WO2023165142A1 - Her2结合多肽及其用途 - Google Patents

Her2结合多肽及其用途 Download PDF

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WO2023165142A1
WO2023165142A1 PCT/CN2022/126577 CN2022126577W WO2023165142A1 WO 2023165142 A1 WO2023165142 A1 WO 2023165142A1 CN 2022126577 W CN2022126577 W CN 2022126577W WO 2023165142 A1 WO2023165142 A1 WO 2023165142A1
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seq
her2
amino acid
acid sequence
sequence shown
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PCT/CN2022/126577
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French (fr)
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须涛
杨艳玲
齐浩
王超
孙艳
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苏州智核生物医药科技有限公司
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Publication of WO2023165142A1 publication Critical patent/WO2023165142A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • This application relates to the field of biomedicine, in particular to a HER2-binding polypeptide and its application.
  • HER2 Human epidermal growth factor receptor 2
  • EGFR epidermal growth factor receptor
  • radioactive targeted therapy drugs can perform radioactive physical killing of tumor cells through the radiation released by radionuclides.
  • the object of the present invention is to provide a HER2-binding polypeptide, as well as further obtained radionuclide markers for diagnosis and treatment, as well as preparation methods and applications.
  • the application provides a HER2-binding polypeptide that competes with a reference antibody for binding to the HER2 protein, wherein the reference antibody comprises a heavy chain variable region (VH); wherein the VH comprises SEQ ID NO: 10 The amino acid sequence shown.
  • VH heavy chain variable region
  • the HER2-binding polypeptide is capable of binding to the HER2 protein with a KD value of no more than about 1.20E-09 in an ELISA assay.
  • the HER2-binding polypeptide has one or more of the following properties:
  • the HER2-binding polypeptide comprises an antibody or antigen-binding fragment thereof.
  • the antibodies include monoclonal antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, and/or fully human antibodies.
  • the antigen-binding fragments comprise Fab, Fab', Fv fragments, F(ab')2, scFv, VHH and/or dAb.
  • VHH is camelid, chimeric, human, partially humanized or fully humanized.
  • it comprises at least one CDR in the amino acid sequence shown in any one of SEQ ID NO: 158, SEQ ID NO: 2 to SEQ ID NO: 12.
  • it comprises SEQ ID NO: 158, HCDR1, HCDR2 and HCDR3 in any one of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 12.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises SEQ ID NO: 159, SEQ ID NO: 14, SEQ ID NO: 15 and The amino acid sequence shown in any one of SEQ ID NO:21.
  • HCDR1 comprises the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 86 and SEQ ID NO: 87.
  • said HCDR1 comprises SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID The amino acid sequence shown in NO:23 and SEQ ID NO:24.
  • HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:160, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:34.
  • HCDR2 comprises the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:88 and SEQ ID NO:89.
  • said HCDR2 comprises SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:32, SEQ ID NO: The amino acid sequence shown in any one of NO:33, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.
  • said HCDR3 comprises SEQ ID NO: 161, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID The amino acid sequence shown in any one of NO:44, SEQ ID NO:45 and SEQ ID NO:46.
  • said HCDR3 comprises SEQ ID NO:38, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID The amino acid sequence shown in NO:95 or SEQ ID NO:96.
  • HCDR3 comprises the amino acid sequence shown in SEQ ID NO:47 or SEQ ID NO:48.
  • VH comprises: HCDR1, HCDR2 and HCDR3, wherein said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:159, and said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:160
  • the amino acid sequence of, and described HCDR3 comprises the amino acid sequence shown in SEQ ID NO:161; Or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:14
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:39; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:15
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:27
  • the HCDR3 comprises SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO :42, the amino acid sequence shown in any one of SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:34
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:46.
  • VH comprises: HCDR1, HCDR2 and HCDR3, wherein said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:13, SEQ ID NO:86 or SEQ ID NO:87 , the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:25, SEQ ID NO:88 or SEQ ID NO:89, and the HCDR3 comprises SEQ ID NO:38, SEQ ID NO:90, SEQ ID NO:91 , the amino acid sequence shown in SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95 or SEQ ID NO:96.
  • VH comprises: HCDR1, HCDR2 and HCDR3, wherein said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16, and said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:28
  • the amino acid sequence of, and described HCDR3 comprises the amino acid sequence shown in SEQ ID NO:40;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:29
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:41; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:18
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:42; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:19
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:31
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:43; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:32
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:44; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:33
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:45; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:22
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:35
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:47; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:23
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:36
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:48; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:24
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:37
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:47.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 158, SEQ ID NO: 2 to SEQ ID NO: 12.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:118 to SEQ ID NO:157.
  • the HER2-binding polypeptide comprises at least one VH.
  • the HER2-binding polypeptide comprises a Nanobody comprising the amino acid sequence shown in any one of SEQ ID NO: 158, SEQ ID NO: 2 to SEQ ID NO: 12.
  • the HER2-binding polypeptide comprises a Nanobody comprising the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 118 to SEQ ID NO: 157.
  • the application provides an isolated nucleic acid molecule or molecules encoding the HER2-binding polypeptides described herein.
  • the present application provides a construct comprising the nucleic acid molecule described in the present application.
  • the present application provides a cell comprising the nucleic acid molecule described herein or the construct described herein.
  • the present application provides a method for preparing the HER2-binding polypeptide described in the present application, comprising culturing the cells described in the present application under conditions that allow the expression of the HER2-binding polypeptide.
  • the method further comprises recovering the HER2-binding polypeptide expressed by the cells.
  • the method further comprises purifying and/or modifying the HER2-binding polypeptide.
  • the present application provides an immunoconjugate comprising the HER2-binding polypeptide described in the present application.
  • the immunoconjugate comprises:
  • a conjugation moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, viral coat proteins or VLPs, or combinations thereof.
  • the detectable label is one or more reagents selected from the group consisting of radionuclides, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes.
  • radionuclide is suitable for medical imaging and/or therapy.
  • the radionuclides include 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 67 Cu, 67 Ga, 68 Ga, 68 Ge , 86 Y, 90 Y, 89 Zr, 94m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52m Mn, 72 As, 75 Br, 76 Br, 82m Rb, 83 Sr or other ⁇ -, ⁇ -, or positron emitters.
  • HER2-binding polypeptide is directly or indirectly conjugated to said detectable label.
  • HER2-binding polypeptide is conjugated to said detectable label via a chelating agent.
  • the chelating agent is selected from DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, One or more of HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, HEHA and their derivatives.
  • the present application provides a composition comprising the HER2-binding polypeptide described in the present application, the nucleic acid molecule described in the present application, the construct described in the present application, the cell described in the present application and/or the present application.
  • the immunoconjugate described in the application and optionally a pharmaceutically acceptable carrier.
  • the composition comprises the HER2-binding polypeptide described herein or the immunoconjugate described herein, and the composition is a detection agent or a therapeutic agent.
  • the detection agent is a reagent for detecting HER2 protein.
  • the detection agent is a contrast agent.
  • the contrast agent is a contrast agent for detecting HER2 protein.
  • the therapeutic agent is used to treat tumors.
  • the therapeutic agent is used to treat HER2 positive tumors.
  • the application provides the HER2 binding polypeptide described in the application, the nucleic acid molecule described in the application, the construct described in the application, the cell described in the application, the immunoconjugate described in the application and /or the use of the composition described in this application in the preparation of medicaments, reagents, detection plates or kits;
  • the reagent, detection plate or kit is used to detect HER2 protein in the sample
  • the medicament is used for detecting the expression of HER2 protein and/or treating tumors expressing HER2.
  • the present application provides a recombinant protein comprising the HER2-binding polypeptide described in the present application.
  • the recombinant protein comprises: (i) the HER2-binding polypeptide described herein; and (ii) an optional tag sequence that assists expression and/or purification.
  • the application provides a method for detecting the presence and/or amount of HER2 in a biological sample, comprising: contacting the biological sample with the HER2-binding polypeptide described in the application, the immunoconjugate described in the application Compounds, compositions described herein.
  • the biological sample is tissue
  • tissue is selected from blood tissue, lymphoid tissue and tumor tissue.
  • the method comprises detecting the presence and/or amount of HER2-positive cells in a biological sample.
  • the presence and/or amount of HER2-positive cells in the biological sample is determined by imaging.
  • the presence and/or amount of HER2-positive cells in the biological sample is determined by flow cytometry.
  • the present application provides a method for detecting and/or diagnosing a disease or disorder related to abnormal expression of HER2, comprising administering the HER2-binding polypeptide described in the present application, the HER2-binding polypeptide described in the present application to a subject in need thereof.
  • said method further comprises imaging said subject.
  • said imaging comprises ECT imaging.
  • said ECT imaging comprises SPECT imaging or PET imaging.
  • the disease or disorder associated with abnormal expression of HER2 comprises a tumor.
  • the present application provides a method for treating and/or preventing tumors, the method comprising administering the HER2-binding polypeptide described in the present application, the immunoconjugate described in the present application or Compositions described herein.
  • the application provides a method for monitoring the efficacy of anti-tumor therapy in a subject, the method comprising:
  • the presence and/or amount of HER2-positive cells in the subject's tumor is determined by imaging.
  • the tumor comprises a HER2 positive tumor.
  • the tumor comprises a solid tumor.
  • the tumor is selected from breast cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, ovarian cancer, pancreatic cancer, endometrial cancer, cervical squamous cell carcinoma, salivary adenoma, bladder cancer, lung cancer, At least one of tumors such as colorectal cancer, head and neck cancer, prostate cancer, osteosarcoma, and medulloblastoma in children.
  • the present application provides a kit comprising the HER2-binding polypeptide described in the present application, the immunoconjugate described in the present application or the composition described in the present application.
  • Figures 1A-1C show the binding of the anti-Her2 antibody described in the present application to N87 cells
  • Figure 2 shows the KD affinity detection results of the anti-Her2 antibody H1 described in the present application
  • Figure 3 shows the KD affinity test results of the anti-Her2 antibody H2 described in the present application
  • Figures 4A-4B show the results of epitope competition between the anti-Her2 antibody described in the present application and Herceptin;
  • Figure 5 shows the combination of the anti-Her2 antibody H1 (Her2-Nb1) described in the present application with BT474 cells;
  • Figure 6 shows the combination of the anti-Her2 antibody H1 described in the present application with MCF7 cells
  • Figure 7 shows the single-point binding of the anti-Her2 antibody H1 described in the present application to BT474 cells
  • Figure 8 shows the internalization results of the anti-Her2 antibody H1 described in the present application.
  • Figure 9 shows the saturation binding curve of the 125 I-labeled anti-Her2 antibody ( 125 I-Her2-Nbs) described in this application in cells (SKOV3);
  • Figure 10 shows the biodistribution of 125 I-Her2-Nb1 described in this application in BT474 tumors
  • Figure 11 shows the dynamic imaging of 125 I-Her2-Nb1 described in this application in BT474 tumor
  • Figure 12 shows the SPE CT/CT imaging of 99m TC-Her2-Nb1 described in this application in BT474 tumor
  • Figure 13 shows the biodistribution of 177Lu -Her2-Nb1 in MCF7 tumors described in this application;
  • Figure 14 shows the anti-tumor effect of 177 Lu-Her2-Nb1 described in this application.
  • Figures 15A-15B show the results of SDS-PAGE analysis of the antibody before and after mutation of the H1 antibody described in the present application and after antibody purification.
  • HER2 generally refers to a type I transmembrane protein belonging to the epidermal growth factor receptor family, also known as c-erbB2, ErbB2 or Neu.
  • the term “HER2” also encompasses homologs, variants and isoforms of HER2, including splice isoforms.
  • HER2 is associated with neoplastic transformation in human breast cancer cells, as overexpression of HER2 protein has been detected in patients with breast, gastric, pancreatic, ovarian, peritoneal or colon cancer.
  • the terms “HER2 positive” and “expressing HER2” are used interchangeably in this application.
  • a "HER2 positive” tumor contains tumor cells that have higher than normal levels of HER2.
  • HER2-positive tumors include HER2-positive breast cancer and HER2-positive gastric cancer.
  • HER2-positive is a cancer that overexpresses HER2, and in certain embodiments, a HER-2-positive cancer has an immunohistochemistry (IHC) score and/or in situ hybridization (ISH) amplification of 2+ or 3+. Growth rate ⁇ 2.0.
  • IHC immunohistochemistry
  • ISH in situ hybridization
  • HER2-binding polypeptide means any polypeptide capable of specifically binding to HER2.
  • the binding polypeptide is an antibody.
  • the binding polypeptide is, for example, an antibody mimetic, cytokine, or growth factor.
  • a "HER2-binding polypeptide” may alternatively refer to a monovalent HER2-binding polypeptide (ie, a polypeptide that binds one epitope of HER2), as well as a bivalent or multivalent binding polypeptide (ie, a binding polypeptide that binds more than one epitope).
  • the "HER2-binding polypeptide” of the present application may comprise at least one variable domain that binds HER2.
  • a "HER2-binding polypeptide" of the present application may comprise 2, 3, 4 or more variable domains that bind HER2.
  • the HER2-binding polypeptides of the present application may also comprise a linker and/or a portion with effector functions, such as a half-life extending portion (such as a variable domain binding to serum albumin), and/or a fusion partner, in addition to the variable domain binding to HER2 body (such as serum albumin) and/or conjugated polymer (such as PEG) and/or Fc region.
  • a "HER2-binding polypeptide" of the present application also encompasses bispecific antibodies, which contain variable domains that bind different antigens.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (eg, bispecific antibodies) , and antibody fragments, as long as they show the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861), that is, bind HER2.
  • Antibodies can be murine, human, humanized, chimeric, or derived from other species.
  • a full-length antibody typically refers to an antibody consisting of two “full-length antibody heavy chains” and two “full-length antibody light chains”.
  • a “full-length antibody heavy chain” is generally a polypeptide consisting, in the N-terminal to C-terminal direction, of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), an antibody hinge region (HR) , antibody heavy chain constant domain 2 (CH2), and antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and in the case of antibodies of the IgE subclass, optionally It also includes the antibody heavy chain constant domain 4 (CH4).
  • VH antibody heavy chain variable domain
  • CH1 antibody constant heavy chain domain 1
  • HR antibody hinge region
  • CH2 antibody heavy chain constant domain 2
  • CH3 antibody heavy chain constant domain 3
  • a "full-length antibody heavy chain” is a polypeptide consisting of VH, CH1, HR, CH2, and CH3 in an N-terminal to C-terminal direction.
  • a “full-length antibody light chain” is generally a polypeptide consisting of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) in the N-terminal to C-terminal direction, abbreviated as VL-CL.
  • the antibody light chain constant domain (CL) may be kappa (kappa) or lambda (lambda).
  • the two full-length antibody chains are linked together by an inter-polypeptide disulfide bond between the CL domain and the CH1 domain and between the hinge region of the full-length antibody heavy chain.
  • Typical examples of full-length antibodies are natural antibodies such as IgG (eg, IgGl and IgG2), IgM, IgA, IgD, and IgE).
  • an antigen binding domain generally refers to a portion of an antibody molecule comprising the amino acids responsible for the specific binding between the antibody and the antigen.
  • the portion of an antigen that is specifically recognized and bound by an antibody is called an "epitope" as described above.
  • An antigen binding domain will typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it need not comprise both.
  • Fd fragments for example, have two VH regions and typically retain some antigen-binding function of the full antigen-binding domain.
  • antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments having VL, VH, constant light chain (CL) and CH1 domains; (2) F(ab') 2 fragments, having two Bivalent fragment of two Fab fragments connected by sulfur bridge; (3) Fd fragment with two VH and CH1 domains; (4) Fv fragment with VL and VH domains of antibody single arm, (5) dAb fragment (Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature 341:544-546 (1989), which is hereby incorporated by reference in its entirety), which has a VH domain; (6) Isolated Complementarity Determining Regions (CDRs); (7) Single-chain Fv (scFv), eg derived from a scFv-library.
  • Fab fragments monovalent fragments having VL, VH, constant light chain (CL) and CH1 domain
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows it to be produced as a single protein in which the VL and VH regions pair to form a monovalent molecule chain (termed single-chain Fv (scFv)) (see, e.g., Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc.
  • scFv single-chain Fv
  • VHH relates to variable antigens from heavy chain antibodies of the family Camelidae (camel, dromedary, llama, alpaca, etc.) Binding domain (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and review Vanlandschoot P. et al., 2011, Antiviral Research 92 , 389-407). VHHs may also be referred to as Nanobodies (Nb) and/or Single Domain Antibodies. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the function of the fragments is evaluated in the same manner as intact antibodies.
  • variable domain generally refers to the variable domain of an antibody capable of specifically binding an antigenic epitope.
  • antibody variable domains VH and VL VH domain and VL domain.
  • VHH domain a variable domain (or simply "VHH").
  • VHH domains also known as heavy chain single domain antibodies, VHH, VHH domains, VHH antibody fragments, and VHH antibodies, are antigens known as “heavy chain antibodies” (ie, “antibodies lacking light chains") Binding variable domains of immunoglobulins (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363,446- 448 (1993)).
  • VHH domain is used to distinguish the variable domain from the heavy chain variable domain present in conventional 4 chain antibodies (which is referred to in this application as a "VH domain”) and the variable domain present in conventional 4 chain antibodies.
  • light chain variable domains (which are referred to in this application as "VL domains") in antibodies.
  • a VHH domain specifically binds an epitope without the need for another antigen binding domain (in contrast to the VH or VL domains in conventional 4-chain antibodies, in which case their epitope is recognized by the VL domain together with the VH domain).
  • variable domains generally have the same general structure, and each domain contains four framework (FR) regions with highly conserved sequences, wherein the FR regions include “framework region 1" or “FR1", Four “framework regions” of “framework region 2" or “FR2”, “framework region 3” or “FR3”, and “framework region 4" or “FR4", FR region “complementarity determining region 1" or “CDR1”
  • the three “complementarity determining regions” or “CDRs” of , “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3" or “CDR3” are connected.
  • the general structure or sequence of a variable domain can be expressed as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • Antibody variable domains endow the antibody with specificity for an antigen by having an antigen-binding site.
  • CDR generally refers to the complementarity determining regions within the variable sequences of antibodies.
  • CDR1, CDR2 and CDR3 are 3 CDRs in each variable region of the heavy and light chains, which are called CDR1, CDR2 and CDR3 for each variable region.
  • the precise boundaries of these CDRs have been defined differently according to different systems.
  • the system described by Kabat Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody , but also provides the precise residue boundaries defining these three CDRs.
  • These CDRs can be referred to as Kabat CDRs.
  • Padlan FASEB J 9:133-139(1995)
  • MacCallum JMoI Biol 262(5):732-45(1996)
  • Other CDR bounds may not strictly follow one
  • the system described above still overlaps with the Kabat CDRs, and although they can be shortened or lengthened according to the following predictions or experimental findings, specific residues or groups of residues or even the entire CDR do not significantly affect antigen binding.
  • CDR CDR
  • HCDR1 HCDR2
  • LCDR3 LCDR3
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies in the population are identical except for minor natural mutations that may be present.
  • Monoclonal antibodies are usually highly specific against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants.
  • monoclonal antibodies have the advantage that they can be synthesized by hybridoma cultures without contamination from other immunoglobulins.
  • monoclonal denote the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method.
  • monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR region of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulins. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids may also be permissible so long as they still retain the ability of the antibody to bind a particular antigen.
  • a humanized antibody optionally will comprise at least a portion of a human immunoglobulin constant region.
  • a "humanized antibody” retains antigen specificity similar to the original antibody.
  • “Humanized” forms of non-human (eg, murine) antibodies may contain, at a minimum, chimeric antibodies of sequence derived from non-human immunoglobulin.
  • CDR region residues in a human immunoglobulin can be replaced with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or capabilities. , rabbit or non-human primate) residue substitution in the CDR region.
  • donor antibody such as mouse, rat
  • rabbit or non-human primate residue substitution in the CDR region such as mouse, rat
  • FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues.
  • humanized antibodies can comprise amino acid modifications that are absent in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody properties, such as binding affinity.
  • Fully human antibody generally refers to the antibody that is expressed by transferring the gene encoding the human antibody into a genetically engineered animal lacking the antibody gene. All portions of the antibody, including the variable and constant regions of the antibody, are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies on the human body. Methods for obtaining fully human antibodies in this field include phage display technology, transgenic mouse technology, ribosome display technology and RNA-polypeptide technology.
  • reference antibody generally refers to an antibody with which an antigen binding protein described herein competes for binding to an antigen (eg, HER2).
  • consortium when used in the context of antigen binding proteins competing for the same epitope generally refers to competition between antigen binding proteins, as prevented or inhibited by the antigen binding proteins (e.g., antibodies or immunologically functional fragments thereof) which are tested As determined by an assay for (eg, reduced) specific binding of a reference antigen binding protein (eg, a ligand or a reference antibody) to a common antigen (eg, HER2 or a fragment thereof).
  • antigen binding proteins e.g., antibodies or immunologically functional fragments thereof
  • ⁇ solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • sandwich competition assay See, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid phase direct biotin-avidin EIA see, e.g., Kirkland et al., 1986, J. Immunol.
  • solid phase Direct labeling assay solid-phase direct-labeling sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct-labeled RIA using I-125 labeling (see, e.g., Morel et al. , 1988, Molec.Immunol.25: 7-15); solid-phase direct biotin-avidin EIA (see, for example, Cheung et al., 1990, Virology 176: 546-552); and direct label RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82).
  • such assays involve the use of purified antigen bound to a solid surface or a unit carrying either of these, an unlabeled test antigen binding protein and a labeled reference antigen binding protein.
  • Competitive inhibition is measured by determining the amount of label bound to the solid surface or unit in the presence of the test antigen binding protein.
  • the test antigen binding protein is present in excess.
  • Antigen-binding proteins identified by competition assays include antigen-binding proteins that bind the same epitope as the reference antigen-binding protein and neighbors that bind the epitope of the reference antigen-binding protein in close enough proximity to be sterically hindered. Epitope binding antigen binding protein.
  • the competing antigen binding protein when it is present in excess, it inhibits (e.g., reduces) specific binding of the reference antigen binding protein to the common antigen by at least about 40-45%, about 45-50%, about 50-55%, About 55-60%, about 60-65%, about 65-70%, about 70-75%, or about 75% or more. In some instances, binding is inhibited by at least about 80-85%, about 85-90%, about 90-95%, about 95-97%, or about 97% or more.
  • sequence identity generally refers to the same nucleic acid or amino acid sequence when two or more aligned sequences are aligned using a sequence alignment program.
  • % sequence identity generally refers to the level of nucleic acid or amino acid sequence identity between two or more aligned sequences when aligned using a sequence alignment program. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity.
  • sequence identity For determination of sequence identity, see for example: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York , 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, 20 von Heinje, G., Academic Press, 1987 and Se quence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
  • amino acid residues will be referred to according to the standard three-letter or one-letter amino acid codes as known and agreed upon in the art.
  • amino acid difference generally refers to an insertion, deletion or substitution of a specified number of amino acid residues at a position in a reference sequence as compared to another sequence.
  • the substitution is a conservative amino acid substitution, which means that an amino acid residue is replaced by another amino acid residue with a similar chemical structure, and it has little impact on the function, activity or other biological properties of the polypeptide or essentially no effect.
  • conservative amino acid substitutions are well known in the art, for example a conservative amino acid substitution is the substitution of an amino acid within the following groups (i)-(v) by another amino acid residue within the same group: (i) smaller lipid Family of nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (iii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; Sexually positively charged residues: His, Arg, and Lys; (iv) larger aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (v) aromatic residues: Phe, Tyr, and Trp .
  • Particularly preferred conservative amino acid substitutions are as follows: Ala by Gly or Ser; Arg by Lys; Asn by Gln or His; Asp by Glu; Cys by Ser; Gln by Asn; Glu by Asp; Gly by Ala or Pro; His by Asn or Gln; Ile by Leu or Val; Leu by Ile or Val; Lys by Arg, Gln or Glu; Met by Leu, Tyr or Ile; Phe by Met, Leu or Tyr Substitution; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu.
  • the substitution is a non-conservative amino acid substitution, for example, Ala is substituted by Asp, Asn, Glu or Gin.
  • affinity generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (eg, polypeptide or antibody) and its binding partner (eg, target or antigen).
  • the affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, such as surface plasmon resonance, and also include those methods reported in this application. A higher affinity of molecule X for its binding partner Y can be seen in lower Kd values and/or EC50 values.
  • isolated generally refers to a molecule (eg, antibody, nucleic acid, etc.) that is at least partially separated from other molecules with which it is normally associated in its native state.
  • isolated polypeptide is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, cell debris, and growth media.
  • isolated nucleic acid generally exists in a form or context other than that in which it is found in nature.
  • the term "construct” generally refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein.
  • the coding sequence or "encoding nucleic acid sequence” may include initiation and termination signals operably linked to regulatory elements, including promoters and polynucleotides capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. adenylation signal. Examples include, but are not limited to, circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from bacteriophage lambda), viral genomes containing non-native nucleic acid sequences, and the like.
