WO2023066133A1 - 抗间皮素纳米抗体及其用途 - Google Patents

抗间皮素纳米抗体及其用途 Download PDF

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WO2023066133A1
WO2023066133A1 PCT/CN2022/125135 CN2022125135W WO2023066133A1 WO 2023066133 A1 WO2023066133 A1 WO 2023066133A1 CN 2022125135 W CN2022125135 W CN 2022125135W WO 2023066133 A1 WO2023066133 A1 WO 2023066133A1
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Prior art keywords
antigen
nanobody
binding fragment
seq
sequence
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PCT/CN2022/125135
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English (en)
French (fr)
Chinese (zh)
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张振清
缪小牛
吴凡
李志远
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Biotheus Inc
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Biotheus Inc
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Priority to MX2024004702A priority Critical patent/MX2024004702A/es
Priority to KR1020247016339A priority patent/KR20240099295A/ko
Priority to JP2024523232A priority patent/JP2024542944A/ja
Priority to AU2022373543A priority patent/AU2022373543A1/en
Priority to EP22882743.2A priority patent/EP4421095A4/en
Priority to CA3235697A priority patent/CA3235697A1/en
Priority to US18/702,317 priority patent/US20240417452A1/en
Publication of WO2023066133A1 publication Critical patent/WO2023066133A1/zh
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Definitions

  • the present invention relates to Nanobodies or antigen-binding fragments thereof that specifically bind to MSLN, compositions containing said Nanobodies or antigen-binding fragments thereof, nucleic acids encoding said antibodies or antigen-binding fragments thereof and host cells comprising them, and related use. Furthermore, the invention relates to therapeutic and diagnostic uses of these antibodies or antigen-binding fragments thereof.
  • MSLN Mesothelin
  • MSLN Mesothelin
  • MSLN is a 40kDa cell surface glycoprotein highly expressed in pancreatic cancer, ovarian cancer, mesothelioma and some other cancers. Because MSLN can also exist in the blood of a small number of patients in the secreted form of sMSLN, the determination of MSLN in blood may be useful for diagnosis and follow-up of the patient's condition.
  • the MSLN gene encodes a 69-kDa precursor protein that is processed into a 40-kDa membrane-bound protein (called MSLN) and a 31-kDa shedding fragment called megakaryocyte-potentiating factor (MPF) ), the fragment is released from the cell.
  • MSLN 40-kDa membrane-bound protein
  • MPF megakaryocyte-potentiating factor
  • MSLN is not a cancer-specific antigen, it can be expressed in mesothelial cells of normal pleura, pericardium and peritoneum, but it is highly expressed in a variety of cancer cells.
  • the limited distribution of MSLN in normal tissues makes it a potential target for tumor-specific therapy.
  • Anetumab ravtansine an antibody-conjugated drug developed by companies such as Bayer, ImmunoGen, and Morphosys
  • Amatuximab a monoclonal antibody drug targeting MSLN developed by Morphotek
  • the trial has also advanced to clinical phase II for the clinical treatment of solid tumors; in addition, the Military Medical College of the Chinese People's Liberation Army and TCR2 have also developed CAR-T therapy targeting MSLN, which are currently in the clinical trial stage.
  • Existing clinical trials mostly show that the safety of mesothelin-targeted therapy is acceptable, but the therapeutic effect is mediocre.
  • Single domain antibody also known as nanobody (nanobody) or heavy chain antibody (hcAb) is an antibody isolated from the serum of camelids and sharks, and its volume is about 1/10 of traditional antibodies.
  • single-domain antibodies are only composed of heavy chains, and their antigen-binding region is only a single domain connected to the Fc region through a hinge region, and this antigen-binding region still has the ability to bind antigen after it is separated from the antibody. Function.
  • Single-domain antibodies have the characteristics of small molecular weight and good stability. Compared with traditional common normal antibodies in drug development and diagnostic reagent development, they have good tissue infiltration, flexible administration methods, high degree of humanization, and easy protein recombination. renovation and many other advantages.
  • the inventors of the present application screened and obtained nanobodies against MSLN, which have high binding activity to MSLN and have cross-reactivity with human, monkey and/or mouse MSLN.
  • the nanobody also has the characteristics of small molecular weight and good stability. Compared with traditional common normal antibodies in drug development and diagnostic reagent development, it has good tissue infiltration, flexible administration methods, and a high degree of humanization. , easy recombinant protein transformation and many other advantages.
  • the present application also provides a composition containing the Nanobody or an antigen-binding fragment thereof, a nucleic acid encoding the Nanobody or an antigen-binding fragment thereof, a host cell comprising the same, and related uses.
  • the present application provides Nanobodies or antigen-binding fragments thereof capable of specifically binding to MSLN.
  • the Nanobodies described herein generally consist of four framework regions (FRs) and three complementarity determining regions (CDRs), termed FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, the antigen-binding fragment comprising the At least a portion of a Nanobody sufficient to confer on the fragment the ability to specifically bind MSLN.
  • the Nanobodies according to the invention may be truncated at the N-terminus or C-terminus so that they only comprise part of FR1 and/or FR4, or lack one or both of those framework regions, as long as they are substantially It is sufficient to maintain antigen binding and specificity.
