WO2022116079A1 - Anticorps anti-ceacam5 humanisé et son procédé de préparation et son utilisation - Google Patents

Anticorps anti-ceacam5 humanisé et son procédé de préparation et son utilisation Download PDF

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WO2022116079A1
WO2022116079A1 PCT/CN2020/133549 CN2020133549W WO2022116079A1 WO 2022116079 A1 WO2022116079 A1 WO 2022116079A1 CN 2020133549 W CN2020133549 W CN 2020133549W WO 2022116079 A1 WO2022116079 A1 WO 2022116079A1
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antibody
antigen
seq
cancer
binding fragment
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PCT/CN2020/133549
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Chinese (zh)
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牟男
于跃
袁纪军
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上海吉倍生物技术有限公司
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Priority to PCT/CN2020/133549 priority Critical patent/WO2022116079A1/fr
Priority to TW109144531A priority patent/TW202222838A/zh
Publication of WO2022116079A1 publication Critical patent/WO2022116079A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/68—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

  • the present application belongs to the technical field of biological immunity, and specifically relates to a humanized antibody that can specifically bind to human carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5) and an antigen-binding fragment thereof.
  • CEACAM5 human carcinoembryonic antigen cell adhesion molecule 5
  • the present application also relates to the preparation methods and uses of the antibodies and antigen-binding fragments thereof.
  • CEACAM human carcinoembryonic antigen cell adhesion molecule
  • CEACAM proteins are located outside the cell membrane, among which CEACAM1, CEACAM3 and CEACAM4 are linked to the cell membrane through hydrophobic transmembrane domains; CEACAM5-8 are linked to the cell membrane through glycosylphosphoinositides. These extracellular domains typically serve as adhesion molecules between cells (eg, epithelial, endothelial, dendritic, and leukocytes).
  • CEACAM is involved in a variety of cellular functions, based on the function of intercellular adhesion, regulates cell growth and differentiation through signal transduction, and plays an important role in insulin homeostasis, angiogenesis, and immune regulation.
  • Members of the CEACAM gene family are involved in a wide variety of pathophysiological roles, including as receptors for microbial pathogens. They play important roles in carcinogenesis, especially in cancer detection, progression and metastasis.
  • CEACAM5 (abbreviated as CEA, also known as CD66e) is a glycoprotein with a molecular weight of about 180 kDa.
  • CEACAM5 contains seven domains linked to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor.
  • GPI glycosylphosphatidylinositol
  • the seven domains include a single N-terminal Ig variable domain and six domains (A1-B1-A2-B2-A3-B3) homologous to the Ig constant domains.
  • CEACAM5 Originally thought to be a protein expressed in fetal tissue, CEACAM5 has now been identified in several normal adult tissues. Overexpression of CEACAM5 is observed in many types of cancer. For example, CEACAM5 can be detected in the blood of colon cancer patients. And, further studies have determined that its overexpression is associated with many malignancies, often with poor prognosis. Overexpression of CEACAM5 has been shown to act as a tumor biomarker in prostate and colorectal cancers.
  • CEACAM5/CEACAM6 was also found to be overexpressed in various malignant tumors, such as breast, pancreatic, ovarian, colon, lung and gastric gland tumors, and was associated with tumor aggressiveness and metastasis.
  • CEACAM5 binds to its receptor CEAr, and their interaction leads to the activation and production of pro-inflammatory cytokines, mainly IL-1, IL-6, IL-10, and TNF- ⁇ .
  • pro-inflammatory cytokines mainly IL-1, IL-6, IL-10, and TNF- ⁇ .
  • these cytokines alter the microenvironment of hepatocytes and Kupffer cells, as well as their interactions with hepatic sinusoids. These interactions not only affect tumor cells or other hepatocytes, but also appear to promote the viability of CSCs and other circulating tumor cells in the cerebrospinal fluid.
  • CEACAM5 has become a Potentially useful tumor-associated antigens for targeted therapy.
  • Two main approaches have been reported using CEACAM5-targeted immunotherapy for cancer.
  • CEACAM5 antibodies to elicit the lytic activity of immune cells, particularly through antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), to eliminate CEACAM5-expressing tumor cells;
  • ADCC antibody-dependent cytotoxicity
  • CDC complement-dependent cytotoxicity
  • CEACAM5 antibodies or antibody fragments are conjugated to effector molecules such as drugs, toxins, radionucleotides, immunomodulators, or cytokines to specifically target CEACAM5-expressing tumor cells, thereby exerting the therapeutic effect of the effector molecules.
  • effector molecules such as drugs, toxins, radionucleotides, immunomodulators, or cytokines
  • CEACAM5 will help inhibit the metastatic process of tumors.
  • antibodies with strong specificity to CEACAM5 can better avoid the toxic and side effects caused by off-target antibodies.
  • CEACAM1 and CEACAM3 are widely distributed in the human immune system and bone marrow, such as neutrophils, etc.
  • the specific CEACAM5 antibody can reduce the possible side effects of drugs and improve the therapeutic window.
  • antibody in this application includes any immunoglobulin, monoclonal, polyclonal, multispecific or bispecific (bivalent) antibody that binds to a particular antigen.
  • a native intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second and third constant regions; each light chain consists of a variable region and a constant region. Mammalian heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , and mammalian light chains can be classified as ⁇ or ⁇ .
  • Antibodies are in the "Y" shape, and the neck of the Y-shaped structure consists of the second and third constant regions of two heavy chains, which are bound by disulfide bonds.
  • Each arm of the "Y"-shaped structure includes the variable and first constant regions of one of the heavy chains, which bind to the variable and constant regions of one of the light chains.
  • the variable regions of the light and heavy chains determine antigen binding.
  • the variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs).
  • the CDRs of the light chain (L) include VLCDR1, VLCDR2, and VLCDR3, and the CDRs of the heavy chain (H) include VHCDR1, VHCDR2, and VHCDR3.
  • the CDR boundaries of the antibodies and antigen-binding fragments disclosed in this application can be named or identified by Kabat, Chothia or Al-Lazikani nomenclature.
  • the three CDRs are separated by a lateral contiguous portion called the framework region (FR), which is more highly conserved than the CDRs and forms a scaffold to support the hypervariable loop.
  • the constant regions of the heavy and light chains are not involved in antigen binding but have multiple effector functions.
  • Antibodies can be divided into several classes based on the amino acid sequence of the heavy chain constant region. Antibodies can be divided into five major classes or isoforms, depending on whether they contain alpha, delta, epsilon, gamma, and mu heavy chains: IgA, IgD, IgE, IgG, and IgM, respectively.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • IgA2 ⁇ 2 heavy chain
  • antigen-binding fragment in this application refers to an antibody fragment formed from an antibody portion containing one or more CDRs or any other antibody fragment that binds an antigen but does not have the entire antibody structure.
  • antigen-binding fragments include, but are not limited to, such as diabodies, Fab, Fab', F(ab') 2 , Fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv) 2 , Bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabodies (ds diabody), single chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bivalent single chain antibodies (BsFv), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies and bivalent domain antibodies.
  • Antigen-binding fragments can bind to the same antigen as the parent antibody.
  • an antigen-binding fragment may contain one or more CDR
  • Fab fragment of an antibody refers to that portion of an antibody molecule that consists of a light chain (including variable and constant regions) and the variable and part of the constant region of a heavy chain joined by disulfide bonds.
  • a “Fab'" fragment refers to a Fab fragment that contains part of the hinge region.
  • F(ab') 2 refers to a dimer of Fab.
  • the Fc region of an antibody is responsible for various effector functions such as ADCC and CDC, but is not involved in antigen binding.
  • Fv segment of an antibody refers to the smallest antibody fragment that contains the entire antigen-binding site. Fv fragments consist of the variable region of one light chain and the variable region of one heavy chain.
  • Single-chain Fv antibody or “scFv” refers to an engineered antibody composed of a light chain variable region directly linked to a heavy chain variable region or linked by a peptide chain (Huston JS et al., Proc Natl Acad Sci USA, 85: 5879 (1988)).
  • Single chain antibody Fv-Fc or “scFv-Fc” refers to an engineered antibody consisting of an scFv linked to the Fc segment of an antibody.
  • Nanobody refers to an antibody fragment consisting of a VH domain from a heavy chain antibody and two constant regions, CH2 and CH3.
  • a “diabody” includes an antibody fragment with two antigen-binding sites, wherein the fragment contains the VH and VL domains linked on the same polypeptide chain (see, Holliger P. et al., Proc Natl Acad Sci US A. 90(14): 6444-8 (1993); EP404097; WO 93/11161).
  • the short linker between the two domains prevents the two domains on the same chain from pairing with each other, forcing the two domains to pair with the complementary domains of the other chain to form two antibody binding sites.
  • the two antibody binding sites can be targeted for binding to the same or different antigens (or epitopes).
  • Domain antibody refers to antibody fragments that contain only one heavy chain variable region or one light chain variable region.
  • two or more VH domains are covalently joined by a polypeptide linker and form a bivalent domain antibody.
  • the two VH domains of bivalent domain antibodies can be targeted to the same or different antigens.
  • "(dsFv) 2" contains three peptide chains: the two VH genes are linked by a polypeptide linker, and the two VL genes are bound by a disulfide bond.
