WO2023241493A1 - Anticorps se liant spécifiquement à trop2 ou à son fragment de liaison à l'antigène, son procédé de préparation et son utilisation - Google Patents

Anticorps se liant spécifiquement à trop2 ou à son fragment de liaison à l'antigène, son procédé de préparation et son utilisation Download PDF

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WO2023241493A1
WO2023241493A1 PCT/CN2023/099573 CN2023099573W WO2023241493A1 WO 2023241493 A1 WO2023241493 A1 WO 2023241493A1 CN 2023099573 W CN2023099573 W CN 2023099573W WO 2023241493 A1 WO2023241493 A1 WO 2023241493A1
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seq
trop2
sequence
cells
chain variable
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PCT/CN2023/099573
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Chinese (zh)
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王彦
王海鹰
熊青卉
胡红明
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上海恒润达生生物科技股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the present invention relates to the technical field of chimeric antigen receptors, and in particular to an antibody that specifically binds Trop2 or an antigen-binding fragment thereof and its preparation method and application.
  • Targeted therapies that have appeared in recent years design corresponding therapeutic drugs at the cellular and molecular level for clear carcinogenic sites. When the drugs enter the body, they will specifically select the carcinogenic sites to combine and act, causing tumor cells to specifically die. Normal tissue cells surrounding the tumor will not be harmed.
  • Trop2 is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene, whose full name is human trophoblast cell surface glycoprotein antigen 2 (Trophoblast Cell Surface Antigens 2).
  • Trop2 consists of a hydrophobic leader peptide, an extracellular domain, a transmembrane domain and a cytoplasmic tail. It is a single-pass transmembrane glycoprotein with a size of 35.7KD. It is a type of calcium ion channel signal converter.
  • the N-terminus of the Trop2 protein is the extracellular domain (Trop2 EC), which is connected to the intracellular short tail (Trop2 IC) through a unidirectional transmembrane helix (TM) and is thus fixed to the cell membrane.
  • Trop2 phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequence, indicating that PIP2 plays an important role in Trop2 signal transduction. In addition to the PIP2 binding motif, it contains conserved tyrosine and serine phosphorylation sites. Trop2 is not expressed or has low expression in normal tissues. It is overexpressed in various malignant tumors such as breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer, and can promote tumor occurrence and invasion. , metastasis and spread, etc., play a key role in the process of tumor growth, so Trop2 is considered a candidate target for tumor immunity.
  • PIP2 phosphatidylinositol 4,5-bisphosphate
  • Chimeric Antigen Receptor-T cell (CAR-T) T cells refer to T cells that have been genetically modified to recognize specific target antigens in an MHC-unrestricted manner and to continuously activate and expand.
  • the 2012 International Cell Therapy Association Annual Meeting pointed out that biological immune cell therapy has become the fourth method of treating tumors in addition to surgery, radiotherapy, and chemotherapy. It is a new immunotherapy method that targets specific antigens on the surface of tumor cells.
  • a large number of studies have shown that CAR-T cells can effectively recognize tumor antigens, induce specific anti-tumor immune responses, and significantly improve the survival status of patients.
  • Chimeric Antigen Receptor is the core component of CAR-T, which gives T cells the ability to recognize tumor antigens in an HLA-independent manner. This allows CAR-modified T cells to be more capable than the natural T cell surface receptor TCR. Identify broader goals.
  • the basic design of CAR includes a tumor-associated antigen (TAA) binding region (usually derived from the scFv segment of the monoclonal antibody antigen-binding region), an extracellular hinge region, a transmembrane region and an intracellular signal area.
  • TAA tumor-associated antigen
  • the selection of the target antigen is a key determinant of the specificity and effectiveness of the CAR as well as the safety of the genetically modified T cells themselves.
  • HAMA human anti-mouse antibody reaction
  • the invention provides a Trop2-binding molecule, which comprises an anti-Trop2 antibody or an antigen-binding fragment thereof.
  • the Trop2-binding molecule includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes SEQ ID NO:
  • the light chain variable region includes the light chain complementarity determining region LCDR shown in any one of SEQ ID NO: 13-24,
  • HCDR1 with the sequence shown in SEQ ID NO:1, HCDR2 with the sequence shown in SEQ ID NO:2, HCDR3 with the sequence shown in SEQ ID NO:3 and LCDR1 with the sequence shown in SEQ ID NO:13 , the LCDR2 with the sequence shown in SEQ ID NO:14, the LCDR3 with the sequence shown in SEQ ID NO:15;
  • HCDR1 whose sequence is shown in SEQ ID NO:4, HCDR2 whose sequence is shown in SEQ ID NO:5, HCDR3 whose sequence is shown in SEQ ID NO:6 and LCDR1 whose sequence is shown in SEQ ID NO:16 , the LCDR2 with the sequence shown in SEQ ID NO:17, the LCDR3 with the sequence shown in SEQ ID NO:18;
  • sequences of the heavy chain variable region and light chain variable region are selected from any of the following:
  • the antibody is at least one of a monoclonal antibody, a humanized antibody, a chimeric antibody, and a bispecific antibody;
  • the antigen-binding fragment is Fab, F(ab'), At least one of F(ab')2, Fd, single chain antibody scFv, disulfide bonded Fv (sdFv), or single domain antibody.
  • Another aspect of the invention provides a chimeric antigen receptor, comprising an optional signal peptide sequence, a Trop2 binding molecule as described in any embodiment herein, a hinge region, a transmembrane region and an intracellular region.
  • the chimeric antigen receptor sequentially contains a signal peptide, a Trop2-binding molecule described in any embodiment herein, a hinge region, a transmembrane region, and intracellular costimulation. domain and intracellular signaling domain.
  • the invention also provides nucleic acid molecules having a sequence selected from any of the following:
  • the invention also provides a nucleic acid construct comprising a nucleic acid molecule described herein.
