WO2022257868A1 - Antigen binding protein against human serum albumin - Google Patents

Antigen binding protein against human serum albumin Download PDF

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Publication number
WO2022257868A1
WO2022257868A1 PCT/CN2022/097052 CN2022097052W WO2022257868A1 WO 2022257868 A1 WO2022257868 A1 WO 2022257868A1 CN 2022097052 W CN2022097052 W CN 2022097052W WO 2022257868 A1 WO2022257868 A1 WO 2022257868A1
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seq
amino acid
acid sequence
sequence shown
binding protein
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PCT/CN2022/097052
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French (fr)
Chinese (zh)
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顾春银
王宗达
曹晓丹
刘培培
邓俗俊
潘忠宗
王学萍
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上海济煜医药科技有限公司
江西济民可信集团有限公司
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Application filed by 上海济煜医药科技有限公司, 江西济民可信集团有限公司 filed Critical 上海济煜医药科技有限公司
Priority to CN202280040871.9A priority Critical patent/CN117545504A/en
Publication of WO2022257868A1 publication Critical patent/WO2022257868A1/en

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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
    • 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
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present application relates to the field of biomedicine, in particular to an antigen-binding protein capable of binding to human serum albumin.
  • protein drugs have been successfully and widely used in clinical treatment, and many protein drugs have provided effective therapeutic effects for some diseases.
  • half-life of most proteins and peptides is very short.
  • protein drugs will be degraded by proteases in the body and quickly cleared, and the other is that drugs with a molecular weight of less than 60KD are easily eliminated in renal metabolism due to the filtration of glomeruli.
  • high-dose or multiple, long-term medication regimens are often used, which will increase its toxic and side effects on the one hand, and bring great inconvenience to patients while taking medication.
  • Human serum albumin is the main protein in human blood and the protein with the longest half-life in the human body. It can combine with some drug molecules to provide protection for drug molecules, and is an ideal drug carrier molecule. Therefore, there is an urgent need to develop molecules that can bind to human serum albumin to provide more effective drug carriers.
  • This application provides an antigen-binding protein capable of recognizing human serum albumin, which can be screened to obtain a binding protein with high affinity.
  • the antigen-binding protein has stable physical and chemical properties, can bind to human serum albumin, and prolong the life of biological drugs. Half-life, which favors the beneficial effects of biologics in therapy.
  • the application provides an isolated antigen-binding protein comprising at least one CDR in the variable region VH of an antibody heavy chain, said VH comprising the amino acid sequence shown in SEQ ID NO: 26 or 38.
  • the isolated antigen binding protein comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:29.
  • the HCDR3 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 28.
  • the isolated antigen binding protein comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:37.
  • the isolated antigen binding protein comprises HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, 31 or 32.
  • the isolated antigen binding protein comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:36.
  • the isolated antigen binding protein comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3 or 30.
  • the isolated antigen-binding protein comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:36, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:37 , and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:29.
  • the HCDR1 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:3 or 30, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, 31 or 32 , and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 28.
  • the HCDR1 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 comprises The amino acid sequence shown in SEQ ID NO:1; or the HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO : the amino acid sequence shown in 28; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 30, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1 or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:31, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1 or the HCDR1 comprises the amino acid amino acid sequence shown
  • the isolated antigen binding protein comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1
  • the H-FR1 comprises SEQ ID NO: The amino acid sequence shown in 22.
  • the H-FR1 of the isolated antigen binding protein comprises any one of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:11 amino acid sequence.
  • the isolated antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:23 .
  • the H-FR2 of the isolated antigen binding protein comprises any one of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:15 amino acid sequence.
  • said isolated antigen binding protein comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 comprises SEQ ID NO:24 amino acid sequence.
  • the H-FR3 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:13.
  • the isolated antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO: The amino acid sequence shown in 25.
  • said H-FR4 of said isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:14 and SEQ ID NO:16.
  • the antigen binding protein of described separation comprises H-FR1, H-FR2, H-FR3 and H-FR4, and described H-FR1 comprises the aminoacid sequence shown in SEQ ID NO:22, described H-FR2 comprises the amino acid sequence shown in SEQ ID NO:23, said H-FR3 comprises the amino acid sequence shown in SEQ ID NO:24, and said H-FR4 comprises the amino acid sequence shown in SEQ ID NO:25.
  • the antigen binding protein of described isolation comprises H-FR1, H-FR2, H-FR3 and H-FR4, and described H-FR1 comprises SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:8, SEQ ID NO:
  • the amino acid sequence shown in any one of ID NO:9 and SEQ ID NO:11, described H-FR2 comprises SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:15
  • the amino acid sequence shown in any one the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:13
  • the H-FR4 comprises SEQ ID NO:7, SEQ ID NO:14 and the amino acid sequence shown in any one of SEQ ID NO:16.
  • the isolated antigen binding protein comprises any group of H-FR1, H-FR2, H-FR3 and H-FR4 selected from the group consisting of:
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6
  • the amino acid sequence shown, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:8
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6
  • the amino acid sequence shown, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6
  • the amino acid sequence shown, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:11
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:12
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:13
  • the amino acid sequence shown and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 14;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:11
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:15
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:13
  • the amino acid sequence shown and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:16.
  • the isolated antigen binding protein comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 26 or 38.
  • said VH of said isolated antigen binding protein comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 21, SEQ ID NO: The amino acid sequence shown in any one of ID NO:33, SEQ ID NO:34, and SEQ ID NO:35.
  • the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.
  • the isolated antigen binding protein comprises an antibody heavy chain constant region.
  • the antibody heavy chain constant region of the isolated antigen binding protein is derived from a human antibody heavy chain constant region.
  • the antibody heavy chain constant region of the isolated antigen binding protein is derived from a human IgG heavy chain constant region.
  • the antibody heavy chain constant region of the isolated antigen binding protein is derived from a human IgG1 heavy chain constant region.
  • the antigen-binding fragment comprises Fab, Fab', Fv fragment, F(ab') 2 , scFv, di-scFv, dAb and/or VHH .
  • the antigen-binding fragment is a VHH .
  • the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the isolated antigen binding protein is capable of binding human serum albumin.
  • the present application also provides a fusion protein comprising the isolated antigen-binding protein.
  • the present application also provides an isolated nucleic acid molecule encoding the isolated antigen-binding protein or the fusion protein.
  • the present application also provides a vector comprising the nucleic acid molecule.
  • the present application also provides a cell comprising the isolated antigen-binding protein, the fusion protein, the nucleic acid molecule or the carrier.
  • the present application also provides a method for preparing the isolated antigen-binding protein, the method comprising culturing the cell under the condition that the isolated antigen-binding protein is expressed.
  • the present application also provides a pharmaceutical composition, which comprises the isolated antigen-binding protein, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable carrier.
  • the present application also provides a method for detecting or measuring human serum albumin, said method comprising said isolated antigen-binding protein or said fusion protein.
  • the present application also provides a kit, which comprises the isolated antigen-binding protein, the fusion protein, the nucleic acid molecule, the carrier, the cell and/or the said pharmaceutical composition.
  • the present application also provides the isolated antigen-binding protein, the fusion protein, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and/or the Use of the above-mentioned kit in the preparation of medicines for preventing and/or treating diseases or diseases.
  • Figure 1 shows the construction method of yeast display library.
  • Human Serum Albumin may be used interchangeably with “HSA”.
  • HSA Human Serum Albumin
  • the term also covers variants, homologues, analogs, derivatives and functionally active fragments of human serum albumin.
  • isolated generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.
  • isolated antigen-binding protein generally refers to a protein having antigen-binding ability that is removed from its naturally occurring state.
  • isolated antigen binding protein may comprise an antigen-binding moiety and, optionally, a framework or framework portion that permits the antigen-binding moiety to adopt a conformation that facilitates binding of said antigen-binding moiety to antigen.
  • Antigen binding proteins may comprise, for example, antibody-derived protein framework regions (FR) or alternative protein framework regions or artificial framework regions with grafted CDRs or CDR derivatives.
  • Such frameworks include, but are not limited to, antibody-derived framework regions comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antigen binding protein, and fully synthetic framework regions comprising, eg, biocompatible polymers. See eg Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004).
  • antigen binding proteins include, but are not limited to: human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single chain antibodies; diabodies; triabodies; tetrabodies; (ab') 2 , F(ab) 2 , scFv, di-scFv, dAb, IgD antibody; IgE antibody; IgM antibody; IgGl antibody; IgG2 antibody; IgG3 antibody; or IgG4 antibody and fragments thereof.
  • CDR also referred to as “complementarity determining region” generally refers to the region in the variable domain of an antibody, the sequence of which is highly variable and/or forms a structure-defining loop.
  • naturally occurring camelid antibodies consisting only of heavy chains are capable of functioning and stabilizing in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).
  • Antibody CDRs can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia, etc.
  • IMGT the international ImMunoGeneTics information system@imgt.cines.fr; http://imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res.
  • the CDRs of the antigen binding protein can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • variable domains of native heavy and light chains each comprise four FR regions, four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL. (L-FR1, L-FR2, L-FR3 and L-FR4).
  • variable domain and “variable region” are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain.
  • the variable domains of the heavy and light chains may be referred to as “ VH “ and “ VL “, respectively (or “VH” and “VL”, respectively). These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class) and comprise the antigen binding site.
  • variable generally means that some segments of the variable domain may have large differences in sequence between antibodies.
  • the variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed throughout the variable domains. It is usually concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains.
  • CDRs or HVRs hypervariable regions
  • the more highly conserved portions of variable domains are called the framework regions (FR).
  • the variable domains of native heavy and light chains each comprise four FR regions, most adopting a ⁇ -sheet configuration, connected by three CDRs, which form a circular connection and in some cases form part of the ⁇ -sheet structure .
  • the CDRs in each chain are held in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antibody's antigen-binding site (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • antibody generally refers to an immunoglobulin or fragment or derivative thereof, encompassing any polypeptide that includes an antigen combining site, whether produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid, mutated, and transplanted antibodies.
  • the term “antibody” also includes antibody fragments, such as Fab, F(ab') 2 , Fv, scFv, Fd, dAbs and other antibody fragments that retain antigen binding function (eg, specifically bind HSA). Typically, such fragments will include the antigen binding domain.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibodies consist of 5 basic heterotetrameric units and another polypeptide called the J chain, and contain 10 antigen-binding sites, while IgA antibodies include 2-5 that can be combined with the J chain to form a multivalent A basic 4-chain unit for combinations.
  • the 4-chain unit is typically about 150,000 Daltons.
  • Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for the alpha and gamma chains each, and four CH domains for the mu and epsilon isoforms.
  • Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen-binding site.
  • immunoglobulins can be assigned to different classes, or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively.
  • the term "antigen-binding fragment” generally refers to one or more fragments that have the ability to specifically bind an antigen (eg, HSA).
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, dAb and/or VHH .
  • Fab generally refers to an antigen-binding fragment of an antibody.
  • Intact antibodies can be digested using papain as described above. Papain digestion of antibodies yields two identical antigen-binding fragments, the "Fab” fragment, and a residual "Fc” fragment (ie, the Fc region, supra).
  • Fab fragments may consist of a complete L chain with the variable region of a heavy chain and the first constant region (CH 1 ) of the H chain ( VH ).
  • Fab' fragment generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which fragment is slightly larger than a Fab fragment.
  • a Fab' fragment may include all of the light chain, all of the variable domains of the heavy chain, and all or part of the first and second constant domains of the heavy chain.
  • a Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.
  • F(ab')2 generally refers to antibody fragments produced by pepsin digestion of intact antibodies.
  • the F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds.
  • F(ab')2 fragments have bivalent antigen binding activity and are capable of cross-linking antigen.
  • Fv fragment generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody comprising all or part of the heavy and light chain variable regions and lacking the heavy and light chain constant regions.
  • the heavy and light chain variable regions include, for example, CDRs.
  • an Fv fragment includes all or part of the approximately 110 amino acid amino-terminal variable regions of the heavy and light chains.
  • the term "scFv” generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chains are variable
  • the regions are contiguous (eg via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single chain polypeptide wherein the scFv retains the specificity of the intact antibody from which it was derived.
  • a scFv can have the VL and VH variable regions in any order (for example, relative to the N-terminal and C-terminal of the polypeptide), and the scFv can include VL-linker-VH or VH-Linker-VL can be included.
  • the term “dAb” generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having a VH domain or a VL domain, see e.g. Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6) , with reference to Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and to other published patent applications such as WO 06/030220, WO 06/003388 and Domantis Ltd.
  • the term “dAb” may include sdAb
  • the term “sdAb” generally refers to a single domain antigen binding molecule, and in this application the term may include single domain antibody, as well as VHH .
  • monoclonal antibody generally refers to a preparation of antibody molecules of single molecular composition.
  • 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.
  • the modifier "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.
  • chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
  • the variable regions are derived from an antibody of a laboratory animal, such as a rodent ("parent antibody”), and the constant regions are derived from a human antibody, such that the resulting chimeric antibody is more specific in a human individual than the parental (e.g., alpaca-derived) antibody. Less likely to trigger an adverse immune response.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (such as an alpaca antibody) are replaced by 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, llama) 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 alpaca, mouse, etc.) having the desired properties, affinity and/or capabilities. , rat, rabbit or non-human primate) CDR region residue replacement.
  • donor antibody such as alpaca, mouse, etc.
  • 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 an antibody that comprises only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains if they are produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Similarly, a “mouse antibody” or “rat antibody” refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology methods. Exemplary techniques that can be used to make antibodies are described in US Patents: 6,150,584, 6,458,592, 6,420,140. Other techniques, such as using libraries, are known in the art.
  • nucleic acid molecule generally refers to nucleotides of any length in isolated form, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • the vector can be expressed by transforming, transducing or transfecting the host cell, so that the genetic material elements carried by it can be expressed in the host cell.
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein coats, but not only.
  • the term "cell” generally refers to a single cell, cell line or cell culture that can be or has been the recipient of a subject's plasmid or vector, which includes a nucleic acid molecule as described herein or a nucleic acid molecule as described herein. Carrier.
  • Cells can include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell. Cells may include cells transfected in vitro with the vectors described herein.
  • the term "pharmaceutical composition” generally refers to a composition for preventing/treating a disease or condition.
  • the pharmaceutical composition may comprise the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and/or the cell described herein, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may also comprise one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. preparations. Acceptable ingredients of the compositions are generally nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the term "pharmaceutically acceptable carrier” generally includes a pharmaceutically acceptable carrier, excipient or stabilizer that is inert to the cells or mammals to which it is exposed at the dosage and concentration employed. poisonous.
  • Physiologically acceptable carriers can include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions such as sodium; and/or nonionic surfactants.
  • the term "specifically binds" or “specific” generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, that can occur in a heterogeneous population of molecules, including biomolecules. Presence determines the presence of a target.
  • an antibody that specifically binds a target (which may be an epitope) can be an antibody that binds that target with greater affinity, avidity, greater ease, and/or for a greater duration than it binds other targets .
  • an antibody specifically binds an epitope on a protein that is conserved among proteins of different species.
  • specific binding can include, but does not require exclusive binding.
  • 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.
  • protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues having the same or similar functions as the protein or polypeptide.
  • the variant may be, for example, substituted, deleted or added one or more A protein or polypeptide of amino acids.
  • the functional variant may comprise at least 1, such as 1-30, 1-20 or 1-10, further such as 1, 2, 3, 4 or 5 amino acid substitutions , proteins or polypeptides with amino acid changes by deletion and/or insertion.
  • Said functional variant may substantially retain the biological properties of said protein or said polypeptide prior to alteration (eg, substitution, deletion or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the protein or polypeptide prior to the alteration.
  • the substitutions may be conservative substitutions.
  • the homologue may be at least about 85% (eg, having at least about 85%) of the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds to HSA). %, about 87%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) Proteins or polypeptides with sequence homology.
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • Perfectage of sequence homology can be calculated in the following manner: compare the two sequences to be aligned in the comparison window, and determine that there are identical nucleic acid bases (for example, A, T, C, G, I) in the two sequences ) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) Number of positions To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size), and the result was multiplied by 100 to yield the percent sequence identity.
  • Alignment for purposes of determining percent sequence homology can be accomplished in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of sequence of interest.
  • the homology can also be determined by the following methods: FASTA and BLAST.
  • FASTA FASTA and BLAST.
  • a description of the FASTA algorithm can be found in "An Improved Tool for Biological Sequence Comparison" by W.R.Pearson and D.J. Lipman, Proc. Natl. Acad. Sci., 85:2444-2448, 1988; and D.J.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the CDR of an antibody also known as the complementarity determining region, is part of the variable region.
  • the amino acid residues in this region may make contacts with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, comprehensive consideration of Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems.
  • the CDR covers the CDR sequence divided according to any CDR division method; also covers its variants, the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • amino acids For example 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or or insertions; homologues thereof, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences having about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology.
  • the CDRs are divided by the Chothia coding system.
  • the application provides an isolated antigen-binding protein comprising at least one CDR in the variable region VH of an antibody heavy chain, said VH comprising the amino acid sequence shown in SEQ ID NO: 26 or 38.
  • the isolated antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:29.
  • the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:28.
  • the isolated antigen-binding protein may comprise HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:37.
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, 31 or 32.
  • the isolated antigen-binding protein may comprise HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:36.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3 or 30.
  • the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:36, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:37, And the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:29.
  • the HCDR1 of the isolated antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 1.
  • the HCDR1 of the isolated antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 28.
  • said HCDR1 of said isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:30
  • said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2
  • said HCDR3 comprises SEQ ID NO:1 Amino acid sequence shown.
  • said HCDR1 of said isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:3
  • said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:31
  • said HCDR3 comprises SEQ ID NO:1 Amino acid sequence shown.
  • said HCDR1 of said isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:3
  • said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:32
  • said HCDR3 comprises SEQ ID NO:1 Amino acid sequence shown.
  • the isolated antigen-binding protein may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise SEQ ID NO: The amino acid sequence shown in 22.
  • the H-FR1 of the isolated antigen-binding protein comprises the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 11.
  • the isolated antigen binding protein may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise SEQ ID NO:23 amino acid sequence.
  • the H-FR2 of the isolated antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:15 .
  • the isolated antigen-binding protein may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise SEQ ID NO:24 amino acid sequence.
  • the H-FR3 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:13.
  • the isolated antigen-binding protein may comprise H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the H-CDR3, and the H-FR4 may comprise SEQ ID The amino acid sequence shown in NO:25.
  • the H-FR4 of the isolated antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:14 and SEQ ID NO:16.
  • the isolated antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 22
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 23
  • the H- FR3 may comprise the amino acid sequence shown in SEQ ID NO:24
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:25.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:4, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:8
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:14.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 15
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:16.
  • the VH of the isolated antigen-binding protein comprises amino acid mutations at one or more of the following positions compared with the amino acid shown in SEQ ID NO: 17: H1, H13, H44, H45, H49, H74 , H76, H79, H81, H82B, H83, H84, H108, the positions are determined by the Chothia coding system.
  • the mutation comprises a substitution of aspartic acid D for glutamic acid E at position H1.
  • the mutation comprises a substitution of glutamic acid E by glutamine Q at position H13.
  • the mutation comprises a substitution of glutamic acid E for glycine G at position H44.
  • the mutation comprises a substitution of an arginine R for a leucine L at position H45.
  • the mutation comprises a substitution of alanine A for serine S at position H49.
  • the mutation comprises a substitution of arginine R for serine S at position H74.
  • the mutation comprises a substitution of lysine K for asparagine N at position H76.
  • the mutation comprises a substitution of serine S for tyrosine Y at position H79.
  • the mutation comprises a substitution of aspartic acid D for glutamine Q at position H81.
  • the mutation comprises a substitution of aspartic acid D for serine S at position H82B.
  • the mutation comprises a substitution of lysine K for arginine R at position H83.
  • the mutation comprises a substitution of proline P for alanine A at position H84.
  • the mutation comprises a substitution of glutamine Q for methionine M at position H108.
  • the mutation comprises a substitution of glutamine Q for leucine L at position H108.
  • the isolated antigen-binding protein may comprise VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 26 or 38.
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 17.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 18.
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 19.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:20.
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 21.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:33.
  • the VH can comprise the amino acid sequence shown in SEQ ID NO:34.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:35.
  • the isolated antigen binding protein may comprise an antibody heavy chain constant region.
  • the antibody heavy chain constant region is derived from a human antibody heavy chain constant region.
  • the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.
  • the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.
  • the human IgG1 heavy chain constant region comprises human IgG1 Fc.
  • the human IgGl Fc comprises an amino acid modification of N297A.
  • the isolated antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragment may include Fab, Fab', Fv fragment, F(ab') 2 , scFv, di-scFv, dAb and/or VHH .
  • the antigen-binding fragment is a VHH .
  • the antibody may be selected from the group consisting of monoclonal antibody, chimeric antibody, humanized antibody and fully human antibody.