  • bispecific ds diabodies contain VL1-VH2 (linked by a polypeptide linker) and VH1-VL2 (also linked by a polypeptide linker), both connected by two Sulfur bond.
  • VH1-VH2 genes the heavy chains of which are linked by a polypeptide linker (eg, a long elastic linker), and which are connected to each other by disulfide bonds.
  • the VL1 and VL2 genes are combined, and each pair of heavy and light chains paired by disulfide bonds has a different antigenic specificity.
  • scFv dimers are bivalent diabodies or bivalent single chain antibodies (BsFv) containing two VH-VL (linked by polypeptide linker) genes dimerized, one of which The VH of a gene cooperates with the VL of another gene to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes).
  • scFv dimers are bispecific diabodies containing VL1-VH2 (linked by a polypeptide linker) and VH1-VL2 (linked by a polypeptide linker), wherein VH1 and VL1 cooperates, VH2 and VL2 cooperate, and each cooperative pair has a different antigen specificity.
  • fully human when applied to an antibody or antigen-binding fragment, means that the antibody or antigen-binding fragment has or consists of an amino acid sequence corresponding to a sequence derived from a human or amino acid sequences of antibodies produced by human immune cells, or derived from non-human sources such as transgenic non-human animals using human antibody libraries, or other sequences encoding human antibodies.
  • fully human antibodies do not contain amino acid residues (particularly antigen-binding residues) derived from non-human antibodies.
  • humanized when applied to an antibody or antigen-binding fragment, is meant to include CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans (where applicable) ) antibodies or antigen-binding fragments. Since humanized antibodies or antigen-binding fragments have reduced immunogenicity, they are useful in certain embodiments as therapeutics in humans.
  • the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.
  • the humanized antibody or antigen-binding fragment consists essentially entirely of human sequences, except for the CDR sequences, which are non-human.
  • the human-derived FR region may include the same amino acid sequence as the human antibody from which it is derived, or it may include some amino acid changes, eg, no more than 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid change.
  • the amino acid change may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains.
  • the humanized antibodies include human FR1-3 and human JH and JK.
  • chimeric refers to having a portion of a heavy and/or light chain derived from one species in combination with an antibody or antigen that has the remainder of the heavy and/or light chain derived from a different species Fragment.
  • a chimeric antibody may include constant regions derived from a human and variable regions derived from a non-human animal such as a mouse.
  • CEACAM5 Carcinoembryonic antigen cell adhesion molecule 5
  • CD66e also known as CD66e; see eg, AAA51967.1/GI:180223, 702 amino acids
  • CEACAM5 contains seven Ig-like domains including a single N-terminal Ig variable domain and six domains (A1-B1-A2-B2-A3-B3) homologous to Ig constant domains.
  • CEACAM is attached to the cell membrane via a carboxy-terminal glycosylphosphatidylinositol (GPI) anchor.
  • GPI glycosylphosphatidylinositol
  • Specific binding or “specific binding” in this application refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen.
  • the antibody or antigen-binding fragment thereof of the present application specifically binds to human and/or monkey carcinoembryonic antigen cell adhesion molecule 5 with a binding affinity (KD) ⁇ 10 -6 M.
  • KD in this application refers to the ratio of dissociation velocity to association velocity (koff/kon), which can be determined by means of surface plasmon resonance, eg using instruments such as Biacore.
  • “Selectively binds” in the present application means that the antibody or antigen-binding fragment thereof of the present application specifically binds to the CEACAM5 protein, but does not substantially bind or binds at a significantly lower level to other CEACAM proteins, such as CEACAM1 protein, CEACAM3 protein, CEACAM6 protein.
  • the heavy chain constant regions of the antibodies described herein are of the human IgGl type. In certain embodiments, the light chain constant region of the antibodies described herein is a kappa chain. In certain embodiments, the heavy and light chain constant regions of the antibodies described herein are human IgGl and kappa chains, respectively.
  • conservative substitution refers to the replacement of one amino acid residue with another amino acid residue of a side chain having similar physicochemical properties. For example, between hydrophobic side chain amino acid residues (eg Met, Ala, Val, Leu and Ile), neutral hydrophilic side chain residues (eg Cys, Ser, Thr, Asn and Gln), acidic side chain residues Conservative substitutions are made between bases (eg Asp, Glu), basic side chain amino acids (eg His, Lys and Arg) or aromatic side chain residues (eg Trp, Tyr and Phe). It is known in the art that conservative substitutions generally do not result in significant changes in protein conformational structure, thus preserving the biological activity of the protein.
  • percent sequence identity when “percent sequence identity” is used for amino acid sequences (or nucleic acid sequences), it means that, in the candidate sequence, after the sequence alignment is performed, and if necessary, spacing is introduced to maximize the number of identical amino acids (or nucleic acids) The percentage of amino acid (or nucleic acid) residues in the candidate sequence that are identical to the sequence. Conservative substitutions of such amino acid residues may or may not be considered identical residues. Sequences can be aligned by tools disclosed in the art to determine percent sequence identity of amino acid (or nucleic acid) sequences. Those skilled in the art can use the default parameters of the tool or adjust the parameters appropriately according to the needs of the alignment, for example by picking a suitable algorithm.
  • T cells include CD4 + T cells, CD8 + T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, and suppressor T cells.
  • effector function refers to the biological activity of the Fc region of an antibody in binding to its effectors, such as the C1 complex and Fc receptors.
  • exemplary effector functions include complement-dependent cytotoxicity (CDC) induced by the interaction of the antibody with C1q on the C1 complex, antibody-dependent cell-mediated cytotoxicity (CDC) induced by binding of the Fc region of the antibody to Fc receptors on effector cells Toxicity (ADCC) and phagocytosis.
  • Cancer or “cancer disorder” as used herein refers to any medical condition mediated by the growth, proliferation or metastasis of tumors or malignant cells, and which results in solid and non-solid tumors such as leukemia.
  • Tumor in this application refers to a solid substance of tumor and/or malignant cells.
  • Treatment or “therapy” of a condition includes preventing or alleviating a condition, reducing the rate at which a condition arises or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition , reduce or stop symptoms associated with a condition, produce a complete or partial reversal of a condition, cure a condition, or a combination of the above.
  • treatment or “therapy” can refer to inhibiting or slowing tumor or malignant cell growth, proliferation, or metastasis, or some combination of the above.
  • treatment or “therapy” includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor progression, or some combination of the above.
  • “Separated” matter has been artificially altered from its natural state. If a “separated” substance or component occurs in nature, it has been altered or removed from its original state, or both.
  • a “separated” substance or component occurs in nature, it has been altered or removed from its original state, or both.
  • naturally occurring polynucleotides or polypeptides in a living animal are not isolated, but can be considered “separated”.
  • antibodies and antigen-binding fragments are at least 90%, 93%, 95%, 96%, 97%, 98%, 99% pure, which can be determined by electrophoretic methods (eg, SDS-PAGE, isoelectric focusing , capillary electrophoresis), or chromatography (eg, ion-exchange chromatography or reverse-phase HPLC).
  • vector refers to a vehicle into which a polynucleotide encoding a protein can be operatively inserted and the protein can be expressed.
  • Vectors can be used to transform, transduce or transfect host cells so that elements of the genetic material they carry are expressed in the host cells.
  • vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses, etc.
  • Vectors Animal virus species used as vectors are retroviruses (including lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40)).
  • Vectors may contain a variety of elements to control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain an origin of replication site.
  • the carrier may also include components to facilitate its entry into the cell, including, but not limited to, viral particles, liposomes, or protein coats.
  • a "host cell” in the present application is a cell into which exogenous polynucleotides and/or vectors are introduced.
  • Host cells described herein include, but are not limited to, prokaryotic cells such as E. coli or B. subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells , COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • chimeric antigen receptor is abbreviated as CAR, which refers to a cell surface receptor that can recognize a specific antigen (such as a tumor antigen) and contains an extracellular domain that can recognize a specific antigen (such as an antibody that recognizes and binds to a specific antigen).
  • antigen-binding fragments) and intracellular domains also known as intracellular signal transduction domains, such as the delta chain of CD3 or the intracellular portion of Fc ⁇ RI ⁇ capable of transmitting extracellular signals to the interior of the cell.
  • CAR-T cells T cells that carry and express such chimeric antigen receptors are called CAR-T cells, which can recognize and bind specific antigens and cells (such as tumor cells) expressing the specific antigen through the extracellular domain, and pass the intracellular
  • the signal transduction function of the domain activates the immune response, releases a large number of various effectors, and efficiently kills cells (such as tumor cells) expressing the specific antigen, thereby exerting a therapeutic effect (such as tumor treatment).
  • one of the main purposes of this application is to provide an anti-CEACAM5 antibody with higher specificity and better selectivity.
  • the present application also provides the preparation method and use of the antibody, and the anti-CEACAM5 antibody of the present application can be used to detect and/or treat tumors.