  • the nucleic acid construct is a cloning vector, an expression vector, or an integration vector.
  • the invention also provides a host cell selected from:
  • the host cells are immune effector cells, preferably T cells.
  • the invention also provides a method for producing a Trop2-binding molecule according to any embodiment herein, comprising: in a process suitable for producing a Trop2-binding molecule (e.g., an anti-Trop2 antibody or an antigen-binding fragment thereof, a monovalent or multivalent anti-Trop2 antibody, or a multispecific anti-Trop2 antibody).
  • a Trop2-binding molecule e.g., an anti-Trop2 antibody or an antigen-binding fragment thereof, a monovalent or multivalent anti-Trop2 antibody, or a multispecific anti-Trop2 antibody.
  • the host cells described herein are cultured under conditions with Trop2 antibodies), and the Trop2-binding molecules are optionally purified from the culture.
  • the present invention also provides a pharmaceutical composition, comprising the Trop2 binding molecule, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment herein, and pharmaceutically acceptable excipients.
  • the pharmaceutical composition is used to treat a disease or condition associated with Trop2 expression.
  • the present invention also provides the use of Trop2 binding molecules, chimeric antigen receptors, nucleic acid molecules, nucleic acid constructs or host cells according to any embodiment herein in the preparation of medicaments for preventing or treating diseases or conditions related to Trop2 expression.
  • the disease or condition is selected from one or more of the following: breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, lung cancer, esophageal cancer, Kidney, bladder, uterine and ovarian cancer.
  • the present invention also provides a method for treating or preventing diseases or conditions related to Trop2 expression, which method includes administering to a patient in need a therapeutically effective amount of the Trop2-binding molecule or host cell according to any embodiment of the present invention, or the present invention.
  • the kit further includes reagents for detecting binding of Trop2 to the Trop2 binding molecule.
  • the bound reagent is detected, for example, by enzyme-linked immunoassay.
  • the reagent that detects binding is a detectable label, such as biotin, that can be linked to a Trop2 binding molecule.
  • the detectable label is linked to the Trop2 binding molecule or is present separately in the kit.
  • the present invention also provides a non-diagnostic method for detecting the presence of Trop2 in a sample.
  • the method includes: incubating the sample with the Trop2-binding molecule described in any embodiment of this document, and detecting the binding of Trop2 to the Trop2-binding molecule, This determines the presence of Trop2 in the sample.
  • the detection is an enzyme-linked immunoreaction assay.
  • the present invention also provides the use of the Trop2-binding molecule described in any embodiment herein in the preparation of a kit for detecting Trop2 in a sample, evaluating the effect of drug treatment, or diagnosing cancer.
  • the present invention provides a new antibody that specifically recognizes Trop2 and a CAR-modified cell containing the antibody.
  • the antibody and cells have good therapeutic effect and safety targeting Trop2, and provide treatment for diseases related to Trop2 expression. or ways to improve.
  • Figure 1 shows the SDS electrophoresis pattern of recombinant human Trop2-avi-his antigen protein.
  • Figure 3 shows the CAR positive rate of Trop2 CAR-T cells of different clones.
  • Figure 4 shows the CD107a expression of Trop2 CAR-T cells of different clones.
  • Figure 5 shows the INF ⁇ secretion of Trop2 CAR-T cells of different clones when the effect-to-target ratio is 10:1.
  • Figure 6 shows the INF ⁇ secretion of Trop2 CAR-T cells of different clones when the effect-to-target ratio is 2:1.
  • Figure 8 shows the IL-2 secretion of Trop2 CAR-T cells of different clones when the effect-to-target ratio is 2:1.
  • Figure 9 shows the results of the killing experiment of different clone Trop2 CAR-T cells on the target cell BxPC3-LUC-GFP in Example 5.
  • Figure 13 is a statistical graph of the 29H and 59H killing rates of different clone Trop2 CAR-T cells on target cells in Example 6.
  • antibody includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2, Fd and Fv.
  • antibody and “immunoglobulin” are used interchangeably.
  • Antibodies contain the basic 4-chain antibody unit, which is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (for each alpha and gamma chain, CH1, CH2, and CH3) and four (for the mu and epsilon isoforms, CH1, CH2, and CH3). CH2, CH3 and CH4) constant domains (CH) and the hinge region (Hinge) located between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end.
  • VH variable domain
  • CL constant domain
  • variable region refers to the amino-terminal domain of the heavy or light chain of the antibody.
  • variable domains of the heavy and light chains may be referred to as "VH” and “VL” respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain the antigen-binding site.
  • variable domains of the native heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a ⁇ -sheet conformation by forming loop connections and in some cases forming a ⁇ -sheet structure Part of the three HVR connections.
  • the HVRs in each chain are held in close proximity by the FR region and together with the HVRs of the other chain contribute to the formation of the antibody's antigen-binding site.
  • the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4
  • the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4.
  • Fc region (crystallizable fragment region) or “Fc domain” or “Fc” refers to the C-terminal region of the antibody heavy chain, which mediates immunoglobulin Binding to host tissues or factors includes binding to Fc receptors located on various cells of the immune system (eg, effector cells), or to the first component (Clq) of the classical complement system.
  • the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of the two heavy chains of the antibody; the Fc regions of IgM and IgE are present in each polypeptide chain. Contains three heavy chain constant domains (CH domains 2-4).
  • Human antibody refers to an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human and/or is produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues. body. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.
  • the invention also includes such antibody derivatives and analogs.
  • “Derivatives” and “analogues” refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the invention.
  • Derivatives or analogs of the present invention may be (i) a polypeptide having substituent groups in one or more amino acid residues, or (ii) a mature polypeptide combined with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol).
  • polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence such as a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a protein sequence, or with a 6His tag fusion protein formed.
  • those skilled in the art can change one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to obtain variants of the antibody or functional fragment sequence thereof.
