Antigen-binding protein and use thereof

Abstract

A nanobody specifically binding to HER2 or an antigen-binding fragment thereof, a composition comprising the nanobody or the antigen-binding fragment thereof, and a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof. In addition, the present invention further relates to the use of the nanobody or the antigen-binding fragment thereof in the treatment and diagnosis of diseases.

Description

Antigen binding proteins and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311639416.3 filed on December 1, 2023, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to Nanobodies or antigen-binding fragments thereof that specifically bind to HER2, compositions containing such Nanobodies or antigen-binding fragments thereof, and nucleic acid molecules encoding such antibodies or antigen-binding fragments thereof. Furthermore, the present invention also relates to the use of such Nanobodies or antigen-binding fragments thereof in the treatment and diagnosis of diseases. Background Art

[0004] HER2, or human epidermal growth factor receptor-2, also known as ErbB-2, is a 185 kDa receptor tyrosine kinase (RTK) in the epidermal growth factor receptor (EGFR) family. HER2 forms homodimers or heterodimers with three other members of the EGFR family: HER1 (EGFR, ErbB-1), HER3 (ErbB-3), and HER4 (ErbB-4). This leads to phosphorylation of receptor tyrosine residues, activating multiple signaling pathways associated with cell proliferation and tumor development, such as the PI3K-AKT-mTOR pathway. Currently, no ligands that directly bind to HER2 have been identified. Furthermore, ErbB2 can reduce the expression of cyclin D and c-myc. These proteins sequester the cyclin-dependent kinase (CDK) inhibitor p27kip1, preventing it from inhibiting CDK2 activity and ultimately leading to increased cell proliferation.

[0005] As research continues to expand and deepen, HER2 has been found to be expressed and overexpressed in a variety of tumors. Currently, HER2 has been reported to be overexpressed in a variety of cancer types, including breast cancer, gastric cancer, lung cancer, intestinal cancer, and ovarian cancer. HER2 overexpression is also associated with a poor prognosis in breast cancer. Therefore, there is an urgent need for effective HER2-targeting drugs to treat malignant tumors. Currently available monoclonal antibodies targeting HER2 include trastuzumab and pertuzumab.

[0006] Trastuzumab is a humanized monoclonal antibody targeting HER2 developed by Genentech in the United States (patent numbers US5677171A and US5821337A). Trastuzumab exerts its anti-tumor activity by specifically binding to the extracellular domain IV of HER2, inducing antibody-dependent cellular cytotoxicity (ADCC) and inhibiting HER2-based signaling. Trastuzumab has shown good therapeutic effects on tumors that overexpress HER2. In 1998, the FDA approved trastuzumab in combination with paclitaxel for the first-line treatment of patients with HER2-overexpressing advanced breast cancer, or as a single agent for the treatment of HER2-overexpressing metastatic breast cancer that has undergone at least one cycle of chemotherapy.

[0007] Pertuzumab, also developed by Genentech (Patent No. US7862817B2), binds to the extracellular domain II of the HER2 receptor, inhibiting dimer formation and thereby suppressing receptor-mediated signaling pathways. In 2012, the US FDA approved pertuzumab in combination with trastuzumab and docetaxel for the treatment of patients with HER2-positive advanced metastatic breast cancer.

[0008] Over the past 20 years, HER2-targeted therapy has significantly improved the prognosis of patients with HER2-overexpressing breast cancer. However, only approximately 15% of breast cancer patients overexpress HER2, while the remaining approximately 85% of patients with HER2-low-expressing or negative breast cancer are in urgent need of more effective HER2-targeted therapy. Secondly, HER2-positive breast cancer is prone to metastasis, especially in the brain. The molecular weight of conventional antibodies is approximately 150kDa. Single-domain antibodies / nanoantibodies derived from alpacas, after fusion with human Fc, have a molecular weight of only approximately 75kDa, only half that of conventional antibodies. Compared with standard monoclonal antibodies, they have a smaller structure and a stronger ability to penetrate the blood-brain barrier, allowing them to penetrate into the core of the tumor, maximizing the chances of antibody drugs binding to tumor cells, and producing better results in diagnosing and treating tumors.

[0009] Therefore, providing a single-domain antibody against HER2 will have better prospects for diagnosing and treating tumors. Summary of the Invention

[0010] After extensive research, the inventors of this application have screened and obtained a series of anti-HER2 nanobodies and polypeptide constructs. These nanobodies or polypeptide constructs have high binding activity to HER2 and do not compete with trastuzumab or pertuzumab for binding to the HER2 antigen. In particular, the nanobodies or polypeptide constructs of the present invention have endocytosis activity and can mediate the entry of toxins into cells through endocytosis, thereby playing a role in killing tumor cells. On this basis, humanized nanobodies or polypeptide constructs prepared from these nanobodies still have these outstanding activities. In addition, the nanobodies or polypeptide constructs also have the characteristics of small molecular weight and easy production.

[0011] Based on this, the present application also provides a conjugate containing the nanobody or its antigen-binding fragment, a nucleic acid molecule encoding the nanobody or its antigen-binding fragment and a host cell containing the same, as well as related uses.

[0012] Nanobodies and their antigen-binding fragments

[0013] In one aspect, the invention provides nano antibodies or antigen-binding fragments thereof that specifically bind to HER2. The nano antibodies described herein are generally composed of four framework regions (FRs) and three complementary determining regions (CDRs), referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and the antigen-binding fragment comprises at least a portion of the nano antibody that is sufficient to confer the ability of the fragment to specifically bind to HER2. In some embodiments, the nano antibodies described herein can be truncated at the N-terminus or C-terminus so that it comprises only part of FR1 and / or FR4, or lack one or two of those framework regions, as long as it substantially maintains antigen binding and specificity.

[0014] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises: CDR1, CDR2 and CDR3 contained in the VHH shown in any one of SEQ ID NOs: 15-18.

[0015] In certain embodiments, the CDRs are defined according to the Kabat, Chothia, or IMGT numbering systems.

[0016] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises: a CDR1 as shown in SEQ ID NO: 1; a CDR2 as shown in SEQ ID NO: 2 or 3; and a CDR3 as shown in SEQ ID NO: 4; wherein the CDRs are defined according to the Kabat numbering system.

[0017] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises: a CDR1 as shown in SEQ ID NO: 5; a CDR2 as shown in SEQ ID NO: 6; and a CDR3 as shown in SEQ ID NO: 7; wherein the CDRs are defined according to the IMGT numbering system.

[0018] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises: a CDR1 as shown in SEQ ID NO: 8; a CDR2 as shown in SEQ ID NO: 9 or 10; and a CDR3 as shown in SEQ ID NO: 11; wherein the CDRs are defined according to the Kabat numbering system; or

[0019] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises: a CDR1 as shown in SEQ ID NO: 12; a CDR2 as shown in SEQ ID NO: 13; and a CDR3 as shown in SEQ ID NO: 14; wherein the CDRs are defined according to the IMGT numbering system.

[0020] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises the framework region sequence of a camelid antibody.

[0021] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 15, 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 several amino acid substitutions, deletions or additions (for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) thereto. In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 15.

[0022] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 17, 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 several amino acid substitutions, deletions or additions (for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) thereto. In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 17.

[0023] In certain embodiments, such Nanobodies or antigen-binding fragments thereof are humanized, ie wherein one or more framework regions have been essentially replaced by human framework regions.

[0024] In certain embodiments, the Nanobody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (for example, a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline antibody gene), which heavy chain framework region optionally comprises one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to camel residues.