  • the application provides a humanized antibody or antigen-binding fragment thereof that specifically binds to CEACAM5 protein, the antibody or antigen-binding fragment thereof comprising:
  • VH heavy chain variable region
  • CDRs 3 complementarity determining regions
  • VH CDR1 consisting of a heavy chain as set forth in SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 or 42 VH CDR1 sequences contained in the variable region (VH), or sequences having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom ;
  • VH CDR2 consisting of a heavy chain as set forth in SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 or 42 VH CDR2 sequences contained in the variable region (VH), or sequences having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom ;and
  • VH CDR3 consisting of a heavy chain as set forth in SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 or 42 VH CDR3 sequences contained in the variable region (VH), or sequences having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom ;
  • VL light chain variable region
  • CDRs 3 complementarity determining regions
  • VL CDR1 consisting of a light chain as set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43 VL CDR1 sequences contained in the variable region (VL), or sequences having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom ;
  • VL CDR2 consisting of a light chain as set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43 VL CDR2 sequences contained in the variable region (VL), or sequences having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom ;and
  • VL CDR3 consisting of a light chain as set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43 VL CDR3 sequences contained in the variable region (VL), or sequences having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom .
  • the 3 CDRs contained in the VH and/or the 3 CDRs contained in the VL are defined according to the Kabat, IMGT or Chothia numbering system. In certain embodiments, the 3 CDRs contained in the VH and/or the 3 CDRs contained in the VL are defined according to the Kabat numbering system.
  • substitutions of any of (i)-(vi) are conservative substitutions.
  • the application provides a humanized antibody or antigen-binding fragment thereof that specifically binds to a CEACAM5 protein, the antibody or antigen-binding fragment thereof comprising:
  • VH heavy chain variable region
  • CDRs 3 complementarity determining regions
  • VH CDR1 consisting of the following sequence: SEQ ID NO: 1, or having one or several amino acid substitutions, deletions or additions therefrom (e.g. 1, 2 or 3 amino acid substitutions, deletions) or added) sequence,
  • VH CDR2 consisting of the following sequence: SEQ ID NO: 2, or with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or addition)
  • VH CDR3 consisting of the following sequence: SEQ ID NO: 3, 44, or 45, or with one or several amino acid substitutions, deletions, or additions (e.g., 1 , 2 or 3 amino acid substitutions, deletions or additions) sequences;
  • VL light chain variable region
  • CDRs 3 complementarity determining regions
  • VL CDR1 consisting of the following sequence: SEQ ID NO: 4, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or added) sequence,
  • VL CDR2 consisting of the following sequence: SEQ ID NO: 5, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or addition), and
  • the present application provides a humanized antibody or antigen-binding fragment thereof that specifically binds to the CEACAM5 protein, the antibody or antigen-binding fragment thereof comprising: such as SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 or 42 contained in the 3 CDRs contained in the variable region of the heavy chain (VH); and/or, as in SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43.
  • the three CDRs contained in the variable region (VL) of the light chain is defined by the Kabat, IMGT or Chothia numbering systems.
  • the 3 CDRs contained in the VH and/or the 3 CDRs contained in the VL are defined according to the Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises: the following 3 heavy chain CDRs: VH CDR1 of sequence SEQ ID NO: 1, VH CDR2 of sequence SEQ ID NO: 2, sequence of SEQ ID NO: 2 VH CDR3 of ID NO: 3, 44 or 45; and/or, the following 3 light chain CDRs: VL CDR1 of sequence SEQ ID NO: 4, VL CDR2 of sequence SEQ ID NO: 5, sequence of SEQ ID NO : 6 VL CDR3.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • sequences shown in (i) have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% %, at least 98%, at least 99%, or 100% sequence identity;
  • VL light chain variable region
  • sequences shown in (vi) and (iv) have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% %, at least 98%, at least 99%, or 100% sequence identity.
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH having the sequence shown in SEQ ID NO:28 and VL having the sequence shown in SEQ ID NO:29;
  • VH having the sequence shown in SEQ ID NO:30 and VL having the sequence shown in SEQ ID NO:31;
  • VH having the sequence shown in SEQ ID NO:42 and VL having the sequence shown in SEQ ID NO:43.
  • An antibody or antigen-binding fragment thereof of the present application may comprise a mammalian (eg, murine or human) immunoglobulin-derived constant region sequence or variant thereof having one or more of the sequences from which it is derived amino acid substitutions, deletions or additions or any combination thereof (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions or any combination thereof; e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof).
  • a mammalian eg, murine or human
  • immunoglobulin-derived constant region sequence or variant thereof having one or more of the sequences from which it is derived amino acid substitutions, deletions or additions or any combination thereof (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions or any combination thereof; e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof).
  • the heavy chain of an antibody or antigen-binding fragment thereof of the present application comprises a heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof, which, compared to the sequence from which it is derived, has One or more amino acid substitutions, deletions, or additions or any combination thereof (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions, or any combination thereof; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof).
  • CH heavy chain constant region
  • the light chain of an antibody or antigen-binding fragment thereof of the present application comprises a light chain constant region (CL) of a human immunoglobulin, or a variant thereof, which, compared to the sequence from which it is derived, has One or more amino acid substitutions, deletions, or additions or any combination thereof (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions, or any combination thereof; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof).
  • CL light chain constant region
  • the variant of the heavy chain constant region (CH) may have conservative substitutions of one or more amino acids (eg, 1, 2, 3, 4 or 5 amino acid conservative substitutions).
  • the variant of the light chain constant region (CL) may have conservative substitutions of one or more amino acids (eg, 1, 2, 3, 4 or 5 amino acid conservative substitutions).
  • the heavy chain constant region is an IgG heavy chain constant region, eg, an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region. In certain embodiments, the heavy chain constant region is a human IgGl, IgG2, IgG3 or IgG4 heavy chain constant region.
  • the light chain constant region is a kappa light chain constant region. In certain embodiments, the light chain constant region is a human kappa light chain constant region.
  • the antibody or antigen-binding fragment thereof of the present application comprises a heavy chain constant region (CH) set forth in SEQ ID NO:9; and/or, a light chain constant set forth in SEQ ID NO:11 District (CL).
  • CH heavy chain constant region
  • CL light chain constant
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabodies, and single domain antibodies (sdAbs).
  • the antibody is a bispecific antibody or a multispecific antibody.
  • the antibodies or antigen-binding fragments thereof of the present application are labeled.
  • the antibody or antigen-binding fragment thereof bears a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance) or Biotin.
  • the application provides exemplary anti-CEACAM5 humanized antibodies UM05-6L-1 to UM05-6L-14.
  • CDR sequences can be modified to include one or more amino acid substitutions, thereby resulting in increased biological activity such as increased binding affinity to human carcinoembryonic antigen cell adhesion molecule 5.
  • a library of antibody variants eg, Fab or FcFv variants
  • computer software can be used to simulate the binding of the antibody to CEACAM5 and to identify amino acid residues on the antibody that form the binding interface. Substitution of these residues can be avoided to prevent reduced binding affinity, or can be targeted for substitution for stronger binding.
  • at least one (or all) substitutions in a CDR sequence are conservative substitutions.
  • the antibody or antigen-binding fragment comprises one or more CDR sequences having at least 80% of the sequence of SEQ ID NO: 1, 2, 3 or 44 or 45, 4, 5 or 6 (eg at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity while retaining its parental Antibodies with similar or even higher binding affinity to CEACAM5.
  • the parental antibodies have substantially the same sequence, but their corresponding CDR sequences have 100% sequence identity to the sequences set forth in SEQ ID NO: 1, 2, 3 or 44 or 45, 4, 5 or 6.
  • the antibodies or antigen-binding fragments described herein are capable of specifically binding to CEACAM5 with a binding affinity (KD) of <10<" 7 > M, as measured by surface plasmon resonance.
  • the binding affinity value can be expressed as a KD value, which is calculated by the ratio of the off-rate to on-rate (koff/kon) when the binding of the antigen and the antigen-binding molecule reaches equilibrium.
  • Said antigen binding affinity (eg KD) may suitably be determined by suitable methods known in the art, eg including plasmon resonance binding using an instrument such as Biacore.
  • the antibodies or antigen-binding fragments described herein bind to CEACAM5 with an EC50 (ie, half-binding concentration) of 1 ng/mL to 10 ⁇ g/mL. Binding of an antibody or antigen-binding fragment to CEACAM5 can be determined by methods known in the art such as sandwich assays such as ELISA, Western blot, FACS or other binding assays.
  • the antibody to be tested ie, the primary antibody
  • the primary antibody is bound to immobilized CEACAM5 or cells expressing CEACAM5, followed by washing away the unbound antibody and introducing a labeled secondary antibody, which is capable of binding to the primary antibody and thus capable of Bound secondary antibody is detected.
  • the assay can be performed on a microtiter plate when using immobilized carcinoembryonic antigen cell adhesion molecule 5, or using FACS analysis when using cells expressing CEACAM5.
  • the antibodies or antigen-binding fragments described herein bind to CEACAM5 with an EC50 (ie, an effective concentration of 50%) of 10 ng/mL to 10 ⁇ g/mL (determined using FACS analysis).
  • the antibody is specific for CEACAM5.
  • the antibody optionally does not bind CEACAM1, CEACAM3, CEACAM6, and binds CEACAM8 with a significantly lower binding affinity than CEACAM5.
  • the antibodies described herein can be used in combination with immunogenic substances, such as tumor cells, purified tumor antigens and cells transfected with encoding immunostimulatory factors, tumor vaccines.
  • immunogenic substances such as tumor cells, purified tumor antigens and cells transfected with encoding immunostimulatory factors, tumor vaccines.
