Anti-mesothelin nanobodies and uses thereof

Abstract

The present invention relates to Nanobodies or antigen-binding fragments thereof that specifically bind to MSLN, compositions comprising the Nanobodies or antigen-binding fragments thereof, nucleic acids encoding the antibodies or antigen-binding fragments thereof, and host cells comprising same, and related uses. Furthermore, the present invention relates to therapeutic and diagnostic uses of these antibodies or antigen-binding fragments thereof.

Description

[Technical field]

[0001] Technical Field The present application relates to nanobodies or antigen-binding fragments thereof that specifically bind to MSLN, compositions comprising the nanobodies or antigen-binding fragments thereof, nucleic acids encoding the antibodies or antigen-binding fragments thereof, and host cells comprising the same, and uses related thereto. Furthermore, the present invention relates to therapeutic and diagnostic uses of the antibodies or antigen-binding fragments thereof. [Background technology]

[0002] Background technology Mesothelin (MSLN) is a 40 kDa cell surface glycoprotein that is highly expressed in pancreatic cancer, ovarian cancer, mesothelioma, and several other cancers. MSLN may also be present in the blood of a small number of patients in the form of secreted soluble sMSLN, so measurement of MSLN in blood may be useful for diagnosing and tracking the condition of patients. 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 cleavage fragment called megakaryocyte potentiating factor (MPF), which is secreted from the cell. MSLN is not a cancer-specific antigen and can also be expressed in normal pleural, pericardial, and peritoneal mesothelial cells, but is highly expressed in a variety of cancer cells. The limited distribution of MSLN in normal tissues makes it a promising target for tumor-specific therapy.

[0003] Currently, various antibody drugs or cell therapies targeting MSLN have progressed to the clinical stage. For example, the antibody conjugate drug anetumab-ravtansine developed by Bayer, Immunogen, Morphosys and other companies is being used in Phase II clinical trials as a treatment for solid tumors (NCT03926143); in addition, the monoclonal antibody drug amatuximab targeting MSLN developed by Morphotek has also been used in clinical treatment of solid tumors and is currently in Phase II clinical trials; in addition, the Chinese People's Liberation Army Military Medical University and TCR 2Therapeutics is also developing a CAR-T therapy targeting MSLNs, both of which are currently in clinical trials. Most existing clinical trials have shown that mesothelin-targeting therapies are safe, but the therapeutic effects are modest.

[0004] Single domain antibodies (sdAbs), also known as nanobodies or heavy chain antibodies (hcAbs), are antibodies isolated from camel and shark serum, and their volume is about 1 / 10 of that of conventional antibodies. Unlike conventional antibodies, single domain antibodies are composed of only heavy chains, and their antigen-binding region is only a single domain connected to the Fc region via a hinge region, and this antigen-binding region still has the ability to bind to antigens after being separated from the antibody. Single domain antibodies are characterized by small molecular weight and excellent stability. In drug development and diagnostic reagent development, compared with conventional normal antibodies, they have good tissue penetration, flexible administration, high degree of humanization, easy conversion to recombinant proteins, and many other advantages. Summary of the Invention [Problem to be solved by the invention]

[0005] Contents of the Application After extensive research, the inventors of the present application screened and obtained an anti-MSLN nanobody. This nanobody has high binding activity to MSLN and cross-reactivity with human, monkey and / or mouse MSLN. In addition, the nanobody also has the characteristics of small molecular weight and good stability. In the development of pharmaceuticals and diagnostic reagents, compared with conventional general antibodies, it has good tissue penetration, flexible administration, high degree of humanization, easy conversion to recombinant protein, and many other advantages.

[0006] Based on this, the application also provides compositions comprising a Nanobody or an antigen-binding fragment thereof, nucleic acids encoding a Nanobody or an antigen-binding fragment thereof, host cells comprising same, and uses related thereto. [Means for solving the problem]

[0007] Nanobodies and antigen-binding fragments thereof Thus, in a first aspect, the present application provides a nanobody or an antigen-binding fragment thereof capable of specifically binding to MSLN. The nanobodies described herein are generally composed of four framework regions (FRs) and three complementarity determining regions (CDRs), referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. An antigen-binding fragment comprises at least a portion of the nanobody sufficient to confer the fragment's ability to specifically bind to MSLN. In some embodiments, the nanobodies of the present application can be truncated at the N-terminus or C-terminus to include only a portion of FR1 and / or FR4, or to lack one or two of these framework regions, so long as they substantially retain their ability and specificity to bind to the antigen.

[0008] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: CDR1 or a variant thereof, CDR2 or a variant thereof, or CDR3 or a variant thereof contained in a variable region (VHH) set forth in any one of SEQ ID NOs: 4 and 6 to 9; Including, A variant has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0009] In certain embodiments, the CDRs are defined according to the IMGT, Kabat or Chothia numbering systems.

[0010] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: CDR1 or a variant thereof having the sequence set forth in SEQ ID NO:1, CDR2 or a variant thereof having the sequence set forth in SEQ ID NO:2, and CDR3 or a variant thereof having the sequence set forth in SEQ ID NO:3; Including, A variant has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0011] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises a CDR1 having the sequence set forth in SEQ ID NO:1, a CDR2 having the sequence set forth in SEQ ID NO:2, and a CDR3 having the sequence set forth in SEQ ID NO:3.

[0012] In certain embodiments, the CDRs are defined by the IMGT numbering system. In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (i) the sequence set forth in SEQ ID NO:4; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two, three, four or five amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO:4; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:4. The amino acid sequence is selected from the group consisting of:

[0013] In certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the Nanobody or antigen-binding fragment thereof is humanized.

[0014] In certain embodiments, the Nanobody, or antigen-binding fragment thereof, further comprises a human immunoglobulin heavy chain framework region (e.g., a heavy chain framework region comprised in an amino acid sequence encoded by a human immunoglobulin heavy chain germline gene), said heavy chain framework region optionally comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) back mutations from human residues to camelid residues.

[0015] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (i) FR1 as set forth in SEQ ID NO: 10, or FR1 having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 10, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to SEQ ID NO: 10; (ii) FR2 set forth in any one of SEQ ID NOs: 11 to 13, or a FR2 having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to any one of SEQ ID NOs: 11 to 13, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to any one of SEQ ID NOs: 11 to 13; (iii) FR3 set forth in any one of SEQ ID NOs: 14 to 16, or an FR3 having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to any one of SEQ ID NOs: 14 to 16, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to any one of SEQ ID NOs: 14 to 16; and / or (iv) FR4 as set forth in SEQ ID NO: 17, or a FR4 having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 17, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to SEQ ID NO: 17. Includes.