  • the Nanobody or antigen-binding fragment thereof comprises:
  • VHH variable region
  • the variant has one or several amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived.
  • the substitutions are conservative substitutions.
  • the CDRs are defined according to the IMGT, Kabat or Chothia numbering system.
  • the Nanobody or antigen-binding fragment thereof comprises:
  • the sequence is the CDR1 of SEQ ID NO: 1 or a variant thereof, the sequence is the CDR2 of SEQ ID NO: 2 or a variant thereof, and the sequence is the CDR3 of SEQ ID NO: 3 or a variant thereof;
  • the variant has one or several amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived.
  • the substitutions are conservative substitutions.
  • the Nanobody or antigen-binding fragment thereof comprises: CDR1 having the sequence of SEQ ID NO:1, CDR2 having the sequence of SEQ ID NO:2, and CDR3 having the sequence of SEQ ID NO:3.
  • the CDRs are defined by the IMGT numbering system.
  • the Nanobody or antigen-binding fragment thereof comprises an amino acid sequence selected from:
  • the substitutions are conservative substitutions.
  • the Nanobody or antigen-binding fragment thereof is humanized.
  • the Nanobody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region comprised in the amino acid sequence encoded by a human heavy chain germline antibody gene ), said heavy chain framework region optionally comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) from Back mutation of human residues to camel residues.
  • a human immunoglobulin e.g., a heavy chain framework region comprised in the amino acid sequence encoded by a human heavy chain germline antibody gene
  • said heavy chain framework region optionally comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) from Back mutation of human residues to camel residues.
  • the Nanobody or antigen-binding fragment thereof comprises:
  • the Nanobody or antigen-binding fragment thereof comprises an amino acid sequence selected from:
  • (iii) 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% of the sequence shown in any one of SEQ ID NOs: 6-9 , at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the substitutions are conservative substitutions.
  • the present application also provides a polypeptide construct specifically binding to MSLN, which comprises a Nanobody or an antigen-binding fragment thereof as described above, and an immunoglobulin Fc domain.
  • the Fc domain is also referred to as Fc region, and refers to a part of the heavy chain constant region including CH2 and CH3.
  • the Fc domain comprises a hinge, CH2 and CH3.
  • the hinge mediates dimerization between two Fc-containing polypeptides.
  • the Fc domain can be of any antibody heavy chain constant region isotype.
  • the Fc domain is IgGl, IgG2, IgG3 or IgG4.
  • the Fc domain comprised by the polypeptide construct of the present invention is a native Fc region comprising an amino acid sequence identical to that of an Fc region found in nature.
  • the Fc domain can be a native sequence human IgGl Fc region, a native sequence human IgG2 Fc region, a native sequence human IgG3 Fc region, or a native sequence human IgG4 Fc region.
  • a native Fc region may have effector functions. Exemplary "effector functions" include binding to Fc receptors; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of B cell receptors); and B cell activation, among others.
  • Effector function can be altered by substituting at least one amino acid residue in the native Fc region with a different residue or by chemical modification, e.g., altering the affinity of the antibody for an effector ligand such as FcR or complement C1q (e.g. decrease or increase).
  • the Fc domain comprised by the polypeptide construct of the present invention may also be a variant Fc region, which may comprise one or more (for example 1-10, for example 1- 5) Amino acid mutation or chemical modification to change one or more of the following properties of the antibody of the invention: Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function or complement function, etc. .
  • the Fc domain contained in the polypeptide construct of the present invention has ADCC activity. In some embodiments, the Fc domain contained in the polypeptide construct of the present invention does not have ADCC activity.
  • said immunoglobulin Fc domain is linked to the N-terminus and/or C-terminus (eg, C-terminus) of said Nanobody or antigen-binding fragment thereof, optionally via a peptide linker.
  • the immunoglobulin Fc domain is an IgG Fc domain (eg, an IgGl Fc domain).
  • the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 5, or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92% compared thereto , 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, or have one or several amino acid substitutions compared thereto , a sequence deleted or added (eg, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions).
  • the Nanobody or polypeptide construct of the present invention can be prepared by various methods known in the art, for example, by genetic engineering and recombination techniques.
  • a DNA molecule encoding a Nanobody or polypeptide construct of the invention is 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 polypeptide construct of the present invention.
  • the antigen-binding fragments of the present invention can be obtained by hydrolyzing the complete Nanobody molecule (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985) ). In addition, these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000 )). Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.
  • the present application also provides an isolated nucleic acid molecule encoding a Nanobody or antigen-binding fragment thereof as described above or a polypeptide construct as described above.
  • the present application also provides a vector comprising the nucleic acid molecule as described above.
  • the vector is a cloning vector or an expression vector.
  • the present application also provides a host cell comprising the nucleic acid molecule or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as E. coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells, and animal cells (such as mammalian cells, such as small mouse cells, human cells, etc.).
  • the present application also provides a method for preparing a Nanobody or an antigen-binding fragment thereof as described above or a polypeptide construct as described above, which comprises, under conditions that allow protein expression, culturing the host as described above cells, and recovering said Nanobody or antigen-binding fragment thereof or said polypeptide construct from cultured host cell culture.
