WO2023109953A1 - 靶向Claudin18.2的抗体-药物偶联物 - Google Patents

靶向Claudin18.2的抗体-药物偶联物 Download PDF

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WO2023109953A1
WO2023109953A1 PCT/CN2022/139637 CN2022139637W WO2023109953A1 WO 2023109953 A1 WO2023109953 A1 WO 2023109953A1 CN 2022139637 W CN2022139637 W CN 2022139637W WO 2023109953 A1 WO2023109953 A1 WO 2023109953A1
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antibody
amino acid
acid sequence
drug conjugate
solvate
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WO2023109953A9 (zh
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何开杰
周帅祥
刘晓丹
陆佳
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Innovent Biologics Suzhou Co Ltd
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Innovent Biologics Suzhou Co Ltd
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Priority to AU2022416272A priority Critical patent/AU2022416272A1/en
Priority to CN202280083295.6A priority patent/CN119173534A/zh
Priority to EP22906698.0A priority patent/EP4450521A1/en
Priority to KR1020247023670A priority patent/KR20240133715A/ko
Priority to CA3242970A priority patent/CA3242970A1/en
Priority to JP2024535848A priority patent/JP7796230B2/ja
Application filed by Innovent Biologics Suzhou Co Ltd filed Critical Innovent Biologics Suzhou Co Ltd
Priority to US18/719,023 priority patent/US20250073347A1/en
Publication of WO2023109953A1 publication Critical patent/WO2023109953A1/zh
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
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    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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Definitions

  • the present invention relates to antibody-drug conjugates (ADCs) targeting Claudin18.2 (CLDN18.2) and compositions containing said molecules.
  • ADCs antibody-drug conjugates
  • CLDN18.2 Claudin18.2
  • the invention also relates to the therapeutic and diagnostic uses of these molecules.
  • Claudins are a family of proteins that are essential building blocks that make up the tight junctions of cells. They create cell-to-cell barriers that control the flow of molecules between cells. Claudins family proteins have tetratransmembrane domains, both N-terminal and C-terminal are included in the cytoplasm. Different Claudins proteins are expressed in different tissues, and their functional changes are related to the formation of cancer in various tissues. For example, Claudin-1 is expressed in colon cancer and has prognostic value. Claudin-18 is highly expressed in gastric cancer and pancreatic cancer. Claudin- 10 expression is highly expressed in hepatocellular carcinoma. As cell membrane surface proteins, Claudins are useful targets for various therapeutic strategies.
  • Isoform 2 of Claudin-18 (Claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker whose expression in normal tissues is strictly restricted to differentiated epithelial cells of the gastric mucosa but not to the gastric stem cell zone.
  • CLDN18.2 is expressed in a considerable part of primary gastric cancer, and its expression level is retained in gastric metastatic cancer tissues.
  • CLDN18.2 expression is also found in pancreatic cancer, which is an ideal target molecule for the treatment of these cancers (Singh,P.,Toom,S.&Huang,Y.Anti-CLDN18.2 antibody as new targeted therapy for advanced gastric cancer. J Hematol Oncol 10, 105 (2017). https://doi.org/10.1186/s13045-017-0473-4 ).
  • ADCs antibody-drug conjugates
  • the present invention provides an antibody-drug conjugate (ADC) targeting Claudin18.2, which has the following advantages:
  • the ADC of the present invention has high endocytosis efficiency
  • CLDN18.2 is expressed or overexpressed on the cell surface; in some embodiments, the target cells are CHO cells or 293 cells expressing CLDN18.2, such as CHO-S cells or HEK293 cells; in some embodiments wherein the target cell is a cancer cell expressing CLDN18.2, such as a cell expressing CLDN18.2 naturally or artificially transfected to express CLDN18.2 or having an increased expression level of CLDN18.2 through artificial transfection, such as expressing CLDN18.2 gastric or pancreatic cancer cell lines or colon or colorectal cancer cell lines.
  • the target cell is a cell line with an intermediate expression level of hCLDN18.2, eg, NUGC-4, SNU620.
  • the target cells are cell lines with high expression levels, such as DAN-G cells overexpressing hCLDN18.2.
  • Figure 1 shows that the HB37A6 antibody specifically binds to CLDN18.2 on the cell surface.
  • Figure 2 shows that the HB37A6 antibody does not bind cell surface CLDN18.1.
  • Figure 3 shows the binding of HB37A6 antibody to gastric cancer cell line NUGC-4, gastric cancer cell line KATO III-hCLDN18.2 and pancreatic cancer cell line DAN-G-hCLDN18.2.
  • Figure 4 shows the anti-tumor effect of HB37A6 antibody in a mouse model of pancreatic cancer.
  • Figure 5 shows the anti-tumor effect of the HB37A6 antibody in a mouse model of gastric cancer.
  • Figure 6 shows the cell binding of the IEX019 molecule.
  • Figure 7 shows the endocytosis of IEX019 molecules on DANG-hCLDN18.2 cells.
  • Figure 8 shows the killing effect of IEX019 molecules on cell lines with low expression of hCLDN18.2 ( Figure 8A), the killing effect of IEX019 molecules on cell lines with medium expression levels of hCLDNA8.2 ( Figure 8B) and the killing effect of IEX019 molecules on cell lines with high expression of hCLDN18.2 Killing effect of cell lines (Fig. 8C).
  • Figure 9 shows the side-killer effect of the IEX019 molecule.
  • Figure 10 shows the tumor suppressive efficacy (Figure 10A) and body weight change (Figure 10B) of the IEX019 molecule in mice.
  • Figure 11 shows the tumor suppressive efficacy (Figure 11A) and body weight change (Figure 11B) of the IEX019 molecule in mice.
  • Figure 12 shows the tumor suppressive efficacy (Figure 12A) and body weight change (Figure 12B) of the IEX019 molecule in mice.
  • the term “comprising” or “comprising” means including stated elements, integers or steps, but not excluding any other elements, integers or steps.
  • the term “comprising” or “comprising” is used, unless otherwise specified, it also covers the situation of combining the mentioned elements, integers or steps.
  • an antibody variable region that "comprises” a particular sequence it is also intended to encompass an antibody variable region that consists of that particular sequence.
  • Claudin protein is the most important scaffold protein determining the structure of tight junctions between cells, which is involved in adherens junctions, and plays an important role in the metastasis and invasion of tumor cells.
  • Claudin protein widely exists in mammalian epithelial and endothelial cells, and its distribution is mainly on the side of epithelial cells and on the plasma membrane of basal cells. Different Claudin proteins have their own specific expression in different tissues.
  • the Claudin18 (CLDN18) gene is located at 3q22.3, with a molecular weight of 24kDa and 261 amino acid residues.
  • CLDN18 or Claudin18 protein contains two extracellular ring and 4 transmembrane regions.
  • the two subtypes of human CLDN18 or Claudin18 protein are Claudin18.1 or CLDN18.1 (UniProt ID: P56856-1), and Claudin18.2 or CLDN18.2 (UniProt ID: P56856-2), respectively.
  • CLDN18.1 and CLDN18.2 UniProt ID: P56856-2
  • CLDN18.2 the sequence of extracellular loop 1 of CLDN18.2 is completely consistent in different species such as human, mouse, and macaque, and the homology of CLDN18.2 protein between human and mouse reaches 84%, indicating that the protein sequence of CLDN18.2 is extremely conserved (O.Tureci. et al., Gene 481:83-92, 2011).
  • CLDN18.2 or any variants and isoforms thereof may be isolated from cells or tissues that naturally express them, or produced recombinantly using techniques well known in the art and/or those described herein.
  • the CLDN18.2 described herein is human CLDN18.2.
  • anti-CLDN18.2 antibody As used herein, the term "anti-CLDN18.2 antibody”, “anti-CLDN18.2”, “CLDN18.2 antibody” or “antibody that binds to CLDN18.2” or “antibody that specifically binds to CLDN18.2” refers to such an antibody , said antibody is capable of binding (human) CLDN18.2 with sufficient affinity such that said antibody can be used as a therapeutic agent targeting (human) CLDN18.2. In one embodiment, said (human) CLDN18.2 antibody binds (human) CLDN18.2 with high affinity in vitro or in vivo. In one embodiment, said (human) CLDN18.2 antibody does not bind CLDN18.1.
  • said (human) CLDN18.2 antibody binds to cells expressing CLDN18.2 but not to cells expressing CLDN18.1.
  • the binding is measured, eg, by radioimmunoassay (RIA), biofilm thin layer interferometry (BLI), MSD assay, or surface plasmon resonance (SPR), or flow cytometry.
  • RIA radioimmunoassay
  • BLI biofilm thin layer interferometry
  • MSD assay MSD assay
  • SPR surface plasmon resonance
  • the expression of CLDN18.2 in cells can be determined by various means, such as anti-CLDN18.2 antibody.
  • the binding strength of the "highly expressing CLDN18.2" cells to the anti-CLDN18.2 antibody may be 500, 600, 700 times the binding strength of the anti-CLDN18.2 antibody to the cells not expressing CLDN18.2 , 800, 900 or preferably 1000 times or more, for example 1100 times, 1200, 1300, 1400 times or more or higher.
  • cells that "intermediately express CLDN18.2" can bind to an anti-CLDN18.2 antibody (e.g., as determined by FACS) 5-500 times more strongly than an anti-CLDN18.2 antibody to cells that do not express CLDN18.2 , such as 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 times or higher, but not more than 500 times.
  • an anti-CLDN18.2 antibody e.g., as determined by FACS
  • 5-500 times more strongly than an anti-CLDN18.2 antibody to cells that do not express CLDN18.2 such as 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 times or higher, but not more than 500 times.
  • full-length antibody refers to an antibody molecule having the molecular structure of a native immunoglobulin.
  • a full-length antibody comprises two heavy chains (H) and two light chains (L) inter-connected by disulfide bonds.