  • the isolated antigen binding protein may comprise a single domain antibody.
  • the single domain antibodies described in the present application include the single domain antibodies numbered Ab0716.1, Ab0716.2, Ab0716.4, Ab0716.Hz1 and/or Ab0716.Hz2 prepared in the examples of the present application.
  • the single domain antibody described in the present application includes the single domain antibody numbered Ab0716Am01, Ab0716Am02, or Ab0716Am06 prepared in the examples of the present application.
  • the single domain antibody may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:29.
  • the HCDR3 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the HCDR3 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:28.
  • the single domain antibody may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the single domain antibody may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the single domain antibody may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:36, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:37, And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:29.
  • the HCDR1 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1 amino acid sequence.
  • the HCDR1 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:28 amino acid sequence.
  • the HCDR1 of the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:30
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1 amino acid sequence.
  • the HCDR1 of the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:31, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1 amino acid sequence.
  • the HCDR1 of the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:32, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1 amino acid sequence.
  • the single domain antibody may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may comprise SEQ ID NO:22
  • the amino acid sequence shown may comprise the amino acid sequence shown in SEQ ID NO:4.
  • the H-FR1 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:8.
  • the H-FR1 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:9.
  • the H-FR1 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO: 11.
  • the single domain antibody may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:23 .
  • the H-FR2 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:5.
  • the H-FR2 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:10.
  • the H-FR2 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO: 12.
  • the H-FR2 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:15.
  • the single domain antibody may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:24 .
  • the H-FR3 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the H-FR3 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO: 13.
  • the single domain antibody may comprise H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the H-CDR3, and the H-FR4 may comprise SEQ ID NO: The amino acid sequence shown in 25.
  • the H-FR4 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR4 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:14.
  • the H-FR4 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO: 16.
  • the single domain antibody may comprise H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:4
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6
  • the amino acid sequence shown and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:8, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:14.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 15
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:16.
  • the single domain antibody may comprise a VHH , and the VHH may comprise the amino acid sequence shown in SEQ ID NO:26 or 38.
  • the VHH may comprise the amino acid sequence shown in SEQ ID NO:17.
  • the VHH may comprise the amino acid sequence shown in SEQ ID NO:18.
  • the VHH may comprise the amino acid sequence shown in SEQ ID NO:19.
  • the VHH may comprise the amino acid sequence shown in SEQ ID NO:20.
  • the VHH may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the VHH may comprise the amino acid sequence shown in SEQ ID NO:33.
  • the VHH may comprise the amino acid sequence shown in SEQ ID NO:34.
  • the VHH may comprise the amino acid sequence shown in SEQ ID NO:35.
  • the amino acid sequence of the HCDR1 of the single domain antibody Ab0716.1 is shown in SEQ ID NO: 3
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2
  • the amino acid sequence of the HCDR3 As shown in SEQ ID NO: 1
  • the amino acid sequence of the H-FR1 is shown in SEQ ID NO: 4
  • the amino acid sequence of the H-FR2 is shown in SEQ ID NO: 5
  • the amino acid sequence of the H-FR3 The sequence is shown in SEQ ID NO:6
  • the amino acid sequence of H-FR4 is shown in SEQ ID NO:7.
  • the amino acid sequence of the VHH of the single domain antibody Ab0716.1 is shown in SEQ ID NO:17.
  • the amino acid sequence of the HCDR1 of the single domain antibody Ab0716.2 is shown in SEQ ID NO: 3
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2
  • the amino acid sequence of the HCDR3 As shown in SEQ ID NO: 28
  • the amino acid sequence of the H-FR1 is shown in SEQ ID NO: 8
  • the amino acid sequence of the H-FR2 is shown in SEQ ID NO: 5
  • the amino acid sequence of the H-FR3 The sequence is shown in SEQ ID NO:6
  • the amino acid sequence of H-FR4 is shown in SEQ ID NO:7.
  • the amino acid sequence of the VHH of the single domain antibody Ab0716.2 is shown in SEQ ID NO:18.
  • the amino acid sequence of the HCDR1 of the single domain antibody Ab0716.4 is shown in SEQ ID NO: 3
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2
  • the amino acid sequence of the HCDR3 As shown in SEQ ID NO: 1
  • the amino acid sequence of the H-FR1 is shown in SEQ ID NO: 9
  • the amino acid sequence of the H-FR2 is shown in SEQ ID NO: 10
  • the amino acid sequence of the H-FR3 The sequence is shown in SEQ ID NO:6
  • the amino acid sequence of H-FR4 is shown in SEQ ID NO:7.
  • the amino acid sequence of the VHH of the single domain antibody Ab0716.4 is shown in SEQ ID NO:19.
  • the amino acid sequence of the HCDR1 of the single domain antibody Ab0716.Hz1 is shown in SEQ ID NO:3, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of the HCDR3
  • the amino acid sequence of the H-FR1 is shown in SEQ ID NO: 11
  • the amino acid sequence of the H-FR2 is shown in SEQ ID NO: 12
  • the amino acid sequence of the H-FR3 The sequence is shown in SEQ ID NO:13
  • the amino acid sequence of H-FR4 is shown in SEQ ID NO:14.
  • the amino acid sequence of the VHH of the single domain antibody Ab0716.Hz1 is shown in SEQ ID NO:20.
  • the amino acid sequence of the HCDR1 of the single domain antibody Ab0716.Hz2 is shown in SEQ ID NO: 3
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2
  • the amino acid sequence of the HCDR3 As shown in SEQ ID NO: 1, the amino acid sequence of the H-FR1 is shown in SEQ ID NO: 11, the amino acid sequence of the H-FR2 is shown in SEQ ID NO: 15, the amino acid sequence of the H-FR3
  • the sequence is shown in SEQ ID NO:13, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:16.
  • the amino acid sequence of the VHH of the single domain antibody Ab0716.Hz2 is shown in SEQ ID NO:21.
  • the HCDR1 of the single domain antibody Ab0716Am01 includes the amino acid sequence shown in SEQ ID NO:30
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:1 The amino acid sequence shown.
  • the HCDR1 of the single domain antibody Ab0716Am02 includes the amino acid sequence shown in SEQ ID NO:3
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:31
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:1 The amino acid sequence shown.
  • the HCDR1 of the single domain antibody Ab0716Am06 includes the amino acid sequence shown in SEQ ID NO:3
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:32
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:1 The amino acid sequence shown.
  • the isolated antigen binding protein is capable of binding human serum albumin.
  • the antigen binding protein described in the present application can be in the form of less than or equal to 2.0 ⁇ 10 -9 M, less than or equal to 1.9 ⁇ 10 -9 M, less than or equal to 1.8 ⁇ 10 -9 M, less than or equal to 1.7 ⁇ 10 -9 M , less than or equal to 1.6 ⁇ 10 -9 M, less than or equal to 1.5 ⁇ 10 -9 M, less than or equal to 1.4 ⁇ 10 -9 M, less than or equal to 1.3 ⁇ 10 -9 M, less than or equal to 1.2 ⁇ 10 -9 M , less than or equal to 1.1 ⁇ 10 -9 M, less than or equal to 1.0 ⁇ 10 -9 M, less than or equal to 9 ⁇ 10 -10 M, less than or equal to 8 ⁇ 10 -10 M, less than or equal to 7 ⁇ 10 -10 M , less than or equal to 6 ⁇ 10 -10 M, less than or equal to 5 ⁇ 10 -10 M, less than or equal to 4.15 ⁇ 10 -10 M
  • the Tm value of the isolated antigen-binding protein can be greater than or equal to 59.5°C, greater than or equal to 59.6°C, greater than or equal to 59.7°C, greater than or equal to 59.8°C, greater than or equal to 59.9°C, greater than or equal to 60.0 °C, greater than or equal to 60.1°C, greater than or equal to 60.2°C, greater than or equal to 60.5°C, greater than or equal to 61.0°C, greater than or equal to 61.5°C, greater than or equal to 62°C, greater than or equal to 62.5°C, greater than or equal to 62.9°C, Greater than or equal to 63°C, greater than or equal to 63.5°C, greater than or equal to 64°C.
  • Fusion proteins nucleic acid molecules, vectors, cells and pharmaceutical compositions
  • the application provides a fusion protein, which may comprise the antigen binding protein described in the application.
  • the application provides isolated nucleic acid molecules that encode the isolated antigen binding proteins described herein.
  • it may be produced or synthesized by: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification; (ii) recombinantly produced by cloning; (iii) purified or (iv) synthetic, such as by chemical synthesis.
  • PCR polymerase chain reaction
  • the present application provides a vector, which may comprise the nucleic acid molecule described in the present application.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that permit proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering.
  • the vector is an expression vector.
  • the vector may also include components that facilitate its entry into cells, such as, but not exclusively, viral particles, liposomes or protein coats.
  • the present application provides a cell, which may comprise the nucleic acid molecule or the vector described in the present application.
  • each or each host cell may comprise one or more of the nucleic acid molecules or vectors described herein.
  • each or each host cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into the host cells, such as eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like.
  • the cells may be bacterial cells (e.g., E.
  • yeast cells or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2/0 cells, NSO cells or myeloma cells.
  • COS cells Chinese hamster ovary (CHO) cells
  • CHO-K1 cells Chinese hamster ovary (CHO) cells
  • LNCAP cells CHO-K1 cells
  • HeLa cells HeLa cells
  • 293T cells HeLa cells
  • COS-1 cells COS-1 cells
  • SP2/0 cells SP2/0 cells
  • NSO cells myeloma cells
  • the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable carrier .
  • the pharmaceutical composition may further comprise one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or or a suitable formulation of preservatives.
  • Acceptable ingredients of the compositions are generally nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the pharmaceutical compositions may also contain more than one active compound, generally those with complementary activities that do not adversely affect each other.
  • the type and effective amount of such drug may depend, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.
  • the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents compatible with pharmaceutical administration, generally safe, nontoxic .
  • the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the pharmaceutical composition can be administered without interruption. Such uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the influx of the therapeutic agent into the patient, as described in WO2015/036583.
  • the present application provides methods for preparing the isolated antigen-binding protein.
  • the method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed. For example, by using appropriate medium, appropriate temperature and incubation time, etc., these methods are understood by those of ordinary skill in the art.
  • Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application. For example, by panning and amplifying the nanobody phage library, constructing a yeast display library to screen the single domain antibody.
  • the isolated antigen-binding protein can be prepared by selecting a Nanobody phage library for panning against biotin-labeled HSA. Using the panned plasmid as a template to amplify the single-chain antibody, the amplified fragment and the yeast display plasmid are co-transfected into Saccharomyces cerevisiae, and the single-chain antibody is displayed on the surface of the yeast cell wall.
  • the present application provides a method for detecting or measuring human serum albumin, the method comprising using the isolated antigen-binding protein of the present application or the fusion protein of the present application.
  • the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
  • the present application provides a human serum albumin kit, which may include the use of the isolated antigen-binding protein or the fusion protein.
  • the kit may further include instructions for use, which describe the method for detecting the presence and/or content of human serum albumin.
  • the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
  • the present application provides a use of the isolated antigen-binding protein or the fusion protein in the preparation of a kit, which can be used to detect the presence and/or content of human serum albumin method.
  • the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
  • the application provides the isolated antigen-binding protein, the fusion protein, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and/or the
  • the kits are used for preventing, alleviating and/or treating a disease or condition.
  • the present application provides a kind of said isolated antigen-binding protein, said fusion protein, said nucleic acid molecule, said carrier, said cell, said pharmaceutical composition and/or Use of the kit in the preparation of medicaments for preventing, alleviating and/or treating diseases or conditions.
  • the present application provides a method for preventing and/or treating a disease or disorder, which comprises administering the isolated antigen-binding protein, the fusion protein, the nucleic acid to a subject in need
  • the molecule, the carrier, the cell, the pharmaceutical composition and/or the kit comprises administering the isolated antigen-binding protein, the fusion protein, the nucleic acid to a subject in need.
  • compositions, pharmaceutical combinations and methods described herein may be used in conjunction with other types of cancer therapy, such as chemotherapy, surgery, radiation, gene therapy, and the like.
  • the pharmaceutical compositions and methods described in this application can be used in other disease conditions that depend on immune responses.
  • the subject may include humans or non-human animals.
  • the non-human animal can be selected from the group consisting of monkeys, chickens, geese, cats, dogs, mice and rats.
  • non-human animals may also include any animal species other than humans, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals.
  • the human can be Caucasian, African, Asian, Semitic, or other ethnicity, or a hybrid of various ethnicities.
  • the human can be elderly, adult, adolescent, child or infant.
  • the effective amount in humans can be inferred from the effective amount in experimental animals.
  • Freireich et al. describe the correlation of doses in animals and humans (based on milligrams per square meter of body surface) (Freiheim et al., Cancer Chemother. Rep. 50, 219 (1966)).
  • Body surface area can be approximately determined from the patient's height and weight. See, eg, Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970).
  • a self-built nanobody phage library was selected to perform two rounds of panning against biotin-labeled human serum albumin (hereinafter referred to as HSA-Biotin, acro, catalog number: HSA-H82E3) to obtain positive enrichment.
  • HSA-Biotin biotin-labeled human serum albumin
  • V H H single-chain antibody
  • PCR polymerase chain reaction
  • Saccharomyces cerevisiae strain EBY100 purchased from ATCC
  • the V H H gene was inserted into the yeast display plasmid through homologous recombination of Saccharomyces cerevisiae, and then the single-chain antibody was displayed on the surface of the yeast cell wall, as shown in Figure 1.
  • the yeast-displayed single-chain antibody library was named JYYDL032.
  • library JYYDL032 was cultured overnight in 100 mL SD-Trp medium (Clontech, product number: 630308) at 30°C and 225 rpm; 1.0 ⁇ 108 cells were resuspended in 20 mL YPGP liquid medium (2% galactose, 2% peptone, 1% yeast extract, 0.54% Na 2 HPO 4 , 0.86% NaH 2 PO 4 ⁇ H 2 O), cultured at 20°C, 225 rpm for 24 hours, and placed in a 4°C refrigerator for use.
  • the second-round screening product of JYYDL032 was spread on the SD-Trp plate and cultured statically at 30°C for 3 days to grow a yeast monoclonal colony. 92 single clones were picked for sequencing analysis, and finally 3 unique single-chain antibody sequences were obtained (Table 1).
  • the sequence of the control antibody (derived from Ablynx, ALX-0761, see SEQ ID NO: 27 for the amino acid sequence) was displayed on the surface of the yeast as a positive control antibody, as shown in Table 2.
  • Antibody number Amino acid sequence (SEQ ID NO: ) Ab0716.1 SEQ ID NO:17 Ab0716.2 SEQ ID NO:18 Ab0716.4 SEQ ID NO:19
  • VHH sequence of each clone was fused with IgG1Fc N297A, codon optimization was performed, and the gene was synthesized and loaded into the expression vector pcDNA3.4 (Life Technologies). After expression plasmid amplification and plasmid extraction, the plasmid was transferred into ExpiCHO cells (ThermoFisher Scientific, A29133), and the antibody was transiently expressed according to the supplier’s ExpiCHO expression system method.
  • ExpiCHO cells Cultivate Expi CHO cells at a concentration of % carbon dioxide to a density of 6 ⁇ 10 6 /mL, and use ExpiFectamine transfection reagent to transfect 10 ⁇ g of antibody light and heavy chain expression plasmids into the cells; one day after transfection, take 150 ⁇ L and 4 mL of ExpiCHO enhancer and ExpiCHO adjuvant Add it to the cultured cells, continue culturing for 9 days, centrifuge at 4°C at 3500 rpm to get the supernatant.
  • Each cycle includes the following steps: 1) immersion in the buffer for 60s; 2) detection of non-specific binding of the antigen to the sensor; 3) regeneration of 10mM glycine solution at pH 1.7; 4) immersion in the buffer for 60s; 5) antibody immobilization on the sensor Above, the time is 30s; 6) The sensor is immersed in the buffer solution for 180s; 7) The combination of antigen and antibody, the time is 180s; 8) The dissociation of antigen and antibody, the time is 10 minutes; 9) Sensor regeneration.
  • the physical and chemical indicators such as purity, thermal stability, and hydrophilicity of the candidate antibody Ab0716.1 are relatively ideal, and Ab0716.1 is used as a candidate molecule to continue humanization to improve the homology with human antibodies, so as to Reduce immunogenicity.
  • the V H H sequence of the candidate antibody Ab0716.1 was finally selected for humanization.
  • IMGT/Domain Gap Align was performed on the V H H sequence of Ab0716.1, and the human germline with the highest homology was found to be IGHV3-23*04.
  • the sequence of Ab0716.1 was numbered according to the Chothia rules, and the H1, H13, H44, H45, H49, H74, H76, H79, H81, H82B, H83, H84, H108 and other sites were mutated according to Table 7, and the amino acids at other sites remained unchanged. Change.
  • the VHH sequence of Ab0716Hz1 is shown in SEQ ID NO:20
  • the VHH sequence of Ab0716Hz2 is shown in SEQ ID NO:21.
  • VHH sequences of Ab0716Hz1 and Ab0716Hz2 in Table 7 were analyzed by IMGT/Domain Gap Align to compare the homology with IGHV3-23*04 before and after humanization; in addition, the humanized VHH and IgG1Fc N297A For fusion expression, refer to Example 2.1 for the expression and purification of the humanized antibody, see Table 8 for specific results. It can be seen from Table 8 that the homology between the antibody and IGHV3-23*04 was increased to about 87% after humanization, and the expression level of the humanized antibody was relatively ideal, and the follow-up evaluation was continued.
  • the humanized antibody molecules Ab0716Hz1 and Ab0716Hz2 were evaluated for their druggability, as follows:
  • the antibody Ab0716Hz2 was finally selected for further affinity maturation to improve its affinity with monkey serum albumin.
  • Amino acids at the antigen-binding determinant (CDR) site of Ab0716Hz2 were randomly mutated, and a mutation library of each CDR region was constructed. Yeast display technology was used to screen high-throughput sequences with strong antigen-specific binding.
  • the amino acid sequence of the variable region of Ab0716Hz2 is coded according to Chothia coding rules, and the CDR region is defined according to Chothia.
  • NNK mutation primers were designed to carry out polymerase chain reaction (PCR) to amplify gene fragments of each CDR mutation library.
  • Example 1 to construct an affinity maturation mutation library, library number JYYDL184-187; after electroporation, the library JYYDL184-187 was cultured in 100 mL SD-Trp medium at 30°C and 225 rpm overnight; each took 1.0 ⁇ 10 8 bacteria , resuspended in 20mL YPGP induction medium, cultured at 20°C, 225 rpm for 24 hours, and placed in a 4°C refrigerator for use. At the same time, the parental sequence of Ab0716Hz2 was displayed on the yeast surface and used as a parental control.
  • the cells were cultured and induced again, and the same as the second round, 2.0 ⁇ 10 7 cells were incubated in 10nM CSA-Biotin for the third round of flow sorting, and after sorting, some cells were coated.
  • static culture at 30°C for 3 days.
  • V H H sequence of each clone was fused with IgG1Fc N297A, and Shanghai Baiying Biotechnology Co., Ltd. was entrusted to perform transient HEK293 cell expression and purification, and finally obtained affinity maturation candidate antibodies are shown in Table 13.
  • the affinities of the affinity matured antibodies Ab0716Am01, Ab0716Am02, Ab0716Am06 and the parental antibody Ab0716Hz2 to serum albumin from human, monkey, and mouse were continuously measured, and the results are shown in Table 14. It can be seen from Table 14 that after affinity maturation, the affinity of antibodies Ab0716Am01, Ab0716Am02, Ab0716Am06 to monkey serum albumin was greatly improved compared with the parental antibody; the affinity to human serum albumin was maintained between 1.41-5.0nM, which was acceptable; the affinity Neither before nor after antibody binds to mouse serum albumin.
  • Example 8 the physicochemical properties of the antibodies Ab0716Am01, Ab0716Am02, and Ab0716Am06 after affinity maturation were evaluated, and the results are shown in Table 15. It can be seen from Table 15 that the physical and chemical indicators of the antibodies Ab0716Am01, Ab0716Am02, and Ab0716Am06 after affinity maturation are ideal, such as purity, thermal stability, hydrophilicity, and charge isomers, and meet internal druggability standards.

Abstract

A separated antigen-binding protein capable of binding to human serum albumin, which comprises at least one CDR in a heavy chain variable region VH, the VH comprising an amino acid sequence represented by SEQ ID NO: 26 or 38. Further provided are a preparation method for the antigen-binding protein and a use thereof.

Description

抗人血清白蛋白的抗原结合蛋白Antigen binding protein against human serum albumin 技术领域technical field
本申请涉及生物医药领域，具体的涉及一种能够结合人血清白蛋白的抗原结合蛋白。The present application relates to the field of biomedicine, in particular to an antigen-binding protein capable of binding to human serum albumin.
