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)
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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 AU2022373543A priority patent/AU2022373543A1/en
Priority to EP22882743.2A priority patent/EP4421095A4/en
Priority to JP2024523232A priority patent/JP2024542944A/ja
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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Abstract

本发明涉及特异性结合MSLN的纳米抗体或其抗原结合片段，含有所述纳米抗体或其抗原结合片段的组合物，编码所述抗体或其抗原结合片段的核酸及包含其的宿主细胞，以及相关用途。此外，此发明涉及这些抗体或其抗原结合片段的治疗和诊断用途。

Description

抗间皮素纳米抗体及其用途 技术领域
本发明涉及特异性结合MSLN的纳米抗体或其抗原结合片段，含有所述纳米抗体或其抗原结合片段的组合物，编码所述抗体或其抗原结合片段的核酸及包含其的宿主细胞，以及相关用途。此外，此发明涉及这些抗体或其抗原结合片段的治疗和诊断用途。
背景技术
间皮素(Mesothelin，MSLN)是一种40kDa的细胞表面糖蛋白，在胰腺癌、卵巢癌、间皮瘤和其他一些癌症中高度表达。由于MSLN还可以分泌形式sMSLN存在于少数患者的血液中，因而血液中MSLN的测定可能对诊断和跟踪患者病情有用。MSLN基因可编码一种分子量为69kDa的前体蛋白，该蛋白被加工成一个40kDa的膜结合蛋白(称为MSLN)和一个31kDa的脱落片段，称为巨核细胞增强因子(megakaryocyte-potentiating factor，MPF)，该片段从细胞中释放出来。MSLN不是癌症特异性抗原，可表达于正常胸膜、心包和腹膜的间皮细胞中，但在多种癌症细胞中高表达。MSLN在正常组织上的有限分布使其成为肿瘤特异性治疗很有潜力的靶标。
目前，多种靶向MSLN抗原的抗体药物或者细胞治疗都已进展到临床阶段。如Bayer、ImmunoGen、Morphosys等公司研发的抗体偶联药物Anetumab ravtansine，该药物在II期临床实验中用于治疗实体瘤(NCT03926143)；Morphotek公司开发的靶向MSLN的单克隆抗体药物Amatuximab，目前临床试验也已推进到临床II期，用于实体肿瘤的临床治疗；此外，中国人民解放军军事医学院和TCR2公司还开发了靶向MSLN的CAR-T疗法，目前均处于临床试验阶段。现有的临床试验多表明间皮素靶向治疗安全性尚可，但治疗效果一般。
单域抗体(sdAb)，又称纳米抗体(nanobody)或重链抗体(hcAb)，是从骆驼科动物和鲨鱼的血清中分离出的一种抗体，其体积约为传统抗体的1/10。与传统抗体不同的是，单域抗体仅由重链构成，其抗原结合区仅是一个通过铰链区与Fc区连接的单结构域，而且这个抗原结合区自抗体上分离后仍具有结合抗原的功能。单域抗体具有分子量小，稳定性好等特点，在药物研发和诊断试剂开发等方面相比于传统常见的正常抗体有着组织浸润性好、给药方式灵活、人源化程度高、容易重组蛋白改造等诸多优势。
发明内容
本申请的发明人经过大量的研究，筛选获得了抗MSLN的纳米抗体，所述纳米抗体与MSLN具有高结合活性，并且具备与人、猴和/或小鼠MSLN的交叉反应活性。此外，所述纳米抗体还具有分子量小，稳定性好等特点，在药物研发和诊断试剂开发等方面相比于传统常见的正常抗体有着组织浸润性好、给药方式灵活、人源化程度高、容易重组蛋白改造等诸多优势。
基于此，本申请还提供了含有所述纳米抗体或其抗原结合片段的组合物，编码所述纳米抗体或其抗原结合片段的核酸及包含其的宿主细胞，以及相关用途。
纳米抗体及其抗原结合片段
因此，在第一方面，本申请提供了能够特异性结合MSLN的纳米抗体或其抗原结合片段。本文中所述的纳米抗体通常由4个构架区(FRs)和3个互补决定区(CDRs)组成，称为FR1、CDR1、FR2、CDR2、FR3、CDR3及FR4，所述抗原结合片段包含该纳米抗体的至少一部分，该部分足以赋予该片段特异性结合MSLN的能力。在一些实施方案中，本发明所述的纳米抗体可在N端或C端处截短以使其仅包含部分FR1和/或FR4，或缺少那些骨架区中的一个或两个，只要其实质上保持抗原结合和特异性即可。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：
如SEQ ID NOs:4、6-9任一项所示的可变区(VHH)中含有的CDR1或其变体、CDR2或其变体、CDR3或其变体；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)。在某些实施方案中，所述的置换为保守置换。
在某些实施方案中，所述CDR根据IMGT、Kabat或Chothia编号系统定义。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：
序列为SEQ ID NO：1或其变体的CDR1，序列为SEQ ID NO：2或其变体的CDR2，以及，序列为SEQ ID NO：3或其变体的CDR3；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)。在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：序列为SEQ ID NO：1的CDR1，序列为SEQ ID NO：2的CDR2，以及，序列为SEQ ID NO：3的CDR3。
在某些实施方案中，所述CDR由IMGT编号系统定义。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含选自下列的氨基酸序列：
(i)如SEQ ID NO:4所示的序列；
(ii)与SEQ ID NO：4所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：4所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述纳米抗体或其抗原结合片段是人源化的。
在某些实施方案中，所述纳米抗体或其抗原结合片段进一步包含人免疫球蛋白的重链框架区(例如，人重链胚系抗体基因所编码的氨基酸序列中所包含的重链框架区)，所述重链框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至驼源残基的回复突变。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：