[0016] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (i) a sequence set forth in any one of SEQ ID NOs: 6 to 9; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two, three, four or five amino acid substitutions, deletions or additions) compared to the sequence set forth in any one of SEQ ID NOs: 6 to 9; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to any one of the sequences set forth in SEQ ID NOs: 6 to 9. The amino acid sequence is selected from the group consisting of:

[0017] In certain embodiments, the substitutions are conservative substitutions. Polypeptide Constructs In a second aspect, the present application also provides a polypeptide construct capable of specifically binding to MSLN, comprising an aforementioned Nanobody or an antigen-binding fragment thereof and an immunoglobulin Fc domain.

[0018] In this context, Fc domain, also referred to as Fc region, refers to a portion of the heavy chain constant region that includes CH2 and CH3 domains. In some embodiments, the Fc domain includes a hinge, a CH2 domain, and a CH3 domain. When the Fc domain includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc domain can be any antibody heavy chain constant region isotype. In some embodiments, the Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain.

[0019] In certain embodiments, the Fc domain contained in the polypeptide construct of the present application is a native Fc region that comprises an amino acid sequence that matches the amino acid sequence of the Fc region found in nature.For example, the Fc domain can be a human IgG1 Fc region with native sequence, a human IgG2 Fc region with native sequence, a human IgG3 Fc region with native sequence, or a human IgG4 Fc region with native sequence.The native Fc region can have effector functions.Exemplary "effector functions" include binding to Fc receptors; C1q binding (Clq binding) and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and activation of B cells, etc. Functional alterations can be made by replacing at least one amino acid residue in the native Fc region with a different residue or by chemical modification, including, for example, altering the affinity of the antibody for an effector ligand (e.g., FcR or complement component C1q), thereby altering (e.g., decreasing or enhancing) effector function.

[0020] Thus, in certain embodiments, the Fc domain comprised in the polypeptide construct of the present application may be a variant Fc region that may contain one or more (e.g., 1 to 10, such as 1 to 5) amino acid mutations or chemical modifications compared to the native Fc region to alter one or more of the following properties of the antibodies of the present application: Fc receptor binding, glycosylation of the antibody, the number of cysteine ​​residues, effector cell function, or complement function.

[0021] In certain embodiments, the Fc domain contained in the polypeptide construct of the present application has ADCC activity. In certain embodiments, the Fc domain contained in the polypeptide construct of the present application does not have ADCC activity.

[0022] In certain embodiments, the immunoglobulin Fc domain is linked to the N-terminus and / or C-terminus (e.g., the C-terminus) of the Nanobody or antigen-binding fragment thereof, optionally via a peptide linker.

[0023] In certain embodiments, the immunoglobulin Fc domain is an IgG Fc domain (eg, an IgG1 Fc domain).

[0024] In certain embodiments, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO:5, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) thereto.

[0025] Preparation of Nanobody and Polypeptide Constructs The Nanobody or polypeptide construct of the present application can be prepared by various methods known in the art, for example, obtained by recombinant genetic engineering techniques. For example, a DNA molecule encoding the Nanobody or polypeptide construct of the present application can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and transfected into a host cell. The transfected host cell is then cultured under specific conditions to express the antibody or polypeptide construct of the present application.

[0026] The antigen-binding fragments of the present application can be obtained by hydrolysis of intact Nanobody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Furthermore, these antigen-binding fragments can also be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). The skilled artisan will be familiar with other techniques for preparing such antigen-binding fragments.

[0027] nucleic acid molecule In a third aspect, the application also provides an isolated nucleic acid molecule encoding the above-mentioned Nanobody or antigen-binding fragment thereof, or the above-mentioned polypeptide construct.

[0028] vector In a fourth aspect, the present application also provides a vector comprising the above-mentioned nucleic acid molecule. In certain embodiments, the vector is a cloning vector or an expression vector.

[0029] host cell In a fifth aspect, the present application also provides a host cell comprising the above-mentioned nucleic acid molecule or vector. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells such as mouse cells, human cells, etc.).

[0030] Preparation method In a sixth aspect, the present application also provides a method for preparing a Nanobody or antigen-binding fragment thereof as described above, or a polypeptide construct as described above, comprising culturing a host cell as described above under conditions allowing expression of the protein, and recovering the Nanobody or antigen-binding fragment thereof, or the polypeptide construct from the culture of the cultured host cells.

[0031] Bispecific or multispecific antibodies In a seventh aspect, 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 a Nanobody or an antigen-binding fragment thereof or a polypeptide construct of the present application, or a nucleic acid molecule, vector or host cell encoding same, in the manufacture of a bispecific or multispecific antibody.

[0032] In certain embodiments, the bispecific antibody or said multispecific antibody specifically binds to MSLN and also specifically binds to one or more other targets.

[0033] In certain embodiments, the bispecific antibody or said multispecific antibody further comprises at least one second antibody having a second binding specificity for a second target.

[0034] Conjugates In an eighth aspect, the present application also provides a conjugate comprising said Nanobody or antigen-binding fragment thereof or said polypeptide construct and a therapeutic agent conjugated to said Nanobody or antigen-binding fragment thereof or said polypeptide construct. The present application also provides the use of the Nanobody or antigen-binding fragment thereof or polypeptide construct of the present application, or a nucleic acid molecule, vector or host cell encoding it, in the manufacture of a conjugate.

[0035] In certain embodiments, the therapeutic agent is selected from the group consisting of antineoplastic agents, such as cytotoxic agents, hormonal agents, biological response modifiers, additional antibodies or antigen-binding fragments thereof.

[0036] Chimeric Antigen Receptor In a ninth aspect, 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 (e.g. a primary signaling domain and / or a costimulatory signaling domain), wherein the antigen binding domain comprises a Nanobody or antigen binding fragment thereof as described above. The present application also provides the use of a Nanobody or antigen binding fragment thereof or polypeptide construct of the present application, or a nucleic acid molecule, vector or host cell encoding it, in the manufacture of a chimeric antigen receptor or an immune cell expressing a chimeric antigen receptor.

[0037] In certain embodiments, the antigen-binding domain confers on the chimeric antigen receptor the ability to recognize MSLN; the spacer domain promotes structural flexibility of the protein, allowing movement of one or two domains relative to one another; the transmembrane domain may be thermodynamically stable when associated with a cell membrane (particularly the membrane of a eukaryotic cell); and the intracellular signaling domain is involved in transmitting an effective antigen receptor-binding signal to the interior of the immune effector cell, activating at least one normal effector function of the CAR-expressing immune effector cell, or enhancing secretion of at least one cytokine by the CAR-expressing immune effector cell.

[0038] In certain embodiments, the chimeric antigen receptor is expressed by an immune effector cell (e.g., a T cell).

[0039] In a tenth aspect, the present application also provides an isolated nucleic acid molecule encoding the above-mentioned chimeric antigen receptor.