背景技术Background technique
目前，蛋白质药物已成功广泛应用于临床治疗，许多蛋白质药物为一些疾病提供了有效的治疗效果。然而大多数蛋白和多肽的半衰期很短，一是因为蛋白质药物会被体内的蛋白酶降解而快速清除，二是由于肾小球的过滤作用是分子量小于60KD的药物在肾脏代谢中易被清除。为了达到有效治疗，往往采用高剂量或多次、长时间用药的方案，这样一方面会增加其毒副作用，同时也给患者用药带来极大的不便。At present, protein drugs have been successfully and widely used in clinical treatment, and many protein drugs have provided effective therapeutic effects for some diseases. However, the half-life of most proteins and peptides is very short. One is that protein drugs will be degraded by proteases in the body and quickly cleared, and the other is that drugs with a molecular weight of less than 60KD are easily eliminated in renal metabolism due to the filtration of glomeruli. In order to achieve effective treatment, high-dose or multiple, long-term medication regimens are often used, which will increase its toxic and side effects on the one hand, and bring great inconvenience to patients while taking medication.
为延长蛋白质药物在体内的半衰期，通过与具有较长半衰期的人体蛋白直接融合是一种有效方法。人血清白蛋白是人体血液中的主要蛋白，也是人体内半衰期最长的蛋白，它可以与一些药物分子结合，对药物分子提供保护作用，是一种理想的药物载体分子。因此，亟需开发能够结合人血清白蛋白的分子，提供更多有效的药物载体。In order to prolong the half-life of protein drugs in vivo, direct fusion with human proteins with longer half-life is an effective method. Human serum albumin is the main protein in human blood and the protein with the longest half-life in the human body. It can combine with some drug molecules to provide protection for drug molecules, and is an ideal drug carrier molecule. Therefore, there is an urgent need to develop molecules that can bind to human serum albumin to provide more effective drug carriers.
发明内容Contents of the invention
本申请提供了一种能够识别人血清白蛋白的抗原结合蛋白，可通过筛选得到具有高亲和力的结合蛋白，所述抗原结合蛋白具有稳定的理化性质，能够与人血清白蛋白结合，延长生物药物半衰期，有利于生物药在治疗中的有益效果。This application provides an antigen-binding protein capable of recognizing human serum albumin, which can be screened to obtain a binding protein with high affinity. The antigen-binding protein has stable physical and chemical properties, can bind to human serum albumin, and prolong the life of biological drugs. Half-life, which favors the beneficial effects of biologics in therapy.
一方面，本申请提供了一种分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:26或38所示的氨基酸序列。In one aspect, the application provides an isolated antigen-binding protein comprising at least one CDR in the variable region VH of an antibody heavy chain, said VH comprising the amino acid sequence shown in SEQ ID NO: 26 or 38.
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR3，所述HCDR3包含SEQ ID NO:29所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:29.
在某些实施方式中，所示分离的抗原结合蛋白的HCDR3包含SEQ ID NO:1或SEQ ID NO:28所示的氨基酸序列。In certain embodiments, the HCDR3 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 28.
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，所述HCDR2包含SEQ ID NO:37所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:37.
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，所述HCDR2包含SEQ ID NO:2、31或32所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein comprises HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, 31 or 32.
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，所述HCDR1包含SEQ ID  NO:36所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:36.
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，所述HCDR1包含SEQ ID NO:3或30所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3 or 30.
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:36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:29所示的氨基酸序列。In some embodiments, the isolated antigen-binding protein comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:36, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:37 , and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:29.
在某些实施方式中，所述分离的抗原结合蛋白的所述HCDR1包含SEQ ID NO:3或30所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2、31或32所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1或SEQ ID NO:28所示的氨基酸序列。In certain embodiments, the HCDR1 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:3 or 30, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, 31 or 32 , and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 28.
在某些实施方式中，所述分离的抗原结合蛋白的所述HCDR1包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列；或者所述HCDR1包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:28所示的氨基酸序列；或者所述HCDR1包含SEQ ID NO:30所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列；或者所述HCDR1包含SEQ ID NO:3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:1所示的氨基酸序列；或者所述HCDR1包含SEQ ID NO:3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:1所示的氨基酸序列。In certain embodiments, the HCDR1 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 comprises The amino acid sequence shown in SEQ ID NO:1; or the HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO : the amino acid sequence shown in 28; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 30, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1 or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:31, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1 or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:32, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1.
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，所述H-FR1的C端与所述HCDR1的N端直接或间接相连，且所述H-FR1包含SEQ ID NO:22所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises SEQ ID NO: The amino acid sequence shown in 22.
在某些实施方式中，所述分离的抗原结合蛋白的所述H-FR1包含SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:11中任一项所示的氨基酸序列。In certain embodiments, the H-FR1 of the isolated antigen binding protein comprises any one of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:11 amino acid sequence.
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR2，所述H-FR2位于所述HCDR1和HCDR2之间，且所述H-FR2包含SEQ ID NO:23所示的氨基酸序列。In some embodiments, the isolated antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:23 .
在某些实施方式中，所述分离的抗原结合蛋白的所述H-FR2包含SEQ ID NO:5、SEQ ID NO:10、SEQ ID NO:12和SEQ ID NO:15中任一项所示的氨基酸序列。In some embodiments, the H-FR2 of the isolated antigen binding protein comprises any one of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:15 amino acid sequence.
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3包含SEQ ID NO:24所示的氨基酸序列。In certain embodiments, said isolated antigen binding protein comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 comprises SEQ ID NO:24 amino acid sequence.
在某些实施方式中，所述分离的抗原结合蛋白的所述H-FR3包含SEQ ID NO:6或SEQ  ID NO:13所示的氨基酸序列。In certain embodiments, the H-FR3 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:13.
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR4，所述H-FR4的N端与所述HCDR3的C端直接或间接相连，且所述H-FR4包含SEQ ID NO:25所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO: The amino acid sequence shown in 25.
在某些实施方式中，所述分离的抗原结合蛋白的所述H-FR4包含SEQ ID NO:7、SEQ ID NO:14和SEQ ID NO:16中任一项所示的氨基酸序列。In certain embodiments, said H-FR4 of said isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:14 and SEQ ID NO:16.
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:22所示的氨基酸序列，所述H-FR2包含SEQ ID NO:23所示的氨基酸序列，所述H-FR3包含SEQ ID NO:24所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:25所示的氨基酸序列。In certain embodiments, the antigen binding protein of described separation comprises H-FR1, H-FR2, H-FR3 and H-FR4, and described H-FR1 comprises the aminoacid sequence shown in SEQ ID NO:22, described H-FR2 comprises the amino acid sequence shown in SEQ ID NO:23, said H-FR3 comprises the amino acid sequence shown in SEQ ID NO:24, and said H-FR4 comprises the amino acid sequence shown in SEQ ID NO:25.
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:11中任一项所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5、SEQ ID NO:10、SEQ ID NO:12和SEQ ID NO:15中任一项所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6或SEQ ID NO:13所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7、SEQ ID NO:14和SEQ ID NO:16中任一项所示的氨基酸序列。In certain embodiments, the antigen binding protein of described isolation comprises H-FR1, H-FR2, H-FR3 and H-FR4, and described H-FR1 comprises SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:8, SEQ ID NO: The amino acid sequence shown in any one of ID NO:9 and SEQ ID NO:11, described H-FR2 comprises SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:15 The amino acid sequence shown in any one, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:13, and the H-FR4 comprises SEQ ID NO:7, SEQ ID NO:14 and the amino acid sequence shown in any one of SEQ ID NO:16.
在某些实施方式中，所述分离的抗原结合蛋白包含选自下组的任一组H-FR1，H-FR2，H-FR3和H-FR4：In certain embodiments, the isolated antigen binding protein comprises any group of H-FR1, H-FR2, H-FR3 and H-FR4 selected from the group consisting of:
1)所述H-FR1包含如SEQ ID NO:4所示的氨基酸序列，所述H-FR2包含如SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含如SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含如SEQ ID NO:7所示的氨基酸序列；1) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
2)所述H-FR1包含如SEQ ID NO:8所示的氨基酸序列，所述H-FR2包含如SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含如SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含如SEQ ID NO:7所示的氨基酸序列；2) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:8, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
3)所述H-FR1包含如SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含如SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含如SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含如SEQ ID NO:7所示的氨基酸序列；3) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
4)所述H-FR1包含如SEQ ID NO:11所示的氨基酸序列，所述H-FR2包含如SEQ ID NO:12所示的氨基酸序列，所述H-FR3包含如SEQ ID NO:13所示的氨基酸序列，且所述H-FR4包含如SEQ ID NO:14所示的氨基酸序列；以及4) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:11, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:12, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:13 The amino acid sequence shown, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 14; and
5)所述H-FR1包含如SEQ ID NO:11所示的氨基酸序列，所述H-FR2包含如SEQ ID NO:15所示的氨基酸序列，所述H-FR3包含如SEQ ID NO:13所示的氨基酸序列，且所述H-FR4包含如SEQ ID NO:16所示的氨基酸序列。5) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:11, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:15, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:13 The amino acid sequence shown, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:16.
在某些实施方式中，所述分离的抗原结合蛋白包含VH，所述VH包含SEQ ID NO:26或38所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 26 or 38.
在某些实施方式中，所述分离的抗原结合蛋白的所述VH包含SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:33、SEQ ID NO:34、和SEQ ID NO:35中任一项所示的氨基酸序列。In certain embodiments, said VH of said isolated antigen binding protein comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 21, SEQ ID NO: The amino acid sequence shown in any one of ID NO:33, SEQ ID NO:34, and SEQ ID NO:35.
在某些实施方式中，所述分离的抗原结合蛋白包括抗体或其抗原结合片段。In certain embodiments, the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.
在某些实施方式中，所述分离的抗原结合蛋白包含抗体重链恒定区。In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region.
在某些实施方式中，所述分离的抗原结合蛋白的抗体重链恒定区源自人抗体重链恒定区。In certain embodiments, the antibody heavy chain constant region of the isolated antigen binding protein is derived from a human antibody heavy chain constant region.
在某些实施方式中，所述分离的抗原结合蛋白的抗体重链恒定区源自人IgG重链恒定区。In certain embodiments, the antibody heavy chain constant region of the isolated antigen binding protein is derived from a human IgG heavy chain constant region.
在某些实施方式中，所述分离的抗原结合蛋白的抗体重链恒定区源自人IgG1重链恒定区。In certain embodiments, the antibody heavy chain constant region of the isolated antigen binding protein is derived from a human IgG1 heavy chain constant region.
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，di-scFv，dAb和/或V HH。 In certain embodiments, the antigen-binding fragment comprises Fab, Fab', Fv fragment, F(ab') 2 , scFv, di-scFv, dAb and/or VHH .
在某些实施方式中，所述抗原结合片段为V HH。 In certain embodiments, the antigen-binding fragment is a VHH .
在某些实施方式中，所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。在某些实施方式中，所述分离的抗原结合蛋白能够结合人血清白蛋白。In certain embodiments, the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. In certain embodiments, the isolated antigen binding protein is capable of binding human serum albumin.
另一方面，本申请还提供了融合蛋白，其包含所述分离的抗原结合蛋白。In another aspect, the present application also provides a fusion protein comprising the isolated antigen-binding protein.
另一方面，本申请还提供了分离的核酸分子，其编码所述分离的抗原结合蛋白或所述融合蛋白。In another aspect, the present application also provides an isolated nucleic acid molecule encoding the isolated antigen-binding protein or the fusion protein.
另一方面，本申请还提供了载体，其包含所述核酸分子。On the other hand, the present application also provides a vector comprising the nucleic acid molecule.
另一方面，本申请还提供了细胞，其包含所述分离的抗原结合蛋白，所述融合蛋白，所述核酸分子或所述载体。In another aspect, the present application also provides a cell comprising the isolated antigen-binding protein, the fusion protein, the nucleic acid molecule or the carrier.
另一方面，本申请还提供了制备所述分离的抗原结合蛋白的方法，所述方法包括在使得所述分离的抗原结合蛋白表达的条件下，培养所述的细胞。On the other hand, the present application also provides a method for preparing the isolated antigen-binding protein, the method comprising culturing the cell under the condition that the isolated antigen-binding protein is expressed.
另一方面，本申请还提供了药物组合物，其包含所述分离的抗原结合蛋白，所述核酸分子、所述载体和/或所述细胞，以及任选地药学上可接受的载剂。On the other hand, the present application also provides a pharmaceutical composition, which comprises the isolated antigen-binding protein, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable carrier.
另一方面，本申请还提供了一种用于检测或测定人血清白蛋白的方法，所述方法包括所述的分离的抗原结合蛋白或所述的融合蛋白。On the other hand, the present application also provides a method for detecting or measuring human serum albumin, said method comprising said isolated antigen-binding protein or said fusion protein.
另一方面，本申请还提供了一种试剂盒，其包含所述的分离的抗原结合蛋白，所述的融合蛋白，所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物。On the other hand, the present application also provides a kit, which comprises the isolated antigen-binding protein, the fusion protein, the nucleic acid molecule, the carrier, the cell and/or the said pharmaceutical composition.
另一方面，本申请还提供了所述的分离的抗原结合蛋白，所述的融合蛋白，所述的核酸分子、所述的载体、所述的细胞、所述的药物组合物和/或所述的试剂盒在制备预防和/或治疗疾病或病症的药物中的用途。On the other hand, the present application also provides the isolated antigen-binding protein, the fusion protein, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and/or the Use of the above-mentioned kit in the preparation of medicines for preventing and/or treating diseases or diseases.
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。Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.
附图说明Description of drawings
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：The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:
图1显示的是酵母展示文库的构建方法。Figure 1 shows the construction method of yeast display library.
具体实施方式Detailed ways
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。The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.
术语定义Definition of Terms
在本申请中，术语“人血清白蛋白(Human Serum Albumin)”可以与“HSA”互换使用。在本申请中，该术语还涵盖人血清白蛋白的变体、同源物、类似物、衍生物以及功能活性片段。In this application, the term "Human Serum Albumin" may be used interchangeably with "HSA". In the present application, the term also covers variants, homologues, analogs, derivatives and functionally active fragments of human serum albumin.
在本申请中，术语“分离的”通常指从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。In the present application, the term "isolated" generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of. The term "isolated" does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.
在本申请中，术语“分离的抗原结合蛋白”通常指脱离了其天然存在状态的具有抗原结合能力的蛋白。该“分离的抗原结合蛋白”可以包含结合抗原的部分和任选地，允许抗原结合部分采用促进所述抗原结合部分结合抗原的构象的框架或构架部分。抗原结合蛋白可以包含例如抗体来源的蛋白框架区(FR)或具有移植的CDR或CDR衍生物的备选蛋白框架区或人工框架区。此类框架包括，但不限于包含被引入例如以稳定抗原结合蛋白的三维结构的突变的抗体来源的框架区以及包含例如生物相容性聚合物的完全合成的框架区。参见例如Korndorfer等,2003,Proteins:Structure,Function,andBioinformatics,53(1):121-129(2003)；Roque等,Biotechnol.Prog.20:639-654(2004)。抗原结合蛋白的实例包括但不限于：人抗体、人源化抗体；嵌合抗体；重组抗体；单链抗体；双功能抗体；三功能抗体；四功能抗体；Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv，dAb，IgD抗体；IgE抗体；IgM抗体；IgG1抗体；IgG2抗体；IgG3抗体；或IgG4抗体以及其片段。 In this application, the term "isolated antigen-binding protein" generally refers to a protein having antigen-binding ability that is removed from its naturally occurring state. The "isolated antigen binding protein" may comprise an antigen-binding moiety and, optionally, a framework or framework portion that permits the antigen-binding moiety to adopt a conformation that facilitates binding of said antigen-binding moiety to antigen. Antigen binding proteins may comprise, for example, antibody-derived protein framework regions (FR) or alternative protein framework regions or artificial framework regions with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to, antibody-derived framework regions comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antigen binding protein, and fully synthetic framework regions comprising, eg, biocompatible polymers. See eg Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Examples of antigen binding proteins include, but are not limited to: human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single chain antibodies; diabodies; triabodies; tetrabodies; (ab') 2 , F(ab) 2 , scFv, di-scFv, dAb, IgD antibody; IgE antibody; IgM antibody; IgGl antibody; IgG2 antibody; IgG3 antibody; or IgG4 antibody and fragments thereof.
在本申请中，术语“CDR”也称“互补决定区”，通常指抗体可变结构域中的区域，其序列是高度可变的和/或形成结构定义环。在某些实施方案中，仅由重链组成的天然存在的骆驼抗体在缺乏轻链的情况下，其功能也能够正常且稳定。参见，例如，Hamers-Casterman et al.,Nature 363:446-448(1993)；Sheriff et al,Nature Struct.Biol.3:733-736(1996)。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。例如，所述抗原结合蛋白的氨基酸序列编号可以按照IMGT编号方案(IMGT,the international ImMunoGeneTics information system@imgt.cines.fr；http：//imgt.cines.fr；Lefranc等,1999,Nucleic Acids Res.27:209-212；Ruiz等,2000Nucleic Acids Res.28:219-221；Lefranc等,2001,Nucleic Acids Res.29:207-209；Lefranc等,2003,Nucleic Acids Res.31:307-310；Lefranc等,2005,DevComp Immunol 29:185-203)。例如，所述抗原结合蛋白的CDR可以根据Kabat编号系统确定(参见例如Kabat EA&Wu TT(1971)Ann NY AcadSci 190:382-391和Kabat EAet al.,(1991)Sequences of Proteins of Immunological Interest,FifthEdition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)。In this application, the term "CDR", also referred to as "complementarity determining region", generally refers to the region in the variable domain of an antibody, the sequence of which is highly variable and/or forms a structure-defining loop. In certain embodiments, naturally occurring camelid antibodies consisting only of heavy chains are capable of functioning and stabilizing in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996). Antibody CDRs can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. For example, the amino acid sequence numbering of the antigen-binding protein can be numbered according to the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system@imgt.cines.fr; http://imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res. 27:209-212; Ruiz et al., 2000 Nucleic Acids Res.28:219-221; Lefranc et al., 2001, Nucleic Acids Res.29:207-209; Lefranc et al., 2003, Nucleic Acids Res.31:307-310; Lefranc et al., 2005, DevComp Immunol 29:185-203). For example, the CDRs of the antigen binding protein can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
在本申请中，术语“FR”通常指抗体可变结构域的更高度保守的部分，其被称为框架区。通常，天然重链和轻链的可变结构域各自包含四个FR区，即在VH中四个(H-FR1，H-FR2，H-FR3和H-FR4)，和在VL中四个(L-FR1，L-FR2，L-FR3和L-FR4)。In this application, the term "FR" generally refers to the more highly conserved portions of antibody variable domains, known as the framework regions. Typically, the variable domains of native heavy and light chains each comprise four FR regions, four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL. (L-FR1, L-FR2, L-FR3 and L-FR4).
在本申请中，术语“可变结构域”与“可变区”可以互换使用，通常指抗体重链和/或轻链的一部分。重链和轻链的可变结构域可以分别称为“V H”和“V L”(或者分别称为“VH” 和“VL”)。这些结构域通常是抗体的变化最大的部分(相对于相同类型的其它抗体)，且包含抗原结合位点。 In this application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain. The variable domains of the heavy and light chains may be referred to as " VH " and " VL ", respectively (or "VH" and "VL", respectively). These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class) and comprise the antigen binding site.
在本申请中，术语“可变”通常指在抗体之间可变结构域的某些区段在序列上可能存在较大差异。可变结构域介导抗原结合并决定特定抗体对其特定抗原的特异性。然而，可变性并非在整个可变结构域范围内均匀分布。它通常集中在轻链和重链可变结构域中称为高变区(CDR或HVR)的三个区段中。可变结构域的更高度保守的部分称为框架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区，大多数采用β-折叠构型，通过三个CDR连接，其形成环形连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDR通过FR区紧密靠近地保持在一起，并且来自另一条链的CDR一同促进抗体的抗原结合位点的形成(参见Kabat et al,Sequences of Immunological Interest,Fifth Edition,National Institute of Health,Bethesda,Md.(1991))。In the present application, the term "variable" generally means that some segments of the variable domain may have large differences in sequence between antibodies. The variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains. It is usually concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, most adopting a β-sheet configuration, connected by three CDRs, which form a circular connection and in some cases form part of the β-sheet structure . The CDRs in each chain are held in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antibody's antigen-binding site (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
在本申请中，术语“抗体”通常指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、单特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，为了本发明的目的，术语“抗体”也包括抗体片段，比如Fab、F(ab') 2、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合HSA)的其它抗体片段。通常，这样的片段应当包括抗原结合结构域。基本的4链抗体单元是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般为约150,000道尔顿。每个L链通过一个共价二硫键与H链连接，而两个H链通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还具有规则间隔的链内二硫化桥键。每个H链在N末端具有可变结构域(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端具有可变结构域(VL)，在其另一端具有恒定结构域。VL与VH对应，且CL与重链的第一恒定结构域(CH1)相对应。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。VH和VL配对一起形成单个抗原结合位点。对于不同类别抗体的结构和性质，参见例如Basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw(eds),Appleton&Lange,Norwalk,Conn.,1994，第71页和第6章。来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。根据 重链(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。目前存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。 In this application, the term "antibody" generally refers to an immunoglobulin or fragment or derivative thereof, encompassing any polypeptide that includes an antigen combining site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid, mutated, and transplanted antibodies. Unless otherwise modified by the term "intact", as in "intact antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, F(ab') 2 , Fv, scFv, Fd, dAbs and other antibody fragments that retain antigen binding function (eg, specifically bind HSA). Typically, such fragments will include the antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetrameric units and another polypeptide called the J chain, and contain 10 antigen-binding sites, while IgA antibodies include 2-5 that can be combined with the J chain to form a multivalent A basic 4-chain unit for combinations. For IgG, the 4-chain unit is typically about 150,000 Daltons. Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for the alpha and gamma chains each, and four CH domains for the mu and epsilon isoforms. Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen-binding site. For the structure and properties of the different classes of antibodies see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and Chapter 6. L chains from any vertebrate species can be classified into one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain of the heavy chain (CH), immunoglobulins can be assigned to different classes, or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively.