[0025] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 16, 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 several amino acid substitutions, deletions or additions (for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) thereto. In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 16.

[0026] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 18, 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 several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) thereto. In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 18.

[0027] polypeptide constructs

[0028] In another aspect, the present invention also provides a polypeptide construct that specifically binds to HER2, comprising a Nanobody or antigen-binding fragment thereof of the present invention and an immunoglobulin Fc domain.

[0029] As used herein, the Fc domain, also referred to as the Fc region, refers to the portion of the heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc domain comprises a hinge, CH2, and CH3. When the Fc domain comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc domain can be of any antibody heavy chain constant region isotype. In some embodiments, the Fc domain is IgG1, IgG2, IgG3, or IgG4.

[0030] In certain embodiments, the Fc domain included in the polypeptide construct of the present invention is a native Fc region, which comprises an amino acid sequence consistent with the amino acid sequence of the Fc region found in nature. For example, the Fc domain can be a native sequence human IgG1 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 cell surface receptors (e.g., B cell receptors); and B cell activation, etc. Functional changes can be produced by replacing at least one amino acid residue in the native Fc region with a different residue or chemical modification, for example, changing the affinity of the antibody for effector ligands (such as FcR or complement Clq), thereby changing the effector function (e.g., reducing or enhancing).

[0031] Therefore, in certain embodiments, 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 (e.g., 1-10, such as 1-5) amino acid mutations or chemical modifications compared to a native Fc region to alter one or more of the following properties of the antibody of the present invention: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function, etc. In certain embodiments, the Fc domain comprised by the polypeptide construct of the present invention has reduced or eliminated effector function, such as an Fc domain comprising the LALA mutation.

[0032] In certain embodiments, the immunoglobulin Fc domain is linked to the N-terminus and / or C-terminus (eg, the C-terminus) of the Nanobody or antigen-binding fragment thereof, optionally via a peptide linker.

[0033] In certain embodiments, the immunoglobulin Fc domain is an Fc domain of IgG (eg, an Fc domain of IgG1, IgG2, IgG3, or IgG4).

[0034] In certain embodiments, the immunoglobulin Fc domain is a human immunoglobulin Fc domain, such as an Fc domain of human IgG (eg, an Fc domain of human IgG1, IgG2, IgG3, or IgG4).

[0035] In certain embodiments, the immunoglobulin Fc domain comprises the sequence of SEQ ID NO: 19 or 20, 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. In certain embodiments, the immunoglobulin Fc domain comprises the sequence of SEQ ID NO: 19 or 20.

[0036] In certain embodiments, the polypeptide construct comprises a sequence as shown in any one of SEQ ID NOs: 21-24, 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 several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) thereto.

[0037] Preparation of nanobodies and peptide constructs

[0038] The Nanobodies or polypeptide constructs of the present invention can be prepared using various methods known in the art, for example, by genetic engineering recombinant technology. For example, a DNA molecule encoding a Nanobody or polypeptide construct of the present 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. The transfected host cell is then cultured under specific conditions and expresses the antibody or polypeptide construct of the present invention.

[0039] In another aspect, the invention also provides an isolated nucleic acid molecule encoding a Nanobody or antigen-binding fragment thereof of the invention or a polypeptide construct of the invention.

[0040] In another aspect, the present invention also provides a vector comprising the nucleic acid molecule of the present invention. In certain embodiments, the vector is a cloning vector or an expression vector.

[0041] In another aspect, the present invention also provides a host cell comprising a nucleic acid molecule or vector of the present invention. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., Escherichia coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.).

[0042] In another aspect, the invention also provides a method for preparing a Nanobody or an antigen-binding fragment thereof of the invention or a polypeptide construct of the invention, comprising culturing a host cell of the invention under conditions that allow protein expression, and recovering said Nanobody or its antigen-binding fragment or said polypeptide construct from the culture of the cultivated host cell.

[0043] Bispecific or multispecific antibodies

[0044] In another aspect, the invention also provides bispecific or multispecific antibodies comprising a Nanobody or antigen-binding fragment thereof of the invention or a polypeptide construct of the invention. The invention also provides the use of a Nanobody or antigen-binding fragment thereof or polypeptide construct of the invention, or a nucleic acid molecule, vector or host cell encoding the same, for the preparation of a bispecific or multispecific antibody.

[0045] In certain embodiments, the bispecific or multispecific antibody specifically binds HER2 and additionally specifically binds one or more other targets.

[0046] In certain embodiments, the bispecific or multispecific antibody further comprises at least one second antibody with a second binding specificity for a second target.

[0047] Conjugate

[0048] In another aspect, the invention also provides a conjugate comprising a Nanobody or antigen-binding fragment thereof of the invention or a polypeptide construct of the invention or a bispecific or multispecific antibody of the invention and a coupling portion linked thereto.

[0049] In certain embodiments, the conjugated moiety is selected from a therapeutic agent (eg, a cytotoxic agent, a cytokine, a toxin, or a radionuclide).

[0050] In certain embodiments, the conjugate is an antibody-drug conjugate (ADC).

[0051] Pharmaceutical composition

[0052] In another aspect, the invention also provides a pharmaceutical composition comprising a Nanobody or antigen-binding fragment thereof, a polypeptide construct, an isolated nucleic acid molecule, a vector, a host cell, a bispecific or multispecific antibody or conjugate of the invention, and a pharmaceutically acceptable carrier and / or excipient.

[0053] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as an anti-tumor agent.

[0054] In certain embodiments, in the pharmaceutical composition, the Nanobody or antigen-binding fragment thereof, polypeptide construct, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific antibody or conjugate of the invention and the additional pharmaceutically active agent can be provided as separate components or as mixed components.

[0055] Nanobodies or antigen-binding fragments thereof, polypeptide constructs, isolated nucleic acid molecules, vectors, host cells, bispecific or multispecific antibodies, conjugates or pharmaceutical compositions of the invention 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 injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, a sterile injection solution can be prepared by the following method: incorporating the necessary dose of an antibody or antigen-binding fragment thereof of the invention into an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use.

[0056] In certain exemplary embodiments, the pharmaceutically 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 sterile injectable liquid is selected from 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 solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0057] The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the Nanobodies or antigen-binding fragments thereof, isolated nucleic acid molecules, vectors, host cells, bispecific or multispecific antibodies, or conjugates described herein. A "prophylactically effective amount" is an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0058] Detection Application

[0059] Reagent test kit

[0060] In another aspect, the invention also provides a kit comprising a Nanobody or antigen-binding fragment thereof of the invention or a polypeptide construct of the invention.

[0061] In certain embodiments, the kit comprises a conjugate comprising a Nanobody of the invention or an antigen-binding fragment thereof or a polypeptide construct of the invention and a detectable label linked to said Nanobody or antigen-binding fragment thereof or polypeptide construct, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as an acridinium ester, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (such as fluorescein or a fluorescent protein), a radionuclide or biotin.

[0062] In certain embodiments, the kit comprises a Nanobody or antigen-binding fragment thereof of the invention or a polypeptide construct of the invention, and a second antibody that specifically recognizes said Nanobody or antigen-binding fragment thereof or polypeptide construct; optionally, the second antibody further comprises a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (such as fluorescein or a fluorescent protein), a radionuclide or biotin.

[0063] Chimeric antigen receptor

[0064] On the other hand, the present application also provides a chimeric antigen receptor, which comprises the antigen-binding domain of the aforementioned nanobody or antigen-binding fragment thereof or the aforementioned polypeptide construct.