[0040] In an eleventh aspect, the present application also provides a vector comprising the isolated nucleic acid molecule described above. In a specific embodiment, the isolated nucleic acid molecule is used for the preparation of chimeric antigen receptor T cells.

[0041] In a twelfth aspect, the present application also provides a host cell comprising the above-mentioned isolated nucleic acid molecule or vector.

[0042] In certain embodiments, the host cell is an immune effector cell (eg, a T cell or a NK cell).

[0043] In certain embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0044] Pharmaceutical Compositions In a thirteenth aspect, the application also provides a pharmaceutical composition comprising the Nanobody or antigen-binding fragment thereof of the first aspect, the polypeptide construct of the second aspect, the bispecific or multispecific antibody of the seventh aspect, the conjugate of the eighth aspect, the chimeric antigen receptor of the ninth aspect, the isolated nucleic acid molecule of the third or tenth aspect, the vector of the fourth or eleventh aspect, or the host cell of the fifth or twelfth aspect.

[0045] In certain embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier and / or excipient.

[0046] In certain exemplary embodiments, the pharma- ceutically acceptable carrier and / or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a surfactant-containing solution (e.g., a solution containing 0.01% polysorbate 20), a pH buffer (e.g., phosphate buffer), Ringer's solution, and any combination thereof.

[0047] In certain embodiments, 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 vector of the fourth aspect, or the host cell of the fifth aspect.

[0048] In certain embodiments, the pharmaceutical composition comprises the bispecific or multispecific antibody of the seventh aspect above.

[0049] In certain embodiments, the pharmaceutical composition comprises a conjugate according to the eighth aspect above. In certain embodiments, the pharmaceutical composition comprises the chimeric antigen receptor of the ninth aspect, the isolated nucleic acid molecule of the tenth aspect, the vector of the eleventh aspect, or the host cell of the twelfth aspect above.

[0050] Medical Use In a fourteenth aspect, the application also provides the use of the Nanobody or antigen-binding fragment thereof of the first aspect, the polypeptide construct of the second aspect, the bispecific or multispecific antibody of the seventh aspect, the conjugate of the eighth aspect, the chimeric antigen receptor of the ninth aspect, the isolated nucleic acid molecule of the third or 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 in the manufacture of a medicament for preventing and / or treating a tumor in a subject.

[0051] In certain embodiments, the tumor is an MSLN-positive tumor. In certain embodiments, the tumor is selected from the group consisting of 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, for example, Aurore et al., Cancer Discovery, 2016).

[0052] In certain embodiments, the subject is a mammal, such as a human. In certain embodiments, 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 pharmaceutical composition is used alone or in combination with an additional pharma- ceutical active agent (e.g., an anti-neoplastic agent).

[0053] Methods for preventing and / or treating tumors In a fifteenth aspect, the application also provides a method for preventing and / or treating a tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of the nanobody or antigen-binding fragment thereof of the first aspect, the polypeptide construct of the second aspect, the bispecific or multispecific antibody of the seventh aspect, the conjugate of the eighth aspect, the chimeric antigen receptor of the ninth aspect, the isolated nucleic acid molecule of the third or 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.

[0054] In certain embodiments, the tumor is an MSLN-positive tumor. In certain embodiments, the tumor is selected from the group consisting of solid tumors, such as gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma or breast cancer.

[0055] In certain embodiments, the subject is a mammal, such as a human. The nanobody or antigen-binding fragment thereof, polypeptide construct, bispecific or multispecific antibody, or pharmaceutical composition of the present application may be formulated into any dosage form known in the medical art, such as tablets, pills, suspensions, emulsions, liquids, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including solutions for injection, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic application. The nanobody or antigen-binding fragment thereof, polypeptide construct, bispecific or multispecific antibody, or pharmaceutical composition of the present application should be sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injectable solution. Such an injectable solution may be a sterile injectable solution. For example, sterile injectable solutions can be prepared by incorporating the required amount of the nanobody or antigen-binding fragment thereof, polypeptide construct, bispecific or multispecific antibody, or pharmaceutical composition of the present application in an appropriate solvent, optionally together with other desired ingredients (including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, isotonicity agents, preservatives, diluents, or combinations thereof), followed by filter sterilization. Moreover, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or lyophilization) for ease of storage and use. Such sterile, lyophilized powders may be dispersed in a suitable carrier prior to use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a surfactant-containing solution (e.g., a solution containing 0.01% polysorbate 20), a pH buffer (e.g., phosphate buffer), Ringer's solution, and any combination thereof.

[0056] The 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, ophthalmic, topical, parenteral, rectal, intrathecal, intracisternal, inguinal, intravesical, body surface (e.g., powder, ointment, or infusion), or nasal routes. However, for many therapeutic applications, the preferred route / mode of administration is parenteral administration (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, the nanobodies or antigen-binding fragments thereof, polypeptide constructs, bispecific or multispecific antibodies, or pharmaceutical compositions of the present application are administered by intravenous injection or bolus injection.

[0057] Detection Applications Conjugates In a sixteenth aspect, 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 detectable label conjugated to the Nanobody or an antigen-binding fragment thereof or the polypeptide construct. In certain embodiments, the detectable label is, for example, an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide or biotin.

[0058] kit In a seventeenth aspect, the present application also provides a kit comprising a Nanobody or an antigen-binding fragment thereof as described above, or a polypeptide construct as described above, or a conjugate of the sixteenth aspect described above.

[0059] In certain embodiments, the kit comprises a conjugate according to the sixteenth aspect above. In certain embodiments, the kit comprises a Nanobody or antigen-binding fragment thereof as described above, or a polypeptide construct as described above, and a second antibody capable of specifically recognizing the Nanobody or antigen-binding fragment thereof, or the polypeptide construct; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0060] Detection Method In an 18th aspect, the present application also provides a method for detecting the presence or level of MSLN in a sample, the method comprising using a nanobody or an antigen-binding fragment thereof as described above, or a polypeptide construct as described above, or a conjugate of the 16th aspect above. In certain embodiments, the method is used for therapeutic, diagnostic or non-therapeutic non-diagnostic purposes.

[0061] In certain embodiments, 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.

[0062] In certain embodiments, the method comprises using a conjugate of the sixteenth aspect above.

[0063] In certain embodiments, the method comprises using a Nanobody or an antigen-binding fragment thereof as described above, or a polypeptide construct as described above, and the method further comprises using a second Nanobody with a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin) to detect the Nanobody or antigen-binding fragment thereof, or the polypeptide construct.

[0064] In certain embodiments, the method comprises (1) contacting a sample with a Nanobody or antigen-binding fragment thereof of the present application, a polypeptide construct of the present application, or a conjugate of the sixteenth aspect of the present application; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex is indicative of the presence of MSLN or an MSLN-expressing cell.

