or more (e.g., 1, 2, 3 or 4) of C10. 1-6 Substitution of alkyl and polyol groups; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3 or 4). 1-6 Substituents of alkyl and polyol groups; or (iii) none; wherein L a3 When present, N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connection, C end and L a4 Connection or when La4 If it does not exist, connect it to D1;

[0408] L a4 The connectors either do not exist or are cuttable or self-destructive connectors.

[0409] L b It is a connector;

[0410] D2 is the drug component, preferably the anti-tumor drug component;

[0411] m is 1, 2, 3, 4, 5, or 6; and

[0412] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0413] It should be understood that m refers to the -L atom linked to Ab in antibody-drug conjugate molecules of formula (III). a1 -L a2 -L a3 -L a4 The number of -D1s can also be referred to as the DAR value of the ADC molecule with respect to D1. n refers to the number of -Ls attached to Abs in antibody-drug conjugates of formula (III). b The number of -D2 can also be called the DAR value of the ADC molecule relative to D2.

[0414] In some implementations, the antibody-drug conjugate of formula (III) may have the structure of (IIIa):

[0415] The variables are defined as described in this paper, for example, for equation (II).

[0416] In some implementations, L a1 L a2 L a3 L a4 D1 and D1 are each as defined herein in the various embodiments and examples and / or aspects of Equation (II).

[0417] In some implementations, L a1 yes: One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0418] In some implementations, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0419] In some implementations, La1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0420] In some implementations, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0421] In some implementations, -L a1 -L a2 -yes Where s1 and s2 are each independently 1, 2, 3, 4, 5, 6, 7 or 8, preferably 4, 5 or 6, t is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, 6, 7 or 8, and one bit is connected to Ab, and two bits are connected to L. a3 connect.

[0422] In some implementations, -L a1 -L a2 -yes Where s1 is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, and one bit is connected to Ab, and two bits are connected to L. a3 connect.

[0423] The drug moiety D2 can be any antitumor drug, as long as it can be coupled to Ab via a linker. Inside tumor cells, part or all of the linker can be cleaved, releasing the antitumor drug moiety and thus exhibiting an antitumor effect.

[0424] In some embodiments, drug fraction D2 is an antitumor drug fraction having the same mechanism of action as D1. In other embodiments, drug fraction D2 is an antitumor drug fraction having a different mechanism of action than D1.

[0425] In some embodiments, drug fraction D2 can be a cytotoxic agent, a cell growth inhibitor, or an immunosuppressive agent. For example, drug fraction D2 can be a tubulin inhibitor, a DNA synthesis inhibitor, a topoisomerase inhibitor, a DNA minor groove binder, a DNA replication inhibitor, an alkylating agent, an antibiotic, an antifolate agent, an antimetabolite, a chemotherapy sensitizer, vinca alkaloids, etc. Preferably, drug fraction D2 is a tubulin inhibitor (including tubulin polymerization enhancers and tubulin polymerization inhibitors), a DNA synthesis inhibitor, or a topoisomerase inhibitor (including topoisomerase I inhibitors and topoisomerase II inhibitors). More preferably, drug fraction D2 is a topoisomerase inhibitor, preferably a topoisomerase I inhibitor.

[0426] In some implementations, drug fraction D2 is a camptothecin compound (e.g., ethiotacone, Dxd, SN38), anorectatine compound such as MMAE or MMAF, maytansine compound such as DM1 and DM4, cazithromycin antitumor antibiotic, or atrazomycin antitumor antibiotic.

[0427] In some implementations, the drug portion D2 has the following structure: -QL 2 -L 1 -D 2a

[0428] in:

[0429] Q is -O-, -S-, or -NR 7 -;

[0430] L 2 It does not exist, *-(C 1-10 alkylene)-C(O)N(R 5 )- or *-(C 1-10 alkylene)-N(R 5 )C(O)-; where * indicates that the end is covalently connected to Q, and R 5 Is it H or C? 1-6 alkyl;

[0431] L 1 Is it non-existent or -(C) 1-10 alkylene);

[0432] D 2a It has the structure shown in equation (D-1):

[0433] Among them, R 1 and R 6 Selected from H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 2-6 Haloalkenyl and C 2-6 Halogenated alkynyl group; or R 1 and R 6 Together with the carbon atoms they are attached to, they form 5-9 membered rings, such as 5-8 membered rings;

[0434] R 2 It is H, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, -OR 4 or -SR 4 ;

[0435] R 3 It is H, halogen, CN, C1-6 Alkyl, C 1-6 Halogenated alkyl or -OR 4 ;

[0436] Or R 2 and R 3 Together they form -O(CH2) p O- or -O(CF2) p O-, where p is 1 or 2;

[0437] R 4 Is it H or C? 1-4 Alkyl; and

[0438] R 7 Is it H or C? 1-6 alkyl.

[0439] It should be understood that the wavy bond that intersects the benzene ring in formula (D-1) means that formula (D-1) is connected to -QL in any feasible position. 2 -L 1 -connect.

[0440] In some implementation schemes, D 2a It has the structure shown in formula (D-1a) or (D-1b):

[0441] The remaining symbols (e.g., R) 1 R 2 R 3 R 6 As defined in this document. It should be understood that the wavy line represents -QL. 2 -L 1 - The location of the connection.

[0442] In some implementation schemes, D 2a It has the structure shown in equation (D-2):

[0443] Among them, each symbol (e.g., R) 1 R 2 R 3 and R 6 As defined above.

[0444] In some implementation schemes, D 2a It has the structure shown in equation (D-2a) or (D-2b):

[0445] Among them, each symbol (e.g., R) 1 R 2 R 3 R 6 (n1 and n2) are as defined above.

[0446] In some implementation schemes, R 1 and R 6 For H, R 2 C 1-6 Alkyl or C 1-6 Alkoxy, R 3 Halogen, preferably -F.

[0447] In some implementations, -L 2 -L 1 -is-(C 1-6 alkylene)-, *-(C 1-6 alkylene)-C(O)N(R 5 )-(C 1-6 alkylene)- or *-(C 1-6 alkylene)-N(R 5 )C(O)-(C 1-6 (alkylene)-, where * indicates that the terminus is covalently connected to Q; and R 5 Is it H or C? 1-6 alkyl.

[0448] In some implementations, -L 2 -L 1 -is-(C 1-6 Alkylene)-.

[0449] In some implementations, -QL 2 -L 1 - can be -OCH2-CH2-CH2-CH2-, -OCH2-CH2-CH2-, or -NH-. It should be understood that the left side of this group is related to L... b Connect, right side with D 2a connect.

[0450] In some implementations, the drug portion D2 or -QL 2 -L 1 -D 2a Selected from:

[0451] In some implementations, connector L b It has the following structure: -ZEX-

[0452] in:

[0453] Connect Z to Ab, and connect X to D;

[0454] Z is selected from

[0455] Where m a1 and m a2Independently selected integers from 0 to 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; and m is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, or 8;

[0456] The carbonyl group on the right end of Z is covalently connected to E;

[0457] E is a short chain containing 1-10 amino acids, such as a single amino acid residue or a peptide residue containing 2-10 amino acids, wherein the short chain is optionally selected by one or more (e.g., 2, 3, or 4) from C. 1-6 Group substitution of alkyl and polyol groups, wherein the N-terminus of the short chain is covalently linked to Z; and

[0458] X is selected from non-existent, -NH-CH2-, or... Where R a1 Selected from H, C 1-6 Alkyl, C 1-6 Alkoxy, pentose, penturonic acid, hexose, hexuronic acid, disaccharide and trisaccharide; the left end of the group is connected to E and the right end is connected to D2.

[0459] In some implementation schemes, Z is Where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5.

[0460] In some implementation schemes, Z is Where, m a2 It is an integer selected from 0 to 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, preferably 4, and m is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably 2.

[0461] In some implementations, E is a single amino acid residue or a peptide residue containing 2, 3, or 4 amino acids.

[0462] In some embodiments, the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, leucine, tyrosine, lysine, citrulline, serine, aspartic acid, asparagine, isoleucine, arginine, and proline, wherein the amino acid (preferably glutamine or glutamic acid) is optionally substituted with one polyol group and optionally substituted with one carbon atom. 1-6 Alkyl substitution.

[0463] In some embodiments, the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted with one polyol group and optionally with one carbon atom. 1-6 Alkyl substitution.

[0464] In some embodiments, the polyol group is

[0465] In some embodiments, the substituted glutamine or glutamic acid has the following structure:

[0466] Preferred Where R 8 Is it H or C? 1-6 alkyl.

[0467] In some implementations, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Ala-, or Where R 8 Is it H or C? 1-6 alkyl.

[0468] It should be understood that the above-mentioned E group is covalently connected to Z through the left-side N-terminus.

[0469] In some implementations, X is -NH-CH2- or

[0470] In some implementation schemes, R a1 Selected from: H,

[0471] In some implementation schemes, R a1 yes

[0472] In some implementations, X is -NH-CH2-,

[0473] In some implementations, connector L b -ZEX- has the following structure:

[0474] In some implementations, connector L b -ZEX- has the following structure:

[0475] It is understood that the left end of the structure is connected to part Ab.

[0476] In some implementations, -L b The -D2 part is:

[0477] The wavy line indicates a connection to Ab.

[0478] In some implementations, -L b The -D2 part is:

[0479] The wavy line indicates a connection to Ab.

[0480] In some implementations, -L b -D2 can be derived from:

[0481] In some implementations, -L b -D2 can be derived from:

[0482] NT3 as the L-linker toxin of NT3' b The -D2 portion can be obtained as described in WO2021 / 173773. NT2 is the L-type linker toxin of NT2'. b The -D2 portion can be obtained as described in PCT / CN2023 / 086909.

[0483] In some embodiments, the antibody-drug conjugate of formula (III) of the present invention has the following subform:

[0484] Wherein, Ab is the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment, such as mAb7, mAb8, mAb9 or mAb10;

[0485] m is 1, 2, 3, 4, 5, or 6 (preferably, m is 1; more preferably, m is 2); and

[0486] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 (preferably, n is 4; even more preferably, n is 8).

[0487] In some embodiments, the antibody-drug conjugate of formula (III) of the present invention has the following subform:

[0488] Wherein, Ab is the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment, such as mAb7, mAb8, mAb9 or mAb10;

[0489] m is 1, 2, 3, 4, 5, or 6 (preferably, m is 1; more preferably, m is 2); and

[0490] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 (preferably, n is 4; even more preferably, n is 8).

[0491] In some embodiments, m is 1, 2, 3, or 4. Preferably, m is 1 or 2. In some embodiments, m is 1. In other embodiments, m is 2.

[0492] In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. Preferably, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, n is 4. In other embodiments, n is 8.

[0493] In some implementations, m is 1 and n is 4. In some implementations, m is 2 and n is 4. In some implementations, m is 1 and n is 8. In some implementations, m is 2 and n is 8.

[0494] In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or its pharmaceutically acceptable salt or ester, solvate, or isotopic label, is in the form of a composition comprising one or more of the antibody-drug conjugate or its pharmaceutically acceptable salt or ester, solvate, or isotopic label, and the composition has an average DAR of 0.6-6, preferably 0.8-4.0, more preferably 0.8-2.2 for drug portion D1 and an average DAR of 2.0-14.0, preferably 4.0-12.0, more preferably 6.0-10.0 for drug portion D2.

[0495] In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR selected from any of the following ranges with respect to D1: 1 ± 0.4, 2 ± 0.4, 3 ± 0.4, or 4 ± 0.4. In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR of about 1.0 or 2.0 with respect to D1.

[0496] In some embodiments, the antibody-drug conjugate of formula (III) or its sub-formulas of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR of about 2.0-14.0, preferably 4.0-12.0, more preferably 6.0-10.0 with respect to the drug moiety D2, for example, about 2.0, 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, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, and the range with any two of these values ​​as endpoints.

[0497] In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR of 1.0 ± 0.4 or 2.0 ± 0.4 for D1 and an average DAR of 4.0 ± 0.4 or 8.0 ± 0.4 for D2. In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR of 1 ± 0.4 for D1 and an average DAR of 4 ± 0.4 for D2. In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR of 2 ± 0.4 for D1 and an average DAR of 4 ± 0.4 for D2. In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR of 1 ± 0.4 for D1 and an average DAR of 8 ± 0.4 for D2. In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of the antibody-drug conjugates, has an average DAR of 2 ± 0.4 for D1 and an average DAR of 8 ± 0.4 for D2.

[0498] In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of the antibody-drug conjugates, are those in the examples.

[0499] The various embodiments and examples described herein, and any combination thereof, are equivalent to the linker toxin compounds and antibody-drug conjugates of the present invention, or their pharmaceutically acceptable salts or esters, solvates or isotope labels, etc. For example, the various embodiments and examples described in formula (II), and any combination thereof, are equivalent to the subforms of formula (II) such as formulas (IIa), (II-a1) to (II-a11), (II-1) to (II-11), and to formula (III) and its subforms such as formulas (IIIa), (III-a1) to (III-a24), (III-1) to (III-24).

[0500] In some embodiments, the Ab in the ADC of the present invention (including those of formula (II) or (III)) comprises a cysteine ​​residue. In some embodiments, in the ADC of the present invention, the Ab is linked to a linker via its cysteine ​​residue (e.g., naturally occurring and / or introduced by a cysteine ​​mutation), for example, the linker is coupled to the Ab via a thiol group of the cysteine ​​residue.

[0501] In some embodiments, the Ab is coupled to a linker via a natural cysteine ​​residue, for example, the linker is coupled to the Ab via the thiol group of a cysteine ​​residue with a disulfide bond open (e.g., random coupling).

[0502] In some embodiments, the Ab is coupled to the linker via a cysteine ​​introduced by a cysteine ​​mutation, for example, the Ab is coupled to the linker via a cysteine ​​residue that is not paired or otherwise part of an intramolecular or intermolecular disulfide bond (e.g., cysteine ​​obtained after a cysteine ​​mutation) (e.g., site-directed coupling), or the linker is coupled to the Ab via the thiol group of a cysteine ​​with a disulfide bond open (e.g., random coupling or site-directed coupling).

[0503] In some embodiments, in the ADC molecule of the present invention, the Ab is coupled to a toxin via a linker (e.g., site-directed coupling) by a cysteine ​​introduced by a mutation in its cysteine, and coupled to another toxin via its native cysteine ​​(e.g., random coupling).

[0504] In some embodiments, the Ab in the ADC molecule of the present invention comprises a cysteine ​​introduced by a cysteine ​​mutation, such that the linker is coupled to the antibody via the thiol group of the mutated cysteine. In some embodiments, the Ab comprises, on its heavy or light chain, one or more non-cysteine ​​residues mutated to cysteine, such that the linker is coupled to the cysteine ​​residue, for example, site-directed coupling. Antibodies or antigen-binding fragments thereof comprising the cysteine ​​mutation suitable for use in the ADC of the present invention are described in detail herein, wherein the mutated cysteine ​​residue is coupled to the linker, for example, site-directed coupling.

[0505] In some embodiments, in the ADC molecule of the present invention, the cysteine ​​residue obtained by the Ab through the following mutation is coupled to the linker:

[0506] (i) a cysteine ​​obtained after a mutation at position 160 in one or both Lamda light chain constant regions; or

[0507] (ii) Cysteine ​​obtained after a mutation of cysteine ​​at position 239 in one or two heavy chain constant regions.

[0508] In some specific embodiments, the ADC molecule of the present invention contains an Ab with a cysteine ​​mutation comprising two heavy chains and two light chains, and the cysteine ​​obtained through the following mutation is coupled to the linker:

[0509] (i) A cysteine ​​obtained by a heavy chain constant region after a cysteine ​​mutation at position 239;

[0510] (ii) A cysteine ​​residue acquired after a mutation at position 160 in the constant region of a Lambda light chain; or

[0511] (iii) Cysteine ​​obtained after the cysteine ​​mutation at position 160 in the constant regions of the two Lambda light chains.

[0512] Any antibody or antigen-binding fragment mentioned herein, or any cysteine ​​obtained by any cysteine ​​mutation obtained herein, can be used for conjugation with a linker, such as site-directed conjugation, to obtain the ADC molecule of the present invention.

[0513] In some embodiments, the linker toxin-L of the ADCs of the present invention a1 -L a2 -L a3 -L a4 -D1 is covalently coupled to the sulfhydryl group of cysteine ​​in the Ab (natural or modified, e.g., introduced via cysteine ​​mutation). Specifically, the linker toxin -L a1 -L a2 -L a3 -L a4 -D1 is coupled to the sulfhydryl group of cysteine ​​introduced by the cysteine ​​mutation in Ab, preferably by site-directed coupling.

[0514] In some embodiments, the linker toxin-L of the ADCs of the present invention b -D2 covalently couples with the thiol group formed by the interchain disulfide bond of the native or modified cysteine ​​residues of Ab (e.g., the thiol group formed after the interchain disulfide bond of the native cysteine ​​residue in Ab is opened). For example, linker toxin -L b -D2 is coupled to the thiol group generated by the opening of the interchain disulfide bond of the natural cysteine ​​in the Ab, preferably by random coupling.

[0515] The anti-CEACAM5 antibody or its antigen-binding fragment antibody of the present invention exhibits sufficient stability in blood circulation, good targeting, excellent tumor-killing activity, and good safety and tolerability. Furthermore, due to the dual-cleavage mechanism of the linker toxin of the present invention, the shedding of toxin molecules in blood circulation is reduced, mitigating off-target toxicity and dose-limiting toxicity, further improving safety and tolerability. Specifically, some ADCs of the present invention contain glycoside units, allowing the ADC to release toxin molecules only after reaching the target antigen, further reducing off-target toxicity and dose-limiting toxicity.

[0516] The inventors have also discovered that the dual-toxin ADC drug comprising the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention, as well as two toxins, also exhibits excellent tumor-killing activity and good tolerability, even showing significantly superior tumor-killing activity compared to single-toxin ADC drugs. The dual-toxin ADC drug of the present invention has good blood circulation stability, low off-target toxicity, can effectively target tumor cells, release an effective dose of toxin molecules, exert excellent tumor-killing activity, and exhibits good tolerability and low drug resistance. In particular, the dual-toxin ADC drug of the present invention has shown significantly superior tumor-killing activity compared to single-toxin ADCs in various cell and tumor models (e.g., CDX or PDX).