在本申请中，术语“抗原结合片段”通常指具有特异结合抗原(例如，HSA)能力的一个或多个片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2，scFv，di-scFv，dAb和/或V HH。 In this application, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind an antigen (eg, HSA). In the present application, the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, dAb and/or VHH .
在本申请中，术语“Fab”通常指抗体的抗原结合片段。如上所述，可以使用木瓜蛋白酶消化完整的抗体。抗体经木瓜蛋白酶消化后产生两个相同的抗原结合片段，即“Fab”片段，和残余的“Fc”片段(即Fc区，同上)。Fab片段可以由一条完整的L链与一条重链的可变区和该H链(V H)的第一恒定区(C H1)组成。 In this application, the term "Fab" generally refers to an antigen-binding fragment of an antibody. Intact antibodies can be digested using papain as described above. Papain digestion of antibodies yields two identical antigen-binding fragments, the "Fab" fragment, and a residual "Fc" fragment (ie, the Fc region, supra). Fab fragments may consist of a complete L chain with the variable region of a heavy chain and the first constant region (CH 1 ) of the H chain ( VH ).
在本申请中，术语“Fab′片段”通常指人单克隆抗体的单价抗原结合片段，该片段比Fab片段稍大。例如，Fab′片段可以包括所有轻链，所有重链可变区以及重链的所有或部分第一和第二恒定区。例如，Fab′片段还可包括重链的部分或所有的220-330个氨基酸残基。In this application, the term "Fab' fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which fragment is slightly larger than a Fab fragment. For example, a Fab' fragment may include all of the light chain, all of the variable domains of the heavy chain, and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.
在本申请中，术语“F(ab')2”通常指通过胃蛋白酶消化完整抗体所产生的抗体片段。F(ab')2片段含有由二硫键维持在一起的两个Fab片段和部分铰链区。F(ab')2片段具有二价抗原结合活性并且能够交联抗原。In this application, the term "F(ab')2" generally refers to antibody fragments produced by pepsin digestion of intact antibodies. The F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds. F(ab')2 fragments have bivalent antigen binding activity and are capable of cross-linking antigen.
在本申请中，术语“Fv片段”通常指人单克隆抗体的单价抗原结合片段，包括所有或部分重链可变区和轻链可变区，并且缺乏重链恒定区和轻链恒定区。重链可变区和轻链可变区包括例如CDR。例如，Fv片段包括重链和轻链的约110个氨基酸的所有或部分氨基端可变区。In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody comprising all or part of the heavy and light chain variable regions and lacking the heavy and light chain constant regions. The heavy and light chain variable regions include, for example, CDRs. For example, an Fv fragment includes all or part of the approximately 110 amino acid amino-terminal variable regions of the heavy and light chains.
在本申请中，术语“scFv”通常指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非特别说明，否则如本申请中使用的那样，scFv可以以任何顺序(例如相对于多肽的N末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chains are variable The regions are contiguous (eg via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single chain polypeptide wherein the scFv retains the specificity of the intact antibody from which it was derived. Unless otherwise specified, as used in this application, a scFv can have the VL and VH variable regions in any order (for example, relative to the N-terminal and C-terminal of the polypeptide), and the scFv can include VL-linker-VH or VH-Linker-VL can be included.
在本申请中，术语“dAb”通常是指具有VH域、VL域或具有VH域或VL域的抗原结合片段，参考例如Ward等人(Nature,1989Oct 12；341(6242)：544-6)，参考Holt等人,Trends Biotechnol.,2003,21(11)：484-490；以及参考例如WO 06/030220、WO 06/003388和DomantisLtd的其它公布的专利申请。在本申请中，术语“dAb”可以包括sdAb，术语“sdAb”通常指单结构域抗原结合分子，在本申请中，该术语可以包括单域抗体，以及V HH。 In the present application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having a VH domain or a VL domain, see e.g. Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6) , with reference to Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and to other published patent applications such as WO 06/030220, WO 06/003388 and Domantis Ltd. In this application, the term "dAb" may include sdAb, the term "sdAb" generally refers to a single domain antigen binding molecule, and in this application the term may include single domain antibody, as well as VHH .
在本申请中，术语“单克隆抗体”通常指单分子组成的抗体分子制备物。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。In this application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules of single molecular composition. 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. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma cultures without contamination from other immunoglobulins. The modifier "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. For example, monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如羊驼来源)抗体相比，在人类个体中引发不良免疫反应的可能性降低。In this application, the term "chimeric antibody" generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species. Typically, the variable regions are derived from an antibody of a laboratory animal, such as a rodent ("parent antibody"), and the constant regions are derived from a human antibody, such that the resulting chimeric antibody is more specific in a human individual than the parental (e.g., alpaca-derived) antibody. Less likely to trigger an adverse immune response.
在本申请中，术语“人源化抗体”通常是指非人抗体(例如羊驼抗体)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(例如羊驼)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)(诸如羊驼，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。In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (such as an alpaca antibody) are replaced by 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, llama) antibodies may contain, at a minimum, chimeric antibodies of sequence derived from non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) can be replaced with a non-human species (donor antibody) (such as alpaca, mouse, etc.) having the desired properties, affinity and/or capabilities. , rat, rabbit or non-human primate) CDR region residue replacement. In certain instances, FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, 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.
术语“全人源抗体”通常指仅包含人类免疫球蛋白蛋白质序列的抗体。如果其是在小鼠中、在小鼠细胞中或在衍生自小鼠细胞的杂交瘤中生产，那么全人源抗体可能含有鼠糖链。类似地，“小鼠抗体”或“大鼠抗体”分别指仅包含小鼠或大鼠免疫球蛋白序列的抗体。可通过噬菌体展示或其它分子生物学方法，在人体内、在具有人类免疫球蛋白种系序列的转基因动物体内生成全人源抗体。可用于制造抗体的示例性技术在美国专利：6,150,584、6,458,592、6,420,140中描述。其它技术，如使用文库，是本领域已知的。The term "fully human antibody" generally refers to an antibody that comprises only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains if they are produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Similarly, a "mouse antibody" or "rat antibody" refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology methods. Exemplary techniques that can be used to make antibodies are described in US Patents: 6,150,584, 6,458,592, 6,420,140. Other techniques, such as using libraries, are known in the art.
在本申请中，术语“核酸分子”通常指任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length in isolated form, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.
在本申请中，术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can be expressed by transforming, transducing or transfecting the host cell, so that the genetic material elements carried by it can be expressed in the host cell. A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein coats, but not only.
在本申请中，术语“细胞”通常指可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物，其包括本发明所述的核酸分子或本发明所述的载体。细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。细胞可包括用本申请所述的载体在体外转染的细胞。In this application, the term "cell" generally refers to a single cell, cell line or cell culture that can be or has been the recipient of a subject's plasmid or vector, which includes a nucleic acid molecule as described herein or a nucleic acid molecule as described herein. Carrier. Cells can include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell. Cells may include cells transfected in vitro with the vectors described herein.
在本申请中，术语“药物组合物”通常指用于预防/治疗疾病或病症的组合物。所述药物组合物可以包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。此外，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下通常对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。In this application, the term "pharmaceutical composition" generally refers to a composition for preventing/treating a disease or condition. The pharmaceutical composition may comprise the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and/or the cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also comprise one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. preparations. Acceptable ingredients of the compositions are generally nontoxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
在本申请中，术语“药学上可接受的载剂”通常包括药剂学可接受的载体、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。生理学可接受的载体可包括例如缓冲剂，抗氧化剂，低分子量(少于约10个残基)多肽，蛋白质，亲水性聚合物，氨基酸，单糖，二糖和其它碳水化合物，螯合剂，糖醇，成盐反荷离子，例如钠；和/或非离子表面活性剂。As used herein, the term "pharmaceutically acceptable carrier" generally includes a pharmaceutically acceptable carrier, excipient or stabilizer that is inert to the cells or mammals to which it is exposed at the dosage and concentration employed. poisonous. Physiologically acceptable carriers can include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions such as sodium; and/or nonionic surfactants.
在本申请中，术语“特异性结合”或“特异性的”通常指可测量的和可再现的相互作用，例如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体可以是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标的抗体。在某些实施方案中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在某些实施方案中，特异性结合可以包括但不要求排他性地结合。In this application, the term "specifically binds" or "specific" generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, that can occur in a heterogeneous population of molecules, including biomolecules. Presence determines the presence of a target. For example, an antibody that specifically binds a target (which may be an epitope) can be an antibody that binds that target with greater affinity, avidity, greater ease, and/or for a greater duration than it binds other targets . In certain embodiments, an antibody specifically binds an epitope on a protein that is conserved among proteins of different species. In certain embodiments, specific binding can include, but does not require exclusive binding.
在本申请中，术语“受试者”通常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。In this application, the term "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.
在本申请中，涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。In this application, the protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues having the same or similar functions as the protein or polypeptide.
在本申请中，所述变体可以为，例如在所述蛋白质和/或所述多肽(例如，特异性结合HSA的抗体或其片段)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。In the present application, the variant may be, for example, substituted, deleted or added one or more A protein or polypeptide of amino acids. For example, the functional variant may comprise at least 1, such as 1-30, 1-20 or 1-10, further such as 1, 2, 3, 4 or 5 amino acid substitutions , proteins or polypeptides with amino acid changes by deletion and/or insertion. Said functional variant may substantially retain the biological properties of said protein or said polypeptide prior to alteration (eg, substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the protein or polypeptide prior to the alteration. For example, the substitutions may be conservative substitutions.
在本申请中，所述同源物可以为与所述蛋白质和/或所述多肽(例如，特异性结合HSA的抗体或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约87％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。In the present application, the homologue may be at least about 85% (eg, having at least about 85%) of the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds to HSA). %, about 87%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) Proteins or polypeptides with sequence homology.
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。In this application, the homology generally refers to the similarity, similarity or association between two or more sequences. "Percentage of sequence homology" can be calculated in the following manner: compare the two sequences to be aligned in the comparison window, and determine that there are identical nucleic acid bases (for example, A, T, C, G, I) in the two sequences ) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) Number of positions To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size), and the result was multiplied by 100 to yield the percent sequence identity. Alignment for purposes of determining percent sequence homology can be accomplished in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in "An Improved Tool for Biological Sequence Comparison" by W.R.Pearson and D.J. Lipman, Proc. Natl. Acad. Sci., 85:2444-2448, 1988; and D.J. Lipman and W.R. Pearson "Fast and sensitive protein similarity search", Science, 227:1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers, and D. Lipman, "A Basic Local Alignment Search Tool", Journal of Molecular Biology, 215: 403-410 , 1990.
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。In this application, the term "comprises" generally refers to the meanings comprising, encompassing, comprising or encompassing. In some cases, it also means "for" and "consisting of".
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0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。In this application, the term "about" generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
发明详述Detailed description of the invention
分离的抗原结合蛋白isolated antigen binding protein
抗体的CDR又称互补决定区，是可变区的一部分。该区域的氨基酸残基可以与抗原或抗原表位接触。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、AbM、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。本领域技术人员可以根据抗体的序列和结构，用不同的编码系统确定出CDR区。使用不同的编码系统，CDR区可能存在差别。在本申请中，所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在某些实施方式中，所述CDR通过Chothia编码系统来划分。The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region may make contacts with the antigen or antigenic epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, comprehensive consideration of Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems. In this application, the CDR covers the CDR sequence divided according to any CDR division method; also covers its variants, the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids . For example 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or or insertions; homologues thereof, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences having about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology. In certain embodiments, the CDRs are divided by the Chothia coding system.
一方面，本申请提供了一种分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:26或38所示的氨基酸序列。In one aspect, the application provides an isolated antigen-binding protein comprising at least one CDR in the variable region VH of an antibody heavy chain, said VH comprising the amino acid sequence shown in SEQ ID NO: 26 or 38.
在本申请中，所述分离的抗原结合蛋白可以包含HCDR3，所述HCDR3可以包含SEQ ID NO:29所示的氨基酸序列。例如，所述分离的抗原结合蛋白的HCDR3可以包含SEQ ID NO:1所示的氨基酸序列。例如，所述分离的抗原结合蛋白的HCDR3可以包含SEQ ID NO:28所示的氨基酸序列。In the present application, the isolated antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:29. For example, the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:1. For example, the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:28.
在本申请中，所述分离的抗原结合蛋白可以包含HCDR2，所述HCDR2包含SEQ ID NO:37所示的氨基酸序列。例如，所述HCDR2包含SEQ ID NO:2、31或32所示的氨基酸序列。In the present application, the isolated antigen-binding protein may comprise HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:37. For example, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, 31 or 32.
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，所述HCDR1包含SEQ ID NO:36所示的氨基酸序列。例如，所述HCDR1包含SEQ ID NO:3或30所示的氨基酸序列。In the present application, the isolated antigen-binding protein may comprise HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:36. For example, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3 or 30.
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:36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:29所示的氨基酸序列。In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:36, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:37, And the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:29.
例如，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列。例如，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:28所示的氨基酸序列。例如，所述分离的抗原结合蛋白的所述HCDR1包含SEQ ID NO:30所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。例如，所述分离的抗原结合蛋白的所述HCDR1包含SEQ ID NO:3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:1所示的氨基酸序列。例如，所述分离的抗原结合蛋白的所述HCDR1包含SEQ ID NO:3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:1所示的氨基酸序列。For example, the HCDR1 of the isolated antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 1. For example, the HCDR1 of the isolated antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 28. For example, said HCDR1 of said isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:30, said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and said HCDR3 comprises SEQ ID NO:1 Amino acid sequence shown. For example, said HCDR1 of said isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:3, said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:31, and said HCDR3 comprises SEQ ID NO:1 Amino acid sequence shown. For example, said HCDR1 of said isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:3, said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:32, and said HCDR3 comprises SEQ ID NO:1 Amino acid sequence shown.
在本申请中，所述分离的抗原结合蛋白可以包含H-FR1，所述H-FR1的C端与所述HCDR1的N端直接或间接相连，且所述H-FR1可以包含SEQ ID NO:22所示的氨基酸序列。In the present application, the isolated antigen-binding protein may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise SEQ ID NO: The amino acid sequence shown in 22.
在本申请中，所述分离的抗原结合蛋白的H-FR1包含SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:11中任一项所示的氨基酸序列。In the present application, the H-FR1 of the isolated antigen-binding protein comprises the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 11.
在本申请中，所述分离的抗原结合蛋白可以包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2可以包含SEQ ID NO:23所示的氨基酸序列。In the present application, the isolated antigen binding protein may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise SEQ ID NO:23 amino acid sequence.
在本申请中，所述分离的抗原结合蛋白的H-FR2可以包含SEQ ID NO:5、SEQ ID NO:10、SEQ ID NO:12和SEQ ID NO:15中任一项所示的氨基酸序列。In the present application, the H-FR2 of the isolated antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:15 .
在本申请中，所述分离的抗原结合蛋白可以包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3可以包含SEQ ID NO:24所示的氨基酸序列。In the present application, the isolated antigen-binding protein may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise SEQ ID NO:24 amino acid sequence.
在本申请中，所述分离的抗原结合蛋白的H-FR3可以包含SEQ ID NO:6或SEQ ID NO:13所示的氨基酸序列。In the present application, the H-FR3 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:13.
在本申请中，所述分离的抗原结合蛋白可以包含H-FR4，所述H-FR4的N端与所述H-CDR3的C端直接或间接相连，且所述H-FR4可以包含SEQ ID NO:25所示的氨基酸序列。In the present application, the isolated antigen-binding protein may comprise H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the H-CDR3, and the H-FR4 may comprise SEQ ID The amino acid sequence shown in NO:25.
在本申请中，所述分离的抗原结合蛋白的H-FR4可以包含SEQ ID NO:7、SEQ ID NO:14和SEQ ID NO:16中任一项所示的氨基酸序列。In the present application, the H-FR4 of the isolated antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:14 and SEQ ID NO:16.
在本申请中，所述分离的抗原结合蛋白可以包含H-FR1、H-FR2、H-FR3和H-FR4。例如，所述分离的抗原结合蛋白的所述H-FR1可以包含SEQ ID NO:22所示的氨基酸序列，所 述H-FR2可以包含SEQ ID NO:23所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:24所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:25所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:4所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:7所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:8所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:7所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:7所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:11所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:12所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:13所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:14所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:11所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:15所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:13所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:16所示的氨基酸序列。In the present application, the isolated antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4. For example, the H-FR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 22, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 23, and the H- FR3 may comprise the amino acid sequence shown in SEQ ID NO:24, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:25. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:4, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:8, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13 The amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:14. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 15, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13 The amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:16.
在本申请中，所述分离的抗原结合蛋白的VH包含与SEQ ID NO:17所示的氨基酸相比，在以下一个或多个位置的氨基酸突变：H1，H13，H44，H45，H49，H74，H76，H79，H81，H82B，H83，H84，H108，所述位置由Chothia编码系统确定。例如，所述突变包含在位置H1由天冬氨酸D替换为谷氨酸E。例如，所述突变包含在位置H13由谷氨酸E替换为谷氨酰胺Q。例如，所述突变包含在位置H44由谷氨酸E替换为甘氨酸G。例如，所述突变包含在位置H45由精氨酸R替换为亮氨酸L。例如，所述突变包含在位置H49由丙氨酸A替换为丝氨酸S。例如，所述突变包含在位置H74由精氨酸R替换为丝氨酸S。例如，所述突变包含在位置H76由赖氨酸K替换为天冬酰胺N。例如，所述突变包含在位置H79由丝氨酸S替换为酪氨酸Y。例如，所述突变包含在位置H81由天冬氨酸D替换为谷氨酰胺Q。例如，所述突变包含在位置H82B由天冬氨酸D替换为丝氨酸S。例如，所述突变包含在位置H83由赖氨酸K替换为精氨酸R。例如，所述突变包含在位置H84由脯氨酸P替换为丙氨酸A。例如，所述突变包含在位置H108由谷氨酰胺Q替换为甲硫氨酸M。例如，所述突变包含在位置H108由谷氨酰胺Q替换为亮氨酸L。In the present application, the VH of the isolated antigen-binding protein comprises amino acid mutations at one or more of the following positions compared with the amino acid shown in SEQ ID NO: 17: H1, H13, H44, H45, H49, H74 , H76, H79, H81, H82B, H83, H84, H108, the positions are determined by the Chothia coding system. For example, the mutation comprises a substitution of aspartic acid D for glutamic acid E at position H1. For example, the mutation comprises a substitution of glutamic acid E by glutamine Q at position H13. For example, the mutation comprises a substitution of glutamic acid E for glycine G at position H44. For example, the mutation comprises a substitution of an arginine R for a leucine L at position H45. For example, the mutation comprises a substitution of alanine A for serine S at position H49. For example, the mutation comprises a substitution of arginine R for serine S at position H74. For example, the mutation comprises a substitution of lysine K for asparagine N at position H76. For example, the mutation comprises a substitution of serine S for tyrosine Y at position H79. For example, the mutation comprises a substitution of aspartic acid D for glutamine Q at position H81. For example, the mutation comprises a substitution of aspartic acid D for serine S at position H82B. For example, the mutation comprises a substitution of lysine K for arginine R at position H83. For example, the mutation comprises a substitution of proline P for alanine A at position H84. For example, the mutation comprises a substitution of glutamine Q for methionine M at position H108. For example, the mutation comprises a substitution of glutamine Q for leucine L at position H108.