  • the term "immunoconjugate” generally refers to a conjugate formed by linking an antibody or antibody fragment thereof to other active agents, such as chemotherapeutic agents, toxins, immunotherapeutic agents, radioactive elements, imaging probes, spectroscopic Probes and more.
  • the linkage may be a covalent bond, or a non-covalent interaction such as through electrostatic forces.
  • linkers known in the art can be used to form immunoconjugates.
  • the conjugate can deliver the other agent to the target cell (eg, a tumor cell) through specific binding of the antibody or antigen-binding fragment thereof to an antigen on the target cell.
  • the immunoconjugate can be provided as a fusion protein that can be expressed from a polynucleotide encoding the immunoconjugate.
  • chelating agent generally refers to an organic molecule capable of forming a complex with a metal ion. Chelating agents are commonly used to label proteins or peptides. The final product of the metal ion conjugate is used in radioimmunoassay, radioimmunotherapy, magnetic resonance imaging, photodynamic therapy or other similar modalities.
  • Non-limiting examples of chelating or complexing agents are DTPA (diethylenetriaminepentaacetic anhydride) and its derivatives, NOTA (1,4,7-triazacyclononane-N,N',N "-triacetic acid) and its derivatives such as NODA-GA (NODAGA), Maleimide-NODAGA, DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N"' -tetraacetic acid) (conjugated radioactive metal ions) and its derivatives, TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid) and its derivatives, DTTA (N-(p-benzylisothiocyanate)-diethylenetriamine-N,N',N",N"'-tetraacetic acid). These and other chelating agents are readily available from commercial sources available.
  • the term "pharmaceutically acceptable carrier” generally refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient.
  • Such formulations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents.
  • Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents or encapsulating substances suitable for human administration.
  • Other contemplated carriers, excipients, and/or additives that may be used in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, Protein excipients (such as serum albumin, gelatin, casein), salt-forming counterions (such as sodium), etc.
  • the term "administer" and similar terms are generally not limited to bodily administration, suitable methods include in vitro, ex vivo or in vivo methods.
  • suitable methods include in vitro, ex vivo or in vivo methods.
  • any method of administration known to those skilled in the art for contacting cells, organs or tissues with the composition may be employed.
  • the compounds may be introduced into the body of a subject in need of treatment by any route of introduction or delivery.
  • the compositions of the present application may be administered orally, topically, intranasally, intramuscularly, subcutaneously, intradermally, intrathecally, intraperitoneally, or transdermally.
  • ex vivo is used interchangeably with “in vitro” and generally refers to an activity in a controlled environment in cells, tissues and/or organs that have been removed from a subject.
  • diagnosis generally refers to detecting a disease or condition, or determining the state or extent of a disease or condition.
  • the term “diagnosing” can also include detecting the cause of a disease or condition, determining the therapeutic effect of a drug treatment, or predicting the pattern of response to a drug treatment.
  • treating generally refers to: (i) preventing a disease, disorder, and/or condition from occurring in a patient who may be predisposed to it but has not been diagnosed with it; (ii) inhibiting the disease , disorder or condition, i.e. arresting its development; and (iii) relieving the disease, disorder or condition, i.e. making the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition subside.
  • tumor and cancer are used interchangeably and generally refer to neoplastic or malignant cell growths.
  • the tumors of this application may be benign or malignant.
  • the tumors of this application may be solid or non-solid.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as about 0.5%, about 1%, about 1.5%, about 2% above or below the specified value , about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10%.
  • a/an and “the” include plural referents.
  • the term “a/a (a)” (or “one/an (an)”), and the terms “one or more/one or more” and “at least one/at least one (atleast one)” can be used interchangeably here.
  • a cell can refer to a single cell or a population of cells.
  • the application provides a HER2-binding polypeptide that competes with a reference antibody for binding to the HER2 protein, wherein the reference antibody comprises a heavy chain variable region (VH); wherein the VH comprises SEQ ID NO: 10 The amino acid sequence shown.
  • VH heavy chain variable region
  • the HER2-binding polypeptide in an ELISA assay, can be expressed at no more than about 1.20E-09, about 1.10E-09, about 1.0E-09, about 9.0E-10, about 8.0E -10, about 7.0E-10, about 6.0E-10, about 5.0E-10, about 4.0E-10, about 3.0E-10, about 2.0E-10, about 1.0E-10, about 9.0E-11 KD value for binding to HER2 protein.
  • the HER2-binding polypeptide comprises an antibody or antigen-binding fragment thereof.
  • the antibodies include monoclonal antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, and/or fully human antibodies.
  • the antigen-binding fragments comprise Fab, Fab', Fv fragments, F(ab')2, scFv, VHH and/or dAb.
  • the antibody or antigen-binding fragment thereof may be VHH.
  • the VHHs are not limited to a particular biological source or to a particular method of preparation.
  • the VHH can generally be obtained in the following ways: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3 ) by "humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding such a humanized VHH domain; (4) by natural expression from any animal species, such as from a mammalian species, such as from a human "Camelization" of existing VH domains, or by expression of nucleic acids encoding such camelized VH domains; (5) "Camelization” by "domain antibodies” or “Dabs” as described in the art , or by the expression of nucleic acids encoding such camelized VH domains; (6) by using synthetic or semi-synthetic techniques to prepare proteins, polypeptides or other amino acid sequences
  • VHH is camelid, chimeric, human, partially humanized or fully humanized.
  • the HER2-binding polypeptide may comprise "humanized", that is, one or more amino acid residues in the amino acid sequence (and specifically in the framework sequence) of a naturally occurring VHH sequence VHH in which the base is substituted with one or more amino acid residues present at the corresponding positions in the VH domain of a conventional 4-chain antibody derived from humans.
  • humanization can be done using humanization techniques known in the art.
  • potential humanizing substitutions or combinations of humanizing substitutions can be determined by methods known in the art, for example, by comparing the sequence of the VHH with the sequence of a naturally occurring human VH domain Compare.
  • the humanizing substitutions are selected such that the resulting humanized VHH retains favorable functional properties.
  • the VHHs of the present application can become more "human-like" compared to corresponding naturally occurring VHH domains, while still retaining advantageous properties, such as reduced immunogenicity.
  • the humanized VHH of the present application can be obtained by any suitable means known in the art, and thus is not strictly limited to a polypeptide that has been obtained using a polypeptide comprising a naturally occurring VHH domain as a starting material.
  • it comprises at least one CDR in the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 12.
  • it comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:12.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and The amino acid sequence shown in any one of SEQ ID NO:21.
  • said HCDR1 comprises SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID The amino acid sequence shown in NO:23 and SEQ ID NO:24.
  • HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:34.
  • said HCDR2 comprises SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:32, SEQ ID NO: The amino acid sequence shown in any one of NO:33, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.
  • said HCDR3 comprises SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID The amino acid sequence shown in any one of NO:44, SEQ ID NO:45 and SEQ ID NO:46.
  • HCDR3 comprises the amino acid sequence shown in SEQ ID NO:47 or SEQ ID NO:48.
  • VH comprises: HCDR1, HCDR2 and HCDR3, wherein said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:13, and said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:25
  • the amino acid sequence of, and described HCDR3 comprises the amino acid sequence shown in SEQ ID NO:38; Or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:14
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:39; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:15
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:27
  • the HCDR3 comprises SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO :42, the amino acid sequence shown in any one of SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:34
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:46.
  • VH comprises: HCDR1, HCDR2 and HCDR3, wherein said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16, and said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:28
  • the amino acid sequence of, and described HCDR3 comprises the amino acid sequence shown in SEQ ID NO:40;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:29
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:41; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:18
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:30
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:42; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:19
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:31
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:43; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:32
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:44; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:33
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:45; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:22
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:35
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:47; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:23
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:36
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:48; or
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:24
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:37
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:47.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 12.
  • the amino acid sequence of the variable domain may have one or several amino acid substitutions, Deletion or addition; the substitution, deletion or addition does not substantially reduce the ability of the HER2-binding polypeptides of the present application to specifically bind to HER2.
  • amino acid mutations may be in the CDRs of the targeting moiety (eg, CDR1 region, CDR2 region, or CDR3 region).
  • amino acid changes may be in the framework regions (FRs) of the targeting moiety (eg, the FR1 region, FR2 region, FR3 region, or FR4 region).
  • the HER2-binding polypeptide comprises at least one VH.
  • the HER2 binding polypeptide may comprise one, or two, or three variable domains.
  • the HER2-binding polypeptide comprises a Nanobody comprising the amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 12.
  • the HER2-binding polypeptides of the present application also include variant forms of polypeptides comprising the above CDR regions that have the same function as the HER2-binding polypeptides of the present application. These variations include (but are not limited to): deletions, insertions, and/or substitutions of one or more (usually 1-50, 1-30, 1-20, or 1-10) amino acids, and Add one or several (usually within 20, within 10, or within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA hybrids that can hybridize with the DNA encoding the antibody of the present invention under high or low stringency conditions
  • the encoded protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
  • the application also provides other polypeptides, such as fusion proteins comprising Nanobodies or fragments thereof.
  • the invention also includes fragments of the Nanobodies of the invention.
  • the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the HER2-binding polypeptides of the present application.
  • “conservative variant” generally refers to that compared with the amino acid sequence of the HER2-binding polypeptide of the present application, there are at most 10, at most 8, at most 5, or at most 3 amino acids that are similar or similar in nature Amino acids are substituted to form polypeptides.
  • the substitution is a conservative amino acid substitution, which means that an amino acid residue is replaced by another amino acid residue with a similar chemical structure, and it has little impact on the function, activity or other biological properties of the polypeptide or essentially no effect.
  • conservative amino acid substitutions are well known in the art, for example a conservative amino acid substitution is the substitution of an amino acid within the following groups (i)-(v) by another amino acid residue within the same group: (i) smaller lipid Family of nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (iii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; Sexually positively charged residues: His, Arg, and Lys; (iv) larger aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cy
  • Particularly preferred conservative amino acid substitutions are as follows: Ala by Gly or Ser; Arg by Lys; Asn by Gln or His; Asp by Glu; Cys by Ser; Gln by Asn; Glu by Asp; Gly by Ala or Pro; His by Asn or Gln; Ile by Leu or Val; Leu by Ile or Val; Lys by Arg, Gln or Glu; Met by Leu, Tyr or Ile; Phe by Met, Leu or Tyr Substitution; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu.
  • the substitution is a non-conservative amino acid substitution, for example, Ala is substituted by Asp, Asn, Glu or Gin.
  • the application provides a HER2-binding polypeptide
  • the HER2-binding polypeptide may comprise at least one VH, and the amino acid sequence of the VH may have one or more than the amino acid sequence shown in SEQ ID NO:1. Amino acid substitutions, deletions or additions.
  • the HER2-binding polypeptide may comprise one, or two, or three VHs.
  • the VH may comprise one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen, or fifteen, or sixteen, or seventeen, or eighteen, or ten Amino acid sequence of nine, or twenty amino acid substitutions, deletions or additions.
  • the amino acid sequence of the VH may have one or several amino acid substitutions, and may include conservative and/or non-conservative substitutions.
  • amino acid mutations may be in the CDRs of the targeting moiety (eg, CDR1 region, CDR2 region, or CDR3 region).
  • amino acid changes may be in the framework regions (FRs) of the targeting moiety (eg, the FR1 region, FR2 region, FR3 region, or FR4 region).
  • Amino acid sequence modification can be achieved using any technique known in the art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989; and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989 .
  • substitutions, deletions or additions do not substantially reduce the ability of the HER2-binding polypeptides of the present application to specifically bind to HER2.
  • the equilibrium dissociation constant (KD) can be used to describe the full length and/or mature form and/or isoform and/or splice variant and/or splice variant and/or fragment and/or any other natural Existing or synthetic analogs, variants or mutants (including monomeric, dimeric, heterodimeric, multimeric and/or related forms) of binding affinity.
  • the HER2 binding polypeptide may comprise a targeting moiety which binds to the full length and/or mature form and/or isoform and/or splice variant and/or fragment and/or any other native HER2 of human HER2 Existing or synthetic analogues, variants or mutants (including monomeric, dimeric, heterodimeric, multimeric and/or related forms) wherein the KD is less than about 1 uM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, or about 5 nM, or About 1nM.
  • the HER2-binding polypeptide has one or more of the following properties:
  • the expression of the HER2-binding polypeptide of the present application can be about 5%, 10%, 20%, 30%, 40% compared with the reference antibody whose amino acid sequence is shown in SEQ ID NO: 1 , 50%, 60%, 70%, 80%, 90% or higher.
  • the HER2-binding polypeptide can have a PI value of about 5.5, about 6.0, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.
  • the HER2-binding polypeptide comprises an antibody or antigen-binding fragment thereof.
  • the antibodies can include monoclonal antibodies, multispecific antibodies (eg, bispecific antibodies), chimeric antibodies, humanized antibodies, and/or fully human antibodies.
  • the antigen-binding fragment may comprise a Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the antibody or antigen-binding fragment thereof may be VHH.
  • the VHHs are not limited to a particular biological source or to a particular method of preparation.
  • the VHH can generally be obtained in the following ways: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3 ) by "humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding such a humanized VHH domain; (4) by natural expression from any animal species, such as from a mammalian species, such as from a human "Camelization" of existing VH domains, or by expression of nucleic acids encoding such camelized VH domains; (5) "Camelization” by "domain antibodies” or “Dabs” as described in the art , or by the expression of nucleic acids encoding such camelized VH domains; (6) by using synthetic or semi-synthetic techniques to prepare proteins, polypeptides or other amino acid sequences
  • the HER2-binding polypeptide may comprise a camelized polypeptide that has been "camelized", that is, by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody with a camelid VHH of one or more amino acid residues present at corresponding positions in the VHH domain of a heavy chain antibody.
  • "camelizing" substitutions are inserted at amino acid positions forming and/or present at the VH-VL interface and/or at so-called Camelidae hallmark residues (see, e.g., WO9404678, which describes The entire contents of the documents are incorporated into this application by reference).
  • the VH sequence used as starting material or starting point for generating or designing a camelized VHH is a VH sequence from a mammal, eg, a human VH sequence, such as a VH3 sequence.
  • Said camelized VHHs may be obtained in any suitable manner known in the art (i.e. as indicated in points (1) to (8) above), and are therefore not strictly limited to those already used comprising naturally occurring VH domains.
  • a polypeptide obtained from a polypeptide as a starting material is a VH sequence from a mammal, eg, a human VH sequence, such as a VH3 sequence.
  • VHH can be camelid, chimeric, human, partially humanized or fully humanized.
  • the HER2-binding polypeptide may comprise "humanized", that is, one or more amino acid residues in the amino acid sequence (and specifically in the framework sequence) of a naturally occurring VHH sequence VHH in which the base is substituted with one or more amino acid residues present at the corresponding positions in the VH domain of a conventional 4-chain antibody derived from humans.
  • humanization can be done using humanization techniques known in the art.
  • potential humanizing substitutions or combinations of humanizing substitutions can be determined by methods known in the art, for example, by comparing the sequence of the VHH with the sequence of a naturally occurring human VH domain Compare.
  • the humanizing substitutions are selected such that the resulting humanized VHH retains favorable functional properties.
  • the VHHs of the present application can become more "human-like" compared to corresponding naturally occurring VHH domains, while still retaining advantageous properties, such as reduced immunogenicity.
  • the humanized VHH of the present application can be obtained by any suitable means known in the art, and thus is not strictly limited to a polypeptide that has been obtained using a polypeptide comprising a naturally occurring VHH domain as a starting material.
  • VH may comprise CDR1, CDR2 and CDR3 in the amino acid sequence shown in SEQ ID NO:1.
  • the CDR can be Kabat CDR, AbM CDR, Chothia CDR or Contact CDR. In some embodiments, the CDRs are Chothia CDRs.
  • the application provides a HER2-binding polypeptide comprising VH, when compared with an antibody comprising VH of the amino acid sequence shown in SEQ ID NO: 1, the HER2-binding polypeptide has one or more of the following VH Contains amino acid substitutions at positions: L2, I28, V31, A41, E43, Q44, R45, A57, D61, V67, D73, A74, S76, V78, N84, D98, W101, D103, D104, F106, E107, and Q117 .
  • the HER2-binding polypeptide is compared with the reference antibody whose amino acid sequence is shown in SEQ ID NO: 1, and the VH of the HER2-binding polypeptide comprises one or more amino acids selected from the group replace:
  • the HER2 binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 159, and the HCDR2 comprises SEQ ID NO: The amino acid sequence shown in 160, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 161.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises any of SEQ ID NO:13, SEQ ID NO:86, and SEQ ID NO:87
  • the HCDR2 includes SEQ ID NO:25, SEQ ID NO:88, the amino acid sequence shown in any one of SEQ ID NO:89
  • the HCDR3 includes SEQ ID NO:38, SEQ ID NO:38, SEQ ID NO:89
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 25
  • the amino acid sequence of the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:38.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:86, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:25
  • the amino acid sequence of the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:38.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:87, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:25
  • the amino acid sequence of the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:38.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 88
  • the amino acid sequence of the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:38.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 89
  • the amino acid sequence of the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:38.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 25 The amino acid sequence of the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:90.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 25 The amino acid sequence of the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:91.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 25 The amino acid sequence of the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:92.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 25 The amino acid sequence of the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:93.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 88
  • the amino acid sequence of the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:94.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 88 The amino acid sequence of the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:95.
  • the HER2-binding polypeptide comprises VH, wherein the VH comprises: HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 88 The amino acid sequence of the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:96.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 comprises the amino acid sequence shown in SEQ ID NO: 162, and the HFR2 comprises SEQ ID NO: 162
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, and the HFR1 comprises the amino acid sequence shown in SEQ ID NO:49 or SEQ ID NO:51 , the HFR2 comprises SEQ ID NO:55, the amino acid sequence shown in any one of SEQ ID NO:97-SEQ ID NO:106, and the HFR3 comprises SEQ ID NO:60, SEQ ID NO:107-SEQ ID The amino acid sequence shown in any one of NO:116, the HFR4 comprises the amino acid sequence shown in SEQ ID NO:70 or SEQ ID NO:117.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO: 49, and the HFR2 may comprise SEQ ID NO: The amino acid sequence shown in any one of 55, the HFR3 can comprise the amino acid sequence shown in any one of SEQ ID NO:60, and the HFR4 can comprise the amino acid sequence shown in SEQ ID NO:70.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO: 49, and the HFR2 may comprise SEQ ID NO: The amino acid sequence shown in 97, the HFR3 can include the amino acid sequence shown in SEQ ID NO:60, and the HFR4 can include the amino acid sequence shown in SEQ ID NO:70.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO: 49, and the HFR2 may comprise SEQ ID NO: The amino acid sequence shown in 98, the HFR3 can include the amino acid sequence shown in SEQ ID NO:60, and the HFR4 can include the amino acid sequence shown in SEQ ID NO:70.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO: 49, and the HFR2 may comprise SEQ ID NO: The amino acid sequence shown in 99, the HFR3 can include the amino acid sequence shown in SEQ ID NO:60, and the HFR4 can include the amino acid sequence shown in SEQ ID NO:70.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO: 49, and the HFR2 may comprise SEQ ID NO: The amino acid sequence shown in 100, the HFR3 can include the amino acid sequence shown in SEQ ID NO:60, and the HFR4 can include the amino acid sequence shown in SEQ ID NO:70.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO: 49, and the HFR2 may comprise SEQ ID NO: The amino acid sequence shown in 55, the HFR3 can include the amino acid sequence shown in SEQ ID NO: 107, and the HFR4 can include the amino acid sequence shown in SEQ ID NO: 70.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO: 49, and the HFR2 may comprise SEQ ID NO: The amino acid sequence shown in 55, the HFR3 can include the amino acid sequence shown in SEQ ID NO: 108, and the HFR4 can include the amino acid sequence shown in SEQ ID NO: 70.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO: 49, and the HFR2 may comprise SEQ ID NO: The amino acid sequence shown in 55, the HFR3 can include the amino acid sequence shown in SEQ ID NO: 109, and the HFR4 can include the amino acid sequence shown in SEQ ID NO: 70.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO: 49, and the HFR2 may comprise SEQ ID NO: The amino acid sequence shown in 55, the HFR3 can include the amino acid sequence shown in SEQ ID NO:110, and the HFR4 can include the amino acid sequence shown in SEQ ID NO:70.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO: 49, and the HFR2 may comprise SEQ ID NO: The amino acid sequence shown in 55, the HFR3 can include the amino acid sequence shown in SEQ ID NO: 111, and the HFR4 can include the amino acid sequence shown in SEQ ID NO: 70.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO: 49, and the HFR2 may comprise SEQ ID NO: The amino acid sequence shown in 55, the HFR3 can include the amino acid sequence shown in SEQ ID NO:112, and the HFR4 can include the amino acid sequence shown in SEQ ID NO:70.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO:51, and the HFR2 may comprise SEQ ID NO: The amino acid sequence shown in 55, the HFR3 can include the amino acid sequence shown in SEQ ID NO:60, and the HFR4 can include the amino acid sequence shown in SEQ ID NO:117.
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO:51, and the HFR2 may comprise SEQ ID NO:
  • the HER2-binding polypeptide comprises VH, wherein the VH further comprises: HFR1, HFR2, HFR3 and HFR4, the HFR1 may comprise the amino acid sequence shown in SEQ ID NO: 49, and the HFR2 may comprise SEQ ID NO:
  • the HER2-binding polypeptide is compared with the reference antibody whose amino acid sequence is shown in SEQ ID NO: 1, and the VH of the HER2-binding polypeptide comprises one or more amino acids selected from the group Replaces: L2V, A41P, E43K, Q44G, R45L, A57T, V67F, D73N, A74S, S76N, V78L, N84S and Q117L.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: A41P, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 122, and the HER2 binding polypeptide is named as H1-A41P.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: E43K, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 125, and the HER2 binding polypeptide is named as H1-E43K.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: Q44G, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 123, and the HER2 binding polypeptide is named as H1-Q44G.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: R45L, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 124, and the HER2 binding polypeptide is named as H1-R45L.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: A57T, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 126, and the HER2 binding polypeptide is named as H1-A57T.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: V67F, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 127, and the HER2 binding polypeptide is named as H1-V67F.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: D73N, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 118, and the HER2 binding polypeptide is named as H1-D73N.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from the group consisting of: A74S of the HER2-binding polypeptide VH may comprise the amino acid sequence shown in SEQ ID NO: 119, and the HER2 binding polypeptide is named as H1-A74S.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: S76N, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 120, and the HER2 binding polypeptide is named as H1-S76N.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: V78L, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 128, and the HER2 binding polypeptide is named as H1-V78L.
  • the VH of the HER2-binding polypeptide may include amino acid substitutions selected from: N84S, and the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 129, and the HER2 binding polypeptide is named H1-N84S.
  • the HER2-binding polypeptide may contain amino acid substitutions selected from the group consisting of: L2V and Q117L.
  • the HER2 The VH of the binding polypeptide may comprise the amino acid sequence shown in SEQ ID NO: 121, and the HER2 binding polypeptide is named as H1-VL.
  • the HER2-binding polypeptide is compared with the reference antibody whose amino acid sequence is shown in SEQ ID NO: 1, and the VH of the HER2-binding polypeptide comprises one or more amino acids selected from the group Replaces: D61A, D103A, D104A, D98A and E107S.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: D61A, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 140, and the HER2 binding polypeptide is named as H1-D61A.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: D103A, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 141, and the HER2 binding polypeptide is named as H1-D103A.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: D104A, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 142, and the HER2 binding polypeptide is named as H1-D104A.
  • the VH of the HER2-binding polypeptide may include amino acid substitutions selected from: D98A, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 143, and the HER2 binding polypeptide is named as H1-D98A.
  • the HER2-binding polypeptide may contain amino acid substitutions selected from the group consisting of: E107S, the HER2-binding polypeptide
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 144, and the HER2 binding polypeptide is named as H1-E107S.
  • the HER2-binding polypeptide is compared with the reference antibody whose amino acid sequence is shown in SEQ ID NO: 1, and the VH of the HER2-binding polypeptide comprises one or more amino acids selected from the group Replaces: I28T, V31G, A57T, N84S, W101G, W101S and F106Y.
  • the HER2-binding polypeptide may contain amino acid substitutions selected from the following amino acid substitutions: A57T and N84S, and the HER2
  • the VH of the binding polypeptide may comprise the amino acid sequence shown in SEQ ID NO: 145, and the HER2 binding polypeptide is named as H1-AN-TS.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from the group consisting of: V31G, A57T and N84S, the The VH of the HER2-binding polypeptide may comprise the amino acid sequence shown in SEQ ID NO: 146, and the HER2-binding polypeptide is named as H1-VG.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: I28T, A57T and N84S, so The VH of the HER2-binding polypeptide may comprise the amino acid sequence shown in SEQ ID NO: 147, and the HER2-binding polypeptide is named as H1-IT.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: A57T, N84S and W101G, so The VH of the HER2-binding polypeptide may comprise the amino acid sequence shown in SEQ ID NO: 148, and the HER2-binding polypeptide is named H1-WG.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: A57T, N84S and W101S
  • the The VH of the HER2-binding polypeptide may comprise the amino acid sequence shown in SEQ ID NO: 149, and the HER2-binding polypeptide is named H1-WS.