  • These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein. For example, amino acids with similar properties are substituted in the FR and/or Fc region.
  • amino acid residues that are subject to conservative substitutions are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. As another example, adding one or more amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein. They are all considered to be included in the scope of protection of the present invention.
  • Variant forms of the antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, hybridization to the DNA encoding the antibody of the invention under high or low stringency conditions Proteins encoded by DNA, and polypeptides or proteins obtained using antiserum against the antibodies of the present invention.
  • the sequence of a variant described herein may be at least 95%, 96%, 97%, 98% or 99% identical to the sequence from which it is derived.
  • the sequence identity described in the present invention can be measured using sequence analysis software.
  • the computer program BLAST uses default parameters, especially BLASTP or TBLASTN.
  • the present invention also includes those molecules having antibody heavy chain variable regions or light chain variable regions with CDRs as long as their CDRs are more than 90% (preferably more than 95%, optimally 98%) identical to the CDRs identified herein above) homology.
  • the present invention also provides a chimeric antigen receptor (CAR) targeting Trop2.
  • the CAR contains an optional signal peptide sequence, an antigen recognition region, which is the anti-Trop2 binding molecule described herein, a hinge region, a transmembrane region and an intracellular region.
  • the intracellular region includes one or more intracellular costimulatory domains and/or one or more intracellular signaling domains.
  • the "hinge region”, “transmembrane region” and “intracellular region” in this article can all be selected from the sequences of the hinge region, transmembrane region and intracellular region in known CAR-T technology.
  • a signal peptide is a peptide sequence that targets a polypeptide to a desired location in a cell.
  • the signal peptide targets the polypeptide to the secretory pathway of the cell and will allow the polypeptide to integrate and anchor into the lipid bilayer; the signal peptide may also be a membrane-localized signal peptide.
  • Exemplary signal peptides such as CD8 signal peptide, CD28 signal peptide, CD4 signal peptide or light chain signal peptide, the sequences of which are within the knowledge of those skilled in the art.
  • the CD8 signal peptide suitable for the present invention can be various human CD8 signal peptide sequences commonly used for CAR in the art.
  • the amino acid sequence of the CD8 signal peptide includes the sequence shown in SEQ ID NO: 33.
  • the hinge region of the chimeric antigen receptor is located between the extracellular antigen-binding region and the transmembrane region.
  • the hinge region is a segment of amino acids that usually exists between two domains of a protein and can allow for the flexibility of the protein and the separation of the two domains. move relative to each other.
  • the hinge region may be that of a naturally occurring protein or a portion thereof.
  • Hinge regions of antibodies such as IgG, IgA, IgM, IgE or IgD antibodies may also be used in the chimeric antigen receptors described herein.
  • Non-naturally occurring peptides may also be used as hinge regions of the chimeric antigen receptors described herein.
  • the hinge region of the CAR is selected from the group consisting of a CD8 ⁇ hinge region, an IgD hinge region, an IgG1FcCH2CH3 hinge region, or an IgG4FcCH2CH3 hinge region, the sequences of which are within the knowledge of those skilled in the art.
  • the CD8 ⁇ hinge region suitable for the present invention can be various human CD8 ⁇ hinge region sequences commonly used for CAR in the art.
  • the human CD8 alpha hinge region comprises the sequence set forth in SEQ ID NO: 34.
  • the transmembrane region of a chimeric antigen receptor can form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of spanning the cellular phospholipid bilayer.
  • the transmembrane region may be of natural or synthetic origin.
  • the transmembrane region can be selected from the transmembrane regions of the following proteins: CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, alpha, beta or zeta chain of T cell receptor.
  • the human CD8 ⁇ transmembrane region suitable for the present invention can be various human CD8 ⁇ transmembrane region sequences commonly used for CAR in the art.
  • the amino acid sequence of the human CD8 ⁇ transmembrane region comprises the sequence set forth in SEQ ID NO:35.
  • the intracellular signaling domain (or intracellular signaling domain) is responsible for the activation of at least one normal effector function of the immune effector cell expressing the chimeric antigen receptor.
  • the effector function of a T cell may be cytolytic activity or auxiliary activity, including secretion of cytokines.
  • cytolytic activity or auxiliary activity, including secretion of cytokines.
  • auxiliary activity including secretion of cytokines.
  • an intracellular signaling domain includes any truncated form of an intracellular signaling domain that is sufficient to transduce an effector function signal.
  • the intracellular signaling domain of the CAR can be selected as needed, including but not limited to those derived from at least one of CD3 ⁇ , FcR ⁇ (FCER1G), FcR ⁇ (Fc ⁇ Rib), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b and CD66d Intracellular signaling domain.
  • the intracellular signal region is derived from the human CD3 ⁇ intracellular signal region.
  • the human CD3 ⁇ intracellular signal region has the amino acid sequence shown in SEQ ID NO: 37.
  • costimulatory domain may be the cytoplasmic portion of a costimulatory molecule.
  • costimulatory molecule refers to an associated binding partner on an immune cell, such as a T cell, that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the immune cell, such as, but not limited to, proliferation and survival. .
  • Suitable intracellular costimulatory domains can be selected as needed, including intracellular domains with costimulatory signaling molecules, such as those derived from 4-1BB, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40 , CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, Fc ⁇ RI ⁇ , MyD88, and at least one of the intracellular domains of 41BBL.
  • the amino acid sequence of the 4-1BB costimulatory domain includes the sequence set forth in SEQ ID NO: 36.
  • Linker sequences can contain 1, 2, 3, 4 or 5 repeating motifs.
  • the length of the linker can be 3 to 25 amino acid residues, such as 3 to 15, 5 to 15, or 10 to 20 amino acid residues.
  • the linker sequence is a polyglycine linker sequence.
  • the number of glycines in the linker sequence is not particularly limited, but is usually 2 to 20, such as 2 to 15, 2 to 10, or 2 to 8.