[0065] In certain embodiments, the methods are used to detect whether a tumor is treatable by an anti-tumor therapy that targets MSLN.

[0066] Use for the preparation of detection reagents In a nineteenth aspect, the present application also provides the use of a nanobody or an antigen-binding fragment thereof as described above, or a polypeptide construct as described above, or a conjugate of the sixteenth aspect as described above, in the manufacture of a detection reagent for detecting the presence or level of MSLN in a sample, or for detecting whether a tumor is treatable by an anti-tumor therapy targeting MSLN.

[0067] In certain embodiments, the detection reagent detects the presence or level of MSLN in a sample, and optionally detects whether a tumor is treatable with an anti-tumor therapy that targets MSLN by the method of the 18th aspect above.

[0068] In certain embodiments, the sample is a cell sample (eg, a sample containing tumor cells) or a bodily fluid sample (eg, blood) from a subject (eg, a mammal, eg, a human).

[0069] Definition of Terms In this application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.In addition, the experimental procedures of virology, biochemistry and immunology used herein are routine procedures widely used in the corresponding fields.Meanwhile, in order to better understand this application, the definitions and explanations of related terms are provided below.

[0070] When the terms "eg," "such as," "for example," "comprise," "include," or variations thereof are used in this specification, these terms are not to be regarded as limiting terms and instead are to be interpreted to mean "without limitation" or "not limited to."

[0071] Unless otherwise indicated herein or clearly contradicted by context, in the context of describing this application (and particularly in the context of the claims which follow), the terms "a," "an," and "the" and similar references are to be construed as encompassing the singular and the plural.

[0072] As used herein, the term "camelid antibody" refers to an antibody against an antigen produced by immunizing or challenging a camelid (including camels, alpacas, and llamas (L.glama)) with the antigen. It is known to those skilled in the art that among the antibodies produced by camelids, there are "camelid heavy chain antibodies (HCAb)" that lack light chains. Such antibodies contain only one HCAb heavy chain variable domain (VHH) and two conventional CH2 and CH3 regions, and the VHH regions, which are cloned and expressed separately, have good structural stability and antigen binding activity. VHH is currently known as the smallest unit capable of binding to a target antigen.

[0073] As used herein, the term "nanobody" has its meaning commonly understood by those skilled in the art and refers to an antibody fragment that is usually derived from the variable region of a heavy chain antibody (e.g., a camel or shark antibody) and consists of a single monomeric variable antibody domain (e.g., a single heavy chain variable region). Typically, nanobodies are composed of four framework regions and three complementarity determining regions and have the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus to include only a portion of FR1 and / or FR4, or to lack one or two of these framework regions, so long as they substantially retain antigen binding ability and specificity. Nanobodies are also called single domain antibodies (sdAbs), the two being used interchangeably.

[0074] As used herein, the term "antigen-binding fragment" of a Nanobody refers to a polypeptide comprising a fragment of a Nanobody, also referred to as an "antigen-binding portion", that retains the ability to specifically bind to the same antigen as the Nanobody binds and / or competes with the Nanobody for specific binding of an antigen. See generally Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, New York (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the antibodies of the present application may be obtained by recombinant DNA techniques or by enzymatic or chemical cleavage of the Nanobodies of the present application. In some embodiments, compared to full-length Nanobodies, "antigen-binding fragments" of Nanobodies may be truncated at the N-terminus or C-terminus to include only a portion of FR1 and / or FR4, or to lack one or two of these framework regions, as long as they substantially retain antigen-binding ability and specificity.

[0075] Antigen-binding fragments of a Nanobody can be obtained from a given Nanobody (e.g., a Nanobody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and antigen-binding fragments of a Nanobody can be screened for specificity in the same way as intact Nanobodies.

[0076] As used herein, when the term "nanobody" is mentioned, it includes not only intact nanobodies but also antigen-binding fragments of nanobodies, unless the context clearly indicates otherwise.

[0077] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Nanobodies contain three CDRs, named CDR1, CDR2, and CDR3. The exact 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., (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given Nanobody, one of skill in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those of skill in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0078] As used herein, the term "framework region" or "FR" residues refers to the amino acid residues of an antibody variable domain other than the CDR residues defined above.

[0079] As used herein, the term "Fc domain" or "Fc region" refers to a portion of the heavy chain constant region, including the CH2 and CH3 domains. The Fc fragment of an antibody has many different functions, but is not involved in antigen binding. "Effector functions" mediated by the Fc region include Fc receptor binding; C1q binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation. In some embodiments, the Fc region includes a hinge, a CH2 domain, and a CH3 domain. When the Fc region includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of an antibody heavy chain constant region isotype, such as IgG1, IgG2, IgG3, or IgG4.

[0080] The Fc domain may comprise either a native Fc region or a variant Fc region. A native Fc region comprises an amino acid sequence that corresponds to the amino acid sequence of an Fc region found in nature, for example, a native human Fc region comprises a human IgG1 (non-A and A allotype) Fc region with a native sequence; a human IgG2 Fc region with a native sequence; a human IgG3 Fc region with a native sequence; and a human IgG4 Fc region with a native sequence, as well as naturally occurring variants thereof. A variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of a native Fc region by at least one amino acid modification. In some embodiments, a variant Fc region may have an effector function (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function) that is altered compared to a native Fc region.

[0081] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered and has an altered amino acid sequence to enhance sequence homology with that of a human antibody. Generally speaking, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., FRs and / or constant regions of the variable region) are derived from a human immunoglobulin (receptor antibody). In certain embodiments, the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., FRs and / or constant regions of the variable region) are derived from a human immunoglobulin (receptor antibody). A humanized antibody generally retains the expected properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, etc. In the present application, the donor antibody may be a camelid antibody having the desired properties (e.g., antigen specificity, affinity, reactivity, etc.). To prepare a humanized antibody, the CDR regions of the antibody of an immunized animal can be inserted into a human framework sequence using methods known in the art. In the context of nanobodies, a humanized antibody can refer to a humanized VHH, i.e., a VHH in which one or more framework regions are substantially replaced with human framework regions. In some cases, certain framework regions (FR) of a human immunoglobulin are replaced with corresponding non-human residues. Furthermore, a humanized VHH may contain residues that are not found in the original VHH or in the human framework sequence, but are included to further improve and optimize the performance of the VHH or VHH-containing polypeptide.

[0082] As used herein, the term "identity" is used to mean the sequence match between two polypeptides or two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the first amino acid sequence or nucleic acid sequence to best match the second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of overlapping identical positions / total number of positions×100%). In certain embodiments, both sequences are the same length.