[0065] In certain embodiments, the antigen binding domain is expressed by immune effector cells (e.g., T cells).

[0066] Isolated nucleic acid molecules

[0067] In another aspect, the present application also provides an isolated nucleic acid molecule encoding the chimeric antigen receptor as described above.

[0068] carrier

[0069] In another aspect, the present application also provides a vector comprising the isolated nucleic acid molecule as described above. In certain embodiments, the vector is used to prepare chimeric antigen receptor T cells.

[0070] host cells

[0071] On the other hand, the application also provides a host cell comprising an isolated nucleic acid molecule as described above or a vector as described above. In certain embodiments, the host cell is an immune effector cell (e.g., T cell or NK cell). In certain embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0072] Pharmaceutical uses

[0073] In another aspect, the invention also provides the use of the Nanobodies or antigen-binding fragments thereof, polypeptide constructs, isolated nucleic acid molecules, vectors, host cells, bispecific or multispecific antibodies, conjugates or pharmaceutical compositions of the invention for the preparation of a medicament for preventing and / or treating a tumor in a subject.

[0074] In certain embodiments, the tumor is a HER2-positive tumor.

[0075] In certain embodiments, the medicament is used to inhibit the growth of tumor cells expressing HER2 and / or kill the tumor cells.

[0076] In certain embodiments, the medicament further comprises an additional pharmaceutically active agent.

[0077] In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

[0078] In certain embodiments, the tumor is selected from a solid tumor, such as gastric cancer, lung cancer, liver cancer, head and neck cancer, skin cancer, colorectal cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, colorectal cancer, or oral squamous cell carcinoma.

[0079] In certain embodiments, the subject is a mammal, such as a human.

[0080] Methods for preventing and / or treating tumors

[0081] In another aspect, the invention also provides a method for preventing and / or treating a tumor in a subject, comprising administering to a subject in need thereof an effective amount of a Nanobody or antigen-binding fragment thereof, polypeptide construct, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific antibody, conjugate or pharmaceutical composition of the invention.

[0082] In certain embodiments, the tumor is a HER2-positive tumor.

[0083] In certain embodiments, the tumor is selected from a solid tumor, such as gastric cancer, lung cancer (such as non-small cell lung cancer), liver cancer, head and neck cancer, skin cancer, colorectal cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, colorectal cancer, or oral squamous cell carcinoma.

[0084] In certain embodiments, the subject is a mammal, such as a human.

[0085] The nanobodies or antigen-binding fragments thereof, polypeptide constructs, bispecific or multispecific antibodies, conjugates or pharmaceutical compositions of the invention 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 injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The nanobodies or antigen-binding fragments thereof, polypeptide constructs, bispecific or multispecific antibodies, conjugates or pharmaceutical compositions of the invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injectable solutions can be prepared by the following method: incorporating the necessary dose of the nanobody or antigen-binding fragment thereof, polypeptide construct, bispecific or multispecific antibody, conjugate or pharmaceutical composition of the invention in an appropriate solvent, and optionally, other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof) in a suitable solvent, followed by filtration sterilization. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for ease of storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before 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 solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0086] Nanobodies or antigen-binding fragments thereof, polypeptide constructs, bispecific or multispecific antibodies, conjugates or pharmaceutical compositions of the invention can 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 (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled person will appreciate that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, Nanobodies or antigen-binding fragments thereof, polypeptide constructs, bispecific or multispecific antibodies, conjugates or pharmaceutical compositions of the invention are administered by intravenous or bolus injection.

[0087] Detection method

[0088] In another aspect, the invention also provides methods for detecting the presence or level of HER2 in a sample, comprising using nano antibodies of the invention or its antigen-binding fragment or polypeptide construct of the invention. In certain embodiments, the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic non-diagnostic purposes.

[0089] In certain embodiments, the method is an immunological assay, such as immunoblotting, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0090] In certain embodiments, the methods comprise the use of a conjugate comprising a Nanobody of the invention or an antigen-binding fragment thereof or a polypeptide construct of the invention and a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as an acridinium ester, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (such as fluorescein or a fluorescent protein), a radionuclide or biotin, linked to said Nanobody or antigen-binding fragment thereof or polypeptide construct.

[0091] In certain embodiments, the method comprises the use of a Nanobody or antigen-binding fragment thereof of the invention or a polypeptide construct of the invention, and the method further comprises detecting the Nanobody or antigen-binding fragment thereof or polypeptide construct using a second antibody carrying a detectable label (e.g. an enzyme (e.g. horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g. an acridinium ester, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g. fluorescein or a fluorescent protein), a radionuclide or biotin).

[0092] In certain embodiments, the method comprises: (1) contacting the sample with a Nanobody of the invention or an antigen-binding fragment thereof, a polypeptide construct of the invention, or a conjugate comprising a Nanobody of the invention or an antigen-binding fragment thereof or a polypeptide construct of the invention and a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin, linked to the Nanobody or its antigen-binding fragment or polypeptide construct; 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 indicates the presence of HER2 or a cell expressing HER2.

[0093] In certain embodiments, the method is used to detect whether a tumor can be treated by the anti-tumor therapy of targeting HER2. In such embodiments, the sample is from a subject, and the subject suffers from a tumor, suspects suffering from a tumor, or has a risk of suffering from a tumor. In certain embodiments, the sample is a cell sample (such as a sample comprising a tumor cell) or a body fluid sample (such as blood) from a subject (such as a mammal, such as people). In certain embodiments, when there is HER2 or the cell expressing HER2, and / or compared with a reference level (such as compared with a patient without a tumor disease), when the amount of HER2 or the cell expressing HER2 increases, it is shown that the subject is suitable for the anti-tumor therapy of targeting HER2.

[0094] Preparation of detection reagents

[0095] In another aspect, the present invention also provides the use of a Nanobody or antigen-binding fragment thereof of the invention or a polypeptide construct of the invention in the preparation of a detection reagent for detecting the presence or level of HER2 in a sample or for detecting whether a tumor can be treated by an anti-tumor therapy targeting HER2.

[0096] In certain embodiments, the detection reagent detects the presence or level of HER2 in a sample and optionally detects whether a tumor can be treated with an anti-tumor therapy targeting HER2 by the method of the present invention as described above.

[0097] In certain embodiments, the sample is a cell sample (eg, a sample comprising tumor cells) or a body fluid sample (eg, blood) from a subject (eg, a mammal, eg, a human).

[0098] Definition of terms

[0099] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0100] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0101] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0102] As used herein, the term "camel-derived antibodies" refers to antibodies produced by Camelidae animals (including camels, alpacas, and llamas) against an antigen following immunization or antigen invasion. It is known to those skilled in the art that camelid antibodies contain light chain-deficient "heavy-chain antibodies" (HCAbs). These antibodies consist of only a single heavy chain variable domain (VHH) and two conventional CH2 and CH3 regions. The individually cloned and expressed VHH region exhibits excellent structural stability and antigen-binding activity. VHH is the smallest unit currently known to bind to a target antigen.

[0103] As used herein, the terms "nanobody" or "single domain antibody" have the meanings commonly understood by those skilled in the art and are used interchangeably. They refer to antibody fragments consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, a nanobody consists of four framework regions and three complementarity determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A nanobody can be truncated at the N-terminus or C-terminus so that it comprises only part of FR1 and / or FR4, or lacks one or two of those framework regions, as long as it substantially maintains antigen binding and specificity.