  • the VH of the HER2-binding polypeptide may comprise amino acid substitutions selected from: A57T, N84S and F106Y, so The VH of the HER2-binding polypeptide may comprise the amino acid sequence shown in SEQ ID NO: 150, and the HER2-binding polypeptide is named as H1-FY.
  • the HER2-binding polypeptide is compared with the reference antibody whose amino acid sequence is shown in SEQ ID NO: 130, and the VH of the HER2-binding polypeptide comprises one or more amino acids selected from the group Replaces: P41A, K43E, G44Q, L45R, T57A, F67V, L78V, S84N, V2L and L117Q.
  • the VH of the HER2-binding polypeptide may comprise the following amino acid substitution: P41A, and the HER2-binding polypeptide comprises SEQ ID
  • the amino acid sequence shown in NO:131, the HER2 binding polypeptide is named FH1-P41A.
  • the VH of the HER2-binding polypeptide may comprise the following amino acid substitution: K43E, and the HER2-binding polypeptide comprises SEQ ID
  • the amino acid sequence shown in NO:132, the HER2 binding polypeptide is named FH1-K43E.
  • the VH of the HER2-binding polypeptide may comprise the following amino acid substitution: G44Q, and the HER2-binding polypeptide comprises SEQ ID
  • the amino acid sequence shown in NO:133, the HER2 binding polypeptide is named FH1-G44Q.
  • the VH of the HER2-binding polypeptide may comprise the following amino acid substitution: L45R, and the HER2-binding polypeptide comprises SEQ ID
  • the amino acid sequence shown in NO:134, the HER2 binding polypeptide is named FH1-L45R.
  • the VH of the HER2-binding polypeptide may comprise the following amino acid substitution: T57A, and the HER2-binding polypeptide comprises SEQ ID
  • the amino acid sequence shown in NO:131, the HER2 binding polypeptide is named FH1-T57A.
  • the VH of the HER2-binding polypeptide may comprise the following amino acid substitution: F67V, and the HER2-binding polypeptide comprises SEQ ID
  • the amino acid sequence shown in NO:136, the HER2 binding polypeptide is named FH1-F67V.
  • the VH of the HER2-binding polypeptide may comprise the following amino acid substitution: L78V, and the HER2-binding polypeptide comprises SEQ ID
  • the amino acid sequence shown in NO:137, the HER2 binding polypeptide is named FH1-L78V.
  • the HER2-binding polypeptide may include the following amino acid substitution: S84N, and the HER2-binding polypeptide includes SEQ ID NO: 130.
  • the amino acid sequence shown in NO:138, the HER2 binding polypeptide is named FH1-S84N.
  • the VH of the HER2-binding polypeptide may include the following amino acid substitutions: V2L and L117Q, and the HER2-binding polypeptide includes The amino acid sequence shown in SEQ ID NO: 139, the HER2 binding polypeptide is named as FH1-V2L-L117Q.
  • the HER2-binding polypeptide is compared with the reference antibody whose amino acid sequence is shown in SEQ ID NO: 145, and the VH of the HER2-binding polypeptide comprises one or more amino acids selected from the group Replaces: A41P, E43K, Q44G, R45L, R66V, D73N, A74S, S76N, K86R and P87A.
  • the VH of the HER2-binding polypeptide comprises the following amino acid substitutions: A41P, Q44G, R45L, R66V, D73N, A74S and S76N
  • the HER2-binding polypeptide comprises the amino acid sequence shown in SEQ ID NO: 151
  • the HER2-binding polypeptide is named FH1-H.
  • the VH of the HER2-binding polypeptide comprises the following amino acid substitutions: A41P, E43K, Q44G, R45L, R66V, D73N , A74S and S76N
  • the HER2-binding polypeptide comprises the amino acid sequence shown in SEQ ID NO: 152
  • the HER2-binding polypeptide is named FH1-3KE.
  • the VH of the HER2-binding polypeptide comprises the following amino acid substitutions: A41P, D73N, A74S, S76N, K86R and P87A
  • the HER2-binding polypeptide comprises the amino acid sequence shown in SEQ ID NO: 153
  • the HER2-binding polypeptide is named hH1-AN-TS.
  • the HER2-binding polypeptide contains the following amino acid substitutions: A41P, K86R and P87A, and the HER2-binding polypeptide Comprising the amino acid sequence shown in SEQ ID NO: 154, the HER2-binding polypeptide is named hH1-DA.
  • the VH of the HER2-binding polypeptide contains the following amino acid substitutions: A41P, E43K and Q44G, and the HER2-binding polypeptide Comprising the amino acid sequence shown in SEQ ID NO: 155, the HER2-binding polypeptide is named hH1-KP.
  • the HER2-binding polypeptide is compared with the reference antibody whose amino acid sequence is shown in SEQ ID NO: 1, and the VH of the HER2-binding polypeptide comprises one or more amino acids selected from the group Replaces: Y37V, A41P, R45L, A57T, D73N, A74S, N84S, K86R and/or P87A.
  • the VH of the HER2-binding polypeptide may include the following amino acid substitutions Y37V, A41P, R45L, D73N, A74S, K86R and P87A
  • the HER2-binding polypeptide comprises the amino acid sequence shown in SEQ ID NO: 156
  • the HER2-binding polypeptide is named FH1-N.
  • the VH of the HER2-binding polypeptide may include the following amino acid substitutions R45L, A57T and N84S, and the HER2-binding polypeptide Comprising the amino acid sequence shown in SEQ ID NO: 157, the HER2 binding polypeptide is named H1-RL-VH.
  • the application provides an isolated nucleic acid molecule or molecules encoding the HER2-binding polypeptides described herein.
  • the nucleic acid may encode an amino acid sequence comprising the VHH of the antibody.
  • an isolated nucleic acid encoding an anti-HER2 antibody heavy chain variable region is provided, wherein the nucleic acid comprises a nucleic acid sequence encoding any one of SEQ ID NO:74 to SEQ ID NO:84 having Sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.
  • the present application provides a construct comprising the nucleic acid molecule described in the present application.
  • the present application provides a cell comprising the nucleic acid molecule described herein or the construct described herein.
  • Nucleic acids encoding HER2-binding polypeptides of the present application can be incorporated (linked) into constructs that can be introduced into host cells by transfection, transformation or transduction techniques.
  • a nucleic acid encoding a HER2-binding polypeptide of the present application can be introduced into a host cell by retroviral transduction.
  • the host cell is eukaryotic, such as Chinese Hamster Ovary (CHO) cells or lymphoid cells (eg, Y0, NSO, Sp20 cells).
  • the host cell is prokaryotic, such as an E. coli cell.
  • the present application provides a method for preparing the HER2-binding polypeptide described in the present application, comprising culturing the cells described in the present application under conditions that allow the expression of the HER2-binding polypeptide.
  • the engineered gene is first cloned into an expression vector by placing it downstream of a suitable bacterial promoter such as Trp or Tac and a prokaryotic signal sequence.
  • a suitable bacterial promoter such as Trp or Tac
  • a prokaryotic signal sequence such as Trp or Tac
  • the engineered gene is to be expressed in eukaryotic cells, such as CHO cells, it is first inserted into expression in the carrier. Genetic constructs can be introduced into host cells using transfection, transformation or transduction techniques.
  • the method further comprises recovering the HER2-binding polypeptide expressed by the cells.
  • the method further comprises purifying and/or modifying the HER2-binding polypeptide.
  • HER2-binding polypeptides of the present application can also be expressed in vivo, eg, in a patient.
  • a HER2-binding polypeptide of the present application can be administered in the form of a nucleic acid encoding a HER2-binding polypeptide of the present application.
  • the nucleic acid may be DNA or RNA.
  • a HER2-binding polypeptide of the present application is encoded by a modified mRNA, ie, an mRNA comprising one or more modified nucleotides.
  • the present application relates to gene therapy vectors comprising said modified mRNA.
  • the present application relates to gene therapy methods comprising said gene therapy vectors.
  • the nucleic acid is in the form of an oncolytic virus, such as an adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus, or vaccinia.
  • the present application provides an immunoconjugate comprising the HER2-binding polypeptide described in the present application.
  • the immunoconjugate comprises:
  • a conjugation moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, viral coat proteins or VLPs, or combinations thereof.
  • the detectable label is one or more reagents selected from the group consisting of radionuclides, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes.
  • radionuclide is suitable for medical imaging and/or therapy.
  • Fluorescent agents that can be used for conjugation include, but are not limited to, fluorescein isothionine, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine; chemiluminescent agents that can be used for conjugation Including, but not limited to, luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalates; bioluminescent agents that can be used for conjugation include, but are not limited to, luciferin, luciferase, and jellyfish luminescence protein.
  • Paramagnetic ions that can be used for conjugation include, but are not limited to, chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III) ), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III) and erbium(III), or radiopaque materials, Such as dam, diatrizoate, ethyl iodide, gallium citrate, iocaric acid, iodointamic acid, iodamide, ciliary acid, ioxacic acid, iodactamide, iohexol, iopamidol, iodine Panic acid, ioprosic acid, iociferic acid, io
  • the detectable label may be a radionuclide.
  • the radionuclides that can be used for conjugation can be radionuclides with energy between 20-4000KeV, including but not limited to 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 67 Cu, 67 Ga, 68 Ga, 68 Ge, 86 Y, 90 Y, 89 Zr, 94m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52m Mn, 72 As, 75 Br, 76 Br, 82m Rb, 83 Sr or other ⁇ -, ⁇ -, or positron emitters.
  • the radionuclide may be99mTc , 177Lu or125I .
  • HER2-binding polypeptide is directly or indirectly conjugated to said detectable label.
  • Methods of conjugating detectable labels to polypeptides are well known to those skilled in the art.
  • the HER2-binding polypeptide can be conjugated to the detectable label via a chelating agent.
  • the chelating agent is selected from DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, One or more of HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, HEHA and their derivatives.
  • the present application provides a method for preparing a radionuclide-labeled immunoconjugate of the present application, such as 177 Lu, comprising 1) conjugating the HER2-binding polypeptide of the present application with a chelating agent to generate the HER2 binding the conjugated precursor of the polypeptide and the chelating agent; and 2) contacting the conjugated precursor of step 1) with a radionuclide such as 177 Lu, whereby the radionuclide such as 177 Lu is labeled by chelation of the chelating agent HER2-binding polypeptides of the present application.
  • the chelator is NOTA
  • the HER2-binding polypeptide is generated in step 1) by reacting the HER2-binding polypeptide with p-SCN-Bn-NOTA or p-NH2-Bn-NOTA Conjugate precursor with NOTA.
  • the present application can also use the IODO-BEADS solid-phase labeling method to label the HER2-binding polypeptide described in the present application with radionuclides such as 125 I.
  • the present application provides a composition comprising the HER2-binding polypeptide described in the present application, the nucleic acid molecule described in the present application, the construct described in the present application, the cell described in the present application and/or the present application.
  • the immunoconjugate described in the application and optionally a pharmaceutically acceptable carrier.
  • compositions described herein are in the form of pharmaceutically acceptable salts.
  • the HER2 binding polypeptides described herein may have sufficiently basic functional groups reactive with inorganic or organic acids, or carboxyl groups reactive with inorganic or organic bases, to form pharmaceutically acceptable salts.
  • compositions described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier.
  • Such compositions may optionally contain suitable amounts of pharmaceutically acceptable excipients in order to provide a form for proper administration.
  • Pharmaceutical excipients can be liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the pharmaceutical excipients may be, for example, physiological saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea, and the like.
  • auxiliaries, stabilizers, thickeners, lubricants and colorants may be used.
  • the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any of the agents described herein is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions.
  • suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol , Propylene, Ethylene Glycol, Water, Ethanol, etc. Further examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (ed. Alfonso R. Gennaro, 19th ed., 1995), which is incorporated herein by reference.
  • compositions and/or other therapeutic agents
  • inventive compositions described herein may be in the form of solutions, suspensions, emulsions, drops, tablets, pills, pellets, capsules, capsules containing liquids, gelatin capsules, powders, In the form of sustained release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powders, frozen suspensions, dry powders, or any other combined form.
  • the composition is in the form of a capsule.
  • the composition is in the form of a lozenge.
  • the composition is formulated in the form of a soft gel capsule.
  • the composition is formulated in the form of a gelatin capsule.
  • the composition is formulated as a liquid.
  • composition described herein is formulated according to routine procedures into a composition suitable for the mode of administration described herein.
  • Routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intrathecal, transdermal, rectal, by inhalation or topically.
  • Administration can be local or systemic.
  • the administering is accomplished orally.
  • said administering is by parenteral injection.
  • the mode of administration can be left to the discretion of the physician and will depend in part on the site of the medical condition.
  • the composition comprises the HER2-binding polypeptide described herein or the immunoconjugate described herein, and the composition is a detection agent or a therapeutic agent.
  • the detection agent is a reagent for detecting HER2 protein.
  • the detection agent is a contrast agent.
  • the contrast agent is a contrast agent for detecting HER2 protein.
  • the contrast agent may be an ECT contrast agent.
  • the ECT contrast agent may include a SPECT contrast agent or a PET contrast agent.
  • the therapeutic agent is used to treat tumors.
  • the therapeutic agent is used to treat HER2 positive tumors.
  • the application provides the HER2 binding polypeptide described in the application, the nucleic acid molecule described in the application, the construct described in the application, the cell described in the application, the immunoconjugate described in the application and /or the use of the composition described in this application in the preparation of medicaments, reagents, detection plates or kits;
  • the reagent, detection plate or kit is used to detect HER2 protein in the sample
  • the medicament is used for detecting the expression of HER2 protein and/or treating tumors expressing HER2.
  • the present application provides a recombinant protein comprising the HER2-binding polypeptide described in the present application.
  • the recombinant protein comprises: (i) the HER2-binding polypeptide described herein; and (ii) an optional tag sequence that assists expression and/or purification.
  • the application provides a method for detecting the presence and/or amount of HER2 in a biological sample, comprising: contacting the biological sample with the HER2-binding polypeptide described in the application, the immunoconjugate described in the application Compounds, compositions described herein.
  • the biological sample is tissue
  • tissue is selected from blood tissue, lymphoid tissue and tumor tissue.
  • the method comprises detecting the presence and/or amount of HER2-positive cells in a biological sample.
  • the presence and/or amount of HER2-positive cells in the biological sample is determined by imaging.
  • the presence and/or amount of HER2-positive cells in the biological sample is determined by flow cytometry.
  • the present application provides a method for detecting and/or diagnosing a disease or disorder related to abnormal expression of HER2, comprising administering the HER2-binding polypeptide described in the present application, the HER2-binding polypeptide described in the present application to a subject in need thereof.
  • said method further comprises imaging said subject.
  • said imaging comprises ECT imaging.
  • said ECT imaging comprises SPECT imaging or PET imaging.
  • the disease or disorder associated with abnormal expression of HER2 comprises a tumor.
  • the present application provides a method for treating and/or preventing tumors, the method comprising administering the HER2-binding polypeptide described in the present application, the immunoconjugate described in the present application or Compositions described herein.
  • the application provides a method for monitoring the efficacy of anti-tumor therapy in a subject, the method comprising:
  • the presence and/or amount of HER2-positive cells in the subject's tumor is determined by imaging.
  • the tumor comprises a HER2 positive tumor.
  • the tumor comprises a solid tumor.
  • the tumor is selected from breast cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, ovarian cancer, pancreatic cancer, endometrial cancer, cervical squamous cell carcinoma, salivary adenoma, bladder cancer, lung cancer, At least one of tumors such as colorectal cancer, head and neck cancer, prostate cancer, osteosarcoma, and medulloblastoma in children.
  • the present application provides a kit comprising the HER2-binding polypeptide described in the present application, the immunoconjugate described in the present application or the composition described in the present application.
  • Instructions for use can be included in the kit. Instructions for use typically include tangible representations describing techniques to be employed in using the kit components to achieve a desired therapeutic result, such as in the treatment of cancer.
  • the kit also contains other useful components readily understood by those skilled in the art, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting tools or measuring tools , dressing material or other useful accessories.
  • the Her2-cHis fusion protein used for immunization was expressed by 293F cells and purified by nickel column affinity chromatography. Select an alpaca for immunization. After the immunization, the lymphocytes in the peripheral blood of the alpaca were extracted and the RNA was extracted according to the instructions of Trizol, and 1 ug of the RNA sample was taken for electrophoresis to test its purity. use III First-Strand Synthesis System for RT-PCR kit reverse-transcribed the extracted RNA into cDNA according to the instructions. Nucleic acid fragments encoding the variable regions of heavy chain antibodies were amplified by nested PCR.
  • the VHH fragment was purified using a DNA product purification kit, the vector and the fragment were digested with the restriction endonuclease sifi at 50°C overnight, the digested fragment was recovered by gel cutting, and cloned into the vector pComb3XSS for phage display.
  • the product was then electrotransformed into Escherichia coli electroporation competent cell TG1, and a phage display library of heavy chain single domain antibody against Her2 was constructed and tested. By serial dilution plating, the calculated stock size is 5.04 ⁇ 10 9 .
  • 48 clones were randomly selected for identification. The results show that the insertion rate has reached 100% and the size is correct.
  • the plate was coated with 5 ⁇ g/well of Her2-cHis fusion protein and left overnight at 4°C. The next day, block with 3% BSA at 37°C for 1 hour, wash the plate 3 times with PBST (0.01% Tween 20 in PBS), add 100 ⁇ l phage library, and incubate at 37°C for 1 hour. Then wash with PBST (PBS containing 0.01% Tween 20) 6 times to wash away unbound phages.
  • PBST 0.01% Tween 20 in PBS
  • Recovery rate recovery amount/library input amount
  • clones No. 22 and No. 30 were not sent for sequencing, and the rest of the clones were sent for sequencing.
  • the protein sequences of each clone were analyzed according to the sequence comparison software DNAMAN.
  • Clones with the same CDR1, CDR2, and CDR3 sequences were regarded as the same antibody strain, while clones with different CDR sequences were regarded as different antibody strains.
  • 12 candidate sequences were selected for subsequent experiments.
  • the coding sequence of the single domain antibody obtained by sequencing analysis was subcloned into the expression vector PSNA008, and transfected into HEK293 cells for antibody expression. Dilute the recombinant expression plasmid with Freestyle293 medium and add PEI (Polyethylenimine) solution required for transformation, add the plasmid/PEI mixture to HEK293 cell suspension, and place it at 37°C, 10% CO 2 , 90rpm for culture. Four hours later, add EX293 medium, 2mM glutamine, and culture at 135rpm. After 24 hours 3.8 mM VPA was added. After culturing for 6-7 days, the transient expression culture supernatant was collected and purified with a nickel column. Finally, the antibody protein with a purity of more than 90% is obtained.
  • PEI Polyethylenimine
  • the amino acid sequence of the Her2-Fc fusion protein is:
  • amino acid sequence of a single domain antibody is:
  • a pair of primers were designed for the nucleotide sequence of the anti-Her2 single domain antibody screened, and the nucleotide sequence of each antibody (including the His tag) was amplified by PCR using the bacterial solution as a template, and then cloned into PSNA008 [pCDNA4 (Invitrogen , Cat V86220)] vector, the gene sequence was determined to confirm the correctness of the obtained target clone gene sequence.
  • Antibody number Relative activity (%) 36 139 12 137 2RS15D 118 2 116 5F7 115 31 100 11 49.2 45 47.8 38 0.85 27 0.28 14 0.22 32 0.15 15 0.08
  • Collect N87 cells and pipette into single cell suspension divide into 2x10 ⁇ 5 cells/200uL/tube; add antibody to make the final concentration 5ug/mL, mix well and incubate on ice for 1 hour; centrifuge and wash 3 times, add anti- His secondary antibody, mix well and incubate on ice for 1 hour; centrifuge and wash 3 times, resuspend in 300uL and put on the machine.
  • SA sensor (LOT#1906062) to load biotin-labeled antibody for 120s; after baseline100s, association antigen Her2-chis for 180s; dissociation for 600s.
  • Collect BT474 cells divide into 2x10 ⁇ 5 cells/tube, 200uL/tube, incubate on ice for 30min; 0.04ug/mL), incubated on ice for 1 hour; after centrifugation and washing twice, add his-PE secondary antibody (Miltenyi, batch number 5200303504), and incubate on ice for 1 hour; after centrifugation and washing twice, resuspend in 300uL on the machine.
  • Figure 5 shows that the Her2-1 antibody (Her2-Nb1) binds to the Her2 antigen better than 2RS15D.
  • the density of MCF7 cells was adjusted to 1*10 ⁇ 6 cells/mL; each 100uL was dispensed into EP tubes and applied on ice for 20min.
  • the final reaction concentration of 5F7-cHis, Her2-1-cHis, 2RS15D-cHis was 5 ⁇ g/mL, and 1.15 ⁇ L, 0.5 ⁇ L, 0.69 ⁇ L of corresponding antibodies were added to each tube, and incubated on ice for 30 minutes. Add 300 ⁇ L of 1XPBS+0.5% BSA, centrifuge at 1200 rpm for 5 minutes, suck off the supernatant, and then wash with 300 ⁇ L of 1XPBS+0.5%.
  • Figure 6 shows that Her2-1 can also bind to the Her2 low expression cell line.
  • Collect BT474 cells divide into 2x10 ⁇ 5 cells/tube, 100uL/tube, incubate on ice for 20min; add Her2-1-chis (final concentration is 1ug/mL, 0.1ug/mL), incubate on ice for 30min; wash by centrifugation After 3 times, add his-PE secondary antibody and incubate on ice for 30min; centrifuge and wash 3 times and resuspend in 200uL on the machine.
  • Figure 7 shows that the Her2-1 antibody binds well to the cell line with high expression of Her2.
  • Her2-1 can specifically bind Her2 antigen, and its binding ability is positively correlated with the expression level of Her2 antigen.
  • Her2-1 and 2RS15D can approach the internalization peak within 1 hour, the internalization is fast, and the internalization of 5F7 is slow;
  • SK-OV-3 cells were cultured in a culture dish, and the cells were added to a 6-well plate (3 plates) at approximately 2 ⁇ 10 5 Cells/well with a volume of 1000 ⁇ L/well. Incubate adherent cells overnight at 37°C, 5% CO 2 .
  • the 6-well plate cells are divided into 6 groups, and each group has 3 parallel samples. Add 900 ⁇ L of medium to the cells, and then add 100 ⁇ L of diluted 125 I-004-nbs, and count about 100,000. One group is the block group, and 100 times the corresponding The cold antibody (the corresponding antibody of unlabeled 125 I). The remaining groups are experimental groups. A total volume of 1mL was incubated at 4°C for 1h.
  • the labeling rate of 125 I-Her2-Nb1 was 100%.
  • 125 I-Her2-Nb1 was injected into the tail vein of three BT474-tumor-bearing nude mice, 60 ⁇ Ci/125 ⁇ L each. After 90 minutes of administration, the mice were euthanized, and each tissue was dissected, weighed, and counted on a gamma counter to determine the % injected dose per gram of each tissue (%ID/g).
  • FIG. 10 show that 125 I-labeled Her2--Nb1 has high labeling efficiency, can be highly enriched in the tumor site, and can be metabolized out of the body through the kidney.
  • BT474-BALB/c-nude nude mice were given 125 I-Her2-Nb1 solution 230 ⁇ Ci/125 ⁇ L/mouse iv, and SPECT-CT (IVIS spectrum) dynamic scanning for 0-4h.
  • the results show that 125 I-labeled Her2--Nb1 can be highly enriched in the tumor site, and the ratio of tumor/muscle is high, showing good tumor specificity. There is also high uptake in the kidneys, suggesting that it is mainly metabolized by the kidneys.
  • the labeling rate of 99m Tc-Her2-Nb1 after purification was 100%. According to the labeling ratio, the specific activity of 99m Tc-Her2-Nb1 was calculated to be 31.85 ⁇ Ci/ ⁇ g.
  • 99m Tc-Her2-Nb1 was administered intravenously to BT474 tumor-bearing mice, and about 200 ⁇ Ci of the purified protein was injected into Balb/c nude mice through the tail vein, and then SPECT/CT imaging was performed. ROIs delineate uptake in different tissues.
  • 177 Lu-DOTA-HER2-1 The specific activity of 177 Lu-DOTA-HER2-1 was 6 ⁇ Ci/ ⁇ g.
  • Three MCF7 mice were euthanized 1.5 hours after tail vein injection of 177Lu -DOTA-HER2-1 (10MBq), and the heart, lung, liver, spleen, kidney, stomach, intestine, bone, meat, and tumor were taken to measure the weight and ⁇ count.
  • BT474 tumor-bearing mice were injected with normal saline (control group), 177Lu -DOTA-Her2-1 10Mbq and 20Mbq (once a week, intravenous injection) and Herceptin 10mg/kg (twice a week, intraperitoneal injection) respectively. ), all animals had free access to food and water during the experiment, and the tumor was monitored 2 to 3 times a week.