在本申请中，所述分离的抗原结合蛋白可以包含VH，且所述VH可以包含SEQ ID NO:26或38所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:17所示的氨基酸序列。例 如，所述VH可以包含SEQ ID NO:18所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:19所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:20所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:21所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:33所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:34所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:35所示的氨基酸序列。In the present application, the isolated antigen-binding protein may comprise VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 26 or 38. For example, the VH can comprise the amino acid sequence shown in SEQ ID NO: 17. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 18. For example, the VH can comprise the amino acid sequence shown in SEQ ID NO: 19. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:20. For example, the VH can comprise the amino acid sequence shown in SEQ ID NO: 21. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:33. For example, the VH can comprise the amino acid sequence shown in SEQ ID NO:34. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:35.
在本申请中，所述分离的抗原结合蛋白可以包含抗体重链恒定区。在某些实施方式中，所述抗体重链恒定区源自人抗体重链恒定区。在某些实施方式中，所述抗体重链恒定区源自人IgG重链恒定区。在某些实施方式中，所述抗体重链恒定区源自人IgG1重链恒定区。在某些实施方式中，所述人IgG1重链恒定区包含人IgG1Fc。在某些实施方式中，所述人IgG1Fc包含N297A的氨基酸修饰。In the present application, the isolated antigen binding protein may comprise an antibody heavy chain constant region. In certain embodiments, the antibody heavy chain constant region is derived from a human antibody heavy chain constant region. In certain embodiments, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region. In certain embodiments, the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region. In certain embodiments, the human IgG1 heavy chain constant region comprises human IgG1 Fc. In certain embodiments, the human IgGl Fc comprises an amino acid modification of N297A.
在本申请中，所述分离的抗原结合蛋白可以包括抗体或其抗原结合片段。In the present application, the isolated antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof.
在本申请中，所述抗原结合片段可以包括Fab，Fab’，Fv片段，F(ab’) 2，scFv，di-scFv，dAb和/或V HH。在某些实施方式中，所述抗原结合片段为V HH。 In the present application, the antigen-binding fragment may include Fab, Fab', Fv fragment, F(ab') 2 , scFv, di-scFv, dAb and/or VHH . In certain embodiments, the antigen-binding fragment is a VHH .
在本申请中，所述抗体可以选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。In the present application, the antibody may be selected from the group consisting of monoclonal antibody, chimeric antibody, humanized antibody and fully human antibody.
在本申请中，所述分离的抗原结合蛋白可以包含单域抗体。例如，本申请所述的单域抗体包含在本申请的实施例中制备的编号为Ab0716.1、Ab0716.2、Ab0716.4、Ab0716.Hz1和/或Ab0716.Hz2的单域抗体。例如，本申请所述的单域抗体包含在本申请的实施例中制备的编号为Ab0716Am01、Ab0716Am02、或Ab0716Am06的单域抗体。In the present application, the isolated antigen binding protein may comprise a single domain antibody. For example, the single domain antibodies described in the present application include the single domain antibodies numbered Ab0716.1, Ab0716.2, Ab0716.4, Ab0716.Hz1 and/or Ab0716.Hz2 prepared in the examples of the present application. For example, the single domain antibody described in the present application includes the single domain antibody numbered Ab0716Am01, Ab0716Am02, or Ab0716Am06 prepared in the examples of the present application.
在本申请中，所述单域抗体可以包含HCDR3，且所述HCDR3可以包含SEQ ID NO:29所示的氨基酸序列。例如，所述单域抗体的HCDR3可以包含SEQ ID NO:1所示的氨基酸序列。例如，所述单域抗体的HCDR3可以包含SEQ ID NO:28所示的氨基酸序列。In the present application, the single domain antibody may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:29. For example, the HCDR3 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:1. For example, the HCDR3 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:28.
在本申请中，所述单域抗体可以包含HCDR2，且所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列。In the present application, the single domain antibody may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2.
在本申请中，所述单域抗体可以包含HCDR1，且所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列。In the present application, the single domain antibody may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3.
在本申请中，所述单域抗体可以包含HCDR1，HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:36所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:37所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:29所示的氨基酸序列。例如，所述单域抗体的HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所 示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列。例如，所述单域抗体的HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:28所示的氨基酸序列。例如，所述单域抗体的所述HCDR1包含SEQ ID NO:30所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。例如，所述单域抗体的所述HCDR1包含SEQ ID NO:3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:1所示的氨基酸序列。例如，所述单域抗体的所述HCDR1包含SEQ ID NO:3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:1所示的氨基酸序列。In the present application, the single domain antibody may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:36, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:37, And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:29. For example, the HCDR1 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1 amino acid sequence. For example, the HCDR1 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:28 amino acid sequence. For example, the HCDR1 of the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:30, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1 amino acid sequence. For example, the HCDR1 of the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:31, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1 amino acid sequence. For example, the HCDR1 of the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:32, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1 amino acid sequence.
在本申请中，所述单域抗体可以包含H-FR1，所述H-FR1的C端与所述HCDR1的N端直接或间接相连，且所述H-FR1可以包含SEQ ID NO:22所示的氨基酸序列。例如，所述单域抗体的H-FR1可以包含SEQ ID NO:4所示的氨基酸序列。例如，所述单域抗体的H-FR1可以包含SEQ ID NO:8所示的氨基酸序列。例如，所述单域抗体的H-FR1可以包含SEQ ID NO:9所示的氨基酸序列。例如，所述单域抗体的H-FR1可以包含SEQ ID NO:11所示的氨基酸序列。In the present application, the single domain antibody may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may comprise SEQ ID NO:22 The amino acid sequence shown. For example, the H-FR1 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:4. For example, the H-FR1 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:8. For example, the H-FR1 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:9. For example, the H-FR1 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO: 11.
在本申请中，所述单域抗体可以包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2可以包含SEQ ID NO:23所示的氨基酸序列。例如，所述单域抗体的H-FR2可以包含SEQ ID NO:5所示的氨基酸序列。例如，所述单域抗体的H-FR2可以包含SEQ ID NO:10所示的氨基酸序列。例如，所述单域抗体的H-FR2可以包含SEQ ID NO:12所示的氨基酸序列。例如，所述单域抗体的H-FR2可以包含SEQ ID NO:15所示的氨基酸序列。In the present application, the single domain antibody may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:23 . For example, the H-FR2 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:5. For example, the H-FR2 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:10. For example, the H-FR2 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO: 12. For example, the H-FR2 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:15.
在本申请中，所述单域抗体可以包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3可以包含SEQ ID NO:24所示的氨基酸序列。例如，所述单域抗体的H-FR3可以包含SEQ ID NO:6所示的氨基酸序列。例如，所述单域抗体的H-FR3可以包含SEQ ID NO:13所示的氨基酸序列。In the present application, the single domain antibody may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:24 . For example, the H-FR3 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:6. For example, the H-FR3 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO: 13.
在本申请中，所述单域抗体可以包含H-FR4，所述H-FR4的N端与所述H-CDR3的C端直接或间接相连，且所述H-FR4可以包含SEQ ID NO:25所示的氨基酸序列。例如，所述单域抗体的H-FR4可以包含SEQ ID NO:7所示的氨基酸序列。例如，所述单域抗体的H-FR4可以包含SEQ ID NO:14所示的氨基酸序列。例如，所述单域抗体的H-FR4可以包含SEQ ID NO:16所示的氨基酸序列。In the present application, the single domain antibody may comprise H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the H-CDR3, and the H-FR4 may comprise SEQ ID NO: The amino acid sequence shown in 25. For example, the H-FR4 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:7. For example, the H-FR4 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO:14. For example, the H-FR4 of the single domain antibody may comprise the amino acid sequence shown in SEQ ID NO: 16.
在本申请中，所述单域抗体可以包含H-FR1，H-FR2，H-FR3和H-FR4。例如，所述H- FR1可以包含SEQ ID NO:4所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:7所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:8所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:7所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:7所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:11所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:12所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:13所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:14所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:11所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:15所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:13所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:16所示的氨基酸序列。In the present application, the single domain antibody may comprise H-FR1, H-FR2, H-FR3 and H-FR4. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:4, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:8, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13 The amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:14. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 15, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13 The amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:16.
在本申请中，所述单域抗体可以包含V HH，所述V HH可以包含SEQ ID NO:26或38所示的氨基酸序列。例如，所述V HH可以包含SEQ ID NO:17所示的氨基酸序列。例如，所述V HH可以包含SEQ ID NO:18所示的氨基酸序列。例如，所述V HH可以包含SEQ ID NO:19所示的氨基酸序列。例如，所述V HH可以包含SEQ ID NO:20所示的氨基酸序列。例如，所述V HH可以包含SEQ ID NO:21所示的氨基酸序列。例如，所述V HH可以包含SEQ ID NO:33所示的氨基酸序列。例如，所述V HH可以包含SEQ ID NO:34所示的氨基酸序列。例如，所述V HH可以包含SEQ ID NO:35所示的氨基酸序列。 In the present application, the single domain antibody may comprise a VHH , and the VHH may comprise the amino acid sequence shown in SEQ ID NO:26 or 38. For example, the VHH may comprise the amino acid sequence shown in SEQ ID NO:17. For example, the VHH may comprise the amino acid sequence shown in SEQ ID NO:18. For example, the VHH may comprise the amino acid sequence shown in SEQ ID NO:19. For example, the VHH may comprise the amino acid sequence shown in SEQ ID NO:20. For example, the VHH may comprise the amino acid sequence shown in SEQ ID NO:21. For example, the VHH may comprise the amino acid sequence shown in SEQ ID NO:33. For example, the VHH may comprise the amino acid sequence shown in SEQ ID NO:34. For example, the VHH may comprise the amino acid sequence shown in SEQ ID NO:35.
例如，在本申请中，单域抗体Ab0716.1的所述HCDR1的氨基酸序列如SEQ ID NO:3所示，所述HCDR2的氨基酸序列如SEQ ID NO:2所示，所述HCDR3的氨基酸序列如SEQ ID NO:1所示，所述H-FR1的氨基酸序列如SEQ ID NO:4所示，所述H-FR2的氨基酸序列如SEQ ID NO:5所示，所述H-FR3的氨基酸序列如SEQ ID NO:6所示，且所述H-FR4的氨基酸序列如SEQ ID NO:7所示。例如，所述单域抗体Ab0716.1的V HH的氨基酸序列如SEQ ID NO:17所示。 For example, in this application, the amino acid sequence of the HCDR1 of the single domain antibody Ab0716.1 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR3 As shown in SEQ ID NO: 1, the amino acid sequence of the H-FR1 is shown in SEQ ID NO: 4, the amino acid sequence of the H-FR2 is shown in SEQ ID NO: 5, the amino acid sequence of the H-FR3 The sequence is shown in SEQ ID NO:6, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:7. For example, the amino acid sequence of the VHH of the single domain antibody Ab0716.1 is shown in SEQ ID NO:17.
例如，在本申请中，单域抗体Ab0716.2的所述HCDR1的氨基酸序列如SEQ ID NO:3所示，所述HCDR2的氨基酸序列如SEQ ID NO:2所示，所述HCDR3的氨基酸序列如SEQ ID NO:28所示，所述H-FR1的氨基酸序列如SEQ ID NO:8所示，所述H-FR2的氨基酸序列如SEQ ID NO:5所示，所述H-FR3的氨基酸序列如SEQ ID NO:6所示，所述H-FR4的氨基酸 序列如SEQ ID NO:7所示。例如，所述单域抗体Ab0716.2的V HH的氨基酸序列如SEQ ID NO:18所示。 For example, in this application, the amino acid sequence of the HCDR1 of the single domain antibody Ab0716.2 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR3 As shown in SEQ ID NO: 28, the amino acid sequence of the H-FR1 is shown in SEQ ID NO: 8, the amino acid sequence of the H-FR2 is shown in SEQ ID NO: 5, the amino acid sequence of the H-FR3 The sequence is shown in SEQ ID NO:6, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:7. For example, the amino acid sequence of the VHH of the single domain antibody Ab0716.2 is shown in SEQ ID NO:18.
例如，在本申请中，单域抗体Ab0716.4的所述HCDR1的氨基酸序列如SEQ ID NO:3所示，所述HCDR2的氨基酸序列如SEQ ID NO:2所示，所述HCDR3的氨基酸序列如SEQ ID NO:1所示，所述H-FR1的氨基酸序列如SEQ ID NO:9所示，所述H-FR2的氨基酸序列如SEQ ID NO:10所示，所述H-FR3的氨基酸序列如SEQ ID NO:6所示，且所述H-FR4的氨基酸序列如SEQ ID NO:7所示。例如，所述单域抗体Ab0716.4的V HH的氨基酸序列如SEQ ID NO:19所示。 For example, in this application, the amino acid sequence of the HCDR1 of the single domain antibody Ab0716.4 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR3 As shown in SEQ ID NO: 1, the amino acid sequence of the H-FR1 is shown in SEQ ID NO: 9, the amino acid sequence of the H-FR2 is shown in SEQ ID NO: 10, the amino acid sequence of the H-FR3 The sequence is shown in SEQ ID NO:6, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:7. For example, the amino acid sequence of the VHH of the single domain antibody Ab0716.4 is shown in SEQ ID NO:19.
例如，在本申请中，单域抗体Ab0716.Hz1的所述HCDR1的氨基酸序列如SEQ ID NO:3所示，所述HCDR2的氨基酸序列如SEQ ID NO:2所示，所述HCDR3的氨基酸序列如SEQ ID NO:1所示，所述H-FR1的氨基酸序列如SEQ ID NO:11所示，所述H-FR2的氨基酸序列如SEQ ID NO:12所示，所述H-FR3的氨基酸序列如SEQ ID NO:13所示，且所述H-FR4的氨基酸序列如SEQ ID NO:14所示。例如，在本申请中，所述单域抗体Ab0716.Hz1的V HH的氨基酸序列如SEQ ID NO:20所示。 For example, in this application, the amino acid sequence of the HCDR1 of the single domain antibody Ab0716.Hz1 is shown in SEQ ID NO:3, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of the HCDR3 As shown in SEQ ID NO: 1, the amino acid sequence of the H-FR1 is shown in SEQ ID NO: 11, the amino acid sequence of the H-FR2 is shown in SEQ ID NO: 12, the amino acid sequence of the H-FR3 The sequence is shown in SEQ ID NO:13, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:14. For example, in this application, the amino acid sequence of the VHH of the single domain antibody Ab0716.Hz1 is shown in SEQ ID NO:20.
例如，在本申请中，单域抗体Ab0716.Hz2的所述HCDR1的氨基酸序列如SEQ ID NO:3所示，所述HCDR2的氨基酸序列如SEQ ID NO:2所示，所述HCDR3的氨基酸序列如SEQ ID NO:1所示，所述H-FR1的氨基酸序列如SEQ ID NO:11所示，所述H-FR2的氨基酸序列如SEQ ID NO:15所示，所述H-FR3的氨基酸序列如SEQ ID NO:13所示，且所述H-FR4的氨基酸序列如SEQ ID NO:16所示。例如，在本申请中，所述单域抗体Ab0716.Hz2的V HH的氨基酸序列如SEQ ID NO:21所示。 For example, in this application, the amino acid sequence of the HCDR1 of the single domain antibody Ab0716.Hz2 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the HCDR3 As shown in SEQ ID NO: 1, the amino acid sequence of the H-FR1 is shown in SEQ ID NO: 11, the amino acid sequence of the H-FR2 is shown in SEQ ID NO: 15, the amino acid sequence of the H-FR3 The sequence is shown in SEQ ID NO:13, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:16. For example, in this application, the amino acid sequence of the VHH of the single domain antibody Ab0716.Hz2 is shown in SEQ ID NO:21.
例如，所述单域抗体Ab0716Am01的所述HCDR1包含SEQ ID NO:30所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。For example, the HCDR1 of the single domain antibody Ab0716Am01 includes the amino acid sequence shown in SEQ ID NO:30, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:1 The amino acid sequence shown.
例如，所述单域抗体Ab0716Am02的所述HCDR1包含SEQ ID NO:3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:1所示的氨基酸序列。For example, the HCDR1 of the single domain antibody Ab0716Am02 includes the amino acid sequence shown in SEQ ID NO:3, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:31, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:1 The amino acid sequence shown.
例如，所述单域抗体Ab0716Am06的所述HCDR1包含SEQ ID NO:3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:1所示的氨基酸序列。For example, the HCDR1 of the single domain antibody Ab0716Am06 includes the amino acid sequence shown in SEQ ID NO:3, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:32, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:1 The amino acid sequence shown.
在本申请中，所述分离的抗原结合蛋白能够结合人血清白蛋白。例如，本申请所述抗原 结合蛋白可以以小于或等于2.0×10 -9M，小于或等于1.9×10 -9M，小于或等于1.8×10 -9M，小于或等于1.7×10 -9M，小于或等于1.6×10 -9M，小于或等于1.5×10 -9M，小于或等于1.4×10 -9M，小于或等于1.3×10 -9M，小于或等于1.2×10 -9M，小于或等于1.1×10 -9M，小于或等于1.0×10 -9M，小于或等于9×10 -10M，小于或等于8×10 -10M，小于或等于7×10 -10M，小于或等于6×10 -10M，小于或等于5×10 -10M，小于或等于4.15×10 -10M，小于或等于4×10 -10M，小于或等于3.93×10 -10M，小于或等于3×10 -10M，小于或等于2×10 -10M的KD值与HSA结合。 In the present application, the isolated antigen binding protein is capable of binding human serum albumin. For example, the antigen binding protein described in the present application can be in the form of less than or equal to 2.0×10 -9 M, less than or equal to 1.9×10 -9 M, less than or equal to 1.8×10 -9 M, less than or equal to 1.7×10 -9 M , less than or equal to 1.6×10 -9 M, less than or equal to 1.5×10 -9 M, less than or equal to 1.4×10 -9 M, less than or equal to 1.3×10 -9 M, less than or equal to 1.2×10 -9 M , less than or equal to 1.1×10 -9 M, less than or equal to 1.0×10 -9 M, less than or equal to 9×10 -10 M, less than or equal to 8×10 -10 M, less than or equal to 7×10 -10 M , less than or equal to 6×10 -10 M, less than or equal to 5×10 -10 M, less than or equal to 4.15×10 -10 M, less than or equal to 4×10 -10 M, less than or equal to 3.93×10 -10 M , less than or equal to 3×10 -10 M, the KD value of less than or equal to 2×10 -10 M binds to HSA.
在本申请中，所述分离的抗原结合蛋白的Tm值能够大于或等于59.5℃，大于或等于59.6℃，大于或等于59.7℃，大于或等于59.8℃，大于或等于59.9℃，大于或等于60.0℃，大于或等于60.1℃，大于或等于60.2℃，大于或等于60.5℃，大于或等于61.0℃，大于或等于61.5℃，大于或等于62℃，大于或等于62.5℃，大于或等于62.9℃，大于或等于63℃，大于或等于63.5℃，大于或等于64℃。In the present application, the Tm value of the isolated antigen-binding protein can be greater than or equal to 59.5°C, greater than or equal to 59.6°C, greater than or equal to 59.7°C, greater than or equal to 59.8°C, greater than or equal to 59.9°C, greater than or equal to 60.0 ℃, greater than or equal to 60.1°C, greater than or equal to 60.2°C, greater than or equal to 60.5°C, greater than or equal to 61.0°C, greater than or equal to 61.5°C, greater than or equal to 62°C, greater than or equal to 62.5°C, greater than or equal to 62.9°C, Greater than or equal to 63°C, greater than or equal to 63.5°C, greater than or equal to 64°C.
融合蛋白、核酸分子、载体、细胞和药物组合物Fusion proteins, nucleic acid molecules, vectors, cells and pharmaceutical compositions
另一方面，本申请提供了融合蛋白，其可以包含本申请所述的抗原结合蛋白。In another aspect, the application provides a fusion protein, which may comprise the antigen binding protein described in the application.
另一方面，本申请提供了分离的核酸分子，其可以编码本申请所述的分离的抗原结合蛋白。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的；(ii)通过克隆重组产生的；(iii)纯化的，例如通过酶切和凝胶电泳分级分离；或者(iv)合成的，例如通过化学合成。In another aspect, the application provides isolated nucleic acid molecules that encode the isolated antigen binding proteins described herein. For example, it may be produced or synthesized by: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification; (ii) recombinantly produced by cloning; (iii) purified or (iv) synthetic, such as by chemical synthesis.
另一方面，本申请提供了一种载体，其可以包含本申请所述的核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。所述载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。In another aspect, the present application provides a vector, which may comprise the nucleic acid molecule described in the present application. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may also contain expression control elements that permit proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like. The vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries can be expressed in the host cell. Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector may also include components that facilitate its entry into cells, such as, but not exclusively, viral particles, liposomes or protein coats.
另一方面，本申请提供了一种细胞，其可以包含本申请所述的核酸分子或本申请所述的载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例 如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如真核细胞，如来自植物的细胞、真菌或酵母细胞等。在某些实施方式中，所述细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、CHO-K1细胞、LNCAP细胞、HeLa细胞、293T细胞、COS-1细胞、SP2/0细胞、NS0细胞或骨髓瘤细胞。In another aspect, the present application provides a cell, which may comprise the nucleic acid molecule or the vector described in the present application. In certain embodiments, each or each host cell may comprise one or more of the nucleic acid molecules or vectors described herein. In certain embodiments, each or each host cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into the host cells, such as eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like. In certain embodiments, the cells may be bacterial cells (e.g., E. coli), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2/0 cells, NSO cells or myeloma cells.