(i)如SEQ ID NO:10所示或与SEQ ID NO:10相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的FR1；
(ii)如SEQ ID NOs:11-13任一项所示或与SEQ ID NOs:11-13任一项相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的FR2；
(iii)如SEQ ID NOs:14-16任一项所示或与SEQ ID NOs:14-16任一项相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少 93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的FR3；
和/或，
(iv)如SEQ ID NO:17所示或与SEQ ID NO:17相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的FR4。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含选自下列的氨基酸序列：
(i)如SEQ ID NOs:6-9任一项所示的序列；
(ii)与SEQ ID NOs：6-9任一项所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NOs：6-9任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述的置换是保守置换。
多肽构建体
在第二方面，本申请还提供了特异性结合MSLN的多肽构建体，其包含如上所述的纳米抗体或其抗原结合片段，以及免疫球蛋白Fc结构域。
在本文中，所述Fc结构域也称为Fc区，是指包含CH2和CH3的重链恒定区的一部分。在一些实施方案中，Fc结构域包含铰链、CH2和CH3。当Fc结构域包含铰链时，铰链调节两个含Fc的多肽之间的二聚作用。Fc结构域可为任何抗体重链恒定区同型。在一些实施方案中，Fc结构域是IgG1、IgG2、IgG3或IgG4。
在某些实施方案中，本发明多肽构建体所包含的Fc结构域是天然Fc区，其包含与自然界中发现的Fc区的氨基酸序列一致的氨基酸序列。例如，所述Fc结构域可以是天然序列人类IgG1Fc区、天然序列人类IgG2Fc区、天然序列人类IgG3Fc区或天然序列人类IgG4Fc区。天然Fc区可具有效应子功能。示例性“效应子功能”包括与Fc受体结合；Clq结合和补体依赖性细胞毒性(CDC)；抗体依赖性细胞介导的细胞毒性(ADCC)；噬菌作用；对细胞表面受体(例如B细胞受体)的下调；和B细胞活化等。可以通过将天然Fc区中的至少一个氨基酸残基替换为不同残基或化学修饰，产生功能改 变，例如，改变抗体对效应子配体(如FcR或补体C1q)的亲和力，从而改变效应子功能(例如降低或增强)。
因此，在某些实施方案中，本发明多肽构建体所包含的Fc结构域也可以是变异Fc区，其与天然Fc区相比可以包含一个或多个(例如1-10个，例如1-5个)氨基酸突变或化学修饰以改变本发明抗体的下列中的一个或更多个特性：Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应细胞功能或补体功能等。
在某些实施方案中，本发明多肽构建体所包含的Fc结构域具备ADCC活性。在某些实施方案中，本发明多肽构建体所包含的Fc结构域不具备ADCC活性。
在某些实施方案中，所述免疫球蛋白Fc结构域任选地通过肽接头连接至所述纳米抗体或其抗原结合片段的N端和/或C端(例如C端)。
在某些实施方案中，所述免疫球蛋白Fc结构域是IgG的Fc结构域(例如IgG1的Fc结构域)。
在某些实施方案中，所述免疫球蛋白Fc结构域包含SEQ ID NO：5所示的序列，或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个、4个或5个氨基酸置换、缺失或添加)的序列。
纳米抗体及多肽构建体的制备
本发明的纳米抗体或多肽构建体可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明纳米抗体或多肽构建体的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体或多肽构建体。
本发明的抗原结合片段可以通过水解完整的纳米抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
核酸分子
在第三方面，本申请还提供了分离的核酸分子，其编码如上所述的纳米抗体或其抗原 结合片段或如上所述的多肽构建体。
载体
在第四方面，本申请还提供了载体，其包含如上所述的核酸分子。在某些实施方案中，所述载体为克隆载体或表达载体。
宿主细胞
在第五方面，本申请还提供了宿主细胞，其包含如上所述的核酸分子或载体。此类宿主细胞包括但不限于，原核细胞例如细菌细胞(如大肠杆菌细胞)，以及真核细胞例如真菌细胞(例如酵母细胞)，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。
制备方法
在第六方面，本申请还提供了制备如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体的方法，其包括，在允许蛋白表达的条件下，培养如上所述的宿主细胞，和从培养的宿主细胞培养物中回收所述纳米抗体或其抗原结合片段或所述多肽构建体。
双特异性或多特异性抗体
在第七方面，本申请还提供了双特异性或多特异性抗体，其包含如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体。本申请还提供了本发明的纳米抗体或其抗原结合片段或多肽构建体、或编码它们的核酸分子、载体或宿主细胞用于制备双特异性或多特异性抗体的用途。
在某些实施方案中，所述双特异性或多特异性抗体特异性结合MSLN，并且额外地特异性结合一个或多个其他靶标。
在某些实施方案中，所述双特异性或多特异性抗体还包含至少一种具有针对第二靶标的第二结合特异性的第二抗体。
缀合物
在第八方面，本申请还提供了缀合物，其包含如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体，以及与所述纳米抗体或其抗原结合片段或多肽构建体连接的治疗剂。本申请还提供了本发明的纳米抗体或其抗原结合片段或多肽构建体、或编码它们的核酸分子、载体或宿主细胞用于制备缀合物的用途。
在某些实施方案中，所述治疗剂选自抗肿瘤药物，例如细胞毒剂、激素类药物、生物反应调节剂、另外的抗体或其抗原结合片段。
嵌合抗原受体
在第九方面，本申请还提供了嵌合抗原受体，其包含抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域(例如初级信号传导结构域和/或共刺激信号传导结构域)，其中所述抗原结合结构域包含如上所述的纳米抗体或其抗原结合片段。本申请还提供了本发明的纳米抗体或其抗原结合片段或多肽构建体、或编码它们的核酸分子、载体或宿主细胞用于制备嵌合抗原受体或表达所述嵌合抗原受体的免疫细胞的用途。