  • the anti-CEACAM5 antibodies and antigen-binding fragments thereof can be included in combination therapy, including standard chemotherapy and radiation therapy, target-based small molecule therapy, and other emerging immune checkpoint modulator therapies.
  • the antibodies and antigen-binding fragments thereof can be used as base molecules for antibody-drug conjugates, bispecific or multivalent antibodies.
  • the antibodies and antigen-binding fragments thereof described herein are camelized single chain antibody domain antibodies, diabodies, scFv, scFv dimers, BsFv, dsFv, ( dsFv) 2 , dsFv-dsFv', Fv fragments, Fab, Fab', F(ab') 2 , ds diabodies, Nanobodies, domain antibodies or bivalent domain antibodies.
  • the antibodies described herein include immunoglobulin constant regions.
  • the immunoglobulin constant regions include heavy and/or light chain constant regions.
  • the heavy chain constant region includes a CH1, CH1-CH2 or CH1-CH3 region.
  • the immunoglobulin constant region may further comprise one or more modifications to obtain desired properties.
  • the constant region can be modified to reduce or eliminate one or more effector functions, enhance FcRn receptor binding or introduce one or more cysteine residues.
  • the antibodies and antigen-binding fragments thereof further comprise a conjugate.
  • the antibodies or antigen-binding fragments thereof of the present application may be linked to a variety of conjugates (see, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology, J.M. Cruuse and R.E. Lewis, Jr. (eds.), Carger Press , New York (1989)).
  • conjugates can be linked to the antibody or antigen-binding fragment by other means such as covalent binding, affinity binding, intercalation, coordinate binding, complexation, conjugation, mixing or addition.
  • the antibodies and antigen-binding fragments disclosed herein can be engineered to contain specific sites other than epitope binding moieties that can be used to bind one or more conjugates.
  • such sites may contain one or more reactive amino acid residues, such as cysteine residues and histidine residues, to facilitate covalent attachment to the conjugate.
  • the antibody may be attached to the conjugate indirectly, or through another conjugate.
  • the antibody or antigen-binding fragment thereof can bind biotin and then indirectly bind a second conjugate, which is linked to avidin.
  • the conjugate can be a detectable label, a pharmacokinetic modifying moiety, a purification moiety or a cytotoxic moiety.
  • detectable labels may include fluorescent labels (eg, fluorescein, rhodamine, dansyl, phycoerythrin, or Texas red), enzyme substrate labels (eg, horseradish peroxidase, alkaline phosphatase) , luciferase, glucoamylase, lysozyme, carbohydrate oxidase or ⁇ -D galactosidase), stable or radioisotopes, chromophore moieties, digoxigenin, biotin/avidin, DNA molecules or gold for detection.
  • the conjugate may be a pharmacokinetic modifying moiety such as PEG, which helps prolong the half-life of the antibody.
  • the conjugate may be a purification moiety such as a magnetic bead.
  • a "cytotoxic moiety" can be any agent that is detrimental to cells or that may damage or kill cells.
  • cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecine, mitomycin, etopoxi, tenipogan, vincristine, Vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraxin dione, mitoxantrone, mithramycin, actinomycin D, l-dehydrotestosterone, glucocorticoids, Procaine, tetracaine, lidocaine, putrolol, puromycin and its analogs, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-mercaptoguanine, arabinoside cytidine, 5-fluorouracil dacarb), alkylating agents (e.g.
  • the antibodies of the present application can be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques.
  • DNA molecules encoding the antibodies of the present application or antigen-binding fragments thereof can be obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. Then, the transfected host cells are cultured under specific conditions and express the antibody or antigen-binding fragment thereof of the present application.
  • Antigen-binding fragments of the present application can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
  • Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from recombinant host cell culture medium. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.
  • the application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the application, or a heavy chain variable region and/or a light chain variable region thereof.
  • the nucleic acid molecule comprises, for example, SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 , 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 or 73 of the nucleotide sequence.
  • the heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of:
  • sequences shown in (i) have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% %, at least 98%, at least 99%, or 100% sequence identity.
  • the nucleotide sequence encoding the heavy chain variable region is selected from SEQ ID NOs: 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, or 72.
  • the light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of:
  • sequences shown in (vi) and (iv) have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% %, at least 98%, at least 99%, or 100% sequence identity.
  • the nucleotide sequence encoding the light chain variable region is selected from the group consisting of SEQ ID NOs: 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, or 73.
  • the isolated nucleic acid molecule comprises:
  • the application provides a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above.
  • a vector eg, a cloning vector or an expression vector
  • the vectors of the present application are plasmids, cosmids, phages, and the like.
  • the vector comprises a first nucleotide sequence encoding a heavy chain variable region of an antibody or antigen-binding fragment thereof of the present application, and/or a light source encoding an antibody or antigen-binding fragment thereof of the present application
  • the second nucleotide sequence of the chain variable region In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.
  • the application provides a host cell comprising the isolated nucleic acid molecule or vector as described above.
  • the host cell is a mammalian cell.
  • the host cell is a human, murine, ovine, equine, dog or feline cell.
  • the host cell is a Chinese hamster ovary cell.
  • an antibody or antigen-binding fragment thereof of the present application comprising, culturing a host cell as described above under conditions that permit expression of the antibody or antigen-binding fragment thereof, and extracting from the cultured The antibody or antigen-binding fragment thereof is recovered from the host cell culture.
  • the application also provides bispecific or multispecific molecules comprising said antibodies or antigen-binding fragments thereof.
  • the bispecific or multispecific molecule specifically binds CEACAM5, and additionally specifically binds one or more other targets.
  • the bispecific or multispecific molecule further comprises at least one molecule (eg, a second antibody) having a second binding specificity for a second target.
  • the application also provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.
  • the therapeutic agent is selected from cytotoxic agents.
  • the therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, antineoplastic antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and the like random combination.
  • the immunoconjugate is an antibody-drug conjugate (ADC).
  • amino acid sequences of the antibodies and antigen-binding fragments thereof described herein can be converted into corresponding DNA coding sequences using genetic engineering techniques well known in the art. Due to the degeneracy of the genetic code, the resulting DNA sequences may not be completely identical, while the encoded protein sequences remain unchanged.
  • Vectors comprising polynucleotides encoding the antibodies and antigen-binding fragments thereof can be introduced into host cells for cloning (amplification of DNA) or gene expression using recombinant techniques well known in the art.
  • the antibodies and antigen-binding fragments thereof can be prepared by methods known in the art by homologous recombination.
  • vectors are available.
  • Vector components typically include, but are not limited to, two or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter (eg: SV40, CMV, EF-1a) and Transcription termination sequence.
  • the vector system includes mammalian, bacterial, yeast systems, etc., and will include plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pELpGEMEX, pGEX, pCLpCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2 and other vectors that can be obtained from laboratories or commercially available.
  • Suitable vectors may include plasmid or viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses).
  • Vectors comprising polynucleotides encoding the antibodies and antigen-binding fragments thereof can be introduced into host cells for cloning or gene expression.
  • the host cells suitable for cloning or expressing the DNA in the vector in the present application are prokaryotic cells, yeast or the above-mentioned higher eukaryotic cells.
  • Prokaryotic cells suitable for use herein include eubacteria, such as Gram-negative or Gram-positive bacteria, such as Enterobacteriaceae (eg E.
  • eukaryotic microorganisms such as filamentous fungi or yeast can also be used as host cells for cloning or expression of vectors encoding antibodies.
  • Saccharomyces cerevisiae, or baker's yeast is the most commonly used lower eukaryotic host microorganism.
  • Kluyveromyces hosts such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC12424), Kluyveromyces bulgaricus (ATCC16045), Kluyveromyces wischei (ATCC24178), Kluyveromyces (ATCC56500), Kluyveromyces Drosophila (ATCC36906), Kluyveromyces thermotolerant and Kluyveromyces marxianus: Yarrowia lipolytica (EP402226); Pichia pastoris (EP183070); Candida: Trichoderma reesei (EP244234); Alternaria; Fungi, such as Neurospora, Penicillium, Curvus, and Aspergillus, such as Aspergillus nidulans and Aspergillus niger.
  • Fungi such as Neurospora, Penicillium, Curvus
  • Aspergillus such as Aspergillus nidulans and Aspergill
  • Suitable host cells for expressing glycosylated antibodies or antigen-binding fragments thereof provided herein are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Various baculoviral strains and their variants, as well as corresponding permissive insect host cells, have been found from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito ), Aedes albopictus (mosquito), Drosophila melanogaster (Drosophila melanogaster) and silkworm.
  • a variety of viral strains for transfection are publicly available, such as the Autographa californica nuclear polyhedrosis virus and the Bm-5 variant of the silkworm nuclear polyhedrosis virus, all of which can be used in this application, particularly For transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts.
  • vertebral cells are of greatest interest, and culturing (tissue culture) of vertebral cells has become routine.
  • useful mammalian host cells are the SV40 transformed monkey kidney cell line CV1 (COS-7, ATCC CRL1651); the human embryonic kidney cell line (293 or a suspension cultured 293 cell subclone, Graham et al., Gen Virol. 36: 59 (1977)); baby hamster kidney cells (ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)) Mouse Sertoli cells (TM4, Mather J.P., Biol. Reprod.