另一方面，本申请还提供了药物组合物，其可以包含所述分离的抗原结合蛋白，所述核酸分子、所述载体和/或所述细胞，以及任选地药学上可接受的载剂。On the other hand, the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable carrier .
在某些实施方案中，所述药物组合物还可以包含一种或多种(药学上有效的)佐剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下通常对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。In certain embodiments, the pharmaceutical composition may further comprise one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or or a suitable formulation of preservatives. Acceptable ingredients of the compositions are generally nontoxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
在某些实施方案中，所述药物组合物还可含有多于一种活性化合物，通常为不会不利地影响彼此的具有互补活性的那些活性化合物。此类药物的类型和有效量可以取决于例如制剂中存在的拮抗剂的量和类型，以及受试者的临床参数。In certain embodiments, the pharmaceutical compositions may also contain more than one active compound, generally those with complementary activities that do not adversely affect each other. The type and effective amount of such drug may depend, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.
在某些实施方案中，所述药学上可接受的载剂可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂，通常安全、无毒。In certain embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents compatible with pharmaceutical administration, generally safe, nontoxic .
在某些实施方案中，所述药物组合物可以包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述药物组合物可以通过输注或注射施用于患者或者受试者。在某些实施方案中，所述药物组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。在某些实施方案中，所述药物组合物可以不间断施用。所述不间断(或连续)施用可以通过患者佩戴的小泵系统来实现，以测量流入患者体内的治疗剂，如WO2015/036583所述。In certain embodiments, the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition can be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition can be administered without interruption. Such uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the influx of the therapeutic agent into the patient, as described in WO2015/036583.
制备方法Preparation
另一方面，本申请提供了制备所述分离的抗原结合蛋白的方法。所述方法可包括，在使得所述的抗原结合蛋白表达的条件下，培养所述本申请所述的宿主细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。In another aspect, the present application provides methods for preparing the isolated antigen-binding protein. The method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed. For example, by using appropriate medium, appropriate temperature and incubation time, etc., these methods are understood by those of ordinary skill in the art.
任何适于产生单克隆抗体的方法都可用于产生本申请的抗原结合蛋白。例如，通过纳米抗体噬菌体文库淘选、扩增，构建酵母展示文库筛选所述单域抗体。Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application. For example, by panning and amplifying the nanobody phage library, constructing a yeast display library to screen the single domain antibody.
在本申请中，所述分离的抗原结合蛋白可以通过以下方法制备：选用纳米抗体噬菌体文库针对生物素标记的HSA进行淘选。以淘选后的质粒为模板扩增单域抗体，将扩增的片段与 酵母展示质粒共转入酿酒酵母，在酵母细胞壁表面展示单链抗体。In this application, the isolated antigen-binding protein can be prepared by selecting a Nanobody phage library for panning against biotin-labeled HSA. Using the panned plasmid as a template to amplify the single-chain antibody, the amplified fragment and the yeast display plasmid are co-transfected into Saccharomyces cerevisiae, and the single-chain antibody is displayed on the surface of the yeast cell wall.
例如，本申请提供了一种用于检测或测定人血清白蛋白的方法，所述方法包括使用本申请的分离的抗原结合蛋白或本申请的融合蛋白。在本申请中，所述方法可包括体外方法，离体方法，非诊断或非治疗目的的方法。For example, the present application provides a method for detecting or measuring human serum albumin, the method comprising using the isolated antigen-binding protein of the present application or the fusion protein of the present application. In this application, the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
另一方面，本申请提供了一种人血清白蛋白的试剂盒，其可包括使用所述分离的抗原结合蛋白或所述的融合蛋白。In another aspect, the present application provides a human serum albumin kit, which may include the use of the isolated antigen-binding protein or the fusion protein.
在本申请中，所述试剂盒还可包含使用说明，所述使用说明记载用于检测人血清白蛋白的存在和/或含量的方法。例如，所述方法可包括体外方法，离体方法，非诊断或非治疗目的的方法。In the present application, the kit may further include instructions for use, which describe the method for detecting the presence and/or content of human serum albumin. For example, the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
另一方面，本申请提供了一种所述的分离的抗原结合蛋白或所述的融合蛋白在制备试剂盒中的用途，所述试剂盒可用于检测人血清白蛋白的存在和/或含量的方法。例如，所述方法可包括体外方法，离体方法，非诊断或非治疗目的的方法。In another aspect, the present application provides a use of the isolated antigen-binding protein or the fusion protein in the preparation of a kit, which can be used to detect the presence and/or content of human serum albumin method. For example, the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
另一方面，本申请提供了所述的分离的抗原结合蛋白，所述的融合蛋白，所述的核酸分子、所述的载体、所述的细胞、所述的药物组合物和/或所述的试剂盒用于预防、缓解和/或治疗疾病或病症。In another aspect, the application provides the isolated antigen-binding protein, the fusion protein, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and/or the The kits are used for preventing, alleviating and/or treating a disease or condition.
另一方面，本申请提供了一种所述的分离的抗原结合蛋白，所述的融合蛋白，所述的核酸分子、所述的载体、所述的细胞、所述的药物组合物和/或所述的试剂盒在制备药物中的用途，所述药物用于预防、缓解和/或治疗疾病或病症。In another aspect, the present application provides a kind of said isolated antigen-binding protein, said fusion protein, said nucleic acid molecule, said carrier, said cell, said pharmaceutical composition and/or Use of the kit in the preparation of medicaments for preventing, alleviating and/or treating diseases or conditions.
另一方面，本申请提供了一种预防和/或治疗疾病或病症的方法，其包括向有需要的受试者施用所述的分离的抗原结合蛋白，所述的融合蛋白，所述的核酸分子、所述的载体、所述的细胞、所述的药物组合物和/或所述的试剂盒。In another aspect, the present application provides a method for preventing and/or treating a disease or disorder, which comprises administering the isolated antigen-binding protein, the fusion protein, the nucleic acid to a subject in need The molecule, the carrier, the cell, the pharmaceutical composition and/or the kit.
本申请所述药物组合物、药物组合及方法可与其他类型的癌症疗法结合使用，诸如化学疗法、手术、放射、基因疗法等。本申请中所描述的药物组合物及方法可用于其他依赖于免疫反应的疾病病状。The pharmaceutical compositions, pharmaceutical combinations and methods described herein may be used in conjunction with other types of cancer therapy, such as chemotherapy, surgery, radiation, gene therapy, and the like. The pharmaceutical compositions and methods described in this application can be used in other disease conditions that depend on immune responses.
在本申请中，所述受试者可以包括人或非人动物。例如，所述非人动物可以选自下组：猴、鸡、鹅、猫、狗、小鼠和大鼠。此外，非人动物也可以包括任何除人以外的动物物种，例如家畜动物，或啮齿类动物，或灵长类动物，或家养动物，或家禽动物。所述人可以是高加索人、非洲人、亚洲人、闪族人，或其他种族，或各种种族的杂合体。又例如，所述人可以是老年、成年、青少年、儿童或者婴儿。In the present application, the subject may include humans or non-human animals. For example, the non-human animal can be selected from the group consisting of monkeys, chickens, geese, cats, dogs, mice and rats. In addition, non-human animals may also include any animal species other than humans, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals. The human can be Caucasian, African, Asian, Semitic, or other ethnicity, or a hybrid of various ethnicities. As another example, the human can be elderly, adult, adolescent, child or infant.
可以根据在实验动物中的有效量推测在人类中的有效量。例如，Freireich等人描述了动 物和人的剂量的相互关系(基于每平方米身体表面的毫克数)(Freireich et al.，Cancer Chemother.Rep.50，219(1966))。身体表面积可以从患者的身高和体重近似确定。参见例如Scientific Tables，Geigy Pharmaceuticals，Ardsley，N.Y.，537(1970)。The effective amount in humans can be inferred from the effective amount in experimental animals. For example, Freireich et al. describe the correlation of doses in animals and humans (based on milligrams per square meter of body surface) (Freireich et al., Cancer Chemother. Rep. 50, 219 (1966)). Body surface area can be approximately determined from the patient's height and weight. See, eg, Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970).
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抗原结合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。Without intending to be limited by any theory, the following examples are only for explaining the antigen-binding protein of the present application, the preparation method and application, etc., and are not intended to limit the scope of the invention of the present application.
实施例Example
实施例1酵母展示文库构建与筛选Example 1 Yeast Display Library Construction and Screening
选用自建的纳米抗体噬菌体文库，针对生物素标记的人血清白蛋白(以下简称HSA-Biotin，acro，货号：HSA-H82E3)进行两轮淘选，获得阳性富集。以噬菌体两轮淘选后质粒为模板，设计引物进行聚合酶链式反应(PCR)扩增单链抗体(V HH)；PCR扩增的V HH基因片段回收后与酵母展示质粒共转入酿酒酵母菌株EBY100(购自ATCC)，通过酿酒酵母的同源重组使V HH基因插入至酵母展示质粒中，进而实现在酵母细胞壁表面展示单链抗体，如图1。酵母展示单链抗体库命名为JYYDL032。文库JYYDL032电转后在100mL的SD-Trp培养基(Clontech，货号：630308)，30℃、225转/分钟培养过夜；取1.0×10 8细胞重悬于20mL YPGP液体培养基(2％半乳糖，2％蛋白胨，1％酵母提取物，0.54％Na 2HPO 4，0.86％NaH 2PO 4·H 2O)，20℃、225转/分钟培养24小时，置于4℃冰箱待用。 A self-built nanobody phage library was selected to perform two rounds of panning against biotin-labeled human serum albumin (hereinafter referred to as HSA-Biotin, acro, catalog number: HSA-H82E3) to obtain positive enrichment. Using the plasmid after two rounds of phage panning as a template, design primers to amplify the single-chain antibody (V H H) by polymerase chain reaction (PCR); the PCR-amplified V H H gene fragment is recovered and co-transfected with the yeast display plasmid Saccharomyces cerevisiae strain EBY100 (purchased from ATCC), the V H H gene was inserted into the yeast display plasmid through homologous recombination of Saccharomyces cerevisiae, and then the single-chain antibody was displayed on the surface of the yeast cell wall, as shown in Figure 1. The yeast-displayed single-chain antibody library was named JYYDL032. After electroporation, library JYYDL032 was cultured overnight in 100 mL SD-Trp medium (Clontech, product number: 630308) at 30°C and 225 rpm; 1.0×108 cells were resuspended in 20 mL YPGP liquid medium (2% galactose, 2% peptone, 1% yeast extract, 0.54% Na 2 HPO 4 , 0.86% NaH 2 PO 4 ·H 2 O), cultured at 20°C, 225 rpm for 24 hours, and placed in a 4°C refrigerator for use.
文库诱导后菌液，测定菌液的OD 600，按1OD为1.0×10 7细胞数计算，取1.0×10 8细胞，用磁珠分选系统进行第一轮富集：1.用20mL 1×PBSA(1×PBS+1％BSA)洗涤一次，3000转/分钟离心3分钟(以下离心均为此条件)弃上清；2.与50mL含1nM HSA-Biotin的1×PBSA，室温孵育30分钟；3.洗涤后加入抗生物素的磁珠(miltenyi，货号：130-090-485)混匀孵育10min，过磁力柱(Quadro MACS Starting Kit)收集阳性细胞。 After library induction, measure the OD 600 of the bacterial liquid, and calculate the number of 1.0×10 7 cells based on 1OD, take 1.0×10 8 cells, and use the magnetic bead sorting system for the first round of enrichment: 1. Use 20mL 1× Wash once with PBSA (1×PBS+1%BSA), centrifuge at 3000 rpm for 3 minutes (this is the condition for centrifugation below) and discard the supernatant; 2. Incubate with 50mL 1×PBSA containing 1nM HSA-Biotin for 30 minutes at room temperature 3. Add anti-biotin magnetic beads (miltenyi, product number: 130-090-485) after washing, mix and incubate for 10 min, and collect positive cells through a magnetic column (Quadro MACS Starting Kit).
阳性细胞经过再次培养、诱导后，取4.0×10 7细胞进行第二轮流式分选：1)用1mL 1×PBSA，离心弃上清；2)与1mL含1nM HSA-Biotin及鼠抗V5抗体(Invitrogen，货号2156578，1:1000稀释)的1×PBSA冰上孵育30min；3)离心弃上清，加入1mL 1×PBSA洗涤一次；4)加入200μL含荧光抗体的1×PBSA(SA-PE厂家eBioscience，货号：12-4317-8，按1:200稀释；羊抗鼠-647厂家Invitrogen，货号：A21235，按1:400稀释)，避光冰上孵育20分钟；5)重复步骤3，加入2mL 1×PBSA重悬细胞，通过流式分选仪器收集647荧光信号与PE荧光信号均强的细胞群。分选后取部分细胞涂布于SD-Trp固体培养基(Clontech，货号：630309)平板，30℃静置培养3天。 After the positive cells were cultured and induced again, 4.0×10 7 cells were taken for the second round sorting: 1) with 1mL 1×PBSA, centrifuge and discard the supernatant; 2) with 1mL containing 1nM HSA-Biotin and mouse anti-V5 antibody (Invitrogen, Cat. No. 2156578, diluted 1:1000) in 1×PBSA for 30 minutes; 3) Centrifuge to discard the supernatant, add 1 mL 1×PBSA to wash once; 4) Add 200 μL of 1×PBSA (SA-PE Manufacturer eBioscience, product number: 12-4317-8, diluted 1:200; goat anti-mouse-647 manufacturer Invitrogen, product number: A21235, diluted 1:400), incubate on ice for 20 minutes in the dark; 5) Repeat step 3, Add 2 mL of 1×PBSA to resuspend the cells, and collect the cell population with strong 647 fluorescence signal and PE fluorescence signal by flow cytometry. After sorting, some cells were spread on SD-Trp solid medium (Clontech, product number: 630309) plate, and cultured statically at 30°C for 3 days.
实施例2单克隆酵母菌落测序与流式染色鉴定Example 2 Monoclonal Yeast Colony Sequencing and Flow Staining Identification
JYYDL032第二轮筛选产物，涂布于SD-Trp平板，30℃静置培养3天生长出酵母单克隆菌落。挑取92个单克隆进行测序分析，最终获得3个独一的单链抗体序列(表1)。将对照抗体的序列(源自Ablynx，ALX-0761，氨基酸序列见SEQ ID NO:27)展示于酵母表面，作为阳性对照抗体，如表2对相应的酵母单克隆菌落进行流式染色分析，各取1×10 6个细胞按表2方案进行染色评估：方案1评估各克隆与HSA-Biotin结合水平，PE平均荧光信号强度(MFI)越强代表结合能力越强；方案2评估各克隆与无关抗原的非特异性结合水平，PE的MFI值越低说明非特异性越弱；方案3评估各克隆与对照抗体(内部制备)的竞争信号，APC的MFI值越低说明竞争性越强。 The second-round screening product of JYYDL032 was spread on the SD-Trp plate and cultured statically at 30°C for 3 days to grow a yeast monoclonal colony. 92 single clones were picked for sequencing analysis, and finally 3 unique single-chain antibody sequences were obtained (Table 1). The sequence of the control antibody (derived from Ablynx, ALX-0761, see SEQ ID NO: 27 for the amino acid sequence) was displayed on the surface of the yeast as a positive control antibody, as shown in Table 2. The corresponding yeast monoclonal colonies were analyzed by flow staining, each Take 1× 106 cells for staining evaluation according to the scheme in Table 2: Scheme 1 evaluates the binding level of each clone to HSA-Biotin, and the stronger the mean fluorescence signal intensity (MFI) of PE represents the stronger binding ability; Scheme 2 evaluates the binding ability of each clone to HSA-Biotin. For the non-specific binding level of antigen, the lower the MFI value of PE, the weaker the non-specificity; Scheme 3 evaluates the competition signal between each clone and the control antibody (prepared in-house), and the lower the MFI value of APC, the stronger the competition.
表1单链抗体序列测定Table 1 Single-chain antibody sequence determination
抗体编号Antibody number 氨基酸序列(SEQ ID NO：)Amino acid sequence (SEQ ID NO: )
Ab0716.1Ab0716.1 SEQ ID NO:17SEQ ID NO:17
Ab0716.2Ab0716.2 SEQ ID NO:18SEQ ID NO:18
Ab0716.4Ab0716.4 SEQ ID NO:19SEQ ID NO:19
表2单克隆酵母菌落流式染色鉴定方案Table 2 Identification scheme of monoclonal yeast colonies by flow cytometry staining
Figure PCTCN2022097052-appb-000001
Figure PCTCN2022097052-appb-000001
根据各克隆按方案1、2、3的染色结果，综合各克隆序列的相似性，最终挑取克隆Y24A2、Y24A5、Y24B2的序列进行抗体表达，如表3。According to the staining results of each clone according to schemes 1, 2, and 3, and considering the similarity of the sequences of each clone, the sequences of clones Y24A2, Y24A5, and Y24B2 were finally selected for antibody expression, as shown in Table 3.
表3 JYYDL032文库二轮筛选后酵母单克隆菌落流式染色分析结果Table 3 Flow cytometric analysis results of yeast monoclonal colonies after the second round of screening of the JYYDL032 library
Figure PCTCN2022097052-appb-000002
Figure PCTCN2022097052-appb-000002
Figure PCTCN2022097052-appb-000003
Figure PCTCN2022097052-appb-000003
实施例3候选抗体表达Example 3 Candidate antibody expression
将各克隆的V HH序列与IgG1Fc N297A融合，进行密码子优化，基因合成后装入表达载体pcDNA3.4(Life Technologies)。表达质粒扩增和质粒抽提后质粒转入ExpiCHO细胞(ThermoFisher Scientific，A29133)，根据供应商ExpiCHO表达系统方法进行抗体瞬转表达，过程如下：在培养总体积25mL培养基中，36.5℃，8％二氧化碳浓度下培养Expi CHO细胞到密度6×10 6/mL，使用ExpiFectamine转染试剂化转各10μg抗体轻重链表达质粒到细胞；转染一天后，各取150μL和4mL ExpiCHO增强剂和ExpiCHO辅料添加到培养细胞中，继续培养至9天，4度，3500转离心取上清。混合AmMagTM Protein A磁珠(Genscript，L00695)和抗体表达上清，室温孵育2小时，PBS洗涤两次弃上清，加入适量洗脱缓冲液Protein G or A SefinoseTM Elution buffer(Sangon,C600481)，充分混匀后置于试管架上静止孵育5分钟，孵育期间重悬磁珠2-3次，重复洗脱2次，洗脱后，立即加入适量中和液1M Tris-HCl，pH7.5(Sangon,B548124)中和备用，得到纯化的抗体见表4。 The VHH sequence of each clone was fused with IgG1Fc N297A, codon optimization was performed, and the gene was synthesized and loaded into the expression vector pcDNA3.4 (Life Technologies). After expression plasmid amplification and plasmid extraction, the plasmid was transferred into ExpiCHO cells (ThermoFisher Scientific, A29133), and the antibody was transiently expressed according to the supplier’s ExpiCHO expression system method. Cultivate Expi CHO cells at a concentration of % carbon dioxide to a density of 6×10 6 /mL, and use ExpiFectamine transfection reagent to transfect 10 μg of antibody light and heavy chain expression plasmids into the cells; one day after transfection, take 150 μL and 4 mL of ExpiCHO enhancer and ExpiCHO adjuvant Add it to the cultured cells, continue culturing for 9 days, centrifuge at 4°C at 3500 rpm to get the supernatant. Mix AmMagTM Protein A magnetic beads (Genscript, L00695) and antibody expression supernatant, incubate at room temperature for 2 hours, wash twice with PBS, discard the supernatant, add an appropriate amount of elution buffer Protein G or A SefinoseTM Elution buffer (Sangon, C600481), fully After mixing, place it on a test tube rack and incubate for 5 minutes. During the incubation period, resuspend the magnetic beads 2-3 times, and repeat the elution twice. After elution, immediately add an appropriate amount of neutralizing solution 1M Tris-HCl, pH7.5 (Sangon , B548124) for neutralization, and the purified antibodies are shown in Table 4.