[0104] As used herein, 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 bound by the Nanobody and / or competes with the Nanobody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the antibodies of the invention can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the Nanobodies of the invention. In some embodiments, the "antigen-binding fragment" of such a Nanobody may be truncated at the N-terminus or C-terminus compared to a full-length Nanobody so that it only comprises part of FR1 and / or FR4, or lacks one or both of those framework regions, as long as it substantially retains antigen binding and specificity.

[0105] Antigen-binding fragments of Nanobodies can be obtained from a given Nanobody (such as the Nanobodies provided herein) using conventional techniques known to those skilled in the art (for example, recombinant DNA techniques or enzymatic or chemical cleavage methods), and screened for specificity in the same manner as for intact Nanobodies.

[0106] Herein, unless the context clearly indicates otherwise, when referring to the term "single domain antibody" or "Nanobody", it includes not only complete Nanobodies but also antigen-binding fragments of Nanobodies.

[0107] 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. In nanobodies, there are three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, according to 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, a person skilled 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 skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). In this context, the CDRs of a Nanobody are preferably determined by the Kabat, Chothia and / or IMGT numbering systems.

[0108] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0109] As used herein, the term "Fc domain" or "Fc region" means a portion 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. The "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 cell surface receptors (e.g., B cell receptors); and B cell activation, among others. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. When the Fc region comprises a hinge, the hinge regulates the dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype, such as IgG1, IgG2, IgG3, or IgG4.

[0110] The Fc domain may include both a native Fc region and a variant Fc region. A native Fc region comprises an amino acid sequence that is consistent with the amino acid sequence 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 IgG3 Fc region; and a native sequence human IgG4 Fc region, 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 sequence Fc region due to at least one amino acid modification. In some embodiments, a variant Fc region may have altered effector functions (e.g., Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function) compared to a native Fc region.

[0111] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). In certain embodiments, the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc. In the present application, the donor antibody can be a camel-derived antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, etc.). To prepare humanized antibodies, the CDR region of an immune animal can be inserted into a human framework sequence using methods known in the art. In the context of nanobodies, humanized antibodies may refer to humanized VHHs, i.e., VHHs in which one or more framework regions have been substantially replaced by human framework regions. In some cases, certain framework regions (FRs) of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized VHHs may contain residues that are not found in the original VHH or human framework sequence, but are included to further improve and optimize the performance of the VHH or VHH-containing polypeptide.

[0112] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order 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., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). 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, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0113] The determination of percent identity between two sequences can also be achieved 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. Natl. Acad. Sci. USA 87: 2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0114] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates. In the present invention, 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 the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0115] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant K D(See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D , kon and kdis values. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0116] As used herein, the detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, and examples include, but are not limited to, 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 32 P), 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 terpyridine ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0117] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to 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), or P1-derived artificial chromosomes (PACs); bacteriophages, 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 (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0118] 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 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.

[0119] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or entirely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared to the expected survival (if not receiving treatment), extending the survival period.

[0120] As used herein, the terms "cancer" and "tumor" are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0121] Advantageous Effects of the Invention

[0122] The anti-HER2 nanobodies or polypeptide constructs provided herein have high binding activity to HER2. In particular, the nanobodies or polypeptide constructs of the present invention have endocytosis activity, which can mediate the entry of toxins into cells through endocytosis, thereby playing a role in killing tumor cells. On this basis, humanized nanobodies or polypeptide constructs prepared from the nanobodies or polypeptide constructs still have these outstanding activities. In addition, the nanobodies or polypeptide constructs also have the characteristics of small molecular weight and easy production.

[0123] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] FIG1 shows the binding ability of the candidate antibodies to human HER2 antigen detected by ELISA.

[0125] FIG2 shows the binding activity of the selected antibodies to SK-BR-3 cells in a FACS experiment.

[0126] FIG3A shows the internalization percentage of the selected antibodies in SK-BR-3 cells.

[0127] FIG3B shows the MFI differences of the selected antibodies on the surface of SK-BR-3 cells.

[0128] FIG4 shows the killing activity of the candidate antibody - Fab-ZAP against SK-BR-3 cells.

[0129] FIG5A shows the test results of whether the candidate antibodies compete with trastuzumab for binding to human recombinant HER2.

[0130] FIG5B shows the test results of whether the candidate antibodies compete with Pertuzumab for binding to human recombinant HER2.

[0131] FIG6 shows the ELISA binding curves of MHAB14-10 humanized antibody and human recombinant HER2.

[0132] FIG7 shows the ELISA binding curves of MHAB14-14 humanized antibody and human recombinant HER2.

[0133] FIG8 shows the flow cytometric binding curves of MHAB14-10 humanized antibody and SK-BR-3 cells.

[0134] FIG9 shows the flow cytometric binding curves of MHAB14-14 humanized antibody and SK-BR-3 cells.

[0135] FIG10 shows the internalization percentage of humanized antibodies in SK-BR-3 cells.

[0136] FIG11 shows the killing curve of humanized antibody-Fab-ZAP in SK-BR-3 cells.

[0137] FIG12 shows the test results of whether the antibody MHAB14-14 competes with trastuzumab or pertuzumab for binding to cell surface HER2 antigen.

[0138] FIG13 shows the ELISA binding curves of antibody MHAB14-14-6 to HER2 from different species.

[0139] FIG14 shows the ELISA binding curve of antibody MHAB14-14-6 to EGFR family proteins.

[0140] Sequence information

[0141] Information on the partial sequences involved in the present invention is provided in Table 1 below.

[0142] Table 1: Description of sequences DETAILED DESCRIPTION

[0143] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0144] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0145] Example 1. Acquisition of anti-human HER2 nanobody sequences

[0146] 1-1 Alpaca immunization and serum titer detection

[0147] A blank alpaca was immunized with the Her2 / ERBB2 Protein, Human, Recombinant (ECD, His Tag) antigen (Sinobiological, Cat. No. 10004-H08H). Before the initial immunization, 10 mL of blood was collected as negative serum. For the initial immunization, a mixture of 1 mg of HER2 antigen and complete Freund's adjuvant in a 1:1 ratio was administered subcutaneously at multiple sites. Subsequently, at weeks three, five, seven, nine, and eleven, 0.5 mg of HER2 antigen was mixed with incomplete Freund's adjuvant in a 1:1 ratio and injected subcutaneously at multiple sites. Before the third, fourth, fifth, and sixth immunizations, 10 mL of peripheral blood was collected, serum was isolated, and immune responses were monitored using ELISA. Five days after the sixth immunization, 50 mL of peripheral blood was collected for subsequent nanobody library construction.

[0148] 1-2 Screening and identification of specific nanoantibodies

[0149] Peripheral blood was collected from immunized alpacas, RNA was extracted, and cDNA samples were prepared. VHH antibody-encoding genes were cloned by PCR to construct a single-domain antibody phage display library. Randomly selected clones were then analyzed for library capacity and diversity.

[0150] Using Her2 / ERBB2 Protein, Human, Recombinant (ECD, His Tag) antigen (Sinobiological, Cat. No. 10004-H08H), a single-domain antibody phage display library was subjected to panning and enrichment experiments. After screening, single clones were selected for Phage-ELISA detection, and candidate clones with different amino acid sequences in the CDR region were selected. Finally, 40 unique sequences were selected.

[0151] 1-3 Unique sequence solubility verification and antibody expression

[0152] Plasmids from the 40 positive clones were extracted and transformed into E. coli Rosetta, where expression was induced overnight with 0.4 mM IPTG at 20°C, 200 rpm. The next day, the pellets were centrifuged, resuspended in PBS, and ultrasonically disrupted. The supernatants from the 40 clones were then centrifuged to obtain the supernatants. Binding activity of the supernatants from the 40 clones to the human HER2 antigen was assayed using ELISA (OD450 values ​​are shown in Table 2 below).