  • the full length antibody comprises two heavy chains (H) interconnected by disulfide bonds.
  • the full-length antibody heavy chain usually consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region, wherein the heavy chain constant region contains at least three domains CH1, CH2 and CH3.
  • a full-length antibody light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain, CL.
  • Each heavy chain variable region VH and each light chain variable region consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • antibody fragment includes a portion of an intact antibody.
  • antibody fragments are antigen-binding fragments.
  • Antigen-binding fragment refers to a molecule, distinct from an intact antibody, that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; dAb (domain antibody); linear antibodies; single chain antibodies (e.g. scFv); ; a diabody or a fragment thereof; or a camelid antibody.
  • antigen refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • epipe refers to the portion of an antigen (eg, CLDN18.2) that specifically interacts with an antibody molecule.
  • a “complementarity determining region” or “CDR region” or “CDR” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ( "antigen contact point”).
  • the CDRs are primarily responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus.
  • the CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S.
  • a CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).
  • the heavy chain variable region CDR of the antibody of the present invention is determined according to the following rules:
  • VH CDR1 is determined according to AbM rules; and VH CDR2 and 3 are both determined according to Kabat rules.
  • the light chain variable region CDRs of the antibodies of the present invention are determined according to Kabat's rules.
  • the heavy chain variable region CDR of the antibody of the present invention is determined according to the following rules: VH CDR1 is determined according to the AbM rule; and VH CDR2 and 3 are determined according to the Kabat rule; and the light chain variable region CDR is determined according to the Kabat rule .
  • the boundaries of the CDRs of the variable region of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems are different.
  • the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • Antibodies with different specificities have different binding sites for different antigens
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding.
  • a minimal binding unit may be a subsection of a CDR.
  • the residues of the remainder of the CDR sequences can be determined from the structure and protein folding of the antibody. Accordingly, the invention also contemplates variations of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conserved amino acid residues.
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a native immunoglobulin "Fc domain” comprises two or three constant domains, a CH2 domain, a CH3 domain and optionally a CH4 domain.
  • the immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) derived from the two heavy chains of antibodies of the IgG, IgA and IgD classes; The second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) from the two heavy chains of antibodies of the IgM and IgE classes.
  • amino acid residue numbering in the Fc region or heavy chain constant region is according to, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, The EU numbering system described in 1991 (also known as the EU Index) is used for numbering.
  • the term "Fc region” does not include the heavy chain variable region VH and the light chain variable region VL and the heavy chain constant region CH1 and the light chain constant region CL of an immunoglobulin, but may be included in the heavy chain in some cases.
  • the hinge region at the N-terminus of the chain constant region is according to, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, The EU numbering system described in 1991 (also known as the EU Index) is used for numbering.
  • the term "Fc region” does not include the heavy chain variable region V
  • an antibody in IgG form refers to the IgG form to which the heavy chain constant region of the antibody belongs.
  • the heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different.
  • an antibody in IgG4 form means that its heavy chain constant region is from IgG4
  • an antibody in IgGl form means that its heavy chain constant region is from IgGl.
  • bind or “specifically bind” means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antigen binding site to bind a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD method or surface plasmon resonance (SPR).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • MSD method surface plasmon resonance
  • antibody-drug conjugate refers to a structure obtained by linking an antibody and a drug.
  • sucrose indicates monosaccharides such as glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc).
  • saccharide indicates monosaccharides such as glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc).
  • sugar derivative means a derivative of a monosaccharide, ie a monosaccharide comprising substituents and/or functional groups.
  • sugar derivatives include amino sugars and sugar acids such as glucosamine (GlcN), galactosamine (GalN), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetyl Neuraminic acid (NeuNAc) and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA) and iduronic acid (IdoA).
  • sugar derivatives also include compounds denoted herein as E(A)x, where E is a sugar or a sugar derivative, and where E includes x functional groups A.
  • a core-N-acetylglucosamine substituent is defined herein as a GlcNAc bonded to the antibody's GlcNAc through C1, preferably through and the N of the amide nitrogen atom on the side chain of the antibody's asparagine amino acid. -Glycosidic bond.
  • the core-GlcNAc substituent can be present at the antibody's native glycosylation site, but it can also be introduced at a different site on the antibody.
  • the core-N-acetylglucosamine substituent is a monosaccharide substituent, or (if the core-GlcNAc substituent is fucosylated) a disaccharide core-(Fuc ⁇ 1-6)GlcNAc substituent— Also known as GlcNAc(Fuc).
  • glycosylation engineering refers to the process of changing the sugar chains of antibodies by glycosylation engineering. Glycosylation of antibodies can be further engineered to obtain antibodies with novel glycosylation for various purposes, for example, removal of glycosylation to eliminate Fc ⁇ R affinity and complement fixation/effector functions, to enhance Fc-mediated
  • the ADCC and CDC effects decreased fucose and sialic acid groups and increased bisected N-acetylglucosamine, galactose and mannose.
  • glycosylation engineering for example, by changing the glycosylation site of the antibody to increase or decrease the sugar chains on the surface of the antibody, or by chemically or enzymatically modifying the sugar chains in vitro, or by expressing
  • the glycosylation pathway of the system eg, composed of enzymes such as glycosidases and glycosyltransferases
  • the glycosylation pathway of the system is changed to catalyze the glycosylation of antibodies, and the glycosylation of antibodies can also be changed by the influence of cell culture conditions.
  • the "glycosylation modification" of the present invention is carried out by in vitro enzymatic modification of sugar chains.
  • the glycosylation modification of the present invention is carried out by modifying sugar chains with glycosidases (such as endoglycosidases or glycosyltransferases).
  • an antibody with engineered glycosylation of the present invention refers to an antibody with an engineered glycosylation pattern compared to an antibody with a natural glycosylation pattern.
  • the antibody with engineered glycosylation means that the antibody expressed in an expression system (such as mammalian cells) undergoes enzymatic modification of sugar chains in vitro (such as by glycosidases (such as endoglycosidases). or glycosyltransferase) to modify the sugar chain) antibody obtained.
  • the antibody with engineered glycosylation of the present invention refers to an antibody comprising a core-GlcNAc and a sugar derivative E(A)x linked thereto, wherein GlcNAc is bonded to the antibody through C1, preferably through and the antibody's N-glycosidic bond to the amide nitrogen atom on the side chain of the asparagine amino acid. If the -GlcNAc substituent in the GlcNAc-E(A)x substituent is fucosylated, typically fucose is linked to C6 of the -GlcNAc substituent via ⁇ -1,6.
  • a fucosylated -GlcNAc substituent refers to core-GlcNAc(Fuc), and a fucosylated GlcNAc-E(A)x substituent refers to GlcNAc(Fuc)-E(A)x.
  • site-specific conjugation refers to conjugation in which a drug/active substance is specifically linked to a specific site of an antibody through a linker.
  • alkyl refers to a fully saturated branched or unbranched hydrocarbon group.
  • the alkyl group preferably contains 1-24 carbon atoms, more preferably 1-16 carbon atoms, 1-12 carbon atoms, 1-6 carbon atoms or 1-4 carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, Base, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.
  • aryl refers to a monocyclic or bicyclic aromatic hydrocarbon group having 6-20, eg 6-12 carbon atoms in the ring portion.
  • aryl is (C 6 -C 10 )aryl.
  • Non-limiting examples include phenyl, biphenyl, naphthyl or tetrahydronaphthyl, each of which may be optionally substituted with 1-4 substituents such as alkyl, trifluoromethyl, cycloalkyl , halogen, hydroxyl, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, mercapto, alkyl-S-, aryl-S- , nitro, cyano, carboxyl, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonylamino, heterocyclyl, etc
  • cycloalkyl is a cyclic alkyl group, that is, a monovalent, saturated or unsaturated hydrocarbon group having a ring structure. Cycloalkyl includes all saturated or partially saturated (containing 1 or 2 double bonds) hydrocarbon groups having a cyclic structure. Cycloalkyl groups may contain 3 or more, eg, 3-18, 3-10, or 3-8 carbon atoms, and typically, according to the invention, 3 to 6 atoms in the ring. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
  • heteroaryl refers to a 5-20 membered (eg 5-14, 5-8, 5-6) membered unit containing 1-8 heteroatoms selected from N, O or S Cyclic - or bicyclic - or fused polycyclic ring systems.
  • heteroaryl is a 5-10 membered ring system.
  • heteroaryl groups include 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4- or 5-imidazolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-isothiazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-iso Oxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3- or 4-pyridyl, 3 - or 4-pyridazinyl, 3-, 4- or 5-pyrazinyl, 2-pyrazinyl, 2-, 4- or 5-pyrimidinyl.
  • pharmaceutically acceptable salt refers to a salt that maintains the biological effects and properties of the ADC conjugate of the present invention, and the salt is not biologically or otherwise undesirable.
  • the ADC conjugates of the present invention may exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts.
  • a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed between the ADC conjugate in the present invention and an organic or inorganic acid
  • the organic or inorganic acid includes but is not limited to hydrochloric acid, sulfuric acid, hydrobromic acid , hydriodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, Benzoic acid, p-toluenesulfonic acid, malic acid, etc.
  • the pharmaceutically acceptable non-toxic base addition salt refers to the salt formed by the ADC conjugate in the present invention and an organic or inorganic base, 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; salts of organic bases, such as ammonium salts formed with organic bases containing N groups.
  • alkali metal salts such as lithium, sodium or potassium salts
  • alkaline earth metal salts such as calcium or magnesium salts
  • salts of organic bases such as ammonium salts formed with organic bases containing N groups.
  • solvate means an association formed by one or more solvent molecules and the ADC conjugate of the present invention.
  • Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethylsulfoxide, and the like.
  • DAR drug:antibody ratio
  • DAR refers to the ratio of small molecule drug moiety (D) to Ab moiety conjugated to the Ab moiety described herein.