  • the present application also provides a bispecific or multispecific antibody comprising a Nanobody or an antigen-binding fragment thereof as described above or a polypeptide construct as described above.
  • the present application also provides the use of the Nanobodies or their antigen-binding fragments or polypeptide constructs of the present invention, or nucleic acid molecules, vectors or host cells encoding them, for the preparation of bispecific or multispecific antibodies.
  • the bispecific or multispecific antibody specifically binds MSLN and additionally specifically binds one or more other targets.
  • the bispecific or multispecific antibody further comprises at least one second antibody having a second binding specificity for a second target.
  • the present application also provides a conjugate comprising a Nanobody or an antigen-binding fragment thereof as described above or a polypeptide construct as described above, and a Nanobody or an antigen-binding fragment thereof or a polypeptide construct as described above. body-linked therapeutics.
  • the present application also provides the use of Nanobodies or antigen-binding fragments thereof or polypeptide constructs of the invention, or nucleic acid molecules, vectors or host cells encoding them, for the preparation of conjugates.
  • the therapeutic agent is selected from antineoplastic agents, such as cytotoxic agents, hormonal agents, biological response modifiers, additional antibodies or antigen-binding fragments thereof.
  • the present application also provides a chimeric antigen receptor comprising an antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain (such as a primary signaling domain and/or costimulatory domain). signaling domain), wherein said antigen binding domain comprises a Nanobody or an antigen binding fragment thereof as described above.
  • the present application also provides Nanobodies or antigen-binding fragments thereof or polypeptide constructs of the present invention, or nucleic acid molecules encoding them, vectors or host cells for use in preparing chimeric antigen receptors or expressing said chimeric antigen receptors for immunization The purpose of the cells.
  • the antigen binding domain confers on the chimeric antigen receptor the ability to recognize MSLN;
  • the spacer domain allows the protein to be flexible and allows movement of one or both domains relative to each other;
  • the transmembrane domain can be thermodynamically stable in the cell membrane (especially the eukaryotic cell membrane);
  • the intracellular signaling domain participates in transducing effective antigen-receptor binding signals into the immune effector cells, and activates the immune effector that expresses CAR At least one normal effector function of the cell, or enhanced secretion of at least one cytokine by the CAR-expressing immune effector cell.
  • the chimeric antigen receptor is expressed by immune effector cells (eg, T cells).
  • immune effector cells eg, T cells
  • the present application also provides an isolated nucleic acid molecule encoding a chimeric antigen receptor as described above.
  • the present application also provides a vector comprising the isolated nucleic acid molecule as described above.
  • the isolated nucleic acid molecules are used to generate chimeric antigen receptor T cells.
  • the present application also provides a host cell comprising the isolated nucleic acid molecule or vector as described above.
  • 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
  • the present application also provides a pharmaceutical composition, which comprises the Nanobody or antigen-binding fragment thereof of the first aspect, the polypeptide construct of the second aspect, and the bispecific or multispecific antibody of the seventh aspect as described above.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutically acceptable carrier and/or excipient comprises a sterile injectable liquid (eg, aqueous or non-aqueous suspension or solution).
  • a sterile injectable liquid eg, aqueous or non-aqueous suspension or solution.
  • sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (eg 0.9% (w/v) NaCl), dextrose solutions (eg, 5% glucose), solutions containing surfactants (eg, 0.01% polysorbate 20), pH buffered solutions (eg, phosphate buffered saline), Ringer's solutions, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution eg 0.9% (w/v) NaCl
  • dextrose solutions eg, 5% glucose
  • surfactants eg, 0.01% polysorbate 20
  • the pharmaceutical composition comprises the Nanobody or antigen-binding fragment thereof of the first aspect, the polypeptide construct of the second aspect, the isolated nucleic acid molecule of the third aspect, the A vector or host cell of the fifth aspect.
  • the pharmaceutical composition comprises the bispecific or multispecific antibody of the seventh aspect as described above.
  • the pharmaceutical composition comprises the conjugate of the eighth aspect as described above.
  • the pharmaceutical composition comprises the chimeric antigen receptor of the ninth aspect, the isolated nucleic acid molecule of the tenth aspect, the carrier of the eleventh aspect, or the host of the twelfth aspect as described above cell.
  • the present application also provides the Nanobody or antigen-binding fragment thereof in the first aspect, the polypeptide construct in the second aspect, the bispecific or multispecific antibody in the seventh aspect, the eighth aspect
  • the conjugate of the aspect, the chimeric antigen receptor of the ninth aspect, the isolated nucleic acid molecule of the third aspect or the tenth aspect, the vector of the fourth aspect or the eleventh aspect, the host of the fifth aspect or the twelfth aspect Use of the cell, or the pharmaceutical composition of the thirteenth aspect for the preparation of a medicament for preventing and/or treating tumors in a subject.
  • the tumor is a MSLN positive tumor.
  • the tumor is selected from solid tumors such as gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma or breast cancer.
  • solid tumors such as gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma or breast cancer.