[0517] Preparation method

[0518] In one aspect, the antibody-drug conjugate of formula (II) of the present invention, or its pharmaceutically acceptable salt or ester, solvate, or isotope label, can be prepared by a method comprising the steps of:

[0519] (a) The linker toxin compound of formula (I) is coupled to the thiol group of cysteine ​​of the antibody or its antigen-binding fragment (Ab).

[0520] In another aspect, the antibody-drug conjugate of formula (III) of the present invention, or its pharmaceutically acceptable salt or ester, solvate, or isotope-labeled form, can be prepared by sequential coupling, for example, by sequential site-directed coupling and conventional coupling. The dual-toxin ADCs of formula (III) prepared by the present invention exhibit good blood circulation stability and contain appropriate proportions of two toxin molecules, demonstrating good safety and tolerability. In particular, the antibody-drug conjugate of formula (III) of the present invention, or its pharmaceutically acceptable salt or ester, solvate, or isotope-labeled form, can be prepared by a method comprising the following steps:

[0521] (a) coupling the linker toxin compound of formula (I) to the thiol group of the cysteine ​​residue of the antibody or its antigen-binding fragment (Ab); and

[0522] (b) Forming a thiol group from the interchain disulfide bond of the antibody or its antigen-binding fragment (Ab), and coupling a linker toxin containing D2 to the thiol group.

[0523] In some implementations, step (a) can be performed via site-specific conjugation, such as thiomab conjugation. Through site-specific conjugation, the linker toxin compound is targeted to the sulfhydryl group of a cysteine ​​residue at a specific site on the antibody or its antigen-binding fragment, forming a site-specific antibody-drug conjugate. This significantly increases the homogeneity and stability of the ADC molecule, reduces off-target toxicity and dose-limiting toxicity, and improves clinical response and safety.

[0524] In some implementations, step (a) may include the following steps:

[0525] (a1) Reduce the antibody or its antigen-binding fragment (Ab) with a reducing agent;

[0526] (a2) Add an oxidizing agent to the reactants in step (a1) to carry out oxidation;

[0527] (a3) Add the linker toxin compound of formula (I) to the product of step (a2) to conjugate and obtain the antibody-drug conjugate.

[0528] In some embodiments, step (a1) is performed in a buffer solution. In some embodiments, the buffer solution has a pH of 5.0-9.0, preferably 6.0-8.0. In some embodiments, the buffer solution is a histidine buffer or a phosphate buffer.

[0529] In some embodiments, the reaction in step (a1) is carried out at a suitable temperature, preferably 15-45°C, more preferably 20-25°C, such as ambient temperature.

[0530] In some embodiments, the antibody concentration in step (a1) is 3-30 mg / mL, preferably 5-15 mg / mL. In some embodiments, the reducing agent in step (a1) is TCEP, such as an aqueous TCEP solution. In some embodiments, the molar ratio of reducing agent to antibody in step (a1) is 5 to 40, preferably 10 to 30, more preferably 10 to 20.

[0531] In some embodiments, the reaction in step (a2) is carried out at pH 5.0-9.0, preferably pH 6.0-8.0.

[0532] In some embodiments, the reaction in step (a2) is carried out at a suitable temperature, preferably 15-45°C, more preferably 20-37°C, such as ambient temperature.

[0533] In some embodiments, the antibody concentration in step (a2) is 3-30 mg / mL, preferably 5-15 mg / mL. In some embodiments, the oxidant in step (a2) is dehydroascorbic acid (dhAA), preferably dissolved in DMSO. In some embodiments, the molar ratio of oxidant to antibody in step (a2) is 5 to 60, preferably 20 to 40.

[0534] In some embodiments, the reaction in step (a3) ​​is carried out at a suitable temperature, preferably 15-45°C, more preferably 20-37°C, such as ambient temperature.

[0535] In some embodiments, in step (a3), the linker toxin compound of formula (I) is added to the reaction system in excess. In some embodiments, the molar ratio of linker toxin compound to antibody in step (a3) ​​is 3 to 20, for example 3 to 8, 2 to 6, or 3 to 6.

[0536] In some embodiments, step (b) can be performed using conventional coupling methods. For example, conventional coupling methods can be used to open the interchain disulfide bonds of the antibody or its antigen-binding fragment and connect the toxin molecule D2. In some embodiments, step (b) may include the following steps:

[0537] (b1) Add a reducing agent to the antibody-drug conjugate obtained in step (a) to reduce it;

[0538] (b2) Add a linker toxin containing D2 to the reactants of step (b1) for conjugation to obtain a bitoxin antibody-drug conjugate.

[0539] In some implementations, step (b1) is performed at pH 5.0-9.0, preferably pH 6.0-8.0.

[0540] In some embodiments, the reaction in step (b1) is carried out at a suitable temperature, preferably 15-45°C, more preferably 20-37°C, such as ambient temperature or 25°C.

[0541] In some embodiments, the antibody concentration (of the antibody-drug conjugate) in step (b1) is 3-30 mg / mL, preferably 5-15 mg / mL. In some embodiments, the reducing agent in step (b1) is TCEP, such as an aqueous TCEP solution. In some embodiments, the reducing agent:antibody molar ratio in step (b1) is 5 to 40, for example 10 to 20, or 8 to 20.

[0542] In some implementations, step (b2) is performed at a suitable temperature, preferably 15-45°C, more preferably 20-37°C, such as ambient temperature or room temperature.

[0543] In some embodiments, in step (b2), the linker toxin containing D2 is added to the reaction system in excess. In some embodiments, the toxin:antibody molar ratio in step (b2) is 5 to 40, for example 10 to 20 or 10 to 16.

[0544] In some embodiments, the above steps are performed under the reaction conditions disclosed in the examples. It should be noted that embodiments obtained by varying the range or specific values ​​of the reaction conditions disclosed in the examples by 100%, 80%, 60%, 40%, 20%, or 10% are also considered in this invention.

[0545] In some implementations, antibody-drug conjugates can be purified using conventional methods, such as spin desalting, ultrafiltration, or dialysis.

[0546] IV. Pharmaceutical Compositions

[0547] In some embodiments, the present invention provides compositions comprising molecules of the present invention (e.g., the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment or its immunoconjugate, such as an ADC molecule) or a pharmaceutically acceptable salt thereof, preferably pharmaceutical compositions or pharmaceutical formulations. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, for example, a pharmaceutical composition, comprises molecules of the present invention (e.g., the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment or its immunoconjugate, such as an ADC molecule) and a combination of one or more other therapeutic agents.

[0548] The present invention also includes compositions (including pharmaceutical compositions) comprising molecules of the present invention (e.g., the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment or its immunoconjugate such as an ADC molecule) or its pharmaceutically acceptable salts. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0549] As used in this article, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents.

[0550] For information on the use and applications of pharmaceutical excipients, see "Handbook of Pharmaceutical Excipients", 8th edition, R.C. Rowe, P.J. Seskey and S.C. Swen, Pharmaceutical Press, London, Chicago.

[0551] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0552] A pharmaceutical product comprising the molecules of the present invention (e.g., the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment or its immunoconjugate, such as an ADC molecule) can be prepared by mixing the molecules of the present invention having the desired purity (e.g., the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment or its immunoconjugate, such as an ADC molecule) with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0553] In some embodiments, the pharmaceutical composition is a formulation. In some embodiments, the pharmaceutical composition is in the form of an injectable formulation. More preferably, the pharmaceutical composition is in a form suitable for intravenous injection or infusion, such as an aqueous solution for injection or a lyophilized powder for injection. Most preferably, the pharmaceutical composition is in the form of a lyophilized powder formulation, which is prepared for injection with a sterile injection solvent (e.g., water for injection or sterile saline) just before use.

[0554] The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). The active ingredients are suitably combined in amounts effective for the intended use.

[0555] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being a shaped article, such as a film or microcapsule.

[0556] V. Drug combinations and pillboxes

[0557] In some embodiments, the present invention also provides pharmaceutical combinations or pharmaceutical combination products comprising the molecules of the present invention (e.g., the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment or its immunoconjugate such as an ADC molecule), and one or more other therapeutic agents.

[0558] Another object of the present invention is to provide a complete pillbox containing the drug combination of the present invention, preferably said pillbox in the form of drug dosage units. This allows dosage units to be provided according to a dosing regimen or drug administration interval.

[0559] In one embodiment, the complete medicine box of the present invention comprises, within the same package:

[0560] - A first container containing a pharmaceutical composition comprising molecules of the present invention (e.g., the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment or its immunoconjugate, such as an ADC molecule);

[0561] - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0562] In some embodiments, when the molecules of the present invention (e.g., the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment or its immunoconjugate, such as an ADC molecule) are used to treat tumors, other therapeutic agents that may be combined with or used in combination with the molecules of the present invention cover a variety of therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0563] Other exemplary antibodies include those that specifically bind to immune checkpoints.

[0564] VI. Uses and Methods

[0565] This invention provides, in one aspect, a method for preventing or treating a disease or condition in a subject, comprising administering to the subject an effective amount of a molecule of the invention (e.g., an anti-CEACAM5 antibody of the invention or its antigen-binding fragment or its immunoconjugate, such as an ADC molecule), a pharmaceutical composition, a drug combination, or a kit. In some embodiments, the molecule of the invention is used as a monotherapy or in combination to prevent or treat a disease or condition in an individual.

[0566] In some embodiments, the disease or condition is a CEACAM5-related disease and / or condition. In some embodiments, the disease is, for example, a tumor.

[0567] In some embodiments, the present invention relates to molecules of the invention (e.g., the anti-CEACAM5 antibody of the invention or its antigen-binding fragment or its immunoconjugate such as an ADC molecule), pharmaceutical compositions, pharmaceutical combinations or kits, for use in therapies, such as for treating CEACAM5-related diseases and / or conditions.

[0568] In some embodiments, the present invention relates to methods for treating diseases, such as those mentioned herein, using molecules of the invention (e.g., the anti-CEACAM5 antibody of the invention or its antigen-binding fragment or its immunoconjugate, such as an ADC molecule), pharmaceutical compositions, pharmaceutical combinations, or kits, or to uses for said treatment, or to uses for preparing medicaments for said treatment. In some embodiments, the disease is a CEACAM5-related disease and / or condition. In some embodiments, the disease is, for example, a tumor.

[0569] In some embodiments, the tumor is a solid tumor or a hematologic malignancy, as well as a metastatic lesion. In one embodiment, examples of solid tumors include malignant tumors such as cancer. The cancer can be in the early, intermediate, or late stage, or it can be metastatic. In some embodiments, the tumor is a cancer that has tumor immune evasion.

[0570] In some embodiments, the tumor is a CEACAM5-positive tumor or cancer. In some embodiments, the tumor is associated with abnormal expression or abnormal activity of CEACAM5.

[0571] In some embodiments, a CEACAM5-positive tumor or cancer refers to abnormal expression or activity of CEACAM5 (e.g., CEACAM5 overexpression or activating mutation) in a subject suffering from said tumor or cancer. In some embodiments, the subject (particularly an adult subject) has abnormal CEACAM5 expression. In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels or activity) of CEACAM5 (e.g., compared to a healthy subject). In some embodiments, the subject's biological sample (e.g., tumor cells or tumor tissue) has (e.g., elevated levels, such as nucleic acid or protein levels or activity) of CEACAM5 (e.g., compared to a biological sample from a healthy subject (e.g., corresponding tissue or cells in a healthy subject), or compared to CEACAM5 in adjacent healthy tissue or cells of the subject).

[0572] In some embodiments, a CEACAM5-positive tumor or cancer refers to tumor cells in an individual suffering from said tumor or cancer that abnormally express CEACAM5. In some embodiments, the tumor cells of said individual abnormally express CEACAM5, for example, moderately or highly. In some embodiments, abnormal expression of CEACAM5 means that the expression of CEACAM5 on tumor cells is higher than that in control cells (e.g., healthy cells in the corresponding tissue of a healthy individual, or healthy cells adjacent to tumor cells).

[0573] In a preferred embodiment, the tumor cells of CEACAM5-positive tumors have moderate or high CEACAM5 expression.

[0574] In some implementations, the tumor or cancer is lung cancer or a gastrointestinal tumor, such as intestinal cancer, stomach cancer, pancreatic cancer, or colon cancer.

[0575] In one specific embodiment, the molecules of the present invention, such as any antibody or its antigen-binding fragment or immune conjugate, such as ADC molecules, are capable of killing tumor cells and / or inhibiting tumor cell proliferation, for example, tumor cells that overexpress CEACAM5 or express CEACAM5 with an activating mutation.

[0576] In some embodiments, the tumor is a tumor that has already been treated with other treatments such as chemotherapy and / or radiotherapy.

[0577] Depending on their therapeutic use, the antibodies or antigen-binding fragments or immunoconjugates of the present invention, such as ADC molecules or pharmaceutical compositions, may also be administered in combination with one or more other therapies, such as other treatment modalities and / or other therapeutic agents, for the purposes described herein, such as for the prevention and / or treatment of the related diseases or conditions mentioned herein.

[0578] In some embodiments, when the molecules of the present invention (e.g., anti-CEACAM5 antibody or its antigen-binding fragment or its immunoconjugate, etc.) are used to treat tumors, the treatment methods include surgery; radiotherapy, local irradiation or focused irradiation, etc.

[0579] In other respects, the present invention provides the use of the molecules of the present invention (e.g., anti-CEACAM5 antibodies or their antigen-binding fragments or their immunoconjugates, etc.) or compositions or combinations thereof in the manufacture or preparation of medicaments for the purposes described herein, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0580] In other respects, the present invention also provides molecules of the present invention (e.g., anti-CEACAM5 antibodies or their antigen-binding fragments or their immunoconjugates such as ADC molecules), or compositions or pharmaceuticals or formulations or combination products comprising the present invention for therapeutic purposes, such as for treating the related diseases or conditions mentioned herein.

[0581] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., an individual suffering from or at risk of suffering from the diseases described herein). In one embodiment, the subject suffers from or is at risk of suffering from the diseases described herein (e.g., cancer). In some embodiments, the subject has received or has received other treatments, such as chemotherapy and / or radiation therapy. In some embodiments, the subject has previously received or is currently receiving immunotherapy.

[0582] The combination therapy of the present invention covers combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations) and separate administration, in which case the administration of the molecules of the present invention (e.g., anti-CEACAM5 antibody or its antigen-binding fragment or its immunoconjugate such as ADC molecule, etc.) or compositions or pharmaceutical preparations containing the present invention may occur before, simultaneously with, and / or after the administration of other therapeutic agents and / or pharmaceutical agents.

[0583] The drug composition can be administered via known methods, such as oral, intravenous injection, intraperitoneal, intracerebral (internal parenchyma), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; via a continuous release system or via an implantable device. In some embodiments, the composition can be administered by bolus injection, continuous infusion, or via an implantable device.

[0584] The composition can also be administered topically via an implantable membrane, sponge, or another suitable material on which the desired molecule is absorbed or encapsulated. In some embodiments, when using an implantable device, the device can be implanted into any suitable tissue or organ and the desired molecule can be delivered via diffusion, timed release of a pellet, or continuous administration. In some embodiments, when the molecules of the present invention (e.g., the anti-CEACAM5 antibody of the present invention or its antigen-binding fragment or its immunoconjugate, such as an ADC molecule) are used to treat tumors, other therapeutic agents that can be combined with or administered in combination with the molecules of the present invention cover a wide range of therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). Other exemplary antibodies include antibodies that specifically bind to immune checkpoints.

[0585] In one aspect, the present invention also relates to methods for diagnosing and detecting antibodies of the present invention or antigen-binding fragments thereof, and compositions comprising such antibodies for diagnosing and detecting.

[0586] In some implementations, any anti-CEACAM5 antibody or its antigen-binding fragment provided herein can be used to detect the presence of CEACAM5 in biological samples.

[0587] When used herein, the term "detection" includes both quantitative and qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, ELISA assays, and PCR techniques (e.g., RT-PCR). In some embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In some embodiments, the biological sample comprises cells or tissues.

[0588] In one implementation, an anti-CEACAM5 antibody or its antigen-binding fragment is provided for use in diagnostic or detection methods.

[0589] In another aspect, a method for detecting the presence of CEACAM5 in a biological sample is provided. In some embodiments, the method comprises detecting the presence of the CEACAM5 protein in the biological sample. In some embodiments, CEACAM5 is human CEACAM5. In some embodiments, the method comprises contacting the biological sample with an anti-CEACAM5 antibody or a fragment thereof as described herein under conditions that allow the anti-CEACAM5 antibody or a fragment thereof to bind to CEACAM5, and detecting whether a complex is formed between the anti-CEACAM5 antibody or a fragment thereof and CEACAM5. The formation of the complex indicates the presence of CEACAM5. The method may be an in vitro or in vivo method. In one embodiment, the anti-CEACAM5 antibody or a fragment thereof is used to select subjects suitable for treatment using the anti-CEACAM5 antibody or a fragment thereof, for example, where CEACAM5 is a biomarker for selecting said subjects.

[0590] In some embodiments, a labeled anti-CEACAM5 antibody or a fragment thereof is provided. Labeling includes, but is not limited to, labels or portions that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.

[0591] In some embodiments provided herein, the sample is obtained prior to treatment with the molecules of the invention (e.g., the anti-CEACAM5 antibody of the invention or its antigen-binding fragment or its immunoconjugate, such as an ADC molecule), pharmaceutical composition, drug combination, or kit. In some embodiments, the sample is obtained prior to treatment with other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

[0592] In some implementations, CEACAM5 is detected before treatment, for example, before initiating treatment or before a treatment after a treatment interval.

[0593] In some embodiments, a method for treating the disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) for the presence of CEACAM5, thereby determining a CEACAM5 value; comparing the CEACAM5 value with a control value; and if the CEACAM5 value is greater than the control value, administering to the subject a therapeutically effective amount of a molecule of the present invention (e.g., an anti-CEACAM5 antibody of the present invention or its antigen-binding fragment or its immunoconjugate, such as an ADC molecule), pharmaceutical composition, drug combination, or kit, optionally in combination with one or more other therapies, thereby treating the disease.