[0083] Determining percent identity between two sequences can also be accomplished using a mathematical algorithm.A non-limiting example of a mathematical algorithm for comparing two sequences is Karlin and Altschul, 1990, Proc.Natl. Acad.Sci. USA 87:2264-2268, which was improved by Karlin and Altschul, 1993, Proc.Natl. Acad.Sci. USA 90:5873-5877.Such an algorithm is integrated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0084] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction is determined by the equilibrium dissociation constant (K D In this application, the term "K D" 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 binding between the antibody and the antigen and the higher the affinity between the antibody and the antigen.

[0085] The specific binding properties between two molecules can be determined using methods known in the art. One approach is to measure the rates of formation and dissociation of the antigen-binding site / antigen complex. The "association rate constant" (k a or k on ) and "dissociation rate constant" (k dis or k off ) can both be calculated from the concentrations and the actual association and dissociation rates (see Malmqvist M, Nature, 1993, 361:186-187). dis / k on is the dissociation constant K D (Davies et al., Annual Rev Biochem, 1990; 59:439-473). D , k on , and k dis The value of can be measured by any effective method. In certain embodiments, the dissociation constant can be measured by surface plasmon resonance (SPR) using a Biacore instrument. Bioluminescence interferometry or Kinexa can also be used to measure the dissociation constant.

[0086] As used herein, a detectable label of the present application can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art and examples include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I, 35 S, 14 C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as ruthenium terpyridine), calorimetric markers such as magnetic beads (e.g., Dynabeads®), colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin that binds to avidin (e.g., streptavidin) modified with the above labels.

[0087] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. If the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, transfection so that the genetic material element it carries can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), P1-derived artificial chromosomes (PACs); phages 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, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40). Vectors may contain a variety of expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may also contain an origin of replication.

[0088] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as E. coli, Bacillus subtilis, yeast cells, fungal cells such as 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, HEK293 cells or human cells.

[0089] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the expected properties of a protein / polypeptide that contains the amino acid sequence. For example, 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 those in which an amino acid residue is replaced with another amino acid residue that has a similar side chain, such as one that is physically or functionally similar to the corresponding amino acid residue (e.g., one that has similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to substitute a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0090] The twenty conventional amino acids included herein are described according to conventional usage. See, for example, Immunology-A Synthesis (2nd ed., ES Golub and DR Gren, eds., Sinauer Associates, Sunderland, Massachusetts (1991)), which is incorporated herein by reference. In this application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And, in this application, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0091] As used herein, the term "pharmaceutical acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th ed., Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, 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 enhancing agents 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 for maintaining osmolarity include, but are not limited to, sugars, NaCl, etc. Agents for delaying absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (e.g., buffered saline), alcohols, and polyols (e.g., glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art and are capable of stabilizing the desired activity of the active ingredient in a pharmaceutical product, and include, but are not limited to, sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein), or degradation products thereof (e.g., lactalbumin hydrolysate), and the like.In certain exemplary embodiments, the pharma- ceutically acceptable carrier or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a surfactant-containing solution (e.g., a solution containing 0.01% polysorbate 20), a pH buffer (e.g., phosphate buffer), Ringer's solution, and any combination thereof.

[0092] As used herein, the term "prevention" refers to a method carried out to prevent or delay the occurrence of a disease or condition or symptom in a subject. As used herein, the term "treatment" refers to a method carried out to obtain a beneficial or desired clinical outcome. For the purposes of this application, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief of symptoms (whether partial or complete). In addition, "treatment" can mean prolonging survival compared to the expected survival if not treated.

[0093] As used herein, the term "subject" refers to a mammal, such as a human, monkey, mouse, etc. In certain embodiments, the subject (e.g., a human, monkey, mouse) has or is at risk for a disease associated with MSLN (e.g., an MSLN-positive tumor).

[0094] As used herein, the term "effective amount" refers to an amount sufficient to at least partially obtain a desired effect. For example, a prophylactically effective amount is an amount sufficient to prevent, inhibit or delay the onset of a disease (e.g., MSLN-positive tumor); a therapeutically effective amount is an amount sufficient to cure or at least partially prevent an existing disease and its complications in a patient suffering from the disease. It is well within the capabilities of a person skilled in the art to determine such an effective amount. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient, such as age, weight and sex, the method of drug administration, other therapeutic agents administered at the same time, etc. Effect of the Invention

[0095] Beneficial Effects of the Invention The present application provides a nanobody with high binding activity to MSLN, which has cross-reactivity with human, monkey and / or mouse MSLN. In addition, nanobodies are characterized by small molecular weight and good stability. In the development of pharmaceuticals and diagnostic reagents, compared with conventional general antibodies, they have good tissue penetration, flexible administration, high degree of humanization, easy conversion to recombinant proteins, and many other advantages.

[0096] Thus, the nanobodies of the present application can be used for various purposes, including but not limited to, inhibition of tumor growth and detection of MSLN. Furthermore, the fully humanized antibodies of the present application can be safely administered to human subjects without eliciting an immunogenic response. Thus, the antibodies of the present application have significant clinical value.

[0097] Hereinafter, the embodiments of the present application will be described in detail with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to explain the present application and do not limit the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments. [Brief description of the drawings]

[0098] [Figure 1] FIG. 1 shows the results of detection of the affinity of anti-MSLN nanobodies to CHO-hMSLN cells. [Diagram 2] FIG. 2 shows the results of detection of the affinity of humanized anti-MSLN nanobodies to CHO-hMSLN cells. [Diagram 3] FIG. 3 shows the results of affinity detection of humanized anti-MSLN nanobodies for CHO-cyMSLN cells. [Figure 4] FIG. 4 shows the results of affinity detection of humanized anti-MSLN nanobodies for CHO-mMSLN cells. [Diagram 5] FIG. 5 shows the results of detecting the inhibitory activity of humanized anti-MSLN nanobodies that inhibit the binding of human MSLN to its ligand CA125. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0099] Sequence information The sequences covered in this application are set out in the table below.

[0100] [Table 1-1]

[0101] [Table 1-2]

[0102] Specific Model for Implementing the Present Application The present application will now be described with reference to the following examples which are intended to illustrate, but not limit, the present application.

[0103] Unless otherwise specified, the molecular biological experiments and immunoassays used in this application were essentially performed with reference to the methods described in J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989 and FM Ausubel et al., Compiled Laboratory Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; restriction enzymes were used according to the conditions recommended by the product manufacturers. Those skilled in the art will understand that the examples are illustrative of the present application and are not intended to limit the scope of the present application as claimed.

[0104] Example 1: Immunization and screening of anti-MSLN nanobodies After immunization of an alpaca (Llama) with human MSLN (purchased from AcroBiosystems, catalog number: MSN-H522a), total RNA was extracted from the alpaca's peripheral lymphocytes and reverse transcribed to obtain cDNA. The PCR products of the cDNA were ligated into a yeast display vector and then electrotransformed into Saccharomyces cerevisiae (purchased from ATCC, catalog number: 208289) to construct an anti-MSLN nanobody library.