  • the linker can also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine Acid (F), arginine (R), glutamine (Q), etc.
  • the linker sequence is a (GGGGS)n linkage, where n is an integer from 1 to 5.
  • the CAR contains CD8 signal peptide, anti-Trop2 antibody or antigen-binding fragment thereof as described herein, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB costimulatory domain, CD3 ⁇ in sequence from N-terminus to C-terminus. Intracellular signaling domain.
  • an exemplary CAR having the above structure is as shown in any of SEQ ID NOs: 38-41.
  • the amino terminus or carboxyl terminus of the CAR of the present invention may also contain one or more polypeptide fragments as protein tags.
  • Any suitable tag may be used for this article.
  • the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE and Ty1. These tags can be used to purify proteins.
  • the antigen recognition region in the CAR of the present invention can be a variant of the aforementioned anti-Trop2 antibody or its functional fragment sequence.
  • other parts of the CAR can also undergo sequence changes, and the resulting mutant has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with the CAR and retains the sequence identity.
  • Biological activity of CAR (such as activated T cells). Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp.
  • Mutants also include amino acid sequences that have one or several mutations (insertions, deletions, or substitutions) in the amino acid sequence of the CAR described in any embodiment while still retaining the biological activity of the CAR.
  • the number of mutations usually refers to within 1-10, such as 1-8, 1-5 or 1-3.
  • Substitutions are preferably conservative substitutions.
  • conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein or polypeptide.
  • amino acids with similar or similar properties include, for example, families of amino acid residues with similar side chains.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • amino acids with acidic side chains chain amino acids (e.g., aspartic acid, glutamic acid)
  • amino acids with uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine amino acids
  • amino acids with non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • Amino acids with ⁇ -branched side chains eg threonine, valine, isoleucine
  • amino acids with aromatic side chains eg tyrosine, phenylalanine, tryptophan, histidine
  • the invention also provides polynucleotides encoding the above-mentioned antibodies or CARs.
  • the polynucleotides of the invention may be in DNA form or RNA form. Forms of DNA include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.
  • the present invention also encompasses degenerate variants of the polynucleotide sequence encoding the fusion protein, ie, nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides of the invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding water during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90%, more It is best when hybridization occurs only when the ratio is above 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombinant or artificial synthesis.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them.
  • the coding sequence of the heavy chain or light chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • the sequence of the CAR can also be obtained as above.
  • the sequences of each part of the CAR (signal peptide, antigen recognition region, hinge region, transmembrane region or intracellular region) can be obtained as above and then connected to obtain the full length of the CAR.
  • Biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.
  • the DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the invention through chemical synthesis.
  • Each part of the CAR can be cloned sequentially into the vector or can be integrated into the full-length CAR and then cloned.
  • the present invention also relates to nucleic acid constructs containing the polynucleotide sequences described herein, and one or more regulatory sequences operably linked to these sequences.
  • the polynucleotide sequences of the invention can be manipulated in various ways to ensure the expression of the antibody or CAR. Before inserting the nucleic acid construct into the vector, the nucleic acid construct can be manipulated according to the differences or requirements of the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.
  • the control sequence may be a suitable promoter sequence.
  • the promoter sequence is usually operably linked to the coding sequence of the protein to be expressed.
  • the promoter can be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular genes that are homologous or heterologous to the host cell. or genetic acquisition of intracellular polypeptides.
  • An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto.
  • EF-1 ⁇ elongation growth factor-1 ⁇
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV avian leukemia virus promoter
  • Epstein-Barr virus immediate early promoter Epstein-Barr virus immediate early promoter
  • Ruth's sarcoma virus promoter and human gene promoters such as but not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter.
  • inducible promoters may also be considered.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.
  • the regulatory sequence may also be a suitable leader sequence, an untranslated region of the mRNA important for translation by the host cell. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.
  • the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integration vector.
  • Expression of a polynucleotide sequence of the invention is generally accomplished by operably linking the polynucleotide sequence of the invention to an expression vector.
  • Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.
  • Integration vectors contain components that integrate target sequences into the cellular genome. These vectors can be used to transform appropriate host cells to enable expression of the protein.
  • Vectors typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
  • the sequences (collectively referred to in certain embodiments as “flanking sequences”) generally include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a donor-containing sequence and acceptor splice site, a sequence encoding a leader for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, and a polylinker for insertion of nucleic acid encoding the antibody to be expressed. area and optional marker elements.
  • the type of vector is not limited, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids, and may vary depending on the host cell to be introduced.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • the vector introduced into the cell may also contain either a selectable marker gene or a reporter gene.
  • a selectable marker gene or a reporter gene.
  • One or both to facilitate the identification and selection of expressing cells from a population of cells seeking to be transfected or infected by a viral vector.
  • Host cells suitable for introducing the nucleic acid constructs described herein can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, especially immune cells, Immune effector cells are preferred.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells, especially immune cells
  • Immune effector cells are preferred.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Immuno effector cells are immune cells that perform immune effector functions.
  • the immune effector cells express at least Fc ⁇ RIII and perform ADCC effector functions.
  • Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer
  • monocytes cytotoxic T cells
  • neutrophils neutrophils
  • eosinophils eosinophils.
  • the immune effector cells are selected from: at least one of immune cells cultured and differentiated from pluripotent stem cells or embryonic stem cells, T lymphocytes, NK cells, peripheral blood mononuclear cells (PBMC) and hematopoietic stem cells. More preferably, the immune effector cells are T lymphocytes (the same as T cells).
  • T cells can be CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-, or combinations thereof.
  • T cells produce IL-2, IFN, and/or TNF when expressing chimeric antigen receptors and binding to target cells.
  • CD8+ T cells lyse antigen-specific target cells when expressing chimeric antigen receptors and binding to target cells.
  • T cells suitable for use in the present invention can be various types of T cells from various sources.