  • Such tumors are known in the art to highly express MSLN (see e.g. Aurore et al., cancer discovery, 2016).
  • the subject is a mammal, such as a human.
  • the Nanobody or antigen-binding fragment thereof, polypeptide construct, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific antibody, conjugate, chimeric antigen receptor, Or the pharmaceutical composition is used alone, or used in combination with another pharmaceutically active agent (such as an antineoplastic agent).
  • another pharmaceutically active agent such as an antineoplastic agent.
  • the present application also provides a method for preventing and/or treating tumors in a subject, which includes: administering an effective amount of the above-mentioned first aspect to a subject in need thereof Nanobody or antigen-binding fragment thereof, polypeptide construct of the second aspect, bispecific or multispecific antibody of the seventh aspect, conjugate of the eighth aspect, chimeric antigen receptor of the ninth aspect, third aspect Or the isolated nucleic acid molecule of the tenth aspect, the vector of the fourth or eleventh aspect, the host cell of the fifth or twelfth aspect, or the pharmaceutical composition of the thirteenth aspect.
  • the tumor is a MSLN positive tumor.
  • the tumor is selected from solid tumors such as gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma or breast cancer.
  • the subject is a mammal, such as a human.
  • Nanobodies or antigen-binding fragments thereof, polypeptide constructs, bispecific or multispecific antibodies, or pharmaceutical compositions of the present application can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, Emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injections and concentrated solutions for injections), inhalants, sprays, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • Nanobodies or antigen-binding fragments thereof, polypeptide constructs, bispecific or multispecific antibodies, or pharmaceutical compositions of the invention should be sterile and stable under the conditions of manufacture and storage.
  • a preferred dosage form is injection.
  • Such injections can be sterile injectable solutions.
  • sterile injectable solutions can be prepared by incorporating the necessary doses of a Nanobody or antigen-binding fragment thereof, polypeptide construct, bispecific or multispecific antibody, or drug of the invention in an appropriate solvent composition, and optionally, simultaneously incorporate other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination), followed by filter sterilization.
  • sterile injectable solutions can be prepared as sterile lyophilized powder (eg, by vacuum drying or freeze-drying) for ease of storage and use.
  • Such sterile lyophilized powders can be dispersed in suitable carriers before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), Dextrose solution (eg 5% glucose), surfactant containing solution (eg 0.01% polysorbate 20), pH buffered solution (eg phosphate buffered saline), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution such as 0.9% (w/v) NaCl
  • Dextrose solution eg 5% glucose
  • surfactant containing solution eg 0.01% polysorbate 20
  • pH buffered solution eg phosphate buffered saline
  • Ringer's solution any combination thereof.
  • Nanobodies or antigen-binding fragments thereof, polypeptide constructs, bispecific or multispecific antibodies, or pharmaceutical compositions of the present application may be administered by any suitable method known in the art, including but not limited to, oral, buccal , sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (eg, powder, ointment, or drops), or nasal routes.
  • the preferred route/mode of administration is parenteral (eg, intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection).
  • a Nanobody or antigen-binding fragment thereof, polypeptide construct, bispecific or multispecific antibody, or pharmaceutical composition of the invention is administered by intravenous injection or bolus injection.
  • the present application also provides a conjugate comprising a Nanobody or an antigen-binding fragment thereof as described above or a polypeptide construct as described above, and a nanobody or an antigen-binding fragment thereof or a polypeptide as described above.
  • Construct-linked detectable labels such as enzymes (such as horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives) , fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin.
  • the present application also provides a kit comprising the Nanobody or antigen-binding fragment thereof as described above or the polypeptide construct as described above or the conjugate of the sixteenth aspect as described above.
  • the kit comprises the conjugate of the sixteenth aspect as described above.
  • the kit comprises a Nanobody, or an antigen-binding fragment thereof, or a polypeptide construct as described above, and a protein that specifically recognizes said Nanobody, or an antigen-binding fragment thereof, or a polypeptide construct.
  • Second antibody also includes a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescence reagent (such as acridinium esters, luminamine fluorescein and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin.
  • an enzyme such as horseradish peroxidase or alkaline phosphatase
  • chemiluminescence reagent such as acridinium esters, luminamine fluorescein and its derivatives, or ruthenium derivatives
  • fluorescent dyes such as fluorescein or fluorescent protein
  • the present application also provides a method for detecting the presence or level of MSLN in a sample, comprising using a Nanobody as described above or an antigen-binding fragment thereof or a polypeptide construct as described above or as described above The conjugate of the sixteenth aspect.
  • the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic non-diagnostic purposes.
  • the method is an immunological assay, such as a western blot, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.
  • the method comprises using the conjugate of the sixteenth aspect as described above.
  • the method comprises the use of a Nanobody or antigen-binding fragment thereof as described above or a polypeptide construct as described above, and the method further comprises the use of a nanobody carrying a detectable label (e.g. an enzyme (e.g. paprika) root peroxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radioactive nuclei or biotin) to detect the Nanobody or antigen-binding fragment or polypeptide construct thereof.