[0153] Twenty-six unique sequences with strong human HER2 binding activity were selected, and the VHH sequences were integrated into the Fc (LALA) sequence of human IgG1 (SEQ ID NO: 19). Transient transfection was performed in CHO cells to produce MHAB14 candidate antibodies (the correspondence between the application numbers and the screened unique sequences is shown in Table 3. Subsequent descriptions of the MHAB14 candidate antibodies will use the application numbers, i.e., MHAB14-1 to 26). After expression, the antibodies were purified using Protein A and dissolved in PBS. The antibody solutions had a purity (SEC, 280 nm) of >95% and an endotoxin level (EU / mg) of <1. Aliquots were stored at -80°C.

[0154] Table 2. OD450 results of 40 clones

[0155] Table 3. Number correspondence

[0156] Example 2. Anti-human HER2 nanobody activity detection

[0157] 2-1 ELISA binding assay

[0158] The binding ability of the candidate MHAB14 antibody (anti-human HER2 VHH Fc fusion protein) to human HER2 antigen was tested using an ELISA method. Her2 / ERBB2 Protein, Human, Recombinant (ECD, His Tag) antigen (Sinobiological, Cat. No. 10004-H08H) was diluted to 0.5 μg / mL in PBS and added to an ELISA plate (Corning, Cat. No. 9018) at 100 μL / well. The plate was coated overnight at 2°C-8°C. Rinse 3 times with PBST, add 1% BSA / PBST to block at room temperature for 1 hour, then rinse 5 times with PBST, add the test antibody (test antibody number is MHAB14, positive control antibody is Trastuzumab, initial concentration is 10μg / mL, 7-fold serial dilution), incubate for 1 hour, rinse 7 times with PBST to wash away unbound antibody, add 1:50,000 diluted Goat Anti-Human IgG Fc (HRP) (Abcam, Cat.No.ab97225), incubate at room temperature for 30 minutes. After washing away excess secondary antibody, add 1-Step TMUltra TMB-ELISA Substrate Solution (Absin, Cat. No. 9178) (100 μL / well) was added and developed at room temperature in the dark for 10 min. The reaction was terminated by adding 100 μL of TMB Stop Solution (Absin, Cat. No. abs9472). The absorbance was read at 450 nm using a microplate reader, and a four-parameter curve was plotted using GraphPad.

[0159] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0160] The results are shown in Figure 1 and Table 4. The binding activity of the MHAB14 candidate antibodies to the human HER2 antigen: except for MHAB14-18 and MHAB14-26, the EC50 of the remaining sequences were all lower than that of the positive control Trastuzumab.

[0161] Table 4. EC 50 result

[0162] Continued Table 4.EC 50 result

[0163] Continued Table 4.EC 50 result

[0164] 2-2 FACS binding experiment

[0165] In FACS experiments, human breast cancer SK-BR-3 cells endogenously expressing human HER2 (SK-BR-3 complete culture medium consists of ATCC-formulated McCoy's 5a Medium Modified, Catalog No. 30-2007 + 10% FBS, Sigma-Aldrich, Cat. no. F8687 + 1% P / S, Gibco, Cat. no. 15140-122) were used to detect binding of candidate antibodies. The test antibody was serially diluted (30,000 ng / mL, 7,500 ng / mL, 1,875 ng / mL, 468.8 ng / mL, 117.2 ng / mL, 29.3 ng / mL, 7.3 ng / mL and 1.8 ng / mL) and 100 μL of FITC anti-human IgG Fc The binding of the test antibody was detected by secondary antibody (Biolegend, Cat. no. 366916), and the FITC fluorescence intensity (Mean Fluorescence Intensity, MFI) was measured by flow cytometry.

[0166] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0167] The FACS binding results are shown in Figure 2 . Except for MHAB14-26, the EC50s of the binding of the MHAB14 candidate antibodies to SK-BR-3 cells were all lower than that of Trastuzumab.

[0168] 2-3 Endocytosis Experiment

[0169] Endocytosis activity was detected using human breast cancer SK-BR-3 cells that endogenously express human HER2. 1×10 6 / mL cell suspension and 10μg / mL anti-HER2 test antibody solution, add the cell suspension to a 1.5mL EP tube, centrifuge at 1200rpm, 4℃ for 3 minutes, and discard the supernatant. Pipette 1.5mL of the test antibody solution and add it to the cells, incubate on ice for 30 minutes, and wash four times with pre-cooled 200μL / well FACS buffer. Resuspend the cells with 1.5mL complete medium, mix well, and add the cells to a 96-well plate at 100μL / well. Incubate at 4℃ and 37℃ for 0h, 1h, 2h, and 4h (duplicate wells). After incubation, centrifuge at 1200 rpm and 4°C for 3 minutes, add 100 μL of FITC anti-human IgG Fc Antibody diluent (Biolegend, Cat. no. 366916), and incubate at 4°C for 30 minutes. After washing away unbound secondary antibody, use flow cytometry to measure FITC fluorescence intensity (mean fluorescence intensity, MFI) and calculate the internalization rate using the following formula: Internalization (%) = 100% - MFI T / MFI T0 *100%

[0170] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0171] The endocytosis percentage curves at each time period are shown in FIG3A . At 37° C., the MHAB14 candidate antibodies have good endocytosis activity in SK-BR-3 cells (among which MHAB14-10 has the best endocytosis activity), and the endocytosis activity is comparable to that of Trastuzumab.

[0172] The MFI difference curve of the candidate antibodies on the surface of SK-BR-3 cells is shown in Figure 3B. The results show that at 37°C, MHAB14-14 has the largest MFI difference in SK-BR-3 cell internalization (i.e., the absolute number of antibodies internalized in SK-BR-3 cells is the largest).

[0173] 2-4 Fab-ZAP assay to detect antibody endocytosis-mediated cell killing

[0174] Human breast cancer SK-BR-3 cells were digested and resuspended in complete culture medium (ATCC-formulated McCoy's 5a Medium Modified, Catalog No. 30-2007 + 10% FBS, Sigma-Aldrich, Cat. no. F8687 + 1% P / S, Gibco, Cat. no. 15140-122). The cell density was adjusted to 4E4 cells / mL and 50 μL was added to each well of a 96-well cell culture plate. The cell culture plate was incubated at 37°C for 16 hours. The next day, ZAP diluent containing 9 nM ZAP-Fab was prepared in culture medium. The antibody was then serially diluted with ZAP diluent to a working solution ranging from 4000 pM to 0.26 pM (7 concentration steps, 1:5 dilution). After incubation at 37°C for 15 minutes, the working solution was added to the cell culture plate at 50 μL per well, mixed, and the cell culture plate was incubated at 37°C for an additional 120 hours. 7.5 μL Triton-X 100 was added to the cell wells not treated with antibody-Fab-ZAP in advance and incubated for 30 minutes. This well served as a positive control. The cell wells not treated with antibody-Fab-ZAP or Triton-X100 (NT) served as negative controls. 20 μL MTS (Promega, Cat: G3598B) was then added to each well of the cell culture plate and incubated in a 37°C cell culture incubator for 2 hours. The cells were shaken for 10 seconds in a microplate reader and the data were read at the detection wavelength A490. The killing rate (%) was calculated using the formula: 100% - (OD sample -OD Triton-X100) / (OD NT -OD Triton-X100)*100% to calculate the cell killing efficiency.