在某些实施方案中，所述抗原结合结构域赋予所述嵌合抗原受体识别MSLN的能力；所述间隔结构域允许蛋白质具有柔性并且允许一个或两个结构域相对于彼此的运动；所述跨膜结构域能够在细胞膜(特别是真核细胞膜)中热力学稳定；所述胞内信号传导结构域参与将有效的抗原受体结合的信号传导进免疫效应细胞内部，激活表达CAR的免疫效应细胞的至少一种正常效应子功能，或增强表达CAR的免疫效应细胞的至少一种细胞因子的分泌。
在某些实施方案中，所述嵌合抗原受体由免疫效应细胞(例如T细胞)所表达。
在第十方面，本申请还提供了分离的核酸分子，其编码如上所述的嵌合抗原受体。
在第十一方面，本申请还提供了载体，其包含如上所述的分离的核酸分子。在某些实施方案中，所述分离的核酸分子用于制备嵌合抗原受体T细胞。
在第十二方面，本申请还提供了宿主细胞，其包含如上所述的分离的核酸分子或载体。
在某些实施方案中，所述宿主细胞是免疫效应细胞(例如，T细胞或NK细胞)。
在某些实施方案中，所述宿主细胞是嵌合抗原受体T细胞(CAR-T)。
药物组合物
在第十三方面，本申请还提供了药物组合物，其包含如上所述的第一方面的纳米抗体或其抗原结合片段、第二方面的多肽构建体、第七方面的双特异性或多特异性抗体、第八方面的缀合物、第九方面的嵌合抗原受体、第三方面或第十方面的分离的核酸分子、第四方面或第十一方面的载体、或第五方面或第十二方面的宿主细胞。
在某些实施方案中，所述药物组合物还包含药学上可接受的载体和/或赋形剂。
在某些示例性实施方案中，所述药学上可接受的载体和/或赋形剂包含无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH 缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
在某些实施方案中，所述药物组合物包含如上所述的第一方面的纳米抗体或其抗原结合片段、第二方面的多肽构建体、第三方面的分离的核酸分子、第四方面的载体或第五方面的宿主细胞。
在某些实施方案中，所述药物组合物包含如上所述的第七方面的双特异性或多特异性抗体。
在某些实施方案中，所述药物组合物包含如上所述的第八方面的缀合物。
在某些实施方案中，所述药物组合物包含如上所述的第九方面的嵌合抗原受体、第十方面的分离的核酸分子、第十一方面的载体、或第十二方面的宿主细胞。
制药用途
在第十四方面，本申请还提供了如上所述的第一方面的纳米抗体或其抗原结合片段、第二方面的多肽构建体、第七方面的双特异性或多特异性抗体、第八方面的缀合物、第九方面的嵌合抗原受体、第三方面或第十方面的分离的核酸分子、第四方面或第十一方面的载体、第五方面或第十二方面的宿主细胞、或第十三方面的药物组合物用于制备药物的用途，所述药物用于在受试者中预防和/治疗肿瘤。
在某些实施方案中，所述肿瘤为MSLN阳性的肿瘤。
在某些实施方案中，所述肿瘤选自实体瘤，例如胃癌、肺癌、卵巢癌、食道癌、胰腺癌、宫颈癌、间皮瘤或乳腺癌。本领域已知所述肿瘤高表达MSLN(参见例如Aurore et al.,cancer discovery,2016)。
在某些实施方案中，所述受试者为哺乳动物，例如人。
在某些实施方案中，所述纳米抗体或其抗原结合片段、多肽构建体、分离的核酸分子、载体、宿主细胞、双特异性或多特异性抗体、缀合物、嵌合抗原受体、或药物组合物单独使用，或与另外的药学活性剂(例如抗肿瘤剂)联合使用。
预防和/或治疗肿瘤的方法
在第十五方面，本申请还提供了用于在受试者中预防和/或治疗肿瘤的方法，其包括：给有此需要的受试者施用有效量的如上所述的第一方面的纳米抗体或其抗原结合片段、第二方面的多肽构建体、第七方面的双特异性或多特异性抗体、第八方面的缀合物、第九方面的嵌合抗原受体、第三方面或第十方面的分离的核酸分子、第四方面或第十一方面的载体、第五方面或第十二方面的宿主细胞、或第十三方面的药物组合物。
在某些实施方案中，所述肿瘤为MSLN阳性的肿瘤。
在某些实施方案中，所述肿瘤选自实体瘤，例如胃癌、肺癌、卵巢癌、食道癌、胰腺癌、宫颈癌、间皮瘤或乳腺癌。
在某些实施方案中，所述受试者为哺乳动物，例如人。
本申请的纳米抗体或其抗原结合片段、多肽构建体、双特异性或多特异性抗体、或药物组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的纳米抗体或其抗原结合片段、多肽构建体、双特异性或多特异性抗体、或药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。例如，可通过下述方法来制备无菌注射溶液：在适当的溶剂中掺入必需剂量的本发明的纳米抗体或其抗原结合片段、多肽构建体、双特异性或多特异性抗体、或药物组合物，以及任选地，同时掺入其他期望的成分(包括但不限于，pH调节剂，表面活性剂，佐剂，离子强度增强剂，等渗剂、防腐剂、稀释剂，或其任何组合)，随后过滤除菌。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
本申请的纳米抗体或其抗原结合片段、多肽构建体、双特异性或多特异性抗体、或药物组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射或推注，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在某些实施方案中，本发明的纳米抗体或其抗原结合片段、多肽构建体、双特异性或多特异性抗体、或药物组合物通过静脉注射或推注给予。
检测应用
缀合物
在第十六方面，本申请还提供了缀合物，其包含如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体，以及与所述纳米抗体或其抗原结合片段或多肽构建体连接的可检测的标记，例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
试剂盒
在第十七方面，本申请还提供了试剂盒，其包括如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体或如上所述的第十六方面的缀合物。
在某些实施方案中，所述试剂盒包含如上所述的第十六方面的缀合物。
在某些实施方案中，所述试剂盒包含如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体，以及特异性识别所述纳米抗体或其抗原结合片段或多肽构建体的第二抗体；任选地，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
检测方法
在第十八方面，本申请还提供了用于检测MSLN在样品中的存在或其水平的方法，其包括使用如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体或如上所述的第十六方面的缀合物。在某些实施方案中，所述方法用于治疗目的，诊断目的，或者非治疗非诊断目的。