  • DAR can be determined by p and r in formula I, for example DAR can be 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, eg 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • DAR can also be calculated as the average DAR of the population of molecules in the product, i.e., the Ab moieties conjugated to the Ab moieties described herein, measured by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assays, electrophoresis and/or HPLC).
  • detection methods e.g., by conventional methods such as mass spectrometry, ELISA assays, electrophoresis and/or HPLC.
  • the overall ratio of the small molecule drug fraction (D) to the Ab fraction, this DAR is referred to as the average DAR in the text.
  • the conjugates of the invention have an average DAR value of 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4 -6, 6-10, such as 1.0-8.0, 2.0-6.0, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 ,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4,4.1,4.2,4.3,4.4,4.5,4.6 , 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 ,7.2,7.3,7.4,7.5,7.6,7.7,7.8.0,7
  • therapeutic agent encompasses any substance effective in the prevention or treatment of tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immunosuppressants).
  • cytotoxic agent is used in the present invention to refer to a substance that inhibits or prevents cell function and/or causes cell death or destruction.
  • “Chemotherapeutic agents” include chemical compounds useful in the treatment of cancer or disorders of the immune system.
  • small molecule drug refers to low molecular weight organic compounds capable of modulating biological processes.
  • Small molecules are defined as molecules having a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 1 kD.
  • Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules can be more cell permeable, less susceptible to degradation, and less prone to eliciting an immune response than larger molecules.
  • the term "immunomodulator” refers to a natural or synthetic active agent or drug that suppresses or modulates an immune response.
  • the immune response can be a humoral or cellular response.
  • Immunomodulators include immunosuppressants.
  • the immunomodulators of the invention include immune checkpoint inhibitors or immune checkpoint agonists.
  • an effective amount refers to such an amount or dose of the antibody or fragment or composition or combination of the present invention, which produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in single or multiple doses.
  • a “therapeutically effective amount” refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (e.g. tumor volume) by at least about 30%, even more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, relative to an untreated subject. %, 70%, 75%, 80%, 85%, 90% or even 100%.
  • prophylactically effective amount refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired prophylactic result. Typically, a prophylactically effective amount will be less than a therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity in originally transformed cells are included herein.
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a polynucleotide probe or antibody, and facilitates detection of the agent to which it is conjugated or fused.
  • Labels can themselves be detectable (eg, radioisotopic or fluorescent labels) or, in the case of enzymatic labels, can catalyze the chemical alteration of a detectable substrate compound or composition.
  • the term is intended to encompass both direct labeling of a probe or antibody by conjugating (ie, physically linking) a detectable substance to the probe or antibody as well as indirect labeling of a probe or antibody by reacting with another reagent that is directly labeled.
  • “Individual” or “subject” includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., , mice and rats). In some embodiments, the individual or subject is a human.
  • an “isolated” antibody or other molecule is one that has been separated from a component of its natural environment or the environment in which it is expressed.
  • the antibody or ADC molecule is purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reverse phase HPLC.
  • anti-tumor effect refers to a biological effect that can be exhibited by various means, including but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival.
  • tumor and cancer are used interchangeably herein to encompass both solid tumors and hematological tumors.
  • cancers suitable for treatment by the antibodies of the invention include gastric, pancreatic, or gastroesophageal junction cancers, including metastatic forms of those cancers.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancerous and cancerous cells and tissues.
  • pharmaceutical excipient refers to a diluent, adjuvant (such as Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., which are administered together with the active substance.
  • adjuvant such as Freund's adjuvant (complete and incomplete)
  • excipient carrier or stabilizer, etc.
  • composition refers to a composition that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional substances that are unacceptably toxic to the subject to which the composition is administered. ingredients.
  • non-fixed combination means that the active ingredients (e.g., (i) the ADC molecule of the invention, and (ii) the other therapeutic agent) are combined as separate entities simultaneously, with no specific time limit or at the same or different time intervals, Sequential administration to the patient wherein such administration provides prophylactically or therapeutically effective levels of the two or more active agents in the patient.
  • ADC molecules of the invention and other therapeutic agents used in pharmaceutical combinations are administered at levels no greater than when they are used alone.
  • fixed combination means that two or more active agents are administered to a patient simultaneously as a single entity.
  • Dosages and/or intervals of two or more active agents are preferably selected such that the combined use of the parts produces a greater effect in treating a disease or condition than can be achieved by any one component alone.
  • the components may each be in the form of separate formulations, which may be the same or different.
  • combination therapy refers to the administration of two or more therapeutic agents or treatment modalities, such as radiation therapy or surgery, to treat a disease described herein.
  • administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, eg, in a single capsule with fixed ratios of the active ingredients.
  • administration includes co-administration for each active ingredient in multiple or in separate containers (eg tablets, capsules, powders and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dosage before administration.
  • such administration also includes using each type of therapeutic agent in a sequential manner at about the same time or at different times. In either case, the treatment regimen will provide for the beneficial effect of the drug combination in treating the disorders or conditions described herein.
  • treating means slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes the inhibition of the occurrence or development of a disease or disorder or a symptom of a particular disease or disorder.
  • subjects with a family history of cancer are candidates for prophylactic regimens.
  • prevention refers to the administration of a drug prior to the onset of signs or symptoms of cancer, especially in subjects at risk of cancer.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • Subject/patient/individual sample refers to a collection of cells or fluid obtained from a patient or subject.
  • the source of the tissue or cell sample can be solid tissue like from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood components; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid ), peritoneal fluid (ascites), or interstitial fluid; cells from any time during pregnancy or development of a subject.
  • Tissue samples may contain compounds that are not naturally intermingled with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • the present invention provides an antibody-drug conjugate having formula (I):
  • Ab is an antibody or fragment thereof that binds to CLDN18.2 (eg, human CLDN18.2);
  • L is a linker
  • D is a drug, including a prodrug, preferably an antineoplastic compound
  • p is 1 to 10, eg 1-9, 2-8, 3-7, 4-6, 2-6, eg 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • r is 1-5, such as 1, 2, 3, 4 or 5, preferably 1 or 2.
  • the Ab in formula (I) of the present invention is a human antibody or a humanized antibody, preferably a human antibody.
  • the Ab in formula (I) of the present invention is an antibody fragment, preferably an antigen-binding fragment, such as Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; dAb (domain antibody) a linear antibody; a single chain antibody (eg scFv); a single domain antibody such as a VHH; a diabody or a fragment thereof; or a camelid antibody.
  • the Ab of formula (I) of the invention is a bispecific antibody or a multispecific antibody.
  • the Ab comprises three complementarity determining regions (HCDRs), HCDR1, HCDR2 and HCDR3, derived from the heavy chain variable region.
  • HCDRs complementarity determining regions
  • the Ab comprises three complementarity determining regions (LCDRs), LCDR1, LCDR2 and LCDR3, derived from the light chain variable region.
  • LCDRs complementarity determining regions
  • the Ab comprises 3 complementarity determining regions (HCDRs) from the heavy chain variable region and 3 complementarity determining regions (LCDRs) from the light chain variable region.
  • HCDRs complementarity determining regions
  • LCDRs complementarity determining regions
  • the Ab comprises a heavy chain variable region (VH). In some aspects, the Ab comprises a light chain variable region (VL). In some aspects, the Ab comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises three complementarity determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, from the heavy chain variable region. In some embodiments, the light chain variable region comprises three complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, from the light chain variable region.
  • HCDRs complementarity determining regions
  • LCDRs three complementarity determining regions
  • VH VH
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 4 or consists of said amino acid sequence;
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO:4;
  • amino acid sequence (iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 4 , more preferably amino acid conservative substitutions), the amino acid sequence consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR region.
  • VL VL
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 9 or consists of said amino acid sequence; or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 9;
  • amino acid sequence (iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 9 , more preferably amino acid conservative substitutions), the amino acid sequence consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR region.
  • the three complementarity determining regions (HCDRs) from VH of the invention HCDR1, HCDR2 and HCDR3 are
  • the sequence contains at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions.
  • the three complementarity determining regions (LCDRs) from VL of the invention, LCDR1, LCDR2 and LCDR3 are
  • the sequence contains at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions.
  • HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:1, or HCDR1 comprises one, two or three changes (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.
  • HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO:2, or HCDR2 comprises one, two or three changes (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.
  • HCDR3 comprises the amino acid sequence of SEQ ID NO:3, or consists of said amino acid sequence, or HCDR3 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO:3 (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.
  • LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:6, or LCDR1 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO:6 (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.
  • the LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 7, or the LCDR2 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO: 7 (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.
  • the LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 8, or the LCDR3 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO: 8 (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.
  • the Ab of formula (I) of the invention comprises a heavy chain constant region. In some embodiments, the Ab of formula (I) of the invention comprises a light chain constant region. In some embodiments, the Ab of formula (I) of the invention further comprises a heavy chain constant region and a light chain constant region.
  • the heavy chain constant region HC of the present invention is an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, preferably an IgG1 heavy chain constant region, such as a wild-type IgG1 heavy chain constant region.
  • the antibody light chain constant region LC of the present invention is a lambda or kappa light chain constant region.
  • the heavy chain constant region HC of the present invention is a heavy chain constant region HC of the present invention.
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO:5;
  • amino acid sequence comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 5 ( Preferably amino acid substitutions, more preferably amino acid conservative substitutions), or consist of amino acid sequences.
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 10;
  • amino acid sequence comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 10 ( Preferably amino acid substitutions, more preferably amino acid conservative substitutions), or consist of amino acid sequences.
  • the Ab of formula (I) of the invention comprises a heavy chain. In some specific embodiments of the invention, the Ab of formula (I) of the invention comprises a light chain. In some specific embodiments of the present invention, the Ab in formula (I) of the present invention comprises a heavy chain and a light chain.
  • the heavy chain of the invention comprises or consists of a heavy chain variable region and a heavy chain constant region.