[0594] These and other aspects and embodiments of the invention are described in the accompanying drawings and the following detailed description of the invention and are exemplified in the following embodiments. Any or all features discussed above and throughout this application may be combined in various embodiments of the invention. The following embodiments further illustrate the invention; however, it should be understood that the embodiments are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art. Example

[0595] The following embodiments are provided to further illustrate the present invention. It should be understood that they are merely for the purpose of better understanding the present invention and are not intended to limit the scope of the present invention in any way.

[0596] In this application, when the chemical name and structural formula are inconsistent, the structural formula shall prevail, unless the chemical name rather than the structural formula can be inferred from the context as correct. For simplicity, not all hydrogen atoms are explicitly labeled in the structural formulas of some compounds given in this application. When a vacant valence exists in a compound, it indicates the presence of unlabeled hydrogen atoms. It is understood that the element may be replaced by its isotope. For example, hydrogen in a compound or ADC may be H. 1 H 2 or H 3 .

[0597] Unless otherwise specified, all experimental materials and reagents used in the examples are commercially available or can be readily prepared using methods known to those skilled in the art. Specifically, monomethyl auristatin E (MMAE) was obtained from Jinan Chengquan Chemical Co., Ltd., batch number CQ20230301. DMSO was obtained from Sigma, catalog number 276855. TCEP was obtained from Aldrich, catalog number 646547. Dehydroascorbic acid (dhAA) was obtained from Aldrich, catalog number 261556.

[0598] The RP-HPLC conditions used to determine the DAR value of ADC are as follows:

[0599] The SEC-HPLC conditions used to determine ADC purity are as follows:

[0600] Unless otherwise specified, solvent ratios in this document are by volume. Abbreviations used herein (e.g., for chemical groups and compounds) generally have meanings well-known in the art, unless otherwise indicated.

[0601] Example 1. Development of anti-CEACAM5 antibody using the Single B technology platform

[0602] immunity

[0603] Balb / c mice (purchased from Vital River) were immunized with CEACAM5-overexpressing Chinese hamster ovary cells (glutamine system) GS-CHO cell line and extracellular fragment proteins. A total of seven immunizations were administered, with subcutaneous injections at two-week intervals between each immunization.

[0604] SingleB-based antibody screening

[0605] Once the serum titer met the requirements, the spleen of mice was harvested to obtain suspended lymphocytes. Next, B cells in the cell suspension were enriched using the EasySep Mouse Pan-B Cell Isolation Kit (Stemcell, 19844A). The enriched B cells were then sorted using an Aria III flow cytometer. After removing Human CEACAM1-positive cells, single B cells with bispecific Human CEACAM5 and Cyno CEACAM5 were sorted into 96-well PCR plates containing lysis buffer. The amplification products of the antibody heavy and light chain variable region genes were obtained through RT-PCR and nested PCR. The paired amplification products were purified, and the purified products were used to construct a linear expression cassette (LEC) via overlapping PCR and transfected into Expi293 cells. Simultaneously, the PCR products were used for Sanger sequencing to obtain the antibody variable region sequence.

[0606] Five days after LEC transfection and culture, the supernatant was collected for initial screening. First, ELISA was used to detect binding to the extracellular domains of Human CEACAM5 and Cyno CEACAM5, and flow cytometry was used to detect binding to overexpressing Human CEACAM5 and Cyno CEACAM5 cell lines. Simultaneously, negative screening was performed using family proteins (CEACAM1, CEACAM3, CEACAM6, and CEACAM8) and the overexpressing cell line (CEACAM1) to remove clones with cross-activity with other receptor molecules in the same family. Positive clones that bound to Human CEACAM5 and Cyno CEACAM5 in both ELISA and flow cytometry but not to other molecules in the same family were selected for evaluating endocytosis assays, and affinity was determined using thin-layer interferometry (BLI).

[0607] Expression and detection of candidate molecular chimeric antibodies

[0608] After analyzing the sequences based on diversity and PTM sites, 22 pairs of heavy and light chain variable region genes were selected for the construction of chimeric antibody eukaryotic expression plasmids. The heavy chain variable region was constructed into the Human IgG1 Fc (SEQ ID NO:24) expression vector, and the light chain variable region was constructed into the Human IgK (SEQ ID NO:30) expression vector. The plasmids were co-transfected into Expi293 cells for expression and purification.

[0609] The successfully expressed and purified antibody was functionally evaluated using cell binding and endocytosis assays. According to the cell experiments, mAb1 showed strong binding ability to the HT55 tumor cell line (IC50 = 3.31 nM), and also exhibited strong endocytic activity in the HCT116-CEACAM5 cell line (IC50 = 0.027 nM), making it an ideal candidate molecule.

[0610] Affinity assays for candidate molecules were performed using Fortebio to determine their affinity for human CEACAM5 (UniProt database accession number P06731), cynomolgus monkey CEACAM5, and related family proteins (human CEACAM1, CEACAM3, CEACAM6, and CEACAM8). Binning assays were also performed to detect their epitopes.

[0611] ForteBio affinity assays were performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody antigen affinity and epitope binning. MAbs, 2013.5(2): 270-8).

[0612] Half an hour before the experiment, according to the number of samples, take an appropriate number of AHC (18-5060, Sartorius) sensors and immerse them in SD buffer (PBS 1×, BSA 0.1%, Tween 20 0.05%).

[0613] 100 μl of SD buffer, antibody, and antigen [including human CEACAM5 (UniProt database accession number P06731), cynomolgus monkey CEACAM5 (Cyno CEACAM5), and related proteins (human CEACAM1, CEACAM3, CEACAM6, and CEACAM8)] were added to 96-well black polystyrene semi-mass microplates (Greiner, 675076). The plate was arranged according to the sample location, and the sensor position was selected. The instrument settings were as follows: Run steps: Baseline, Loading ~1 nm, Baseline, Association, and Dissociation; the run time for each step depended on the sample binding and dissociation rates, the rotation speed was 1000 rpm, and the temperature was 30℃. KD values ​​were analyzed using ForteBio analysis software.

[0614] As a result, mAb1 exhibited nM-level affinity for both human CEACAM5 and cynomolgus monkey CEACAM5, and did not bind to other homologous receptors in the same family. Therefore, mAb1 was ultimately selected as the candidate molecule for humanization.

[0615] Table 1. Affinity results of CEACAM5 candidate clones

[0616] "+" represents different bins, and "-" represents the same bin.

[0617] Example 2. Antibody Humanization

[0618] Using Discovery Studio software, the chimeric antibody mAb1 obtained above was humanized through the following steps: determining the CDR region of the chimeric antibody; screening for homologous sequences closest to the V / J regions of the heavy and light chains of the chimeric antibody in human germline sequence databases; constructing the CDR region of the chimeric antibody onto a human Germline backbone; determining the amino acid positions in the backbone region that maintain the CDR function based on sequence and structural characteristics, and performing reversion mutations (returning to the input amino acid type) at the identified important sequence positions; finally, synthesizing the sequence to prepare antibody mAb6. The amino acid sequences of the heavy and light chain variable regions of the humanized antibody mAb6 are shown in the sequence listing. mAb6 was prepared and purified as described in Example 1.

[0619] After obtaining the protein, the ability of the chimeric antibody and humanized antibody in this application to bind CEACAM5 was determined using the thin-layer interferometry (BLI) technique as described in Example 1 (see Table 2).

[0620] Table 2. Affinity constants (M) for ForteBio detection of human antigen-antibody binding

[0621] Based on the affinity data in Table 2, it can be concluded that the humanized molecule maintained an affinity comparable to that of the chimeric antibody.

[0622] Example 3. Preparation of linker toxin compounds

[0623] Example 3.1

[0624] (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8- Diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A1)

[0625] Step 1: Triacetic acid (2S,3R,4S,5S,6S)-2-(5-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester A1-2

[0626] Compound A1-1 (17.8 g, 44.8 mmol, supplier: Adamas-beta) and 3-hydroxy-4-nitrobenzaldehyde (5 g, 29.9 mmol) were dissolved in anhydrous acetonitrile (200 mL). Ag₂O (27.8 g, 119.8 mmol) was added at 0 °C. The mixture was heated to 25 °C and stirred in the dark for 24 hours. The reaction solution was filtered through a diatomaceous earth layer, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to give compound A1-2 (9.0 g, yield: 62%). LC-MS: ESI-MS (m / z): [M+H] + =484.1.

[0627] Step 2: Triacetic acid (2S,3R,4S,5S,6S)-2-(2-amino-5-hydroxymethylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester A1-3

[0628] Compound A1-2 (9.0 g, 18.6 mmol) was dissolved in ethyl acetate (400 mL), and palladium on carbon (10 wt%, 2.5 g) and triethylamine (376 mg, 3.7 mmol) were added. The reaction system was purged three times with hydrogen and stirred at room temperature for 24 hours under a hydrogen atmosphere. The reaction solution was filtered through a diatomaceous earth layer, and the filtrate was concentrated under reduced pressure to give compound A1-3 (8.0 g, yield: 94%). LC-MS: ESI-MS (m / z): [M+H] + =456.1.

[0629] Step 3: Triacetic acid (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)-5-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester Al-4

[0630] Compound A1-3 (419 mg, 1.0 mmol) and (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionic acid (425 mg, 0.93 mmol) were dissolved in dichloromethane (5 mL) and methanol (1 mL). N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (390 mg, 1.7 mmol) was added, and the mixture was stirred at room temperature for 16 hours. Dichloromethane (50 mL) was added to the reaction mixture, and the solution was washed successively with dilute hydrochloric acid (1 M, 50 mL), water (50 mL), and saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 9) to give compound A1-4 (500 mg, yield: 63%). LC-MS: ESI-MS (m / z): [M+H] + =848.3.

[0631] Step 4: Triacetic acid (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)-5-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester Al-5

[0632] Compound A1-4 (9.6 g, 11.3 mmol) and bis(4-nitrophenyl) carbonate (5.2 g, 17.1 mmol) were dissolved in dry N,N-dimethylformamide (70 mL), and N,N-diisopropylethylamine (4.4 g, 34.1 mmol) was added dropwise. The mixture was stirred at room temperature for 6 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to give compound A1-5 (10.0 g, yield: 87%). LC-MS: ESI-MS (m / z): [M+H] + =1013.3.

[0633] Step 5: Triacetic acid (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)-5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2) ... R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester A1-6

[0634] Compound A1-5 (6.8 g, 6.7 mmol), monomethylaurestatin E (MMAE, 4.0 g, 5.6 mmol), and 1-hydroxybenzotriazole (1.5 g, 11.1 mmol) were dissolved in dry N,N-dimethylformamide (50 mL), and N,N-diisopropylethylamine (2.2 g, 16.7 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to give compound A1-6 (8.0 g, yield: 89%). LC-MS: ESI-MS (m / z): [M+H] + =1591.8.

[0635] Step 6: (2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-amino-3-methylbutyrylamino)propionylamino)-5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1- Phenylacetyl-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid Al-7

[0636] Compound A1-6 (10.0 g, 6.5 mmol) was dissolved in a mixture of tetrahydrofuran and methanol (1 / 1 v / v, 100 mL). Lithium hydroxide solution (800 mg, 32.5 mmol, 100 mL water) was slowly added dropwise over 3 hours in an ice bath. The reaction mixture was stirred at room temperature for 3 hours. Diethylamine (20 mL) was added, and the mixture was stirred at room temperature for 6 hours. The reaction mixture was concentrated under reduced pressure to 160 mL. Glacial acetic acid (20 mL) was added, and the mixture was stirred at room temperature for 0.5 hours until the turbid solution turned into a yellow, clear solution. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 160 mL of methanol. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A1-7 (5.6 g, yield: 75%). LC-MS: ESI-MS (m / z): [M+H] + =1229.7.

[0637] Step 7: (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5 ,8-Diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A1

[0638] Compound A1-7 (4.1 g, 3.3 mmol) and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid 2,5-dioxopyrrolidine-1-yl ester (1.23 g, 4.0 mmol) were dissolved in dry dimethyl sulfoxide (15 mL), and N,N-diisopropylethylamine (645 mg, 5.0 mmol) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A1 (3.0 g, yield: 64%). LC-MS: ESI-MS (m / z): [M+H] + =1422.8.

[0639] Example 3.2

[0640] ((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((1S,2R)-1-phenyl-1-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propane-2-yl)amino)propyl) )pyrrolidine-1-yl)-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl ester (A2)

[0641] Step 1: ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (9H-fluorene-9-yl)methyl ester A2-2

[0642] Compound A2-1 (MMAE, 500 mg, 0.7 mmol) was dissolved in dry dichloromethane (10 mL), and 9-fluorenyl chloroformate (216 mg, 0.8 mmol) was added. N,N-diisopropylethylamine (135 mg, 1.0 mmol) was added under ice bath conditions, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was then washed with dichloromethane (50 mL) and brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to give compound A2-2 (620 mg, yield: 95%). LC-MS: ESI-MS (m / z): [M+H] + =940.2.

[0643] Step 2: Triacetic acid (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-((1S,2R)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-sec-butyl)-1-(9H-fluorene-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradecane-14-acyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionamide)-1-phenylpropoxy)tetrahydro-2H-pyran-3,4,5-triyl ester A2-3

[0644] Compound A2-2 (200 mg, 0.2 mmol) and triacetic acid (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl ester (524 mg, 1.1 mmol) were dissolved in a dry mixture of dichloromethane and acetonitrile (2 mL, 1 / 1 v / v). The reaction mixture was cooled to -20 °C, and trifluoromethanesulfonic acid (32 mg, 0.2 mmol) was slowly added dropwise at this temperature. The reaction mixture was slowly heated to 0 °C over 2 hours and quenched with triethylamine (0.1 mL). The mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to give compound A2-3 (200 mg, yield: 75%). LC-MS: ESI-MS (m / z): [M+H] + =1270.7.

[0645] Step 3: Triacetic acid (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-((1S,2R)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-1-phenylpropoxy)tetrahydro-2H-pyran-3,4,5-triyl ester A2-4

[0646] Compound A2-3 (200 mg, 0.16 mmol) was dissolved in dichloromethane (5 mL), and ethylenediamine (1 mL) was added. The reaction mixture was stirred at room temperature for 1 hour, and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to give compound A2-4 (132 mg, yield: 80%). LC-MS: ESI-MS (m / z): [M+H] + =1048.6.

[0647] Step 4: Triacetic acid (2R,3R,4S,5S,6R)-2-((1S,2R)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-1-(4-((S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)phenyl)-11 -((S)-sec-butyl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradecane-14-acyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-1-phenylpropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl ester A2-5

[0648] Compound A2-4 (132 mg, 0.13 mmol) and (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino))-1-oxopropyl-2-yl)amino)-1-oxobut-2-yl)carbamate (128 mg, 0.19 mmol) were dissolved in dry N,N-dimethylformamide (2 mL), and 1-hydroxybenzotriazole (25 mg, 0.19 mmol) and N,N-diisopropylethylamine (66 mg, 0.5 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give compound A2-5 (120 mg, yield: 50%). LC-MS: ESI-MS (m / z): [M+H]+ =1588.8.

[0649] Step 5: ((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((1S,2R)-1-phenyl-1-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran- 2-yl)oxy)propane-2-yl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 4-((S)-2-((S)-2-amino-3-methylbutyrylamino)propionylamino)benzyl ester A2-6

[0650] Compound A2-5 (100 mg, 0.06 mmol) was dissolved in a mixed solution of tetrahydrofuran and methanol (4 mL, 1 / 1 v / v). Lithium hydroxide aqueous solution (9 mg, 0.04 mmol, 1 mL) was slowly added dropwise under ice bath conditions. The reaction mixture was slowly brought to room temperature and stirred for 1 hour. Glacial acetic acid (0.3 mL) was added dropwise, and stirring continued for 20 minutes. The crude product was purified by silica gel column chromatography (C18 column, acetonitrile / water = 3 / 10, 0.05% formic acid as additive) to give compound A2-6 (37 mg, yield: 50%). LC-MS: ESI-MS (m / z): [M+H] + =1199.7.

[0651] Step 6: ((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((1S,2R)-1-phenyl-1-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propane-2-yl)amino )propyl)pyrrolidone-1-yl)-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolidone-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl ester A2

[0652] Compound A2-6 (37 mg, 0.03 mmol) was dissolved in dry dimethyl sulfoxide (1 mL), and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid ester (12 mg, 0.04 mmol) and N,N-diisopropylethylamine (6 mg, 0.05 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour, and the reaction was quenched dropwise with glacial acetic acid (0.1 mL). The crude product was purified by silica gel column chromatography (C18 column, acetonitrile / water = 3 / 10, 0.05% formic acid as additive) to give compound A2 (25 mg, yield: 60%). LC-MS: ESI-MS (m / z): [M+H] + =1392.8.

[0653] Example 3.3

[0654] (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((1S,2R)-1-phenyl-1-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy) Propane-2-yl)amino)propyl)pyrrolidine-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A3)

[0655] Step 1: Triacetic acid (2R,3R,4S,5S,6R)-2-((1S,2R)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-1-(4-((S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)-3-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6- (methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-11-((S)-sec-butyl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradecane-14-acyl)pyrrolin-2-yl)-3-methoxy-2-methylpropionamide)-1-phenylpropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl ester A3-1

[0656] Compounds A2-4 (100 mg, 0.095 mmol) and A1-5 (145 mg, 0.14 mmol) were dissolved in dry N,N-dimethylformamide (1 mL), and 1-hydroxybenzotriazole (19 mg, 0.14 mmol) and N,N-diisopropylethylamine (49 mg, 0.4 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give compound A3-1 (88 mg, yield: 48%). LC-MS: ESI-MS (m / z): [M+H] + =1920.9.