  • the host cell is a 293F cell.
  • the host cells used in this application to produce the antibodies and antigen-binding fragments thereof can be cultured in a variety of media known in the art.
  • the medium may also contain any other necessary additives at appropriate concentrations known in the art.
  • Conditions of the medium such as temperature, pH, and the like, are those previously used to select host cells for expression, and are well known to those of ordinary skill.
  • the antibodies can be produced intracellularly, in the parietal space, or secreted directly into the culture medium. If the antibody is produced intracellularly, the particulate debris of the host cells or lysed fragments is first removed, eg, by centrifugation or sonication. Carter et al., Bio/Technology 10: 163-167 (1992) describe a method for the isolation of antibodies secreted into the parietal space of E. coli. Briefly, the cell paste was dissolved in the presence of uranyl acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for more than about 30 minutes. Cell debris was removed by centrifugation.
  • uranyl acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonyl fluoride
  • the supernatant of the expression system is typically first concentrated using a commercially available protein concentration filter, such as an IAmicon or Millipore Pellicon ultrafiltration unit.
  • Protease inhibitors such as PMSF to inhibit protein degradation, and antibiotics to prevent the growth of incidental contaminants can be added in any of the preceding steps.
  • Antibodies prepared from the cells can be purified using purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, wherein Affinity chromatography is the preferred purification technique.
  • the identity of the antibody and the presence of any immunoglobulin Fc domains in the antibody determine the suitability of Protein A as an affinity ligand.
  • Protein A can be used to purify antibodies based on human ⁇ 1, ⁇ 2 or ⁇ 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:13 (1983)).
  • Protein G is available for all murine isoforms and human gamma 3 (Guss et al., EMBO J. 5:1567-1575 (1986)).
  • Agarose is the most commonly used matrix for affinity ligand attachment, but other matrices are also available.
  • Mechanically stable matrices such as controlled pore glass or poly(styrene)benzene allow for faster flow rates and shorter processing times than with agarose. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX.TM resin (J.T. Baker, Phillipsburg, N.J.).
  • protein purification such as fractionation in ion exchange columns, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin sepharose chromatography based on anion or cation exchange resins (such as polyaspartum), can also be determined depending on the antibody obtained as needed. amino acid column), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation.
  • the mixture containing the antibody of interest and impurities can be treated by means of low pH hydrophobic interaction chromatography with an elution buffer at pH about 2.5-4.5, preferably at low salt concentrations (eg, from about 0 to 0.25M salt concentration).
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present application, the bispecific or multispecific molecule of the present application, or the immunoconjugate of the present application, and a pharmaceutically acceptable Accepted carriers and/or excipients.
  • the pharmaceutical composition may further comprise additional pharmaceutically active agents.
  • the additional pharmaceutically active agent is a drug with antitumor activity, eg, alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • a drug with antitumor activity eg, alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • the antibody or antigen-binding fragment, bispecific or multispecific molecule or immunoconjugate thereof and the additional pharmaceutically active agent are separate components or as components of the same composition supply.
  • the antibody or antigen-binding fragment thereof of the present application and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.
  • the application further provides pharmaceutical compositions comprising the antibodies and one or more pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial substances, etc. Osmotic substances, buffers, antioxidants, anesthetics, suspending/dispersing agents, integrating agents, diluents, adjuvants, adjuvants or non-toxic auxiliary substances, other components known in the art or various combinations of the above.
  • Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, emulsifiers or stabilizers such as Sugar and Cyclodextrin.
  • Useful antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene and/or propyl gallate.
  • Including one or more antioxidants, such as methionine, in a composition containing the antibodies disclosed herein will reduce the oxidation of the antibodies. The reduction in oxidation can prevent or reduce the loss of binding affinity, thereby improving antibody stability and extending shelf life.
  • pharmaceutically acceptable carriers may include, for example, aqueous media such as Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, or Dextrose and Lactate Lattice injection, non-aqueous medium such as: fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacteriostatic or fungal inhibitory concentrations, isotonic agents such as sodium chloride or glucose, Buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (Tween-80), integrating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-aminoethyl)
  • Antibacterial agents as carriers can be incorporated into pharmaceutical compositions in multi-dose containers including phenols or cresols, mercury, benzyl alcohol, chlorobutanol, methyl and propyl parabens, Thiomersal, Chlorphenethanium and Chlorphenethanium.
  • Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol.
  • Suitable non-toxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or sodium acetate, sorbitan laurate, triethanolamine oleate, or cyclodextrins. substance.
  • the pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder.
  • Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • the pharmaceutical composition is formulated as an injectable composition.
  • injectable pharmaceutical compositions can be prepared in any conventional form, eg, liquid solvents, suspending agents, emulsifying agents, or solid forms suitable for the production of liquid solvents, suspending agents, or emulsifying agents.
  • Injectable preparations may include ready-to-use sterile and/or pyrogen-free solutions, sterile-dried solubles, such as lyophilized powders, including subcutaneous tablets, ready-to-use sterile suspensions, which are combined with a solvent prior to use. Sterile dry insoluble products, and sterile and/or pyrogen-free emulsions, for constitution with vehicle prior to use.
  • the solvent can be an aqueous phase or a non-aqueous phase.
  • the unit-dose injectable formulation is packaged in an ampoule, a vial, or a syringe with a needle. It is known in the art that all formulations for injectable administration should be sterile and pyrogen-free.
  • sterile lyophilized powders can be prepared by dissolving an antibody or antigen-binding fragment thereof disclosed herein in an appropriate solvent.
  • the solvent may contain an additional pharmacological component that enhances the stability of the powder or reconstituted solution prepared from the powder, or improves the powder or reconstituted solution. Suitable excipients include, but are not limited to, water, glucose, tribitol, fructose, corn syrup, xylitol, glycerol, glucose, brown sugar or other suitable substances.
  • the solvent may contain a buffer, such as a citrate buffer, sodium or potassium phosphate buffer, or other buffers known to those skilled in the art, in one embodiment, the pH of the buffer is neutral.
  • each vial can contain a single dose or multiple doses of the anti-CEACAM5 antibody or antigen-binding fragment thereof, or a composition thereof.
  • the volume in each vial can be slightly higher than required for each dose or for multiple doses (eg, a 10% excess) to ensure accurate sampling and accurate dosing.
  • the lyophilized powder can be stored under appropriate conditions, eg, in the range of about 4°C to room temperature.
  • the lyophilized powder is reconstituted with water for injection to obtain a formulation for injection administration.
  • the lyophilized powder can be reconstituted by adding sterile pyrogen-free water or other suitable liquid carrier. The exact amount is determined by the therapy chosen and can be determined empirically.
  • An antibody or antigen-binding fragment thereof of the present application can be derivatized, eg, linked to another molecule (eg, another polypeptide or protein).
  • another molecule eg, another polypeptide or protein.
  • derivatization eg, labeling
  • the antibodies or antigen-binding fragments thereof of the present application are also intended to include such derivatized forms.
  • an antibody or antigen-binding fragment thereof of the present application can be functionally linked (by chemical coupling, genetic fusion, non-covalent attachment, or otherwise) to one or more other molecular moieties, such as another antibody (eg, to form bispecific antibodies), detection reagents, pharmaceutical reagents, and/or proteins or polypeptides (eg, avidin or polyhistidine tags) capable of mediating binding of an antibody or antigen-binding fragment to another molecule.
  • the antibodies or antigen-binding fragments thereof of the present application can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.
  • the application provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof described herein and a therapeutic agent linked to the antibody or antigen-binding fragment thereof;
  • the therapeutic agent is selected from cytotoxic agents
  • the therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, antineoplastic antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and the like random combination;
  • the immunoconjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the antibody or antigen-binding fragment thereof of the present application is capable of specifically binding to CEACAM5 protein and substantially not to CEACAM1, CEACAM3, CEACAM6 proteins, and to CEACAM8 with significantly lower binding affinity to CEACAM5. Therefore, the antibody or antigen-binding fragment thereof of the present application has higher specificity and accuracy in detection.
  • the present application provides a kit comprising an antibody or antigen-binding fragment thereof of the present application, or a conjugate of the present application.
  • the antibody or antigen-binding fragment thereof bears a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance) or Biotin.
  • a detectable label such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance) or Biotin.
  • the kit further includes a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof.
  • the second antibody further includes a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.
  • a detectable label such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.
  • kits of the present application may further comprise reagents for causing the corresponding detectable labels to be detected.
  • the detectable label is an enzyme
  • the kit may also include a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethyl-linked Aniline (TMB), ABTS or luminol compounds, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase.
  • OPD o-phenylenediamine
  • TMB tetramethyl-linked Aniline
  • ABTS ABTS
  • luminol compounds p-nitrophenyl phosphate
  • p-NPP p-nitrophenyl phosphate
  • AMPPD p-nitrophenyl phosphate
  • the detectable label is a chemiluminescent reagent (eg, an acridine ester compound)
  • the kit may further comprise a pre-excitation solution
  • the present application also provides use of the antibody or antigen-binding fragment thereof in the preparation of a kit for detecting whether a tumor can be treated by an anti-tumor therapy targeting CEACAM5.
  • the antibody or antigen-binding fragment thereof is detectably labeled.
  • the CEACAM5 is human CEACAM5.