  • the light chain of the invention comprises a light chain variable region and a light chain constant region, or consists of said light chain variable region and a light chain constant region.
  • the Ab in the formula (I) of the present invention specifically binds to CLDN18.2, and comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3, and/or the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:9.
  • the Ab in formula (I) of the present invention comprises HCDR1, HCDR2, HCDR3 shown in the following amino acid sequences respectively: SEQ ID NO:1, 2 and 3, and/or respectively as shown in LCDR1, LCDR2 and LCDR3 shown in the following amino acid sequences: SEQ ID NOs: 6, 7 and 8.
  • the Ab in formula (I) of the present invention comprises
  • VH comprising the amino acid sequence shown in SEQ ID NO: 4 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto amino acid sequence or consists of said amino acid sequence, and/or
  • VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 90% identity therewith.
  • the Ab in formula (I) of the present invention comprises VH and VL, wherein the amino acid sequence of VH is represented by SEQ ID NO:4, and the amino acid sequence of VL is represented by SEQ ID NO:9 Show.
  • the Ab in formula (I) of the invention is an IgG antibody, ie it comprises a heavy chain and a light chain that binds to CLDN18.2.
  • the Abs of formula (I) of the invention are whole antibodies.
  • the heavy chain of the Ab in formula (I) is the heavy chain of the Ab in formula (I).
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 11;
  • amino acid sequence comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 11 ( Preferably amino acid substitutions, more preferably amino acid conservative substitutions), or consist of amino acid sequences.
  • the light chain of the Ab in formula (I) is the light chain of the Ab in formula (I)
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 12;
  • amino acid sequence comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 12 ( Preferably amino acid substitutions, more preferably amino acid conservative substitutions), or consist of amino acid sequences.
  • the Ab in the formula (I) of the present invention comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is represented by SEQ ID NO: 11, and the amino acid sequence of the light chain is represented by SEQ ID NO: NO:12 shown.
  • the amino acid changes described herein include amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid changes described herein occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes of the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region. In some embodiments, the amino acid changes described herein occur in the Fc region of the antibody heavy chain constant region.
  • substitutions are conservative substitutions.
  • a conservative substitution is one in which an amino acid is replaced by another within the same class, such as one acidic amino acid by another acidic amino acid, one basic amino acid by another basic amino acid, or one neutral amino acid by another neutral amino acid replacement.
  • substitutions occur in the CDR regions of the antibody.
  • the resulting variant is modified (eg, improved) relative to the parent antibody in certain biological properties (eg, increased affinity) and/or will have certain biological properties of the parent antibody that are substantially retained.
  • Exemplary substitutional variants are affinity matured antibodies.
  • the antibody Ab in formula (I) of the present invention may be an antibody with engineered glycosylation.
  • the antibody is an antibody obtained by enzymatically modifying sugar chains in vitro (for example, sugar chains are modified by glycosidases such as endoglycosidases or glycosyltransferases).
  • the antibody with modified glycosylation refers to that the sugar chain on the glycosylation site of the antibody is modified from a non-uniform structure N sugar chain to a reactive group (for example, capable of interacting with a linker moiety). Any reactive group that reacts, such as azido, keto, and alkynyl) is a single-structure N-glycan antibody.
  • the N-glycosylation site of the antibody is a conserved N-glycosylation site on the Fc domain of the antibody, such as Asn297.
  • an antibody of the invention having engineered glycosylation is an antibody comprising a GlcNAc-E(A) x substituent, wherein GlcNAc is N-acetylglucosamine, wherein E(A)x is A sugar derivative comprising x functional groups A, wherein A is independently selected from azido, keto and alkynyl and x is 1, 2, 3 or 4; wherein the GlcNAc-E(A)x substituent is obtained by C1 of the N-acetylglucosamine of the GlcNAc-E(A)x substituent is bonded to the antibody, wherein the N-acetylglucosamine is optionally fucosylated. Where N-acetylglucosamine is fucosylated, it is bonded to fucose (Fuc) via C6.
  • the antibody with engineered glycosylation of the present invention is an antibody of formula (III), wherein Ab represents an antibody, GlcNAc is N-acetylglucosamine, Fuc is fucose, b is 0 or 1 , y is 1 to 20, wherein E(A)x is a sugar derivative containing x functional groups A, wherein A is azido, keto and alkynyl, and x is 1, 2, 3 or 4.
  • y is 1 to 10, more preferably, y is 1, 2, 3, 4, 5, 6, 7 or 8, even more preferably, y is 1, 2, 3 or 4, Most preferably, y is 1 or 2.
  • the sugar derivative E(A)x in the GlcNAc-E(A)x substituent of an antibody with engineered glycosylation may be bonded to C4 of the GlcNAc, for example, via a ⁇ (1,4)-glycosidic bond or via An ⁇ (1,3)-glycosidic bond is bonded to C3 of the GlcNAc, preferably via a ⁇ (1,4)-glycosidic bond to C4 of the GlcNAc.
  • the N-acetylglucosamine of the GlcNAc-E(A)x substituent is C1 bonded to the antibody, preferably N-glycosidically bonded to the side chain of the antibody's asparagine Amide nitrogen atom (GlcNAc ⁇ 1-Asn).
  • the GlcNAc on the GlcNAc-E(A)x substituent is optionally fucosylated.
  • GlcNAc-E in the antibody-drug conjugate when present, can also be linked as previously described.
  • the functional group A is an azido group.
  • A is azido, preferably A is bonded to C2, C3, C4 or C6.
  • one or more azide substituents in E(A)x may be bonded to C2, C3, C4 or C6 of the sugar or sugar derivative E in place of the hydroxyl (OH). It is understood that the bonding position of the functional group A corresponds to the position at which the A-containing sugar is attached to the linker.
  • the sugar derivative E(A)x is derived from a sugar or sugar derivative E.
  • E is a sugar or a sugar derivative selected from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactose Amine (GalNAc), glucuronic acid (Gcu), fucose (Fuc) and N-acetylneuraminic acid (sialic acid), preferably Gal, GlcNAc, glucose and GalNAc, most preferably GalNAc.
  • the E(A)x is GalNAc-N 3 , preferably, E(A)x is 6-azido-6-deoxy-2-acetamidogalactose.
  • the reactive group (such as functional group A) is attached to the sugar chain of the antibody during the glycosylation of the antibody, therefore "an antibody with engineered glycosylation" It is defined as the antibody containing the reactive group, but when the antibody-drug conjugate is formed, the reactive group may react with the linker part, and then form a new group with the linker part, so that in the antibody In drug conjugates, this new group is considered as part of the linker.
  • the antibody of the present invention is a monoclonal antibody, more preferably an IgG antibody (such as a four-chain IgG antibody), most preferably an IgG1 antibody.
  • the antibody is a whole antibody.
  • the antibody preferably comprises two or more, more preferably two, GlcNAc-E(A)x substituents, the GlcNAc-E(A)x substituents is optionally fucosylated.
  • the modified antibody is an antibody fragment, such as a Fab or Fc fragment, the antibody may have only one GlcNAc-E(A)x substituent, which is optionally fucosylated.
  • the GlcNAc-E(A)x substituents can be located anywhere on the antibody, provided that the substituents do not interfere with the binding of antigen to the antigen binding site of the antibody.
  • the GlcNAc-E(A)x substituent is located in the Fc domain of the antibody, more preferably, in the CH2 domain.
  • the antibody with engineered glycosylation of the present invention comprises more than one GlcNAc-E(A)x substituent, for example, two GlcNAc-E(A)x substituents.
  • the GlcNAc-E(A)x substituent is present at a native N-glycosylation site (e.g., a naturally conserved N-glycosylation site) of the antibody, e.g., the Fc region (more Preferably at the glycosylation site of the CH2 domain).
  • the antibody is an IgG antibody and the GlcNAc-E(A)x substituent is present at the natural N-glycosylation site of the IgG antibody (naturally conserved N-glycosylation site) superior.
  • said native site is the Asn297-glycosylation site of an IgG antibody.
  • the Asn297-glycosylation site is present in the Fc region of the heavy chain of an IgG antibody.
  • said GlcNAc-E(A)x substituent is present at the Asn297-glycosylation site of both heavy chains of the antibody.
  • L in formula (I) of the invention is a linker. Any linker known in the art can be used to link with the anti-human CLDN18.2 of the present invention, preferably, the linker can realize site-specific coupling of ADC.
  • a linker suitable for use in the present invention may be any linker that enables conjugation of the antibody to the drug.
  • the linker may be a linker used in a technique that enables site-specific conjugation.
  • the linker of the invention is a linker attached to an antibody oligosaccharide.
  • the "linker linked to the antibody oligosaccharide” as defined herein refers to any linker linked to the reactive group on the sugar chain at the glycosylation site of the antibody to couple the antibody to the drug.
  • the sugar chains on the glycosylation sites of antibodies are generally N-sugar chains, and are usually engineered to transform non-uniform structural N-sugar chains into single-structure N-sugar chains with reactive groups, and then use the sugar chains to carry The reactive group of the drug is connected to the "linker" to realize the site-specific coupling of the drug and the antibody to obtain an antibody-drug conjugate.
  • the N-glycosylation site of the antibody is an antibody Fc domain, preferably a conserved N-glycosylation site on the CH2 domain, such as Asn297. Therefore, in one embodiment, the "linker linked to antibody oligosaccharides" of the present invention is especially linked to the N-glycosylation site at the conserved N-glycosylation site (such as Asn297) on the Fc domain of the antibody. Any linker whose reactive group enables site-specific coupling, for example the linker described in PCT/NL2013/050744 or the linker of PCT/EP2021/075401, which is incorporated herein in its entirety.
  • the reactive group of the invention is an azido, keto or alkynyl group.
  • a linker of the invention is a linker comprising an alkynyl group.
  • the reactive group of the invention is an azido, keto or alkynyl group, preferably an azido group, and the linker of the invention is a linker comprising an alkynyl group.