  • H1-cHis was selected as the final antibody sequence among the candidate antibodies, and a tag-free recombinant plasmid H1 was constructed at the same time.
  • the recombinantly constructed single domain antibody H1 fusion protein plasmid was transfected into HEK293 cells for antibody expression. And it was purified by A3 affinity chromatography. After identification, it was found that the H1 antibody did not bind to the A3 affinity chromatography filler, which made it difficult to obtain the protein in the subsequent purification. Therefore, it is necessary to optimize the sequence of the H1 antibody so that it can be purified by affinity Purify by chromatography.
  • Figure 15A-15B It is the result of SDS-PAGE analysis after antibody purification before and after mutation.
  • Figure 15A shows that the H1 antibody does not bind to the A3 filler, and the protein is flowing through.
  • Figure 15B shows that the H1-AN-TS binds to the A3 filler, and the protein is eluted in buffer C.
  • this part uses a recombinant plasmid with a histidine tag for transient transfection. After the transient transfection, the recombinant protein is purified by affinity chromatography with the corresponding Ni+ resin gel, and the obtained target protein can be used for expression. and detection of binding activity.
  • Detection of affinity activity 0.5 ⁇ g/well of Her2-Fc fusion protein was coated on a plate overnight at 4°C, washed with PBST and then added with 3% BSA for blocking, and then a serial dilution series of each mutant H1 single domain antibody protein was added, at room temperature React for 1 hour. After washing, add anti-his horseradish peroxidase-labeled antibody and react at room temperature for 1 hour. After washing, add chromogenic solution, and read the absorbance at 450nm wavelength.
  • the application software SotfMaxPro v5.4 was used for data processing and graph analysis.
  • Calculation of expression amount Calculate the expression amount based on the total amount of the target protein obtained after one-step affinity chromatography purification.
  • Calculation of expression amount Calculate the expression amount based on the total amount of the target protein obtained after one-step affinity chromatography purification.
  • Antibody molecules generally require an isoelectric point higher than 7, so as to ensure that the molecule can be applied to ion exchange chromatography in the downstream purification process platform. Moreover, the isoelectric point of the host DNA is mainly distributed between 4 and 4.5. If the PI value of the antibody is close to it, it is difficult to achieve the removal effect. Select the negatively charged amino acid in the CDR3 region for mutation. On the one hand, it is considered that the PI value of the antibody can be improved through mutation. On the other hand, the affinity of the antibody is directly related to the CDR3 region of the antibody. Therefore, it is hoped that the H1 antibody can be improved while changing the PI value. affinity. Based on the above reasons, a total of 5 mutants were carried out in this part.
  • Table 12 The binding situation and expression level of each combination mutant to Her2 antigen
  • Camelid antibodies have advantages that traditional antibodies do not have, such as small molecular weight, good in vivo permeability, and easy passage through blood vessels or tissues to reach target sites. These advantages make nanobodies widely used as tools for disease diagnosis and detection. However, if multivalent antibodies are used clinically for a long time, different degrees of immune responses may be generated, which may affect the therapeutic effect. Therefore, humanization of H1 antibodies is required. Humanization is accomplished by the method of humanizing amino acids on the surface of the protein and the transplantation of the general framework of VHH humanization.
  • the humanization steps are as follows: Obtain the general humanized VHH framework h-NbBcIII0FGLA (PDB number: 3EAK) designed by Cecile Vincke et al. based on sequence homology.
  • the framework design is based on the nanobody NbBcIII0 antibody (PDB number: 3DWT), refer to the human antibody to humanize the amino acid on the surface of the protein, and select the site that needs to be humanized according to the specific conditions of the H1 nanobody sequence.
  • the H1 antibody was humanized and mutated using the H1 antibody and the H1-AN-TS antibody as templates, and a total of 7 antibody humanized variants were obtained.
  • Table 13 and Table 14 list the amino acid changes of these humanized variants and the names of the mutated antibodies, wherein the numbering of amino acid residues is in accordance with the Kabat numbering, and the specific names and mutation positions are shown in the table.

Abstract

本申请涉及一种HER2结合多肽和诊疗核素标记物及其制备方法与应用。本申请的HER2结合多肽序列具有良好的体内稳定性、药代动力学性质，其与诊断性或者治疗性放射性核素标记合成的免疫缀合物对HER2分子均具有良好的亲和力和功能活性，有望成为具有良好应用前景的靶向HER2显像用及肿瘤治疗用放射性药物。

Description

HER2结合多肽及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种HER2结合多肽及其用途。
背景技术
人类表皮生长因子受体2(Human epidermal growth factor receptor 2，HER2)，为表皮生长因子受体(epithelial growth factor receptor，EGFR)家族成员之一，在多种肿瘤组织中均有不同程度的表达。HER2扩增或过表达与肿瘤高侵袭性、易复发、死亡率增加密切相关。
癌症的治疗常规治疗方法包括手术、放化疗等方式，在产生一定治疗效果的同时也对其他正常细胞产生了杀伤毒性。虽然在近年出现了靶向治疗，也在一定程度上延长了患者的生存时间，但靶向治疗容易出现耐药性，比如乳腺癌采用曲妥珠单抗治疗后一年左右会出现比较明显的耐药性。因此需要一种有不同杀伤机制的放射性靶向治疗药物作为另一种治疗选择。在靶向结合到肿瘤部位后，放射性靶向治疗药物可通过放射性核素释放的射线对肿瘤细胞进行放射性物理杀伤。
本领域需要能够用于检测表达HER2的细胞的检测剂以及靶向HER2阳性肿瘤的治疗剂，为HER2阳性肿瘤患者提供有效的分子影像学手段和靶向治疗手段。
发明内容
本发明的目的是提供一种HER2结合多肽，以及进一步得到的诊疗核素标记物及制备方法与应用。
一方面，本申请提供了一种HER2结合多肽，其与参比抗体竞争结合HER2蛋白，其中所述参比抗体包含重链可变区(VH)；其中所述VH包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，其在ELISA试验中，所述的HER2结合多肽能够以不超过约1.20E-09的K D值与HER2蛋白相结合。
在某些实施方式中，其中所述HER2结合多肽具有以下性质中的一种或多种：
(i)其能够与A3亲和层析柱的填料结合；
(ii)与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，其能够以相同或更高的亲和力与HER2结合；
(iii)与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，其具有增加的表达量；和
(iv)与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，其等电点提高。
在某些实施方式中，所述的HER2结合多肽包括抗体或其抗原结合片段。
在某些实施方式中，所述抗体包括单克隆抗体、多特异性抗体、嵌合抗体、人源化抗体和/或全人源抗体。
在某些实施方式中，所述抗原结合片段包括Fab、Fab’、Fv片段、F(ab’)2、scFv、VHH和/或dAb。
在某些实施方式中，其中所述VHH是骆驼科的、嵌合的、人类的、部分人源化的或完全人源化的。
在某些实施方式中，其包含SEQ ID NO:158，SEQ ID NO:2至SEQ ID NO:12中任一项所示氨基酸序列中的至少一个CDR。
在某些实施方式中，其包含SEQ ID NO:158，SEQ ID NO:2至SEQ ID NO:12中任一项所示氨基酸序列中的HCDR1、HCDR2和HCDR3。
在某些实施方式中，所述的HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:159、SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:21中任一项所示的氨基酸序列。
在某些实施方式中，其中所述HCDR1包含SEQ ID NO:13、SEQ ID NO:86和SEQ ID NO:87所示的氨基酸序列。
在某些实施方式中，其中所述HCDR1包含SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，其中所述HCDR2包含SEQ ID NO:160、SEQ ID NO:26、SEQ ID NO:27和SEQ ID NO:34中任一项所示的氨基酸序列。
在某些实施方式中，其中所述HCDR2包含SEQ ID NO:25、SEQ ID NO:88和SEQ ID NO:89所示的氨基酸序列。
在某些实施方式中，其中所述HCDR2包含SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:36和SEQ ID NO:37中任一项所示的氨基酸序列。
在某些实施方式中，其中所述HCDR3包含SEQ ID NO:161、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45 和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，其中所述HCDR3包含SEQ ID NO:38、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95或SEQ ID NO:96所示的氨基酸序列。
在某些实施方式中，其中所述HCDR3包含SEQ ID NO:47或SEQ ID NO:48所示的氨基酸序列。
在某些实施方式中，其包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:159所示的氨基酸序列，所述HCDR2包含SEQ ID NO:160所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:161所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:14所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:39所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:15所示的氨基酸序列，所述HCDR2包含SEQ ID NO:27所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44和SEQ ID NO:45中任一项所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含SEQ ID NO:34所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:46所示的氨基酸序列。
在某些实施方式中，其包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:13，SEQ ID NO:86或SEQ ID NO:87所示的氨基酸序列，所述HCDR2包含SEQ ID NO:25，SEQ ID NO:88或SEQ ID NO:89所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:38，SEQ ID NO:90，SEQ ID NO:91，SEQ ID NO:92，SEQ ID NO:93，SEQ ID NO:94，SEQ ID NO:95或SEQ ID NO:96所示的氨基酸序列。
在某些实施方式中，其包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:28所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:40所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:17所示的氨基酸序列，所述HCDR2包含SEQ ID NO:29所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:41所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:42所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:19所示的氨基酸序列，所述HCDR2包含SEQ ID NO:31 所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:43所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:32所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:44所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:33所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:45所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含SEQ ID NO:35所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:47所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:23所示的氨基酸序列，所述HCDR2包含SEQ ID NO:36所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:48所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:24所示的氨基酸序列，所述HCDR2包含SEQ ID NO:37所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:47所示的氨基酸序列。
在某些实施方式中，所述的HER2结合多肽包含VH，其中所述VH包含SEQ ID NO:158，SEQ ID NO:2至SEQ ID NO:12中任一项所示的氨基酸序列。
在某些实施方式中，所述的HER2结合多肽包含VH，其中所述VH包含SEQ ID NO:1，SEQ ID NO:118至SEQ ID NO:157中任一项所示的氨基酸序列。
在某些实施方式中，所述HER2结合多肽包含至少一个VH。
在某些实施方式中，所述HER2结合多肽包括纳米抗体，所述纳米抗体包含SEQ ID NO:158，SEQ ID NO:2至SEQ ID NO:12中任一项所示的氨基酸序列。
在某些实施方式中，所述HER2结合多肽包括纳米抗体，所述纳米抗体包含SEQ ID NO:1，SEQ ID NO:118至SEQ ID NO:157中任一项所示的氨基酸序列。
另一方面，本申请提供了一种分离的一种或多种核酸分子，其编码本申请所述的HER2结合多肽。
另一方面，本申请提供了一种构建体，其包含本申请所述的核酸分子。
另一方面，本申请提供了一种细胞，其包含本申请所述的核酸分子或本申请所述的构建体。
另一方面，本申请提供了一种制备本申请所述的HER2结合多肽的方法，包括在允许所述HER2结合多肽表达的条件下培养本申请所述的细胞。
在某些实施方式中，所述方法还包括回收由所述细胞表达的HER2结合多肽。
在某些实施方式中，所述方法还包括纯化和/或修饰所述HER2结合多肽。
另一方面，本申请提供了一种免疫缀合物，其包含本申请所述的HER2结合多肽。
在某些实施方式中，所述免疫缀合物包含：
i)本申请所述的HER2结合多肽；
ii)选自下组的缀合部分：可检测标记物、药物、毒素、细胞因子、病毒外壳蛋白或VLP、或其组合。
在某些实施方式中，其中所述可检测标记物选自下组的一种或多种试剂：放射性核素、荧光剂、化学发光剂、生物发光剂、顺磁离子和酶。
在某些实施方式中，其中所述放射性核素适用于医学成像和/或治疗。
在某些实施方式中，其中所述放射性核素包括 110In、 111In、 177Lu、 18F、 52Fe、 62Cu、 67Cu、 67Ga、 68Ga、 68Ge、 86Y、 90Y、 89Zr、 94mTc、 120I、 123I、 124I、 125I、 131I、 154-158Gd、 32P、 11C、 13N、 15O、 186Re、 188Re、 51Mn、 52mMn、 72As、 75Br、 76Br、 82mRb、 83Sr或其他γ-、β-、或正电子发射体。
在某些实施方式中，其中所述HER2结合多肽直接或间接与所述可检测标记物缀合。
在某些实施方式中，其中所述HER2结合多肽通过螯合剂与所述可检测标记物缀合。
在某些实施方式中，其中所述螯合剂选自DTPA、EDTA、NOTA、DOTA、TRAP、TETA、NETA、CB-TE2A、Cyclen、Cyclam、Bispidine、TACN、ATSM、SarAr、AmBaSar、MAG3、MAG2、HYNIC、DADT、EC、NS3、H2dedpa、HBED、DFO、PEPA、HEHA及它们的衍生物中的一种或多种。
另一方面，本申请提供了一种组合物，其包含本申请所述的HER2结合多肽、本申请所述的核酸分子、本申请所述的构建体、本申请所述的细胞和/或本申请所述的免疫缀合物，以及任选地药学上可接受的载体。
在某些实施方式中，所述组合物包含本申请所述的HER2结合多肽或本申请所述的免疫缀合物，所述组合物为检测剂或治疗剂。
在某些实施方式中，其中所述检测剂为用于检测HER2蛋白的试剂。
在某些实施方式中，其中所述检测剂为造影剂。
在某些实施方式中，其中所述造影剂为检测HER2蛋白的造影剂。
在某些实施方式中，其中所述治疗剂用于治疗肿瘤。
在某些实施方式中，其中所述治疗剂用于治疗HER2阳性肿瘤。
另一方面，本申请提供了本申请所述的HER2结合多肽、本申请所述的核酸分子、本申 请所述的构建体、本申请所述的细胞、本申请所述的免疫缀合物和/或本申请所述的组合物在制备药剂、试剂、检测板或试剂盒中的用途；
其中所述试剂、检测板或试剂盒用于检测样品中HER2蛋白；
其中所述药剂用于检测HER2蛋白的表达和/或治疗表达HER2的肿瘤。
另一方面，本申请提供了重组蛋白，所述的重组蛋白包含本申请所述的HER2结合多肽。
在某些实施方式中，其中所述的重组蛋白包含：(i)本申请所述的HER2结合多肽；以及(ii)任选的协助表达和/或纯化的标签序列。
另一方面，本申请提供了一种检测生物学样品中HER2的存在和/或量的方法，包括：使所述生物学样品接触本申请所述的HER2结合多肽、本申请所述的免疫缀合物、本申请所述的组合物。
在某些实施方式中，其中所述接触在体外或离体进行。
在某些实施方式中，其中所述生物学样品为组织。
在某些实施方式中，其中所述组织选自血液组织、淋巴组织和肿瘤组织。
在某些实施方式中，所述方法包括检测生物学样品中HER2阳性细胞的存在和/或量。
在某些实施方式中，其中通过成像确定生物学样品中HER2阳性细胞的存在和/或量。
在某些实施方式中，其中通过流式细胞术确定生物学样品中HER2阳性细胞的存在和/或量。
另一方面，本申请提供了一种检测和/或诊断与HER2表达异常相关的疾病或病症方法，包括向有此需要的受试者施用本申请所述的HER2结合多肽、本申请所述的免疫缀合物或本申请所述的组合物。
在某些实施方式中，其中所述方法还包括对所述受试者进行成像。
在某些实施方式中，其中所述成像包括ECT成像。
在某些实施方式中，其中所述ECT成像包括SPECT成像或PET成像。
在某些实施方式中，其中所述与HER2表达异常相关的疾病或病症包括肿瘤。
另一方面，本申请提供了一种治疗和/或预防肿瘤的方法，所述方法包括向有需要的受试者施用本申请所述的HER2结合多肽、本申请所述的免疫缀合物或本申请所述的组合物。
另一方面，本申请提供了一种用于监测抗肿瘤疗法在受试者中的功效的方法，所述方法包含：
(i)给患有肿瘤且采用抗肿瘤疗法治疗的受试者施用本申请所述的HER2结合多肽、本申 请所述的免疫缀合物或本申请所述的组合物；和
(ii)确定所述受试者的肿瘤中HER2阳性细胞的量。
在某些实施方式中，通过成像确定所述受试者的肿瘤中HER2阳性细胞的存在和/或量。
在某些实施方式中，其中所述肿瘤包括HER2阳性肿瘤。
在某些实施方式中，其中所述肿瘤包括实体瘤。
在某些实施方式中，其中所述肿瘤选自乳腺癌、胃癌、食管癌、胆管癌、卵巢癌、胰腺癌、子宫内膜癌、子宫颈鳞状细胞癌、唾液腺瘤、膀胱癌、肺癌、结直肠癌、头颈癌、前列腺癌、骨肉瘤、儿童成神经管细胞瘤等肿瘤中的至少一种。
另一方面，本申请提供了一种试剂盒，其包含本申请所述的HER2结合多肽、本申请所述的免疫缀合物或本申请所述的组合物。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1A-1C显示的是本申请所述抗Her2抗体与N87细胞的结合情况；
图2显示的是本申请所述抗Her2抗体H1的KD亲和力检测结果；
图3显示的是本申请所述抗Her2抗体H2的KD亲和力检测结果；
图4A-4B显示的是本申请所述抗Her2抗体与Herceptin的表位竞争结果；
图5显示的是本申请所述抗Her2抗体H1(Her2-Nb1)与BT474细胞的结合情况；
图6显示的是本申请所述抗Her2抗体H1与MCF7细胞的结合情况；
图7显示的是本申请所述抗Her2抗体H1与BT474细胞的单点结合情况；
图8显示的是本申请所述抗Her2抗体H1的内化结果；
图9显示的是本申请所述 125I标记的抗Her2抗体( 125I-Her2-Nbs)的在细胞(SKOV3) 饱和结合曲线；
图10显示的是本申请所述 125I-Her2-Nb1在BT474肿瘤的生物分布情况；
图11显示的是本申请所述 125I-Her2-Nb1在BT474肿瘤的动态显像；
图12显示的是本申请所述 99mTC-Her2-Nb1在BT474肿瘤的SPE CT/CT显影；
图13显示的是本申请所述 177Lu-Her2-Nb1在MCF7肿瘤的生物分布情况；
图14显示的是本申请所述 177Lu-Her2-Nb1的抗肿瘤效应。
图15A-15B显示的是本申请所述H1抗体突变前后抗体纯化后SDS-PAGE分析结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“HER2”通常指属于表皮生长因子受体家族的I型跨膜蛋白，又称为c-erbB2、ErbB2或Neu。术语“HER2”还涵盖HER2的同系物、变体和同等型，包括剪接同等型。HER2与人乳腺癌细胞中的肿瘤转化相关，如已在乳腺癌、胃癌、胰腺癌、卵巢癌、腹膜癌或结肠肠癌的患者中检测到HER2蛋白的过量表达。术语“HER2阳性”和“表达HER2”在本申请中可互换使用。“HER2阳性”肿瘤包含具有高于正常水平的HER2的肿瘤细胞。HER2阳性肿瘤的实例包括HER2阳性乳腺癌和HER2阳性胃癌。任选地，HER2阳性是过度表达HER2的癌症，并且在某些实施方案中，HER-2阳性癌症具有2+或3+的免疫组织化学(IHC)评分和/或原位杂交(ISH)扩增率≥2.0。
在本申请中，术语“HER2结合多肽”意指任何能够特异性结合HER2的多肽。在一些实施方案中，结合多肽是抗体。在其它实施方案中，结合多肽是例如抗体模拟物、细胞因子或生长因子。例如本申请的特异性结合HER2的单域抗体。“HER2结合多肽”或者可以指结合HER2的单价多肽(即与HER2的一个表位结合的多肽)，以及二价或多价结合多肽(即结合一个以上表位的结合多肽)。本申请的“HER2结合多肽”可以包含至少一个结合HER2的可变结构域。在一些实施方案中，本申请的“HER2结合多肽”可以包含2、3、4或更多个结合HER2的可变结构域。本申请的HER2结合多肽除结合HER2的可变结构域外也可包含接头和/或具有效应器功能的部分，例如半衰期延长部分(如结合血清白蛋白的可变结构域)、和/或融合配偶体(如血清白蛋白)和/或缀合的聚合物(如PEG)和/或Fc区。在一些实施方案中， 本申请的“HER2结合多肽”还涵盖双特异性抗体，其含有结合不同抗原的可变结构域。
在本申请中，术语“抗体”是在最广泛的意义上加以使用并且具体地涵盖单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体(例如，双特异性抗体)、和抗体片段，只要它们显示所期望的生物活性(Milleretal(2003)Jour.ofImmunology170:4854-4861)，即结合HER2。抗体可以是鼠、人、人源化、嵌合抗体，或源于其它物种。
全长抗体典型地是指由两条“全长抗体重链”和两条“全长抗体轻链”组成的抗体。“全长抗体重链”通常是这样的多肽，其在N端到C端方向由抗体重链可变结构域(VH)、抗体恒定重链结构域1(CH1)，抗体铰链区(HR)，抗体重链恒定结构域2(CH2)，和抗体重链恒定结构域3(CH3)组成，缩写为VH-CH1-HR-CH2-CH3；并且在IgE亚类的抗体的情形中，任选地还包括抗体重链恒定结构域4(CH4)。在一些实施方式中，“全长抗体重链”是在N端到C端方向由VH，CH1，HR，CH2和CH3组成的多肽。“全长抗体轻链”通常是在N端到C端方向由抗体轻链可变结构域(VL)，和抗体轻链恒定结构域(CL)组成的多肽，缩写为VL-CL。所述抗体轻链恒定结构域(CL)可以是κ(kappa)或λ(lambda)。两条全长抗体链通过在CL结构域和CH1结构域之间的多肽间二硫键和全长抗体重链的铰链区之间的多肽间二硫键连接在一起。典型的全长抗体的实例是天然抗体如IgG(例如，IgG1和IgG2)，IgM，IgA，IgD，和IgE)。