  • T cells can be derived from PBMCs of patients with malignant solid tumors, such as pancreatic cancer.
  • T cells after T cells are obtained, they can be first stimulated and activated with an appropriate amount (for example, 30-80ng/ml, such as 50ng/ml) of CD3 antibodies, and then containing an appropriate amount (for example, 30-80IU/ml, such as 50ng/ml) of CD3 antibody for activation. 50IU/ml) IL2 medium for culture and use.
  • nucleic acids or vectors into mammalian cells
  • the vectors may be introduced into the cells by physical, chemical, or biological means.
  • the host is a prokaryotic organism such as E. coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method.
  • the steps used are well known in the art.
  • the host is a eukaryotic organism
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • transduced or transfected immune effector cells are propagated ex vivo following introduction of nucleic acid or vector.
  • the obtained transformants can be cultured using conventional methods to express the antibody or CAR encoded by the gene of the present invention.
  • the medium used in culture can be selected from various conventional media. Cultivate under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.
  • All aspects of the antibodies, CARs, coding sequences, nucleic acid constructs, and cells described herein can be used to prepare medicaments for the prevention or treatment of various conditions and diseases described herein that are associated with Trop2 expression.
  • Diseases or conditions caused by abnormal expression of Trop2 Diseases caused directly or indirectly usually refer to diseases caused by Trop2 overexpression, such as cancer, including but not limited to: breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, lung cancer, and Ovarian cancer.
  • the invention also encompasses a type of cell therapy that involves expressing a CAR as described herein in immune cells (eg, T cells) and administering to a recipient in need thereof a therapeutically effective amount of the cells capable of killing tumor cells in the recipient.
  • immune cells eg, T cells
  • CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to sustained tumor control.
  • the anti-tumor immune response caused by CAR-T cells can be an active or passive immune response.
  • a CAR-mediated immune response can be part of an adoptive immunotherapy step, in which CAR-T cells induce an immune response specific for the antigen-binding portion of the CAR.
  • the antibodies, nucleic acids or CAR-modified cells of the invention can be administered alone or as pharmaceutical compositions in combination with diluents and/or with other components such as relevant cytokines or cell populations.
  • the pharmaceutical composition may be prepared in the form of a lyophilized preparation or aqueous solution by mixing the active agent with the desired purity and optionally a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are nontoxic to the recipient at the dosage and concentration used and may include buffers (e.g., neutral buffered saline, sulfate buffered saline), antioxidants, preservatives, isotonic agents, stabilizing agents at least one of an agent, a chelating agent (such as EDTA or glutathione), an adjuvant (such as aluminum hydroxide), and a surfactant.
  • buffers e.g., neutral buffered saline, sulfate buffered saline
  • antioxidants e.g., sulfate buffered saline
  • preservatives e.g., isotonic agents
  • stabilizing agents at least one of an agent e.g., a chelating agent (such as EDTA or glutathione), an adjuvant (such as aluminum hydroxide), and a surfactant.
  • an adjuvant such as aluminum hydroxide
  • surfactant such as aluminum
  • compositions may contain at least one additive from the group consisting of cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, growth inhibitors, and active agents required for the particular indication to be treated.
  • the specific amount of additives can be adjusted according to actual needs.
  • the pharmaceutical composition of the present invention may be administered in an "immunologically effective amount", “anti-tumor effective amount”, “tumor-inhibitory effective amount” or "therapeutic amount”.
  • Treatment refers to a subject taking a treatment regimen described herein to achieve at least one positive therapeutic effect (for example, reduction in the number of cancer cells, reduction in tumor volume, reduction in the rate of cancer cell infiltration into surrounding organs, or reduction in tumor metastasis or tumor growth) rate decreases).
  • an "immunologically effective amount”, “anti-tumor effective amount”, “tumor-suppressive effective amount” or “therapeutic amount” is indicated, the precise amount of the composition of the invention to be administered can be determined by the physician, who takes into account the patient (subject) ) age, weight, tumor size, degree of infection or metastasis, and individual differences in disease.
  • compositions including T cells described herein may be administered at a dose of 10 4 to 10 9 cells/kg body weight, preferably 10 5 to 10 6 cells/kg body weight. T cell compositions can also be administered multiple times at these dosages. Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection, or intraperitoneally.
  • the T cell composition of the invention is administered to the patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by intravenous injection.
  • the composition of T cells can be injected directly into the tumor, lymph node or site of infection.
  • the CAR-T cells of the invention or compositions thereof may be combined with other therapies known in the art.
  • Such therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants.
  • treatment may be combined with radiotherapy or chemotherapy agents known in the art to treat Trop2-mediated diseases.
  • the binding molecules of the invention can be used in assays due to their high affinity for Trop2, for example binding assays to detect and/or quantify Trop2 expressed in tissues or cells. Binding molecules such as single chain antibodies can be used in studies to further investigate the role of Trop2 in disease.
  • the method for detecting Trop2 is roughly as follows: obtain cell and/or tissue samples; detect the level of Trop2 in the samples.
  • Trop2-binding molecules of the invention may be used for diagnostic purposes to detect, diagnose or monitor Trop2-related diseases and/or conditions.
  • the present invention provides for the detection of the presence of Trop2 in a sample using classical immunohistological methods known to those skilled in the art. Detection of Trop2 can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of Trop2 include ELISA, FACS, RIA, etc.
  • binding molecules such as single-chain antibodies are often labeled with detectable labeling groups.
  • Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (e.g., FITC, Rodan fluorophores, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups or a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site for secondary antibody, metal binding domain, epitope tag), MRI (magnetic resonance imaging) or CT (computed X-ray tomography) contrast agent.
  • a secondary reporter e
  • Another aspect of the invention provides a method of detecting the presence of a test molecule that competes with an antibody of the invention for binding to Trop2.