  • a detectable label e.g. an enzyme (e.g. paprika) root peroxidase or alkaline phosphatase)
  • chemiluminescent reagents such as acridinium esters, luminol and its derivatives, or ruthenium derivatives
  • fluorescent dyes such as fluorescein or
  • the method comprises: (1) combining the sample with a Nanobody of the invention or an antigen-binding fragment thereof, a polypeptide construct of the invention, or a conjugate of the sixteenth aspect of the invention contacting; (2) detecting the formation of antigen-antibody immune complexes or detecting the amount of said immune complexes.
  • the formation of such immune complexes indicates the presence of MSLN or cells expressing MSLN.
  • the method is used to detect whether a tumor can be treated by an anti-tumor therapy targeting MSLN.
  • the present application also provides the above-mentioned Nanobody or its antigen-binding fragment or the above-mentioned polypeptide construct or the above-mentioned conjugate of the sixteenth aspect in preparation for detecting MSLN in The presence or level thereof in a sample or use in a detection reagent for detecting whether a tumor can be treated by an anti-tumor therapy targeting MSLN.
  • the detection reagent detects the presence or level of MSLN in the sample by the method of the eighteenth aspect as described above and optionally detects whether the tumor can be cured by anti-tumor therapy targeting MSLN treat.
  • the sample is a sample of cells (eg, a sample comprising tumor cells) or a sample of bodily fluid (eg, blood) from a subject (eg, a mammal, eg, a human).
  • a sample of cells eg, a sample comprising tumor cells
  • a sample of bodily fluid eg, blood
  • the term “camelid antibody” refers to an immunized or antigen-invaded camelid (Camelidae) animal (including camel (Camel), alpaca (Alpaca) and llama (L.glama) ) produced antibodies against the antigen.
  • camelid camel
  • alpaca Alphaaca
  • llama L.glama
  • HCAb heavy chain antibody
  • Nanobody has the meaning commonly understood by those skilled in the art, which refers to an antibody consisting of a single monomer variable antibody domain (for example, a single heavy chain variable region) Fragments, typically derived from the variable region of a heavy chain antibody such as a camelid or shark antibody.
  • a Nanobody consists of 4 framework regions and 3 complementarity determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies may be truncated at the N- or C-terminus so that they comprise only part of FR1 and/or FR4, or lack one or both of those framework regions, so long as they substantially retain antigen binding and specificity. Nanobodies are also known as single-domain antibodies (sdAbs), and the two are used interchangeably.
  • sdAbs single-domain antibodies
  • the term "antigen-binding fragment" of a Nanobody refers to a polypeptide comprising a fragment of a Nanobody that retains the ability to specifically bind to the same antigen to which the Nanobody binds, and/or competes with the Nanobody for antigen binding. It is also called "antigen-binding portion”. See generally, Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. or by enzymatic or chemical cleavage of the Nanobodies of the invention to generate antigen-binding fragments of the antibodies of the invention.
  • the "antigen-binding fragments" of the Nanobodies may be at the N-terminal or C-terminus compared to full-length Nanobodies. Truncated at the terminus so that it comprises only part of FR1 and/or FR4, or lacks one or both of those framework regions, so long as it substantially retains antigen binding and specificity.
  • Antigen-binding fragments of a Nanobody can be obtained from a given Nanobody (such as a Nanobody provided by the invention) using conventional techniques known to those skilled in the art (for example, recombinant DNA techniques or enzymatic or chemical fragmentation methods), and can be obtained as Antigen-binding fragments of Nanobodies are screened for specificity in the same way as for whole Nanobodies.
  • Nanobody includes not only whole Nanobodies but also antigen-binding fragments of Nanobodies.
  • CDR complementarity determining region
  • CDR1 complementarity determining region
  • CDR2 complementarity determining region
  • CDR3 complementarity determining region
  • the precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • the term "Fc domain” or "Fc region” means a part of the heavy chain constant region comprising CH2 and CH3.
  • the Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding.
  • "Effector functions" mediated by the Fc region include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; Downregulation of body (eg, B cell receptor); and B cell activation, etc.
  • the Fc region comprises a hinge, CH2 and CH3. When the Fc region contains a hinge, the hinge mediates dimerization between two Fc-containing polypeptides.
  • the Fc region can be of any antibody heavy chain constant region isotype, eg IgGl, IgG2, IgG3 or IgG4.
  • the Fc domain can include both a native Fc region and a variant Fc region.
  • a native Fc region comprises an amino acid sequence identical to that of an Fc region found in nature, for example, a native sequence human Fc region includes a native sequence human IgG1 Fc region (non-A and A allotypes); a native sequence human IgG2 Fc region; a native sequence human Fc region; an IgG3 Fc region; and a native sequence human IgG4 Fc region, and naturally occurring variants thereof.
  • a variant Fc region comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification.
  • a variant Fc region may possess altered effector functions (e.g., Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function) compared to a native Fc region .
  • the term "humanized antibody” refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody.
  • all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from a human Immunoglobulin (receptor antibody).
  • the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable FR and/or constant regions) are derived from human Immunoglobulin (receptor antibody).
  • Humanized antibodies generally retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, and the like.