在本申请中，术语“抗原结合片段”通常是指抗体分子的一部分，其包含负责抗体与抗原之间的特异性结合的氨基酸。抗原中由抗体特异性地识别和结合的部分是称作如上文所述的“表位”。抗原结合结构域可典型地包含抗体轻链可变区(VL)和抗体重链可变区(VH)；然而，其并非必须包含两者。Fd片段例如具有两个VH区并且通常保留完整抗原结合结构域的一些抗原结合功能。抗体的抗原结合片段的实例包括(1)Fab片段，具有VL、VH、恒定轻链(CL)和CH1结构域的单价片段；(2)F(ab’) 2片段，具有由铰链区的二硫桥连接的两个Fab片段的二价片段；(3)具有两个VH和CH1结构域的Fd片段；(4)具有抗体单臂的VL和VH结构域的Fv片段，(5)dAb片段(Ward等人,“Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli,”Nature 341:544-546(1989)，其以引用的方式整体并入本申请)，其具有VH结构域；(6)分离的互补决定区(CDR)；(7)单链Fv(scFv)，例如源于scFV-文库。尽管Fv片段的两个结构域VL和VH是由独立基因编码，但其可通过合成连接子使用重组方法接合，合成连接子使得其被制备为其中VL和VH区配对以形成单价分子的单一蛋白链(称为单链Fv(scFv))(可参见例如Huston等人,“Protein  Engineering of Antibody Binding Sites:Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli,”Proc.Natl.Acad.Sci.USA 85:5879-5883(1988))；和(8)VHH，“VHH”涉及来自骆驼科(骆驼、单峰骆驼、美洲驼、羊驼等)重链抗体的可变抗原结合结构域(参见Nguyen V.K.等人，2000，The EMBO Journal，19，921-930；Muyldermans S.，2001，J Biotechnol.，74，277-302以及综述Vanlandschoot P.等人，2011，Antiviral Research 92，389-407)。VHH也可称为纳米抗体(Nanobody)(Nb)和/或单域抗体。这些抗体片段使用所属领域的技术人员已知的常规技术获得，且以与完整抗体相同的方式评估所述片段的功能。
在本申请中，术语“可变结构域”通常是指能够特异性结合抗原表位的抗体的可变结构域。例如，抗体可变结构域VH及VL(VH结构域及VL结构域)。可变结构域的另一实例为“VHH结构域”(或简称为“VHH”)。“VHH结构域”，亦称为重链单域抗体、VHH、V HH结构域、VHH抗体片段和VHH抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(Hamers-Casterman C,Atarhouch T,Muyldermans S,Robinson G,Hamers C,Songa EB,Bendahman N,Hamers R.:“Naturally occurring antibodies devoid of light chains”；Nature 363,446-448(1993))。使用术语“VHH结构域”以将所述可变结构域与存在于常规4链抗体中的重链可变结构域(其在本申请中称为“VH结构域”)以及存在于常规4链抗体中的轻链可变结构域(其在本申请中称为“VL结构域”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规4链抗体中的VH或VL结构域相反，在该情况下其表位由VL结构域与VH结构域一起识别)。
在本申请中，“可变结构域”通常具有相同的大体结构，每个结构域包含4个序列高度保守的框架(FR)区，其中，FR区包括“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”，FR区“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”连接。可变结构域的一般结构或序列可表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。抗体可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。
在本申请中，术语“CDR”通常是指抗体可变序列内的互补性决定区。在重链和轻链的每个可变区中存在3个CDR，对于每个可变区，其称为CDR1、CDR2和CDR3。这些CDR的精确界限已经根据不同系统进行了不同定义。Kabat描述的系统(Kabat等人，Sequences of Proteins of Immunological Interest(National Institutes ofHealth，Bethesda，Md.(1987)和(1991))不 仅提供了适用于抗体的任何可变区的明确的残基编号系统，而且还提供了定义这三个CDR的精确残基界限。这些CDR可称为Kabat CDR。Chothia及其同事(Chothia&#x26；Lesk，J.MoI.Biol.196:901-917(1987)和Chothia等人，Nature 342:877-883(1989))发现，Kabat CDR内的一些亚部分几乎采取了相同的肽骨架构象，尽管在氨基酸序列水平上具有很大差异。这些亚部分指定为L1、L2和L3，或者H1、H2和H3，其中“L”和“H”分别是指轻链和重链区域。这些区域可以称为Chothia CDR，其具有与Kabat CDR重叠的界限。Padlan(FASEB J.9:133-139(1995))和MacCallum(JMoI Biol 262(5):732-45(1996))中已经描述了定义与Kabat CDR重叠的CDR的其他界限。其他CDR界限可能没有严格遵照一个上述系统，但是仍然与Kabat CDR重叠，尽管根据以下预测或实验发现，他们可以被缩短或延长，特定残基或残基组或甚至整个CDR没有显著影响抗原结合。除非在说明书中另有明确说明，如本申请所用，术语“CDR”、“HCDR1”、“HCDR2”、“HCDR3”、“LCDR1”、“LCDR2”和“LCDR3”包括上文所述的任何方法(Kabat、Chothia或IMGT)所定义的CDR。
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即集群中的个别抗体是相同的，除了可能存在的少量的自然突变。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，本申请使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的小的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中 或在供体抗体中没有的氨基酸修饰。进行这些修饰可以是为了进一步改进抗体的性能，诸如结合亲和力。
在本申请中，术语“全人源抗体”通常是指将人类编码抗体的基因转移至基因工程改造的抗体基因缺失动物中，使动物表达的抗体。抗体所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码。全人源抗体可以大大减少异源抗体对人体造成的免疫副反应。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术、核糖体展示技术和RNA-多肽技术等。
在本申请中，术语“参比抗体”通常是指本申请所述抗原结合蛋白与之竞争结合抗原(例如HER2)的抗体。
当在竞争相同表位的抗原结合蛋白上下文中使用的术语“竞争”通常表示抗原结合蛋白之间的竞争，如通过其中被测试的抗原结合蛋白(例如，抗体或其免疫功能片段)预防或抑制(例如，减少)参比抗原结合蛋白(例如，配体或参比抗体)与共同抗原(例如，HER2或其片段)特异性结合的测定所确定的。许多类型的竞争性结合测定可用于确定一个抗原结合蛋白是否与另一个竞争，例如：固相直接或间接放射性免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如，Stahli等人，1983，Methods in Enzymology9：242-253)；固相直接生物素-亲和素EIA(参见例如，Kirkland等人，1986，J.Immunol.137：3614-3619)固相直接标记测定、固相直接标记夹心测定(参见例如，Harlow和Lane，1988，Antibodies，A Laboratory Manual，Cold Spring Harbor Press)；使用I-125标记的固相直接标记RIA(参见例如，Morel等人，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如，Cheung等人，1990，Virology176：546-552)；和直接标记RIA(Moldenhauer等人，1990，Scand.J.Immunol.32：77-82)。通常，这种测定涉及与固体表面结合的纯化抗原或者携带这些任何一个的单元、未标记的测试抗原结合蛋白和标记的参比抗原结合蛋白的使用。竞争性抑制通过测定在测试抗原结合蛋白存在下于固体表面或单元结合的标记的量来测量。通常，测试抗原结合蛋白过量存在。通过竞争测定鉴定的抗原结合蛋白(竞争抗原结合蛋白)包括与参比抗原结合蛋白结合相同表位的抗原结合蛋白和与参比抗原结合蛋白结合的表位足够靠近而发生空间位阻的相邻表位结合的抗原结合蛋白。通常，当竞争抗原结合蛋白过量存在时，它使参比抗原结合蛋白与共同抗原的特异性结合抑制(例如，减少)至少约40-45％、约45-50％、约50-55％、约55-60％、约60-65％、约65-70％、约70-75％或约75％或更多。在一些情况下，结合被抑制至少约80-85％、约85-90％、约90-95％、约95-97％或约97％或更多。
在本申请中，术语“序列同一性”通常是指是使用序列比对程序比对时，两个或多个比对的序列相同的核酸或氨基酸序列。在此的术语“％序列同一性”通常指的是使用序列比对程序比对时，两个或多个比对序列之间的核酸或氨基酸序列同一性的水平。用于评价氨基酸或核苷酸之间的序列相同性程度的方法是本领域技术人员已知的。例如，氨基酸序列相同性通常使用序列分析软件来测量。例如，可使用NCBI数据库的BLAST程序来确定相同性。对于序列相同性的确定，可以参见例如：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988；Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993；Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994；Sequence Analysis in Molecular Biology,20von Heinje,G.,Academic Press,1987和Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991。
在本申请中，氨基酸残基将根据如本领域中公知且达成一致的标准三字母或一字母氨基酸编码加以表示。在比较两个氨基酸序列时，术语“氨基酸差异”通常是指与另一序列相比，在参考序列某一位置处指定数目氨基酸残基的插入、缺失或取代。在一些实施方式中，所述取代为保守氨基酸取代，所述保守氨基酸是指氨基酸残基被化学结构类似的另一氨基酸残基置换，且其对多肽的功能、活性或其他生物性质影响较小或基本上无影响。所述保守氨基酸取代在本领域中是公知的，例如保守氨基酸取代是以下组(i)-(v)内的一个氨基酸被同一组内的另一氨基酸残基所取代：(i)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；(ii)极性带负电残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；(iii)极性带正电残基：His、Arg及Lys；(iv)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及(v)芳族残基：Phe、Tyr及Trp。特别优选的保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。在一些实施方式中，所述取代为非保守氨基酸取代，例如，Ala被Asp、Asn、Glu或Gin取代。
在本申请中，术语“亲和力”通常是指分子(例如，多肽或抗体)的单个结合位点与其结合配偶体(例如，靶标或抗原)之间的非共价相互作用的总和的强度。分子X对其配偶体Y的 亲和力通常可以由解离常数(Kd)表示。亲和力可以通过本领域已知的的常用方法测量，诸如表面等离子体共振，并且还包括本申请所报道的那些方法。分子X对其结合配偶体Y的较高的亲和力可见于较低的Kd值和/或EC 50值。
在本申请中，术语“分离的”通常是指至少部分从在其天然状态中通常与其结合的其他分子中分离的分子(例如抗体、核酸等)。“分离的多肽”基本上没有其他生物分子，如核酸、蛋白质、脂质、碳水化合物、细胞碎片和生长培养基。“分离的核酸”通常以不同于其天然发现的形式或背景存在。
在本申请中，术语“构建体”通常是指包含编码蛋白质的核苷酸序列的DNA或RNA分子。编码序列或“编码核酸序列”可包括与调控元件可操作地连接的起始信号和终止信号，所述调控元件包括能够在施用了所述核酸分子的个体的细胞中指导表达的启动子和聚腺苷酸化信号。实例包括但不限于环状、线性、双链、染色体外DNA分子(质粒)、粘粒(含有来自λ噬菌体的COS序列的质粒)、包含非天然核酸序列的病毒基因组等。
在本申请中，术语“免疫缀合物”通常是指抗体或其抗体片段与其它活性剂连接而形成的缀合物，诸如化疗剂，毒素，免疫治疗剂，放射性元素、成像探针，光谱探针等等。所述连接可以是共价键，或例如通过静电力的非共价相互作用。可以使用本领域中已知的多种接头以形成免疫缀合物。该缀合物可以通过所述抗体或其抗原结合片段与靶细胞上的抗原特异性结合，将所述其他试剂递送至靶细胞(例如，肿瘤细胞)。此外，该免疫缀合物可以以融合蛋白的形式提供，所述融合蛋白可以从编码该免疫缀合物的多核苷酸表达。
在本申请中，术语“螯合剂”通常是指能够与金属离子形成络合物的有机分子。螯合剂常用来标记物蛋白质或肽。金属离子缀合物的终产物用于放射免疫检测、放射免疫疗法、磁共振成像、光动力疗法或其他相似的模式。螯合剂或络合剂的非限制性例子是DTPA(二亚乙基三胺五乙酸酐)和其衍生物，NOTA(1,4,7-三氮杂环壬烷-N,N’,N”-三乙酸)和其衍生物如NODA-GA(NODAGA)、Maleimide-NODAGA，DOTA(1,4,7,10-四氮杂环十二烷-N,N’,N”,N”’-四乙酸)(结合放射性金属离子)和其衍生物，TETA(1,4,8,11-四氮杂环十四烷-N,N’,N”,N”’-四乙酸)和其衍生物，DTTA(N-(对-异硫氰酸苄基)-二亚乙基三胺-N,N’,N”,N”’-四乙酸)。这些和其他螯合剂从商业来源轻易可获得。
在本申请中，术语“药学上可接受的载体”通常是指不干扰活性成分的生物活性的有效性的一种或多种非毒性材料。这类制剂常规地可以含有盐、缓冲剂、防腐剂、相容的载体、以及任选地其他治疗剂。这类药学上可接受的制剂还可以含有适合于给予人的相容的固体或 液体填料、稀释剂或包封物质。可以用于在此所描述的配制品中的其他设想的载体、赋形剂、和/或添加剂包括：例如，调味剂、抗微生物剂、增甜剂、抗氧化剂、抗静电剂、脂质、蛋白质赋形剂(如血清白蛋白、明胶、酪蛋白)、成盐平衡离子(如钠)等等。适合用于在此所描述的配制品中的这些和另外已知的药物载体、赋形剂和/或添加剂是本领域中已知的，例如，如“雷明顿药物科学与实践(Remington:The Science &#x26；Practice of Pharmacy)”，第21版，利平科特威廉斯与威尔金斯出版公司(Lippincott Williams &#x26；Wilkins)(2005)以及“医师案头参考(Physician’sDesk Reference)”，第60版，医药经济出版社(Medical Economics)，蒙特维尔(Montvale)，新泽西(2005)中所列出。可以常规地选择对于所希望或所要求的给予方式、溶解度和/或稳定性来说合适的药学上可接受的载体。
在本申请中，术语“施用(administer)”和类似术语通常不限于身体施用，合适的方法包括体外、先体外后体内或体内方法。例如，可以采用本领域技术人员已知的用于使细胞、器官或组织与组合物接触的任何施用方法。例如，可以通过任意引入或递送途径将所述化合物引入需要治疗的受试者的身体中。在一些实施方案中，本申请的组合物可以口服、局部、鼻内、肌内、皮下、皮内、鞘内、腹膜内或经皮施用。
在本申请中，术语“离体”与“体外”可互换，通常是指在受控的环境中在已从受试者体内移除的细胞、组织和/或器官中进行的活动。
在本申请中，术语“诊断”通常是指检测疾病或病症，或测定疾病或病症的状态或程度。术语“诊断”也可以包括检测疾病或病症的诱因，测定药物治疗的治疗效果，或预测对药物治疗的响应模式。
在本申请中，术语“治疗”通常是指：(i)预防可能易患疾病、病症和/或病状、但尚未诊断出患病的患者出现该疾病、病症或病状；(ii)抑制该疾病、病症或病状，亦即遏制其发展；以及(iii)缓解该疾病、病症或病状，亦即使得该疾病、病症和/或病状和/或与该疾病、病症和/或病状相关联的症状消退。
在本申请中，术语“肿瘤”和“癌症”可互换使用，通常是指赘生性或恶性细胞生长。本申请的肿瘤可能是良性的，也可能是恶性的。本申请的肿瘤可能是实体的，也可能是非实体的。
在本申请中，术语“受试者”通常是指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴等。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下， 也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下约0.5％、约1％、约1.5％、约2％、约2.5％、约3％、约3.5％、约4％、约4.5％、约5％、约5.5％、约6％、约6.5％、约7％、约7.5％、约8％、约8.5％、约9％、约9.5％、或约10％的范围内变动。
在本申请中，术语“包含”和其变形形式，包括“含有”、“包括”等其它形式，通常是指包含其它组分、元素、数值、步骤等。
在本说明书和权利要求书中，除非上下文另外明确说明，否则单数形式“一个/种(a/an)”和“该(the)”包括复数参考对象。术语“一个/一种(a)”(或“一个/一种(an)”)、以及术语“一个或多个/一种或多种(one or more)”和“至少一个/至少一种(atleast one)”可以在此可互换地使用。例如，一个细胞可以是指一个单一细胞或一个细胞群。
发明详述
HER2结合多肽
一方面，本申请提供了一种HER2结合多肽，其与参比抗体竞争结合HER2蛋白，其中所述参比抗体包含重链可变区(VH)；其中所述VH包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，其在ELISA试验中，所述的HER2结合多肽能够以不超过约1.20E-09、约1.10E-09、约1.0E-09、约9.0E-10、约8.0E-10、约7.0E-10、约6.0E-10、约5.0E-10、约4.0E-10、约3.0E-10、约2.0E-10、约1.0E-10、约9.0E-11的K D值与HER2蛋白相结合。
在某些实施方式中，所述的HER2结合多肽包括抗体或其抗原结合片段。
在某些实施方式中，所述抗体包括单克隆抗体、多特异性抗体、嵌合抗体、人源化抗体和/或全人源抗体。
在某些实施方式中，所述抗原结合片段包括Fab、Fab’、Fv片段、F(ab’)2、scFv、VHH和/或dAb。例如，所述抗体或其抗原结合片段可以为VHH。
在某些实施方式中，所述VHH不局限于特定的生物学来源或特定的制备方法。举例来说，所述VHH一般可以通过以下方式获得：(1)通过分离天然存在的重链抗体的VHH结构域；(2)通过表达编码天然存在的VHH结构域的核苷酸序列；(3)通过天然存在的VHH结构域的“人源化”或通过编码此类人源化VHH结构域的核酸的表达；(4)通过来自任何动物物种，如来自哺乳动物物种，如来自人类的天然存在的VH结构域的“骆驼化”，或通过编码 此类骆驼化VH结构域的核酸的表达；(5)通过如本领域中描述的“结构域抗体”或“Dab”的“骆驼化”，或通过编码此类骆驼化VH结构域的核酸的表达；(6)通过使用合成或半合成技术来制备本领域中已知的蛋白质、多肽或其它氨基酸序列；(7)通过使用本领域中已知的核酸合成技术来制备编码VHH的核酸，随后表达如此获得的核酸；和/或(8)通过上述一项或多项的任何组合。
在某些实施方式中，其中所述VHH是骆驼科的、嵌合的、人类的、部分人源化的或完全人源化的。
在某些实施方案中，所述HER2结合多肽可以包含已经“人源化”，即通过将天然存在的VHH序列的氨基酸序列中(且具体来说是构架序列中)的一个或多个氨基酸残基置换为来自人类的常规4链抗体的VH结构域中对应位置上存在的一个或多个氨基酸残基的VHH。这可以使用本领域中已知的人源化技术来进行。在一些实施方案中，可能的人源化取代或人源化取代的组合可以通过本领域中已知的方法来确定，例如，通过在VHH的序列与天然存在的人VH结构域的序列之间进行比较。在一些实施方案中，对所述人源化取代进行选择，以使所得人源化VHH仍保留有利的功能性质。一般来说，作为人源化的结果，本申请的VHH相较于对应的天然存在的VHH结构域可以变得更“像人的”，同时仍保留有利性质，如降低的免疫原性。本申请的人源化VHH可以用本领域中已知的任何合适的方式来获得，并且因此不严格限于已经使用包含天然存在的VHH结构域作为起始物质的多肽获得的多肽。
在某些实施方式中，其包含SEQ ID NO:1至SEQ ID NO:12中任一项所示氨基酸序列中的至少一个CDR。
在某些实施方式中，其包含SEQ ID NO:1至SEQ ID NO:12中任一项所示氨基酸序列中的HCDR1、HCDR2和HCDR3。
在某些实施方式中，所述的HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:21中任一项所示的氨基酸序列。
在某些实施方式中，其中所述HCDR1包含SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，其中所述HCDR2包含SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27和SEQ ID NO:34中任一项所示的氨基酸序列。
在某些实施方式中，其中所述HCDR2包含SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:36和SEQ ID NO:37中任一项所示的氨基酸序列。
在某些实施方式中，其中所述HCDR3包含SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，其中所述HCDR3包含SEQ ID NO:47或SEQ ID NO:48所示的氨基酸序列。
在某些实施方式中，其包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2包含SEQ ID NO:25所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:38所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:14所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:39所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:15所示的氨基酸序列，所述HCDR2包含SEQ ID NO:27所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44和SEQ ID NO:45中任一项所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含SEQ ID NO:34所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:46所示的氨基酸序列。
在某些实施方式中，其包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:28所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:40所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:17所示的氨基酸序列，所述HCDR2包含SEQ ID NO:29所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:41所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:42所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:19所示的氨基酸序列，所述HCDR2包含SEQ ID NO:31所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:43所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:32所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:44所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:33所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:45所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含SEQ ID NO:35所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:47所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:23所示的氨基酸序列，所述HCDR2包含SEQ ID NO:36所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:48所示的氨基酸序列；或
所述HCDR1包含SEQ ID NO:24所示的氨基酸序列，所述HCDR2包含SEQ ID NO:37所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:47所示的氨基酸序列。
在某些实施方式中，所述的HER2结合多肽包含VH，其中所述VH包含SEQ ID NO:1至SEQ ID NO:12中任一项所示的氨基酸序列。
在某些实施方案中，所述可变结构域的氨基酸序列与SEQ ID NO:1至SEQ ID NO:12中任一项所示的氨基酸序列相比，可以具有一个或几个氨基酸的取代、缺失或添加；所述取代、缺失或添加基本上不降低本申请的HER2结合多肽特异性地结合至HER2的能力。
在某些实施方案中，氨基酸突变可以在靶向部分的CDR(例如，CDR1区、CDR2区或CDR3区)中。在另一个实施方案中，氨基酸变化可以在靶向部分的构架区(FR)(例如，FR1区、FR2区、FR3区或FR4区)中。
在某些实施方式中，所述HER2结合多肽包含至少一个VH。例如，所述HER2结合多肽可以包含一个、或两个、或三个可变结构域。
在某些实施方式中，所述HER2结合多肽包括纳米抗体，所述纳米抗体包含SEQ ID NO:1至SEQ ID NO:12中任一项所示的氨基酸序列。
变体
本申请的HER2结合多肽还包括具有与本申请HER2结合多肽相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，1-30个，1-20个，或1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，10个以内，或5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突 变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本申请还提供了其他多肽，如包含纳米抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明纳米抗体的片段。通常，该片段具有本申请的HER2结合多肽的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本申请中，“保守性变异体”通常是指与本申请的HER2结合多肽的氨基酸序列相比，有至多10个，至多8个，至多5个，或至多3个氨基酸被性质相似或相近的氨基酸所取代而形成多肽。
在一些实施方式中，所述取代为保守氨基酸取代，所述保守氨基酸是指氨基酸残基被化学结构类似的另一氨基酸残基置换，且其对多肽的功能、活性或其他生物性质影响较小或基本上无影响。所述保守氨基酸取代在本领域中是公知的，例如保守氨基酸取代是以下组(i)-(v)内的一个氨基酸被同一组内的另一氨基酸残基所取代：(i)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；(ii)极性带负电残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；(iii)极性带正电残基：His、Arg及Lys；(iv)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及(v)芳族残基：Phe、Tyr及Trp。特别优选的保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。在一些实施方式中，所述取代为非保守氨基酸取代，例如，Ala被Asp、Asn、Glu或Gin取代。
一方面，本申请提供了一种HER2结合多肽，所述HER2结合多肽可以包含至少一个VH，所述VH的氨基酸序列与SEQ ID NO:1所示的氨基酸序列相比，可以具有一个或几个氨基酸的取代、缺失或添加。
在某些实施方式中，所述HER2结合多肽可以包含一个、或两个、或三个VH。
在某些实施方式中，所述VH可以包含相对于SEQ ID NO:1具有一个、或两个、或三个、或四个、或五个、或六个、或七个、或八个、或九个、或十个、或十一个、或十二个、或十三个、或十四个、或十五个、或十六个、或十七个、或十八个、或十九个、或二十个氨 基酸取代、缺失或添加的氨基酸序列。
在某些实施方案中，所述VH的氨基酸序列与SEQ ID NO:1所示的氨基酸序列相比，可以具有一个或几个氨基酸的取代，并且可以包括保守和/或非保守取代。
在某些实施方案中，氨基酸突变可以在靶向部分的CDR(例如，CDR1区、CDR2区或CDR3区)中。在另一个实施方案中，氨基酸变化可以在靶向部分的构架区(FR)(例如，FR1区、FR2区、FR3区或FR4区)中。
可以使用本领域中的任何已知技术，例如定点诱变或基于PCR的诱变来实现对氨基酸序列的修饰。此类技术描述于例如以下文献中：Sambrook等,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Press,Plainview,N.Y.,1989；和Ausubel等,Current Protocols in Molecular Biology,John Wiley&Sons,New York,N.Y.,1989。
在某些实施方案中，所述取代、缺失或添加基本上不降低本申请的HER2结合多肽特异性地结合至HER2的能力。