表4候选抗体表达、纯化数据Table 4 Candidate antibody expression and purification data
抗体编号Antibody number 理论等电点theoretical isoelectric point 表达量(mg/L)Expression (mg/L)
阳性对照抗体positive control antibody 7.47.4 172172
Ab0716.1Ab0716.1 6.76.7 105105
Ab0716.2Ab0716.2 6.76.7 118118
Ab0716.4Ab0716.4 77 7676
实施例4候选抗体亲和力测定Example 4 Candidate Antibody Affinity Determination
为了测定阳性对照抗体、Ab0716.1、Ab0716.2、Ab0716.4抗体与人血清白蛋白的亲和力，采用Octet RED96e(Fortebio)测定候选抗体与HSA(Acro,货号：HSA-H522a)的亲和力，抗原及抗体均用1×PBST(1×PBS：生工，B548117-0500；0.02％吐温20：sigma，P1379)稀释，抗原使用浓度为100nM，抗体使用浓度为64nM。将候选抗体样品按200μL/孔加入96孔板(Greiner bio-one，655209)，设置软件参数，温度30℃、收集标准动力学信号的频率为5.0Hz；用1×PBST预湿AHC传感器(Fortébio，货号：18-0015)10分钟，然后上机检测。每个循环包含以下步骤：1)浸入缓冲液60s；2)检测抗原是否与传感器有非特异性结合；3)10mM pH1.7的甘氨酸溶液再生；4)浸入缓冲液60s；5)抗体固化在传感器上，时间为 30s；6)传感器浸入缓冲液180s；7)抗原与抗体结合，时间180s；8)抗原抗体的解离，时间10分钟；9)传感器再生。采用Fortebio的Data Analysis 12.0软件，对抗原-抗体以1:1的结合方式，测定结合速率(K on)和解离速率(K off)，以此计算抗体的平衡解离常数(K D)，结果如表5。 In order to determine the affinity of positive control antibody, Ab0716.1, Ab0716.2, Ab0716.4 antibody and human serum albumin, use Octet RED96e (Fortebio) to measure the affinity of candidate antibody to HSA (Acro, catalog number: HSA-H522a), antigen and antibody were diluted with 1×PBST (1×PBS: Sanko, B548117-0500; 0.02% Tween 20: sigma, P1379), the concentration of antigen used was 100 nM, and the concentration of antibody used was 64 nM. Add 200 μL/well of the candidate antibody sample into a 96-well plate (Greiner bio-one, 655209), set the software parameters, the temperature is 30°C, and the frequency of collecting standard kinetic signals is 5.0 Hz; pre-wet the AHC sensor (Fortébio , Item No.: 18-0015) for 10 minutes, and then tested on the machine. Each cycle includes the following steps: 1) immersion in the buffer for 60s; 2) detection of non-specific binding of the antigen to the sensor; 3) regeneration of 10mM glycine solution at pH 1.7; 4) immersion in the buffer for 60s; 5) antibody immobilization on the sensor Above, the time is 30s; 6) The sensor is immersed in the buffer solution for 180s; 7) The combination of antigen and antibody, the time is 180s; 8) The dissociation of antigen and antibody, the time is 10 minutes; 9) Sensor regeneration. Fortebio's Data Analysis 12.0 software was used to measure the binding rate (K on ) and dissociation rate (K off ) of the antigen-antibody in a 1:1 binding mode, and then calculate the equilibrium dissociation constant (K D ) of the antibody, and the result As in Table 5.
表5候选抗体与HSA的亲和力测定Table 5 Affinity Determination of Candidate Antibodies and HSA
抗体编号Antibody number 响应值Response K D(M) K D (M) kon(1/Ms)kon(1/Ms) kdis(1/s)kdis(1/s)
阳性对照抗体positive control antibody 0.620.62 1.33E-081.33E-08 1.88E+051.88E+05 2.50E-032.50E-03
Ab0716.1Ab0716.1 0.930.93 3.93E-103.93E-10 3.66E+053.66E+05 1.44E-041.44E-04
Ab0716.2Ab0716.2 0.930.93 4.15E-104.15E-10 4.12E+054.12E+05 1.71E-041.71E-04
Ab0716.4Ab0716.4 0.810.81 1.26E-091.26E-09 2.17E+052.17E+05 2.75E-042.75E-04
由表5可知，候选抗体均可与HSA结合，且亲和力显著强于阳性对照抗体。It can be seen from Table 5 that all candidate antibodies can bind to HSA, and the affinity is significantly stronger than that of the positive control antibody.
实施例5候选抗体理化性质评估Example 5 Evaluation of Physicochemical Properties of Candidate Antibodies
根据实施例3和实施例4的实验结果，抗体Ab0716.1、Ab0716.2的表达量及亲和力较为理想，作为候选分子继续进行理化成药性评估，具体如下：According to the experimental results of Example 3 and Example 4, the expression levels and affinity of antibodies Ab0716.1 and Ab0716.2 are relatively ideal, and as candidate molecules, the physical and chemical druggability evaluation will continue, as follows:
SEC-HPLC纯度分析SEC-HPLC Purity Analysis
(1)将样品浓度调整至1mg/mL，混匀，12000rpm离心5min，取上清转至样品瓶，放入HPLC样品盘。设置色谱条件如下：(1) Adjust the sample concentration to 1mg/mL, mix well, centrifuge at 12000rpm for 5min, take the supernatant and transfer it to a sample bottle, and put it into the HPLC sample tray. Set the chromatographic conditions as follows:
Figure PCTCN2022097052-appb-000004
Figure PCTCN2022097052-appb-000004
(2)色谱柱采用流动相(200mM磷酸盐缓冲液，pH6.8)平衡后，进样分析，用色谱软件进行数据分析，峰面积归一化法计算各个峰的峰面积百分比，百分比越高说明抗体纯度越高。(2) After the chromatographic column is equilibrated with the mobile phase (200mM phosphate buffer, pH6.8), the sample is injected for analysis, and the data is analyzed with the chromatographic software. The peak area percentage of each peak is calculated by the peak area normalization method. The higher the percentage Indicates the higher the purity of the antibody.
HIC-HPLC分析HIC-HPLC analysis
(1)将样品浓度调整至1mg/ml，离心取上清待测。设置色谱条件如下：(1) Adjust the concentration of the sample to 1mg/ml, centrifuge and take the supernatant for testing. Set the chromatographic conditions as follows:
Figure PCTCN2022097052-appb-000005
Figure PCTCN2022097052-appb-000005
(2)用流动相A(50mM磷酸盐缓冲液/1M硫酸铵，pH 7.0)和流动相B(50mM磷酸盐缓冲液，pH 7.0)进行梯度洗脱，记录主峰保留时间，出峰时间短则抗体亲水性强。(2) Use mobile phase A (50mM phosphate buffer/1M ammonium sulfate, pH 7.0) and mobile phase B (50mM phosphate buffer, pH 7.0) to carry out gradient elution, record the retention time of the main peak, and the shorter the peak time Antibodies are highly hydrophilic.
熔解温度(Tm)值分析Analysis of melting temperature (Tm) value
将样品浓度调整至1mg/mL，然后按照Protein Thermal Shift TM Starter Kit说明书，取供试品溶液13μL加入至PCR管内，加入5μL Protein Thermal shift  TM Buffer，加入2μL10×染色液，使反应体积为20μL，混匀后，12000rpm离心5min以去除气泡。将检测样品置于PCR仪内，进行样品分析，记录样品的Tm值，Tm值越高表示抗体的热稳定性越好。 Adjust the sample concentration to 1mg/mL, then according to the instructions of the Protein Thermal Shift TM Starter Kit, take 13 μL of the test solution and add it to the PCR tube, add 5 μL Protein Thermal shift TM Buffer, add 2 μL 10× staining solution, make the reaction volume 20 μL, After mixing, centrifuge at 12000rpm for 5min to remove air bubbles. Place the test sample in the PCR machine, analyze the sample, and record the Tm value of the sample. The higher the Tm value, the better the thermal stability of the antibody.
根据表6可知，候选抗体Ab0716.1的纯度、热稳定性、亲水性等理化指标较为理想，将Ab0716.1作为候选分子继续进行人源化，提高与人源抗体的同源性，以降低免疫原性。According to Table 6, the physical and chemical indicators such as purity, thermal stability, and hydrophilicity of the candidate antibody Ab0716.1 are relatively ideal, and Ab0716.1 is used as a candidate molecule to continue humanization to improve the homology with human antibodies, so as to Reduce immunogenicity.
表6候选抗体理化性质分析结果Table 6 Analysis results of physicochemical properties of candidate antibodies
Figure PCTCN2022097052-appb-000006
Figure PCTCN2022097052-appb-000006
实施例6候选抗体Ab0716.1人源化与表达Example 6 Humanization and expression of candidate antibody Ab0716.1
综合实施例3-5的实验结果，最终挑取候选抗体Ab0716.1的V HH序列进行人源化。将Ab0716.1的V HH序列进行IMGT/Domain Gap Align，查找与其同源性最高的人源的germline为IGHV3-23*04。Ab0716.1序列按Chothia规则编号，对H1、H13、H44、H45、H49、H74、H76、H79、H81、H82B、H83、H84、H108等位点按表7进行突变，其他位点氨基酸保持不 变。Ab0716Hz1的V HH序列如SEQ ID NO:20所示，Ab0716Hz2的V HH序列如SEQ ID NO:21所示。 Based on the experimental results of Examples 3-5, the V H H sequence of the candidate antibody Ab0716.1 was finally selected for humanization. IMGT/Domain Gap Align was performed on the V H H sequence of Ab0716.1, and the human germline with the highest homology was found to be IGHV3-23*04. The sequence of Ab0716.1 was numbered according to the Chothia rules, and the H1, H13, H44, H45, H49, H74, H76, H79, H81, H82B, H83, H84, H108 and other sites were mutated according to Table 7, and the amino acids at other sites remained unchanged. Change. The VHH sequence of Ab0716Hz1 is shown in SEQ ID NO:20, and the VHH sequence of Ab0716Hz2 is shown in SEQ ID NO:21.
表7候选抗体序列人源化设计Table 7 Candidate antibody sequence humanization design
Figure PCTCN2022097052-appb-000007
Figure PCTCN2022097052-appb-000007
将表7中Ab0716Hz1、Ab0716Hz2的V HH全长序列进行IMGT/Domain Gap Align分析，比较人源化前后与IGHV3-23*04的同源性；此外，将人源化后的VHH与IgG1Fc N297A融合表达，参考实施例2.1进行人源化抗体的表达、纯化，具体结果见表8。由表8可知，人源化后抗体与IGHV3-23*04的同源性提高至87％左右，且人源化抗体的表达量较为理想，继续进行后续评估。 The full-length VHH sequences of Ab0716Hz1 and Ab0716Hz2 in Table 7 were analyzed by IMGT/Domain Gap Align to compare the homology with IGHV3-23*04 before and after humanization; in addition, the humanized VHH and IgG1Fc N297A For fusion expression, refer to Example 2.1 for the expression and purification of the humanized antibody, see Table 8 for specific results. It can be seen from Table 8 that the homology between the antibody and IGHV3-23*04 was increased to about 87% after humanization, and the expression level of the humanized antibody was relatively ideal, and the follow-up evaluation was continued.
表8人源化抗体表达量及同源性Table 8 Humanized antibody expression and homology
抗体编号Antibody number 理论等电点theoretical isoelectric point 表达量(mg/L)Expression (mg/L) 同源性homology
Ab0716.1Ab0716.1 6.76.7 105105 75.3％75.3%
Ab0716Hz1Ab0716Hz1 7.37.3 239239 87.6％87.6%
Ab0716Hz2Ab0716Hz2 7.67.6 364364 86.6％86.6%
实施例7人源化抗体亲和力测定Example 7 Humanized Antibody Affinity Determination
参照实施例4的方法，测定Ab0716.1人源化抗体与人、猴、鼠来源的血清白蛋白的亲和力，结果如表9。由表9可知，虽然人源化后抗体分子Ab0716Hz1、Ab0716Hz2与人血清白蛋白的亲和力均较人源化之前有所下降，但仍与人血清白蛋白保持有较强亲和力；Ab0716Hz1、Ab0716Hz2与猴血清白蛋白亲和力较弱；Ab0716Hz1、Ab0716Hz2与鼠血清白蛋白不结合。Referring to the method of Example 4, the affinity of the Ab0716.1 humanized antibody to human, monkey, and mouse serum albumin was determined, and the results are shown in Table 9. It can be seen from Table 9 that although the affinity of antibody molecules Ab0716Hz1 and Ab0716Hz2 to human serum albumin decreased after humanization, they still maintained a strong affinity with human serum albumin; Ab0716Hz1 and Ab0716Hz2 were compatible with monkey Serum albumin has weak affinity; Ab0716Hz1 and Ab0716Hz2 do not bind to mouse serum albumin.
表9人源化抗体与人、猴、鼠血清白蛋白的亲和力测定Table 9 Affinity determination of humanized antibody and serum albumin of human, monkey and mouse
Figure PCTCN2022097052-appb-000008
Figure PCTCN2022097052-appb-000008
Figure PCTCN2022097052-appb-000009
Figure PCTCN2022097052-appb-000009
实施例8人源化抗体理化性质评估Example 8 Evaluation of Physicochemical Properties of Humanized Antibody
将人源化后抗体分子Ab0716Hz1、Ab0716Hz2进行成药性评估，具体如下：The humanized antibody molecules Ab0716Hz1 and Ab0716Hz2 were evaluated for their druggability, as follows:
同实施例5，SEC-HPLC纯度分析，百分比越高说明抗体纯度越高；熔解温度(Tm)值分析，Tm值越高表示抗体的热稳定性越好；HIC-HPLC分析，出峰时间越短表示抗体的亲水性越好。Same as Example 5, SEC-HPLC purity analysis, the higher the percentage, the higher the antibody purity; the melting temperature (Tm) value analysis, the higher the Tm value, the better the thermal stability of the antibody; HIC-HPLC analysis, the shorter the peak time The shorter the antibody, the more hydrophilic it is.
毛细管等电聚焦(iCIEF)分析Capillary Isoelectric Focusing (iCIEF) Analysis
取样品溶液加入到已经充分混匀的以下体系中：1％的甲基纤维素(MC)70μL，尿素5M80μL，两性电解质Pharmalyte pH 3-10 8μL，pI marker 5.5和9.5各2μL。补加适当体积超纯水至200μL，混匀。离心取上清进样分析。分析结束后，将结果文件导入ChromPerfect软件进行图谱积分处理并计算各峰的等电点以及各峰百分比，分析候选抗体的电荷异构体分布情况。Take the sample solution and add it to the following system that has been thoroughly mixed: 1% methylcellulose (MC) 70 μL, urea 5M 80 μL, ampholyte Pharmalyte pH 3-10 8 μL, pI marker 5.5 and 9.5 2 μL each. Add an appropriate volume of ultrapure water to 200 μL, and mix well. Centrifuge the supernatant for analysis. After the analysis, import the result file into ChromPerfect software for spectrum integration processing and calculate the isoelectric point of each peak and the percentage of each peak, and analyze the distribution of charge variants of candidate antibodies.
表10人源化抗体理化性质分析结果Table 10 Analysis results of physicochemical properties of humanized antibodies
Figure PCTCN2022097052-appb-000010
Figure PCTCN2022097052-appb-000010
综合实施例6-8可知，候选抗体Ab0716.1经过人源化后获得Ab0716Hz1、Ab0716Hz2分子，与IGHV3-23*04的同源性提高至87％左右，并且人源化抗体在表达量、亲和力、理化性质等方面均符合内部成药性标准，可作为候选分子继续开发；但与猴血清白蛋白亲和力较弱，需要继续进行亲和力成熟改造，提高其与猴血清白蛋白的亲和力。Comprehensive examples 6-8 show that the candidate antibody Ab0716.1 obtained Ab0716Hz1 and Ab0716Hz2 molecules after humanization, and the homology with IGHV3-23*04 was increased to about 87%, and the humanized antibody was expressed in terms of expression and affinity It can be used as a candidate molecule for further development; however, its affinity with monkey serum albumin is weak, and it needs to continue to undergo affinity maturation modification to improve its affinity with monkey serum albumin.
实施例9 Ab0716Hz2亲和力成熟文库设计与构建Example 9 Ab0716Hz2 affinity maturation library design and construction
基于实施例1-8综合考虑，最终选择抗体Ab0716Hz2对其继续进行亲和力成熟，提高其与猴血清白蛋白的亲和力。对Ab0716Hz2的抗原结合决定簇(CDR)位点的氨基酸进行随机突变，构建各CDR区的突变文库，利用酵母展示技术高通量筛选与抗原特异性结合力强的序列。将Ab0716Hz2的可变区氨基酸序列按Chothia编码规则进行编码，CDR区按Chothia定义。对重链可变区CDR1、CDR2、CDR3，设计NNK突变引物进行聚合酶链式反应(PCR)扩增各CDR突变文库基因片段。参考实施例1构建亲和力成熟突变文库，文库编号JYYDL184-187；文库JYYDL184-187电转后分别在100mL的SD-Trp培养基，30℃、225 转/分钟培养过夜；各取1.0×10 8菌量，重悬于20mL YPGP诱导培养基，20℃、225转/分钟培养24小时，置于4℃冰箱待用。同时将Ab0716Hz2的亲本序列展示于酵母表面，作为亲本对照使用。 Based on the comprehensive consideration of Examples 1-8, the antibody Ab0716Hz2 was finally selected for further affinity maturation to improve its affinity with monkey serum albumin. Amino acids at the antigen-binding determinant (CDR) site of Ab0716Hz2 were randomly mutated, and a mutation library of each CDR region was constructed. Yeast display technology was used to screen high-throughput sequences with strong antigen-specific binding. The amino acid sequence of the variable region of Ab0716Hz2 is coded according to Chothia coding rules, and the CDR region is defined according to Chothia. For heavy chain variable regions CDR1, CDR2, and CDR3, NNK mutation primers were designed to carry out polymerase chain reaction (PCR) to amplify gene fragments of each CDR mutation library. Refer to Example 1 to construct an affinity maturation mutation library, library number JYYDL184-187; after electroporation, the library JYYDL184-187 was cultured in 100 mL SD-Trp medium at 30°C and 225 rpm overnight; each took 1.0×10 8 bacteria , resuspended in 20mL YPGP induction medium, cultured at 20°C, 225 rpm for 24 hours, and placed in a 4°C refrigerator for use. At the same time, the parental sequence of Ab0716Hz2 was displayed on the yeast surface and used as a parental control.
实施例10 Ab0716Hz2亲和力成熟文库筛选与单克隆鉴定Example 10 Ab0716Hz2 affinity maturation library screening and monoclonal identification
JYYDL184文库诱导后菌液，取1.5×10 9细胞用磁珠分选系统进行第一轮富集：1.用50mL 1×PBSA洗涤一次，3000转/分钟离心3分钟弃上清；2.与20mL含10nM生物素标记的猴血清白蛋白(以下简称CSA-Biotin)的1×PBSA，室温孵育30分钟；3.洗涤后加入抗生物素的磁珠混匀孵育10min，过磁力柱收集阳性细胞；同理，JYYDL185-JYYDL187等文库诱导后菌液，取1.5×10 9细胞用相同方法，在10nM生物素标记的人血清白蛋白条件进行磁珠富集。 After induction of the JYYDL184 library, take 1.5×10 9 cells for the first round of enrichment using a magnetic bead sorting system: 1. Wash once with 50 mL 1×PBSA, centrifuge at 3000 rpm for 3 minutes and discard the supernatant; 2. 20mL 1×PBSA containing 10nM biotin-labeled monkey serum albumin (hereinafter referred to as CSA-Biotin), incubate at room temperature for 30 minutes; 3. After washing, add anti-biotin magnetic beads, mix and incubate for 10 minutes, and collect positive cells through a magnetic column In the same way, 1.5×10 9 cells were taken from the bacterial liquid after induction of JYYDL185-JYYDL187 and other libraries, and enriched with magnetic beads under the condition of 10nM biotin-labeled human serum albumin in the same way.
磁珠筛选后阳性细胞经过再次培养、诱导后，取2.0×10 7细胞进行第二轮流式分选：1)用1mL 1×PBSA，离心弃上清；2)与1mL含3nM CSA-Biotin及鼠抗V5抗体的1×PBSA冰上孵育30min；3)离心弃上清，加入1mL 1×PBSA洗涤一次；4)加入200μL含荧光抗体的1×PBSA(含SA-PE及羊抗鼠-647)，避光冰上孵育20分钟；5)重复步骤3，加入2mL1×PBSA重悬细胞，通过流式分选仪器收集647荧光信号与PE荧光信号均强的细胞群。第二轮流式分选后细胞经过再次培养、诱导后，同第二轮方法，取2.0×10 7细胞在10nM CSA-Biotin孵育后进行第三轮流式分选，分选后取部分细胞涂布于SD-Trp固体培养基平板，30℃静置培养3天。 After the positive cells were screened by magnetic beads, after re-cultivation and induction, 2.0×10 7 cells were taken for the second round of flow sorting: 1) centrifuged with 1mL 1×PBSA and discarded the supernatant; 2) mixed with 1mL containing 3nM CSA-Biotin and Incubate in 1×PBSA with mouse anti-V5 antibody for 30min on ice; 3) Discard supernatant by centrifugation, add 1mL 1×PBSA to wash once; 4) Add 200μL 1×PBSA containing fluorescent antibody (containing SA-PE and goat anti-mouse-647 ), and incubate on ice in the dark for 20 minutes; 5) Repeat step 3, add 2 mL of 1×PBSA to resuspend the cells, and collect the cell populations with strong 647 fluorescence signals and PE fluorescence signals through a flow cytometry instrument. After the second round of flow sorting, the cells were cultured and induced again, and the same as the second round, 2.0×10 7 cells were incubated in 10nM CSA-Biotin for the third round of flow sorting, and after sorting, some cells were coated. On the SD-Trp solid medium plate, static culture at 30°C for 3 days.