  • the present invention refers to this type of linker, because the reactive group reacts with the linker group to form a new group, it can also be formed after the reaction of the reactive group in the antibody-drug conjugate
  • a group is defined as part of a "linker", eg as shown in formula (II) of the present invention.
  • Linkers suitable for use in the present invention also include, for example, cathepsin-degradable linkers, such as Val-Cit linkers (eg, vc-PAB), cBu-Cit linkers, and CX linkers; non-cleavable linkers, such as SMCC linkers or MD Linkers; acid-sensitive linkers, silicone grease-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, and near-infrared-sensitive linkers UV-sensitive linkers such as PC4AP (Antibody–drug conjugates:Recent advances in linkerchemistry, Su, Z., Xiao, D., Xie, F., Liu, L., Wang, Y., Fan, S., ...Li, S.
  • cathepsin-degradable linkers such as Val-Cit linkers (eg, vc-PAB),
  • the linker suitable for the present invention can also be a combination of one or more linkers, for example, a cathepsin-degraded linker can be combined with other types of linkers to form a new linker. Therefore, the "linker” of the present invention covers a single type of linker, or a combination of different types of linkers, as long as it can couple the antibody of the present invention to the drug.
  • a linker suitable for use in the present invention is MC-VC-PAB, vc-PAB, SMCC or MC-GGFG.
  • D in formula (I) of the present invention can be any antitumor compound, as long as it has antitumor effect and has substituents and partial structures that can be connected to the linker structure, there is no particular limitation.
  • an antineoplastic compound may be a pharmaceutically active compound that has an effect on tumors.
  • the antitumor compound it is preferable that a part or all of the linker can be cleaved in the tumor cell to release the antitumor compound part, thereby exhibiting the antitumor effect. When the linker with the drug is cut off, the antitumor compound is released in an unmodified structure, and its original antitumor effect can be exerted.
  • the antineoplastic compound may be, for example, a cytotoxic or chemotherapeutic agent, such as the camptothecin-like compounds Exatecan (a topoisomerase I inhibitor Exatecan), Dxd (a novel topoisomerase I Inhibitor Exatecan derivatives), auristatins such as monomethylauristatin E (MMAE) or maytansinoids such as small molecule microtubule inhibitor DM1.
  • a cytotoxic or chemotherapeutic agent such as the camptothecin-like compounds Exatecan (a topoisomerase I inhibitor Exatecan), Dxd (a novel topoisomerase I Inhibitor Exatecan derivatives), auristatins such as monomethylauristatin E (MMAE) or maytansinoids such as small molecule microtubule inhibitor DM1.
  • a cytotoxic or chemotherapeutic agent such as the camptothecin-like compounds Exatecan (a topoisomerase I inhibitor Exatecan),
  • the present invention provides the antibody-drug conjugate of (II) or a pharmaceutically acceptable salt or solvate thereof
  • L 1 is a linker
  • E is a sugar or a sugar derivative, such as a sugar or sugar derivative as defined above;
  • GlcNAc N-acetylglucosamine
  • Fuc fucose
  • b is 0 or 1, such as 0;
  • x is 1, 2, 3 or 4; preferably 1 or 2;
  • y is 1-20;
  • Fuc is fucose
  • b is 0 or 1, for example 0; and/or x is 1 or 2, more preferably 2, and/or y is 1 or 2, more preferably 2 .
  • E is selected from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc) and N-acetylneuraminic acid (sialic acid), preferably Gal, GlcNAc, glucose and GalNAc, most preferably GalNAc, such as 6-deoxy-2- Acetamidogalactose, which is preferably linked to L1 via the C atom at position 6.
  • the GlcNAc linked to the Ab is present at a natural N-glycosylation site (e.g. a naturally conserved N-glycosylation site) of the antibody, e.g. the glycosylation site of the Fc region superior.
  • the antibody is IgG and the GlcNAc is present at a natural N-glycosylation site of IgG (e.g. a naturally conserved N-glycosylation site), e.g. glycosylation of the Fc region on the spot.
  • said native site is the Asn297-glycosylation site of IgG.
  • the Asn297-glycosylation site is present in the Fc region of the heavy chain of an IgG antibody.
  • said GlcNAc group is present at the Asn297-glycosylation site of both heavy chains of the antibody.
  • L has the structure
  • Q is -N(H)C(O)CH 2 - or -CH 2 -;
  • R 1 is independently selected from hydrogen, halogen, -OR 2 , -NO 2 , -CN, -S(O) 2 R 2 , C 1 -C 12 alkyl, aryl, heteroaryl, C 1 -C 12 Alkylaryl, C 1 -C 12 alkyl heteroaryl, aryl C 1 -C 12 alkyl and heteroaryl C 1 -C 12 alkyl, and wherein the alkyl, aryl, heteroaryl , alkylaryl, alkylheteroaryl, arylalkyl, and heteroarylalkyl are optionally substituted, where two substituents R can be joined together to form a fused, bridged, or spiro-linked ring Alkyl or fused or spiro arene or heteroarene substituent, and wherein R 2 is independently selected from hydrogen, halogen, C 1 -C 24 alkyl, aryl, heteroaryl, C 1 -C 12 alkyl Aryl,
  • R 3 and R 4 are each independently selected from hydrogen, halogen, C 1 -C 24 alkyl, aryl, heteroaryl, C 1 -C 12 alkylaryl, C 1 -C 12 alkylheteroaryl, Aryl C 1 -C 12 alkyl and heteroaryl C 1 -C 12 alkyl;
  • c 0, 1, 2, 3 or 4;
  • n 0 or 1
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • Y is O, S or NR 1 ;
  • L 2 and L 3 are each independently C 1 -C 12 alkyl, one or more (for example 1, 2, 3 or 4) carbon atoms in the alkyl are optionally selected from O, N and S Heteroatom substitution, provided that said O, N and/or S are not directly linked to each other;
  • Each L4 is independently a cleavable linker.
  • each L4 is independently
  • R 5 and R 6 are each independently selected from hydrogen and C 1 -C 12 alkyl
  • Ar is selected from aryl, preferably phenyl
  • each L4 is independently
  • the antibody-drug conjugate has an average DAR of 1-15, such as 1-10, 2-8, 2-6, or 3-5.
  • the antibody-drug conjugate of the present invention is selected from:
  • IEX019-02, IEX019-04, IEX019-05, q is 2-5 such as 3-5, 3-4 or 3.5-4.5, in IEX019-03, q is 5-11 such as 6-10, 7- 9 or 7.5-8.5.
  • One aspect of the present invention provides a method of preparing an antibody with engineered glycosylation, the method comprising
  • glycosylated antibodies Under conditions suitable for antibody expression, culture host cells containing nucleic acids encoding the antibodies (such as any polypeptide chain and/or multiple polypeptide chains) or expression vectors containing the nucleic acids , as provided above, and optionally recovering said antibody from said host cell (or host cell culture medium), obtaining an antibody comprising a core N-acetylglucosamine substituent (core-GlcNAc substituent), wherein said core N-acetylglucosamine and said core N-acetylglucosamine substituent are optionally fucosylated;
  • the nucleic acid encoding the antibody (such as the antibody described above, such as any polypeptide chain and/or multiple polypeptide chains) is isolated and inserted into one or more vectors for use in host cells for further cloning and/or expression.
  • Such nucleic acids are readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes that are capable of binding specifically to genes encoding the antibody heavy and light chains).
  • the endoglycosidase in step (2) can be selected according to the properties of the glycosylated antibody, for example selected from Endo S, Endo A, Endo F, Endo M, Endo D and Endo H enzymes and/or combinations thereof, also for example Be Endo S, Endo S49, Endo F or a combination thereof.
  • the endoglycosidase is the endoglycosidase described in PCT/EP2017/052792, most preferably Endo SH in PCT/EP2017/052792.
  • the glycosyltransferase in step (3) is preferably a glycosyltransferase derived from ⁇ -(1,4)-N acetylgalactosamine transferase, more preferably any ⁇ as described in PCT/EP2016/059194 -(1,4)-GalNAcT enzyme.
  • the ⁇ -(1,4)-GalNAcT enzyme is or is derived from an invertebrate ⁇ -(1,4)-GalNAcT enzyme.
  • the ⁇ -(1,4)-GalNAcT enzyme may be or may be derived from any invertebrate ⁇ -(1,4)-GalNAcT enzyme known to those skilled in the art.
  • the ⁇ -(1,4)-GalNAcT enzyme is or is derived from the phylum Nematoda, preferably from the class Chromadorea or Secernentea, or from the phylum Arthropoda (Arthropoda), preferably ⁇ -(1,4)-GalNAcT enzyme derived from the class Insecta.
  • the ⁇ -(1,4)-GalNAcT enzyme Caenorhabditis elegans, Ascaris suum, Trichoplusia elegans, Drosophila melanogaster, saprophytic fruit nematode, Caeno rhabditis briggsae, Wuze nematode, Loa worm, Pythias elegans ants, Dampwood termites, Florida ants, long oysters, and red spot butterflies.
  • the glycosyltransferase suitable for step (3) is Trichoplusia ⁇ -(1,4)-GalNAcT enzyme named TnGalNAcT disclosed in PCT/EP2016/059194 (eg His-TnGalNacT).
  • ADC in the present invention is defined as an antibody coupled via a linker (L) to an active substance (D) having biological and/or pharmaceutical activity.
  • Said method comprises coupling the antibody of the invention to one or more than one active substance D via one or more linkers (L) as defined in the invention.
  • the linker-active substance is site-specifically coupled to the antibody.
  • a method for preparing the ADC of the present invention comprising making the glycosylated antibody of (Formula III)
  • linker-payload containing an alkynyl group and one or more (eg 1, 2, 3 or 4) drug molecules
  • the linker-drug compound has the formula:
  • the present invention provides a composition, preferably a pharmaceutical composition or pharmaceutical formulation, comprising any ADC molecule described herein or a pharmaceutically acceptable salt thereof.