在某些实施方案中，所述方法是免疫学检测，例如免疫印迹法、酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法。
在某些实施方案中，所述方法包括使用如上所述的第十六方面的缀合物。
在某些实施方案中，所述方法包括使用如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体，并且所述方法还包括使用携带可检测的标记(例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素)的第二抗体来检测所述纳米抗体或其抗原结合片段或多肽构建体。
在某些实施方案中，所述方法包括：(1)将所述样品与本发明的纳米抗体或其抗原结合片段、本发明的多肽构建体、或本发明的第十六方面的缀合物接触；(2)检测抗原-抗体 免疫复合物的形成或检测所述免疫复合物的量。所述免疫复合物的形成表明存在MSLN或表达MSLN的细胞。
在某些实施方案中，所述方法用于检测肿瘤是否能够通过靶向MSLN的抗肿瘤疗法来治疗。
检测试剂制备用途
在第十九方面，本申请还提供了如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体或如上所述的第十六方面的缀合物在制备用于检测MSLN在样品中的存在或其水平或者用于检测肿瘤是否能够通过靶向MSLN的抗肿瘤疗法来治疗的检测试剂中的用途。
在某些实施方案中，所述检测试剂通过如上所述的第十八方面的方法来检测MSLN在样品中的存在或其水平以及任选地检测肿瘤是否能够通过靶向MSLN的抗肿瘤疗法来治疗。
在某些实施方案中，所述样品为来自受试者(例如哺乳动物，例如人)的细胞样品(例如包含肿瘤细胞的样品)或体液样品(例如血液)。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的病毒学、生物化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
当本文使用术语“例如”、“如”、“诸如”、“包括”、“包含”或其变体时，这些术语将不被认为是限制性术语，而将被解释为表示“但不限于”或“不限于”。
除非本文另外指明或根据上下文明显矛盾，否则术语“一个”和“一种”以及“该”和类似指称物在描述本发明的上下文中(尤其在以下权利要求的上下文中)应被解释成覆盖单数和复数。
如本文中所使用的，术语“驼源抗体”是指，经免疫接种或抗原入侵的骆驼科(Camelidae)动物(包括骆驼(Camel)、羊驼(Alpaca)和大羊驼(L.glama))所产生的针对该抗原的抗体。本领域技术人员已知，在骆驼科动物所产生的抗体中存在缺失轻链的“重链抗体”(Camelid heavy-chain antibody,HCAb)，这种抗体只包含一个重链可变 区(variable domain of heavy chain of HCAb,VHH)和两个常规的CH2与CH3区，并且单独克隆并表达出来的VHH区具有很好的结构稳定性与抗原结合活性，VHH是目前己知的可结合目标抗原的最小单位。
如本文中所使用的，术语“纳米抗体(nanobody)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，通常来源于重链抗体(例如骆驼科动物抗体或鲨鱼抗体)的可变区。典型地，纳米抗体由4个构架区和3个互补性决定区组成，具有FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的结构。纳米抗体可在N端或C端处截短以使其仅包含部分FR1和/或FR4，或缺少那些骨架区中的一个或两个，只要其实质上保持抗原结合和特异性即可。纳米抗体也称为单域抗体(single-domain antibody,sdAb)，两者可互换使用。
如本文中所使用的，术语纳米抗体的“抗原结合片段”是指包含纳米抗体的片段的多肽，其保持特异性结合纳米抗体所结合的相同抗原的能力，和/或与纳米抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过本发明纳米抗体的酶促或化学断裂产生本发明抗体的抗原结合片段。在一些实施方案中，所述纳米抗体的“抗原结合片段”与全长纳米抗体相比可在N端或C端处截短以使其仅包含部分FR1和/或FR4，或缺少那些骨架区中的一个或两个，只要其实质上保持抗原结合和特异性即可。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的纳米抗体(例如本发明提供的纳米抗体)获得纳米抗体的抗原结合片段，并且以与用于完整纳米抗体的方式相同的方式就特异性筛选纳米抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“纳米抗体”时，其不仅包括完整纳米抗体，而且包括纳米抗体的抗原结合片段。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在纳米抗体中含有三个CDR，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878- 883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的纳米抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
如本文中所使用的，术语“Fc结构域”或“Fc区”意指，包含CH2和CH3的重链恒定区的一部分。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。由Fc区介导的“效应子功能”包括Fc受体结合；Clq结合和补体依赖性细胞毒性(CDC)；抗体依赖性细胞介导的细胞毒性(ADCC)；噬菌作用；对细胞表面受体(例如B细胞受体)的下调；和B细胞活化等。在一些实施方案中，Fc区包含铰链、CH2和CH3。当Fc区包含铰链时，铰链调节两个含Fc的多肽之间的二聚作用。Fc区可为任何抗体重链恒定区同型，例如IgG1、IgG2、IgG3或IgG4。
Fc结构域既可以包括天然Fc区，也可以包括变异Fc区。天然Fc区包含与自然界中发现的Fc区的氨基酸序列一致的氨基酸序列，例如天然序列人类Fc区包括天然序列人类IgG1Fc区(非A和A同种异型)；天然序列人类IgG2Fc区；天然序列人类IgG3Fc区；及天然序列人类IgG4Fc区，以及其天然存在的变异体。变异Fc区包含因至少一个氨基酸修饰而与天然序列Fc区的氨基酸序列不同的氨基酸序列。在一些实施方案中，变异Fc区可具备相比于天然Fc区改变的效应子功能(例如Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应细胞功能或补体功能)。