  • An example of such an assay would involve detecting the amount of free antibody in a solution containing an amount of Trop2 in the presence or absence of a test molecule. An increase in the amount of free antibody (i.e., antibody that does not bind Trop2) will indicate that the test molecule is able to compete with the antibody for binding to Trop2.
  • the antibody is labeled with a labeling group.
  • the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of antibody.
  • the invention also provides a detection kit for detecting Trop2 levels.
  • the kit includes an antibody that recognizes Trop2 protein, a lysis medium for dissolving the sample, and general reagents and buffers required for detection, such as various buffers, detection Labeling, detection substrate, etc.
  • the detection kit may be an in vitro diagnostic device.
  • Example 1 Construction and eukaryotic expression of recombinant human Trop2 protein expression vector
  • a two-step method is used to construct a human natural antibody phage display library, that is, the human natural antibody light chain variable region gene and heavy chain variable region gene are connected to the phage display vector in two steps.
  • Human immunoglobulin kappa chain variable region forward primer (3z3-1-F) and reverse primer (Huvksc-R) were used to PCR amplify the human immunoglobulin kappa chain variable region gene using human PBMC cDNA as a template.
  • Immunoglobulin lambda chain variable region forward primer (3z3-1-F) and reverse primer (HuvLR-1) use human PBMC cDNA as template to PCR amplify human immunoglobulin lambda chain variable region gene, PCR reaction
  • the conditions are as follows: pre-denaturation at 98°C for 45 seconds and then entering a temperature cycle, denaturation at 98°C for 15 seconds, annealing at 60°C for 20 seconds, extension at 72°C for 23 seconds, 30 cycles, and final extension at 72°C for 5 minutes.
  • a gel recovery kit (Promega) was used to recover about 330 bp of kappa chain variable region gene fragments and lambda chain variable region gene fragments.
  • the kappa chain gene, lambda chain gene and phagemid pcomb3sc were double digested with NheI-HF and SalI-HF DNA endonuclease (NEB).
  • the digested kappa chain gene and lambda chain gene were directly processed using a gel recovery kit. Recycle. After the digested pcomb3sc vector was subjected to 1% agarose gel electrophoresis, a gel recovery kit (Promega) was used to recover the 4100 bp vector fragment.
  • pComb3sc 1000ng, ⁇ and lambda dosage respectively 200ng prepare multiple reaction tubes, 50 ⁇ l/tube, connect overnight at 16°C, take A small amount of the ligation product was analyzed by agarose gel electrophoresis to detect the ligation efficiency.
  • the ligation product is desalted using MECK MILLIPOREF microporous filter membrane. The desalted ligation product was electroporated into self-made TG1 electroporated competent cells to obtain a kappa chain library and a lambda chain library.
  • the electroporated transformed kappa chain library and lambda chain library were amplified overnight, and the next day they were used with a plasmid extraction kit ( NucleoBond Xtra Maxi EF) extracts kappa chain library plasmids and lambda chain library plasmids.
  • a plasmid extraction kit NucleoBond Xtra Maxi EF
  • VH-MIX 1/7) and reverse primer VH-MIX R
  • VH-MIX3 and reverse primer VH-MIX R
  • VH-MIX4 and reverse primer VH-MIX R
  • human immunoglobulin VH variable region gene PCR reaction conditions: 98°C pre-denaturation for 45 seconds, then enter the temperature cycle, 98°C denaturation for 15 seconds, 60°C annealing for 20 seconds, 72°C extension for 23 seconds, 30 cycles, and 72°C final Stretch for 5 minutes.
  • a gel recovery kit Promega
  • VH fragments Mix the recovered VH fragments in equal proportions, and perform single enzyme digestion with SfiI DNA endonuclease with the above-mentioned kappa chain library plasmid and lambda chain library plasmid, and digest at 50°C for 16 hours.
  • the digested VH gene is directly recovered with gel recovery reagent.
  • the cassette was subjected to column recovery, and the digested kappa chain library and lambda chain library were subjected to 1% agarose gel electrophoresis, and a gel recovery kit (Promega) was used to recover vector fragments of approximately 4000 bp.
  • T4 DNA ligase kit (Invitrogen) to connect the VH gene to the kappa chain and lambda chain vector libraries, i.e. pComb3sc-lambda/kappa (SfiI) 1000ng, VH amplify mix (sfiI) 200ng, prepare multiple reaction tubes, 50 ⁇ l/ tube, ligated at 16°C overnight, and a small amount of the ligation product was taken for agarose gel electrophoresis to detect the ligation efficiency.
  • the ligation product is desalted using MECK MILLIPOREF microporous filter membrane.
  • the above-mentioned ligation product was added to the self-made TG1 electroconversion competent cell, and then electroporation was performed using an electroporation instrument. Take out 50 ⁇ L of bacterial solution and perform gradient dilution 10 2 -10 5 times with PBS. Streamline 10 ⁇ L of each gradient dilution on an Amp/2YT plate and incubate at 37°C overnight to count and calculate the size of the phage antibody library. Add 2YT to the remaining electro-transformed bacteria to 500ml, and add ampicillin containing 100 ⁇ g/mL. Incubate overnight at 30°C and 220 rpm. Finally, a scFv immune library exceeding 3E10 was obtained. The antibody library transformed by electroporation was amplified overnight, and the library cells were collected by centrifugation. The final concentration of 20% glycerol was added and stored at -80 degrees.
  • the avi-tag of the recombinant human Trop2 protein was biotin-modified to obtain biotinylated Trop2 protein.
  • TES solution (20% sucrose, 0.1mM EDTA, 50mM Tris-HCl, etc.) to each well. pH 8.0
  • TES solution 20% sucrose, 0.1mM EDTA, 50mM Tris-HCl, etc.
  • pH 8.0 pH 8.0
  • the bacteria then incubate on ice for 30 minutes, then add 200 ⁇ L of ultrapure water and shake for 30 minutes. After shaking, centrifuge at 4000 rpm for 10 minutes. At this time, the supernatant solution in the deep well plate is the antibody-containing solution.