  • the composition further comprises pharmaceutical excipients.
  • a composition eg, a pharmaceutical composition, comprises an ADC molecule of the invention in combination with one or more other therapeutic agents.
  • compositions comprising an ADC molecule of the invention, or a pharmaceutically acceptable salt thereof.
  • compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • compositions of the invention can be in a variety of forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories.
  • liquid solutions eg, injectable solutions and infusible solutions
  • powders or suspensions e.g., liposomes and suppositories.
  • liposomes e.g., liposomes and suppositories.
  • Medicaments comprising the ADCs described herein may be prepared by mixing ADC molecules of the invention of the desired purity with one or more optional pharmaceutical excipients, preferably in the form of lyophilized formulations or aqueous solutions.
  • compositions or formulations of the invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • active ingredients including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (eg, immune checkpoint inhibitors or agonists), among others.
  • the active ingredients are suitably present in combination in amounts effective for the intended use.
  • sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody in the form of shaped articles such as films or microcapsules.
  • the invention also provides a pharmaceutical combination or drug combination product comprising an ADC molecule of the invention, and one or more other therapeutic agents (e.g., therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cellular Factors, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists, etc.).
  • therapeutic agents including chemotherapeutic agents, angiogenesis inhibitors, cellular Factors, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists, etc.).
  • Another object of the present invention is to provide a kit of parts comprising the pharmaceutical combination of the present invention, preferably said kit is in the form of pharmaceutical dosage units. Dosage units may thus be presented according to a dosing regimen or interval between drug administrations.
  • kit of parts of the invention comprises in the same package:
  • One aspect of the present invention provides a method for preventing or treating a tumor (eg, cancer) in a subject, comprising administering to the subject an effective amount of the ADC molecule, pharmaceutical composition, pharmaceutical combination or kit of the present invention.
  • a tumor eg, cancer
  • the tumor (eg, cancer) patient has (eg, elevated levels, eg, at the nucleic acid or protein level) of CLDN18.2.
  • the patient's tumor cells express CLDN18.2, eg, moderately express CLDN18.2, preferably highly express CLDN18.2.
  • the tumor eg, cancer
  • the tumor includes solid tumors and hematological tumors as well as metastatic lesions.
  • examples of solid tumors include malignant tumors. Cancer can be early, middle or advanced or metastatic.
  • the ADC molecule of the present invention can kill tumor cells, and/or inhibit the proliferation of tumor cells, such as tumor cells expressing CLDN18.2, such as digestive tract tumor cells, such as gastric cancer cells or pancreatic cancer cells or colon cancer cells cancer cells or colorectal cancer cells.
  • tumor cells such as tumor cells expressing CLDN18.2
  • digestive tract tumor cells such as gastric cancer cells or pancreatic cancer cells or colon cancer cells cancer cells or colorectal cancer cells.
  • the tumor is an immune escape tumor.
  • the tumor is a cancer, such as an epithelial tumor, such as a tumor of the digestive tract, such as an epithelial cancer or a cancer of the digestive tract, such as gastric cancer or gastroesophageal junction cancer or pancreatic cancer or colorectal cancer or colon cancer.
  • a cancer such as an epithelial tumor, such as a tumor of the digestive tract, such as an epithelial cancer or a cancer of the digestive tract, such as gastric cancer or gastroesophageal junction cancer or pancreatic cancer or colorectal cancer or colon cancer.
  • the subject can be a mammal, eg, a primate, preferably a higher primate, eg, a human (eg, an individual suffering from or at risk of having a disease described herein).
  • the subject has or is at risk of having a disease described herein (eg, cancer).
  • the subject receives or has received other treatments, such as chemotherapy treatment and/or radiation therapy.
  • the subject has previously received or is currently receiving immunotherapy.
  • the present invention provides the use of ADC molecules or pharmaceutical compositions or pharmaceutical combinations or kits in the manufacture or preparation of a medicament for the purposes described herein, for example for the prevention or treatment of the relevant disease or condition.
  • the ADC molecule or pharmaceutical composition or pharmaceutical combination or kit of the invention delays the onset of a disorder and/or symptoms associated with a disorder.
  • the ADC molecules or pharmaceutical compositions of the invention can also be administered in combination with one or more other therapies, e.g., therapeutic modalities and/or other therapeutic agents, for the uses described herein, e.g., for prophylaxis and and/or treat a related disease or condition mentioned herein.
  • therapies e.g., therapeutic modalities and/or other therapeutic agents, for the uses described herein, e.g., for prophylaxis and and/or treat a related disease or condition mentioned herein.
  • treatment modalities include surgery; radiation therapy, localized or focused irradiation, and the like.
  • the therapeutic agent is selected from chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (eg, immune checkpoint inhibitors or agonists).
  • Exemplary other antibodies include antibodies that specifically bind immune checkpoints.
  • Combination therapy according to the invention encompasses combined administration (e.g. two or more therapeutic agents are contained in the same formulation or in separate formulations), and separate administration, in which case the additional therapeutic agents and/or Administration of the ADC molecule of the invention occurs before, simultaneously with, and/or after the agent.
  • the route of administration of the pharmaceutical composition is according to known methods, for example, orally, by intravenous injection, intraperitoneally, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional Route; through a sustained release system or through an implanted device.
  • the compositions can be administered by bolus injection or by continuous infusion or by implanted device.
  • composition may also be administered topically via an implant, a sponge, or another suitable material on which the desired molecule is absorbed or encapsulated.
  • an implant device when used, the device can be implanted into any suitable tissue or organ and can deliver the desired molecule via diffusion, time-release bolus, or continuous administration.
  • a kit (Invitrogen, A1369601) was used to construct a cell line stably expressing human Claudin18.2 (abbreviated as CLDN18.2, the same hereinafter).
  • CLDN18.2 human Claudin18.2
  • the full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed into the vector pCHO1.0 to construct the plasmid, and the constructed plasmid was transformed into CHO- In S cells (Invitrogen, A1369601) and HEK293 cells (Invitrogen, A14527), the transfected cells underwent two rounds of pressurized selection to obtain cell pools expressing CLDN18.2 respectively.
  • the kit (Invitrogen, A1369601) was used to construct a cell line stably expressing human Claudin18.1 (abbreviated as CLDN18.1, the same below).
  • CLDN18.1 human Claudin18.1
  • the full-length gene of human CLDN18.1 (UniProt ID: P56856-1) was constructed into the vector pCHO1.0 (Invitrogen, A1369601) to form a plasmid, and the constructed plasmid was transferred into CHO-S cells by chemical transfection (Invitrogen, A1369601), the transfected cells undergo two rounds of pressurized selection to obtain cell pools expressing CLDN18.1 respectively.
  • the full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed into the vector pWPT-GFP (Addgene, 12255), the GFP sequence was replaced, and the lentiviral packaging vector psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259) was co-transfected into HEK293T (ATCC, CRL-3216) cells for virus packaging. The culture supernatants after 48 hours and 72 hours of culture were collected respectively, and the lentiviruses were concentrated using PEG8000.
  • Pancreatic cancer DAN-G cells (CLS Cell Lines Service GmbH, 300162) and gastric cancer KATO III cells (ATCC, HTB-103) were transfected with the concentrated virus, and then flow cytometry was used to sort (MoFlo XDP, Beckman Coulter) The cells expressing CLDN18.2 were sorted out to obtain the tumor cell lines DAN-G-hCLDN18.2 and KATO III-hCLDN18.2 stably transfected with CLDN18.2.
  • the present invention adopts hybridoma technology, and the cells (CHO-hCLDN18.2) obtained in Example 1 are used to immunize mouse H2L2 fully human antibody transgenic mice (purchased from Harbor BioMed). Spleen cells from mice were then obtained for electrofusion with myeloma cells. Afterwards, the supernatant was collected and screened by flow cytometry (FACS) to select hybridoma cells specifically expressing the anti-CLDN18.2 antibody, and the secreted antibody did not bind to CLDN18.1.
  • the cells to be tested (HEK293-hCLDN18.2) obtained in Example 1 were counted and diluted to 1 ⁇ 10 6 cells/ml, and 100 ⁇ l/well was added to a U-bottom 96-well plate.
  • the cells were resuspended in 50 ⁇ l 1 ⁇ PBS, and detected by FACS.
  • the positive clones were re-screened for CHO-hCLDN18.1 by the same method as above. After two rounds of screening, the fully human antibody clone HB37A6 was obtained.
  • the anti-CLDN18.2 monoclonal antibody HB37A6 (see CN202010570517.X) and the control antibody zolbetuximab (Zmab for short, sequence derived from INN117) were expressed in HEK293 cells (Invitrogen, A14527) in the form of full-length monoclonal antibodies.
  • the expression vector was constructed, and the heavy chain variable region and light chain variable region of HB37A6 and the control antibody (refer to the sequence listing information) were respectively placed in the human IgG1 heavy chain constant region (SEQ ID NO:5) and the light chain kappa constant N-terminus of region (SEQ ID NO: 10). Then it was constructed into the pcDNA3.1 expression vector with N-terminal signal peptide to obtain the light and heavy chain expression vector. The obtained light and heavy chain expression vectors were co-transfected into HEK293 cells by PEI (Polysciences Inc, 23966), and the medium supernatant was collected after 7 days of culture.
  • PEI Polysciences Inc, 23966
  • the supernatant was purified by Protein A column (Hitrap Mabselect Sure, GE 11-0034-95), then ultrafiltered and replaced into PBS (Gibco, 70011-044), the concentration was detected by A280 method and the purity was determined by SEC-HPLC method , to obtain an antibody solution with a purity greater than 95%, and obtain recombinant CLDN18.2 monoclonal antibody HB37A6.