可以用平衡解离常数(KD)来描述本申请的HER2结合多肽对人HER2的全长和/或成熟形式和/或同种型和/或剪接变体和/或片段和/或任何其它天然存在或合成类似物、变体或突变体(包括单体、二聚、异源二聚、多聚和/或相关形式)的结合亲和力。所述HER2结合多肽可以包含靶向部分，所述靶向部分结合至人HER2的全长和/或成熟形式和/或同种型和/或剪接变体和/或片段和/或任何其它天然存在或合成的类似物、变体或突变体(包括单体、二聚、异源二聚、多聚和/或相关形式)，其中KD低于约1uM、约900nM、约800nM、约700nM、约600nM、约500nM、约400nM、约300nM、约200nM、约100nM、约90nM、约80nM、约70nM、约60nM、约50nM、约40nM、约30nM、约20nM、约10nM、或约5nM、或约1nM。
在某些实施方式中，其中所述HER2结合多肽具有以下性质中的一种或多种：
(i)其能够与A3亲和层析柱的填料结合；
(ii)与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，其能够以相同或更高的亲和力与HER2结合；
(iii)与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，其具有增加的表达量；和
(iv)与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，其等电点提高。
在某些实施方式中，本申请的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，其表达可以是约5％、10％、20％、30％、40％、50％、60％、70％、80％、90％或更高。
在某些实施方式中，氨基酸序列如SEQ ID NO:1所示的参比抗体(命名为H1)的等电点(PI)值为5.3，所述HER2结合多肽的PI值不小于5.3。例如，所述HER2结合多肽的PI值可以为约5.5，约6.0，约6.5，约6.6，约6.7，约6.8，约6.9，或约7。
在某些实施方式中，所述HER2结合多肽包括抗体或其抗原结合片段。
在某些实施方式中，所述抗体可以包括单克隆抗体、多特异性抗体(例如，双特异性抗体)、嵌合抗体、人源化抗体和/或全人源抗体。
在某些实施方式中，所述抗原结合片段可以包括Fab、Fab’、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。例如，所述抗体或其抗原结合片段可以为VHH。
在某些实施方式中，所述VHH不局限于特定的生物学来源或特定的制备方法。举例来说，所述VHH一般可以通过以下方式获得：(1)通过分离天然存在的重链抗体的VHH结构域；(2)通过表达编码天然存在的VHH结构域的核苷酸序列；(3)通过天然存在的VHH结构域的“人源化”或通过编码此类人源化VHH结构域的核酸的表达；(4)通过来自任何动物物种，如来自哺乳动物物种，如来自人类的天然存在的VH结构域的“骆驼化”，或通过编码此类骆驼化VH结构域的核酸的表达；(5)通过如本领域中描述的“结构域抗体”或“Dab”的“骆驼化”，或通过编码此类骆驼化VH结构域的核酸的表达；(6)通过使用合成或半合成技术来制备本领域中已知的蛋白质、多肽或其它氨基酸序列；(7)通过使用本领域中已知的核酸合成技术来制备编码VHH的核酸，随后表达如此获得的核酸；和/或(8)通过上述一项或多项的任何组合。
在某些实施方案中，所述HER2结合多肽可以包含已经“骆驼化”，即通过将来自常规4链抗体的天然存在的VH结构域的氨基酸序列中的一个或多个氨基酸残基置换为骆驼重链抗体的VHH结构域中对应位置上存在的一或多个氨基酸残基的VHH。在一些实施方案中，此类“骆驼化”取代被插入在形成和/或存在于VH-VL界面上的氨基酸位置上和/或在所谓的骆驼科标志残基上(参见例如WO9404678，所述文献的全部内容以引用的方式并入本申请)。在一些实施方案中，用作起始物质或起始点以供产生或设计骆驼化VHH用的VH序列是来自哺乳动物的VH序列，例如，人类的VH序列，如VH3序列。所述骆驼化VHH可以用本领域中已知的任何合适的方式获得(即，如以上第(1)至(8)点所指示)，并且因此不严格限于已经使用包含天然存在的VH结构域作为起始物质的多肽而获得的多肽。
在某些实施方式中，其中所述VHH可以是骆驼科的、嵌合的、人类的、部分人源化的或完全人源化的。
在某些实施方案中，所述HER2结合多肽可以包含已经“人源化”，即通过将天然存在的VHH序列的氨基酸序列中(且具体来说是构架序列中)的一个或多个氨基酸残基置换为来自人类的常规4链抗体的VH结构域中对应位置上存在的一个或多个氨基酸残基的VHH。这可以使用本领域中已知的人源化技术来进行。在一些实施方案中，可能的人源化取代或人源化取代的组合可以通过本领域中已知的方法来确定，例如，通过在VHH的序列与天然存在的人VH结构域的序列之间进行比较。在一些实施方案中，对所述人源化取代进行选择，以使所得人源化VHH仍保留有利的功能性质。一般来说，作为人源化的结果，本申请的VHH相较于对应的天然存在的VHH结构域可以变得更“像人的”，同时仍保留有利性质，如降低的免疫原性。本申请的人源化VHH可以用本领域中已知的任何合适的方式来获得，并且因此不严格限于已经使用包含天然存在的VHH结构域作为起始物质的多肽获得的多肽。
在某些实施方式中，其中所述VH可以包含SEQ ID NO:1所示氨基酸序列中的CDR1、CDR2和CDR3。所述CDR可以是Kabat CDR、AbM CDR、Chothia CDR或Contact CDR。在一些实施方案中，所述CDR是Chothia CDR。
一方面，本申请提供了一种HER2结合多肽，其包含VH，当与包含SEQ ID NO:1所示氨基酸序列的VH的抗体相比时，所述HER2结合多肽在VH的下述一个或多个位置处包含氨基酸取代：L2、I28、V31、A41、E43、Q44、R45、A57、D61、V67、D73、A74、S76、V78、N84、D98、W101、D103、D104、F106、E107和Q117。
在某些实施方式中，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH包含选自下组的一个或多个氨基酸取代：
(a)L2V，A41P，E43K，Q44G，R45L，A57T，V67F，D73N，A74S，S76N，V78L，N84S和/或Q117L；
(b)D61A，D103A，D104A，D98A和/或E107S；
(c)I28T，V31G，A57T，N84S，W101G，W101S和/或F106Y；和
(d)Y37V，A41P，R45L，A57T，D73N，A74S，N84S，K86R和/或P87A。
在某些实施方式中，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:159所示的氨基酸序列，所述HCDR2包含SEQ ID NO:160所示的氨基酸序列，所述HCDR3包含SEQ ID NO:161所示的氨基酸序列。
在某些实施方式中，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、 HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:13，SEQ ID NO:86，SEQ ID NO:87中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:25，SEQ ID NO:88，SEQ ID NO:89中任一项所示的氨基酸序列，所述HCDR3包含SEQ ID NO:38，SEQ ID NO:90-SEQ ID NO:96中任一项所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:25所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:38所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:86所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:25所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:38所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:87所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:25所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:38所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:88所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:38所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:89所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:38所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:25所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:90所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:25所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:91所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:25所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:92所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3， 所述HCDR1可以包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:25所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:93所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:88所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:94所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:88所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:95所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:88所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:96所示的氨基酸序列。
在某些实施方式中，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1包含SEQ ID NO:162所示的氨基酸序列，所述HFR2包含SEQ ID NO:163所示的氨基酸序列，所述HFR3包含SEQ ID NO:164所示的氨基酸序列，，所述HFR4包含SEQ ID NO:165所示的氨基酸序列。
在某些实施方式中，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1包含SEQ ID NO:49或SEQ ID NO:51所示的氨基酸序列，所述HFR2包含SEQ ID NO:55，SEQ ID NO:97-SEQ ID NO:106中任一项所示的氨基酸序列，所述HFR3包含SEQ ID NO:60，SEQ ID NO:107-SEQ ID NO:116中任一项所示的氨基酸序列，所述HFR4包含SEQ ID NO:70或SEQ ID NO:117所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1可以包含SEQ ID NO:49所示的氨基酸序列，所述HFR2可以包含SEQ ID NO:55中任一项所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:60中任一项所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:70所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1可以包含SEQ ID NO:49所示的氨基酸序列，所述HFR2可以包含SEQ ID NO:97所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:60所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:70所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和 HFR4，所述HFR1可以包含SEQ ID NO:49所示的氨基酸序列，所述HFR2可以包含SEQ ID NO:98所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:60所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:70所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1可以包含SEQ ID NO:49所示的氨基酸序列，所述HFR2可以包含SEQ ID NO:99所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:60所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:70所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1可以包含SEQ ID NO:49所示的氨基酸序列，所述HFR2可以包含SEQ ID NO:100所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:60所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:70所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1可以包含SEQ ID NO:49所示的氨基酸序列，所述HFR2可以包含SEQ ID NO:55所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:107所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:70所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1可以包含SEQ ID NO:49所示的氨基酸序列，所述HFR2可以包含SEQ ID NO:55所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:108所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:70所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1可以包含SEQ ID NO:49所示的氨基酸序列，所述HFR2可以包含SEQ ID NO:55所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:109所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:70所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1可以包含SEQ ID NO:49所示的氨基酸序列，所述HFR2可以包含SEQ ID NO:55所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:110所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:70所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1可以包含SEQ ID NO:49所示的氨基酸序列，所述HFR2可以包含SEQ ID  NO:55所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:111所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:70所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1可以包含SEQ ID NO:49所示的氨基酸序列，所述HFR2可以包含SEQ ID NO:55所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:112所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:70所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1可以包含SEQ ID NO:51所示的氨基酸序列，所述HFR2可以包含SEQ ID NO:55所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:60所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:117所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1可以包含SEQ ID NO:51所示的氨基酸序列，所述HFR2可以包含SEQ ID NO:101所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:113所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:117所示的氨基酸序列。
例如，所述HER2结合多肽包含VH，其中所述VH还包含：HFR1、HFR2，HFR3和HFR4，所述HFR1可以包含SEQ ID NO:49所示的氨基酸序列，所述HFR2可以包含SEQ ID NO:101所示的氨基酸序列，所述HFR3可以包含SEQ ID NO:113所示的氨基酸序列，所述HFR4可以包含SEQ ID NO:70所示的氨基酸序列。
在某些实施方式中，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH包含选自下组的一个或多个氨基酸取代：L2V，A41P，E43K，Q44G，R45L，A57T，V67F，D73N，A74S，S76N，V78L，N84S和Q117L。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：A41P，所述的HER2结合多肽的VH可以包含SEQ ID NO:122所示的氨基酸序列，所述HER2结合多肽命名为H1-A41P。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：E43K，所述的HER2结合多肽的VH可以包含SEQ ID NO:125所示的氨基酸序列，所述HER2结合多肽命名为H1-E43K。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：Q44G，所述的HER2结合多肽的 VH可以包含SEQ ID NO:123所示的氨基酸序列，所述HER2结合多肽命名为H1-Q44G。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：R45L，所述的HER2结合多肽的VH可以包含SEQ ID NO:124所示的氨基酸序列，所述HER2结合多肽命名为H1-R45L。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：A57T，所述的HER2结合多肽的VH可以包含SEQ ID NO:126所示的氨基酸序列，所述HER2结合多肽命名为H1-A57T。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：V67F，所述的HER2结合多肽的VH可以包含SEQ ID NO:127所示的氨基酸序列，所述HER2结合多肽命名为H1-V67F。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：D73N，所述的HER2结合多肽的VH可以包含SEQ ID NO:118所示的氨基酸序列，所述HER2结合多肽命名为H1-D73N。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：A74S所述的HER2结合多肽的VH可以包含SEQ ID NO:119所示的氨基酸序列，所述HER2结合多肽命名为H1-A74S。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：S76N，所述的HER2结合多肽的VH可以包含SEQ ID NO:120所示的氨基酸序列，所述HER2结合多肽命名为H1-S76N。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：V78L，所述的HER2结合多肽的VH可以包含SEQ ID NO:128所示的氨基酸序列，所述HER2结合多肽命名为H1-V78L。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：N84S，所述的HER2结合多肽的VH可以包含SEQ ID NO:129所示的氨基酸序列，所述HER2结合多肽命名为H1-N84S。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：L2V和Q117L，所述的HER2结合多肽的VH可以包含SEQ ID NO:121所示的氨基酸序列，所述HER2结合多肽命名为H1-VL。
在某些实施方式中，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH包含选自下组的一个或多个氨基酸取代：D61A，D103A，D104A，D98A和E107S。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：D61A，所述的HER2结合多肽的VH可以包含SEQ ID NO:140所示的氨基酸序列，所述HER2结合多肽命名为H1-D61A。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：D103A，所述的HER2结合多肽的VH可以包含SEQ ID NO:141所示的氨基酸序列，所述HER2结合多肽命名为H1-D103A。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：D104A，所述的HER2结合多肽的VH可以包含SEQ ID NO:142所示的氨基酸序列，所述HER2结合多肽命名为H1-D104A。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：D98A，所述的HER2结合多肽的VH可以包含SEQ ID NO:143所示的氨基酸序列，所述HER2结合多肽命名为H1-D98A。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：E107S，所述的HER2结合多肽的VH可以包含SEQ ID NO:144所示的氨基酸序列，所述HER2结合多肽命名为H1-E107S。
在某些实施方式中，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH包含选自下组的一个或多个氨基酸取代：I28T，V31G，A57T，N84S，W101G，W101S和F106Y。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：A57T和N84S，所述的HER2结合多肽的VH可以包含SEQ ID NO:145所示的氨基酸序列，所述HER2结合多肽命名为H1-AN-TS。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：V31G，A57T和N84S，所述的HER2结合多肽的VH可以包含SEQ ID NO:146所示的氨基酸序列，所述HER2结合多肽命名为H1-VG。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：：I28T，A57T和N84S，所述的HER2结合多肽的VH可以包含SEQ ID NO:147所示的氨基酸序列，所述HER2结合多肽命名为H1-IT。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：：A57T，N84S和W101G，所述的HER2结合多肽的VH可以包含SEQ ID NO:148所示的氨基酸序列，所述HER2结合多肽命名为H1-WG。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：A57T，N84S和W101S，所述的HER2结合多肽的VH可以包含SEQ ID NO:149所示的氨基酸序列，所述HER2结合多肽命名为H1-WS。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含选自以下氨基酸取代：：A57T，N84S和F106Y，所述的HER2结合多肽的VH可以包含SEQ ID NO:150所示的氨基酸序列，所述HER2结合多肽命名为H1-FY。
在某些实施方式中，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:130所示的参比抗体相比，所述的HER2结合多肽的VH包含选自下组的一个或多个氨基酸取代：P41A，K43E，G44Q，L45R，T57A，F67V，L78V，S84N，V2L和L117Q。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:130所示的参比抗体相比，所述的HER2结合多肽的VH可以包含以下氨基酸取代：P41A，所述HER2结合多肽包含SEQ ID NO:131所示的氨基酸序列，所述HER2结合多肽命名为FH1-P41A。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:130所示的参比抗体相比，所述的HER2结合多肽的VH可以包含以下氨基酸取代：K43E，所述HER2结合多肽包含SEQ ID NO:132所示的氨基酸序列，所述HER2结合多肽命名为FH1-K43E。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:130所示的参比抗体相比，所述的HER2结合多肽的VH可以包含以下氨基酸取代：G44Q，所述HER2结合多肽包含SEQ ID NO:133所示的氨基酸序列，所述HER2结合多肽命名为FH1-G44Q。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:130所示的参比抗体相比，所 述的HER2结合多肽的VH可以包含以下氨基酸取代：L45R，所述HER2结合多肽包含SEQ ID NO:134所示的氨基酸序列，所述HER2结合多肽命名为FH1-L45R。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:130所示的参比抗体相比，所述的HER2结合多肽的VH可以包含以下氨基酸取代：T57A，所述HER2结合多肽包含SEQ ID NO:131所示的氨基酸序列，所述HER2结合多肽命名为FH1-T57A。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:130所示的参比抗体相比，所述的HER2结合多肽的VH可以包含以下氨基酸取代：F67V，所述HER2结合多肽包含SEQ ID NO:136所示的氨基酸序列，所述HER2结合多肽命名为FH1-F67V。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:130所示的参比抗体相比，所述的HER2结合多肽的VH可以包含以下氨基酸取代：L78V，所述HER2结合多肽包含SEQ ID NO:137所示的氨基酸序列，所述HER2结合多肽命名为FH1-L78V。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:130所示的参比抗体相比，所述的HER2结合多肽的VH可以包含以下氨基酸取代：S84N，所述HER2结合多肽包含SEQ ID NO:138所示的氨基酸序列，所述HER2结合多肽命名为FH1-S84N。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:130所示的参比抗体相比，所述的HER2结合多肽的VH可以包含以下氨基酸取代：V2L和L117Q，所述HER2结合多肽包含SEQ ID NO:139所示的氨基酸序列，所述HER2结合多肽命名为FH1-V2L-L117Q。
在某些实施方式中，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:145所示的参比抗体相比，所述的HER2结合多肽的VH包含选自下组的一个或多个氨基酸取代：A41P，E43K，Q44G，R45L，R66V，D73N，A74S，S76N，K86R和P87A。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:145所示的参比抗体相比，所述的HER2结合多肽的VH包含以下氨基酸取代：A41P，Q44G，R45L，R66V，D73N，A74S和S76N，所述HER2结合多肽包含SEQ ID NO:151所示的氨基酸序列，所述HER2结合多肽命名为FH1-H。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:145所示的参比抗体相比，所述的HER2结合多肽的VH包含以下氨基酸取代：A41P，E43K，Q44G，R45L，R66V，D73N，A74S和S76N，所述HER2结合多肽包含SEQ ID NO:152所示的氨基酸序列，所述HER2结合多肽命名为FH1-3KE。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:145所示的参比抗体相比，所 述的HER2结合多肽的VH包含以下氨基酸取代：A41P，D73N，A74S，S76N，K86R和P87A，所述HER2结合多肽包含SEQ ID NO:153所示的氨基酸序列，所述HER2结合多肽命名为hH1-AN-TS。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:145所示的参比抗体相比，所述的HER2结合多肽的VH包含以下氨基酸取代：A41P，K86R和P87A，所述HER2结合多肽包含SEQ ID NO:154所示的氨基酸序列，所述HER2结合多肽命名为hH1-DA。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:145所示的参比抗体相比，所述的HER2结合多肽的VH包含以下氨基酸取代：A41P，E43K和Q44G，所述HER2结合多肽包含SEQ ID NO:155所示的氨基酸序列，所述HER2结合多肽命名为hH1-KP。