[0105] Human MSLN was labeled according to the product instructions of the biotin labeling kit (purchased from Thermo, catalog number: 90407). The grown anti-MSLN nanobody yeast library was labeled with biotin-labeled MSLN, and then positively labeled yeast was enriched using magnetic beads. The enriched yeast cells were grown and stained with anti-c-Myc antibody (purchased from Thermo, catalog number: MA1-980) diluted 1:200 and an appropriate amount of biotin-labeled MSLN. The yeast cells were washed with PBS, and then goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 (purchased from Invitrogen, catalog number: A32723TR) and streptavidin APC conjugated fluorescent antibody (purchased from Invitrogen, catalog number: SA1005) diluted 1:500 were added and incubated for 15 minutes. The cells were resuspended in PBS and sorted using a BD FACSAria II instrument to obtain yeast cells with high binding ability to human MSLN.

[0106] Yeast cells with high binding ability to human MSLN obtained by magnetic bead enrichment and flow cytometry sorting were cultured overnight at 30°C and 225 rpm in growth medium, and yeast plasmids were extracted according to the procedure of a yeast plasmid extraction kit (purchased from Tiangen (catalog number DP112)). The plasmids were electrotransformed into Top10 competent cells (purchased from Tiangen, catalog number: CB104-02), coated on ampicillin-resistant plates, and cultured overnight at 37°C. Single clones were selected and sequenced to obtain the sequences of the VHH (variable region) genes, and the sequences of the CDR regions were determined according to the IMGT numbering system. The sequence information of the obtained monoclonal nanobody YE-17 is shown in the table below.

[0107] [Table 2]

[0108] Example 2: Construction of expression vectors, protein expression and purification of anti-MSLN nanobodies The coding sequence of the VHH of the screened anti-MSLN antibody YE-17 and the coding sequence of the human IgG1 Fc segment (SEQ ID NO: 5) were assembled by homologous recombination into a fusion protein expression sequence in which the human IgG1 Fc segment was linked to the C-terminus of the VHH. Using the ExpiCHO™ Expression System kit (purchased from Thermo, catalog number: A2910001), the prepared fusion protein expression plasmid was transferred in medium amounts to Expi-CHO cells (purchased from Thermo, catalog number: A2910002) according to the transfection method described in the product instructions. After 5 days of cell culture, the supernatant was collected and the target protein was purified using Protein A magnetic beads (purchased from GenScript, catalog number: L00723). The magnetic beads were resuspended in an appropriate amount (1-4 times the amount of magnetic beads) of binding buffer (PBS + 0.1% Tween 20, pH 7.4), added to the sample to be purified, and incubated at room temperature for 1 hour while gently shaking. The sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer. After adding 3-5 times the amount of elution buffer (0.1 M sodium citrate, pH 3.2) of the magnetic beads, shaking at room temperature for 5-10 minutes, and returning to the magnetic stand, the elution buffer was collected and transferred to a collection tube containing neutralization buffer (1 M Tris, pH 8.54), and mixed well to complete the preparation, and purified anti-MSLN nanobody YE-17 was obtained.

[0109] Example 3: Detection of protein binding affinity of anti-MSLN nanobodies ForteBio affinity measurements were performed according to existing methods (Estep, P et al., High throughput solution based measurement of antibody-antigen affinity and epitope binning, MAbs, 2013.5(2):p.270-8). Briefly, the sensor was equilibrated offline in analysis buffer for 30 min, followed by online detection for 60 s to establish a baseline, and the purified antibody obtained in Example 2 was loaded online onto the AHQ sensor. The sensor was then placed in 100 nM human MSLN (Uniprot ID: Q13421) for 5 min, after which the sensor was transferred to PBS for dissociation for 5 min. Kinetic analysis was performed using a 1:1 binding model. In addition, the antibody amatuximab (CAS number: 931402-35-6) was set up in parallel as a control group. The amino acid sequences of the heavy and light chains of the antibody amatuximab are shown in SEQ ID NOs: 18-19. The results are shown in Table 3.

[0110] [Table 3]

[0111] Example 4: Detection of cell binding affinity of anti-MSLN nanobodies CHO cells overexpressing human MSLN (Uniprot ID: Q13421), i.e., CHO-hMSLN cells, were prepared by transfection with pCHO1.0 vector (purchased from Invitrogen) containing MSLN cDNA. Cultured CHO-MSLN cells were grown at a cell density of 2 × 10 6The concentration of MSLN antibody YE-17 was adjusted to 100 cells / ml, added to a 96-well flow plate at 100 μL per well, and centrifuged for later use. The purified anti-MSLN antibody YE-17 prepared in Example 2 was diluted 3-fold in PBS starting from 400 nM for a total of 12 steps. The diluted samples were added to the 96-well flow plate containing cells at 100 μL per well, incubated at 4° C. for 30 minutes, and washed twice with PBS. Goat F(ab')2 anti-human IgG-Fc(PE) (purchased from Abcam, catalog number: ab98596) diluted 100-fold in PBS was added at 100 μL per well, incubated at 4° C. for 30 minutes, and washed twice with PBS. The cells were resuspended by adding 100 μl of PBS per well, detected using a CytoFlex (Becman) flow cytometer, and the corresponding MFI (mean fluorescence intensity) values ​​were calculated.

[0112] The experimental results are shown in Figure 1 and Table 4. The experimental results showed that the anti-MSLN antibody YE-17 prepared in Example 2 had binding activity to CHO-hMSLN cells, and its binding activity was higher than that of Morphotek's single-chain control antibody amatuximab.

[0113] [Table 4]

[0114] Example 5: Detection of protein binding affinity of humanized anti-MSLN nanobodies In this example, the antibody YE-17 was humanized. Then, the vector was constructed, and the humanized antibody was expressed and purified based on the sequence of the humanized antibody according to the method described in Example 2. Finally, four humanized antibody strains, namely, HZ-P-YE-17-01, HZ-P-YE-17-02, HZ-P-YE-17-03 and HZ-P-YE-17-04, were obtained based on YE-17, and their VHH sequences are shown in SEQ ID NOs: 6 to 9, respectively.

[0115] The protein binding affinity of the purified humanized antibodies was detected according to the method described in Example 3, and the results are shown in Table 5.

[0116] [Table 5]

[0117] Example 6: Detection of binding ability and cross-reactivity of humanized anti-MSLN nanobodies to CHO-hMSLN The purified humanized antibodies were subjected to detection of affinity for CHO-hMSLN cells according to the method described in Example 4.