  • Periplasmic cavity extract Wash the ELISA plate three times with a plate washer, then add 200 ⁇ L 1% BSA/PBS and block at 37°C for 1 hour.
  • amino acid sequence of the heavy chain variable region of L2C8 is shown in SEQ ID NO:25, and the amino acid sequence of the light chain variable region sequence is shown in SEQ ID NO:29.
  • amino acid sequence of the heavy chain variable region of L2C9 is shown in SEQ ID NO:26, and the amino acid sequence of the light chain variable region sequence is shown in SEQ ID NO:30.
  • amino acid sequence of the heavy chain variable region of L3G5 is shown in SEQ ID NO:27, and the amino acid sequence of the light chain variable region sequence is shown in SEQ ID NO:31.
  • amino acid sequence of the heavy chain variable region of K2F5 is shown in SEQ ID NO:28, and the amino acid sequence of the light chain variable region sequence is shown in SEQ ID NO:32.
  • Example 3 Preparation of retrovirus stock solution containing anti-human Trop2 chimeric antigen receptor element
  • a chimeric antigen receptor sequence containing a single-chain antibody scFv against human Trop2 antigen, hinge region, transmembrane region and intracellular signal segment. Its structure is shown in Figure 2. According to the differences in loaded scFv and intracellular signals, the chimeric antigen receptors were named L2C8-BBz, L2C9-BBz, L3G5-BBz and K2F5-BBz, respectively, and their amino acid sequences are SEQ ID NO:38 and SEQ ID NO: 39. SEQ ID NO:40 and SEQ ID NO:41 are shown, and the nucleotide sequences are SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45 respectively.
  • retroviral plasmids expressing chimeric antigen receptors of L2C8, L2C9, L3G5 and K2F5 clones were constructed. Select the correctly sequenced clone, inoculate the bacterial solution into 200ml 2YT medium, shake the culture overnight, and complete the plasmid purification according to the instructions of the NucleoBond Xtra Maxi EF kit.
  • PEI cationic polymer
  • the process is as follows: dilute 36 ⁇ l of PEI and retrovirus packaging plasmid (viral main plasmid 2 ⁇ g, Gag-pol 3.8 ⁇ g, vsvg 1.5 ⁇ g) with 600 ⁇ l of serum-free DMEM respectively; then PEI/DMEM Add plasmid/DMEM mixture, Vortex to mix, and let stand at room temperature for 15 minutes; add the plasmid-PEI complex to the pre-plated 293T cells.
  • PBMC Receive a PBMC, verify that the patient's individual identification number is correct, and then perform resuscitation.
  • PBMC that had been recovered overnight were pipetted gently, filtered with a 70 ⁇ m cell mesh, transferred to a 50 ml centrifuge tube, centrifuged at room temperature, 1500 rpm, for 5 min, and the supernatant was discarded.
  • Remove the magnetic stand from the flow tube take an equal volume of DPBS or X-VIVO15 and resuspend the cell suspension, add the magnetic beads and cells in the cell suspension, mix in a 15ml centrifuge tube, and incubate on a rotating mixer. Incubate at room temperature for 30 minutes. After the incubation is completed, gently transfer the cells to a sterile flow tube, rinse the 15ml centrifuge tube with 1ml DPBS, and merge the rinse solution into the same flow tube. Move the sterile flow tube to the magnetic stand, let it stand for 1 minute, and then aspirate the unadsorbed liquid.
  • CAR-T culture medium to adjust the cell density to 1 ⁇ 10 6 ml, add IL-2 to a final concentration of 200IU/ml, and culture in a 37°C, 5% CO 2 incubator for two days.
  • Adjust the activated T cells to 5 ⁇ 10 5 /mL add 1 ml of T cells and 1 ml of virus stock solution respectively to a 24-well plate, add 2 ⁇ l of polybrene to each well, and centrifuge at 32°C, 2500 rpm for 1.5 h. Discard the supernatant and add 1 ml of T cell culture medium (containing IL-2 300IU/ml) to each well. Place the culture plate in a 37°C, 5% CO2 incubator. 24 hours after infection, transfer to a 6-well plate, observe the cell density every day, and add T cell culture medium containing IL-2 300IU/ml in a timely manner to maintain the density of T cells at about 1 ⁇ 10 6 /ml and allow the cells to expand. increase.
  • the positive rate of CAR was detected in retrovirus-infected T lymphocytes 72 hours after virus infection.
  • the results of flow cytometry analysis of CAR-T positivity rate are shown in Figure 3.
  • Example 5 Functional analysis of CAR-T cells based on anti-human Trop2
  • CAR-T cells containing different antibody clones were compared with target cells (Trop2-positive human pancreatic cancer cell line BxPC3) and control target cells (human glioma cells U251) at an effect-to-target ratio of 1:1. cells and target cells (both 3 ⁇ 10 5 ) were mixed and placed in a 37°C, 5% CO 2 incubator for a total of 4 hours, and the proportion of CD107a in the number of CAR-T cells was detected by flow cytometry. Evaluate the degranulation response of CAR-T cells after stimulation by target cells. The results of flow cytometry analysis of CD107a expression are shown in Figure 4.
  • CAR-T cells containing different antibody clones were compared with target cells (Trop2-positive cell line BxPC3) and control target cells (Trop2 Negatively expressed cell line U251) were incubated for 24 hours at an efficacy-to-target ratio of 10:1 and 2:1 (target cells were 3 ⁇ 10 4 ), and then the supernatant was collected, and the ELISA (enzyme-linked immunoassay) method was used.
  • the secretion of IFN- ⁇ and IL-2 was detected.
  • IFN- ⁇ and IL-2 were detected using the Human IFN-gamma ELISA Kit, Human IL-2 ELISA kit, and the experimental steps were carried out according to the product instructions.