  • the donor antibody may be a camelid antibody with desired properties (eg, antigen specificity, affinity, reactivity, etc.).
  • the CDR regions of the immunized animal can be inserted into human framework sequences using methods known in the art.
  • a humanized antibody may refer to a humanized VHH, i.e. a VHH in which one or more framework regions have been substantially replaced by human framework regions. In some instances, certain framework regions (FRs) of the human immunoglobulin are replaced by corresponding non-human residues.
  • a humanized VHH may contain residues that were not found in either the original VHH or the human framework sequence, but were included to further refine and optimize the properties of the VHH or VHH-containing polypeptide.
  • the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • the sequences are aligned for optimal comparison purposes (for example, gaps may be introduced in a first amino acid sequence or nucleic acid sequence to best align with a second amino acid or nucleic acid sequence).
  • Jiabi pair The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • a non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Modified from .Acad.Sci.U.S.A. 90:5873-5877. Such an algorithm was incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.
  • the term "specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.
  • the strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant ( KD ) for that interaction.
  • KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the specific binding properties between two molecules can be determined using methods well known in the art.
  • One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation.
  • Both the "association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187).
  • the ratio kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473).
  • KD , kon and kdis values can be measured by any effective method.
  • dissociation constants can be measured in Biacore using surface plasmon resonance (SPR).
  • bioluminescent interferometry or Kinexa can be used to measure dissociation constants.
  • a detectable label according to the invention can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means.
  • labels are well known in the art, examples of which include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.), radionuclide Chlorin (for example, 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (for example, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC) , phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa 750)), luminescent substances (such as chemiluminescent substances, such as acridine este
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • Phage such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • papillomaviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg
  • the term "host cell” refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus 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.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions for amino acid residues with amino acid residues that have similar side chains, e.g., are physically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including Substitution of residues with the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acid, proline, phenylalanine, methionine), beta branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g.
  • basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine
  • non-polar side chains such as
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient compatible with the subject and the active ingredient pharmacologically and/or physiologically, These are well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffers.
  • Surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearates and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (eg, buffered saline), alcohols and polyols (eg, glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Stabilizer has the meaning generally understood by those skilled in the art, and it can stabilize the desired activity of the active ingredient in the medicine, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc.
  • the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (eg, aqueous or non-aqueous suspension or solution).
  • such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (eg 0.9% (w/v) NaCl), dextrose solutions (eg, 5% dextrose), solutions containing surfactants (eg, 0.01% polysorbate 20), pH buffered solutions (eg, phosphate buffered saline), Ringer's solutions, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution eg 0.9% (w/v) NaCl
  • dextrose solutions eg, 5% dextrose
  • solutions containing surfactants eg, 0.01% polysorbate 20
  • pH buffered solutions eg, phosphate buffered saline
  • Ringer's solutions e.g, Ringer's solutions, and any combination thereof.
  • prevention refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • a beneficial or desired clinical outcome includes, but is not limited to, relief of symptoms, reduction of the extent of the disease, stabilization (i.e., no longer worsening) of the disease state, delay or slowing of the progression of the disease, amelioration or palliation of the disease status, and relief of symptoms (whether partial or total), whether detectable or undetectable.
  • treating can also refer to prolonging survival as compared to expected survival if not receiving treatment.
  • the term "subject” refers to mammals, such as humans, monkeys, mice.
  • the subject eg, human, monkey, mouse
  • has, or is at risk of having, a disease associated with MSLN eg, involving an MSLN-positive tumor.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • an effective amount for preventing a disease e.g., involving an MSLN-positive tumor
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent an existing disease
  • the amount of the patient's disease and its complications. Determining such an effective amount is well within the capability of those skilled in the art.
  • amounts effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly etc.
  • the present invention provides Nanobodies with high binding activity to MSLN, which have cross-reactivity with human, monkey and/or mouse MSLN.
  • the nanobody also has the characteristics of small molecular weight and good stability. Compared with traditional common normal antibodies in drug development and diagnostic reagent development, it has good tissue infiltration, flexible administration methods, and a high degree of humanization. , easy recombinant protein transformation and many other advantages.
  • the Nanobodies of the invention may be used in a variety of applications including, but not limited to, inhibition of tumor growth and detection of MSLN protein.
  • the fully human antibodies of the present invention can be safely administered to human subjects without eliciting immunogenic responses. Therefore, the antibodies of the present invention have great clinical value.
  • Figure 1 shows the detection results of the affinity of anti-MSLN nanobodies to CHO-hMSLN cells.
  • Figure 2 shows the detection results of the affinity of the humanized anti-MSLN nanobody to CHO-hMSLN cells.
  • Figure 3 shows the detection results of the affinity of the humanized anti-MSLN nanobody to CHO-cyMSLN cells.
  • Figure 4 shows the detection results of the affinity of the humanized anti-MSLN nanobody to CHO-mMSLN cells.
  • Figure 5 shows the detection results of the blocking activity of the humanized anti-MSLN nanobody blocking the binding of human MSLN to its ligand CA125.