在某些实施方式中，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH包含选自下组的一个或多个氨基酸取代：Y37V，A41P，R45L，A57T，D73N，A74S，N84S，K86R和/或P87A。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含以下氨基酸取代Y37V，A41P，R45L，D73N，A74S，K86R和P87A，所述HER2结合多肽包含SEQ ID NO:156所示的氨基酸序列，所述HER2结合多肽命名为FH1-N。
例如，所述的HER2结合多肽与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，所述的HER2结合多肽的VH可以包含以下氨基酸取代R45L，A57T和N84S，所述HER2结合多肽包含SEQ ID NO:157所示的氨基酸序列，所述HER2结合多肽命名为H1-RL-VH。
核酸、构建体和细胞
另一方面，本申请提供了一种分离的一种或多种核酸分子，其编码本申请所述的HER2结合多肽。
在某些实施方案中，所述核酸可以编码包含抗体的VHH的氨基酸序列。在某些实施方案中，提供了编码抗HER2抗体重链可变区的分离的核酸，其中该核酸包含与编码SEQ ID NO:74至SEQ ID NO:84中任一项所示的核酸序列具有至少约90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％序列同一性的序列。
另一方面，本申请提供了一种构建体，其包含本申请所述的核酸分子。
另一方面，本申请提供了一种细胞，其包含本申请所述的核酸分子或本申请所述的构建体。
编码本申请的HER2结合多肽的核酸可以并入(连接)至构建体中，所述构建体可以通过转染、转化或转导技术而引入宿主细胞中。举例来说，可以通过反转录病毒转导将编码本申请的HER2结合多肽的核酸引入至宿主细胞中。在一个实施方案中，宿主细胞是真核的，例如中国仓鼠卵巢(CHO)细胞或淋巴样细胞(例如Y0,NS0，Sp20细胞)。在一个实施方案中，宿主细胞是原核的，例如大肠杆菌细胞。
另一方面，本申请提供了一种制备本申请所述的HER2结合多肽的方法，包括在允许所述HER2结合多肽表达的条件下培养本申请所述的细胞。
具体的表达和纯化条件将取决于所采用的表达系统而变化。举例来说，如果在大肠杆菌中表达基因，那么首先通过将工程化的基因置于合适的细菌启动子例如Trp或Tac和原核信号序列的下游而将它克隆至表达载体中。在另一个实例中，如果将要在真核细胞，例如CHO细胞中表达工程化的基因，那么首先将它插入含有例如合适的真核启动子、分泌信号、增强子和各种内含子的表达载体中。可以使用转染、转化或转导技术将基因构建体引入至宿主细胞中。
在某些实施方式中，所述方法还包括回收由所述细胞表达的HER2结合多肽。
在某些实施方式中，所述方法还包括纯化和/或修饰所述HER2结合多肽。
本申请的HER2结合多肽也可以在体内例如患者中表达。举例来说，在某些实施方案中，本申请的HER2结合多肽可以呈编码本申请的HER2结合多肽的核酸形式施用。所述核酸可以是DNA或RNA。在一些实施方案中，本申请的HER2结合多肽是由经修饰的mRNA，即包含一个或多个经修饰的核苷酸的mRNA编码。在一些实施方案中，本申请涉及包含所述经修饰的mRNA的基因治疗载体。在一些实施方案中，本申请涉及包括所述基因治疗载体的基因治疗方法。在某些实施方案中，所述核酸呈溶瘤病毒，例如腺病毒、呼肠孤病毒、麻疹、单纯疱疹、新城疫病毒或牛痘的形式。
免疫缀合物
另一方面，本申请提供了一种免疫缀合物，其包含本申请所述的HER2结合多肽。
在某些实施方式中，所述免疫缀合物包含：
i)本申请所述的HER2结合多肽；
ii)选自下组的缀合部分：可检测标记物、药物、毒素、细胞因子、病毒外壳蛋白或VLP、或其组合。
在某些实施方式中，其中所述可检测标记物选自下组的一种或多种试剂：放射性核素、 荧光剂、化学发光剂、生物发光剂、顺磁离子和酶。
在某些实施方式中，其中所述放射性核素适用于医学成像和/或治疗。
可用于缀合的荧光剂包括但不限于异硫氨酸荧光素、罗丹明、藻红蛋白、藻蓝蛋白、别藻蓝蛋白、邻苯二醛和荧光胺；可用于缀合的化学发光剂包括但不限于鲁米诺、异鲁米诺、芳族吖啶酯、咪唑、吖啶盐和草酸酯；可用于缀合的生物发光剂包括但不限于荧光素、荧光素酶和水母发光蛋白。可用于缀合的顺磁离子包括但不限于铬(III)、锰(II)、铁(III)、铁(II)、钻(II)、镍(II)、铜(II)、钕(III)、钐(III)、镱(III)、钆(III)、钒(II)、铽(III)、镝(III)、钬(III)和铒(III)，或者是不透辐射的材料，如坝、泛影酸盐、乙碘油、柠檬酸镓、碘卡酸、碘因他酸、碘达胺、胆影酸、碘沙酸、碘古酰胺、碘海醇、碘帕醇、碘番酸、碘普西酸、碘西法酸、碘丝酸、碘砜葡胺、碘酞硫、碘替酸、碘他拉酸、碘曲西酸、碘克沙酸、羟泛影酸、碘泊酸盐、葡甲胺、甲泛葡胶、甲泛影盐、丙碘酣和氧化亚铊。可用于缀合的酶包括但不限于辣根过氧化物酶等。
在某些实施方式中，所述可检测标记可以是放射性核素。可用于缀合的放射性核素可以是能量在20-4000KeV之间的放射性核素，包括但不限于 110In、 111In、 177Lu、 18F、 52Fe、 62Cu、 67Cu、 67Ga、 68Ga、 68Ge、 86Y、 90Y、 89Zr、 94mTc、 120I、 123I、 124I、 125I、 131I、 154-158Gd、 32P、 11C、 13N、 15O、 186Re、 188Re、 51Mn、 52mMn、 72As、 75Br、 76Br、 82mRb、 83Sr或其他γ-、β-、或正电子发射体。
例如，所述放射性核素可以为 99mTc、 177Lu或 125I。
在某些实施方式中，其中所述HER2结合多肽直接或间接与所述可检测标记物缀合。将可检测标记于多肽缀合的方法是本领域技术人员熟知的。例如，所述HER2结合多肽可以通过螯合剂与所述可检测标记缀合。
在某些实施方式中，其中所述螯合剂选自DTPA、EDTA、NOTA、DOTA、TRAP、TETA、NETA、CB-TE2A、Cyclen、Cyclam、Bispidine、TACN、ATSM、SarAr、AmBaSar、MAG3、MAG2、HYNIC、DADT、EC、NS3、H2dedpa、HBED、DFO、PEPA、HEHA及它们的衍生物中的一种或多种。
在另一方面，本申请提供一种制备本申请的放射性核素如 177Lu标记的免疫缀合物的方法，其包含1)使本申请的HER2结合多肽与螯合剂缀合以生成所述HER2结合多肽与螯合剂的缀合物前体；和2)使步骤1)的缀合物前体与放射性核素如 177Lu接触，由此放射性核素如 177Lu通过螯合剂的螯合作用标记本申请的HER2结合多肽。
在一些实施方案中，所述螯合剂是NOTA，所述步骤1)中通过使所述HER2结合多肽与p-SCN-Bn-NOTA或p-NH2-Bn-NOTA反应而生成所述HER2结合多肽与NOTA的缀合物前体。
本申请还可以采用IODO-BEADS固相标记法，用放射性核素如 125I标记本申请所述的HER2结合多肽。
组合物
另一方面，本申请提供了一种组合物，其包含本申请所述的HER2结合多肽、本申请所述的核酸分子、本申请所述的构建体、本申请所述的细胞和/或本申请所述的免疫缀合物，以及任选地药学上可接受的载体。
在一些实施方案中，本申请描述的组合物呈药学上可接受的盐的形式。本申请描述的HER2结合多肽可以具有能与无机或有机酸反应的充分碱性的官能团，或能与无机或有机碱反应的羧基，以形成药学上可接受的盐。
本申请描述的任何组合物都可以作为包含药学上可接受的载体的组合物的组分施用至受试者。此类组合物可以任选地包含适量的药学上可接受的赋形剂，以便提供用于适当施用的形式。
药物赋形剂可以是液体，如水和油，包括石油、动物、植物或合成来源的那些液体，如花生油、大豆油、矿物油、芝麻油等等。所述药物赋形剂可以是例如生理盐水、阿拉伯树胶、明胶、淀粉糊、滑石、角蛋白、胶体二氧化硅、脲等等。另外，可以使用助剂、稳定剂、增稠剂、润滑剂和着色剂。在一个实施方案中，当施用至受试者时，所述药学上可接受的赋形剂是无菌的。当本申请描述的任何剂经静脉内施用时，水是可用的赋形剂。还可以采用生理盐水溶液和葡萄糖水溶液和甘油溶液作为液体赋形剂，具体来说，用于可注射溶液。合适的药物赋形剂还包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙烯、乙二醇、水、乙醇等等。合适的药物赋形剂的其它实例描述于Remington’s Pharmaceutical Sciences 1447-1676(Alfonso R.Gennaro编,第19版,1995)中，所述文献以引用的方式并入本申请。
本申请包括所描述的组合物(和/或其它治疗剂)的各种调配物形式。本申请描述的任何发明组合物(和/或其它治疗剂)可以呈溶液、悬浮液、乳液、滴剂、片剂、丸剂、丹剂、胶囊剂、含有液体的胶囊剂、明胶胶囊、粉剂、持续释放调配物、栓剂、乳液、气雾剂、喷雾剂、悬浮液、冻干粉剂、冷冻悬浮液、干燥粉剂的形式或任何其它合用的形式。在一个实施方案中， 所述组合物呈胶囊剂形式。在另一个实施方案中，所述组合物呈锭剂形式。在另一个实施方案中，所述组合物被配制呈软凝胶胶囊形式。在另一个实施方案中，所述组合物被配制呈明胶胶囊形式。在另一个实施方案中，所述组合物被配制为液体。
本申请描述的任何组合物根据常规程序配制为适合于本申请描述的施用模式的组合物。
施用途径包括例如：口服、皮内、肌肉内、腹膜内、静脉内、皮下、鼻内、硬膜外、舌下、鼻内、脑内、叶鞘内、经皮、直肠、通过吸入或局部。施用可以是局部的或全身的。在一些实施方案中，所述施用是通过口服实现。在另一个实施方案中，所述施用是通过肠胃外注射。施用模式可以留给医师判断，并且部分取决于医学病状的部位。
在某些实施方式中，所述组合物包含本申请所述的HER2结合多肽或本申请所述的免疫缀合物，所述组合物为检测剂或治疗剂。
在某些实施方式中，其中所述检测剂为用于检测HER2蛋白的试剂。
在某些实施方式中，其中所述检测剂为造影剂。
在某些实施方式中，其中所述造影剂为检测HER2蛋白的造影剂。例如，所述造影剂可以是ECT造影剂。又例如，所述ECT造影剂可以包括SPECT造影剂或PET造影剂。
在某些实施方式中，其中所述治疗剂用于治疗肿瘤。
在某些实施方式中，其中所述治疗剂用于治疗HER2阳性肿瘤。
用途
另一方面，本申请提供了本申请所述的HER2结合多肽、本申请所述的核酸分子、本申请所述的构建体、本申请所述的细胞、本申请所述的免疫缀合物和/或本申请所述的组合物在制备药剂、试剂、检测板或试剂盒中的用途；
其中所述试剂、检测板或试剂盒用于检测样品中HER2蛋白；
其中所述药剂用于检测HER2蛋白的表达和/或治疗表达HER2的肿瘤。
另一方面，本申请提供了重组蛋白，所述的重组蛋白包含本申请所述的HER2结合多肽。
在某些实施方式中，其中所述的重组蛋白包含：(i)本申请所述的HER2结合多肽；以及(ii)任选的协助表达和/或纯化的标签序列。
另一方面，本申请提供了一种检测生物学样品中HER2的存在和/或量的方法，包括：使所述生物学样品接触本申请所述的HER2结合多肽、本申请所述的免疫缀合物、本申请所述的组合物。
在某些实施方式中，其中所述接触在体外或离体进行。
在某些实施方式中，其中所述生物学样品为组织。
在某些实施方式中，其中所述组织选自血液组织、淋巴组织和肿瘤组织。
在某些实施方式中，所述方法包括检测生物学样品中HER2阳性细胞的存在和/或量。
在某些实施方式中，其中通过成像确定生物学样品中HER2阳性细胞的存在和/或量。
在某些实施方式中，其中通过流式细胞术确定生物学样品中HER2阳性细胞的存在和/或量。
另一方面，本申请提供了一种检测和/或诊断与HER2表达异常相关的疾病或病症方法，包括向有此需要的受试者施用本申请所述的HER2结合多肽、本申请所述的免疫缀合物或本申请所述的组合物。
在某些实施方式中，其中所述方法还包括对所述受试者进行成像。
在某些实施方式中，其中所述成像包括ECT成像。
在某些实施方式中，其中所述ECT成像包括SPECT成像或PET成像。
在某些实施方式中，其中所述与HER2表达异常相关的疾病或病症包括肿瘤。
另一方面，本申请提供了一种治疗和/或预防肿瘤的方法，所述方法包括向有需要的受试者施用本申请所述的HER2结合多肽、本申请所述的免疫缀合物或本申请所述的组合物。
另一方面，本申请提供了一种用于监测抗肿瘤疗法在受试者中的功效的方法，所述方法包含：
(i)给患有肿瘤且采用抗肿瘤疗法治疗的受试者施用本申请所述的HER2结合多肽、本申请所述的免疫缀合物或本申请所述的组合物；和
(ii)确定所述受试者的肿瘤中HER2阳性细胞的量。
在某些实施方式中，通过成像确定所述受试者的肿瘤中HER2阳性细胞的存在和/或量。
在某些实施方式中，其中所述肿瘤包括HER2阳性肿瘤。
在某些实施方式中，其中所述肿瘤包括实体瘤。
在某些实施方式中，其中所述肿瘤选自乳腺癌、胃癌、食管癌、胆管癌、卵巢癌、胰腺癌、子宫内膜癌、子宫颈鳞状细胞癌、唾液腺瘤、膀胱癌、肺癌、结直肠癌、头颈癌、前列腺癌、骨肉瘤、儿童成神经管细胞瘤等肿瘤中的至少一种。
另一方面，本申请提供了一种试剂盒，其包含本申请所述的HER2结合多肽、本申请所述的免疫缀合物或本申请所述的组合物。
在所述试剂盒中可以包括使用说明书。使用说明书典型地包括有形表示，其描述了使用 所述试剂盒的组分实现所期望的治疗结果，诸如治疗癌症时将要采用的技术。任选地，所述试剂盒还含有本领域技术人员容易了解的其他有用组分，诸如稀释剂、缓冲剂、药学上可接受的载体、注射器、导管、敷涂器、吸移工具或测量工具、包扎材料或其他有用的附件。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的HER2结合多肽、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1抗Her2单域抗体筛选及构建
1.1文库的构建
免疫用的Her2-cHis融合蛋白由293F细胞表达，经镍柱亲和层析纯化得到。选取一只羊驼进行免疫。免疫结束后，提取羊驼外周血的淋巴细胞并按照Trizol使用说明提取RNA，取1ug RNA样品进行电泳检测其纯度。使用
Figure PCTCN2022126577-appb-000001
III First-Strand Synthesis System for RT-PCR试剂盒按照说明书将提取的RNA反转录成cDNA。用巢式PCR扩增编码重链抗体的可变区的核酸片段。
利用DNA产物纯化试剂盒纯化VHH片段，将载体与片段分别使用限制性内切酶sifi 50℃过夜酶切，对酶切后的片段进行切胶回收，将其克隆进入噬菌体展示用载体pComb3XSS中。产物随后电转化至大肠杆菌电转感受态细胞TG1中，构建针对Her2的重链单域抗体噬菌体展示文库并对文库进行检定。通过梯度稀释铺板，计算库容的大小为5.04×10 9。为检测文库的插入率，随机选取48个克隆进行鉴定。结果显示插入率已达到100％且大小正确。
1.2针对抗Her2的重链单域抗体淘选
用Her2-cHis融合蛋白5μg/孔包被平板，4℃放置过夜。第二天用3％的BSA 37℃封闭1小时，PBST(PBS中含有0.01％吐温20)洗板3次后，加入100μl噬菌体文库，37℃下孵育1小时。之后用PBST(PBS中含有0.01％吐温20)洗6遍，以洗掉不结合的噬菌体。之后每孔加入100uL的Gly-Hcl(PH＝2.2)37℃作用6-8min，洗脱特异性结合的噬菌体，将该洗脱液转移至一无菌的离心管中，用15uL的Tris-Hcl(PH＝9.0)溶液中和缓冲液。取10uL进行梯度稀释，测定滴度并计算淘选回收率，并取中和后的噬菌体感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮的筛选。相同筛选过程重复几轮，每一轮淘选需改变淘选条件。由此，阳性的克隆被富集，达到了利用噬菌体展示技术筛取抗体库中Her2特异抗体的目的。表1和表2为亲和淘选的条件以及酸洗脱三轮筛选回收量。
表1 亲和淘选条件
Figure PCTCN2022126577-appb-000002
表2 靶分子的酸洗脱三轮筛选回收量
Figure PCTCN2022126577-appb-000003
回收率＝回收量/文库投入量；
1.3用噬菌体的酶联免疫方法(ELISA)筛选特异性单个阳性克隆
经过几轮淘选后，从最后一轮淘选的平板上随机挑取48个克隆进行鉴定。将随机挑选48个单菌落分别培养，生产并纯化噬菌体。分别用Her2-Fc以及Her2-cHis融合蛋白包被平板4℃过夜，2％脱脂牛奶进行封闭，37℃反应1小时，随后每孔加入50uL噬菌体培养菌液上清及50uL 2％的脱脂牛奶，37℃孵育1h。PBST洗涤之后加入辣根过氧化物标记的抗M13二抗(购于北京义翘神州生物技术有限公司)，37℃反应1小时。洗涤之后加入TMB显色液，450nm波长读取吸收值。选取包被Her2-Fc及Her2-cHis抗原检测OD值均大于阴性对照的样品进行测序。
表3 Her2-cHis抗原phage ELISA鉴定
NO. OD450 NO. OD450 NO. OD450 NO. OD450 NO. OD450 NO. OD450 NO. OD450
1 3.069 9 1.279 17 2.412 25 2.958 33 0.872 41 2.893 Blank 0.252
2 2.058 10 0.702 18 1.001 26 1.936 34 3.16 42 0.739 Blank 0.248
3 1.696 11 2.941 19 3.023 27 2.832 35 2.98 43 2.906 Blank 0.242
4 3.161 12 2.399 20 2.337 28 3.24 36 2.911 44 3.212 Blank 0.286
5 2.338 13 2.456 21 1.151 29 3.08 37 3.176 45 2.564 Blank 0.198
6 2.314 14 3.087 22 0.366 30 0.35 38 1.8 46 2.079 Blank 0.156
7 2.596 15 3.129 23 2.467 31 0.895 39 3.056 47 2.484 Blank 0.183
8 2.054 16 3.39 24 2.327 32 3.089 40 3.285 48 2.482 Blank 0.167
表4 Her2-FC tag抗原phage ELISA鉴定
Figure PCTCN2022126577-appb-000004
Figure PCTCN2022126577-appb-000005
其中22号和30号克隆未送测序，其余克隆均送测序，根据序列比对软件DNAMAN分析各个克隆的蛋白序列。把CDR1、CDR2、CDR3序列均相同的克隆视为同一抗体株，而CDR序列不同的克隆视为不同抗体株。最终挑选12条候选序列进行后续实验。
1.4用哺乳动物细胞制备Her2抗体蛋白
将测序分析所获得单域抗体的编码序列亚克隆至表达载体PSNA008中，转染HEK293细胞进行抗体表达。将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将质粒/PEI混合物分别加入HEK293细胞悬液中，放置在37℃，10％CO 2，90rpm中培养。四小时后再补加EX293培养基，2mM谷氨酰胺，135rpm培养。24小时后加3.8mM VPA。培养6～7天后，收集瞬时表达培养上清液并用镍柱进行纯化。最终得到纯度达90％以上的抗体蛋白。
Her2-Fc融合蛋白氨基酸序列为：
Figure PCTCN2022126577-appb-000006
Figure PCTCN2022126577-appb-000007
单域抗体的氨基酸序列为：
Figure PCTCN2022126577-appb-000008
Figure PCTCN2022126577-appb-000009
注：下划线部分为抗体序列的CDR区
针对筛选得到的抗Her2单域抗体的核苷酸序列设计一对引物，以菌液为模板，通过PCR扩增各抗体的核苷酸序列(包含His标签)，然后克隆至PSNA008【pCDNA4(Invitrogen，Cat V86220)】载体中，经基因序列测定，以确定所获得目的克隆基因序列的正确性。
H1-cHis抗体：
上游引物：CCCAAGCTTGCCGCCACCATGGAGACA
下游引物：CCGGAATTCTTAGTGATGATGGTGGTGGTG
H1-chis的核苷酸序列：
Figure PCTCN2022126577-appb-000010
Figure PCTCN2022126577-appb-000011
H2-cHis抗体：
上游引物：CCCAAGCTTGCCGCCACCATGGAGACA
下游引物：CCGGAATTCTTAGTGATGATGGTGGTGGTG
H2-chis的核苷酸序列：
Figure PCTCN2022126577-appb-000012
H11-cHis抗体：
上游引物：CCCAAGCTTGCCGCCACCATGGAGACA
下游引物：CCGGAATTCTTAGTGATGATGGTGGTGGTG
H11-chis的核苷酸序列：
Figure PCTCN2022126577-appb-000013
H12-cHis抗体：
上游引物：CCCAAGCTTGCCGCCACCATGGAGACA
下游引物：CCGGAATTCTTAGTGATGATGGTGGTGGTG
H12-chis的核苷酸序列：
Figure PCTCN2022126577-appb-000014
H14-cHis抗体：
上游引物：CCCAAGCTTGCCGCCACCATGGAGACA
下游引物：CCGGAATTCTTAGTGATGATGGTGGTGGTG
H14-chis的核苷酸序列：
Figure PCTCN2022126577-appb-000015
H15-cHis抗体：
上游引物：CCCAAGCTTGCCGCCACCATGGAGACA
下游引物：CCGGAATTCTTAGTGATGATGGTGGTGGTG
H15-chis的核苷酸序列：
Figure PCTCN2022126577-appb-000016
H27-cHis抗体：
上游引物：CCCAAGCTTGCCGCCACCATGGAGACA
下游引物：CCGGAATTCTTAGTGATGATGGTGGTGGTG
H27-chis的核苷酸序列：
Figure PCTCN2022126577-appb-000017
H31-cHis抗体：
上游引物：CCCAAGCTTGCCGCCACCATGGAGACA
下游引物：CCGGAATTCTTAGTGATGATGGTGGTGGTG
H31-chis的核苷酸序列：
Figure PCTCN2022126577-appb-000018
Figure PCTCN2022126577-appb-000019
H32-cHis抗体：
上游引物：CCCAAGCTTGCCGCCACCATGGAGACA
下游引物：CCGGAATTCTTAGTGATGATGGTGGTGGTG
H32-chis的核苷酸序列：
Figure PCTCN2022126577-appb-000020
H36-cHis抗体：
上游引物：CCCAAGCTTGCCGCCACCATGGAGACA
下游引物：CCGGAATTCTTAGTGATGATGGTGGTGGTG
H36-chis的核苷酸序列：
Figure PCTCN2022126577-appb-000021
H38-cHis抗体：
上游引物：CCCAAGCTTGCCGCCACCATGGAGACA
下游引物：CCGGAATTCTTAGTGATGATGGTGGTGGTG
H38-chis的核苷酸序列：
Figure PCTCN2022126577-appb-000022
H45-cHis抗体：
上游引物：CCCAAGCTTGCCGCCACCATGGAGACA
下游引物：CCGGAATTCTTAGTGATGATGGTGGTGGTG
H45-chis的核苷酸序列：
Figure PCTCN2022126577-appb-000023
实施例2体外筛选
2.1采用羊驼二抗(HRP)进行ELISA活性筛选
包被Her2-Fc(PP180524)抗原5ug/mL，封闭后加各抗体5ug/mL，37℃孵育1h；加羊驼二抗(1:10000，Lot#18E001484)37℃孵育1h，450nm检测OD值。
表5 羊驼二抗(HRP)ELISA活性筛选
抗体编号 OD 450nm 抗体编号 OD 450nm
12-chis 0.604 1-chis* 0.351
2-chis 1.681 36-chis* 0.231
11-chis 1.465 31-chis* 0.005
45-chis 1.511 Her2-Fc抗原 # 0.006
38-chis 1.579 Her2-chis抗原 # 0.009
27-chis 0.978 Herceptin 1.115
14-chis 1.532 5F7-chis 1.827
32-chis 1.542 2RS15D-chis 1.684
15-chis 1.719    
注： #提示抗原与二抗没有非特异结合；*抗体与羊驼二抗结合较差。
2.2采用His二抗(HRP)进行ELISA活性筛选
包被Her2-Fc(PP180615)5ug/mL，封闭后加样品(1ug/mL起3倍稀释，共12个点)，37℃孵育2h；加入二抗ant-his(HRP)(Lot#GR3248851-4)，37℃孵育1h。
结果显示：以Her2-31为100％，筛选出活性较好的抗体有1#、36#、12#、2#、31#。
表6 His二抗(HRP)进行ELISA活性筛选
抗体编号 相对活性(％)
1 201
抗体编号 相对活性(％)
36 139
12 137
2RS15D 118
2 116
5F7 115
31 100
11 49.2
45 47.8
38 0.85
27 0.28
14 0.22
32 0.15
15 0.08
2.3与N87(Her2+)细胞的结合(流式筛选)
包被Her2-Fc(PP180615)5ug/mL，封闭。
收集N87细胞吹打成单细胞悬液，分至2x10^5个/200uL/管；加抗体使其终浓度为5ug/mL，混匀后冰上孵育1h；离心洗涤3次，再加anti-his二抗，混匀后冰上孵育1h；离心洗涤3次后300uL重悬上机。
结果如图1所示：5ug/mL应是饱和浓度，各抗体均对N87细胞有阳性结合，同时也表明N87为Her2+细胞；细胞结合较强的抗体为1#、2#、12#、36#，与ELISA结果基本一致。
2.4 KD亲和力检测(fortebio octet)
用SA sensor(LOT#1906062)load biotin标记的抗体120s；baseline100s后association抗原Her2-chis 180s；dissociation 600s。
表7 KD亲和力检测
Loading Sample ID KD(M) kon(1/Ms) kdis(1/s) Full R^2
Her2-1-chis 1.20E-09 9.58E+04 1.15E-04 0.9963
Her2-36-chis <1.0E-12 1.21E+04 <1.0E-07 0.8947
Her2-12-chis <1.0E-12 4.66E+04 <1.0E-07 0.9689
Her2-2-chis 1.50E-09 5.26E+04 7.90E-05 0.9697
Her2-31-chis <1.0E-12 1.38E+04 <1.0E-07 0.8756
如表7，图2-3所示：抗体36#、31#的R 2低于0.95，数据不可信；1#、2#2个抗体亲和力均在1nM左右，12#抗体KD值<1.0E -12，不可信(受fortebio octet检测方法所限，不可信数据对应的抗体相对结合活性参照ELISA结果)。
2.5与Herceptin的表位竞争
包被Her2-Fc 0.5ug/well 4℃过夜；封闭后，用1ug/mL biotin-Herceptin作为稀释液稀释Her-31-chis、Her2-2-chis、Her2-12-chis、Her2-36-chis、Her2-1-chis、Her2-11-chis、Her2-45-chis(均10ug/mL起4倍梯度，共12个浓度)，稀释好后以100uL/well加入孔中37℃孵育2h；anti-his(HRP)二抗稀释10000倍、streptavidin(HRP)稀释2000倍后100uL/well 37℃孵育1h；TMB显色并用1M H2SO4终止，450nm读取吸光度。
2.6不同浓度抗体与BT474细胞的结合(vs 2RS15D)
收集BT474细胞，分至2x10^5个/管，200uL/管，冰上孵育30min；分别加入2RS15D-chis、Her2-1-chis(终浓度为5ug/mL、1ug/mL、0.2ug/mL、0.04ug/mL)，冰上孵育1h；离心洗涤2次后，加his-PE二抗(美天旎，批号5200303504)，冰上孵育1h；离心洗涤2次后300uL重悬上机。
图5显示，Her2-1抗体(Her2-Nb1)与Her2抗原结合，优于2RS15D。
2.7与MCF7细胞的结合
MCF7细胞密度调至1*10^6个/mL；每100uL分装到EP管中冰敷20min。5F7-cHis,Her2-1-cHis,2RS15D-cHis反应终浓度为5μg/mL,每管加入对应抗体1.15μL,0.5μL,0.69μL，冰孵30min。加入300μL的1XPBS+0.5％BSA，1200rpm,离心5min，吸去上清，再用300μL的1XPBS+0.5％洗涤。每管加2μL的Anti-His-PE，冰孵30min。加入300μL的1XPBS+0.5％BSA，1200rpm离心5min，吸去上清，再用300μL的1XPBS+0.5％BSA洗涤。用300μL的1XPBS+0.5％BSA重悬，于流式细胞仪上检测。
图6显示，Her2-1也能结合Her2低表达细胞株。
2.8与BT474细胞的单点结合
收集BT474细胞，分至2x10^5个/管，100uL/管，冰上孵育20min；分别加入Her2-1-chis(终浓度为1ug/mL、0.1ug/mL)，冰上孵育30min；离心洗涤3次后，加his-PE二抗，冰上孵育30min；离心洗涤3次后200uL重悬上机。
图7显示，Her2-1抗体与Her2高表达细胞株结合良好。
综合MCF7和BT474数据，Her2-1可特异性结合Her2抗原，且其结合能力与Her2抗原的表达水平呈正相关。
2.9 Her2-Nb1内化(与5F7，2RS15D同期比较)
采用经验证的温控二抗法检测：收集BT474细胞，分至1x10^5个/管，100uL/管，冰上 孵育20min；分别加入终浓度为0.1ug/mL的5F7-chis、Her2-1-chis、2RS15D-chis，冰上孵育30min；离心洗涤3次后，分组：冰上放置0、1、2、4h和37℃培养1、2、4h；加his-PE二抗，冰上孵育30min；离心洗涤3次后200uL重悬上机。流式细胞仪分析检测平均荧光强度MFI。同一时间，(MFI 0℃-MFI 37℃)/MFI 0℃*100％即为该抗体在时间的内化率。
内化结果(图8)显示：
i)亲和力：Her-1＞5F7＞2RS15D；
ii)内化率：5F7呈上升趋势，4h三种抗体内化率无明显差异；
iii)绝对内化量：5F7呈上升趋势，Her2-1稍有上升，2RS15D稍有下降，4h内化量Her2-1＞5F7＞2RS15D；
iv)内化速度：Her2-1与2RS15D在1h即可趋近内化峰值，内化快，5F7内化慢；
v)Her2-1内化效果最佳。
2.10数据汇总
Figure PCTCN2022126577-appb-000024
实施例3
3.1核素标记Her2纳米抗体
125I标记
200μL 1×PBS，5μL Na 125I溶液，加入100μg蛋白混合均匀，最后加入一颗IODO-BEADS。室温下反应0.5h，将液体吸出终止反应即得。
99mTc标记