  • the detection results of IFN- ⁇ secretion are shown in Figure 5 and Figure 6.
  • the detection results of IL-2 secretion are shown in Figure 7 and Figure 8.
  • the CAR-T killing toxicity experiment evaluates the in vitro function of CAR-T cells by detecting the killing effect of CAR-T cells on target cells in vitro.
  • the T cells were treated with Trop2-positive target cells stably expressing firefly luciferase Bxpc3-LUC- at different effective-target ratios (based on 3 ⁇ 10 4 target cells, the effective-target ratios were 10:1 and 2:1 respectively).
  • GFP, and control target cells U251-LUC-GFP were co-cultured, and a positive control with only target cells was set.
  • Killing efficiency (fluorescence value of positive control well - fluorescence value of experimental well)/fluorescence of positive control well value) ⁇ 100%.
  • L1A8 L2G1, K2G1, L1F4, etc.
  • CAR plasmids were respectively constructed, packaged with viruses, and T cells were infected to obtain eight types of CAR-T cells based on different scFvs. Only the scFv sequence of the CAR was different between these CAR-T cells, such as The scFv amino acid sequence of L1F4-BBz is SEQ ID NO:48 (heavy chain CDR1, CDR2, and CDR3 are shown in Nos.
  • Target cell plating Take out the resistance plate and add 100 ⁇ l (1 ⁇ 10 4 ) BxpC3 target cells on a clean bench. Let it rest at room temperature for half an hour, allowing the cells to naturally settle to the bottom of the plate, and then put on the machine.
  • T cell plating The next day, wait until the target cell cell index increases to about 1. Pause the program, take out the resistance plate, and add 100 ⁇ l CAR-T cells according to the effect-to-target ratio of 3:1, 1:1 and 1:3, and add the control at the same time. Empty T cells.
  • the killing kinetic diagram of each clone is shown in Figures 11 and 12 (the green curve represents culture medium plus target cells, the red curve represents T cells plus target cells, and the bottom curve represents CAR-T cells plus target cells). It can be seen from the killing kinetics chart that L2C8, L2C9, L3G5, and K2F5 have obvious inhibitory effects on target cells and kill quickly.
  • L1A8, L2G1, K2G1, and L1F4 have less inhibitory effects on target cells than L2C8, L2C9, and L3G5.
  • K2F5 has a good inhibitory effect on target cells.
  • the killing rates of 29H and 59H were calculated based on the killing kinetic diagram ( Figure 13). It can be seen that L2C8, L2C9, L3G5, and K2F5 have better killing power than other clones at different effect-to-target ratios.

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Abstract

La présente invention concerne un anticorps se liant spécifiquement à Trop2 ou à son fragment de liaison à l'antigène, son procédé de préparation et son utilisation, qui relèvent du domaine technique des immunothérapies biologiques. L'anticorps présente une bonne sécurité et un effet de traitement ciblant Trop2, et l'anticorps peut être utilisé pour préparer une cellule effectrice immunitaire ciblant Trop2, et présente un moyen de traiter ou de soulager les maladies liées à l'expression de Trop2.
PCT/CN2023/099573 2022-06-14 2023-06-12 Anticorps se liant spécifiquement à trop2 ou à son fragment de liaison à l'antigène, son procédé de préparation et son utilisation WO2023241493A1 (fr)

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CN202210664693.9 2022-06-14
CN202210664693.9A CN117264062A (zh) 2022-06-14 2022-06-14 一种特异性结合Trop2的抗体或其抗原结合片段及其制备方法和应用

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108440674A (zh) * 2018-04-28 2018-08-24 杭州荣泽生物科技有限公司 一种Trop-2特异性嵌合抗原受体细胞制备及其用途
CN110317822A (zh) * 2019-07-19 2019-10-11 英威福赛生物技术有限公司 Trop2嵌合抗原受体、其t细胞及其制备方法和用途
CN112771161A (zh) * 2018-07-09 2021-05-07 启德医药科技（苏州）有限公司 滋养层细胞表面抗原2(trop2)特异性抗体

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108440674A (zh) * 2018-04-28 2018-08-24 杭州荣泽生物科技有限公司 一种Trop-2特异性嵌合抗原受体细胞制备及其用途
CN112771161A (zh) * 2018-07-09 2021-05-07 启德医药科技（苏州）有限公司 滋养层细胞表面抗原2(trop2)特异性抗体
CN110317822A (zh) * 2019-07-19 2019-10-11 英威福赛生物技术有限公司 Trop2嵌合抗原受体、其t细胞及其制备方法和用途

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DESAI, A. ET AL.: "Antibody-Drug Conjugates: A Promising Novel Therapeutic Approach in Lung Cancer", LUNG CANCER, vol. 163, 15 December 2021 (2021-12-15), XP086927600, DOI: 10.1016/j.lungcan.2021.12.002 *
QUN YU, MIAO QING-FANG: "Research progress in antitumor molecular target Trop2 ", CHINESE MEDICINAL BIOTECHNOLOGY, ZHONGGUO YIYAO SHENGWU JISHU XIEHUI, CN, vol. 13, no. 4, 10 August 2018 (2018-08-10), CN , pages 353 - 357, XP093118179, ISSN: 1673-713X, DOI: 10.3969/j.issn.1673-713X.2018.04.012 *
ZHAO WEI, JIA LIZHOU, ZHANG MINGJIONG, HUANG XIAOCHEN, QIAN PENG, TANG QI, ZHU JIN, FENG ZHENQING: "The killing effect of novel bi-specific Trop2/PD-L1 CAR-T cell targeted gastric cancer", AMERICAN JOURNAL OF CANCER RESEARCH, E-CENTURY PUBLISHING CORPORATION, US, vol. 9, no. 8, 1 January 2019 (2019-01-01), US , pages 1846 - 1856, XP093118144, ISSN: 2156-6976 *

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