  • the molecular biology experiment methods and immunoassay methods used in the present invention are basically with reference to J.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M.Ausubel et al., Compiled Molecular Biology Experimental Guide, 3rd Edition, John Wiley & Sons, Inc., 1995 by the method described; restriction endonucleases were used in accordance with the conditions recommended by the product manufacturer.
  • restriction endonucleases were used in accordance with the conditions recommended by the product manufacturer.
  • the alpaca (Llama) was immunized with human MSLN antigen (purchased from AcroBiosystems, catalog number: MSN-H522a)
  • human MSLN antigen purchased from AcroBiosystems, catalog number: MSN-H522a
  • the total RNA in the peripheral lymphocytes of the alpaca was extracted and reverse-transcribed to obtain cDNA
  • the PCR product of cDNA was combined with the yeast display vector After ligation, it was electrotransformed into Saccharomyces cerevisiae (purchased from ATCC, catalog number: 208289), and an anti-MSLN nanobody library was constructed.
  • Human MSLN protein was labeled according to the product instruction of the biotin labeling kit (purchased from Thermo, catalog number: 90407). After the amplified anti-MSLN nanobody yeast library was labeled with biotin-labeled MSLN protein, positively labeled yeast were enriched using magnetic beads.
  • the yeast liquid that can be enriched by magnetic beads and flow cytometry and has a higher binding ability to human MSLN antigen was cultured overnight at 30°C and 225rpm in the expansion medium, and the yeast plasmid extraction kit (purchased Yeast plasmids were extracted from Tiangen (product number: DP112).
  • the plasmid was electrotransformed into Top10 competent cells (purchased from Tiangen, Cat. No.: CB104-02), coated with ampicillin-resistant plates, and cultured overnight at 37°C. Single clones were picked and sequenced to obtain the VHH (variable region) gene sequence, and the sequence of the CDR region was determined according to the IMGT numbering system.
  • the sequence information of the obtained monoclonal nanobody YE-17 is shown in the table below.
  • Embodiment 2 Expression vector construction, protein expression and purification of anti-MSLN nanobody
  • VHH coding sequence of anti-MSLN antibody YE-17 and the coding sequence of human IgG1 Fc segment (SEQ ID NO: 5) obtained by screening were constructed into a fusion protein expression sequence through homologous recombination, wherein the human IgG1 Fc segment was connected to the C-terminal of VHH.
  • the medium-prepared fusion protein expression plasmid was transferred into Expi-CHO cells (purchased from Thermo, catalog number: A2910002), and the transfection method was according to the product manual.
  • the supernatant was collected after the cells were cultured for 5 days, and the target protein was purified by sorting with Protein A magnetic beads (purchased from GenScript, catalog number: L00723). Resuspend the magnetic beads with an appropriate volume (1-4 times the volume of magnetic beads) of Binding buffer (PBS+0.1% Tween 20, pH 7.4) and add to the sample to be purified, incubate at room temperature for 1 hour with gentle shaking. The sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed 3 times with Binding buffer.
  • Protein A magnetic beads purchased from GenScript, catalog number: L00723
  • Binding buffer PBS+0.1% Tween 20, pH 7.4
  • the antibody amatuximab (CAS No.: 931402-35-6) was set in parallel as a control group, and the light and heavy chain amino acid sequences of the antibody amatuximab are shown in SEQ ID NOs: 18-19. The results are shown in Table 3.
  • CHO cells (CHO-hMSLN cells) overexpressing human MSLN (Uniprot ID: Q13421) were prepared by transfecting pCHO1.0 vector of MSLN cDNA (purchased from Invitrogen). Adjust the cell density of the expanded CHO-MSLN cells to 2 ⁇ 10 6 cells/ml, add 100 ⁇ L/well to a 96-well flow plate, and centrifuge for later use.
  • the purified anti-MSLN antibody YE-17 prepared in Example 2 was diluted with PBS, starting from 400nM and diluted 3 times, totaling 12 points. Add 100 ⁇ L/well of the above-mentioned diluted sample into the above-mentioned 96-well flow plate with cells, incubate at 4° C.
  • the experimental results are shown in Figure 1 and Table 4.
  • the experimental results show that the anti-MSLN antibody YE-17 prepared in Example 2 has binding activity to CHO-hMSLN cells, and the binding activity is higher than that of the single-chain control antibody amatuximab from Morphotek.
  • the purified humanized antibody was identified at the protein level according to the method described in Example 3, and the results are shown in Table 5.
  • Example 6 Detection of binding force between anti-MSLN humanized nanobody and CHO-hMSLN cells and its species cross-recognition characteristics
  • the purified humanized antibody was tested for affinity at the CHO-hMSLN cell level according to the method described in Example 4.
  • a cell line was constructed according to the method described in Example 4, and the purified humanized antibody was combined with CHO-cyMSLN in the cell level of affinity identification.
  • antibody YE-17 and its humanized antibody have cross-binding activity with human and monkey MSLN
  • antibody YE-17 and its partially humanized antibody also have certain cross-binding activity with human, monkey and mouse MSLN.