  • the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Carcinoma and other hematological malignancies such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and negative PTLD and EBV-associated Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, n
  • CHL
  • kits comprising the antibodies or antigen-binding fragments thereof.
  • the kit is used to detect the presence or level of CEACAM5 in a biological sample.
  • the biological sample can include cells or tissues.
  • the kit includes an antibody or antigen-binding fragment thereof conjugated to a detectable label.
  • the kit includes an unlabeled antibody, and further includes a labeled secondary antibody capable of binding to the unlabeled antibody.
  • the kit may further include instructions for use and packaging that separates each component in the kit.
  • the antibody is linked to a substrate or instrument for use in a sandwich assay such as an ELISA or immunochromatographic assay.
  • a substrate or instrument for use in a sandwich assay such as an ELISA or immunochromatographic assay.
  • Suitable substrates or instruments can be, for example, microplates and test strips.
  • the present application also provides a chimeric antigen receptor comprising the antigen-binding domain of the antibody or antigen-binding fragment thereof.
  • the antigen-binding domain comprises the heavy and light chain variable regions of the antibody or antigen-binding fragment thereof.
  • the antigen binding domain is an scFv.
  • the chimeric antigen receptor comprises an antigen-binding fragment of the antibody.
  • the chimeric antigen receptor is expressed by immune effector cells (eg, T cells).
  • immune effector cells eg, T cells
  • the application also provides an isolated nucleic acid molecule encoding the chimeric antigen receptor.
  • the application also provides a vector comprising the isolated nucleic acid molecule; in certain embodiments, it is used to prepare chimeric antigen receptor T cells.
  • the application also provides a host cell comprising the isolated nucleic acid molecule or vector;
  • the host cells are immune effector cells (eg, T cells or NK cells);
  • the host cell is a chimeric antigen receptor T cell (CAR-T).
  • CAR-T chimeric antigen receptor T cell
  • CEACAM5 can serve as a tumor biomarker. Therefore, the measurement of CEACAM5 in the blood of patients can be used for the prognosis and control of cancer, and the targeted therapy of CEACAM5 has also become a potential cancer treatment method.
  • the antibodies of the present application eg, hUM05-6L-1 to hUM05-6L-14
  • the present application also provides a method for inhibiting the growth and/or killing of tumor cells expressing CEACAM5, comprising combining the tumor cells with an effective amount of the antibody or antigen thereof Binding fragment, or said bispecific or multispecific molecule, or said immunoconjugate, or said pharmaceutical composition, or said chimeric antigen receptor, or said host cell contact .
  • the present application also provides a method for reducing the expression level of CEACAM5 on the cell surface, comprising combining the cell with the antibody or antigen-binding fragment thereof, or the bispecific or Contacting the multispecific molecule, or the immunoconjugate, or the pharmaceutical composition, or the chimeric antigen receptor, or the host cell, reduces the expression level of CEACAM5 on the cell surface; Wherein, the cells express CEACAM5 on their surface.
  • the cell is a CEACAM5-expressing tumor cell.
  • the present application also provides a method for preventing and/or treating a tumor in a subject (eg, a human), the method comprising administering to a subject in need thereof an effective amount of the the antibody or antigen-binding fragment thereof, or the bispecific or multispecific molecule, or the immunoconjugate, or the pharmaceutical composition, or the chimeric antigen receptor, or the described host cells.
  • a subject eg, a human
  • the method comprising administering to a subject in need thereof an effective amount of the the antibody or antigen-binding fragment thereof, or the bispecific or multispecific molecule, or the immunoconjugate, or the pharmaceutical composition, or the chimeric antigen receptor, or the described host cells.
  • the tumor expresses CEACAM5.
  • the tumor involves tumor cells expressing CEACAM5.
  • the CEACAM5 is expressed on the surface of the tumor cell.
  • the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Carcinoma and other hematological malignancies such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and negative PTLD and EBV-associated Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, n
  • CHL
  • the subject is a mammal, such as a human.
  • the method further comprises administering an additional drug with antitumor activity, eg, alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • an additional drug with antitumor activity eg, alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • antitumor activity eg, alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, t
  • the method further comprises administering additional anti-tumor therapy, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative therapy.
  • additional anti-tumor therapy such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative therapy.
  • the present application also provides the antibody or antigen-binding fragment thereof, or the bispecific or multispecific molecule, or the immunoconjugate, or the pharmaceutical composition, Or the use of the chimeric antigen receptor, or the host cell, in the preparation of a medicament for preventing and/or treating a tumor in a subject (eg, a human).
  • the medicament further comprises an additional pharmaceutically active agent.
  • the additional pharmaceutically active agent is a drug with antitumor activity, eg, alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • a drug with antitumor activity eg, alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • the tumor expresses CEACAM5.
  • the tumor involves tumor cells that express CEACAM5; preferably, the CEACAM5 is expressed on the surface of the tumor cells.
  • the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Carcinoma and other hematological malignancies such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and negative PTLD and EBV-associated Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, n
  • CHL
  • the subject is a mammal, such as a human.
  • the present application also provides a method of detecting the presence or amount of CEACAM5 (eg, human CEACAM5) in a sample, comprising the steps of:
  • the antibody or antigen-binding fragment thereof is detectably labeled.
  • the CEACAM5 is human CEACAM5.
  • the present application also provides a method for detecting whether a tumor can be treated by an anti-tumor therapy targeting CEACAM5, comprising the steps of:
  • the antibody or antigen-binding fragment thereof is detectably labeled.
  • the CEACAM5 is human CEACAM5.
  • the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Carcinoma and other hematological malignancies such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and negative PTLD and EBV-associated Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, n
  • CHL
  • the application also provides methods of treatment comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein.
  • Therapeutically effective doses of the antibodies provided in this application depend on a variety of factors well known in the art, such as body weight, age, past medical history, current treatment, the subject's health status and potential for cross-infection, allergies, hypersensitivity and side effects, and administration of drug route and extent of tumor development.
  • One skilled in the art eg, a physician or veterinarian can proportionally reduce or increase the dosage according to these or other conditions or requirements.
  • the antibodies provided herein can be administered at a therapeutically effective dose of between about 0.01 mg/kg and about 100 mg/kg.
  • the antibody is administered at a dose of about 50 mg/kg or less, in certain embodiments at a dose of 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg kg or less, 0.5 mg/kg or less or 0.1 mg/kg or less.
  • a given dose may be administered at multiple intervals, for example, once daily, twice daily or more, twice monthly or more, weekly, biweekly, every three weeks, monthly or every other monthly or more.
  • the dose administered may vary over the course of treatment. For example, in certain embodiments, the initially administered dose may be higher than the subsequently administered dose. In certain embodiments, the administered dose is adjusted over the course of treatment based on the response of the administered subject.
  • the dosing regimen can be adjusted to achieve an optimal response (eg, therapeutic response). For example, a single dose may be administered or multiple divided doses may be administered over a period of time.
  • the antibodies disclosed in this application can be administered by means of administration known in the art, eg, by injection (eg, subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular injection, or intradermal injection) or non-injectionally.
  • injection eg, subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular injection, or intradermal injection
  • Medication eg, oral, nasal, sublingual, rectal, or topical.
  • the antibodies can be used to treat disorders related to their molecular mechanisms, including tumors and cancers, such as non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer Cancer, stomach cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma , mycosis fungoids, Merkel cell carcinoma and other hematological malignancies such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T cell/histiocytic B-cell-rich lymphoma, EBV-positive and negative PTLD and EBV-related diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranod
  • the application further provides methods of using the antibodies.
  • the present application provides a method of treating a condition or disorder associated with the antibody mechanism in an individual comprising administering a therapeutically effective amount of an antibody described herein.
  • the antibodies disclosed herein can be administered alone or in combination with one or more other therapeutic means or substances.
  • the antibodies disclosed herein may be used with chemotherapy, radiation therapy, cancer treatment surgery (eg, tumor resection), antiviral drugs, one or more antiemetic drugs, or other therapies for complications of chemotherapy, or any other Combination of cancer or viral therapeutic substances.
  • the antibodies disclosed herein, when used in combination with one or more therapeutic substances may be administered concurrently with the one or more therapeutic substances, and in certain such embodiments, The antibodies can be administered concurrently as part of the same pharmaceutical composition.
  • antibodies "in combination" with other therapeutic substances need not be administered concurrently or in the same composition as the therapeutic substance.
  • the meaning of “combination” in this application also includes that an antibody administered before or after another therapeutic substance is also considered to be “combined” with that therapeutic substance, even if the antibody and the second substance are administered by different means Dosing.
  • other therapeutic substances used in combination with the antibodies disclosed in this application can be administered with reference to the methods of the product instructions of the other therapeutic substances, or by referring to the Surgeon's Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)), or refer to other methods known in the art.
  • the therapeutic substance is capable of inducing or enhancing an immune response against cancer.
  • tumor vaccines can be used to induce an immune response to certain tumors or cancers.
  • Cytokine therapy can be used to enhance the presentation of tumor antigens to the immune system.
  • cytokine therapy examples include but are not limited to interferons such as interferon alpha, beta and gamma, colony stimulating factors such as macrophage CSF, granulocyte macrophage CSF and granulocyte CSF, interleukins such as IL-1, IL-1a , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12, tumor necrosis factors such as TNF - ⁇ and TNF- ⁇ .