  • Expi293 cells (Invitrogen, A14527) were passaged according to the required transfection volume, and the cell density was adjusted to 1.5 ⁇ 10 6 cells/ml one day before transfection. On the day of transfection, the cell density was about 3 ⁇ 10 6 cells/ml. Take 1/10 of the final volume of Opti-MEM medium (Gibco, 31985-070) as the transfection buffer, add the appropriate plasmid at 1.0 ⁇ g/ml transfected cells, and mix well.
  • Opti-MEM medium Gibco, 31985-070
  • PEI polyethyleneimine
  • the Protein A column (Hitrap Mabselect Sure, GE, 11-0034-95) used for purification was treated with 0.1M NaOH for 2h, and the glass bottles were washed with distilled water and then dried at 180°C for 4h. Before purification, the collected cell feed solution was centrifuged at 4500 rpm for 30 min, and the supernatant was filtered with a 0.22 ⁇ m filter. Use 10 column volumes of binding buffer (sodium phosphate 20mM. NaCl 150mM, pH7.0) to equilibrate the Protein A column. The filtered supernatant was added to the purification column and equilibrated with 10 column volumes of binding buffer.
  • binding buffer sodium phosphate 20mM. NaCl 150mM, pH7.0
  • elution buffer citric acid + sodium citrate 0.1M, pH 3.5
  • elution buffer citric acid + sodium citrate 0.1M, pH 3.5
  • the collected antibody was concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044), and the concentration was detected.
  • the equilibrium dissociation constant (K D ) of HB37A6 binding to human CLDN18.2 was determined by surface plasmon resonance (SPR).
  • Human Claudin18.2 (GenScrip, P50251802) was coupled to the surface of a CM5 chip (GE Healthcare, 29-1496-03) using an amino coupling kit (GE Healthcare, BR-1006-33) according to the manufacturer's instructions, After coupling, 1M ethanolamine was injected to block the remaining active sites.
  • Affinity and kinetic constants were obtained by detecting the binding and dissociation between the chip surface antigen and the antibody in the mobile phase by Biacore (GE Healthcare, T200) according to the manufacturer's instructions.
  • Example 1 The above-mentioned anti-CLDN18.2 monoclonal antibody HB37A6 and the control antibody Zmab were measured by flow cytometry (FACS) respectively with the CHO-S cell line stably transfected with human CLDN18.2 and human CLDN18.1 obtained in Example 1 (ie the combination of CHO-hCLDN18.2 and CHO-hCLDN18.1 prepared as described in Example 1).
  • the cells to be tested (CHO-hCLDN18.2 and CHO-hCLDN18.1) obtained in Example 1 were counted and diluted to 1 ⁇ 10 6 cells/ml, and 100 ⁇ l of /hole. Centrifuge at 500g for 5min to remove the cell culture medium. Add the anti-CLDN18.2 monoclonal antibody HB37A6 and the control antibody Zmab to the U-shaped plate and resuspend the cells. Add 100 ⁇ l to each well. The initial concentration of the antibody is 900 nM, and then serially 3-fold dilution, a total of 10 concentration points. Let stand on ice for 30 minutes. Remove the supernatant at 500 g for 5 min, and wash the cells once with PBS.
  • Example 1.5 the binding of HB37A6 to gastric cancer cell line NUGC-4 (JCRB, JCRB0834), gastric cancer cell line KATO III-hCLDN18.2 and pancreatic cancer cell line DAN-G-hCLDN18.2 was determined by FACS.
  • Figure 3 shows that the fully human antibody HB37A6 has good specific binding to tumor cells, which is better than the control antibody Zmab.
  • the HB37A6 antibody was used to test its anti-tumor effect in NOD-SCID mice bearing human pancreatic cancer (female NOD-SCID mice (15-18 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.).
  • the human pancreatic cancer cells DAN-G-hCLDN18.2 constructed in Example 1 were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and DAN-G-hCLDN18.2 was dispersed with PBS (1 ⁇ ) to obtain a suspension with a cell density of 12 ⁇ 10 ⁇ 5 cells/ml. Mix the cell suspension with matrigel 1:1 to prepare a cell suspension with a cell concentration of 6 ⁇ 10 ⁇ 5 cells/ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of NOD-SCID mice to establish a DAN-G-CLDN18.2 tumor-bearing mouse model.
  • mice with a tumor volume in the range of 43.36 mm 3 to 89.47 mm 3 were selected and grouped into serpentine groups according to tumor volume (8 mice in each group).
  • mice were administered with hIgG (Equitech-Bio, lot number 160308-02) and HB37A6 and the control antibody Zmab at a dose of 10 mg/kg each time, respectively on the 5th, 9th, 12th, and 16th day after inoculation, Mouse tumor volumes were monitored 2-3 times per week.
  • Tumor volume measurement: the maximum long axis (L) and maximum width axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V L ⁇ W 2 /2.
  • Body weight was measured using an electronic balance.
  • the HB37A6 antibody was selected to test its anti-tumor effect in NOG mice bearing human gastric cancer (female NOG mice (15-18 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). Resuscitate PBMC cells (Allcells), collect cells by centrifugation, disperse PBMC cells with PBS (1 ⁇ ), the cell density is 2.5 ⁇ 10 ⁇ 6 cells/ml cell suspension, take 0.2ml cell suspension for NOG on day 0 Mice were injected into the eye vein to establish a NOG humanized mouse model.
  • NUGC-4 cells were routinely recovered and subcultured for subsequent in vivo experiments. Collect the cells by centrifugation, disperse NUGC-4 cells with PBS (1 ⁇ ), the cell density is 12 ⁇ 10 ⁇ 6 cells/ml and mix with matrigel gel 1:1 to prepare a cell suspension with a cell concentration of 6 ⁇ 10 ⁇ 6 cells/ml . On day 5, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of NOG humanized mice to establish a NUCG-4 tumor-bearing mouse model.
  • mice On the first day after tumor cell inoculation, the mice were randomly divided into 7 groups, and hIgG (Equitech-Bio, batch number 160308-02, control group) and HB37A6 (treatment group) and positive control antibody Zmab (treatment group) were administered to each mouse , the dosage is 10 mg/kg each time, administered on the 1st, 5th, 8th, and 12th day after inoculation, respectively, and the tumor volume and body weight of mice are monitored 2-3 times a week.
  • Body weight was measured using an electronic balance.
  • the relative tumor inhibition rate (TGI%) was calculated on the 26th day of inoculation, and the calculation formula was as follows:
  • TGI% 100%*(tumor volume of control group-tumor volume of treatment group)/(tumor volume of control group-tumor volume of control group before administration).
  • HB37A6 and the control antibody Zmab can inhibit tumor growth in the human pancreatic cancer DAN-G-CLDN18.2 mouse model.
  • the TGI of HB37A6 is 28%, and the TGI of Zmab is 24%.
  • HB37A6 showed a better anti-tumor effect than the control antibody Zmab on the human gastric cancer NUGC-4 mouse model, the TGI of HB37A6 was 31%, and the TGI of Zmab was 0%.
  • HB37A6 was expressed and purified in HEK293 cells according to Example 1.3.
  • the resulting HB37A6 (16.4 mg/mL) was incubated with EndoSH (1% w/w) as described in PCT/EP2017/052792 to obtain trimmed HB37A6 with -GlcNAc at position Asn297 or with GlcNAc(Fuc) .
  • the above trimmed HB37A6 was combined with the enzyme His-TnGalNAcT (4.5% w/w) disclosed in PCT/EP2016/059194 and 6-azido-GalNAc-UDP disclosed in PCT/EP2016/059194 (25 eq (eq. )) (in a solution of histidine (20 mM)+NaCl (150 mM) containing 6 mM MnCl 2 ), incubated at 30° C. for 16 hours.
  • the incubation mixture obtained above was purified using a 50 mL protA column (Hitrap Mabselect Sure, GE, 11-0034-95).
  • the incubation mixture obtained above was loaded onto the column and the column was washed with TBS+0.2% Triton and TBS.
  • the column was then eluted with 0.1M acetate buffer pH 2.9 and neutralized with 2.5M Tris-HCl pH 7.2.
  • the obtained (modified) glycosylated antibody was concentrated to 32.6 mg/mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius).
  • the obtained glycosylated antibody is analyzed by mass spectrometry, and the main steps are as follows: before mass spectrometric analysis, the glycosylated antibody is treated with IdeS, so that the Fc/2 fragment can be analyzed.
  • 20 ⁇ g of the (modified) glycosylated antibody solution was incubated with IdeS (Fabricator TM ) (1.25 U/ ⁇ L) in PBS pH 6.6 in a total volume of 10 ⁇ L at 37° C. for 1 hour. Samples were diluted to 80 ⁇ L and then analyzed on a JEOL AccuTOF (ESI-TOF), deconvoluted spectra were obtained using Magtran software.
  • the bioconjugate IEX019-02 is achieved by coupling compound 1 (linker-payload) as a linker-conjugate to azide-modified HB37A6-(GlcNAc(Fuc) 1 -6 as a biomolecule -N 3 -GalNAc) 2 to prepare. Therefore, to a solution of HB37A6-(GlcNAc(Fuc) 1 -6-N 3 -GalNAc) 2 (10.8 mL, 350 mg, 32.6 mg/ml, PBS pH 7.4) was added PBS pH 7.4 (808 ⁇ L), and 1,2 - Propylene glycol (11.3 mL) and compound 1 (350 ⁇ L, 40 mM DMF solution).
  • Mass spectrometry analysis of the IdeS digested samples revealed two major products corresponding to the obtained ADC, the HB37A6-SYNtecan E conjugate.
  • First peak observed mass 26469 Da (calculated mass 26465 Da), corresponding to the coupled Fc/2 fragment (2x blocked lactone form of the payload).
  • Second peak observed mass 26499 Da (calculated mass 26501 Da), corresponding to the bound Fc/2 fragment (2x open carboxylate form of the payload).
  • the resulting structure is as follows.
  • the concentration, DAR value and SEC purity of ADC products were determined by UV, SEC, RP-HPLC and LC-MS.