取500μL 99mTcO 4至ISOLINK KIT试剂盒中，80℃以上反应1.5h；取出100μl 99mTc(H2O)3(CO) 3与100μg Her2-1蛋白混合，37度孵育1.5h即得。
177Lu标记
将200μg DOTA-HER2-1纳米抗体偶联物与100μL 0.1M HCl和500μL 0.25M乙酸铵混合，最后加入 177LuCl 3溶液。于45℃反应1h。如反应体积较小，可加入0.25M乙酸铵增大反应混合物体积。
反应1h后点样进行TLC检测，展开剂为柠檬酸。TLC检测结果，标记率>95％则放行使用。如标记率<95％，使用PD-10柱进行纯化，1％BSA饱和后，以PBS作为洗脱液，每500μL收集并测量放射性活度。合并放射性洗脱液并再次进行TLC检测。放射化学纯度>95％则放行使用。
3.2  125I-Her2-Nbs细胞实验
SK-OV-3细胞培养于培养皿中，细胞按照约2×10 5Cells/孔、体积1000μL/孔添加至6孔板中(3板)。37℃,5％CO 2过夜培养贴壁。6孔板细胞分6组，每组3个平行样品，细胞中添加入900μL培养基，再添加稀释好的 125I-004-nbs 100μL，约100000计数，一组为block组，添加100倍对应的冷抗(未标记 125I的对应抗体)。其余组为实验组。体积共1mL，4℃孵育1h。
4℃孵育完成后1×PBS 1mL洗涤每孔细胞1次，收集孵育完后的细胞上清液及PBS洗液至放免管中，检测每管的cpm值。
细胞中添加新的培养基1mL，37℃孵育。Block组孵育1h，实验组分别孵育0h、1h、2h、4h、24h。孵育完成之后用PBS洗涤1次，用1000μL甘氨酸-盐酸缓冲溶液4℃孵育5min，收集酸洗溶液。γ计数仪检测各管酸洗溶液的cpm值。
加入2M NaOH洗涤20min，收集细胞悬液至放免管中，γ计数仪检测各管细胞悬液的cpm值。
从结果(图9)可看出，NO.36#和31#结合较慢，解离较快，相比之下NO.1，2，12#认为是较优抗体。
3.3 Her2-Nb1的分布及体内显影
125I-Her2-Nb1在BT474肿瘤的生物分布试验
125I-Her2-Nb1供试品标记率100％。将 125I-Her2-Nb1尾静脉注射给予3只BT474-荷瘤裸鼠，每只60μCi/125μL。给药90min后，小鼠安乐死，剖检各组织，称重，且在γ计数器上计数，用于确定各组织每克的％注射剂量(％ID/g)。
结果(图10)显示， 125I标记Her2--Nb1标记效率高，而且能够在肿瘤部位高富集，并 通过肾代谢出体内。
125I-Her2-Nb1在BT474肿瘤的显像
BT474-BALB/c-nude裸鼠，i.v给予 125I-Her2-Nb1溶液230μCi/125μL/只，SPECT-CT(IVIS spectrum)动态扫描0-4h。结果(图11)显示， 125I标记Her2--Nb1能够在肿瘤部位高富集，且肿瘤/肌肉的比值较高，显示出良好的肿瘤特异性。肾脏部位也有高摄取，提示主要通过肾代谢。
99mTc-Her2-Nb1在BT474肿瘤的显像
取500μL 99mTcO4(pH～5.0)至KIT试剂盒中(30mCi)，100℃孵育30分钟；
取出100μl(～5.62mCi)99mTc(H2O)3(CO)3与100μg Her2-1蛋白(溶于PBS中，不少于100μL)混合，37℃孵育约90分钟。
99mTc-Her2-Nb1纯化后标记率为100％。根据标记率计算出样品 99mTc-Her2-Nb1比活度为31.85μCi/μg。
99mTc-Her2-Nb1静脉注射给予BT474荷瘤鼠，取200μCi左右纯化后的蛋白，通过尾静脉注射到Balb/c裸小鼠体内，然后进行SPECT/CT成像。ROI勾画不同组织的摄取。
表8  99mT C-Her2-Nb1—在BT474肿瘤的SPE CT/CT分布
Figure PCTCN2022126577-appb-000025
表8和图12显示， 99mTC-Her2-Nb1在BT474肿瘤中有较高摄取，提示与HER2抗原的体内特异性靶向结合，且至给药后2.5h内肿瘤摄取均持续在较高水平，未随时间的延长而出现明显降低。肾脏的高摄取提示 99mTC-Her2-Nb1主要经肾脏代谢。
177Lu-Her2-Nb1在MCF7小鼠中生物分布
177Lu-DOTA-HER2-1供试品比活度为6μCi/μg。三只MCF7小鼠尾静脉注射 177Lu-DOTA-HER2-1(10MBq)后1.5h，安乐处死，取心、肺、肝、脾、肾、胃、肠、骨、肉、瘤测量重量及γ计数。
结果(图13)显示，Her2-Nb1与Her2低表达细胞在体内也有结合。
177Lu-Her2-Nb1抗肿瘤效应
BT474荷瘤鼠，按照分组分别注射给予生理盐水(对照组)、 177Lu-DOTA-Her2-1 10Mbq和20Mbq(每周一次，静脉注射)和Herceptin 10mg/kg(每周2次，腹腔住注射)，试验期 间所有动物自由摄食及饮水，每周肿瘤监测2～3次。
结果如错误！未找到引用源。所示，与NS组相比， 177Lu-DOTA-Her2-1 20Mbq组肿瘤增长抑制明显，其次为 177Lu-DOTA-Her2-1 10Mbq，后者与Herceptin组的肿瘤生长抑制效应已基本持平。
实施例4
4.1序列优化与鉴定
经过前期筛选与鉴定，在候选抗体中选定H1-cHis为最终的抗体序列，同时构建无标签的重组质粒H1。将上述重组构建的单域抗体H1融合蛋白质粒转染HEK293细胞进行抗体表达。并由A3亲和层析进行纯化，经鉴定发现H1抗体同A3亲和层析填料不结合，为后续纯化得到蛋白增加了困难，因此需要对H1抗体序列进行优化改造，使其可以经由亲和层析进行纯化。
对H1抗体序列的FR(Framework region)框架区进行分析，选取同A3亲和填料结合相关的关键氨基酸进行突变，经过对序列的分析，最终突变体H1-AN-TS可以同A3亲和层析填料进行结合。
H1及其突变体的命名及氨基酸序列
Figure PCTCN2022126577-appb-000026
注：划横线部分为突变点
将重组质粒H1以及突变后的重组质粒H1-AN-TS分别经由HEK293细胞进行抗体表达，并通过A3亲和层析填料进行纯化，来验证突变后同亲和填料的结合情况，图15A-15B为突变前后抗体纯化后SDS-PAGE分析结果。图15A显示的是H1抗体同A3填料不结合，蛋白在流穿中，图15B显示的是H1-AN-TS同A3填料结合，蛋白在buffer C中洗脱。
4.2针对H1抗体的结合活性、表达量进行序列优化
对H1抗体序列的FR(Framework region)框架区和CDR(Complementarity-determing  region)区进行分析，选取同亲和力、表达量相关的氨基酸进行突变尝试。先选取单个氨基酸进行突变，再进行多个氨基酸突变，在同表达量及结合活性相关的部分单点突变共计进行12组。
突变体的命名及氨基酸序列
Figure PCTCN2022126577-appb-000027
Figure PCTCN2022126577-appb-000028
注：下划线部分为突变后的氨基酸
分别对各突变体进行表达量和结合活性的检测。为了检测方便，此部分采用带有组氨酸标签的重组质粒进行瞬时转染，瞬转后用相应的Ni+树脂凝胶对重组蛋白进行亲和层析纯化，得到的目的蛋白即可用于表达量及结合活性的检测。
亲和活性的检测：Her2-Fc融合蛋白0.5μg/孔包被平板4℃过夜，PBST洗涤过后加入3％的BSA进行封闭，之后加入各突变体H1单域抗体蛋白的梯度稀释系列，室温下反应1小时。洗涤之后加入anti-his辣根过氧化物酶标记抗体，室温反应1小时。洗涤之后加入显色液，450nm波长读取吸收值。应用软件SotfMaxPro v5.4进行数据处理和作图分析，通过四参数拟合，得到突变后抗体对Her2-Fc结合曲线，并以H1抗体为对照，比较不同突变体的相对活性，以反映抗体对Her2-Fc的亲和能力。
表达量的计算：根据一步亲和层析纯化后得到的目的蛋白总量来计算表达量。
表9 各突变体对Her2抗原的结合情况及表达量
名称 相对活性(％) 表达量(g/L)
H1 100 0.024
H1-D73N 69.6 0.019
H1-S76N 66.1 0.020
H1-A74S 79.4 0.014
H1-VL 46.6 0.009
H1-A40P 96.1 0.012
H1-Q44G 85.9 0.020
H1-R45L 101.6 0.010
H1-E43K 23.6 0.049
H1-A57T 39.9 0.022
H1-V67F 35.7 0.044
H1-V78L 41.3 0.048
H1-N84S 41.1 0.056
综合各个突变体同Her2-Fc抗原的亲和力及表达量进行分析，选择突变后亲和力及表达量都相对较好的位点进行组合突变，此部分共进行9个组合突变。
突变体命名及氨基酸序列
Figure PCTCN2022126577-appb-000029
注：下划线部分为突变后的氨基酸
以同样的检测方法对此轮突变的9组抗体进行亲和力及表达量的检测，为方便检测，仍然采用带有组氨酸标签的抗体来进行相应的瞬转、纯化及后期检测。聚体的检测结果如表10所示。
表达量的计算：根据一步亲和层析纯化后得到的目的蛋白总量来计算表达量。
表10各组合突变体对Her2抗原的结合情况及表达量
名称 相对活性(％) 表达量(g/L)
H1 100 0.024
FH1-P41A 3.9 0.118
FH1-K43E 2.8 0.088
FH1-G44Q 4.9 0.087
FH1-L45R 10.3 0.092
FH1-T57A 8.0 0.085
FH1-F67V 3.2 0.061
FH1-L78V 18.5 0.055
FH1-S84N 9.4 0.057
FH1-V2L-L117Q 18.8 0.062
4.3针对H1抗体CDR3区负电荷氨基酸进行优化
抗体类分子一般要求等电点高于7，从而保证该分子能够适用于下游纯化工艺平台中的离子交换层析。而且宿主DNA的等电点主要分布在4～4.5，若抗体PI值与其接近，很难达到去除效果。选择CDR3区的负电荷氨基酸进行突变，一方面考虑可以通过突变提高抗体的PI值，另一方面抗体的亲和力同抗体CDR3区有直接的联系，因此希望在改变PI值的同时还能够提高H1抗体的亲和力。综合以上原因，在此部分共进行5个突变体。
突变体命名及氨基酸序列如下：
Figure PCTCN2022126577-appb-000030
注：下划线部分为突变后的氨基酸
突变完成后对改造后的抗体进行亲和力、表达量及PI值的检测，具体结果如表11所示：
表11各组合突变体对Her2抗原的结合情况、表达量及PI值
名称 相对活性(％) 表达量(g/L) PI值
H1 100 0.024 5.3
H1-D61A 82.2 0.060 6.41
H1-D103A 49.3 0.033 6.41
H1-D104A 70.2 0.039 6.41
H1-D98A 69.1 0.030 6.41
H1-E107S 77.5 0.039 6.39
4.4针对H1表面疏水性氨基酸进行优化
纳米抗体在进行放射性显影或治疗时，除肿瘤部位的特异性吸收外，其他非靶器官也会有一定的非特异性吸收，而如果纳米抗体表面存在一些疏水性氨基酸，可能会增加肝脏等器官的摄取，从而在放射性治疗或显影过程中，导致放射性核素在肝脏等器官的聚集，产生肝脏毒性等。为了避免此种情况，对H1抗体的PDB模型进行分析，选取了一些位于抗体表面且疏水性较强的氨基酸进行突变尝试，此部分共进行5个突变体优化。
突变体命名及氨基酸序列如下：
Figure PCTCN2022126577-appb-000031
注：下划线部分为突变后的氨基酸
抗体突变完成后，进行表达及纯化得到相应的目的蛋白，并对其进行表达量及亲和力的检测，具体结果如表12所示：
表12各组合突变体对Her2抗原的结合情况及表达量
名称 相对活性(％) 表达量(g/L)
H1 100 0.024
H1-VG 0 0.018
H1-IT 23.35 0.012
H1-WG 0 0.012
H1-WS 0.6 0.043
H1-FY 72.53 0.047
4.5对H1单域抗体进行人源化
骆驼抗体具有传统抗体不具备的优势，比如分子量小、体内渗透性好，极易穿过血管或者组织到达靶部位，这些优势使得纳米抗体被广泛的用于疾病诊断与检测的工具。但是多价的抗体如果长期用于临床可能会产生不同程度的免疫反应，影响治疗效果，因此需要对H1抗体进行人源化。人源化采用的是蛋白表面氨基酸人源化的方法以及VHH人源化通用框架部分移植法来完成。
人源化步骤如下：获取Cecile Vincke等人根据序列同源性设计完成的通用性人源化VHH框架h-NbBcIII0FGLA(PDB编号为：3EAK)，该框架设计基于纳米抗体NbBcIII0抗体(PDB编号为：3DWT)，参考人源抗体进行蛋白表面氨基酸人源化，根据H1纳米抗体序列的具体情况选择需要人源化的位点。
对H1抗体进行人源化，分为以H1抗体及H1-AN-TS抗体为模板进行突变，共获得7种抗体人源化变体。表13及表14列出了这些人源化变体的氨基酸变化以及突变后抗体的命名，其中氨基酸残基编号比照Kabat编号，具体的命名及突变位置如表所示。
表13氨基酸突变对比表(以H1-AN-TS为模板)
  A41P E43K Q44G R45L R66V D73N A74S S76N K86R P87A
FH1-H √   √ √ √ √ √ √    
FH1-3KE √ √ √ √ √ √ √ √    
hH1-AN-TS √         √ √   √ √
hH1-DA √               √ √
hH1-KP √ √ √              
H1-RL       √            
表14氨基酸突变对比表(以H1为模板)
  Y37V A41P R45L D73N A74S K86R P87A
FH1-N √ √ √ √ √ √ √
突变体具体命名及氨基酸序列如下
Figure PCTCN2022126577-appb-000032
注：划横线部分为突变氨基酸
突变完成后对各突变体进行亲和力以及表达量的分析，具体检测结果如表15所示。
表15人源化变体亲和力及表达量检测
名称 相对活性(％) 表达量(g/L)
H1 100 0.024
FH1-H 48.2 0.085
FH1-3KE 51.6 0.088
hH1-AN-TS 49.5 0.004
hH1-DA 57.01 0.010
hH1-KP 58.08 0.038
FH1-N NA NA
H1-RL 50.63 0.021

Claims (69)

  1. 一种HER2结合多肽，其与参比抗体竞争结合HER2蛋白，其中所述参比抗体包含重链可变区(VH)；其中所述VH包含SEQ ID NO:10所示的氨基酸序列。
  2. 根据权利要求1所述的HER2结合多肽，其在ELISA试验中，能够以不超过约1.20E-09的KD值与HER2蛋白相结合。
  3. 根据权利要求1-2中任一项所述的HER2结合多肽，其中所述HER2结合多肽具有以下性质中的一种或多种：
    (i)其能够与A3亲和层析柱的填料结合；
    (ii)与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，其能够以相同或更高的亲和力与HER2结合；
    (iii)与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，其具有增加的表达量；和
    (iv)与氨基酸序列如SEQ ID NO:1所示的参比抗体相比，其等电点提高。
  4. 根据权利要求1-3中任一项所述的HER2结合多肽，其包括抗体或其抗原结合片段。
  5. 根据权利要求4所述的HER2结合多肽，所述抗体包括单克隆抗体、多特异性抗体、嵌合抗体、人源化抗体和/或全人源抗体。
  6. 根据权利要求4-5中任一项所述的HER2结合多肽，所述抗原结合片段包括Fab、Fab’、Fv片段、F(ab’)2、scFv、VHH和/或dAb。
  7. 根据权利要求6所述的HER2结合多肽，其中所述VHH是骆驼科的、嵌合的、人类的、部分人源化的或完全人源化的。
  8. 根据权利要求1-7中任一项所述的HER2结合多肽，其包含SEQ ID NO:158，SEQ ID NO:2至SEQ ID NO:12中任一项所示氨基酸序列中的至少一个CDR。
  9. 根据权利要求1-8中任一项所述的HER2结合多肽，其包含SEQ ID NO:158，SEQ ID NO:2至SEQ ID NO:12中任一项所示氨基酸序列中的HCDR1、HCDR2和HCDR3。
  10. 根据权利要求1-9中任一项所述的HER2结合多肽，其包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:159、SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:21中任一项所示的氨基酸序列。
  11. 根据权利要求10所述的HER2结合多肽，其中所述HCDR1包含SEQ ID NO:13、SEQ ID NO:86和SEQ ID NO:87所示的氨基酸序列。
  12. 根据权利要求10-11中任一项所述的HER2结合多肽，其中所述HCDR1包含SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的氨基酸序列。
  13. 根据权利要求10-12中任一项所述的HER2结合多肽，其中所述HCDR2包含SEQ ID NO:160、SEQ ID NO:26、SEQ ID NO:27和SEQ ID NO:34中任一项所示的氨基酸序列。
  14. 根据权利要求13所述的HER2结合多肽，其中所述HCDR2包含SEQ ID NO:25、SEQ ID NO:88和SEQ ID NO:89所示的氨基酸序列。
  15. 根据权利要求13-14中任一项所述的HER2结合多肽，其中所述HCDR2包含SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:36和SEQ ID NO:37中任一项所示的氨基酸序列。
  16. 根据权利要求10-15中任一项所述的HER2结合多肽，其中所述HCDR3包含SEQ ID NO:161、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45和SEQ ID NO:46中任一项所示的氨基酸序列。
  17. 根据权利要求16所述的HER2结合多肽，其中所述HCDR3包含SEQ ID NO:38、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95或SEQ ID NO:96所示的氨基酸序列。
  18. 根据权利要求16-17中任一项所述的HER2结合多肽，其中所述HCDR3包含SEQ ID NO:47或SEQ ID NO:48所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的HER2结合多肽，其包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:159所示的氨基酸序列，所述HCDR2包含SEQ ID NO:160所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:161所示的氨基酸序列；或
    所述HCDR1包含SEQ ID NO:14所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:39所示的氨基酸序列；或
    所述HCDR1包含SEQ ID NO:15所示的氨基酸序列，所述HCDR2包含SEQ ID NO:27所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44和SEQ ID NO:45中任一项所示的氨基酸序列；或
    所述HCDR1包含SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含SEQ ID NO:34所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:46所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的HER2结合多肽，其包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:13，SEQ ID NO:86或SEQ ID NO:87所示的氨基酸序列，所述HCDR2包含SEQ ID NO:25，SEQ ID NO:88或SEQ ID  NO:89所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:38，SEQ ID NO:90，SEQ ID NO:91，SEQ ID NO:92，SEQ ID NO:93，SEQ ID NO:94，SEQ ID NO:95或SEQ ID NO:96所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的HER2结合多肽，其包含VH，其中所述VH包含：HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:28所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:40所示的氨基酸序列；或
    所述HCDR1包含SEQ ID NO:17所示的氨基酸序列，所述HCDR2包含SEQ ID NO:29所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:41所示的氨基酸序列；或
    所述HCDR1包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:42所示的氨基酸序列；或
    所述HCDR1包含SEQ ID NO:19所示的氨基酸序列，所述HCDR2包含SEQ ID NO:31所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:43所示的氨基酸序列；或
    所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:32所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:44所示的氨基酸序列；或
    所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:33所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:45所示的氨基酸序列；或
    所述HCDR1包含SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含SEQ ID NO:35所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:47所示的氨基酸序列；或
    所述HCDR1包含SEQ ID NO:23所示的氨基酸序列，所述HCDR2包含SEQ ID NO:36所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:48所示的氨基酸序列；或
    所述HCDR1包含SEQ ID NO:24所示的氨基酸序列，所述HCDR2包含SEQ ID NO:37所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:47所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的HER2结合多肽，其包含VH，其中所述VH包含SEQ ID NO:158，SEQ ID NO:2至SEQ ID NO:12中任一项所示的氨基酸序列。
  23. 根据权利要求1-22中任一项所述的HER2结合多肽，其包含VH，其中所述VH包含SEQ ID NO:1，SEQ ID NO:118至SEQ ID NO:157中任一项所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的HER2结合多肽，所述HER2结合多肽包含至少一个VH。
  25. 根据权利要求1-24中任一项所述的HER2结合多肽，所述HER2结合多肽包括纳米抗体， 所述纳米抗体包含SEQ ID NO:158，SEQ ID NO:2至SEQ ID NO:12中任一项所示的氨基酸序列。
  26. 根据权利要求1-24中任一项所述的HER2结合多肽，所述HER2结合多肽包括纳米抗体，所述纳米抗体包含SEQ ID NO:1，SEQ ID NO:118至SEQ ID NO:157中任一项所示的氨基酸序列。
  27. 分离的一种或多种核酸分子，其编码权利要求1-26中任一项所述的HER2结合多肽。
  28. 构建体，其包含根据权利要求27所述的核酸分子。
  29. 细胞，其包含根据权利要求27所述的核酸分子或根据权利要求28所述的构建体。
  30. 制备权利要求1-26中任一项的HER2结合多肽的方法，包括在允许所述HER2结合多肽表达的条件下培养权利要求29所述的细胞。
  31. 根据权利要求30所述的方法，所述方法还包括回收由所述细胞表达的HER2结合多肽。
  32. 根据权利要求31所述的方法，所述方法还包括纯化和/或修饰所述HER2结合多肽。
  33. 免疫缀合物，其包含权利要求1-26中任一项所述的HER2结合多肽。
  34. 根据权利要求33所述的免疫缀合物，所述免疫缀合物包含：
    i)权利要求1-26中任一项所述的HER2结合多肽；
    ii)选自下组的缀合部分：可检测标记物、药物、毒素、细胞因子、病毒外壳蛋白或VLP、或其组合。
  35. 根据权利要求34所述的免疫缀合物，其中所述可检测标记物选自下组的一种或多种试剂：放射性核素、荧光剂、化学发光剂、生物发光剂、顺磁离子和酶。
  36. 根据权利要求35所述的免疫缀合物，其中所述放射性核素适于医学成像和/或治疗。
  37. 根据权利要求35所述的免疫缀合物，其中所述放射性核素包括 110In、 111In、 177Lu、 18F、 52Fe、 62Cu、 67Cu、 67Ga、 68Ga、 68Ge、 86Y、 90Y、 89Zr、 94mTc、 120I、 123I、 124I、 125I、 131I、 154-158Gd、 32P、 11C、 13N、 15O、 186Re、 188Re、 51Mn、 52mMn、 72As、 75Br、 76Br、 82mRb、 83Sr或其他γ-、β-、或正电子发射体。
  38. 根据权利要求34-37中任一项的免疫缀合物，其中所述HER2结合多肽直接或间接与所述可检测标记物缀合。
  39. 根据权利要求34-38中任一项的免疫缀合物，其中所述HER2结合多肽通过螯合剂与所述可检测标记物缀合。
  40. 根据权利要求39所述的免疫缀合物，其中所述螯合剂选自DTPA、EDTA、NOTA、DOTA、TRAP、TETA、NETA、CB-TE2A、Cyclen、Cyclam、Bispidine、TACN、ATSM、 SarAr、AmBaSar、MAG3、MAG2、HYNIC、DADT、EC、NS3、H2dedpa、HBED、DFO、PEPA、HEHA及它们的衍生物中的一种或多种。
  41. 组合物，其包含权利要求1-26中任一项所述的HER2结合多肽、权利要求27所述的核酸分子、权利要求28所述的构建体、权利要求29所述的细胞和/或权利要求33-40中任一项所述的免疫缀合物，以及任选地药学上可接受的载体。
  42. 根据权利要求41所述的组合物，所述组合物包含权利要求1-26中任一项所述的HER2结合多肽或权利要求33-40中任一项所述的免疫缀合物，所述组合物为检测剂或治疗剂。
  43. 根据权利要求42所述的组合物，其中所述检测剂为用于检测HER2蛋白的试剂。
  44. 根据权利要求42所述的组合物，其中所述检测剂为造影剂。
  45. 根据权利要求44所述的组合物，其中所述造影剂为检测HER2蛋白的造影剂。
  46. 根据权利要求42所述的组合物，其中所述治疗剂用于治疗肿瘤。
  47. 根据权利要求46所述的组合物，其中所述治疗剂用于治疗HER2阳性肿瘤。
  48. 权利要求1-26中任一项所述的HER2结合多肽、权利要求27所述的核酸分子、权利要求28所述的构建体、权利要求29所述的细胞、权利要求33-40任一项的免疫缀合物和/或权利要求41-47任一项的组合物在制备药剂、试剂、检测板或试剂盒中的用途；
    其中所述试剂、检测板或试剂盒用于检测样品中HER2蛋白；
    其中所述药剂用于检测HER2蛋白的表达和/或治疗表达HER2的肿瘤。
  49. 重组蛋白，所述的重组蛋白包含权利要求1-27中任一项所述的HER2结合多肽。
  50. 根据权利要求49所述的重组蛋白，其中所述的重组蛋白包含：(i)权利要求1-26中任一项所述的HER2结合多肽；以及(ii)任选的协助表达和/或纯化的标签序列。
  51. 一种检测生物学样品中HER2的存在和/或量的方法，包括：使所述生物学样品接触权利要求1-26中任一项所述的HER2结合多肽、权利要求33-40中任一项所述的免疫缀合物或权利要求41-47中任一项所述的组合物。
  52. 根据权利要求51所述的方法，其中所述接触在体外或离体进行。
  53. 根据权利要求51中任一项所述的方法，其中所述生物学样品为组织。
  54. 根据权利要求53所述的方法，其中所述组织选自血液组织、淋巴组织和肿瘤组织。
  55. 根据权利要求51中任一项所述的方法，所述方法包括检测生物学样品中HER2阳性细胞的存在和/或量。
  56. 根据权利要求51所述的方法，其中通过成像确定生物学样品中HER2阳性细胞的存在和/或量。
  57. 根据权利要求51所述的方法，其中通过流式细胞术确定生物学样品中HER2阳性细胞的存在和/或量。
  58. 一种检测和/或诊断与HER2表达异常相关的疾病或病症方法，包括向有此需要的受试者施用权利要求1-26中任一项所述的HER2结合多肽、权利要求33-40中任一项所述的免疫缀合物或权利要求41-47中任一项所述的组合物。
  59. 根据权利要求58所述的方法，其中所述方法还包括对所述受试者进行成像。
  60. 根据权利要求59所述的方法，其中所述成像包括ECT成像。
  61. 根据权利要求60所述的方法，其中所述ECT成像包括SPECT成像或PET成像。
  62. 根据权利要求58-61中任一项所述的方法，其中所述与HER2表达异常相关的疾病或病症包括肿瘤。
  63. 一种治疗和/或预防肿瘤的方法，所述方法包括向有需要的受试者施用权利要求1-26中任一项HER2结合多肽、权利要求33-40中任一项的免疫缀合物或权利要求41-47中任一项的组合物。
  64. 一种用于监测抗肿瘤疗法在受试者中的功效的方法，所述方法包含：
    (i)给患有肿瘤且采用抗肿瘤疗法治疗的受试者施用权利要求1-26中任一项所述的HER2结合多肽、权利要求33-40中任一项所述的免疫缀合物或权利要求41-47中任一项所述的组合物；和
    (ii)确定所述受试者的肿瘤中HER2阳性细胞的量。
  65. 根据权利要求64中任一项所述的方法，通过成像确定所述受试者的肿瘤中HER2阳性细胞的存在和/或量。
  66. 根据权利要求64-65中任一项所述的方法，其中所述肿瘤包括HER2阳性肿瘤。
  67. 根据权利要求64-66中任一项所述的方法，其中所述肿瘤包括实体瘤。
  68. 根据权利要求64-67中任一项所述的方法，其中所述肿瘤选自乳腺癌、胃癌、食管癌、胆管癌、卵巢癌、胰腺癌、子宫内膜癌、子宫颈鳞状细胞癌、唾液腺瘤、膀胱癌、肺癌、结直肠癌、头颈癌、前列腺癌、骨肉瘤、儿童成神经管细胞瘤等肿瘤中的至少一种。
  69. 一种试剂盒，其包含权利要求1-26中任一项所述的HER2结合多肽、权利要求33-40中任一项所述的免疫缀合物或权利要求41-47中任一项所述的组合物。
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