[0118] The results are shown in Figure 2 and Table 6. The results demonstrated that all of the humanized anti-MSLN antibodies prepared in Example 5 had binding activity to CHO-hMSLN cells.

[0119] [Table 6]

[0120] To identify the affinity of the humanized antibody to monkey MSLN (cyMSLN, Uniprot ID: F6Q1U7) at the cellular level, a cell line was constructed according to the method described in Example 4, and the affinity of the purified humanized antibody to CHO-cyMSLN cells was detected.

[0121] The results are shown in Figure 3 and Table 7. The results showed that both antibody YE-17 and its humanized anti-MSLN antibody had binding activity to CHO-cyMSLN cells.

[0122] [Table 7]

[0123] To identify the affinity of the antibody to mouse MSLN (mMSLN, Uniprot ID: Q61468) at the cellular level, a cell line was constructed according to the method described in Example 4 to detect the affinity of the purified humanized antibody to CHO-mMSLN cells, and the results are shown in Figure 4 and Table 8.

[0124] [Table 8]

[0125] Combining the results of Tables 6 to 8 and Figures 2 to 4, it was shown that antibody YE-17 and its humanized antibodies have cross-binding activity against human and monkey MSLN cells, and that antibody YE-17 and some of its humanized antibodies (e.g., HZ-P-YE-17-01, HZ-P-YE-17-02, HZ-P-YE-17-03) also have a certain cross-binding activity against human, monkey and mouse MSLN cells.

[0126] Example 7: Inhibition of binding between human MSLN and its ligand CA125 by anti-MSLN humanized nanobodies To identify the inhibitory activity of the antibody that inhibits the binding of human MSLN to its ligand CA125, experimental validation was performed according to the following method: 1 μg / mL human MSLN was coated on a high-adsorption ELISA plate, and the coating was performed overnight at 4°C; then, the coating solution was discarded and blocking was performed with 5% BSA at room temperature for 2 hours; serially diluted humanized antibodies were added and incubated at room temperature for 1 hour; the plate was washed three times, and then 1 μg / mL biotin-CA125 (purchased from Acro, catalog number: CA5-H82F4) was added and incubated at room temperature for 1 hour; the plate was washed three times, and then 1:10000 diluted SA-HRP (purchased from Abcam, catalog number: Ab7403) was added and incubated at room temperature for 30 minutes; the plate was washed six times, and then 50 μl / mL TMB color development solution was added and incubated for 3 to 5 minutes; 50 μl / mL stop solution was added; and the A450 value was read by machine.

[0127] The results are shown in Figure 5 and Table 9. The results showed that both antibody YE-17 and its humanized antibody could effectively inhibit the binding of human MSLN to its ligand CA125, thereby inhibiting cancer invasion and metastasis.

[0128] [Table 9]

[0129] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings disclosed, and these modifications are within the scope of protection of the present application. The entire application is provided by the appended claims and their equivalents.

Claims

1. A nanobody or antigen-binding fragment thereof capable of specifically binding to MSLN, CDR1 or a variant thereof, CDR2 or a variant thereof, and CDR3 or a variant thereof contained in the variable region (VHH) set forth in any one of SEQ ID NOs: 4 and 6 to 9. Including, The variant has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; Preferably, the CDRs are defined according to the IMGT, Kabat or Chothia numbering systems. Nanobodies or antigen-binding fragments thereof.

2. CDR1 or a variant thereof having the sequence set forth in SEQ ID NO: 1, CDR2 or a variant thereof having the sequence set forth in SEQ ID NO: 2, and CDR3 or a variant thereof having the sequence set forth in SEQ ID NO:

3. Including, The variant has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; Preferably, the Nanobody or antigen-binding fragment thereof comprises a CDR1 having the sequence set forth in SEQ ID NO: 1, a CDR2 having the sequence set forth in SEQ ID NO: 2, and a CDR3 having the sequence set forth in SEQ ID NO: 3; Preferably, the CDRs are defined by the IMGT numbering system.

2. The nanobody or antigen-binding fragment thereof of claim 1.

3. (i) the sequence set forth in SEQ ID NO: 4; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two, three, four or five amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 4; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in SEQ ID NO:4 comprising an amino acid sequence selected from Preferably, the substitution is a conservative substitution.

2. The nanobody or antigen-binding fragment thereof of claim 1.

4. It is humanized, Preferably, the antibody further comprises a human immunoglobulin heavy chain framework region (e.g., a heavy chain framework region comprised in an amino acid sequence encoded by a human immunoglobulin heavy chain germline gene), said heavy chain framework region optionally comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) backmutations of human residues to camelid residues.

2. The nanobody or antigen-binding fragment thereof of claim 1, The nanobody or antigen-binding fragment thereof comprises: Preferably, (i) an FR1 as set forth in SEQ ID NO: 10, or an FR1 having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 10, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to SEQ ID NO: 10; (ii) an FR2 set forth in any one of SEQ ID NOs: 11-13, or an FR2 having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to any one of SEQ ID NOs: 11-13, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to any one of SEQ ID NOs: 11-13; (iii) an FR3 set forth in any one of SEQ ID NOs: 14-16, or an FR3 having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to any one of SEQ ID NOs: 14-16, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to any one of SEQ ID NOs: 14-16; and / or (iv) FR4 set forth in SEQ ID NO: 17, or an FR4 having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 17, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:

17. Including, Preferably, (a) a sequence set forth in any one of SEQ ID NOs: 6 to 9; (b) a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) compared to the sequence set forth in any one of SEQ ID NOs: 6 to 9; or (c) comprising an amino acid sequence selected from a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence set forth in any one of SEQ ID NOs: 6-9; Preferably, said substitutions are conservative substitutions.

5. A polypeptide construct capable of specifically binding to MSLN, comprising a Nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 4 and an immunoglobulin Fc domain, Preferably, the immunoglobulin Fc domain is linked to the N-terminus and / or C-terminus (e.g., C-terminus) of the Nanobody or antigen-binding fragment thereof, optionally via a peptide linker; Preferably, the immunoglobulin Fc domain is an IgG Fc domain (e.g., an IgG1 Fc domain); Preferably, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 5, a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) thereto. Polypeptide constructs.

6. An isolated nucleic acid molecule encoding a nanobody or antigen-binding fragment thereof described in any one of claims 1 to 4, or a polypeptide construct comprising said nanobody or antigen-binding fragment thereof and an immunoglobulin Fc domain.

7. A vector comprising the nucleic acid molecule of claim 6; preferably a cloning vector or an expression vector.

8. A host cell comprising the nucleic acid molecule described in claim 6 or a vector containing the nucleic acid molecule.

9. A method for preparing a nanobody or an antigen-binding fragment thereof or a polypeptide construct, comprising culturing a host cell described in claim 8 under conditions allowing protein expression, and recovering the nanobody or an antigen-binding fragment thereof or a polypeptide construct from a culture of the cultured host cells.