  • Interferons such as interferon alpha, beta and gamma
  • colony stimulating factors such as macrophage CSF, granulocyte macrophage CSF and granulocyte CSF
  • interleukins such as IL-1, IL-1a , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12
  • Another group of agents includes those that activate an immune response against tumors or cancer cells, for example, those that increase T cell activation (eg, T cell costimulatory signaling pathways such as CTLA-4, ICOS, OX40, 4-1BB, etc.), As well as those that improve dendritic cell function and antigen presentation.
  • T cell activation eg, T cell costimulatory signaling pathways such as CTLA-4, ICOS, OX40, 4-1BB, etc.
  • the humanized monoclonal antibodies of the present application can bind to CEACAM5 protein or cells expressing CEACAM5 protein with high specificity and selectivity, and they do not substantially bind to CEACAM1, CEACAM3, CEACAM6 proteins, or cells expressing them, bind to CEACAM8 with a significantly lower binding affinity to CEACAM5. Therefore, the humanized monoclonal antibodies of the present application (eg, hUM05-6L-1 to hUM05-6L-14 antibodies) have high clinical application value.
  • Figure 1 shows the overexpression fold of the CEACAM5-overexpressing CHO cell line constructed in this application; compared with conventional CHO cells, the overexpression fold of the CHO cell line constructed in this application is 904 times.
  • Figure 2 shows the results of the ELISA experiment of the binding of antibody UM05-6L to CEACAM5 protein.
  • Figure 3 shows the detection results of the binding ability of antibody UM05-6L to LOVO cells.
  • Figures 4a-4b show the ADCC reporter gene activity of the antibody UM05-6L; wherein, Figure 4a shows the ADCC reporter gene activity of the positive control (Erbitux antibody) and negative control, and Figure 4b shows the ADCC reporter gene activity of UM05-6L.
  • Figures 5a-5b show the detection results of flow cytometry;
  • Figure 5a is the expression efficiency of control T cells (Mock T), and
  • Figure 5b is the expression efficiency of UM05-6L-CAR.
  • Figure 6 shows the results of the secretion of cytokines IFN ⁇ (left panel) and IL-2 (right panel) after UM05-6L-CAR-T was mixed with tumor cells.
  • Figure 7 shows the killing results of UM05-6L-CAR-T cells on tumor cells KATO3.
  • Figure 8 shows the expansion results of UM05-6L-CAR-T cells stimulated by tumor cells LS174T.
  • Figure 9 shows the tumor inhibition results of UM05-6L-CAR-T in mice.
  • Figure 10 shows the release of IFN ⁇ in mice receiving UM05-6L-CAR-T.
  • Figure 11 shows antibodies hUM05-6L-1, hUM05-6L-2, hUM05-6L-3, hUM05-6L-4, hUM05-6L-5, hUM05-6L-6, hUM05-6L-7, hUM05-6L ELISA results of -11 and hUM05-6L-12 binding to CEACAM5 protein.
  • Figure 12 shows the results of ELISA experiments of binding of antibodies hUM05-6L-13, hUM05-6L-14, hUM05-6L-8, hUM05-6L-9 and hUM05-6L-10 to CEACAM5 protein.
  • Figure 13 shows antibodies hUM05-6L-1, hUM05-6L-2, hUM05-6L-3, hUM05-6L-4, hUM05-6L-5, hUM05-6L-6, hUM05-6L-7, hUM05-6L -8.
  • Figure 14 shows graphs of experimental results of binding of antibodies hUM05-6L-10, hUM05-6L-11, hUM05-6L-12, hUM05-6L-13, hUM05-6L-14 and UM05-6L to LS174T cells.
  • FIG. 15 shows graphs of experimental results of binding of antibodies hUM05-6L-1 to hUM05-6L-14 to cells overexpressing CEACAM1, 3, 5, 6 and 8.
  • FIG. 15 shows graphs of experimental results of binding of antibodies hUM05-6L-1 to hUM05-6L-14 to cells overexpressing CEACAM1, 3, 5, 6 and 8.
  • the present application constructed a CHO cell line overexpressing the CEACAM5 protein.
  • the plasmid of CEACAM5 (Beijing Yiqiao Shenzhou Technology Co., Ltd., HG11077-UT) was transfected into CHO cell line (ATCC) and expressed.
  • This plasmid is resistant to hygromycin, so cells stably transfected with this plasmid can be stably passaged in Hygromycin-containing medium.
  • the CHO-CEACAM5 cells detected by FACS have a 904-fold overexpression fold of CEACAM5.
  • mice Six 5-8 week old Balb/c mice (Shanghai Slack) were immunized with CHO-CEACAM5 cells and CEACAM5 protein purified from tumor patients (Shanghai Lingchao Bio, L2C01001) according to the immunization protocol in Table 2.
  • Intraperitoneal immunization CHO-CEACAM5 cells CFA Day 26 Intraperitoneal immunization CHO-CEACAM5 cells IFA Day 49 Intraperitoneal immunization CHO-CEACAM5 cells IFA Day 91 Intraperitoneal immunization CEACAM5 protein Gerbu (MM3001) Day 104 Intraperitoneal immunization CEACAM5 protein Gerbu (MM3001) Day 105 Intraperitoneal immunization CEACAM5 protein Gerbu (MM3001) Day 106 Intraperitoneal immunization CEACAM5 protein Gerbu (MM3001)
  • the splenocytes of the immunized mice were taken for hybridoma fusion with SP2/0-AG14 cells (ATCC), and an appropriate amount of the fused cells were taken and plated into a 96-well plate. About 10 days after fusion, the supernatant of each well was taken, and the binding activity of mouse antibody secreted by hybridoma cells to human CEACAM5 was detected by ELISA.
  • the supernatant in the strong positive wells obtained by ELISA was used to detect the binding activity with LOVO cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) by flow cytometry, and the hybridization with higher binding activity of LOVO cells was obtained. tumor cells. These cells were subcloned to obtain monoclonal cells. Table 3 shows the detection data of some hybridoma cells.
  • Hybridoma cells ELISA OD value LOVO FACS combination 1-2C5 1.0690 12.1 1-9G3 1.0390 7.01 3-2F3 1.4480 11 3-6F5 1.3520 6.51 3-8C10 2.3030 137 3-8G8 1.9270 82.9 2A10A5 2.392 / 2A10C4 2.434 /
  • the hybridoma cell 3-8G8 with better binding activity was selected for sequencing, and an antibody was obtained by sequencing, named UM05-6, using the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991), pp. 647-669) determined the CDR sequence of the UM05-6 antibody, as shown in Table 4.
  • UM05-6L a human-mouse chimeric antibody was designed and expressed, which was named UM05-6L.
  • VH and VL sequences encoding the mouse antibodies were respectively linked to the constant region sequences encoding the human IgG1 heavy chain (the amino acid sequence of which is shown in SEQ ID NO:9 and the nucleotide sequence is shown in SEQ ID NO:10, ) and the constant region sequence of the ⁇ chain (its amino acid sequence is shown in SEQ ID NO: 11, and the nucleotide sequence is shown in SEQ ID NO: 12) to obtain a human-mouse chimeric antibody UM05-6L.
  • the nucleotide sequences encoding the antibody heavy chain and light chain were cloned into mammalian cell expression vector pcDNA3.4, respectively.
  • the heavy chain expression vector and the light chain expression vector were transfected into HEK293 cells with Lipofectamine 2000 transfection reagent (Invitrogen) at a molar ratio of 2:1, and cultured at 37°C and 5% carbon dioxide for 7 days.
  • the culture supernatant was collected, and the antibody in the supernatant was purified by Protein A affinity chromatography.
  • the purified antibody was dialyzed against PBS solution, freeze-dried and concentrated, and stored at -20°C.
  • the human CEACAM5 protein solution with a concentration of 1 ⁇ g/mL was coated with 100 ⁇ L/well on a 96-well high-affinity plate, and shaken overnight at 4°C. The next day, the cells were washed 3 times with 300 ⁇ L PBST (Tween20: 0.5 ⁇ ), then blocked with 100 ⁇ L/well of 5% BSA/PBS for 1 hour, and shaken at room temperature. Wash 3 times with 300 ⁇ L PBST. Prepare serial dilutions of antibody samples in PBS. Add 100 ⁇ L/well to a 96-well plate and shake at room temperature for 1 hour. Wash 3 times with 300 ⁇ L PBST.
  • the binding of the antibody to LOVO tumor cells naturally expressing human CEACAM5 protein or cells overexpressing different CEACAM proteins was detected by flow cytometry (FACS). To put it simply, cells were first collected, washed with PBS, counted, and diluted to 2*10 6 /ml cell suspension; 10 ⁇ l of antibody working solution was added to 100 ⁇ l of cell suspension, and incubated at 4°C for 30 min in the dark; washed twice with PBS After that, the corresponding fluorescently labeled secondary antibody Goat-anti-Human IgG (H+L) (Invitrogen) was added, incubated at 4°C for 30 min in the dark, washed twice with PBS, suspended with 400 ⁇ l FACS buffer, and the antibody and cells were detected by FACS. combination.