JYYDL184-JYYDL187第三轮筛选产物，各挑取46个单克隆进行测序分析，最终获得独一序列的酵母单克隆菌落进行流式染色分析，各取1×10 6个细胞按表11方案进行染色评估：方案1评估各克隆与人血清白蛋白的结合水平，PE平均荧光信号强度(MFI)越强代表结合能力越强；方案2评估各克隆与猴血清白蛋白的结合水平，PE的MFI值越强代表结合能力越强。 In the third round of screening products of JYYDL184-JYYDL187, 46 single clones were selected for sequencing analysis, and finally a yeast single clone colony with a unique sequence was obtained for flow staining analysis, and 1 ×106 cells were stained according to the scheme in Table 11. Evaluation: Scheme 1 evaluates the binding level of each clone to human serum albumin, and the stronger the mean fluorescence signal intensity (MFI) of PE, the stronger the binding ability; Scheme 2 evaluates the binding level of each clone to monkey serum albumin, and the MFI value of PE The stronger represents the stronger binding ability.
表11单克隆酵母菌落流式染色鉴定方案Table 11 Flow cytometry identification scheme for monoclonal yeast colonies
方案Program 一抗Antibody 二抗Secondary Antibodies
11 100nM HSA-Biotin，鼠抗V5100nM HSA-Biotin, mouse anti-V5 SA-PE，羊抗鼠-647SA-PE, goat anti-mouse-647
22 100nM CSA-Biotin，鼠抗V5100nM CSA-Biotin, mouse anti-V5 SA-PE，羊抗鼠-647SA-PE, goat anti-mouse-647
根据各克隆按方案1、2的染色结果，综合各克隆序列的相似性，最终挑取克隆YC225G1、YC222H4、YC226H5的序列进行抗体表达，如表12。According to the staining results of each clone according to schemes 1 and 2, and considering the similarity of the sequences of each clone, the sequences of clones YC225G1, YC222H4, and YC226H5 were finally selected for antibody expression, as shown in Table 12.
表12酵母单克隆菌落流式染色结果Table 12 Yeast monoclonal colony flow cytometric staining results
Figure PCTCN2022097052-appb-000011
Figure PCTCN2022097052-appb-000011
实施例11亲和力成熟候选抗体表达Example 11 Expression of Affinity Maturation Candidate Antibodies
将各克隆的V HH序列与IgG1Fc N297A融合，委托上海百英生物科技有限公司进行瞬转HEK293细胞表达、纯化，最终得到亲和力成熟候选抗体见表13。 The V H H sequence of each clone was fused with IgG1Fc N297A, and Shanghai Baiying Biotechnology Co., Ltd. was entrusted to perform transient HEK293 cell expression and purification, and finally obtained affinity maturation candidate antibodies are shown in Table 13.
表13亲和力成熟候选抗体表达、纯化数据Table 13 Expression and purification data of affinity maturation candidate antibodies
抗体编号Antibody number 理论等电点theoretical isoelectric point 消光系数Extinction coefficient 表达量(mg/L)Expression (mg/L)
Ab0716Am01Ab0716Am01 7.67.6 1.71.7 5656
Ab0716Am02Ab0716Am02 8.28.2 1.551.55 5252
Ab0716Am06Ab0716Am06 7.87.8 1.551.55 8181
实施例12候选抗体亲和力测定Example 12 Candidate Antibody Affinity Determination
同实施例7，继续测定亲和力成熟抗体Ab0716Am01、Ab0716Am02、Ab0716Am06及亲本抗体Ab0716Hz2分别与人、猴、鼠来源的血清白蛋白的亲和力，结果如表14。由表14可知，亲和力成熟后抗体Ab0716Am01、Ab0716Am02、Ab0716Am06与猴血清白蛋白的亲和力相对于亲本抗体均大幅提高；与人血清白蛋白的亲和力维持在1.41-5.0nM之间，均可以接受；亲和力前后抗体与鼠血清白蛋白均不结合。As in Example 7, the affinities of the affinity matured antibodies Ab0716Am01, Ab0716Am02, Ab0716Am06 and the parental antibody Ab0716Hz2 to serum albumin from human, monkey, and mouse were continuously measured, and the results are shown in Table 14. It can be seen from Table 14 that after affinity maturation, the affinity of antibodies Ab0716Am01, Ab0716Am02, Ab0716Am06 to monkey serum albumin was greatly improved compared with the parental antibody; the affinity to human serum albumin was maintained between 1.41-5.0nM, which was acceptable; the affinity Neither before nor after antibody binds to mouse serum albumin.
表14人源化抗体与人、猴、鼠血清白蛋白的亲和力测定Table 14 Affinity determination of humanized antibody and human, monkey, mouse serum albumin
Figure PCTCN2022097052-appb-000012
Figure PCTCN2022097052-appb-000012
实施例13人源化抗体理化性质评估Example 13 Evaluation of Physicochemical Properties of Humanized Antibody
同实施例8，将亲和力成熟后抗体Ab0716Am01、Ab0716Am02、Ab0716Am06进行理化成药性评估，结果如表15。由表15可知，亲和力成熟后抗体Ab0716Am01、Ab0716Am02、Ab0716Am06的纯度、热稳定性、亲水性、电荷异构体等理化指标较为理想，符合内部成药性标准。As in Example 8, the physicochemical properties of the antibodies Ab0716Am01, Ab0716Am02, and Ab0716Am06 after affinity maturation were evaluated, and the results are shown in Table 15. It can be seen from Table 15 that the physical and chemical indicators of the antibodies Ab0716Am01, Ab0716Am02, and Ab0716Am06 after affinity maturation are ideal, such as purity, thermal stability, hydrophilicity, and charge isomers, and meet internal druggability standards.
表15人源化抗体理化性质分析结果Table 15 Analysis results of physicochemical properties of humanized antibodies
Figure PCTCN2022097052-appb-000013
Figure PCTCN2022097052-appb-000013
综合实施例12-13可知，亲和力成熟后抗体Ab0716Am01、Ab0716Am02、Ab0716Am06经过亲和力成熟后与猴血清白蛋白的亲和力大幅提高，同时与人血清白蛋白维持了较好亲和力，且并且亲和力成熟抗体在表达量、亲和力、理化性质等方面均符合内部成药性标准，可作为候选分子继续开发。Based on Examples 12-13, it can be seen that after affinity maturation, the affinity of Ab0716Am01, Ab0716Am02, and Ab0716Am06 to monkey serum albumin is greatly improved, while maintaining a good affinity with human serum albumin, and the affinity matured antibodies are expressing Quantity, affinity, physical and chemical properties, etc. all meet the internal druggability standards, and can be used as candidate molecules for further development.
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。The foregoing detailed description has been offered by way of explanation and example, not to limit the scope of the appended claims. Variations on the presently recited embodiments of this application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims (41)

  1. 分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:26或38所示的氨基酸序列。An isolated antigen binding protein comprising at least one CDR in the variable region VH of an antibody heavy chain, said VH comprising the amino acid sequence shown in SEQ ID NO: 26 or 38.
  2. 根据权利要求1所述的分离的抗原结合蛋白，其包含HCDR3，所述HCDR3包含SEQ ID NO:29所示的氨基酸序列。The antigen binding protein of separation according to claim 1, it comprises HCDR3, and described HCDR3 comprises the aminoacid sequence shown in SEQ ID NO:29.
  3. 根据权利要求1或2所述的分离的抗原结合蛋白，其包含HCDR3，其中所述HCDR3包含SEQ ID NO:1或SEQ ID NO:28所示的氨基酸序列。The isolated antigen-binding protein according to claim 1 or 2, which comprises HCDR3, wherein said HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 28.
  4. 根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其包含HCDR2，所述HCDR2包含SEQ ID NO:37所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-3, which comprises HCDR2, said HCDR2 comprising the amino acid sequence shown in SEQ ID NO:37.
  5. 根据权利要求1-4中任一项所述的分离的抗原结合蛋白，其包含HCDR2，所述HCDR2包含SEQ ID NO:2、31或32所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-4, which comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, 31 or 32.
  6. 根据权利要求1-5中任一项所述的分离的抗原结合蛋白，其包含HCDR1，所述HCDR1包含SEQ ID NO:36所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-5, which comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:36.
  7. 根据权利要求1-6中任一项所述的分离的抗原结合蛋白，其包含HCDR1，所述HCDR1包含SEQ ID NO:3或30所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-6, which comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3 or 30.
  8. 根据权利要求1-7中任一项所述的分离的抗原结合蛋白，其包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:36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:29所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-7, which comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 36, said HCDR2 comprising SEQ ID NO: The amino acid sequence shown in 37, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 29.
  9. 根据权利要求1-8中任一项所述的分离的抗原结合蛋白，其包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:3或30所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2、31或32所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1或SEQ ID NO:28所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-8, which comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3 or 30, said HCDR2 comprising SEQ ID The amino acid sequence shown in NO:2, 31 or 32, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:28.
  10. 根据权利要求1-9中任一项所述的分离的抗原结合蛋白，其包含HCDR1，HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列；或者所述HCDR1包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:28所示的氨基酸序列；或者所述HCDR1包含SEQ ID NO:30所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列；或者所述HCDR1包含SEQ ID NO:3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:1所示的氨基酸序列；或者所述 HCDR1包含SEQ ID NO:3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:1所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-9, which comprises HCDR1, HCDR2 and HCDR3, wherein said HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, and said HCDR2 comprises SEQ ID NO The amino acid sequence shown in: 2, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 1; or the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 3, and the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 2 The amino acid sequence shown, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:28; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:30, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2 sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:31, and The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:32, and the HCDR3 Comprising the amino acid sequence shown in SEQ ID NO:1.
  11. 根据权利要求1-10中任一项所述的分离的抗原结合蛋白，其包含H-FR1，所述H-FR1的C端与HCDR1的N端直接或间接相连，且所述H-FR1包含SEQ ID NO:22所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-10, which comprises H-FR1, the C-terminus of said H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and said H-FR1 comprises Amino acid sequence shown in SEQ ID NO:22.
  12. 根据权利要求11所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:11中任一项所示的氨基酸序列。The isolated antigen-binding protein according to claim 11, wherein said H-FR1 comprises any one of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:11 amino acid sequence.
  13. 根据权利要求1-12中任一项所述的分离的抗原结合蛋白，其包含H-FR2，所述H-FR2位于HCDR1和HCDR2之间，且所述H-FR2包含SEQ ID NO:23所示的氨基酸序列。The isolated antigen binding protein according to any one of claims 1-12, which comprises H-FR2, said H-FR2 is located between HCDR1 and HCDR2, and said H-FR2 comprises SEQ ID NO:23 The amino acid sequence shown.
  14. 根据权利要求13所述的分离的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:5、SEQ ID NO:10、SEQ ID NO:12和SEQ ID NO:15中任一项所示的氨基酸序列。The isolated antigen binding protein according to claim 13, wherein said H-FR2 comprises any one of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:15 amino acid sequence.
  15. 根据权利要求1-14中任一项所述的分离的抗原结合蛋白，其包含H-FR3，所述H-FR3位于HCDR2和HCDR3之间，且所述H-FR3包含SEQ ID NO:24所示的氨基酸序列。The isolated antigen binding protein according to any one of claims 1-14, which comprises H-FR3, said H-FR3 is located between HCDR2 and HCDR3, and said H-FR3 comprises SEQ ID NO:24 The amino acid sequence shown.
  16. 根据权利要求15所述的分离的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:6或SEQ ID NO:13所示的氨基酸序列。The isolated antigen binding protein according to claim 15, wherein said H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:13.
  17. 根据权利要求1-16中任一项所述的分离的抗原结合蛋白，其包含H-FR4，所述H-FR4的N端与HCDR3的C端直接或间接相连，且所述H-FR4包含SEQ ID NO:25所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-16, which comprises H-FR4, the N-terminus of said H-FR4 is directly or indirectly linked to the C-terminus of HCDR3, and said H-FR4 comprises Amino acid sequence shown in SEQ ID NO:25.
  18. 根据权利要求17所述的分离的抗原结合蛋白，其中所述H-FR4包含SEQ ID NO:7、SEQ ID NO:14和SEQ ID NO:16中任一项所示的氨基酸序列。The isolated antigen-binding protein according to claim 17, wherein the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:14 and SEQ ID NO:16.
  19. 根据权利要求1-18中任一项所述的分离的抗原结合蛋白，其包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:22所示的氨基酸序列，所述H-FR2包含SEQ ID NO:23所示的氨基酸序列，所述H-FR3包含SEQ ID NO:24所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:25所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-18, comprising H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising SEQ ID NO:22 The amino acid sequence of the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:23, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:24, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:25 Amino acid sequence shown.
  20. 根据权利要求19所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:11中任一项所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5、SEQ ID NO:10、SEQ ID NO:12和SEQ ID NO:15中任一项所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6或SEQ ID NO:13所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7、SEQ ID NO:14和SEQ ID NO:16中任一项所示的氨基酸序列。The isolated antigen binding protein according to claim 19, wherein said H-FR1 comprises any one of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:11 Amino acid sequence, the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:15, and the H-FR3 comprises SEQ ID NO:6 or the amino acid sequence shown in SEQ ID NO:13, and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:14 and SEQ ID NO:16.
  21. 根据权利要求1-20中任一项所述的分离的抗原结合蛋白，其包含选自下组中的任一组 H-FR1，H-FR2，H-FR3和H-FR4：The isolated antigen-binding protein according to any one of claims 1-20, which comprises any group of H-FR1, H-FR2, H-FR3 and H-FR4 selected from the group consisting of:
    1)所述H-FR1包含如SEQ ID NO:4所示的氨基酸序列，所述H-FR2包含如SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含如SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含如SEQ ID NO:7所示的氨基酸序列；1) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
    2)所述H-FR1包含如SEQ ID NO:8所示的氨基酸序列，所述H-FR2包含如SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含如SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含如SEQ ID NO:7所示的氨基酸序列；2) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:8, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
    3)所述H-FR1包含如SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含如SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含如SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含如SEQ ID NO:7所示的氨基酸序列；3) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
    4)所述H-FR1包含如SEQ ID NO:11所示的氨基酸序列，所述H-FR2包含如SEQ ID NO:12所示的氨基酸序列，所述H-FR3包含如SEQ ID NO:13所示的氨基酸序列，且所述H-FR4包含如SEQ ID NO:14所示的氨基酸序列；以及4) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:11, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:12, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:13 The amino acid sequence shown, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 14; and
    5)所述H-FR1包含如SEQ ID NO:11所示的氨基酸序列，所述H-FR2包含如SEQ ID NO:15所示的氨基酸序列，所述H-FR3包含如SEQ ID NO:13所示的氨基酸序列，且所述H-FR4包含如SEQ ID NO:16所示的氨基酸序列。5) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:11, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:15, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:13 The amino acid sequence shown, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:16.
  22. 根据权利要求1-21中任一项所述的分离的抗原结合蛋白，其包含VH，所述VH包含SEQ ID NO:26或38所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-21, which comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 26 or 38.
  23. 根据权利要求22所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:33、SEQ ID NO:34、和SEQ ID NO:35中任一项所示的氨基酸序列。The isolated antigen binding protein of claim 22, wherein said VH comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID The amino acid sequence shown in any one of NO:33, SEQ ID NO:34, and SEQ ID NO:35.
  24. 根据权利要求1-23中任一项所述的分离的抗原结合蛋白，其包含抗体重链恒定区。The isolated antigen binding protein of any one of claims 1-23, comprising an antibody heavy chain constant region.
  25. 根据权利要求24所述的分离的抗原结合蛋白，其中所述抗体重链恒定区源自人抗体重链恒定区。The isolated antigen binding protein of claim 24, wherein the antibody heavy chain constant region is derived from a human antibody heavy chain constant region.
  26. 根据权利要求24-25中任一项所述的分离的抗原结合蛋白，其中所述抗体重链恒定区源自人IgG重链恒定区。The isolated antigen binding protein of any one of claims 24-25, wherein the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.
  27. 根据权利要求24-26中任一项所述的分离的抗原结合蛋白，其中所述抗体重链恒定区源自人IgG1重链恒定区。The isolated antigen binding protein of any one of claims 24-26, wherein the antibody heavy chain constant region is derived from a human IgGl heavy chain constant region.
  28. 根据权利要求1-27中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。The isolated antigen-binding protein of any one of claims 1-27, which comprises an antibody or antigen-binding fragment thereof.
  29. 根据权利要求28所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’， Fv片段，F(ab’) 2，scFv，di-scFv，dAb和/或V HH。 The isolated antigen binding protein of claim 28, wherein said antigen binding fragment comprises Fab, Fab', Fv fragment, F(ab') 2 , scFv, di-scFv, dAb and/or VHH .
  30. 根据权利要求29所述的分离的抗原结合蛋白，其中所述抗原结合片段为V HH。 The isolated antigen binding protein of claim 29, wherein said antigen binding fragment is a VHH .
  31. 根据权利要求28-30中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。The isolated antigen binding protein according to any one of claims 28-30, wherein said antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.
  32. 根据权利要求1-31中任一项所述的分离的抗原结合蛋白，其能够结合人血清白蛋白。The isolated antigen binding protein according to any one of claims 1-31, which is capable of binding human serum albumin.
  33. 融合蛋白，其包含权利要求1-32中任一项所述的分离的抗原结合蛋白。A fusion protein comprising the isolated antigen binding protein of any one of claims 1-32.
  34. 分离的核酸分子，其编码权利要求1-32中任一项所述的分离的抗原结合蛋白或权利要求33所述的融合蛋白。An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-32 or the fusion protein of claim 33.
  35. 载体，其包含权利要求34所述的核酸分子。A vector comprising the nucleic acid molecule of claim 34.
  36. 细胞，其包含权利要求1-32中任一项所述的分离的抗原结合蛋白，权利要求33所述的融合蛋白，权利要求34所述的核酸分子或权利要求35所述的载体。A cell comprising the isolated antigen binding protein of any one of claims 1-32, the fusion protein of claim 33, the nucleic acid molecule of claim 34 or the vector of claim 35.
  37. 制备权利要求1-32中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得权利要求1-32中任一项所述的分离的抗原结合蛋白表达的条件下，培养权利要求36所述的细胞。A method for preparing the isolated antigen-binding protein of any one of claims 1-32, said method comprising culturing the antigen-binding protein of any one of claims 1-32 under conditions that express The cell of claim 36.
  38. 药物组合物，其包含权利要求1-32中任一项所述的分离的抗原结合蛋白，权利要求33所述的融合蛋白，权利要求34所述的核酸分子、权利要求35所述的载体和/或权利要求36所述的细胞，以及任选地药学上可接受的载剂。A pharmaceutical composition comprising the isolated antigen-binding protein of any one of claims 1-32, the fusion protein of claim 33, the nucleic acid molecule of claim 34, the carrier of claim 35, and /or the cell of claim 36, and optionally a pharmaceutically acceptable carrier.
  39. 一种用于检测或测定人血清白蛋白的方法，所述方法包括使用权利要求1-32中任一项所述的分离的抗原结合蛋白或权利要求33所述的融合蛋白。A method for detecting or measuring human serum albumin, said method comprising using the isolated antigen-binding protein of any one of claims 1-32 or the fusion protein of claim 33.
  40. 一种试剂盒，其包含权利要求1-32中任一项所述的分离的抗原结合蛋白，权利要求33所述的融合蛋白，权利要求34所述的核酸分子、权利要求35所述的载体、权利要求36所述的细胞和/或权利要求38所述的药物组合物。A kit comprising the isolated antigen-binding protein of any one of claims 1-32, the fusion protein of claim 33, the nucleic acid molecule of claim 34, and the carrier of claim 35 , the cell of claim 36 and/or the pharmaceutical composition of claim 38.
  41. 权利要求1-32中任一项所述的分离的抗原结合蛋白，权利要求33所述的融合蛋白，权利要求34所述的核酸分子、权利要求35所述的载体、权利要求36所述的细胞、权利要求38所述的药物组合物和/或权利要求40所述的试剂盒在制备预防和/或治疗疾病或病症的药物中的用途。The isolated antigen-binding protein of any one of claims 1-32, the fusion protein of claim 33, the nucleic acid molecule of claim 34, the carrier of claim 35, the nucleic acid molecule of claim 36 Use of the cells, the pharmaceutical composition according to claim 38 and/or the kit according to claim 40 in the preparation of medicines for preventing and/or treating diseases or conditions.
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