如本文中所使用的，术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。在某些实施方案中，人源化抗体的CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性等。在本申请中，供体抗体可以是有预期性质(例如，抗原特异性、亲和性、反应性等)的驼源抗体。为制备人源化抗体，可以使用本领域已知的方法将免疫动物的CDR区插入人源框架序列。在纳米抗体 的语境下，人源化抗体可指人源化VHH，即其中一或多个骨架区已基本上由人类骨架区替换的VHH。在一些情形中，人类免疫球蛋白的某些骨架区(FR)由相应非人类残基替换。此外，人源化VHH可包含在初始VHH或人类骨架序列中均未发现、但被包括在内以进一步改进和优化VHH或含VHH的多肽的性能的残基。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。为了测定两个氨基酸序列或两个核酸序列的百分比同一性，为了最佳比较目的将序列进行比对(例如，可在第一氨基酸序列或核酸序列中引入缺口以与第二氨基酸或核酸序列最佳比对)。然后比较对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置被与第二序列中的对应位置相同的氨基酸残基或核苷酸占据时，则分子在该位置上是同一的。两个序列之间的百分比同一性是由序列所共享的同一性位置的数目的函数(即，百分比同一性＝同一重叠位置的数目/位置的总数×100％)。在某些实施方案中，两个序列长度相同。
两个序列之间的百分比同一性的测定还可使用数学算法来实现。用于两个序列的比较的数学算法的一个非限制性实例是Karlin和Altschul的算法，1990,Proc.Natl.Acad.Sci.U.S.A.87:2264-2268，如同Karlin和Altschul,1993,Proc.Natl.Acad.Sci.U.S.A.90:5873-5877中改进的。将这样的算法整合至Altschul等人,1990,J.Mol.Biol.215:403的NBLAST和XBLAST程序中。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(K D)表示。在本发明中，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数K D(参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量K D、kon和kdis值。在某些实施方案中，可以使用表面等离子体共振术(SPR)在Biacore中来测量解离常数。除此以外还可用生物发光干涉测量法或Kinexa来测量解离常数。
如本文中所使用的，本发明所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如， 3H、 125I、 35S、 14C或 32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、发光物质(例如化学发光物质，如吖啶酯类化合物、鲁米诺及其衍生物、钌衍生物如三联吡啶钌)、磁珠(例如，
Figure PCTCN2022125135-appb-000001
)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行 的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)， 氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。在某些示例性实施方案中，所述药学上可接受的载体或赋形剂包括无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括(但不限于)减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中所使用的，术语“受试者”是指哺乳动物，例如人、猴、小鼠。在某些实施方案中，所述受试者(例如人、猴、小鼠)患有与MSLN相关的疾病(例如，涉及MSLN阳性的肿瘤)，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，涉及MSLN阳性的肿瘤)有效量是指，足以预防，阻止，或延迟所述疾病的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
本发明提供了与MSLN具有高结合活性的纳米抗体，其具备与人、猴和/或小鼠MSLN的交叉反应活性。此外，所述纳米抗体还具有分子量小，稳定性好等特点，在药物研发和诊断试剂开发等方面相比于传统常见的正常抗体有着组织浸润性好、给药方式灵活、人源化程度高、容易重组蛋白改造等诸多优势。
因此，本发明的纳米抗体可以用于多种用途，包括但不限于抑制肿瘤生长和检测 MSLN蛋白。并且，本发明的全人源抗体可安全地施用给人受试者，而不引发免疫原性反应。因此，本发明的抗体具有重大的临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了抗MSLN纳米抗体与CHO-hMSLN细胞亲和力的检测结果。
图2显示了人源化抗MSLN纳米抗体与CHO-hMSLN细胞亲和力的检测结果。
图3显示了人源化抗MSLN纳米抗体与CHO-cyMSLN细胞亲和力的检测结果。
图4显示了人源化抗MSLN纳米抗体与CHO-mMSLN细胞亲和力的检测结果。
图5显示了人源化抗MSLN纳米抗体阻断人MSLN与其配体CA125结合的阻断活性检测结果。
序列信息
本申请涉及的序列的描述提供于下表中。
表1：序列信息
Figure PCTCN2022125135-appb-000002
Figure PCTCN2022125135-appb-000003
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：抗MSLN纳米抗体的免疫和筛选
利用人MSLN抗原(购自AcroBiosystems，货号：MSN-H522a)免疫羊驼(Llama)后，提取羊驼外周淋巴细胞中的总RNA并进行反转录得到cDNA，将cDNA的PCR产物与酵母展示载体连接后电转化入酿酒酵母(购自ATCC，货号：208289)，构建抗MSLN纳米抗体文库。
按照生物素标记试剂盒(购自Thermo，货号：90407)的产品说明书标记人MSLN蛋白。将扩增后的抗MSLN纳米抗体酵母文库用生物素标记的MSLN蛋白标记后，使用磁珠富集阳性标记酵母。将经过磁珠富集的酵母细胞扩增后，加入1:200稀释的anti-c-Myc抗体(购自Thermo，货号：MA1-980)和适量生物素标记的MSLN抗原染色，PBS清洗酵母后加入1:500稀释的Goat-Anti-mouse IgG(H+L)Alexa Fluor Plus 488(购自Invitrogen，货号：A32723TR)和streptavidin APC Conjugate荧光抗体(购自Invitrogen，货号：SA1005)，孵育15min。用PBS重悬细胞，使用BD FACSAria II仪器进行分选获得可与人MSLN抗原有较高结合能力的酵母。