[0657] Step 2: (2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-amino-3-methylbutyrylamino)propionylamino)-5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((1S,2R)-1-phenyl-1 -((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propane-2-yl)amino)propyl)pyrrolidine-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A3-2

[0658] Compound A3-1 (88 mg, 0.05 mmol) was dissolved in a mixed solution of tetrahydrofuran and methanol (4 mL, 1 / 1 v / v). Lithium hydroxide aqueous solution (9 mg, 0.04 mmol, 1 mL) was slowly added dropwise under ice bath conditions. The reaction mixture was slowly brought to room temperature and stirred for 1 hour. Glacial acetic acid (0.3 mL) was added dropwise, and stirring continued for 20 minutes. The crude product was purified by silica gel column chromatography (C18 column, acetonitrile / water = 3 / 10, 0.05% formic acid as additive) to give compound A3-2 (25 mg, yield: 40%). LC-MS: ESI-MS (m / z): [M+H] + =1391.7.

[0659] Step 3: (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((1S,2R)-1-phenyl-1-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl) (Oxy)propane-2-yl)amino)propyl)pyrrolidine-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A3

[0660] Compound A3-2 (25 mg, 0.02 mmol) was dissolved in dry dimethyl sulfoxide (1 mL), and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid ester (7 mg, 0.02 mmol) and N,N-diisopropylethylamine (3.5 mg, 0.03 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour, and the reaction was quenched dropwise with glacial acetic acid (0.1 mL). The crude product was purified by silica gel column chromatography (C18 column, acetonitrile / water = 3 / 10, 0.05% formic acid as additive) to give compound A3 (10 mg, yield: 35%). LC-MS: ESI-MS (m / z): [M+H] + =1584.8.

[0661] Example 4. Preparation of linker toxin compounds

[0662] Example 4.1

[0663] N-((S)-1-(((S)-1-(((3 2 R,3 3 S,3 4 R,3 5 R,3 6 R,12 2 S,6S,7R,10R,11R,15R,16S,19S,22S)-16-((S)-sec-butyl)-3 3 ,3 4 ,3 5 -Trihydroxy-19,22-diisopropyl-11,15-dimethoxy-7,10,17,23-tetramethyl-4,9,13,18,21,24-hexaoxo-6-phenyl-3 3 ,3 4 ,3 5 ,3 6 -Tetrahydro-3 2 H-2,5,25-trioxa-8,17,20,23-tetraaza-12(2,1)-pyrrolidine-3(2,6)-pyranaza-1(1,3)-benzene-hexaban-1 6 -yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide (A4)

[0664] Compound A1 (30 mg, 0.02 mmol) was dissolved in anhydrous dimethyl sulfoxide (1 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6 mg, 0.0317 mmol) and 1-hydroxybenzotriazole (4 mg, 0.03 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 2 / 3, 0.1% formic acid as additive) to give compound A4 (20 mg, yield: 68%). LC-MS: ESI-MS (m / z): [M+H] + =1404.7

[0665] Example 4.2

[0666] (S)-N-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2-oxa-5,8,11-triazatridecane-13-yl)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)-6-(dipropylamino)hexanoamide (A5)

[0667] Step 1: Compound A5-1 (((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine tert-butyl ester (2 g, 4.72 mmol) was dissolved in methanol (25 mL). Catalytic amounts of glacial acetic acid (0.1 mL), propionaldehyde (680 mg, 11.8 mmol), and sodium cyanoborohydride (890 mg, 14.2 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure and diluted with dichloromethane (100 mL). The solution was washed with saturated sodium bicarbonate solution (100 mL) and saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 97 / 3) to give compound A5-2 (1.96 g, yield: 85%). LC-MS: ESI-MS (m / z): [M+H] + =509.7.

[0668] Step 2: Compound A5-2 (1.96 g, 3.86 mmol) was dissolved in N,N-dimethylformamide (10 mL), and diethylamine (1 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. The crude product was purified by slurrying with methyl tert-butyl ether to give compound A5-3 (1.1 g, yield: 99%). LC-MS: ESI-MS (m / z): [M+H] + =287.5.

[0669] Step 3: Compounds A5-3 (1.1 g, 3.84 mmol) and A5-4 (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (1.3 g, 3.84 mmol) were dissolved in N,N-dimethylformamide (5 mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (1.1 g, 5.76 mmol) and 1-hydroxybenzotriazole (777 mg, 5.76 mmol) were added. The mixture was stirred at room temperature for 4 hours. Ethyl acetate (50 mL) was added to the reaction solution, and the mixture was washed successively with dilute hydrochloric acid (0.5 M, 50 mL), water (50 mL), and saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give compound A5-5 (2 g, yield: 90%). LC-MS: ESI-MS (m / z): [M+H] + =608.8.

[0670] Step 4: Compound A5-5 (2 g, 3.29 mmol) was dissolved in N,N-dimethylformamide (5 mL), and diethylamine (1 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. The crude product was purified by slurrying with methyl tert-butyl ether to give compound A5-6 (1.2 g, yield: 95%). LC-MS: ESI-MS (m / z): [M+H] + =386.6.

[0671] Step 5: Compounds A5-6 (1.2 g, 3.1 mmol) and A5-7 (1.43 g, 4.6 mmol) were dissolved in dry dimethyl sulfoxide (15 mL), and N,N-diisopropylethylamine (645 mg, 5.0 mmol) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was lyophilized to obtain an oily crude product. The crude product was dissolved in dry dichloromethane (10 mL), and trifluoroacetic acid (2 mL) was added. The reaction mixture was removed under reduced pressure. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to obtain compound A5-8 (1.05 g, yield: 65%). LC-MS: ESI-MS (m / z): [M+H] + =523.7.

[0672] Step 6: Compounds A5-8 (26 mg, 0.0503 mmol) and A5-9 (30 mg, 0.0387 mmol) were dissolved in N,N-dimethylformamide (1 mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (15 mg, 0.075 mmol) and 1-hydroxybenzotriazole (10 mg, 0.075 mmol) were added. The mixture was stirred at room temperature for 4 hours. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A5 (24 mg, yield: 64%). LC-MS: ESI-MS (m / z): [M+H] + =998.3.

[0673] Preparation of intermediate A5-9

[0674] Step 1: Compound MMAE (100 mg, 0.139 mmol) and B5-1 (((9H-fluorene-9-yl)methoxy)carbonyl)glycine (50 mg, 0.167 mmol) were dissolved in N,N-dimethylformamide (2 mL). 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (48 mg, 0.251 mmol) and 1-hydroxybenzotriazole (34 mg, 0.251 mmol) were added, and the mixture was stirred at room temperature for 4 hours. Ethyl acetate (50 mL) was added to the reaction mixture, and the solution was washed successively with dilute hydrochloric acid (0.5 M, 50 mL), water (50 mL), and saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give compound B5-2 (125 mg, yield: 90%). LC-MS: ESI-MS (m / z): [M+H] + =998.3.

[0675] Step 2: Compound B5-2 (125 mg, 0.125 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (0.3 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 80 / 20) to give compound A5-9 (78 mg, yield: 80%). LC-MS: ESI-MS (m / z): [M+H] + =776.0.

[0676] Example 4.3

[0677] ((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-(I-1-methoxy-3-oxo-3-(((1S,2R)-1-phenyl-1-(phosphonooxy)propane-2-yl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl ester (A6)

[0678] Step 1: Compound A6-5 (4-aminophenyl)methanol (2 g, 16.2 mmol) and A6-6 (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionic acid (6.6 g, 16.2 mmol, supplier Adamas) were dissolved in dichloromethane (50 mL) and methanol (10 mL). N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (6 g, 24.3 mmol) was added, and the mixture was stirred at room temperature for 16 hours. Dichloromethane (100 mL) was added to the reaction mixture, and the mixture was filtered to obtain a grayish-white solid. The grayish-white solid was washed several times with dichloromethane to obtain compound A6-7 (7.5 g, yield: 90%). LC-MS: ESI-MS (m / z): [M+H] + =516.25.

[0679] Step 2: Compound A6-7 (1 g, 1.94 mmol) was dissolved in N,N-dimethylformamide (5 mL), and diethylamine (1 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. The crude product was purified by slurrying with methyl tert-butyl ether to give compound A6-8 (456 mg, yield: 80%). LC-MS: ESI-MS (m / z): [M+H] + =294.1.

[0680] Step 3: Compounds A6-8 (456 mg, 1.56 mmol) and A5-7 (720 mg, 2.34 mmol) were dissolved in dry dimethyl sulfoxide (5 mL), and N,N-diisopropylethylamine (400 mg, 3.12 mmol) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A6-9 (450 mg, yield: 59%). LC-MS: ESI-MS (m / z): [M+H] + =487.2.

[0681] Step 4: Compound A6-9 (450 mg, 0.924 mmol) and bis(4-nitrophenyl) carbonate (420 mg, 1.39 mmol) were dissolved in dry N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (357 mg, 2.77 mmol) was added dropwise. The mixture was stirred at room temperature for 6 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 97 / 3) to give compound A6-10 (516 mg, yield: 90%). LC-MS: ESI-MS (m / z): [M+H] + =652.3.

[0682] Step 5: Compound A6-10 (49 mg, 0.075 mmol), A6-4 (MMAE-phosphate, 30 mg, 0.038 mmol), and 1-hydroxybenzotriazole (10 mg, 0.075 mmol) were dissolved in dry N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (29 mg, 0.225 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A6 (10 mg, yield: 20%). LC-MS: ESI-MS (m / z): [M+H] + =1310.7.

[0683] Preparation of intermediate A6-4

[0684] Step 1: Compound A3-2 (419 mg, 0.445 mmol) and A6-1 dibenzyldiisopropylphosphonamide (307 mg, 0.891 mmol) were dissolved in dry acetonitrile (5 mL), and 1H-tetrazole (70 mg, 1 mmol) and N,N-diisopropylethylamine (128 mg, 1 mmol) were added. The mixture was stirred at room temperature for 4 hours. Ethyl acetate (50 mL) was added to the reaction solution, and the mixture was washed successively with water (50 mL) and saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 98 / 2) to give compound A6-2 (373 mg, yield: 70%). LC-MS: ESI-MS (m / z): [M+H] + =1201.5.

[0685] Step 2: Compound A6-2 (373 mg, 0.31 mmol) was dissolved in tetrahydrofuran and methanol (5 mL, 1 / 1 v / v), and catalytic amounts of glacial acetic acid (0.1 mL) and palladium on carbon (10% wt, 50 mg) were added. The mixture was stirred at room temperature for 6 hours under hydrogen balloon pressure. The palladium on carbon was filtered through diatomaceous earth, and the resulting reaction solution was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 85 / 15) to give compound A6-3 (218 mg, yield: 69%). LC-MS: ESI-MS (m / z): [M+H] + =1021.2.

[0686] Step 3: Compound A6-3 (218 mg, 0.21 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (0.2 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A6-4 (125 mg, yield: 75%). LC-MS: ESI-MS (m / z): [M+H] + =799.5.

[0687] Example 4.4

[0688] ((2S)-1-(((2S)-1-(((3R,4S,5S)-1-((2S)-2-((1R,2R)-3-(((1S,2R))-1-((hydroxy(phosphono)phosphoyl)oxy)-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl- 1-Oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl ester (A7)

[0689] Step 1: Compound A6-3 (200 mg, 0.196 mmol) and A6-1 dibenzyldiisopropylphosphonamide (135 mg, 0.392 mmol) were dissolved in dry acetonitrile (2 mL). 1H-tetrazole (27 mg, 0.392 mmol) and N,N-diisopropylethylamine (51 mg, 0.392 mmol) were added, and the mixture was stirred at room temperature for 6 hours. Ethyl acetate (20 mL) was added to the reaction mixture, and the solution was washed successively with water (20 mL) and saturated brine (20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 90 / 10) to give compound A7-1 (150 mg, yield: 60%). LC-MS: ESI-MS (m / z): [M+H] + =1281.6.

[0690] Step 2: Compound A7-1 (150 mg, 0.117 mmol) was dissolved in a mixture of tetrahydrofuran and methanol (10 mL, 1 / 1 v / v), and catalytic amounts of glacial acetic acid (0.1 mL) and palladium on carbon (10% wt, 40 mg, 1.7 mmol) were added. The mixture was stirred at room temperature for 2 hours under hydrogen balloon pressure. The palladium on carbon was filtered through diatomaceous earth, and the resulting reaction solution was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 70 / 30) to give compound A7-2 (83 mg, yield: 65%). LC-MS: ESI-MS (m / z): [M+H] + =1100.5.

[0691] Step 3: Compound A7-2 (83 mg, 0.075 mmol) was dissolved in N,N-dimethylformamide (1 mL), and diethylamine (0.2 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A7-3 (46 mg, yield: 70%). LC-MS: ESI-MS (m / z): [M+H] + =878.4.

[0692] Step 4: Compounds A6-10 (59 mg, 0.0912 mmol), A7-3 (MMAE-pyrophosphate, 40 mg, 0.0456 mmol), and 1-hydroxybenzotriazole (12 mg, 0.0912 mmol) were dissolved in dry N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (23 mg, 0.18 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A7 (9 mg, yield: 15%). LC-MS: ESI-MS (m / z): [M+H] + =1390.7.

[0693] Example 4.5

[0694] ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 3-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)acetamido)-4-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)benzyl ester (A8)

[0695] Step 1: Compound A8-1 (500 mg, 1.15 mmol, referencing patents WO2020043129 A1 and WO2024067841A1) and bis(4-nitrophenyl) carbonate (524 mg, 1.72 mmol) were dissolved in dry N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (297 mg, 2.3 mmol) was added dropwise. The mixture was stirred at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 97 / 3) to give compound A8-2 (627 mg, yield: 91%). LC-MS: ESI-MS (m / z): [M+H] + =601.2.

[0696] Step 2: Compound A8-2 (627 mg, 1.05 mmol), monomethylaurestatin E (MMAE, 527 mg, 0.74 mmol), and 1-hydroxybenzotriazole (140 mg, 1.05 mmol) were dissolved in dry N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (270 mg, 2.1 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 96 / 4) to give compound A8-3 (628 mg, yield: 72%). LC-MS: ESI-MS (m / z): [M+H] + =1179.7.

[0697] Step 3: Compound A8-3 (628 mg, 0.533 mmol) was dissolved in dry dichloromethane (5 mL), and trifluoroacetic acid solution (1 mL) was added dropwise at 0 °C. The mixture was stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 90 / 10) to obtain compound A8-4 (511 mg, yield: 89%). LC-MS: ESI-MS (m / z): [M+H] + =1079.6.

[0698] Step 4: Compound A8-4 (511 mg, 0.474 mmol) and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid 2,5-dioxopyrrolidine-1-yl ester (218 mg, 0.71 mmol) were dissolved in dry dimethyl sulfoxide (5 mL), and N,N-diisopropylethylamine (137 mg, 1.07 mmol) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A8 (500 mg, yield: 83%). LC-MS: ESI-MS (m / z): [M+H] + =1272.7.

[0699] Example 4.6

[0700] (2S,3S,4S,5R,6S)-6-(4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2 (-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)propionylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A9)

[0701] Step 1: Compound A1-1 (5 g, 12.6 mmol, supplier: Adamas-beta) and A9-1 4-hydroxy-3-nitrobenzaldehyde (527 mg, 3.2 mmol) were dissolved in anhydrous acetonitrile (30 mL), and Ag₂O (3 g, 12.8 mmol) was added at 0 °C. The mixture was heated to 25 °C and stirred in the dark for 24 hours. The reaction solution was filtered through a diatomaceous earth layer, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to give compound A9-2 (1.0 g, yield: 65%). LC-MS: ESI-MS (m / z): [M+H] + =484.1.

[0702] Step 2: Compound A9-2 (1.0 g, 2.07 mmol) was dissolved in ethyl acetate (50 mL), and palladium on carbon (10 wt%, 100 mg) and triethylamine (42 mg, 0.41 mmol) were added. The reaction system was purged three times with hydrogen and stirred at room temperature for 24 hours under a hydrogen atmosphere. The reaction solution was filtered through a diatomaceous earth layer, and the filtrate was concentrated under reduced pressure to give compound A9-3 (895 mg, yield: 95%). LC-MS: ESI-MS (m / z): [M+H] + =456.1.

[0703] Step 3: Compound A9-3 (895 mg, 1.97 mmol) and A9-4 3-((tert-butoxycarbonyl)amino)propionic acid (446 mg, 2.36 mmol) were dissolved in dichloromethane (5 mL) and methanol (1 mL). N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (741 mg, 3.0 mmol) was added, and the mixture was stirred at room temperature for 16 hours. Dichloromethane (50 mL) was added to the reaction mixture, and the solution was washed successively with dilute hydrochloric acid (0.5 M, 50 mL), water (50 mL), and saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to give compound A9-5 (851 mg, yield: 69%). LC-MS: ESI-MS (m / z): [M+H] + =627.2.

[0704] Step 4: Compound A9-5 (200 mg, 0.319 mmol) and bis(4-nitrophenyl) carbonate (145 mg, 0.479 mmol) were dissolved in dry N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (82 mg, 0.638 mmol) was added dropwise. The mixture was stirred at room temperature for 6 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to give compound A9-6 (219 mg, yield: 87%). LC-MS: ESI-MS (m / z): [M+H] + =792.2.

[0705] Step 5: Compound A9-6 (219 mg, 0.276 mmol), monomethylaurestatin E (MMAE, 139 mg, 0.194 mmol), and 1-hydroxybenzotriazole (37 mg, 0.276 mmol) were dissolved in dry N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (53 mg, 0.414 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 97 / 3) to give compound A9-7 (189 mg, yield: 72%). LC-MS: ESI-MS (m / z): [M+H] + =1370.7.

[0706] Step 6: Compound A9-7 (189 mg, 0.139 mmol) was dissolved in a mixed solution of tetrahydrofuran and methanol (1 / 1 v / v, 6 mL). Under ice bath conditions, lithium hydroxide solution (20 mg, 0.834 mmol, 1 mL water) was slowly added dropwise over 10 minutes. The reaction mixture was stirred at room temperature for 1 hour. Glacial acetic acid was added and stirred for 30 minutes. The solvent was removed by vacuum distillation. The crude product was dissolved in dry dichloromethane (5 mL), and trifluoroacetic acid (1 mL) was added dropwise at 0 °C, with stirring at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A9-8 (77 mg, yield: 50%). LC-MS: ESI-MS (m / z): [M+H] + =1130.6.