[0175] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0176] The endocytosis-mediated cell killing curve is shown in Figure 4. MHAB14-10 and MHAB14-14-Fab-ZAP exhibited strong cytotoxicity against SK-BR-3 cells. Based on the FACS binding and endocytosis results, MHAB14-10 and MHAB14-14 will be humanized and subsequently screened.

[0177] The VHH sequence of MHAB14-10 is shown in SEQ ID NO: 15, the CDR1-CDR3 sequences defined by Kabat are shown in SEQ ID NOs: 1, 2, and 4, respectively, and the CDR1-CDR3 sequences defined by IMGT are shown in SEQ ID NOs: 5-7, respectively. The VHH sequence of MHAB14-14 is shown in SEQ ID NO: 17, the CDR1-CDR3 sequences defined by Kabat are shown in SEQ ID NOs: 8, 9, and 11, respectively, and the CDR1-CDR3 sequences defined by IMGT are shown in SEQ ID NOs: 12-14, respectively.

[0178] 2-5 Competitive ELISA binding experiments

[0179] ELISA was used to test the ability of candidate MHAB14 antibodies, MHAB14-10 and MHAB14-14, to competitively bind to the human HER2 antigen with trastuzumab or pertuzumab. Trastuzumab and pertuzumab were diluted to 0.3 μg / mL and 1 μg / mL, respectively, in PBS. 100 μL / well was added to an ELISA plate (Corning, Cat. No. 9018) and coated overnight at 2°C-8°C. The plate was rinsed three times with PBST, blocked with 1% BSA / PBST for 1 hour at room temperature, and then rinsed five times with PBST. Dilute Her2 / ERBB2 Protein, Human, Recombinant (ECD, His Tag) antigen (Sinobiological, Cat. No. 10004-H08H) to 0.4 μg / mL in 1% BSA / PBST. MHAB14-10, MHAB14-14, and the positive control antibodies, trastuzumab or pertuzumab, were diluted to 60 μg / mL in a 3-fold serial dilution. First, add MHAB14-10 and MHAB14-14 dilutions, followed by 50 μL of HER2, His Tag antigen dilution, for a total of 100 μL / well. Incubate at room temperature for 60 min. Rinse seven times with PBST to remove unbound antibody. Add Anti-His Tag Antibody (HRP), Mouse Monoclonal (Sino Biological, Cat. No. 105327-MM02T-H) at a 1:12,000 dilution and incubate at room temperature for 30 min. After washing away excess secondary antibody, add 1-Step TMUltra TMB-ELISA Substrate Solution (Absin, Cat. No. 9178) (100 μL / well) was added and developed at room temperature in the dark for 10 min. The reaction was terminated by adding 100 μL of TMB Stop Solution (Absin, Cat. No. abs9472). The absorbance was read at 450 nm using a microplate reader, and a four-parameter curve was plotted using GraphPad.

[0180] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0181] The ELISA binding results are shown in Figures 5A and 5B . Neither MHAB14-10 nor MHAB14-14 competes with trastuzumab or pertuzumab for binding to HER2 antigen.

[0182] Example 3. Humanization and activity detection of anti-human HER2 nanobody

[0183] The VHHs of MHAB14-10 and MHAB14-14 were humanized to generate two humanized nanobodies, MHAB14-10-5 (VHH as shown in SEQ ID NO: 16) and MHAB14-14-6 (VHH as shown in SEQ ID NO: 18). These VHH sequences were integrated into the Fc sequence of human IgG1 (SEQ ID NO: 20) and purified for expression in subsequent activity experiments.

[0184] 3-1 ELISA binding assay

[0185] The binding ability of the humanized MHAB14-10 and MHAB14-14 sequences to human HER2 antigen was assessed using an ELISA assay. Her2 / ERBB2 Protein, Human, Recombinant (ECD, His Tag) antigen (Sinobiological, Cat. No. 10004-H08H) was diluted to 0.5 μg / mL in PBS and added to an ELISA plate (Corning, Cat. No. 9018) at 100 μL / well. The plates were coated overnight at 2°C-8°C. Wash with PBST three times, add 1% BSA / PBST to block at room temperature for 1 hour, then wash with PBST five times, add MHAB14-10 and MHAB14-14 humanized antibodies (antibody numbers are MHAB14-10-1~6 and MHAB14-14-1~8, positive control antibody is Trastuzumab), the initial concentration is 10μg / mL, and 7-fold serial dilution is performed), incubate for 1 hour, and then wash with PBST seven times to wash away unbound antibodies, add 1:50,000 dilution of Goat Anti-Human IgG Fc (HRP) (Abcam, Cat.No.ab97225), and incubate at room temperature for 30 minutes. After washing away excess secondary antibody, add 1-Step TM Ultra TMB-ELISA Substrate Solution (Absin, Cat. No. 9178) (100 μL / well) was added and developed at room temperature in the dark for 15 min. The reaction was then terminated by adding 100 μL of TMB Stop Solution (Absin, Cat. No. abs9472). The absorbance was read at 450 nm using a microplate reader, and a four-parameter curve was plotted using GraphPad.

[0186] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0187] ELISA binding results indicate that the binding activity of the humanized MHAB14-10 antibody to human HER2 antigen is comparable to that of the parental MHAB14-10 (Figure 6 and Table 5). The affinity of the humanized MHAB14-14-2, 4, 5, and 7 sequences for HER2 was significantly decreased compared to that of the parental MHAB14-14, while the other sequences were essentially equivalent (Figure 7 and Table 6).

[0188] Table 5. EC 50 result

[0189] Table 6. EC 50 result

[0190] 3-2 FACS binding experiment

[0191] In the FACS assay, the binding of MHAB14-10 and MHAB14-14 humanized antibodies was detected using human breast cancer SK-BR-3 cells endogenously expressing human HER2. The test antibodies were serially diluted (30,000 ng / mL, 7,500 ng / mL, 1,875 ng / mL, 469 ng / mL, 117 ng / mL, 29 ng / mL, 7 ng / mL, and 1.8 ng / mL). Binding of the test antibodies was detected using a Goat anti-Human IgG Fc Secondary Antibody (Invitrogen, Cat. no. 12-4998-82). The mean fluorescence intensity (MFI) of the bound secondary antibody was measured by flow cytometry.

[0192] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0193] Binding curves are shown in Figures 8 and 9. FACS binding results indicate that the binding activity of the humanized MHAB14-10 antibody to SK-BR-3 cells is comparable to that of the parental MHAB14-10 (Figure 8). The affinity of the humanized MHAB14-14-2 sequence for HER2 is significantly lower than that of the parental MHAB14-14 (similar to ELISA results), while the other sequences are comparable to the parental MHAB14-14 (Figure 9).