  • FACS flow cytometry
  • SW620-CEACAM1 is a cell overexpressing CEACAM1 protein
  • SW620-CEACAM3 is a cell overexpressing CEACAM3 protein
  • CHO-CEACAM6 is a cell overexpressing CEACAM6 protein
  • CHO-CEACAM7 is a cell overexpressing CEACAM7 protein
  • CHO-CEACAM8 is Cells overexpressing CEACAM8 protein.
  • the chimeric antibody UM05-6L has particularly outstanding selectivity: it binds CEACAM5 with high affinity, but has no obvious binding to CEACAM1, CEACAM3, CEACAM6, CEACAM7, and only weakly binds to CEACAM8 combine.
  • the common antibody (such as the control antibody UM05-8) can bind to CEACAM1, CEACAM3, CEACAM6, CEACAM7, and CEACAM8 at a higher level while binding to CEACAM5, and has no selectivity.
  • LOVO cells were seeded in a 96-well cell culture plate at a density of 20,000 cells/well, and incubated overnight at 37°C in 5% CO 2 . The next day, the antibody UM05-6L and the positive control antibody Erbitux were prepared in the medium at 20 ⁇ g/ml, and were diluted 3-fold to 8 concentrations. The LOVO cell supernatant medium was aspirated, and the antibody diluted at the indicated concentration (positive control antibody Erbitux, chimeric antibody UM05-6L or negative control irrelevant antibody) was added to LOVO cells at 30 ⁇ L/well.
  • effector cells Jurkat/ADC (Nanjing Nuoaixin Biotechnology Co., Ltd.) were plated into LOVO cell wells at 120,000 cells/30 ⁇ L/well, and incubated at 37° C., 5% CO 2 for 16-20 h. After incubation, Bright-Glo kit (Promega, cat. E2620) was used to detect the expression level of luciferase in effector cells. This luciferase level represents the degree of ADCC activation by effector cells.
  • Figure 4a shows the ADCC reporter activity of the positive control Erbitux antibody and negative control
  • Figure 4b shows the ADCC reporter activity of UM05-6L. Based on the above results, it can be seen that UM05-6L has a strong ADCC activity with an EC50 of 0.57 ⁇ g/mL.
  • the CAR-T recognition antibody used anti-CEACAM5 single-chain antibody as the CAR-T recognition antibody, and used one or more co-stimulatory elements such as 41BB, CD28, OX40, etc. to carry out different CEA-CAR assays.
  • the lentivirus can be used to infect T cells isolated from the peripheral blood of tumor patients to generate CAR-T cells that express the corresponding CAR receptors on the cell membrane surface. This CAR-T cell can effectively recognize and kill tumor cells expressing CEACAM5.
  • the cellular immunotherapy has very good safety and efficacy in both in vitro and in vivo experiments.
  • the single chain antibody UM05-6L scFv shown in SEQ ID NO: 13 was linked to the sequence CD8 ⁇ -CD137-CD3 ⁇ -p2A-tEGFR to construct a chimeric antigen receptor CAR, and then The nucleotide sequence encoding the CAR was cloned into a lentiviral vector (Jikai gene, GV401), and the vector was named UM05-6L-CAR.
  • the expression cassette is EF1a promoter-CAR-2A-tEGFR-WPRE.
  • the UM05-6L-CAR lentiviral vector and packaging plasmid were transiently transfected into 293T cells for 16 hours, the medium was replaced, and the culture was continued for 24-48 hours.
  • the medium supernatant (containing lentivirus) was collected and stored at -80°C.
  • the results are shown in Figure 5.
  • UM05-6L-CAR Figure 5b
  • UM05-6L-CAR-T cells with CEACAM5-expressing tumor cells (e.g. KATO3 and LS174T) and set up a medium control (i.e. use UM05-6L-CAR-T cells only) and a CEACAM5 negative cell control (i.e. use UM05 -6L-CAR-T cells and cells that do not express CEACAM5, such as RKO cells); after culturing the two cells at a 1:1 ratio for 16 hours, the release of IFN ⁇ and IL2 in the supernatant was detected.
  • UM05-6L-CAR-T can recognize cell lines expressing CEACAM5 and release cytokines IFN ⁇ and IL-2.
  • UM05-6L-CAR-T was mixed with CEACAM5-expressing tumor cells (such as KATO3) according to the specified E:T ratio (30:1, 10:1, 3:1, 1:1, 0.3:1) and cultured in 96 well in the culture plate.
  • the release data of lactate dehydrogenase (LDH) in the culture supernatant were detected by Promega LDH detection kit.
  • the calculation results of tumor cell killing are shown in Figure 7. It can be seen from the figure that UM05-6L-CAR-T can kill tumor cells efficiently and in a dose-dependent manner.
  • Example 8 In vivo efficacy experiment of LS174T tumor model
  • LS174T cells (ATCC CL-187) were subcutaneously inoculated into NSG mice (Biocytometer) at 1 ⁇ 10 7 / mouse, and when the tumor volume reached 200-400 mm 3 , PBS control and unmodified cells were administered via the tail vein, respectively.
  • T cell control (Mock T) or UM05-6L CAR-T was administered at a dose of 100 ⁇ l, 1 ⁇ 10 7 cells and 1 ⁇ 10 7 tEGFR+ cells, respectively, and the changes in tumor volume were recorded and the release of IFN ⁇ in peripheral blood was analyzed. .
  • the results are shown in Figure 9 and Figure 10, UM05-6L recognizes LS174T tumor cells in mice, and releases a large amount of IFN ⁇ , resulting in a strong tumor inhibitory effect.
  • humanized antibody UM05-6L According to the sequence of human-mouse chimeric antibody UM05-6L, the humanized antibody was designed. A total of 14 humanized antibody sequences were designed, which were designed and synthesized by Bio-Bio. The 14 humanized antibodies were named hUM05-6L-1 to hUM05-6L-14 respectively, and the specific sequences are shown in Table 6 and Table 1.
  • Example 3 the binding ability of 14 humanized antibodies to CEACAM5 protein was determined, and the experimental steps were the same as those in Example 3. Briefly, human CEACAM5 protein solution (purchased from Leading Bio) was coated on 96-well high-affinity plates. After washing 3 times with PBST, the cells were blocked with BSA/PBS and shaken at room temperature. A gradient dilution solution of the humanized antibody sample was prepared with PBS, added to a 96-well plate and shaken at room temperature for 1 hour. The secondary antibody goat anti-human (goat anti-human) IgG HRP solution was prepared, added to a 96-well plate, and shaken at room temperature for 30 min. Add TMB to develop color for 20min.
  • human CEACAM5 protein solution purchased from Leading Bio
  • Example 11 Binding of humanized antibodies to LS174T cells
  • the human-mouse chimeric antibody UM05-6L and 14 humanized antibodies were diluted with DPBS, respectively.
  • the initial concentration was 50 ⁇ g/ml, which was diluted three times to 5 concentrations, and the negative control was DPBS.
  • LS174T cells ATCC CL-187) were plated into 96-well plates at 5 ⁇ 10 6 cells/ml, 100 ⁇ l per well. Add 10 ⁇ l of the diluted antibody to the well plate, mix well and place at 4°C for 30min. Washed twice with DPBS, resuspended in 100 ⁇ l, and read data by FACS. The results are shown in Figures 13-14.
  • hUM05-6L-7 and hUM05-6L-14 have higher binding EC50 and lower EMax than UM05-6L in humanized antibodies.
  • hUM05-6L-1, hUM05-6L-2, hUM05-6L-5, hUM05-6L-9, hUM05-6L-10 and hUM05-6L-11 have lower combined EC50 and similar EMax.
  • hUM05-6L-12 has lower binding EC50 and lower EMax.
  • RKO cells (ATCC CRL-2577) were infected with a lentiviral vector (purchased from Shanghai Jikai Gene), and an overexpression cell line overexpressing CEACAM1, CEACAM3, CEACAM5, CEACAM6 and CEACAM8 was constructed.
  • the 14 humanized antibodies were each diluted to a concentration of 50 ⁇ g/ml with DPBS.
  • the constructed overexpressed RKO cells were plated into 96-well plates at 5 ⁇ 10 6 cells/ml, 100 ⁇ l per well. Add 10 ⁇ l of the diluted antibody to the well plate, mix well and place at 4°C for 30min. Washed twice with DPBS, resuspended in 100 ⁇ l, and read data by FACS. The results are shown in Figure 15.

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Abstract

La présente invention concerne le domaine technique de l'immunité biologique, et concerne en particulier un anticorps humanisé qui peut se lier spécifiquement à la molécule d'adhésion cellulaire 5 liée à l'antigène carcinoembryonnaire humain (CEACAM5) et un fragment de liaison à l'antigène dudit anticorps, et concerne en outre un procédé de préparation de l'anticorps et du fragment de liaison à l'antigène de celui-ci et l'utilisation de l'anticorps et de son fragment de liaison à l'antigène.
PCT/CN2020/133549 2020-12-03 2020-12-03 Anticorps anti-ceacam5 humanisé et son procédé de préparation et son utilisation WO2022116079A1 (fr)

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CN107743495A (zh) * 2015-03-23 2018-02-27 拜耳制药股份公司 抗ceacam6抗体及其用途
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CN116178556B (zh) * 2023-01-09 2024-01-09 南方医科大学第三附属医院（广东省骨科研究院） 靶向ceacam5的纳米抗体及其制备方法与应用

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