[0707] Step 7: Compound A9-8 (40 mg, 0.0358 mmol) and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid 2,5-dioxopyrrolidine-1-yl ester (17 mg, 0.0538 mmol) were dissolved in dry dimethyl sulfoxide (15 mL), and N,N-diisopropylethylamine (10 mg, 0.0807 mmol) was added in an ice bath. The reaction mixture was stirred at room temperature for 2 hours. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A9 (32 mg, yield: 70%). LC-MS: ESI-MS (m / z): [M+H] + =1323.7.

[0708] Example 4.7

[0709] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-(I-1-methoxy-3-oxo-3-((I-1-phenylprop-2-yl)amino)propyl)pyrrolo-1-yl)-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (A10)

[0710] Step 1: Dissolve MMAE (200 mg, 0.278 mmol) in methanol (10 mL), and add catalytic amounts of glacial acetic acid (0.1 mL) and palladium on carbon (10% wt, 50 mg) to the reaction system. Stir the reaction system under a hydrogen atmosphere for 4 hours. Filter the palladium on carbon through a diatomaceous earth layer, concentrate the filtrate under reduced pressure, and purify the crude product by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to obtain compound A10-1 (173 mg, yield: 89%). LC-MS: ESI-MS (m / z): [M+H] + =702.5.

[0711] Step 2: Compounds A6-10 (55 mg, 0.0856 mmol), A10-1 (MMAE analog, 30 mg, 0.0428 mmol), and 1-hydroxybenzotriazole (11 mg, 0.0856 mmol) were dissolved in dry N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (17 mg, 0.128 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A10 (20 mg, yield: 40%). LC-MS: ESI-MS (m / z): [M+H] + =1214.7.

[0712] Example 4.8

[0713] (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-(I-1-methoxy-3-oxo-3-((I-1-phenylprop-2-yl)amino)propyl)pyrrolidine-1-yl)-2-oxoethyl)-4,10-dimethyl (A11)-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-((S)-2-((S)-2-(6)-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0714] Step 1: Compounds A1-5 (216 mg, 0.214 mmol), A10-1 (100 mg, 0.142 mmol), and 1-hydroxybenzotriazole (28 mg, 0.214 mmol) were dissolved in dry N,N-dimethylformamide (3 mL), and N,N-diisopropylethylamine (41 mg, 0.321 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give compound A11-1 (163 mg, yield: 73%). LC-MS: ESI-MS (m / z): [M+H] + =1575.8.

[0715] Step 2: Compound A11-1 (163 mg, 0.103 mmol) was dissolved in a mixed solution of tetrahydrofuran and methanol (1 / 1 v / v, 6 mL). Under ice bath conditions, lithium hydroxide solution (15 mg, 0.618 mmol, 1 mL water) was slowly added dropwise over 30 minutes. The reaction was brought to room temperature and stirred for 1 hour. Glacial acetic acid was added and stirred for 30 minutes. The solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A11-2 (74 mg, yield: 60%). LC-MS: ESI-MS (m / z): [M+H] + =1213.7.

[0716] Step 3: Compound A11-2 (30 mg, 0.0247 mmol) and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid 2,5-dioxopyrrolidine-1-yl ester (10 mg, 0.03 mmol) were dissolved in dry dimethyl sulfoxide (1 mL), and N,N-diisopropylethylamine (6 mg, 0.045 mmol) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A11 (20 mg, yield: 59%). LC-MS: ESI-MS (m / z): [M+H] + =1406.8.

[0717] Example 5

[0718] Preparation of 15-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-N-((S)-1-(((S)-1-(((3-((S)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-11-yl)propoxy)methyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-3,6,9,12-tetraoxapentadecanamide (NT2)

[0719] Step 1: Zn(OAc)₂ (16 mg, 0.088 mmol) was added to a solution of D1 (50 mg, 0.11 mmol) and compound 8 (79 mg, 0.25 mmol) in 1,4-dioxane (10 mL) at room temperature. The reaction mixture was stirred at 80 °C for 12 hours. The mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM / MeOH = 50:1) to give compound 13 as a yellow solid (55 mg, 0.062 mmol, 57% yield). LC-MS: ESI-MS (m / z): [M+H] + =876.3.

[0720] Step 2: Compound 13 (55 mg, 0.062 mmol) was dissolved in DCM (5 ml), and then diethylamine (2 ml) was added to the solution. The reaction mixture was stirred at room temperature for 4 hours. The mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM / MeOH = 10:1) to give compound 14 as a yellow solid (37 mg, 0.057 mmol, 92% yield). LC-MS: ESI-MS (m / z): [M+H] + =654.3.

[0721] Step 3: Triethylamine (11 mg, 0.114 mmol) was added to a solution of compound 14 (37 mg, 0.057 mmol) and Mal-Peg4-NHS ester (30 mg, 0.068 mmol) in DCM (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 12 hours. The mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM / MeOH = 20:1) to give NT2 as a white solid (24 mg, 0.024 mmol, 42% yield). LC-MS: ESI-MS (m / z): [M+H] + =981.4.

[0722] Example 6. Antibody Modification and Expression

[0723] The antibodies used in this project to prepare the dual-toxin ADC are modified from the humanized antibody mAb6 in Example 2. mAb7 is mAb6 with an LALA mutation. mAb8 is mAb7 with amino acid 160 on both light chains of the mAb7 antibody mutated to cysteine. mAb9 is mAb6 with amino acid 160 on one light chain of the mAb6 antibody mutated to cysteine, and with knock-in-hole and LALA mutations (L234A, L235A) on the heavy chain (Knob: S354C, T366W; Hole: Y349C, T366S, L368A, Y407V). mAb10 is mAb6 with amino acid 239 on one heavy chain of the mAb6 antibody mutated to cysteine, and with knock-in-hole (Knob(S354C+T366W); Hole(Y349C / T366S / L368A / Y407V) and LALA mutations.

[0724] mAb11 is based on the negative control protein anti-GP120. The amino acid at position 160 of one light chain of the antibody is mutated to cysteine, and the heavy chain is knob-in-hole and LALA is mutated. The ADC conjugated with this antibody is used as the negative control group in this study.

[0725] The specific modifications are as follows:

[0726] 6.1 Plasmid construction: The sequence was synthesized by Genewiz and loaded into the pcDNA3.1 (Invitrogen) vector.

[0727] 6.2 Protein Expression: Protein was produced using the ExpiCHO™ Expression system (Gibco, A29133). Specifically, ExpiCHO-STM cells (Gibco) were passaged according to the required transfection volume, and the cell density was adjusted to 3.5 × 10⁶ cells / day before transfection. 6 Cells / ml. On the day of transfection, the cell density was adjusted to 6 × 10⁶ cells / ml. 6Cells / ml. Take a 50ml centrifuge tube and add OptiPRO™ SFM (Gibco, 12309019) transfection buffer at 8% of the cell volume. Calculate the total required plasmid amount based on 0.8μg / ml transfection of cells. The mass ratio of light chain to heavy chain plasmid for mAb7 is 1:1. The parental lines of mAb9 and mAb11 (two double antibodies) are expressed separately, with a light-to-heavy chain mass ratio of 1:1. The mass ratio of light chain to heavy chain 1 to heavy chain 2 for mAb10 is 1:1:1. Filter the transfection buffer containing DNA plasmids through a 0.22μm filter membrane into a new 50ml centrifuge tube. Add ExpiFectamine™ CHO at a DNA:Reagent ratio of 1:4 to the filtered mixture. Mix reagent (Gibco, 100033022) thoroughly. Immediately and slowly add the mixture of transfection reagent and plasmid DNA to the cells while gently shaking the flask. Control the incubation time between the transfection reagent and plasmid to no more than 5 minutes. Incubate at 37°C with 8% CO2. After 18-22 hours, add Enhancer (Gibco, 100033019) at 6 μL / ml cell volume and Feed (Gibco, A29101-01) at 300 μL / ml cell volume. Incubate at 37°C, 120 rpm, and 8% CO2. Continue culturing until day 7 or when cell viability is ≤60%, then collect the cell culture. Mix the cell culture with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per L cell culture), and filter using a 0.22 μm disposable vacuum filter. Use the supernatant for subsequent affinity purification.

[0728] 6.3 Affinity chromatography purification of target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1M NaOH were passed through the tubing and the affinity chromatography column, followed by 10-20 column volumes of distilled water to wash the tubing and the column. The packing column was equilibrated with 5 column volumes of 1×PBS (Gibco). The filtered cell material was passed through the column, and the packing column was washed with 10 column volumes of 1×PBS to remove non-specifically bound proteins. The packing column was washed with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5), the eluent was collected, the pH was adjusted to 6.0 with 2M Tris, and the solution was filtered for sterilization.

[0729] 6.4 In vitro reduction and oxidation of antibodies: Parental mAb9 and mAb11, expressed and purified separately, were mixed at a molar ratio of 1:1, and an appropriate amount of GSH was added. The pH of the reaction was adjusted to 8.0 with 2M Tris. The mixture was incubated overnight at room temperature. The reaction mixture was then transferred to PBS and stored at 4°C for later use.

[0730] 6.5 Ion Exchange Chromatography for Antibody Purification: A Mono S 5 / 50GL ion exchange chromatography column (from GE Healthcare) was used and placed in an AKTApure system (from GE Healthcare). Endotoxins were removed from the AKTApure system equipped with the Mono S 5 / 50GL ion exchange chromatography column using 0.5M NaOH for 2 hours. The system and column were then washed with distilled water. The column was equilibrated with 5-10 column volumes of loading buffer (20mM NaPO4, pH 6.6) until conductivity and pH stabilized. The protein obtained from affinity chromatography was diluted 10-fold with loading buffer and then loaded. The column was reequilibrated with 5 column volumes of loading buffer. Linear elution was performed using a gradient of 0-40% elution buffer (20mM NaPO4, 1M NaCl, pH 6.6) for a total of 30 column volumes. Samples were collected based on UV absorbance.

[0731] The purity of the samples collected in each fraction was determined using size exclusion chromatography (SEC). Samples from fractions with a purity greater than 95% were pooled based on the SEC results.

[0732] The purified bispecific antibody solution was centrifuged in a 15 ml ultrafiltration centrifuge tube at 4500 rpm for 30 minutes. The protein was then diluted with PBS and centrifuged again at 4500 rpm for 30 minutes. This process was repeated several times, changing the buffer solution each time. The antibodies were pooled after each buffer change, and the antibody concentration was measured. Further qualitative and quantitative analysis of the bispecific antibody composition and content was performed using a combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).

[0733] Example 7: Preparation and physicochemical characterization of antibody-drug conjugates targeting CEACAM5

[0734] 7.1 Preparation and characterization of bitoxin ADC1 and monotoxin ADC2

[0735] The single-toxin ADC2 was prepared using Thiomab conjugation. The dual-toxin ADC1 was prepared using a sequential conjugation method: first, A1 was linked to the engineered cysteine ​​residue of the antibody light chain using the Thiomab conjugation method to obtain ADC2; then, the interchain disulfide bonds of ADC2 were opened using a conventional conjugation method, and NT3 was conjugated (e.g., 8) to obtain the dual-toxin ADC1. The specific operation is as follows:

[0736] (a) Dissolve antibody mAb9 in histidine buffer (20 mM, pH 6.5). Add the reducing agent TCEP aqueous solution and incubate the reaction mixture at room temperature for 2 hours, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal TCEP / mAb molar ratio is 10–20; (iii) the optimal reaction temperature is 25 °C; and (iv) the optimal reaction pH is 6.0–8.0. After reduction, remove the reducing agent by desalting, ultrafiltration, or dialysis. Add the oxidizing agent dehydroascorbic acid (dhAA, dissolved in DMSO) and oxidize in a water bath for 2–3 hours, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal dhAA / mAb molar ratio is 20–40; (iii) the optimal reaction temperature is 20–37 °C; and (iv) the optimal reaction pH is 6.0–8.0. An excess of linker-toxin A1 (dissolved in DMSO) was added to the mixture, with the volume ratio of DMSO in the solution being 10%. The reaction mixture was left at room temperature for 1–2 hours, wherein (i) the optimal molar ratio of A1 / mAb was 3–6, and (ii) the optimal reaction temperature was 20–37 °C. The crude product was purified by spin desalting, ultrafiltration, or dialysis to obtain ADC2.

[0737] (b) Add an aqueous solution of the reducing agent TCEP to the ADC2 obtained in step (a), and react the reaction mixture at 20–37°C for 3 hours, wherein: (i) the optimal concentration of ADC2 is 5–15 mg / mL; (ii) the optimal molar ratio of TCEP / mAb is 8–20; and (iii) the optimal pH of the reaction is between 6.0 and 8.0. Add excess NT3 (dissolved in DMSO) to the mixture, and allow the reaction mixture to stand at room temperature for 1–2 hours, wherein: (i) the optimal molar ratio of NT1 / mAb is 10–16; and (ii) the optimal temperature of the reaction is 20–37°C, thereby obtaining crude ADC1. The crude product is purified by spin desalting, ultrafiltration, or dialysis to obtain the final product ADC1.

[0738] The DAR values ​​of ADCs were determined using RP-HPLC. The DAR values ​​were calculated based on the peak areas at UV 280 nm. Figures 1a and 1b show the RP plots used for DAR value analysis and calculation for ADC1, respectively. For A1 DAR value calculation: LC represents the component without drug linkage, and LC+A1 represents the component coupled with one A1 drug. For NT3 DAR value calculation: LC represents the light chain component without drug linkage, LC+NT1 and LC+A1+NT3 represent the light chain components coupled with one drug, and HC+NT1*1, HC+NT3*2, and HC+NT3*3 represent the heavy chain components coupled with 1, 2, and 3 drugs, respectively. The calculated DAR value for A1 of ADC1 was 0.91, and the DAR value for NT3 was 7.5. The DAR value for A1 of ADC2 was 0.87.

[0739] The purity of the ADCs was determined using SEC-HPLC. The purity of the ADC was obtained based on the peak area ratio of monomers, aggregates, and oligomers at UV 280 nm. Figure 1c shows the purity analysis of ADC1. It can be seen that this ADC contains only a small amount of oligomers, with most components being homogeneous monomers, and a purity of 96.32%. The purity of ADC2 was determined to be 97.73%.

[0740] 7.2 Preparation and Characterization of Monotoxin ADC3

[0741] The mAb7 antibody was dissolved in 20 mM histidine buffer, and a 20-fold molar ratio of TCEP reducing agent aqueous solution was added. After reacting the reaction mixture at room temperature for 2 hours, an excess of the linker-toxin NT3 (dissolved in DMSO) was added, and the reaction was continued at room temperature for 1-2 hours. Then, Zeba was used. TM Unreacted small molecule drugs and other impurities were removed using a desalting centrifuge column, and the ADC was then displaced into a 20 mM histidine solution at pH 5.5 for storage. ADC3 was analyzed using RP-HPLC and SEC-HPLC, yielding a DAR of 7.81 and an SEC purity of 95.25%.

[0742] 7.3 Preparation and Characterization of Monotoxin ADC4

[0743] Antibody mAb8 was dissolved in histidine buffer (20 mM, pH 6.5). A reducing agent, TCEP aqueous solution, was added, and the reaction mixture was incubated at room temperature for 2 hours. The antibody concentration was 5 mg / mL, and the TCEP / mAb molar ratio was 20. After reduction, the reducing agent was removed by desalting, ultrafiltration, or dialysis. Oxidizing agent dhAA (dissolved in DMSO) was added, and oxidation was carried out at 37°C for 3 hours. The antibody concentration was 5 mg / mL, and the dhAA / mAb molar ratio was 40. An excess of linker-toxin A1 (dissolved in DMSO) was added to the mixture, with a DMSO volume ratio of 10%. The reaction mixture was incubated at room temperature for 1–2 hours. The A1 / mAb molar ratio was 6, and the reaction temperature was 20–37°C. The resulting crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain ADC4.

[0744] ADC4 was analyzed using RP-HPLC and SEC-HPLC, and the DAR of ADC4 was found to be 1.91, with a purity of 99.34%.

[0745] 7.4 Preparation and Characterization of Bitoxin ADC5

[0746] ADC4 was prepared according to method 7.3. A 20-fold molar ratio of the reducing agent TCEP aqueous solution was added to a solution of ADC4 in histidine buffer. The reaction mixture was incubated at 20–37°C for 3 hours, wherein the concentration of ADC4 was 5 mg / mL, and the optimal pH for the reaction was between 6.0 and 8.0. Excess NT3 (dissolved in DMSO) was added, and the reaction mixture was incubated at room temperature for 1–2 hours, wherein: (i) the optimal molar ratio of NT3 / mAb was 10–16, and (ii) the optimal reaction temperature was 20–37°C, thereby obtaining crude ADC5. The obtained crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain the final product ADC5.

[0747] ADC5 was analyzed using RP-HPLC and SEC-HPLC, and the DAR value of A1 for ADC5 was 1.88, the DAR value of NT3 was 7.83, and the purity of SEC was 97.71%.

[0748] 7.5 Preparation and Characterization of ADC6 and ADC7

[0749] The same procedure as in 7.1 for preparing ADC2 was used, except that A2 was used instead of A1 to obtain ADC6.

[0750] The same procedure as in 7.1 was used to prepare ADC1, except that A2 was used instead of A1 to obtain ADC7.

[0751] ADCs were analyzed using RP-HPLC and SEC-HPLC. The results showed that the DAR(A2) of ADC6 was 0.90 and the SEC purity was 99.06%; the DAR of A2 of ADC7 was 0.90, the DAR of NT3 was 7.64, and the SEC purity was 96.12%.

[0752] 7.6 Preparation and Characterization of ADC8 and ADC9

[0753] The same procedure as in 7.1 was used to prepare ADC2, except that A3 was used instead of A1 to obtain ADC8.

[0754] The same procedure as in 7.1 was used to prepare ADC1, except that A3 was used instead of A1 to obtain ADC9.

[0755] ADCs were analyzed using RP-HPLC and SEC-HPLC. The results showed that the DAR (A3) of ADC8 was 0.87 and the SEC purity was 99.15%; the DAR of A3 for ADC9 was 0.87, the DAR of NT3 was 7.37, and the SEC purity was 96.37%.