[0194] 3-3 Endocytosis Experiment

[0195] Endocytosis activity was detected using human breast cancer SK-BR-3 cells that endogenously express human HER2. 1×10 6 / mL cell suspension and 10μg / mL anti-HER2 test antibody solution, add the cell suspension to a 1.5mL EP tube, centrifuge at 1200rpm, 4℃ for 3 minutes, and discard the supernatant. Pipette 1.5mL of the test antibody solution and add it to the cells, incubate on ice for 30 minutes, and wash four times with pre-cooled 200μL / well FACS buffer. Resuspend the cells with 1.5mL complete medium, mix well, and add the cells to a 96-well plate at 100μL / well. Incubate at 4℃ and 37℃ for 0h, 1h, 2h, and 4h (duplicate wells). After incubation, centrifuge at 1200 rpm and 4°C for 3 minutes, add 100 μL of Goat anti-Human IgG Fc Secondary Antibody (Invitrogen, Cat. no. 12-4998-82) secondary antibody dilution, and incubate at 4°C for 30 minutes. After washing away unbound secondary antibody, use flow cytometry to measure FITC fluorescence intensity (mean fluorescence intensity, MFI) and calculate the internalization rate using the following formula: Internalization (%) = 100% - MFI T / MFI T0 *100%

[0196] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0197] The endocytosis percentage curve is shown in Figure 10. The endocytosis results show that at 37°C, MHAB14-10-5 and MHAB14-14-6 humanized antibodies have good endocytosis activity in SK-BR-3 cells, and the endocytosis activity is comparable to that of parental MHAB14-10 and MHAB14-14.

[0198] 3-4 Fab-ZAP assay to detect antibody endocytosis-mediated cell killing

[0199] Human breast cancer SK-BR-3 cells were digested and resuspended in complete culture medium (ATCC-formulated McCoy's 5a Medium Modified, Catalog No. 30-2007 + 10% FBS, Sigma-Aldrich, Cat. no. F8687 + 1% P / S, Gibco, Cat. no. 15140-122). The cell density was adjusted to 4E4 cells / mL and 50 μL was added to each well of a 96-well cell culture plate. The cell culture plate was incubated at 37°C for 16 hours. The next day, ZAP diluent containing 9 nM ZAP-Fab was prepared in culture medium. The antibody was then serially diluted with ZAP diluent to a working solution ranging from 4000 pM to 0.26 pM (7 concentration steps, 1:5 dilution). After incubation at 37°C for 15 minutes, the working solution was added to the cell culture plate at 50 μL per well, mixed, and the cell culture plate was incubated at 37°C for an additional 120 hours. Pre-treatment with 7.5 μL of Triton-X100 was performed in the wells containing cells not treated with antibody-Fab-ZAP and incubated for 30 minutes. This well served as a positive control. A negative control was performed in the wells containing cells not treated with antibody-Fab-ZAP or Triton-X100 (NT). 20 μL of MTS (Promega, Cat: G3598B) was then added to each well of the cell culture plate. The cells were incubated in a 37°C cell culture incubator for 2 hours. After shaking for 10 seconds, the plate was read using a microplate reader at the detection wavelength A490. Killing rate (%) was calculated using the formula: 100% - (ODsample - ODTriton-X100) / (ODNT - ODTriton-X100) * 100%.

[0200] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0201] The endocytosis-mediated cell killing curve is shown in FIG11 . Both MHAB14-10-5-Fab-ZAP and MHAB14-14-6-Fab-ZAP have excellent killing activity against SK-BR-3.

[0202] Example 4. Competitive FACS binding experiment

[0203] The ability of candidate antibody MHAB14-14 to compete with trastuzumab or pertuzumab for binding to the cell surface HER2 antigen on human breast cancer SK-BR-3 cells endogenously expressing human HER2 was assessed using FACS. SK-BR-3 cells were cultured in complete SK-BR-3 cell culture medium (ATCC-formulated McCoy's 5a Medium Modified, Catalog No. 30-2007 + 10% FBS, Sigma-Aldrich, Cat. no. F8687 + 1% P / S, Gibco, Cat. no. 15140-122). A cell suspension (2.0 × 10 6 First add trastuzumab (final concentration 7.5ug / mL) and pertuzumab (final concentration 7.5ug / mL) or isotype (final concentration 7.5ug / mL), then add the candidate antibody MHAB14-14 or isotype gradient dilution (final concentration gradient 30000ng / mL, 7500ng / mL, 1875ng / mL, 468.8ng / mL, 117.2ng / mL, 29.3ng / mL, 7.3ng / mL and 1.8ng / mL), incubate on ice at 4℃ for 60min, and wash off the excess primary antibody with FACS buffer. Then use 100μL MonoRab TM The binding of the test antibody was detected using Rabbit Anti-Camelid VHH Cocktail [PE] (GenScript, Cat. no. A02018) secondary antibody diluent (diluted with PBS buffer containing 2% FBS, dilution ratio 1:200), and the mean fluorescence intensity (MFI) of PE was measured by flow cytometry.

[0204] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0205] The binding curve is shown in FIG12 . The FACS binding results show that when the concentrations of trastuzumab and pertuzumab are saturated, MHAB14-14 can still bind to the cell surface HER2 antigen.

[0206] Example 5. ELISA species cross-experiment

[0207] The binding ability of MHAB14-14-6 (anti-human HER2 nanobody) to HER2 antigens from different species (human and monkey) was tested using ELISA. Human HER2, His Tag (ACRO, Cat. No. HE2-H5225) and Cynomolgus HER2 Protein, His Tag (Sino Biological, Cat. No. 90295-C08H) were diluted to 0.5 μg / mL in PBS and added to an ELISA plate (Corning, Cat. No. 9018) at 100 μL / well. The plates were coated overnight at 4°C. Rinse 3 times with PBST, add 200 μL 3% BSA / PBST to each well, block at room temperature for 1 hour, then rinse 5 times with PBST, add the antibody to be tested, with an initial concentration of 10 μg / mL, perform 10-fold serial dilution, incubate at room temperature for 1 hour, rinse 7 times with PBST to wash away unbound antibody, add 100 μL 1:50,000 diluted goat anti-human IgG Fc (HRP) (Abcam, Cat.No.ab97225) to each well, incubate at room temperature for 30 minutes. Rinse 7 times with PBST to wash away excess secondary antibody, and add 100 μL 1-Step TM Ultra TMB-ELISA Substrate Solution (Absin, Cat. No. 9178) was added to each well for 15 min at room temperature in the dark. The reaction was terminated by adding 100 μL of TMB Stop Solution (Absin, Cat. No. abs9472) to each well. The absorbance was read at 450 nm using a microplate reader, and a four-parameter curve was plotted using GraphPad.

[0208] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism (GraphPad) software was used for plotting and data analysis.

[0209] The results are shown in FIG13 and Table 7. MHAB14-14-6 has specific binding to both human HER2 and monkey HER2, and the binding activities are comparable.

[0210] Table 7. Binding activity of MHAB14-14-6 to human HER2 and monkey HER2

[0211] Example 6. EGFR family-specific binding ELISA

[0212] The binding ability of MHAB14-14-6 (anti-human HER2 nanobody) to EGFR family proteins (HER1, HER2, HER3, and HER4) was tested by ELISA. Human HER1, His Tag (Sino Biological, Cat. No. 10001-H08H); Human HER2, His Tag (ACRO, Cat. No. HE2-H5225); Human HER3 Protein, His Tag (ACRO, Cat. No. ER3-H5223); and Human HER4 Protein, His Tag (Sino Biological, Cat. No. 10363-H08H) were diluted to 0.5 μg / mL in PBS and added to an ELISA plate (Corning, Cat. No. 9018) at 100 μL / well. The plates were coated overnight at 4°C. Rinse 3 times with PBST, add 200 μL 3% BSA / PBST to each well, block at room temperature for 1 hour, then rinse 5 times with PBST, add the antibody to be tested, with an initial concentration of 10 μg / mL, and perform a 10-fold serial dilution. After incubation at room temperature for 1 hour, rinse 7 times with PBST to wash away unbound antibody, add 100 μL 1:50,000 diluted goat anti-human IgG Fc (HRP) (Abcam, Cat. No. ab97225) to each well, and incubate at room temperature for 30 minutes. Rinse 7 times with PBST to wash away excess secondary antibody, and add 100 μL 1-Step TM Ultra TMB-ELISA Substrate Solution (Absin, Cat. No. 9178) was added to each well for 15 min at room temperature in the dark. The reaction was terminated by adding 100 μL of TMB Stop Solution (Absin, Cat. No. abs9472) to each well. The absorbance was read at 450 nm using a microplate reader, and a four-parameter curve was plotted using GraphPad.