  • the monomer purity detected by SE-HPLC was >99%, and the concentration was 6.12 mg/ml.
  • q represents the average DAR, for example 3-5, 3.2-4.8, or 3.5-4.5, such as 3.52 as determined in Table 2.
  • q represents the average DAR, for example 5-11, 6-10, 7-9 or 7.5-8.5, such as 7.9 as determined in Table 2.
  • the optimal pH value of the reaction is generally between 6.5 and 8.0.
  • step (b) Dissolve excess linker-toxin MC-GGFG-DXD (purchased from Levena biopharma, SET0218, structure can be shown below) in DMSO and react with the thiol group of the antibody reduced in step (a) .
  • the reaction mixture was placed in a shaker for 1-2h, where
  • the ADC crude product was obtained.
  • ADC products were detected by HIC, LC-MS and SEC HPLC to determine the average DAR and SEC purity.
  • Embodiment 2.1.3 Preparation of IEX019-04
  • q represents the average DAR, for example 3-5, 3.2-4.8 or 3.0-4.0, such as 3.5 as determined in Table 2.
  • the optimal pH value of the reaction is generally between 6.5 and 8.0.
  • step (c) After the binding reaction is completed, acetylcysteine solution is added to terminate the reaction of step (b). After mixing, incubate at 20-25°C for 5-15 minutes.
  • q represents the average DAR, for example 3-5, 3.2-4.8, or 3.0-4.5, such as 3.3 as determined in Table 2.
  • This molecule was prepared as follows.
  • the optimal pH value of the reaction is generally between 6.5 and 8.0;
  • the ADC crude product was obtained.
  • the preparation process is similar to IEX019-02, except that the HB37A6 monoclonal antibody is replaced by the control antibody IgG.
  • the preparation process is similar to IEX019-04, except that the HB37A6 monoclonal antibody is replaced by the control antibody IgG.
  • Example 1 In order to detect whether the small molecule coupling will change the binding characteristics of IEX019-01 monoclonal antibody and target cells, we use the DAN-G cell line (hCLDN18.2 negative) and the DAN-G-hCLDN18.2 cell line prepared in Example 1 ( Overexpress hCLDN18.2), and detect the affinity of IEX019-01 and IEX019-02 to the target by flow cytometry, and the experimental method is the same as that in Example 1.5.
  • Both IEX019-01 and IEX019-02 do not bind non-target cell DAN-G, but have strong affinity to DANG-hCLDN18.2, indicating that the binding of the antibody depends on the expression specificity of the target, and the conjugation of Exatecan will not affect Antibody Binding.
  • the control molecule IEX019-06 did not bind to the target cells ( Figure 6).
  • Strong endocytosis is one of the important properties of ADC drugs.
  • the ADC binds to the antigen on the surface of the cell membrane, the ADC-antigen complex enters the cell through endocytosis and kills the target cell. Therefore, the endocytic efficiency of ADC is one of the important indicators to determine the tumor suppressive effect.
  • Example 2.4 In vitro cell killing effect of IEX019 molecule
  • CellTiter-Glo (Promega, G9242) detection kit was used to detect the effect of ADCs on cell viability on various cell lines expressing different levels of hCLDNA8.2 (Table 3).
  • IEX019 molecules had no significant effect on cell viability (Figure 8A); in cell lines with medium expression levels (NUGC-4, SNU620), IEX019 molecules had a certain degree of killing of cells (Figure 8B ); on the highly expressed cell line DAN-G-hCLDN18.2, IEX019 molecules all had significant killing effects (Fig. 8C). It shows that the IEX019 molecule has good selectivity and effectiveness.
  • small molecular compounds can be linked to antibodies through cleavable linkers. After being endocytosed into the cell membrane, the linkers are cleaved and the small molecules are released to kill target cells. After the death of target cells, small molecular compounds are released from the target cells into the intercellular space, further killing non-target cells within a certain range, and this effect is called the side-killer effect. Since the cells inside the tumor will have great differences in the level of target expression (tumor heterogeneity), the side killer effect is very important for effectively killing tumor cells and inhibiting tumor growth.
  • the present invention utilizes non-target cell DAN-G and target cell DANG-hCLDN18.2 to detect the side-killer effect of IEX019 molecule.
  • IEX019-01-negative (i.e. hCLDN18.2-negative) population is DAN-G cells
  • the IEX019-01-positive (i.e. hCLDN18.2-positive) cells are DAN-G- hCLDN18.2.
  • Count the numbers of the two types of cells in each sample calculate the relative cell viability of each cell according to the following equation, and use Graph Pad Prism 8.0 to fit the curve.
  • Relative viability of DANG cells number of DNAG cells in the sample group/number of DNAG cells in the IgG group * 100%
  • DANG-hCLDN18.2 cells number of DNAG-18.2 cells in the sample group/number of DNAG-18.2 cells in the IgG group * 100%
  • the cell viability of IgG group was set as 100%.
  • the negative control sample IEX019-06 had no killing effect on the two cells; the IEX019-05 molecule connected the DM1 toxin to the IEX019-01 monoclonal antibody through a non-cleavable linker, and did not kill the surrounding non-target cells. Therefore, it can only kill DANG-hCLDNA8.2 cells, but has no effect on DANG cells. Only IEX019-02 has a significant bystander effect, which can kill both target cells and non-target cells.
  • Example 2.6 Antitumor efficacy of IEX019 molecule on DAN-G-hCLDN18.2 mouse xenograft tumor model
  • DANG-hCLDN18.2 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the DANG-hCLDN18.2 cells were dispersed with PBS (1 ⁇ ) to prepare a cell suspension with a cell concentration of 3 ⁇ 10 6 cells/ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of CB-17SCID mice to establish a DANG-hCLDN18.2 tumor-bearing mouse model.
  • mice with a tumor volume of 50.16-136.68mm were divided into 3 serpentine groups (6 mice in each group).
  • the tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 10a and 10b, and the monitoring ended after 92 days.
  • TGI% The relative tumor inhibition rate (TGI%) was calculated on the 50th day after inoculation, and the calculation formula was as follows:
  • 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 measurement The maximum long axis (L) and maximum width axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula:
  • V L*W 2 /2.
  • Body weight was measured using an electronic balance.
  • the tumor inhibition rate results are shown in Table 6 and Figure 10A: on the 50th day after inoculation, compared with IEX019-01 monoclonal antibody, the tumor inhibition rate of IEX019-02 single administration reached 103.60%, which was significantly better than IEX019-03 and IEX019 -04, the tumor inhibition rates were 93.70% and 35.20%, respectively.
  • 82 days after inoculation 100% of the mice in the IEX019-02 group had complete tumor regression.
  • we detected the body weight of the mice and the results showed that there was no significant difference in the body weight of the mice as shown in Figure 10B.
  • the experiment used female CB-17-SCID mice of SPF grade (14-17 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and the certificate number is NO.110011201109348141.
  • NUGC-4 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the NUGC-4 cells were dispersed with PBS (1 ⁇ ) to prepare a cell suspension with a cell concentration of 3 ⁇ 10 7 cells/ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of CB-17SCID mice to establish a NUGC-4 tumor-bearing mouse model.
  • mice with a tumor volume of 72.25-140.50mm were divided into 3 serpentine groups (6 mice in each group), and the dosage and method of administration were as shown in Table 7.
  • the tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 11A and 11B, and the monitoring ended after 40 days.
  • TGI% The relative tumor inhibition rate (TGI%) was calculated on the 33rd day after inoculation, and the calculation formula was as follows:
  • 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 measurement The maximum long axis (L) and maximum width axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula:
  • V L*W 2 /2.
  • Body weight was measured using an electronic balance.
  • the tumor inhibition rate results are shown in Tables 8 and 11A: on the 33rd day after inoculation, compared with the negative control IEX019-06, the single-dose tumor inhibition rates of IEX019-02 and IEX019-03 were 80.04% and 54.31%, respectively. At the same time, we tested the body weight of the mice, and the results showed that there was no significant difference in the body weight of the mice as shown in Figure 11B.
  • Example 2.8 Antitumor efficacy of IEX019 molecule in SNU620 mouse xenograft tumor model
  • SNU620 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the SNU620 cells were dispersed with PBS (1 ⁇ ) to prepare a cell suspension with a cell concentration of 3 ⁇ 10 7 cells/ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of CB-17SCID mice to establish the SNU620 tumor-bearing mouse model.
  • mice with a tumor volume of 58.1-117.3mm were divided into 3 serpentine groups (6 mice in each group).
  • the tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 12a and 12b, and the monitoring ended after 39 days.
  • TGI% tumor inhibition rate
  • 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 measurement The maximum long axis (L) and maximum width axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula:
  • V L*W 2 /2.
  • Body weight was measured using an electronic balance.
  • the results of the tumor inhibition rate are shown in Table 10 and Figure 12A: on the 39th day after inoculation, compared with hIgG, the tumor inhibition rate after a single administration of IEX019-02 10 mg/kg was 143.77%, and 100% of the mice tumors completely regressed . There was no significant difference in the body weight of the mice in each administration group compared with the hIgG control group ( FIG. 12B ).

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WO2024109840A1 (zh) * 2022-11-22 2024-05-30 康诺亚生物医药科技（成都）有限公司 稠环类化合物及其偶联物和用途
CN119173534A (zh) * 2021-12-17 2024-12-20 信达生物制药(苏州)有限公司 靶向Claudin18.2的抗体-药物偶联物
CN119345386A (zh) * 2024-10-25 2025-01-24 华润生物医药有限公司 靶向Claudin18.2的抗体药物耦联物及其用途
WO2025051070A1 (zh) * 2023-09-08 2025-03-13 成都临界点生物科技有限公司 抗Claudin18.2人源化单域抗体
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WO2025149029A1 (zh) * 2024-01-12 2025-07-17 信达生物制药(苏州)有限公司 抗cd28抗体及其用途
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