[0756] 7.7 Preparation and Characterization of ADC10 and ADC11

[0757] (a) Dissolve antibody mAb10 in histidine buffer (20 mM, pH 6.5). Add the reducing agent TCEP aqueous solution and incubate the reaction mixture at room temperature for 2 hours, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal TCEP / mAb molar ratio is 10–20; (iii) the optimal reaction temperature is 25 °C; and (iv) the optimal reaction pH is 6.0–8.0. After reduction, remove the reducing agent by desalting, ultrafiltration, or dialysis. Add dhAA oxidant (dissolved in DMSO) and oxidize in a water bath for 2–3 hours, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal dhAA / mAb molar ratio is 20–40; (iii) the optimal reaction temperature is 20–37 °C; and (iv) the optimal reaction pH is 6.0–8.0. An excess of the linker-toxin mc-vc-PAB-MMAE (dissolved in DMSO) was added to the reactants, with the volume ratio of DMSO in the solution being 10%. The reaction mixture was allowed to stand at room temperature for 1–2 hours, wherein: (i) the optimal molar ratio of mc-vcPAB-MMAE / mAb was 3–6, and (ii) the optimal reaction temperature was 20–37 °C. The resulting crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain ADC10, which was stored in histidine buffer.

[0758] (b) Add an aqueous solution of the reducing agent TCEP to the above ADC10 solution, and react the reaction mixture at 20–37°C for 3 hours, wherein: (i) the optimal concentration of ADC10 is 5–15 mg / mL; (ii) the optimal molar ratio of TCEP / mAb is 8–20; and (iii) the optimal pH of the reaction is between 6.0 and 8.0. Add excess NT3 (dissolved in DMSO) to the reactants, and place the reaction mixture at room temperature for 1–2 hours, wherein: (i) the optimal molar ratio of NT3 / mAb is 10–16; and (ii) the optimal temperature of the reaction is 20–37°C, thereby obtaining crude ADC11. Purify the obtained crude ADC product by spin desalting, ultrafiltration, or dialysis to obtain the final product ADC11.

[0759] ADCs were analyzed using RP-HPLC and SEC-HPLC. The results showed that the DAR (MMAE) of ADC10 was 0.91 and the SEC purity was 96.53%; the DAR of MMAE for ADC11 was 0.91, the DAR for NT3 was 7.8, and the SEC purity was 95.89%.

[0760] 7.8 Preparation and Characterization of ADC12 and ADC13

[0761] (a) Dissolve antibody mAb10 in histidine buffer (20 mM, pH 6.5). Add the reducing agent TCEP aqueous solution and react the reaction mixture at room temperature for 2 hours, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal TCEP / mAb molar ratio is 10–20; (iii) the optimal reaction temperature is 25 °C; and (iv) the optimal reaction pH is 6.0–8.0. After reduction, remove the reducing agent by desalting, ultrafiltration, or dialysis. Add dhAA oxidant (dissolved in DMSO) and oxidize in a water bath for 2–3 hours, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal dhAA / mAb molar ratio is 20–40; (iii) the optimal reaction temperature is 20–37 °C; and (iv) the optimal reaction pH is 6.0–8.0. Add excess linker-toxin A1 (dissolved in DMSO) to the reactants, with the volume percentage of DMSO in the solution being 10%. The reaction mixture was placed at room temperature for 1–2 hours, wherein: (i) the optimal molar ratio of Al / mAb was 3–6, and (ii) the optimal reaction temperature was 20–37 °C. The obtained crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain ADC12, which was stored in histidine buffer.

[0762] (b) Add an aqueous solution of TCEP, a reducing agent, to the above ADC12 solution. Incubate the reaction mixture at 20–37°C for 3 hours, where: (i) the optimal concentration of ADC12 is 5–15 mg / mL; (ii) the optimal molar ratio of TCEP / mAb is 8–20; and (iii) the optimal pH of the reaction is between 6.0 and 8.0. Add excess NT3 (dissolved in DMSO), and incubate the reaction mixture at room temperature for 1–2 hours, where: (i) the optimal molar ratio of NT3 / mAb is 10–16; and (ii) the optimal reaction temperature is 20–37°C, thereby obtaining crude ADC13. The obtained crude ADC product is purified by spin desalting, ultrafiltration, or dialysis to obtain the final product ADC13.

[0763] ADCs were analyzed using RP-HPLC and SEC-HPLC. The results showed that the DAR of ADC12 was 0.93 and the SEC purity was 96.39%; the DAR of A1 of ADC13 was 0.93, the DAR of NT3 was 7.7, and the SEC purity was 96.15%.

[0764] 7.9 Preparation of positive control molecule M9140 and negative control molecule ADC14

[0765] M9140 ADC is a positive control ADC. Its antibody sequence is derived from so8G4 in WO2022048883A1, with the heavy chain sequence being SEQ ID NO:13 and the light chain sequence being SEQ ID NO:14. The antibody was dissolved in 20 mM histidine buffer (pH 6.5), and a 20-fold molar ratio of reducing agent TCEP aqueous solution was added. The reaction mixture was incubated at room temperature for 2 hours. Then, an excess of the linker-toxin Mal-Gly-PAB-ethatecan-D-glucuronic acid (MCE, HY-153179) was added, and the reaction was continued at room temperature for 1-2 hours. Finally, Zeba was used. TM Unreacted small molecule drugs and other impurities were removed using a desalting centrifuge column, and the ADC was then displaced into a 20 mM histidine solution at pH 5.5 for storage. The ADC was analyzed using RP-HPLC and SEC-HPLC. The results showed that the DAR value of the M9140 ADC was 7.95, and the SEC purity was 100%.

[0766] The negative control molecule ADC14 was prepared using the same method as ADC1, except that mAb11 was used as the negative control antibody. ADC14 was analyzed using RP-HPLC and SEC-HPLC. The results showed that the DAR for A1 of ADC14 was 0.95, the DAR for NT3 was 7.82, and the SEC purity was 95.25%.

[0767] Example 8. In vitro efficacy validation of CEACAM5-targeting antibody-drug conjugate

[0768] The cytotoxic activity, tumor growth inhibitory activity, and safety of the ADCs described in this application were validated in different cell lines and animal models. The cell lines used in this study (e.g., human gastric cancer cells MKN45, human colon cancer cells HT55, human pancreatic adenocarcinoma cells HPAF-II, and human pancreatic cancer cells HPAC) were all obtained from Nanjing Kebai Biotechnology Co., Ltd. RPMI 1640 medium (22400-071), DMEM medium (11965-092), McCoy's 5a medium (16600-082), MEM Alpha medium (12561-056), and FBS (10091-148) were all obtained from Gibco, unless otherwise instructed.

[0769] 8.1 In vitro toxicity testing of ADCs on HPAF-II, MKN45, HT55 and HPAC cells

[0770] Add 10% FBS (SH30406.05, HYCLONE) to RPMI 1640 (22400-071, Gibco) and mix well; use as growth medium for HPAF-II cells. Add 10% FBS (SH30406.05, HYCLONE) to RPMI 1640 (22400-089, Gibco) and mix well; use as growth medium for MKN45 cells. Add 10% FBS (SH30406.05, HYCLONE) to MEM Alpha (12561-056, Gibco) and mix well; use as growth medium for HT55 cells. Add 10% FBS (SH30406.05, HYCLONE) to DMEM:F-12 (30-2006, ATCC) and mix well; use as growth medium for HPAC cells. Adjust the cell concentration to 20,000 cells / mL using growth medium, and seed 50 μL (1000 cells / well) into each well of a 96-well white plate (167008, NUNC) and incubate overnight at 37°C. Dilute the ADC with medium in a 5-fold serial dilution series (9 dilutions in total). Add 50 μL of the diluted ADC solution or control (blank medium) to each well, for a total volume of 100 μL. Seal the wells with 200 μL of PBS (pH 7.4) and incubate at 37°C for 5 days. Allow the 96-well plate and CTG (VKEY-BIO TECHNOLOGIES, AJC06A) to equilibrate to room temperature. Add an equal volume of 100 μL CTG solution to each well. Shake on a plate for 5 min to fully lyse the cell clusters, and incubate at room temperature for 5 min to stabilize the luminescence signal. The cells were detected using a microplate reader (Spectra MAX i3x, Molecular Divices), and the readings were taken at A450 to calculate cell viability.

[0771] The cell proliferation inhibition rate was calculated using the following formula: Cell viability (%) = a / b * 100, where a: average luminescence value of the experimental sample wells; b: average luminescence value of the control culture medium wells. The IC50 of the drug was calculated by fitting data with GraphPad Prism. 50 .

[0772] Figure 2 and Table 1-2 show the cell survival % and inhibitory IC50 of ADCs in HPAF-II (A), MKN45 (B), HT55 (C), and HPAC (D) cells, respectively. 50 As can be seen, the ADCs of this invention exhibit significant tumor cell killing activity and excellent inhibitory IC50. 50 In particular, in HPAF-II cells, the cytotoxic effects of the ADCs of this invention are comparable to those of the positive control molecule M9140.

[0773] Table 1. Suppression IC of ADCs 50

[0774] Table 2. Suppression IC of ADCs 50

[0775] NA = Not applicable

[0776] 8.2 In vitro toxicity test of ADCs with different linker toxins targeting CEACAM5 on MKN45, HT55 and HPAC cells

[0777] 10% FBS (SH30406.05, HYCLONE) was added to RPMI 1640 (22400-089, Gibco) and mixed thoroughly to serve as the growth medium for MKN45 cells. 10% FBS (SH30406.05, HYCLONE) was added to MEM Alpha (12561-056, Gibco) and mixed thoroughly to serve as the growth medium for HT55 cells. 10% FBS (SH30406.05, HYCLONE) was added to DMEM:F-12 (30-2006, ATCC) and mixed thoroughly to serve as the growth medium for HPAC cells. The cell concentrations of the three types of cells were adjusted to 20,000 cells / mL using the culture media. 50 μL (1000 cells / well) was seeded into each well of a 96-well white plate (167008, NUNC) and incubated overnight at 37°C. ADCs were diluted with culture medium in a 5-fold serial dilution series starting at 500 nM, for a total of 9 dilutions. 50 μL of diluted ADC solution or control was added to each well, for a total volume of 100 μL. The well edges were sealed with 200 μL PBS (pH 7.4), and the plates were incubated at 37°C for 5 days. The cultured 96-well plates were then removed and equilibrated to room temperature with CTG (VKEY-BIO TECHNOLOGIES, AJC06A). An equal volume of 100 μL CTG solution was added to each well. The plates were shaken on a plate for 5 min to ensure complete cell lysis, and then incubated at room temperature for 5 min to stabilize the luminescence signal. Cell viability was calculated using a microplate reader (Spectra MAX i3x, Molecular Divices) at A450.

[0778] As described in Example 8.1, the cell survival (%) and IC50 inhibition of ADCs were measured. 50 The results are shown in Figure 3 and Table 3. It can be seen that the ADCs of this invention exhibit significant tumor cell killing activity and excellent inhibitory IC50. 50On HT55 cells, ADC1 showed stronger cytotoxicity than ADC7 and ADC9. On MKN45 cells, ADC2 exhibited greater cell killing and the lowest IC50 value compared to ADC6 and ADC8; there was no significant difference in cytotoxicity among the three dual toxins ADC1, ADC7, and ADC9. On HPAC cells, ADC2 showed greater cell killing and the lowest IC50 value compared to ADC6 and ADC8, indicating the most significant inhibition of tumor cells; there was no significant difference in cytotoxicity among the three dual toxins ADC1, ADC7, and ADC9.

[0779] Table 3. Suppression IC of ADCs 50

[0780] 8.3 In vitro toxicity test of CEACAM5-targeting ADCs on MKN45, HT55 and HPAC cells

[0781] 10% FBS (SH30406.05, HYCLONE) was added to RPMI 1640 (22400-089, Gibco) and mixed thoroughly to serve as the growth medium for MKN45 cells. 10% FBS (SH30406.05, HYCLONE) was added to MEM Alpha (12561-056, Gibco) and mixed thoroughly to serve as the growth medium for HT55 cells. 10% FBS (SH30406.05, HYCLONE) was added to DMEM:F-12 (30-2006, ATCC) and mixed thoroughly to serve as the growth medium for HPAC cells. The cell concentrations of the three cell types were adjusted to 20,000 cells / mL using the growth media, and 50 μL (1000 cells / well) was seeded into each well of a 96-well white plate (167008, NUNC) and incubated overnight at 37°C. The ADC was diluted with culture medium in a 5-fold serial dilution series starting at 500 nM, for a total of 9 dilutions. 50 μL of the diluted ADC solution or control was added to each well, for a total volume of 100 μL. The well edges were sealed with 200 μL of PBS (pH 7.4), and the plates were incubated at 37°C for 5 days. The 96-well plates and CTG (VKEY-BIO TECHNOLOGIES, AJC06A) were removed and allowed to equilibrate to room temperature. An equal volume of 100 μL CTG solution was added to each well. The plates were shaken on a plate for 5 min to ensure complete cell lysis, and then incubated at room temperature for 5 min to stabilize the luminescence signal. Cell viability was calculated using a microplate reader (Spectra MAX i3x, Molecular Divices) at A450.

[0782] As described in Example 8.1, the cell survival (%) and IC50 inhibition of ADCs were measured.50 The results are shown in Figure 4 and Table 4. It can be seen that the ADCs of the present invention exhibit significant tumor cell killing activity and excellent inhibitory IC50. 50 Figure 4A shows that ADC3, ADC12, and ADC13 exhibit similar cellular activities in MKN45 cells. Figure 4B shows that ADC12 and ADC13 show stronger cytotoxicity in HT55 cells. Figure 4C shows that ADC3, ADC12, and ADC13 show similar cellular activities in HPAC cells. Figure 4D shows that ADC3, ADC10, and ADC11 have similar IC50 values ​​in MKN45 cells, with ADC11 exhibiting stronger maximal killing. Figure 4E shows that ADC10 and ADC11 show stronger cytotoxicity in HT55 cells. Figure 4F shows that ADC3, ADC10, and ADC11 have similar cellular activities in HPAC cells.

[0783] Table 4. Suppression IC of ADCs 50

[0784] Example 9: Therapeutic effect of CEACAM5-ADC drug in HT55 model

[0785] This experiment used HT55 cells to inoculate CB17 SCID mice to determine the antitumor effect of the ADC drug of this invention.

[0786] CB17 SCID mice: Female CB17 SCID mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were SPF grade. The mice were acclimatized for 3 days after arrival before the study began.

[0787] Cells: HT55 cells were obtained from Nanjing Kebai (CAT#: CBP60012) and were passaged strictly according to the manufacturer's instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 25 × 10⁶ cells / mL. 6 Cells / ml. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of CB17 SCID mice to establish an HT55 tumor-bearing mouse model.

[0788] Drug administration: Seven days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volumes ranging from approximately 101 to 198.6 mm were selected. 3Mice were divided into groups of 6 mice each according to tumor volume. The dosage and administration method are shown in Table 5. h-IgG (purchased from EQUITECH-BIO) was used as a negative control and administered on day 7 post-inoculation. Tumor volume and body weight were monitored twice weekly. Body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on day 35 post-inoculation using the following formula: TGI% = 100% × (Tumor volume of control group – Tumor volume of treatment group) / (Tumor volume of control group – Tumor volume of control group before administration). Tumor volume was measured using calipers, measuring the maximum long axis (L) and maximum wide axis (W). The tumor volume was calculated using the following formula: V = L × W 2 / 2. Weight was measured using an electronic balance.

[0789] The tumor inhibition rate results are shown in Figure 5(A) and Table 5: On day 35 post-inoculation, the tumor inhibition rates of the ADC3, ADC10, ADC11, ADC12, and ADC13 groups were 72%, 66%, 79%, 64%, and 98%, respectively. Simultaneously, mouse body weight was monitored, and the results are shown in Figure 5(B). The weight loss in each treatment group was acceptable, indicating that the ADCs of this invention have good tolerability and safety. Among the drugs in this invention, ADC13 showed the best tumor-inhibiting effect.

[0790] Table 5. Experimental design and tumor inhibition rate on day 35.

[0791] Example 10: Therapeutic effect of CEACAM5-ADC drug in HT55 model

[0792] This experiment used HT55 cells to inoculate CB17 SCID mice to determine the antitumor effect of the ADC drug of this invention.

[0793] CB17 SCID mice: Female CB17 SCID mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were SPF grade. The mice were acclimatized for 3 days after arrival before the study began.

[0794] Cells: HT55 cells were obtained from Nanjing Kebai (CAT#: CBP60012) and were passaged strictly according to the manufacturer's instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 25 × 10⁶ cells / mL. 6 Cells / ml. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of CB17 SCID mice to establish an HT55 tumor-bearing mouse model.

[0795] Drug administration: Seven days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volumes ranging from approximately 80.80 to 203.70 mm² were selected. 3Mice were divided into groups of 5 mice each according to tumor volume. The dosage and administration method are shown in Table 6. h-IgG (purchased from EQUITECH-BIO) was used as a negative control. Mice were administered the drug on day 7 post-inoculation, and tumor volume and body weight were monitored twice weekly. Body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on day 32 post-inoculation using the following formula: TGI% = 100% * (Tumor volume of control group – Tumor volume of treatment group) / (Tumor volume of control group – Tumor volume of control group before administration). Tumor volume was measured using calipers, with the maximum long axis (L) and maximum wide axis (W) of the tumor measured. The tumor volume was calculated using the following formula: V = L × W 2 / 2. Weight was measured using an electronic balance.

[0796] The tumor inhibition rate results are shown in Figure 6(A) and Table 6: On day 32 post-inoculation, the tumor inhibition rates of ADC1, ADC7, ADC9, and ADC3 were 95%, 60%, 71%, and 73%, respectively. ADC1 showed the strongest inhibitory activity, with efficacy stronger than the ADC7 and ADC9 groups. Simultaneously, mouse body weight was measured, and the results are shown in Figure 6(B). The weight loss in each treatment group was acceptable, indicating that the ADCs of this invention have good tolerability and safety. ADC1 has a significant inhibitory effect on HT55 tumors.

[0797] Table 6. Experimental design and tumor inhibition rate on day 32.

[0798] Example 11: Therapeutic effect of CEACAM5-ADC drug in HT55 model

[0799] This experiment used HT55 cells to inoculate CB17 SCID mice to determine the antitumor effect of the ADC drug of this invention.