通过磁珠和流式细胞分选富集得到的能与人MSLN抗原有较高结合能力的酵母菌液，在扩增培养基中30℃，225rpm培养过夜，按照酵母质粒抽提试剂盒(购自天根，货号：DP112)操作抽提酵母质粒。质粒通过电转化入Top10感受态细胞(购自天根，货号：CB104-02)，涂布氨苄抗性平板，于37℃培养过夜。挑取单克隆测序获得VHH(可变区)基因序列，并根据IMGT编号系统确定CDR区序列。所获得的单克隆纳米抗体YE-17的序列信息如下表所示。
表2：纳米抗体的VHH以及CDR序列信息
Figure PCTCN2022125135-appb-000004
实施例2：抗MSLN纳米抗体的表达载体构建、蛋白表达和纯化
将筛选所得抗MSLN抗体YE-17的VHH编码序列和人IgG1Fc段(SEQ ID NO:5)编码序列通过同源重组构建成融合蛋白表达序列，其中人IgG1Fc段连接于VHH的C端。采用ExpiCHO TM Expression System试剂盒(购自Thermo，货号：A2910001)，将中量制备的融合蛋白表达质粒转入Expi-CHO细胞(购自Thermo，货号：A2910002)中，转染方法按照商品说明书，细胞培养5天后收集上清，利用Protein A磁珠(购自金斯瑞，货号：L00723)分选法纯化目的蛋白。将磁珠用适当体积(1-4倍磁珠体积)的Binding buffer(PBS+0.1％Tween 20，pH 7.4)重悬后加入至待纯化样品中，室温孵育1小时，期间温柔振荡。样品置于磁力架(购自海狸)上，弃去上清，磁珠用Binding buffer清洗3遍。按照磁珠体积的3-5倍体积加入Elution buffer(0.1M sodium citrate,pH 3.2)室温振荡5-10min，置回磁力架上，收集Elution buffer，转移至已加入Neutralization buffer(1M Tris，pH 8.54)的收集管中混匀，完成制备，获得经纯化的抗MSLN纳米抗体YE-17。
实施例3：抗MSLN纳米抗体在蛋白水平的亲和力检测
ForteBio亲和力测定按照现有的方法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):p.270-8)进行。简言之，将传感器在分析缓冲液中线下平衡30min，然后线上检测60s建立基线，在线加载实施例2获得的经纯化的抗体至AHQ传感器上。再将传感器放入100nM的人MSLN抗原(Uniprot ID：Q13421)中作用5min，之后将传感器转移至PBS中解离5min。使用1:1结合模型进行动力学的分析。此外，平行设置抗体amatuximab(CAS号：931402-35-6)为对照组，所述抗体amatuximab的轻重链氨基酸序列如SEQ ID NOs:18-19所示。结果如表3所示。
表3.候选分子亲和力
抗体编号 KD(M) Kon(1/Ms) Koff(1/s)
YE-17 1.88E-10 5.50E+05 1.04E-04
amatuximab 8.13E-10 1.53E+06 1.24E-03
实施例4：抗MSLN纳米抗体在细胞水平的亲和力检测
通过转染MSLN cDNA的pCHO1.0载体(购自Invitrogen)制备过表达人MSLN (Uniprot ID：Q13421)的CHO细胞(CHO-hMSLN细胞)。将扩大培养的CHO-MSLN细胞调整细胞密度至2×10 6cells/ml,100μL/孔加入96孔流式板，离心备用。将实施例2制备的经纯化的抗MSLN抗体YE-17用PBS稀释，400nM开始3倍稀释，共12个点。将上述稀释好的样品100μL/孔加入上述带有细胞的96孔流式板中，4℃孵育30min，PBS清洗两次。100μL/孔加入用PBS稀释100倍的Goat F(ab')2Anti-Human IgG-Fc(PE)(购自Abcam，货号：ab98596)，4℃孵育30min，PBS清洗两次。100μl/孔加入PBS重悬细胞，在CytoFlex(Bechman)流式细胞仪上进行检测并计算对应的MFI值(平均荧光强度)。
实验结果如图1和表4所示，实验结果表明实施例2制备的抗MSLN抗体YE-17和CHO-hMSLN细胞有结合活性，且结合活性高于来自于Morphotek的单链对照抗体amatuximab。
表4.抗MSLN纳米抗体与CHO-hMSLN细胞水平结和力EC50
抗体编号 EC50(nM)
YE-17 0.9647
amatuximab 1.531
实施例5：抗MSLN人源化纳米抗体在蛋白水平的亲和力检测
本实例中，我们将抗体YE-17完成序列人源化改造。并将人源化的改造的序列按照实施例2中所描述方法完成人源化抗体的载体构建、表达和纯化。最终，通过YE-17获得4株人源化改造抗体，分别为HZ-P-YE-17-01、HZ-P-YE-17-02、HZ-P-YE-17-03和HZ-P-YE-17-04，其VHH序列分别如SEQ ID NOs:6-9所示。
将纯化后的人源化抗体按照实施例3中所述方法完成在蛋白水平的亲和力鉴定，结果如表5所示。
表5.抗MSLN人源化纳米抗体的蛋白水平亲和力检测结果
抗体编号 KD(M) Kon(1/Ms) Koff(1/s)
YE-17 1.40E-09 4.21E+05 5.90E-04
HZ-P-YE-17-01 8.07E-10 4.02E+05 3.25E-04
HZ-P-YE-17-02 2.20E-09 4.03E+05 8.88E-04
HZ-P-YE-17-03 1.91E-08 3.36E+05 6.40E-03
HZ-P-YE-17-04 1.93E-08 3.29E+05 6.36E-03
实施例6：抗MSLN人源化纳米抗体与CHO-hMSLN细胞水平结和力的检测及其种属交叉识别特性
将纯化后的人源化抗体按照实施例4中所述方法完成CHO-hMSLN细胞水平亲和力鉴定。
结果如图2和表6所示，结果表明实施例5制备的人源化抗MSLN抗体和CHO-hMSLN细胞均有结合活性。
表6.人源化纳米抗体与CHO-hMSLN细胞结合力EC50
编号 EC50(nM)
YE-17 0.305
HZ-P-YE-17-01 0.4775
HZ-P-YE-17-02 0.5515
HZ-P-YE-17-03 0.2415
HZ-P-YE-17-04 0.3399
为鉴定人源化抗体与猴MSLN(cyMSLN，Uniprot ID：F6Q1U7)在细胞水平上的亲和力，按照实施例4中所述方法构建细胞株，并完成纯化后的人源化抗体与CHO-cyMSLN在细胞水平上的亲和力鉴定。
结果如图3和表7所示，结果表明抗体YE-17及其人源化抗MSLN抗体和CHO-cyMSLN细胞均有结合活性。
表7.人源化纳米抗体与CHO-cyMSLN细胞结合力EC50
编号 EC50(nM)
YE-17 0.7161
HZ-P-YE-17-01 1.24
HZ-P-YE-17-02 0.6298
HZ-P-YE-17-03 0.7084
HZ-P-YE-17-04 1.009
为鉴定抗体与鼠MSLN(mMSLN，Uniprot ID：Q61468)在细胞水平上的亲和力，按照实施例4中所述方法构建细胞株，并完成纯化后的人源化抗体与CHO-mMSLN在 细胞水平上的亲和力鉴定，结果如图4和表8所示。