  • Example 7 Anti-MSLN humanized Nanobodies block the binding of human MSLN to its ligand CA125

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117843793A (zh) * 2024-03-07 2024-04-09 深圳真实生物医药科技有限公司 抗间皮素抗体、抗原结合片段及其用途

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116466097B (zh) * 2023-06-14 2023-08-29 天津市协和医药科技集团有限公司 一种人胰岛素检测试剂盒

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103819559A (zh) * 2013-12-10 2014-05-28 中国科学院武汉病毒研究所 一种抗间皮素的纳米抗体及其编码基因和该纳米抗体的用途
CN106459989A (zh) * 2013-12-19 2017-02-22 诺华股份有限公司 人间皮素嵌合抗原受体及其用途
CN108129566A (zh) * 2017-12-31 2018-06-08 中国科学院武汉病毒研究所 靶向间皮素的高亲和力c-型单域抗体及其制备方法与应用
CN109467605A (zh) * 2018-11-07 2019-03-15 南京卡提医学科技有限公司 嵌合抗原受体DAP12-T2A-CD8α-MSLN scFv-NKp44及其用途
CN110698562A (zh) * 2019-10-31 2020-01-17 浙江蓝盾药业有限公司 抗人msln单克隆抗体
CA3141085A1 (en) * 2019-06-19 2020-12-24 Brenda Stevens Anti-mesothelin antibodies and immunoconjugates thereof
CN112703205A (zh) * 2018-07-12 2021-04-23 F星贝塔有限公司 抗间皮素抗体

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103819559A (zh) * 2013-12-10 2014-05-28 中国科学院武汉病毒研究所 一种抗间皮素的纳米抗体及其编码基因和该纳米抗体的用途
CN106459989A (zh) * 2013-12-19 2017-02-22 诺华股份有限公司 人间皮素嵌合抗原受体及其用途
CN108129566A (zh) * 2017-12-31 2018-06-08 中国科学院武汉病毒研究所 靶向间皮素的高亲和力c-型单域抗体及其制备方法与应用
CN112703205A (zh) * 2018-07-12 2021-04-23 F星贝塔有限公司 抗间皮素抗体
CN109467605A (zh) * 2018-11-07 2019-03-15 南京卡提医学科技有限公司 嵌合抗原受体DAP12-T2A-CD8α-MSLN scFv-NKp44及其用途
CA3141085A1 (en) * 2019-06-19 2020-12-24 Brenda Stevens Anti-mesothelin antibodies and immunoconjugates thereof
CN110698562A (zh) * 2019-10-31 2020-01-17 浙江蓝盾药业有限公司 抗人msln单克隆抗体

Non-Patent Citations (23)

* Cited by examiner, † Cited by third party
Title
"Immunology-A Synthesis", 1991, SINAUER ASSOCIATES, SUNDERLAND, MASS.
"Uniprot", Database accession no. Q61468
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403
AURORE ET AL., CANCER DISCOVERY, 2016
BRENNAN ET AL., SCIENCE, vol. 229, 1985, pages 81
BRUMMELL ET AL., BIOCHEM., vol. 32, 1993, pages 1180 - 1187
BURKS ET AL., PROC. NATL ACAD. SET USA, vol. 94, 1997, pages 412 - 417
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 878 - 883
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
DAVIES ET AL., ANNUAL REV BIOCHEM, vol. 59, 1990, pages 439 - 473
ESTEP, P ET AL.: "High throughput solution based measurement of antibody-antigen affinity and epitope binning", MABS, vol. 5, no. 2, 2013, pages 270 - 8, XP055105281, DOI: 10.4161/mabs.23049
F. M. AUSUBEL ET AL.: "Compiled Laboratory Guide to Molecular Biology", 1995, PENNSYLVANIA: MACK PUBLISHING COMPANY
HUDSON, CURR. OPIN. IMMUNOL., vol. 11, 1999, pages 548 - 557
J. SAMBROOK ET AL.: "Molecular Cloning: Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
KABAT ET AL.: "Public Health Service", 1991, NATIONAL INSTITUTES OF HEALTH, BETHESDA, article "Sequences of Proteins of Immunological Interest"
KARLINALTSCHUL, PROC. NATL. ACAD. SCI. U.S.A., vol. 87, 1990, pages 2264 - 2268
KARLINALTSCHUL, PROC. NATL. ACAD. SCI. U.S.A., vol. 90, 1993, pages 5873 - 5877
KOBAYASHI ET AL., PROTEIN ENG., vol. 12, no. 10, 1999, pages 879 - 884
LEFRANC ET AL., DEV. COMPARAT. IMMUNOL., vol. 27, 2003, pages 55 - 77
LITTLE ET AL., IMMUNOL. TODAY, vol. 21, 2000, pages 364 - 370
MALMQVIST M, NATURE, vol. 361, 1993, pages 186 - 187
MORIMOTO ET AL., J. BIOCHEM. BIOPHYS. METHODS, vol. 24, 1992, pages 107 - 117
See also references of EP4421095A4

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117843793A (zh) * 2024-03-07 2024-04-09 深圳真实生物医药科技有限公司 抗间皮素抗体、抗原结合片段及其用途

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