10. A bispecific or multispecific antibody comprising a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a polypeptide construct comprising said Nanobody or antigen-binding fragment thereof and an immunoglobulin Fc domain; Preferably, it specifically binds to MSLN and also specifically binds to one or more other targets; Preferably, the antibody further comprises at least one second antibody having a second binding specificity for a second target. Bispecific or multispecific antibodies.

11. A conjugate comprising a nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a polypeptide construct comprising said nanobody or antigen-binding fragment thereof and an immunoglobulin Fc domain, and a therapeutic agent linked to said nanobody or antigen-binding fragment thereof or said polypeptide construct; Preferably, the therapeutic agent is selected from the group consisting of a cytotoxic agent, a hormonal agent, a biological response modifier, an anti-tumor agent such as an additional antibody or antigen-binding fragment thereof. Conjugates.

12. A chimeric antigen receptor comprising an antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding domain comprises the nanobody or antigen-binding fragment thereof of any one of claims 1 to 4; Preferably, the chimeric antigen receptor is expressed by an immune effector cell (e.g., a T cell). Chimeric antigen receptor.

13. An isolated nucleic acid molecule encoding the chimeric antigen receptor described in claim 12.

14. A vector comprising the isolated nucleic acid molecule of claim 13; preferably, the vector is used to prepare chimeric antigen receptor T cells.

15. A host cell comprising the isolated nucleic acid molecule of claim 13 or a vector comprising said isolated nucleic acid molecule; Preferably, it is an immune effector cell (e.g., a T cell or an NK cell); Preferably, it is a chimeric antigen receptor T cell (CAR-T). host cell.

16. A pharmaceutical composition comprising: (i) a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4; (ii) a polypeptide construct comprising said Nanobody or antigen-binding fragment thereof and an immunoglobulin Fc domain; (iii) an isolated nucleic acid molecule, a vector, a host cell comprising a nucleic acid sequence encoding said Nanobody or antigen-binding fragment thereof or said polypeptide construct; (iv) a bispecific or multispecific antibody comprising said Nanobody or antigen-binding fragment thereof or said polypeptide construct; (v) a conjugate comprising said Nanobody or antigen-binding fragment thereof or said polypeptide construct and a therapeutic agent linked thereto; (vi) a chimeric antigen receptor; or (vii) an isolated nucleic acid molecule, a vector, or a host cell comprising a nucleic acid sequence encoding said chimeric antigen receptor; the chimeric antigen receptor comprises an antigen-binding domain comprising the nanobody or an antigen-binding fragment thereof, a spacer domain, a transmembrane domain, and an intracellular signaling domain; Preferably, further comprising a pharmaceutically acceptable carrier and / or excipient; Preferably, the nanobody or antigen-binding fragment thereof, the polypeptide construct, the isolated nucleic acid molecule, the vector, or the host cell comprising a nucleic acid sequence encoding the nanobody or antigen-binding fragment thereof or the polypeptide construct; Preferably, said bispecific or multispecific antibody comprises: Preferably, the conjugate comprises: Preferably, the chimeric antigen receptor, isolated nucleic acid molecule, vector, or host cell comprising a nucleic acid sequence encoding the chimeric antigen receptor. Pharmaceutical compositions.

17. (i) a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, (ii) a polypeptide construct comprising said Nanobody or antigen-binding fragment thereof and an immunoglobulin Fc domain, (iii) an isolated nucleic acid molecule, a vector, a host cell comprising a nucleic acid sequence encoding said Nanobody or antigen-binding fragment thereof, or said polypeptide construct, (iv) a bispecific or multispecific antibody comprising said Nanobody or antigen-binding fragment thereof, or said polypeptide construct, (v) a conjugate comprising said Nanobody or antigen-binding fragment thereof, or said polypeptide construct, and a therapeutic agent linked thereto, (vi) a chimeric antigen receptor, or (vii) a host cell comprising an isolated nucleic acid molecule, a vector, or a nucleic acid sequence encoding said chimeric antigen receptor, or (viii) a pharmaceutical composition comprising any of the above, for preventing and / or treating a tumor in a subject; the chimeric antigen receptor comprises an antigen-binding domain comprising the nanobody or an antigen-binding fragment thereof, a spacer domain, a transmembrane domain, and an intracellular signaling domain; Preferably, the tumor is an MSLN-positive tumor; Preferably, the tumor is selected from the group consisting of solid tumors such as gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma or breast cancer; Preferably, the subject is a mammal, such as a human; Preferably, said Nanobody or antigen-binding fragment thereof, polypeptide construct, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific antibody, conjugate, chimeric antigen receptor, or pharmaceutical composition is administered alone or in combination with an additional pharmaceutically active agent (e.g., an anti-neoplastic agent), Pharmaceutical compositions.

18. A conjugate comprising a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a polypeptide construct comprising said Nanobody or antigen-binding fragment thereof and an immunoglobulin Fc domain, and a detectable label linked to said Nanobody or antigen-binding fragment thereof or said polypeptide construct; for example, wherein said detectable label is an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide or biotin.

19. A kit comprising: (i) a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, (ii) a polypeptide construct comprising said Nanobody or antigen-binding fragment thereof and an immunoglobulin Fc domain, or (iii) a conjugate comprising said Nanobody or antigen-binding fragment thereof or said polypeptide construct and a therapeutic agent linked thereto; Preferably, the conjugate comprises: Preferably, it comprises said Nanobody or antigen-binding fragment thereof or said polypeptide construct and a second antibody capable of specifically recognizing said Nanobody or antigen-binding fragment thereof or said polypeptide construct; optionally, said second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide or biotin. kit.

20. 1. A method for detecting the presence or level of MSLN in a sample and / or for detecting whether a tumor is treatable by an anti-tumor therapy that targets MSLN, comprising the use of (i) a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, (ii) a polypeptide construct comprising said Nanobody or antigen-binding fragment thereof and an immunoglobulin Fc domain, or (iii) a conjugate comprising said Nanobody or antigen-binding fragment thereof or said polypeptide construct and a therapeutic agent linked thereto; Preferably, it is an immunological assay such as a Western blot, an enzyme-linked immunosorbent assay (e.g., ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay; Preferably, the method comprises using said conjugate; Preferably, the method comprises using said Nanobody or antigen-binding fragment thereof or said polypeptide construct, and further comprises using a second antibody with a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide or biotin) to detect said Nanobody or antigen-binding fragment thereof or said polypeptide construct, Preferably, the method, wherein the sample is a cell sample (e.g., a sample containing tumor cells) or a body fluid sample (e.g., blood) from a subject (e.g., a mammal, e.g., a human).

Images