表8.人源化纳米抗体与CHO-mMSLN细胞结合力EC50
编号 EC50(nM)
YE-17 6.312
HZ-P-YE-17-01 12.99
HZ-P-YE-17-02 7.293
HZ-P-YE-17-03 22.35
HZ-P-YE-17-04 ～249165
综合表6-8以及图2-4的结果，表明抗体YE-17及其人源化抗体具备与人和猴MSLN的交叉结合活性，并且，抗体YE-17和其部分人源化抗体(例如HZ-P-YE-17-01、HZ-P-YE-17-02、HZ-P-YE-17-03)还具备一定的与人、猴和鼠MSLN的交叉结合活性。
实施例7：抗MSLN人源化纳米抗体阻断人MSLN与其配体CA125的结合
为鉴定抗体阻断人MSLN与其配体CA125的结合的阻断活性，按照下述方法进行实验验证：首先将1μg/mL人MSLN抗原包被在高吸附ELISA包被板，4摄氏度包被过夜；去除包被液，5％BSA室温封闭2小时；加入梯度稀释的人源化抗体，室温孵育1h；洗板3次，加入1μg/mL Biotin-CA125(购自Acro，货号：CA5-H82F4)，室温孵育1h；洗板3次，加入1:10000稀释的SA-HRP(购自Abcam，货号：Ab7403)，室温孵育30min；洗板6次，加入50μl/mL TMB显色液，3-5分钟；加入50μl/mL终止液；上机读取A450。
结果如图5和表9所示，结果表明抗体YE-17及其人源化抗体均能有效阻断人MSLN与其配体CA125的结合，从而抑制癌症的侵袭和转移。
表9.人源化纳米抗体阻断人MSLN与其配体CA125结合的EC50
编号 EC50(nM)
YE-17 5.958
HZ-P-YE-17-01 3.342
HZ-P-YE-17-02 2.627
HZ-P-YE-17-03 4.753
HZ-P-YE-17-04 3.234
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (24)

  1. 能够特异性结合MSLN的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段包含：
    如SEQ ID NOs:4、6-9任一项所示的可变区(VHH)中含有的CDR1或其变体、CDR2或其变体、CDR3或其变体；
    其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；
    优选地，所述CDR根据IMGT、Kabat或Chothia编号系统定义。
  2. 权利要求1的纳米抗体或其抗原结合片段，其中，所述纳米抗体或其抗原结合片段包含：
    序列为SEQ ID NO：1或其变体的CDR1，序列为SEQ ID NO：2或其变体的CDR2，以及，序列为SEQ ID NO：3或其变体的CDR3；
    其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；
    优选地，所述纳米抗体或其抗原结合片段包含：序列为SEQ ID NO：1的CDR1，序列为SEQ ID NO：2的CDR2，以及，序列为SEQ ID NO：3的CDR3；
    优选地，所述CDR由IMGT编号系统定义。
  3. 权利要求1或2的纳米抗体或其抗原结合片段，其包含选自下列的氨基酸序列：
    (i)如SEQ ID NO:4所示的序列；
    (ii)与SEQ ID NO：4所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO：4所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，所述的置换是保守置换。
  4. 权利要求1-3任一项的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段是人源化的；
    优选地，所述纳米抗体或其抗原结合片段进一步包含人免疫球蛋白的重链框架区(例如，人重链胚系抗体基因所编码的氨基酸序列中所包含的重链框架区)，所述重链框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至驼源残基的回复突变。
  5. 权利要求1-4任一项的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段包含：
    (i)如SEQ ID NO:10所示或与SEQ ID NO:10相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的FR1；
    (ii)如SEQ ID NOs:11-13任一项所示或与SEQ ID NOs:11-13任一项相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的FR2；
    (ii)如SEQ ID NOs:14-16任一项所示或与SEQ ID NOs:14-16任一项相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的FR3；
    和/或，
    (iv)如SEQ ID NO:17所示或与SEQ ID NO:17相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的FR4。
  6. 权利要求1-5任一项的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段包含选自下列的氨基酸序列：
    (i)如SEQ ID NOs:6-9任一项所示的序列；
    (ii)与SEQ ID NOs：6-9任一项所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NOs：6-9任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，所述的置换是保守置换。
  7. 特异性结合MSLN的多肽构建体，其包含权利要求1-6任一项所述的纳米抗体或其抗原结合片段，以及免疫球蛋白Fc结构域；
    优选地，所述免疫球蛋白Fc结构域任选地通过肽接头连接至所述纳米抗体或其抗原结合片段的N端和/或C端(例如C端)；
    优选地，所述免疫球蛋白Fc结构域是IgG的Fc结构域(例如IgG1的Fc结构域)；
    优选地，所述免疫球蛋白Fc结构域包含SEQ ID NO：5所示的序列，或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个、4个或5个氨基酸置换、缺失或添加)的序列。
  8. 分离的核酸分子，其编码权利要求1-6任一项所述的纳米抗体或其抗原结合片段或权利要求7所述的多肽构建体。
  9. 载体，其包含权利要求8所述的核酸分子；优选地，所述载体为克隆载体或表达载体。
  10. 宿主细胞，其包含权利要求8所述的核酸分子或权利要求9所述的载体。
  11. 制备权利要求1-6任一项所述的纳米抗体或其抗原结合片段或权利要求7所述的多肽构建体的方法，其包括，在允许蛋白表达的条件下，培养权利要求10所述的宿主细胞，和从培养的宿主细胞培养物中回收所述纳米抗体或其抗原结合片段或所述多肽构建体。
  12. 双特异性或多特异性抗体，其包含权利要求1-6任一项所述的纳米抗体或其抗原结合片段或权利要求7所述的多肽构建体；
    优选地，所述双特异性或多特异性抗体特异性结合MSLN，并且额外地特异性结合一个或多个其他靶标；
    优选地，所述双特异性或多特异性抗体还包含至少一种具有针对第二靶标的第二结合特异性的第二抗体。
  13. 缀合物，其包含权利要求1-6任一项所述的纳米抗体或其抗原结合片段或权利要求7所述的多肽构建体，以及与所述纳米抗体或其抗原结合片段或多肽构建体连接的治疗剂；
    优选地，所述治疗剂选自抗肿瘤药物，例如细胞毒剂、激素类药物、生物反应调节剂、另外的抗体或其抗原结合片段。
  14. 嵌合抗原受体，其包含抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域，其中所述抗原结合结构域包含权利要求1-6任一项所述的纳米抗体或其抗原结合片段；
    优选地，所述嵌合抗原受体由免疫效应细胞(例如T细胞)所表达。
  15. 分离的核酸分子，其编码权利要求14所述的嵌合抗原受体。
  16. 载体，其包含权利要求15所述的分离的核酸分子；优选地，其用于制备嵌合抗原受体T细胞。
  17. 宿主细胞，其包含权利要求15所述的分离的核酸分子或权利要求16所述的载体；
    优选地，所述宿主细胞是免疫效应细胞(例如，T细胞或NK细胞)；
    优选地，所述宿主细胞是嵌合抗原受体T细胞(CAR-T)。
  18. 药物组合物，其包含权利要求1-6任一项的纳米抗体或其抗原结合片段、权利要求7所述的多肽构建体、权利要求8的分离的核酸分子、权利要求9的载体、权利要求10所述的宿主细胞、权利要求12所述的双特异性或多特异性抗体、权利要求13所述的缀合物、权利要求14所述的嵌合抗原受体、权利要求15的分离的核酸分子、权利要求16的载体、或权利要求17所述的宿主细胞；
    优选地，所述药物组合物还包含药学上可接受的载体和/或赋形剂；
    优选地，所述药物组合物包含权利要求1-6任一项的纳米抗体或其抗原结合片段、权利要求7所述的多肽构建体、权利要求8的分离的核酸分子、权利要求9的载体或权利要求10所述的宿主细胞；
    优选地，所述药物组合物包含权利要求12所述的双特异性或多特异性抗体；
    优选地，所述药物组合物包含权利要求13所述的缀合物；
    优选地，所述药物组合物包含权利要求14所述的嵌合抗原受体、权利要求15的分离的核酸分子、权利要求16的载体、或权利要求17所述的宿主细胞。
  19. 权利要求1-6任一项的纳米抗体或其抗原结合片段、权利要求7所述的多肽构建体、权利要求8的分离的核酸分子、权利要求9的载体、权利要求10所述的宿主细胞、权利要求12所述的双特异性或多特异性抗体、权利要求13所述的缀合物、权利要求14所述的嵌合抗原受体、权利要求15的分离的核酸分子、权利要求16的载体、权利要求17所述的宿主细胞、或权利要求18所述的药物组合物用于制备药物的用途，所述药物用于在受试者中预防和/治疗肿瘤；
    优选地，所述肿瘤为MSLN阳性的肿瘤；
    优选地，所述肿瘤选自实体瘤，例如胃癌、肺癌、卵巢癌、食道癌、胰腺癌、宫颈癌、间皮瘤或乳腺癌；
    优选地，所述受试者为哺乳动物，例如人；
    优选地，所述纳米抗体或其抗原结合片段、多肽构建体、分离的核酸分子、载体、宿主细胞、双特异性或多特异性抗体、缀合物、嵌合抗原受体、或药物组合物单独使用，或与另外的药学活性剂(例如抗肿瘤剂)联合使用。
  20. 用于在受试者中预防和/或治疗肿瘤的方法，其包括：给有此需要的受试者施用有效量的权利要求1-6任一项的纳米抗体或其抗原结合片段、权利要求7所述的多肽构建体、权利要求8的分离的核酸分子、权利要求9的载体、权利要求10所述的宿主细胞、权利要求12所述的双特异性或多特异性抗体、权利要求13所述的缀合物、权利要求14所述的嵌合抗原受体、权利要求15的分离的核酸分子、权利要求16的载体、权利要求17所述的宿主细胞、或权利要求18所述的药物组合物；
    优选地，所述肿瘤为MSLN阳性的肿瘤；
    优选地，所述肿瘤选自实体瘤，例如胃癌、肺癌、卵巢癌、食道癌、胰腺癌、宫颈癌、间皮瘤或乳腺癌；
    优选地，所述受试者为哺乳动物，例如人。
  21. 缀合物，其包含权利要求1-6任一项所述的纳米抗体或其抗原结合片段或权利要求7所述的多肽构建体，以及与所述纳米抗体或其抗原结合片段或多肽构建体连接的可检测的标记，例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
  22. 试剂盒，其包括权利要求1-6任一项所述的纳米抗体或其抗原结合片段或权利要求7所述的多肽构建体或权利要求21所述的缀合物；
    优选地，所述试剂盒包含权利要求21所述的缀合物；
    优选地，所述试剂盒包含权利要求1-6任一项所述的纳米抗体或其抗原结合片段或权利要求7所述的多肽构建体，以及特异性识别所述纳米抗体或其抗原结合片段或多肽构建体的第二抗体；任选地，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
  23. 用于检测MSLN在样品中的存在或其水平的方法，其包括使用权利要求1-6任一项所述的纳米抗体或其抗原结合片段或权利要求7所述的多肽构建体或权利要求21所述的缀合物；
    优选地，所述方法是免疫学检测，例如免疫印迹法、酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法；
    优选地，所述方法包括使用权利要求21所述的缀合物；
    优选地，所述方法包括使用权利要求1-6任一项所述的纳米抗体或其抗原结合片段或权利要求7所述的多肽构建体，并且所述方法还包括使用携带可检测的标记(例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素)的第二抗体来检测所述纳米抗体或其抗原结合片段或多肽构建体。
  24. 权利要求1-6任一项所述的纳米抗体或其抗原结合片段或权利要求7所述的多肽构建体或权利要求21所述的缀合物在制备用于检测MSLN在样品中的存在或其水平或者用于检测肿瘤是否能够通过靶向MSLN的抗肿瘤疗法来治疗的检测试剂中的用途；
    优选地，所述检测试剂通过权利要求23所述的方法来检测MSLN在样品中的存在或其水平以及任选地检测肿瘤是否能够通过靶向MSLN的抗肿瘤疗法来治疗；
    优选地，所述样品为来自受试者(例如哺乳动物，例如人)的细胞样品(例如包含肿瘤细胞的样品)或体液样品(例如血液)。
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