[0800] CB17 SCID mice: Female CB17 SCID mice were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. They were SPF grade. The mice were acclimatized for 3 days after arrival before the study began.

[0801] Cells: HT55 cells were obtained from Nanjing Kebai (CAT#: CBP60012) and were passaged strictly according to the manufacturer's instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 25 × 10⁶ cells / mL. 6 Cells / ml. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of CB17 SCID mice to establish an HT55 tumor-bearing mouse model.

[0802] Drug administration: Six days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volumes ranging from approximately 64.8 to 191.7 mm were selected. 3 Mice were divided into groups of 6 mice each, based on average tumor volume. The dosage and administration method are shown in Table 7. h-IgG (purchased from EQUITECH-BIO) served as a negative control and was administered on day 6 post-inoculation. Tumor volume and body weight were monitored twice weekly. Body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on day 31 post-inoculation using the following formula: TGI% = 100% * (Tumor volume of control group – Tumor volume of treatment group) / (Tumor volume of control group – Tumor volume of control group before administration). Tumor volume was measured using calipers, measuring the maximum long axis (L) and maximum wide axis (W). The tumor volume was calculated using the following formula: V = L × W 2 / 2. Weight was measured using an electronic balance.

[0803] The tumor inhibition rate results are shown in Figure 7(A) and Table 7: On day 31 post-inoculation, the tumor inhibition rates of ADC1, ADC2, ADC3, M9140, and ADC14 were 89%, 68%, 73%, 84%, and 15%, respectively. ADC1 showed the strongest inhibitory activity, with slightly stronger efficacy than the other drug groups. Simultaneously, we measured the mouse body weight, and the results are shown in Figure 7(B). The weight loss in each drug group was acceptable, indicating that the ADCs of this invention have good tolerability and safety. The ADC1 group showed a significant inhibitory effect on HT55 tumors.

[0804] Table 7. Experimental design and tumor inhibition rate on day 31

[0805] Example 12: Therapeutic effect of CEACAM5-ADC drug in HT55 model

[0806] In this experiment, MKN45 cells were inoculated into CB17 SCID mice to determine the antitumor effect of the ADC drug of the present invention.

[0807] CB17 SCID mice: Female CB17 SCID mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were SPF grade. The mice were acclimatized for 3 days after arrival before the study began.

[0808] Cells: MKN45 cells were obtained from Nanjing Kebai (CAT#: CBP60488) and were passaged strictly according to the manufacturer's instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 25 × 10⁶ cells / mL. 6Cells / ml. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of CB17 SCID mice to establish the MKN45 tumor-bearing mouse model.

[0809] Drug administration: Eight days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volumes ranging from approximately 82.1 to 236.6 mm were selected. 3 Mice were divided into groups of 7 mice each, based on average tumor volume. The dosage and administration method are shown in Table 8. h-IgG (purchased from EQUITECH-BIO) served as a negative control and was administered on day 8 post-inoculation. Tumor volume and body weight were monitored twice weekly. Body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on day 46 post-inoculation using the following formula: TGI% = 100% * (Tumor volume of control group – Tumor volume of treatment group) / (Tumor volume of control group – Tumor volume of control group before administration). Tumor volume was measured using calipers, measuring the maximum long axis (L) and maximum wide axis (W). The tumor volume was calculated using the following formula: V = L × W 2 / 2. Weight was measured using an electronic balance.

[0810] The tumor inhibition rate results are shown in Figure 8(A) and Table 8: On day 46 post-inoculation, the tumor inhibition rates of ADC1, ADC2, and ADC3 were 100%, 37%, and 83%, respectively. The ADC1 group showed the strongest inhibitory activity, with efficacy slightly stronger than the individual drug groups. Simultaneously, mouse body weight was measured, and the results are shown in Figure 8(B). Body weight decreased in all groups, but the weight loss in tumor-bearing mice treated with the ADCs of this invention was significantly less than that in the h-IgG group, indicating that the ADCs of this invention have good tolerability and safety. The ADC1 group showed excellent inhibitory effects on MKN45 tumors.

[0811] Table 8. Experimental design and tumor inhibition rate on day 46

[0812] sequence list

Claims

An anti-CEACAM5 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as set forth in SEQ ID NO: 4, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as set forth in SEQ ID NO:

8. An anti-CEACAM5 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3), and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3), wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, respectively. The anti-CEACAM5 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4; or comprises or consists of the amino acid sequence set forth in SEQ ID NO:

4. The anti-CEACAM5 antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL), wherein the light chain variable region comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 8; or comprises or consists of the amino acid sequence set forth in SEQ ID NO:

8. An anti-CEACAM5 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and / or a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. The anti-CEACAM5 antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO:

8. The anti-CEACAM5 antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof comprises an Fc region, for example the Fc region is or is from an Fc region of IgGl, IgG2, IgG3, or IgG4, for example an Fc region of human IgGl, IgG2, IgG3, or IgG4. The anti-CEACAM5 antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region that is or is from a constant region of IgGl, IgG2, IgG3, or IgG4, for instance a constant region of human IgGl, IgG2, IgG3, or IgG4, for example the IgGl heavy chain constant region (i) comprises or consists of the amino acid sequence of SEQ ID NO: 20 or 21; or (ii) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 20 or 21. The anti-CEACAM5 antibody or antigen-binding fragment thereof of any one of claims I -8, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region that is or is from a lambda or Kappa light chain constant region, for example a human lambda or Kappa light chain constant region, preferably the light chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to the amino acid sequence set forth in SEQ ID NO: 28 or 30; or (ii) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28 or 30. ​ The anti-CEACAM5 antibody or antigen-binding fragment thereof of any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof comprises one or two Lambda light chain constant regions and has a cysteine substitution at position 160 (EU numbering) of the Lambda light chain constant region (LLC160C). The anti-CEACAM5 antibody or antigen-binding fragment thereof of claim 10, wherein the Lambda light chain constant region comprising LLC160C comprises the amino acid sequence set forth in SEQ ID NO: 29 or 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: 29 and comprises the amino acid sequence VKAGVCTTTPS (SEQ ID NO: 34). The anti-CEACAM5 antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the antibody or antigen-binding fragment thereof comprises one or two heavy chain constant regions and has a cysteine substitution at position 239 (EU numbering) of the heavy chain constant region (HC239C). The anti-CEACAM5 antibody or antigen-binding fragment thereof of claim 12, wherein the heavy chain constant region having a cysteine mutation at position 239 comprises the amino acid sequence set forth in SEQ ID NO: 22 or has at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 22 and comprises the amino acid sequence GGPCVFLFP (SEQ ID NO: 35). The anti-CEACAM5 antibody or antigen-binding fragment thereof of any one of claims1-13, wherein the antibody or antigen-binding fragment thereof comprises (i) one or two Lambda light chain constant regions having a cysteine mutation at position 160; or (ii) one or two heavy chain constant regions having a cysteine mutation at position 239. The anti-CEACAM5 antibody or antigen-binding fragment thereof of any one of claims 14, wherein the antibody or antigen-binding fragment thereof comprises two heavy chain constant regions and two light chain constant regions, wherein (i) one heavy chain constant region comprises a cysteine mutation at position 239 and the other heavy chain constant region does not comprise a cysteine mutation at position 239 (or the other heavy chain constant region does not comprise a cysteine mutation), and both light chain constant regions do not comprise a cysteine mutation (e.g., the light chain constant regions are Kappa light chain constant regions); (ii) both light chain constant regions are Lambda light chains, and one light chain constant region comprises a cysteine mutation at position 160 and the other light chain constant region does not comprise a cysteine mutation at position 160; or (iii) both light chain constant regions are Lambda light chains, and both light chain constant regions comprise a cysteine mutation at position 160, respectively. The anti-CEACAM5 antibody or antigen-binding fragment thereof of claim 15, wherein when the two heavy chain constant regions are different or the two light chain constant regions are different, the two heavy chain constant regions each comprise a different Fc region that facilitates heterodimerization mutations, the Fc region comprising a Knob mutation such as S354C and T366W (EU numbering) and a Hole mutation such as Y349C, T366S, L368A, and Y407V (EU numbering), respectively. The anti-CEACAM5 antibody or antigen-binding fragment thereof of any one of claims 1-16, wherein the heavy chain constant region or Fc region contained therein further comprises a mutation that reduces binding to an Fc gamma receptor such as L234A / L235A (EU numbering) mutation. The anti-CEACAM5 antibody or antigen-binding fragment thereof of any one of claims 1-17, wherein the antibody is a humanized antibody or a chimeric antibody. The anti-CEACAM5 antibody or antigen-binding fragment thereof of any one of claims 1-18, wherein the antigen-binding fragment is an antibody fragment selected from a Fab, a Fab', a Fab'-SH, a Fv, a single chain antibody (e.g., scFv), a (Fab')2, a dAb (domain antibody), a diabody, or a linear antibody. The anti-CEACAM5 antibody or antigen-binding fragment thereof of any one of claims 1-19, wherein the antibody or antigen-binding fragment thereof (i) comprises a heavy chain 1, a heavy chain 2, a light chain 1, and a light chain 2, wherein the heavy chain 1 comprises or consists 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 set forth in SEQ ID NO: 9, the heavy chain 2 comprises or consists of an amino acid sequence that is at least 85%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 10, the light chain 1 comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, and 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 11; and the light chain 2 comprises or consists of an amino acid sequence that is at least 85%, 86%, 86%, 87%, 88%, 89%, 90%, 91%, and 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, or 99% identical to the amino acid sequence shown in the heavy chain 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9, the heavy chain 2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10, the light chain 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11; and the light chain 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12; ​ ​ ​ ​ (ii) comprises two identical heavy chains and two identical light chains, wherein the heavy chain comprises or consists 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 set forth in SEQ ID NO: 13, and the light chain comprises or consists 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 set forth in SEQ ID NO: 14; or the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14; (iii) comprises two identical heavy chains and two identical light chains, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:13, and the light chain comprises or consists 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: 11; or the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13, and the light chain comprises or consists of the amino acid sequence set forth SEQ ID NO: 11; or (iv) comprises heavy chain 1, heavy chain 2, and two identical light chains, wherein the heavy chain 1 comprises or consists of an amino acid sequence that is at least 85%, 90%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 15, the heavy chain 2 comprises or consists of an amino acid sequence that is at least 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:10, and the light chain comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth SEQ ID NO: 14; or the heavy chain 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, the heavy chain 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:

14. An isolated nucleic acid encoding the anti-CEACAM5 antibody or antigen binding fragment thereof of any one of claims 1-20. An isolated nucleic acid encoding the anti-CEACAM5 antibody or antigen binding fragment of any one of claims 1-20. A vector comprising the nucleic acid of claim 21, preferably the vector is an expression vector. A host cell comprising the nucleic acid of claim 21 or the vector of claim 22, preferably the host cell is prokaryotic or eukaryotic, more preferably selected from a yeast cell, a mammalian cell (e.g. a 293 cell or a CHO cell, e.g. a CHO-K cell or a HEK293 cell) or other cell suitable for making an antibody or antigen binding fragment thereof. A method of making an anti-CEACAM5 antibody or antigen binding fragment thereof, the method comprising a) culturing the host cell of claim 23 under conditions suitable for expression of a nucleic acid encoding the anti-CEACAM5 antibody or antigen binding fragment thereof of any one of claims 1-20, b) optionally isolating the antibody or antigen binding fragment thereof, c) optionally the method further comprises recovering the anti-CEACAM5 antibody or antigen binding fragment thereof from the host cell, optionally the antibody is purified, e.g. by Protein A purification. An immunoconjugate comprising the anti-CEACAM5 antibody or antigen binding fragment thereof of any one of claims 21-20 and another agent, e.g. a chemotherapeutic agent, a toxin, a small molecule drug, a cytotoxic agent, an apoptotic agent, a chelating agent, an immunomodulatory agent, e.g. an immunosuppressive agent. An antibody drug conjugate of Formula (II) or a pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof: wherein Ab is the anti-CEACAM5 antibody or antigen binding fragment thereof of any one of claims 11-20; D1 has the structure of Formula (Ia): wherein R d1 is H, OH, C 1-6 alkyl, C 1-6 alkoxy, pentose, pentosaccharide, hexose, or hexosaccharide, wherein R dm , R dn , and R dp are each independently H or C 1-6 alkyl; or R d1 represents a bond and is connected in a ring with any one of L a2 , L a3 , and L a4 , preferably with L a4 ; R d2 and R d3 each independently is H, OH, C 1-6 alkyl, C 1-6 alkoxy, a pentose, pentosaccharide acid, a hexose, or a hexosaccharide acid; wavy line indicates an optional connection to L a4 connection; L a1 is a linker unit; L a2 is absent or is a bridged spacer; L a3 Yes: (i) a short chain containing 1-10 amino acid residues, wherein said amino acid residues are optionally selected from C10 by one or more (e.g., 1, 2, 3 or 4) of C10. 1-6 Substitution of alkyl and polyol groups; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3 or 4). 1-6 Substituents of alkyl and polyol groups; or (iii) none; wherein L a3 When present, N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1; L a4 is absent or is a cleavable linker or a self-immolative linker; and m is 1, 2, 3, 4, 5 or 6. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to claim 26, wherein the antibody drug conjugate has the structure of formula (IIa): wherein the variables are as defined in claim 26. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to claim 26 or 27, wherein R d1 is H, OH, wherein R dm , R dn and R dp are each independently H or C 1-6 alkyl, preferably H; and 1-4 alkyl, preferably H; and Preferably, R d1 is H, OH, (For example ) or (For example ), wherein R dm , R dn and R dp are each independently H or C 1-6 alkyl, such as C 1- 4alkyl, preferably H; Also preferably, R d1 is H, OH, (For example ), wherein R dm , R dn and R dp are each independently H or C 1-6 alkyl, preferably H; and n is 0, 1 or 2. 1-4 alkyl, preferably H; and n is 0, 1 or 2. Also preferably, R d1 is H, OH, Most preferably, R d1 is H, OH, The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate, or isotopically-labeled material thereof according to any one of claims 26-28, wherein R d2 and R d3 each independently is: H, C 1-6 alkyl, C 1-6 alkoxy, R d2 and R d3 are each independently C 1-6 alkoxy, (For example ) or (For example )； R d2 R d3 each independently is C 1-6 alkyl, for example C 1-4 alkyl, preferably methyl, ethyl, propyl, butyl; most preferably, R d2 R d3 each independently is methoxy. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvent, or isotopically-labeled material thereof according to any one of claims 26-29, wherein: L a1 is: wherein 1 is attached to Ab and 2 is attached to L a2 attached; Preferably, L a1 is wherein 1 is attached to Ab and 2 is attached to L a2 attached; Also preferably, L a1 is wherein 1 is attached to Ab and 2 is attached to L a2 attached; Most preferably, L a1 is wherein 1 is attached to Ab and 2 is attached to L a2 connection. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 26-30, wherein L a2 is absent or is a bridging spacer selected from the group consisting of -(CH2CH2O) p R q ) s1 -(CH2CH2O) t -(CH2CH2O) m R n ) s2 -CO-**; *-(CR p R q ) s1 -L M -(CH2CH2O) t -(CR m R n ) s2 -CO-**; and -(CR p R q ) s1 -(OCH2CH2) t -(CR m R n ) s2 -CO-**; wherein * end to L a1 is connected, and the ** end to L a3 is connected, and wherein R p , R q , R m and R n are each independently H or C 1-6 alkyl, preferably H or C 1-4 alkyl, L M is -NH-CO-, -CO-NH-, -CO-, -NH- or -O, s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, L a2 is absent or is a bridging spacer selected from the group consisting of -(CR p R q ) s1 -(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -NH-CO-(CH2CH2O) t -(CR m R n ) s2 -CO-**; -(CR p R q ) s1 -CO-NH-(CH2CH2O) t -(CR m R n ) s2 -CO-**; -(CR p R q ) s1 -CO-(CH2CH2O) t -(CR m R n ) s2 -CO-**; -(CR p R q ) s1 -NH-(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -O-(CH2CH2O) t -(CR m R n ) s2 -CO-**; and -(CR p R q ) s1 -(OCH2CH2) t -CO-**; wherein the * end is attached to L a1 and the ** end is attached to L a3 and wherein R p , R q , R m , and R n are each independently H or C 1-6 alkyl, preferably H or C 1-4 alkyl, s1, s2, and t are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; Also preferably, L a2 is absent or is a bridging spacer selected from the group consisting of -(CH2) s1 -(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -NH-CO-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -CO-NH-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -CO-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -NH-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -O-(CH2CH2O) t -(CH2) s2 -CO-**; and * -(CH2) s1 - (OCH2CH2) t - CO-**; wherein the * end is attached to L a1 and the ** end is attached to L a3 and wherein s1, s2, and t are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; Also preferably, L a2 is absent or is a bridging spacer selected from the group consisting of p R q ) s1 -CO-**; and -(CH2CH2O) t -(CR m R n ) s2 -CO-**; and -(CR p R q ) s1 -(OCH2CH2) t -CO-**; wherein the * end is connected to L a1 and the ** end is connected to L a3 and wherein R p , R q , R m and R n are each independently H or C 1-6 alkyl, preferably H or C 1-4 alkyl, s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8. Also preferably, L a2 is absent or is a bridging spacer selected from the group consisting of s1 -CO-**; -(CH2CH2O) t -(CH2) s2 -CO-**; and -(CH2) s1 -(OCH2CH2) t -CO-**; wherein the * end is attached to L a1 and the ** end is attached to L a3 and wherein s1, s2, and t are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 26-31, wherein L a2 is absent or is a bridging spacer of the formula *-(CR p R q ) s1 -CO-**, wherein, * end to L a1 connected, and the ** end to L a3 connected, R p and R q each independently H or C 1-6 alkyl, preferably H or C 1-4 alkyl, and s1 is 0, 1, 2, 3, 4, 5, 6, 7, or 8; Preferably, L a2 is absent or is a bridging spacer of the formula *-(CH2) s1 -CO-**, wherein the * end is attached to L a1 and the ** end is attached to L a3 and s1 is 0, 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, 6 or 7, more preferably 5 or 6; Also preferably, L a2 is absent or -CO-, *-(CH2)-CO-**, *-(CH2)2-CO-**, *-(CH2)3-CO-**, *-(CH2)4-CO-**, *-(CH2)5-CO-**, *-(CH2)6-CO-**, *-(CH2)7-CO-** or *-(CH2)8-CO-**, preferably *-(CH2)5-CO-**, wherein the * end is attached to L a1 is absent or -CO-, *-(CH2)-CO-**, *-(CH2)2-CO-**, *-(CH2)3-CO-**, *-(CH2)4-CO-**, *-(CH2)5-CO-**, *-(CH2)6-CO-**, *-(CH2)7-CO-** or *-(CH2)8-CO-**, preferably *-(CH2)5-CO-**, wherein the * end is attached to L a3 is absent or -CO-, *-(CH2)-CO-**, *-(CH2)2-CO-**, *-(CH2)3-CO-**, *-(CH2)4-CO-**, *-(CH2)5-CO-**, *-(CH2)6-CO The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 26-32, wherein -L a1 -L a2 - is wherein s1and s2are each independently 1, 2, 3, 4, 5, 6, 7, or 8, preferably 4, 5, or 6, t is 1, 2, 3, 4, 5, 6, 7, or 8, preferably 5, 6, 7, or 8, and the left side of the group is attached to Aband the right side to L a3 is connected to; Preferably, -L a1 -L a2 - is wherein s1 is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, and the left side of the group is attached to Ab and the right side to L a3 Connection. The antibody-drug conjugate according to any one of claims 26-33, or its pharmaceutically acceptable salt or ester, solvate or isotopic label, wherein L a3 Yes: (i) a short chain containing 1-8 amino acid residues, such as a single amino acid residue or a peptide containing 2, 3, 4, 5, 6, 7 or 8 amino acids, preferably 2, 3 or 4 amino acids, wherein said amino acid is optionally bounded by one or more (e.g. 1, 2, 3 or 4, preferably 1 or 2) C 1-6 Alkyl substitution; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3 or 4, preferably 1 or 2). 1-6 Alkyl substituents; or (iii) none; wherein L a3 When present, N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 26-34, wherein each of the amino acids is independently selected from the group consisting of valine (Val), alanine (Ala), glycine (Gly), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), serine (Ser), aspartic acid (Asp), asparagine (Asn), isoleucine (lie), arginine (Arg), proline (Pro), methionine (Met), tryptophan (Trp), cysteine (Cys), histidine (His), and threonine (Thr), wherein the amino acids are optionally substituted with one or more C 1-6 alkyl groups; Preferably, the amino acid is selected from valine (Val), alanine (Ala), glycine (Gly), lysine (Lys), citrulline (Cit), glutamine (Gin), glutamic acid (Glu), phenylalanine (Phe) and leucine (Leu), wherein the amino acid is optionally substituted with one or more C 1-6 alkyl groups; It is also preferred that the amino acid is selected from valine (Val), alanine (Ala), glycine (Gly), lysine (Lys), citrulline (Cit) and glutamine (Gin), wherein the amino acid (e.g. lysine) is optionally substituted with one or more C 1-6 alkyl groups; It is also preferred that the amino acid is selected from the group consisting of valine (Val), alanine (Ala), glycine (Gly) and lysine (Lys), wherein the lysine is optionally substituted by one or more C 1-6 alkyl, preferably N-substituted. The antibody-drug conjugate according to any one of claims 26-35, or its pharmaceutically acceptable salt or ester, solvate or isotopic label, wherein L a3 Is Gly, Val-Ala, Val-Cit, Phe-Lys, Val-Lys, Leu-Cit, Val-Lys-Gly, Val-(N6,N6-C 1-6 Alkyl group (-Lys)-Gly, -NH-(CH2) 2-6 -CO- or absent, L is preferred. a3 It is Gly, Val-Ala, Val-Lys-Gly, Val-(N6,N6-diisopropyl-Lys)-Gly, or -NH-(CH2). 2-6 -CO-, and L is preferred a3 It is Gly, Val-Ala, Val-Lys-Gly, Val-(N6,N6-diisopropyl-Lys)-Gly or -NH-(CH2)2-CO-; where L a3 The N-terminus and L a2 -Connection or when L a2 When it does not exist and L a1 Connect, and C end is connected to L a4 Connection or when L a4 If it does not exist, connect it to D1. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 26-36, wherein L a3 is (Gly), Or -NH-(CH2)2-CO-, where L a3 The N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 26-37, wherein L a4 is absent or -NH-CH2-, Preferably, L a4 is absent or is wherein the right side of the group is connected to D1and the left side is connected to L a3 connected. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 26-38, wherein, R L selected from H, C 1-6 alkyl, C 1-6 alkoxy, pentose, pentosaccharide, hexose, hexosaccharide, disaccharide, trisaccharide, and 5-8 membered heteroaryl, optionally substituted with one or more substituents selected from C 1-6 alkyl and nitro)-(C 1-4 alkylene)-O-; Preferably, R L is H, Also preferably, R L is H, (As )、 (As ), or Most preferably, R L is H, (As ), or The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 26-39, wherein L a4 is absent or wherein the C-terminus is connected to D1 and the N-terminus is connected to L a3 connected; Preferably, L a4 is absent or is wherein the C-terminus is connected to D1 and the N-terminus is connected to L a3 connected; Also preferably, L a4 is For example wherein the C-terminus is connected to D1 and the N-terminus is connected to L a3 connected. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate of isotopically-labeled material thereof according to any one of claims 26-40, wherein R d1 represents a bond, L a4 is R L is wherein R L is connected to position 1 of R d1 and to position 2 of L a4 , and wherein the C-terminus of L a4 is connected to D1 and the N-terminus is connected to L a3 ; Preferably, R d1 represents a bond, L a4 represents For example wherein L a4 is connected to position 1 of D1, R d1 is connected to position 2 of D1, and L a3 is connected to position 3 of D1. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, or solvate, or isotopically-labeled material thereof according to claim 26, wherein Ab is the anti-CEACAM5 antibody or antigen binding fragment thereof of any one claims 1-20; D1 has the structure of Formula (Ib): wherein R d1 is H, OH, (For example )； R d2 and R d3 each independently is C 1-4 alkoxy, preferably methoxy; wavy line indicates an optional connection to L a4 connection; - L a1 - L a2 - is wherein s1 is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, and the left side of the group is attached to Ab and the right side is attached to L a3 is connected to; L a3 is (Gly), or -NH-(CH2)2-CO-, wherein the N-terminus is attached to L a2 or -NH-(CH2)2-CO-, wherein the N-terminus is attached to L a4 or -NH-(CH2)2-CO-, wherein the N-terminus is attached to L a4 or -NH-(CH2)2-CO-, wherein the N-terminus is attached to L L a4 absent or (For example )、 (For example ), or Preferably For example wherein L a4 when present, is connected to D1 at the C-terminus and to L a3 at the N-terminus. or R d1 represents a bond, L a4 represents For example wherein L a4 is connected to position 1 of D1, to position 2 of R d1 to form a ring, and to position 3 of L a3 ; and m is 1, 2, 3, 4 5 or 6. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to claim 26, wherein the antibody drug conjugate has the following subformula: wherein, Ab is the anti-CEACAM5 antibody or antigen binding fragment thereof of any one of claim 1-20; and m is 1, 2, 3, 4, 5 or 8. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvates or isotopically-labeled material thereof according to any one of claims 26-43, wherein m is 1, 2, 3 or 4, e.g. m is 1, or m is 2. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 26-44 is in the form of a composition comprising one or more antibody drug conjugates or a pharmaceutically acceptable salt or ester, solvate or isotopically-labe led material thereof according to any one of claims 26-44, and the composition has an average DAR of 0.6-6, preferably 0.8-4.0, more preferably 0.8-2.2, for example an average DAR of about 1, or for example an average DAR of about 2. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 26-45, wherein the Ab comprises a naturally occurring or cysteine mutation-introduced cysteine; preferably, the -L a1 -L a2 -L a3 -L a4 The D1 moiety is covalently coupled to the thiol of a cysteine mutation-introduced cysteine of the Ab. An antibody drug conjugate of Formula (III) or a pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof: wherein: Ab is an anti-CEACAM5 antibody or antigen binding fragment thereof according to any one of claims 1-20; D1 has the structure of Formula (Ia): wherein, R d1 is H, OH, C 1-6 alkyl, C 1-6 alkoxy, pentose, pentosaccharide, hexose, or hexosaccharide, wherein R dm , R dn , and R dp are each independently H or C 1-6 alkyl; or R d1 represents a bond and is connected in a ring with any one of L a2 , L a3 , and L a4 , preferably with L a4 ; R d2 and R d3 each independently is H, OH, C 1-6 alkyl, C 1-6 alkoxy, a pentose, pentosaccharide acid, a hexose, or a hexosaccharide acid; wavy line indicates an optional connection to L a4 connection; L a1 is a linker unit; L a2 is absent or is a bridged spacer; L a3 is: (i) a short chain comprising 1-10 amino acid residues, wherein the amino acid residues are optionally substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from C 1-6 alkyl and polyol groups; (ii) -NH-(CH2) 2-6 -CO-, optionally substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from C 1-6 alkyl and polyol groups; or (iii) absent; wherein L a3 is attached to the N-terminus when present or to L a2 when L a2 is absent; and L a1 is attached to the C-terminus or to D1 when L a4 is absent; and D1 is: (i) a short chain comprising 1-10 amino acid residues, wherein the amino acid residues are optionally substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from C a4 alkyl and polyol groups; (ii) -NH-(CH2) -CO-, optionally substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from C alkyl and polyol groups; or (iii) absent. L a4 is absent or is a cleavable linker or a self-immolative linker; L b is a linker; D2 is a drug moiety, preferably an anti-tumor drug moiety; m is 1, 2, 3, 4, 5, or 6; and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to claim 47, wherein said antibody drug conjugate has the structure of (IIIa): wherein the variables are as defined in claim 47. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to claim 47 or 48, wherein L a1 , L a2 , L a3 , L a4 and D1 are as defined in any one of claims 26-46. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate, or isotopically-labeled material thereof according to any one of claims 47-49, wherein the drug moiety D2 has the structure of the following formula: -Q-L 2 -L 1 -D 2a wherein: Q is -0-, -S- or -NR 7 -; L 2 is absent, -(C 1-10 alkylene)-C(O)N(R 5 )- or -(C 1-10 alkylene)-N(R 5 )C(O)-; wherein * indicates that the terminal is covalently attached to Q, and R 5 is H or C 1-6 alkyl; L 1 is absent or -(C 1-10 alkylene)-; D 2a having the structure of Formula (D-1): wherein R 1 and R 6 are selected from H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 2-6 haloalkenyl, and C 2-6 haloalkynyl; or R 1 and R 6 together with the carbon to which they are attached form a 5-9 membered ring, for example a 5-8 membered ring; R 2 is H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, -OR 4 or -SR 4 ; R 3 is H, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl or -OR 4 ; or R 2 and R 3 together form -O(CH2) p O- or -O(CF2) p O-, wherein p is 1 or 2; R 4 is H or C 1-4 alkyl; and R 7 is H or C 1-6 alkyl. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to claim 50, wherein D 2a having the structure of formula (D-1a) or (D-1b): wherein the symbols are as defined in claim 50. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to claim 50, wherein D 2a having the structure of formula (D-2): wherein the symbols are as defined in claim 50. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to claim 52, wherein D 2a having the structure of formula (D-2a) or (D-2b): wherein, the remaining symbols are as defined in any one of claims 47-52. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 47-53, wherein R 1 and R 6 is H, R 2 is C 1-6 alkyl or C 1-6 alkoxy, and R 3 is halogen, preferably -F. The antibody-drug conjugate according to any one of claims 47-54, or its pharmaceutically acceptable salt or ester, solvate or isotopic label, wherein -L 2 -L 1 -is-(C 1-6 alkylene)-, *-(C 1-6 alkylene)-C(O)N(R 5 )-(C 1-6 alkylene)- or *-(C 1-6 alkylene)-N(R 5 )C(O)-(C 1-6 (alkylene)-, where * indicates that the terminus is covalently connected to Q; and R 5 Is it H or C? 1-6 alkyl. The antibody-drug conjugate according to any one of claims 47-55, or its pharmaceutically acceptable salt or ester, solvate or isotopic label, wherein -QL 2 -L 1 - can be -OCH2-CH2-CH2-CH2-, -OCH2-CH2-CH2-, or -NH-, wherein the left side of this group is adjacent to L. b Connect, right side with D 2a connect. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 47-49, wherein the drug moiety D2 is selected from: The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 47-57, wherein linker L b has the structure: -Z-E-X- wherein: Z is attached to Ab and X is attached to D; Z is selected from wherein m a1 and m a2 are independently selected from an integer from 0 to 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; and m is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, or 8; the carbonyl group at the right end of Z is covalently attached to E; E is a short chain comprising 1-10 amino acids, for example a single amino acid residue or a peptide residue comprising 2-10 amino acids, wherein the short chain is optionally substituted with one or more (e.g. 2, 3 or 4) groups selected from C 1-6 groups of an alkyl and a polyol group, wherein the N-terminus of the short chain is covalently linked to Z; and X is selected from the group consisting of absent, -NH-CH2-, or wherein R a1 is selected from H, C 1-6 alkyl, C 1-6 alkoxy, pentose, pentosaccharide acid, hexose, hexosaccharide acid, disaccharide, and trisaccharide; the left end of which is attached to E and the right end is attached to D2. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to claim 58, wherein Z is wherein, m is an integer selected from 1-10, for example 1, 2, 3, 4, 5, 6, 7, or 8, preferably 5; or Z is wherein m a2 is an integer selected from 0-20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, preferably 4, and m is an integer selected from 1 -10, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably 2. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate of isotopically-labeled material thereof according to claim 58 or 59, wherein E is a single amino acid residue or a peptide residue comprising 2, 3, or 4 amino acids. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 60, wherein the amino acid is selected from the group consisting of glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, wherein the glutamine or glutamic acid is optionally substituted with one polyol group and optionally substituted with one C 1-6 alkyl group; preferably, the polyol group is The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solv ate, or isotopically-labeled material thereof according to claim 61, wherein the substituted glutamine or glutamic acid has the structure of the following formula: preferably Where R 8 Is it H or C? 1-6 alkyl. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to claim 58, wherein E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Ala-, or wherein R 8 is H or C 1-6 alkyl. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 58-63, wherein X is -NH-CH2- or R a1 selected from: H, Preferred R a1 yes The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to claim 64, wherein X is -NH-CH2-, The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 47-65, wherein linker L b has the structure: The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 47, wherein -L b The moiety -D2 is: wherein the wavy line indicates attachment to Ab. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to claim 47, wherein the antibody drug conjugate has the structure of: wherein: Ab is an anti-CEACAM5 antibody or antigen binding fragment thereof accord ing to any one of claims 1-20; m is 1, 2, 3, 4, 5, or n is 1, 2, 3, 4, 5, 16, 17, 18, 19, or 20. The antibody drug conjugate or a pharmaceutically acceptable salt or ester, solv ates, or isotopically-labeled material thereof according to any one of claims 47- 68, wherein m is 1, 2, 3, or 4, preferably 1 or 2; and n is 1, 2, 3, 4, 5, 6, The antibody drug conjugate or its pharmaceutically acceptable salt or ester, solvate or isotopically-labeled form according to any one of claims 47-69 is in the form of a composition comprising one or more antibody drug conjugates according to any one of claims 47-69 or its pharmaceutically acceptable salt or ester, solvate or isotopically-labeled form, and wherein the composition has an average DAR of 0.6-6, preferably 0.8-4.0, more preferably 0.8-2.2, still more preferably 1.0 ± 0.4 or 2.0 ± 0.4 for the drug moiety D1 and an average DAR of 2.0-14.0, preferably 4.0-12.0, more preferably 6.0-10.0, for example 4.0 ± 0.4 or 8.0 ± 0.4 for the drug moiety D2. The antibody drug conjugate or pharmaceutically acceptable salt or ester, solvate or isotopically-labeled material thereof according to any one of claims 47-70, wherein the Ab comprises a naturally occurring or cysteine mutation introduced cysteine; preferably, the -L a1 -L a2 -L a3 -L a4 the D1 moiety is covalently coupled to the thiol of a cysteine mutation introduced cysteine of the Ab, and / or the -L b the D2 moiety is covalently coupled to the thiol of a disulfide bond interchain reduced thiol of the Ab. A pharmaceutical composition comprising the anti-CEACAM5 antibody or antigen binding fragment thereof according to any one of claims 1-20 and / or the antibody drug conjugate or its pharmaceutically acceptable salt or ester, solvate or isotope-labeled form according to any one of claims 26-71 and one or more pharmaceutically acceptable carriers. Use of the anti-CEACAM5 antibody or antigen binding fragment thereof according to any one of claims claim 1-20 and / or the antibody drug conjugate or its pharmaceutically acceptable salt or solvate or isotope-labeled form according to any one of claims 26-71 for the manufacture of a medicament for the treatment or prevention of a tumor or cancer; preferably the cancer is a lung cancer or a digestive tract tumor, such as an intestinal cancer, a stomach cancer, a pancreatic cancer or a colon cancer. A method of treatment or prevention of a tumor or cancer, the method comprising administering to a patient in need thereof the anti-CEACAM5 antibody or antigen binding fragment thereof according to any one of claims1-20 and / or the antibody drug conjugate or its pharmaceutically acceptable salt or esters, solvate or isotope-labeled form according to any one of claims 26- 71; preferably the cancer is a lung cancer or a digestive tract tumor, such as an intestinal cancer, The anti-CEACAM5 antibody or antigen binding fragment thereof according to any one of claims 26-71 and / or the antibody drug conjugate or its pharmaceutically acceptable salt or ester, solute or isotope-labeled form according to any one of claims 26-71 for use in the treatment or prevention of a tumor or cancer; preferably the cancer is a lung cancer or a A pharmaceutical combination comprising the anti-CEACAM5 antibody or antigen binding fragment thereof according to any one of claim 1-20 and / or the antibody drug conjugate or its pharmaceutically acceptable salt, ester, solvate or isotope-labeled form according to any one of claims 27-71 and one or more co-agents, such as an anti-tumor agent.

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