[0213] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism (GraphPad) software was used for plotting and data analysis.

[0214] The results, as shown in Figure 14 and Table 8, show that MHAB14-14-6 only binds to HER2 and has no binding activity to other EGFR family members HER1, HER3, and HER4. Therefore, the antibody of the present application has outstanding selective binding activity to EGFR family proteins and has specific binding activity to HER2.

[0215] Table 8. Binding activity of MHAB14-14-6 to human HER1-4

[0216] Although the specific embodiments of the present invention 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 published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A nanobody or an antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor-2 (HER2), wherein: The nanobody or antigen-binding fragment thereof comprises: CDR1, CDR2 and CDR3 contained in the VHH shown in any one of SEQ ID NOs: 15-18; Preferably, the CDRs are defined according to the Kabat, Chothia or IMGT numbering systems.

2. The Nanobody or antigen-binding fragment thereof according to claim 1, comprising: (1) CDR1 as shown in SEQ ID NO: 1; CDR2 as shown in SEQ ID NO: 2 or 3; and CDR3 as shown in SEQ ID NO: 4; wherein, The CDRs are defined according to the Kabat numbering system; (2) CDR1 as shown in SEQ ID NO:5; CDR2 as shown in SEQ ID NO:6; and CDR3 as shown in SEQ ID NO:7; wherein the CDRs are defined according to the IMGT numbering system; (3) CDR1 as shown in SEQ ID NO:8; CDR2 as shown in SEQ ID NO:9 or 10; and CDR3 as shown in SEQ ID NO:11; wherein the CDRs are defined according to the Kabat numbering system; or, (4) CDR1 as shown in SEQ ID NO:12; CDR2 as shown in SEQ ID NO:13; and CDR3 as shown in SEQ ID NO:14; wherein the CDRs are defined according to the IMGT numbering system.

3. The Nanobody or antigen-binding fragment thereof of claim 1 or 2, wherein the Nanobody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 15 or 17, 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 compared thereto, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions, deletions or additions) compared thereto.

4. The Nanobody or antigen-binding fragment thereof of claim 1 or 2, which is humanized; Preferably, the Nanobody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (for example, a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline antibody gene), which heavy chain framework region optionally comprises one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to camel residues.

5. The Nanobody or antigen-binding fragment thereof of claim 4, wherein the Nanobody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 16 or 18, 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 several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) thereto.

6. A polypeptide construct that specifically binds to HER2, comprising a Nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 5, and an immunoglobulin Fc domain; Preferably, the immunoglobulin Fc domain is optionally connected to the N-terminus and / or C-terminus (e.g., the C-terminus) of the Nanobody or antigen-binding fragment thereof via a peptide linker; Preferably, the immunoglobulin Fc domain is an IgG Fc domain, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; Preferably, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 19 or 20, or a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared thereto (e.g., a sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); Preferably, the polypeptide construct has a sequence as shown in any one of SEQ ID NOs: 21-24.

7. An isolated nucleic acid molecule encoding a Nanobody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5 or a polypeptide construct as claimed in claim 6.

8. A vector comprising the nucleic acid molecule according to claim 7; preferably, the vector is a cloning vector or an expression vector.

9. A host cell comprising the isolated nucleic acid molecule of claim 7 or the vector of claim 8.

10. A method for preparing a Nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 5 or a polypeptide construct according to claim 6, comprising culturing a host cell according to claim 9 under conditions that allow protein expression, and recovering the Nanobody or its antigen-binding fragment or the polypeptide construct from the cultured host cell culture.

11. A bispecific or multispecific antibody comprising a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 5 or a polypeptide construct according to claim 6; Preferably, the bispecific or multispecific antibody specifically binds to HER2 and additionally specifically binds to one or more other targets; Preferably, the bispecific or multispecific antibody further comprises at least one second antibody having a second binding specificity for a second target.

12. A conjugate comprising a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 5, a polypeptide construct according to claim 6, or a bispecific or multispecific antibody according to claim 11, and a coupling portion connected thereto; Preferably, the conjugated moiety is selected from a therapeutic agent (such as a cytotoxic agent, a cytokine, a toxin or a radionuclide); Preferably, the conjugate is an antibody-drug conjugate (ADC).

13. A pharmaceutical composition comprising a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 5, a polypeptide construct according to claim 6, an isolated nucleic acid molecule according to claim 7, a vector according to claim 8, a host cell according to claim 9, a bispecific or multispecific antibody according to claim 11, or a conjugate according to claim 12, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises another pharmaceutically active agent, such as an anti-tumor agent.

14. A kit comprising a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 5 or a polypeptide construct according to claim 6; Preferably, the kit comprises a conjugate comprising the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 5 or the polypeptide construct according to claim 6, and a detectable label linked to the Nanobody or antigen-binding fragment thereof or polypeptide construct, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin; Preferably, the kit comprises a Nanobody or an antigen-binding fragment thereof as described in any one of claims 1 to 5 or a polypeptide construct as described in claim 6, and a second antibody that specifically recognizes the Nanobody or its antigen-binding fragment or polypeptide construct; optionally, the second antibody further comprises a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

15. A chimeric antigen receptor comprising a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 5 or a polypeptide construct according to claim 6; Preferably, the antigen binding domain is expressed by immune effector cells (eg, T cells).

16. An isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 15.

17. A vector comprising the isolated nucleic acid molecule of claim 16; preferably, it is used for preparing chimeric antigen receptor T cells.

18. A host cell comprising the isolated nucleic acid molecule of claim 16 or the vector of claim 17; Preferably, the host cell is an immune effector cell (e.g., a T cell or a NK cell); Preferably, the host cell is a chimeric antigen receptor T cell (CAR-T).

19. Use of the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 5, the polypeptide construct according to claim 6, the isolated nucleic acid molecule according to claim 7, the vector according to claim 8, the host cell according to claim 9, the bispecific or multispecific antibody according to claim 11, the conjugate according to claim 12, or the pharmaceutical composition according to claim 13, or the kit according to claim 14, or the chimeric antigen receptor according to claim 15 in the preparation of a medicament for preventing and / or treating a tumor in a subject; Preferably, the tumor expresses HER2; Preferably, the drug is used to inhibit the growth of tumor cells expressing HER2 and / or kill the tumor cells; Preferably, the medicament further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor or an oncolytic virus; Preferably, the tumor is selected from solid tumors, such as gastric cancer, lung cancer, liver cancer, head and neck cancer, skin cancer, colorectal cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, colorectal cancer or oral squamous cell carcinoma; Preferably, the subject is a mammal, such as a human.

20. Use of a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 5 or a polypeptide construct according to claim 6 in the preparation of a kit for detecting whether a tumor can be treated by an anti-tumor therapy targeting HER2, or for detecting the presence or level of HER2 in a sample; Preferably, the sample is a cell sample (eg, a sample comprising tumor cells) or a body fluid sample (eg, blood) from a subject (eg, a mammal, such as a human).

21. A method for detecting the presence or level of HER2 in a sample, comprising using a Nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 5 or a polypeptide construct as described in claim 6; Preferably, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (eg ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay.