WO2022121899A1 - 一种特异性结合Strep-Tag II标签的抗体及其应用 - Google Patents

一种特异性结合Strep-Tag II标签的抗体及其应用 Download PDF

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WO2022121899A1
WO2022121899A1 PCT/CN2021/136113 CN2021136113W WO2022121899A1 WO 2022121899 A1 WO2022121899 A1 WO 2022121899A1 CN 2021136113 W CN2021136113 W CN 2021136113W WO 2022121899 A1 WO2022121899 A1 WO 2022121899A1
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seq
antibody
amino acid
acid sequence
tag
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PCT/CN2021/136113
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English (en)
French (fr)
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鲁薪安
何霆
齐菲菲
黄茹
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北京艺妙神州医药科技有限公司
北京艺妙医疗科技有限公司
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Priority to AU2021395061A priority Critical patent/AU2021395061A1/en
Priority to EP21902604.4A priority patent/EP4261228A1/en
Priority to JP2023558928A priority patent/JP2023553205A/ja
Publication of WO2022121899A1 publication Critical patent/WO2022121899A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1292—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Actinomyces; from Streptomyces (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag

Definitions

  • the present invention relates to an antibody that specifically binds to the Strep-Tag II tag, and discloses the amino acid sequence of the antibody, a cloning or expression vector, a host cell, a method for producing the antibody, and the use of the antibody to purify or detect the expressed Strep-Tag II tag method of biological samples.
  • CAR-T Chimeric antigen receptor-T cells, chimeric antigen receptor T cells
  • TCR-T T cell receptor-T cells, engineered T cell receptor T cells
  • Immunotherapy strategies have received great attention.
  • the expansion level of CAR-T or TCR-T in vivo is an important indicator of its efficacy. Therefore, the development of reagents or methods for rapid and accurate detection of cells will help to effectively monitor its expansion efficiency in vitro and in vivo.
  • CAR-T or TCR-T cells There are currently two methods for detecting CAR-T or TCR-T cells: using quantitative real-time polymerase-linked reaction (Quantitative real time PCR, qPCR) to detect the gene expression of CAR or TCR in T cells; CAR protein or TCR protein expression.
  • quantitative real-time polymerase-linked reaction Quantitative real time PCR, qPCR
  • an antibody that specifically binds to the scFv (Single-chain variable fragment) fragment in the CAR protein is commonly used for flow cytometry detection of T cells, or an antibody that binds to the Fab fragment or protein L, etc. Detection of scFv fragments in CAR proteins.
  • TCR protein in T cells For detecting the expression of TCR protein in T cells, the complex of HLA and polypeptide specific for a certain TCR is commonly used for flow detection of T cells.
  • these methods have obvious limitations: qPCR detection of gene copy number cannot accurately reflect CAR-expressing T cells, because for cells that integrate CAR genes but do not normally express CAR protein, using qPCR detection methods will cause false positives in the detection;
  • the detection background value of Fab antibodies is high, which is easy to cause false positives; protein L is only suitable for the detection of CARs with scFv with ⁇ light chain, and the detection of scFv fragments in CAR proteins.
  • Antibodies, as well as HLA and polypeptide complexes against TCR need to screen out different antibodies or complexes against different CAR proteins or TCRs, and establish relevant detection methods, which are time-consuming, inefficient and costly. If an accurate, sensitive and universal antibody detection CAR-T can be developed, it will greatly improve the detection efficiency of gene-transduced cell therapy drugs and reduce the detection cost.
  • the present invention discloses an isolated antibody that specifically binds to the Strep-Tag II tag, comprising: a heavy chain variable region (hereinafter abbreviated as VH) and a light chain variable region (hereinafter abbreviated as VL), wherein the VH Including: VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2 and VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4;
  • the VL includes: VL-CDR1 comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, VL-CDR2 comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8 and VL-CDR2 comprising the amino acid sequence of SEQ ID NO:8: 9 or the VL-CDR3 of the amino acid sequence of SEQ ID NO: 10.
  • the VH comprises: VH-CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, VH-CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and VH-CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2 3 or the VH-CDR3 composed of the amino acid sequence shown in SEQ ID NO: 4;
  • the VL includes: the VL-CDR1 composed of the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, and the VL-CDR1 composed of the amino acid sequence shown in SEQ ID NO: 6
  • VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8
  • VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10.
  • the VH comprises: a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a VH comprising the amino acid sequence of SEQ ID NO:3 -CDR3.
  • the VH comprises: a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a VH comprising the amino acid sequence of SEQ ID NO:4 -CDR3.
  • the VL comprises: VL-CDR1 comprising the amino acid sequence of SEQ ID NO:5, VL-CDR2 comprising the amino acid sequence of SEQ ID NO:7, and VL comprising the amino acid sequence of SEQ ID NO:9 -CDR3.
  • the VL comprises: VL-CDR1 comprising the amino acid sequence of SEQ ID NO:6, VL-CDR2 comprising the amino acid sequence of SEQ ID NO:8, and VL comprising the amino acid sequence of SEQ ID NO:10 -CDR3.
  • the antibody comprises a VH and a VL
  • the VH comprises: a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:2 and a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:2
  • the VL includes: VL-CDR1 comprising the amino acid sequence of SEQ ID NO:5, VL-CDR2 comprising the amino acid sequence of SEQ ID NO:7 and VL-CDR2 comprising the amino acid sequence of SEQ ID NO:7 :9 amino acid sequence of VL-CDR3.
  • the antibody comprises a VH and a VL
  • the VH comprises: a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:2 and a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:2
  • the VL includes: VL-CDR1 comprising the amino acid sequence of SEQ ID NO:6, VL-CDR2 comprising the amino acid sequence of SEQ ID NO:8 and VL-CDR2 comprising the amino acid sequence of SEQ ID NO:8 : 10 amino acid sequence of VL-CDR3.
  • the antibody comprises VH and VL
  • the VH comprises: VH-CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:1, VH consisting of the amino acid sequence set forth in SEQ ID NO:2 -CDR2 and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3
  • the VL includes: VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 5, represented by SEQ ID NO: 7
  • VL-CDR2 consisting of the amino acid sequence shown
  • VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9; or
  • the VH includes: VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 4 and the VL includes: VL-CDR1 composed of the amino acid sequence shown in SEQ ID NO: 6, VL-CDR2 composed of the amino acid sequence shown in SEQ ID NO: 8 and VL-CDR2 composed of the amino acid sequence shown in SEQ ID NO: 8 : VL-CDR3 composed of the amino acid sequence shown in 10.
  • each CDR in the VH and the VL is identified by the Kabat numbering system.
  • the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 23 or 24, or the VH comprises 85% or more of SEQ ID NO: 23 or 24, Preferably, amino acid sequences of 90% or more, more preferably 95% or more or more preferably 99% or more identical, consist essentially of or consist thereof.
  • the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25 or 26, or the VL comprises more than 85% of SEQ ID NO: 25 or 26, Preferably, amino acid sequences of 90% or more, more preferably 95% or more or more preferably 99% or more identical, consist essentially of or consist thereof.
  • the VH or VL of the antibody further comprises more than one framework region (hereinafter abbreviated as FWR).
  • the VH comprises one, two, three or four FWRs, each FWR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, 14, 15 and The sequence shown in 16, the amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 99% identity thereto, and the amino acid sequence of any one of SEQ ID NOs: 11, 12, 13, 14, 15 and 16 Amino acid sequences in the group consisting of amino acid sequences having more than one amino acid substitution, insertion or deletion are compared.
  • the aforementioned VH comprises four FWRs, wherein FWR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, FWR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13, and FWR3 comprises the amino acid sequence set forth in SEQ ID NO: 13 ID NO: 14 or the amino acid sequence shown in SEQ ID NO: 15, FWR4 comprises the amino acid sequence shown in SEQ ID NO: 16.
  • the VL comprises one, two, three or four FWRs, each FWR comprising an amino acid sequence selected from SEQ ID NOs: 17, 18, 19, 20, 21 and
  • the sequence shown in 22 has an amino acid sequence that is at least 90%, at least 95%, at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 17, 18, 19, 20, 21 and 22
  • the aforementioned VL comprises four FWRs, wherein FWR1 comprises the amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, FWR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, and FWR3 comprises the amino acid sequence set forth in SEQ ID NO: 19 ID NO: 20 or the amino acid sequence shown in SEQ ID NO: 21, FWR4 comprises the amino acid sequence shown in SEQ ID NO: 22.
  • the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 23, and the VL comprises, or consists essentially of the amino acid sequence of SEQ ID NO: 25 or consist of it.
  • the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 24, and the VL comprises, or consists essentially of the amino acid sequence of SEQ ID NO: 26 or consist of it.
  • the heavy chain of the antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:27 or 29, or the heavy chain of the antibody comprises the same sequence as SEQ ID NO:27 or 29 consists essentially of or consists of an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95% or further preferably at least 99% identity.
  • the light chain of the antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:28 or 30, or the light chain of the antibody comprises the same sequence as SEQ ID NO:28 or 30 consists essentially of or consists of an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95% or further preferably at least 99% identity.
  • the heavy chain of the antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:27
  • the light chain of the antibody comprises the amino acid sequence of SEQ ID NO:28, or consist essentially of or consist of it.
  • the heavy chain of the antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:29, and the light chain of the antibody comprises the amino acid sequence of SEQ ID NO:30, or consist essentially of or consist of it.
  • the aforementioned antibodies further comprise at least one of a heavy chain constant region and a light chain constant region.
  • the heavy chain constant region is selected from mouse IgG1, mouse IgG2a, mouse IgG2b or mouse IgG3, and the light chain constant region is selected from kappa chain or lambda chain.
  • the heavy chain constant region is mouse IgG1, and the light chain constant region is the kappa chain of a mouse antibody.
  • the aforementioned antibody is a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, monoclonal antibody, bispecific antibody or multispecific antibody.
  • the present invention also discloses an isolated nucleic acid molecule encoding the antibody as described above.
  • the present invention also discloses a cloning vector or an expression vector, the expression vector comprising the aforementioned nucleic acid molecule.
  • the present invention also discloses a host cell, which comprises one or more cloning vectors or expression vectors as described above.
  • the present invention also discloses a method for producing the aforementioned antibody, comprising culturing the aforementioned host cell, and isolating the antibody.
  • the present invention also discloses a composition or kit, comprising the aforementioned antibody, and one or more pharmaceutically acceptable excipients, diluents or carriers.
  • the present invention also discloses a conjugate comprising the aforementioned antibody linked to a detectable label.
  • detectable labels include, but are not limited to, fluorescent labels, enzyme substrate labels, radioisotopes, digoxigenin, biotin or avidin, DNA molecules for detection, or gold.
  • the fluorescent label includes but is not limited to fluorescein, rhodamine, dansir, phycoerythrin or Texas red.
  • Enzyme substrate tags include, but are not limited to, horseradish peroxidase, alkaline phosphatase, saccharidase, lysozyme, carbohydrate oxidase, or beta-D-galactosidase.
  • Radioisotopes include but are not limited to123I , 124I , 125I , 131I , 35S , 3H , 111In , 112In , 14C , 64Cu , 67Cu,86Y , 88Y , 90Y , 177Lu , 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, other lanthanides.
  • the label can also be a luminescent label or a chromophore.
  • the present invention also discloses a composition or kit comprising the aforementioned conjugate and one or more pharmaceutically acceptable excipients, diluents or carriers.
  • the present invention also discloses the use of the aforementioned antibody in preparing a reagent or a kit for detecting the fusion polypeptide containing the Strep-Tag II tag in a sample.
  • the present invention also discloses a method for detecting a fusion polypeptide containing a Strep-Tag II tag in a sample, comprising (i) in vitro allowing the sample containing the fusion polypeptide to interact with the antibody and the fusion polypeptide under conditions
  • the isolated antibody disclosed herein is contacted, and (ii) the formation of a complex between the antibody and the fusion polypeptide is detected.
  • the method for detecting the complex includes, but is not limited to, at least one of flow cytometry, Western blotting, immunohistochemistry, and immunofluorescence.
  • the sample containing the Strep-Tag II tag is a CAR-T cell containing the Strep-Tag II tag.
  • the present invention also discloses a method for purifying a fusion polypeptide containing a Strep-Tag II tag in a sample, comprising (i) in vitro purifying the fusion polypeptide containing the fusion polypeptide under conditions that allow the antibody to interact with the fusion polypeptide.
  • the sample of the present invention is contacted with the isolated antibody disclosed herein, and (ii) the complex between the purified antibody and the fusion polypeptide is purified.
  • the purification methods include but are not limited to affinity chromatography, immunoprecipitation and the like.
  • the sample is a biological sample comprising at least one of blood, urine, saliva, lymph, cerebrospinal fluid, bone marrow, tissue organs, cells.
  • the blood includes at least one of serum, plasma, and whole blood.
  • the sample comprises chimeric antigen receptor cells or engineered cell receptor cells.
  • the sample is a chimeric antigen receptor cell or an engineered cell receptor cell.
  • the antibody of the present invention has stronger affinity, can more specifically bind to the Strep-Tag II label in the chimeric antigen receptor cells or the engineered cell receptor cells, and effectively monitor the chimeric antigen receptor cells or the engineered cell receptor cells in Amplification efficiency in vitro and in vivo.
  • Figure 1 shows the titer curve of sera from mice after four immunizations.
  • Figure 2 shows the polyacrylamide gel electrophoresis pattern of antibody 8F8D1.
  • Lane M is the molecular weight standard (within 300KD); Lane 1 is the heavy chain (about 55KD) and light chain (about 24KD) of antibody 8F8D1.
  • Figure 3 shows flow cytometry profiles of antibodies 8F8D1 and 8A882.
  • Figure 4 shows the affinity test graph of antibodies 8F8D1 and 8A882.
  • Figure 5 shows a graph of the metabolism of CAR-T cells in mice monitored using the antibody 8F8D1.
  • the term "antigen" as used herein refers to a molecule that elicits an immune response, which may involve antibody production, or activation of specific immunocompetent cells.
  • the immunizing antigen in the present invention can be Strep-Tag II, whose amino acid sequence is NWSHPQFEK, or Strep-Tag II (NWSHPQFEK-KLH) coupled to hemocyanin (Keyhole Limpet Hemocyanin, KLH).
  • Antigens can be generated, synthesized, or can be derived from biological samples including, but not limited to, tissue samples, tumor samples, cells, or biological fluids.
  • detection antigen refers to an antigen used to determine antibody titer, such as Strep-Tag II (NWSHPQFEK-OVA) conjugated to chicken ovalbumin (Ovalbumin, OVA), but those skilled in the art can understand detection
  • NWSHPQFEK-OVA Strep-Tag II conjugated to chicken ovalbumin
  • OVA ovalbumin
  • the antigen is not limited to NWSHPQFEK-OVA, and antigens containing NWSHPQFEK or a part thereof can also be used as detection antigens, so the use of this specific detection antigen should not be considered as a limitation on the antibody preparation method.
  • peptide and “polypeptide” refer to compounds consisting of amino acid residues covalently linked by peptide bonds.
  • a "fusion polypeptide” refers to a polypeptide to which a Strep-Tag II tag is attached, such as the N-terminal or C-terminal of a heavy chain variable region or a light chain variable region in the extracellular domain scFv of a chimeric antigen receptor End-linked Strep-Tag II tag.
  • Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.
  • antibody as used herein is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), full-length antibodies, and antibody fragments ( or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • the antibodies include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, di-scFv, tri-scFv, Fd, and other antibody fragments that retain antigen-binding function; it can also be a dimeric structure Diabody Or the trimeric structure Triabody.
  • Antigen-binding fragments typically include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), which can be further subdivided into: hypervariable regions called complementarity determining regions (CDRs), and interspersed Distributed more conserved regions called framework regions (FWRs).
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • CDRs complementarity determining regions
  • FWRs framework regions
  • the full-length antibody refers to a protein comprising at least two heavy chains and two light chains interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region and a heavy chain constant region
  • each light chain consists of a light chain variable region and a light chain constant region.
  • the heavy chain constant region can be selected from mouse-derived IgG1, IgG2a, IgG2b or IgG3, and the light chain constant region is selected from ⁇ chain or ⁇ chain.
  • chimeric antigen receptor or “CAR” as used herein refers to an artificial receptor engineered to be expressed on immune effector cells and capable of specifically binding an antigen.
  • Chimeric antigen receptor cells refers to immune cells expressing artificial receptors that can specifically bind to antigens on cells, wherein immune cells include lymphocytes, natural killer cells, dendritic cells, monocytes/macrophages, Granulocytes, mast cells, etc., can be used for therapy using adoptive cell transfer.
  • engineered cell receptor or “TCR” used in the present invention, also known as engineered T cell receptor, is a heterodimer composed of a peptide chain of TCR through in vitro genetic engineering to make it more effective. Recognize tumor intracellular antigen polypeptides presented by MHC, and then kill and treat tumors.
  • the fusion polypeptide in the present invention includes CAR or TCR fused with Strep-Tag II tag.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or peptide that occurs naturally in a living animal is not “isolated”, but the same nucleic acid or peptide that is partially or completely separated from coexisting materials in its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-natural environment, eg, a host cell.
  • An "isolated” antibody is one that has been identified and separated from and/or recovered from components of its natural environment.
  • the antibody is purified to greater than 95% or 99% purity, as determined by electrophoresis (eg, SDS-PAGE, isoelectric focusing IEF, capillary electrophoresis) or chromatography (eg, ion exchange or reverse phase HPLC).
  • a host cell as used herein can be any prokaryotic or eukaryotic cell that contains a cloning vector or expression vector, including those prokaryotic or eukaryotic cells that have been genetically engineered to contain a cloned gene in the chromosome or genome of the host cell. Special transgenic animals with modified immune systems can also be used to make antibodies.
  • the immunization antigen is Strep-Tag II (NWSHPQFEK-KLH) conjugated to hemocyanin (Keyhole Limpet Hemocyanin, KLH); the detection antigen is Strep-Tag II (NWSHPQFEK-OVA) conjugated to chicken ovalbumin (Ovalbumin, OVA). All were purchased from Hangzhou Zhongpeptide Biochemical Co., Ltd.
  • the immunizing antigen protein (Strep-Tag II coupled to KLH (NWSHPQFEK-KLH) polypeptide) was dissolved in physiological saline at a concentration of 0.5 mg/mL.
  • mice 100 uL of the diluted antigen protein solution and incomplete Freund's adjuvant (Sigma; Cat#: 1002036152) were mixed at a volume ratio of 1:1, and Balb/c mice were immunized by subcutaneous injection at multiple points in the abdomen for booster immunization. Each mouse was injected with a volume of 200 ⁇ L for a total of 4 antigen immunizations. Serum titers were determined by tail-docking blood on days 22, 36 and 50, respectively.
  • Mouse serum titers were detected by enzyme-linked immunosorbent assay (ELISA) using NWSHPQFEK-OVA polypeptide as follows: 1 ⁇ g/mL OVA-conjugated Strep-Tag II (NWSHPQFEK-OVA) was added to 96 at 4°C Plates (purchased from Corning) were left overnight, and the microtiter plates were washed and washed 5 times with phosphate buffered saline (PBST) containing 0.05% Tween-20. Plates were blocked with 1% bovine serum albumin for 2 hours at room temperature and washed 5 times with PBST.
  • PBST phosphate buffered saline
  • mouse serum was diluted 4 times from 1:1000 to 1:102400 and added to the ELISA plate, incubated at room temperature for 1 hour, and washed 5 times with PBST.
  • 100 ⁇ L/well of HRP-labeled goat anti-mouse secondary antibody (Invitrogen, Cat#: 31430) was added and incubated for 1 hour at room temperature after washing.
  • 50 ⁇ L/well of 3,3',5,5'-tetramethylbenzidine (TMB) was then added to the plate, incubated at room temperature for 15 minutes, then the reaction was stopped and the results read.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • mice #4 were selected for hybridoma fusion, and 3 days before fusion, mice were injected intraperitoneally with 50 ⁇ g of immunizing antigen for shock.
  • the spleen of mouse #4 was extracted, minced and resuspended with Dulbecco's Phosphate Buffered Saline (DPBS) to extract single cells of mouse spleen B cells, centrifuged at 500g for 5 minutes, and the pellet was resuspended in DPBS Then, the cells were washed by centrifugation at 500 g for 5 minutes, and the splenocytes were mixed with mouse myeloma cell line sp2/0 (purchased from Nordic Biotechnology (Beijing) Co., Ltd.) in a ratio of 3:1 (the ratio of the number of cells), and 1 mL was added.
  • DPBS Dulbecco's Phosphate Buffered Saline
  • CAR lentiviral transfer plasmid encoding CD19 and lentiviral preparation 1) The chimeric gene encoding Strep-Tag II and scFv targeting CD19 was synthesized by gene synthesis (Beijing Biomed Gene Technology Co., Ltd. ).
  • step 4) Insert the complete nucleic acid fragment obtained in step 3) into the lentiviral vector pLenti6.3/V5 (Thermo Fisher, Waltham, MA, USA) by the method of restriction enzyme cleavage and ligation to obtain the Strep-Tag II and target Lentiviral transfer plasmid to CD19 CAR gene.
  • the lentiviral packaging plasmids pLP/VSVG, pLP1/MDK, pLP2/RSK (Thermo Fisher, Waltham, MA, USA) and the transfer plasmid obtained in step 4) were mixed with Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA) After transfection into HEK293T cells, the culture medium was collected after 48 hours, centrifuged at 300 g to remove cell debris, and centrifuged at 25,000 rpm for 3 hours with an ultracentrifuge. Dissolve the pellet with 1 mL of physiological saline, which is the desired lentiviral vector.
  • T cells were isolated from peripheral blood mononuclear cells of healthy volunteers (Miaotong (Shanghai) Biotechnology Co., Ltd., China) using CD3/CD28 Dynabeads (Thermo Fisher), The isolated T cells (the T cells at this time were linked with CD3/CD28 Dynabeads) were cultured in a fresh X-VIVO 15 culture system containing IL-2 (500IU/ml) for 48 hours, and the above lentiviral vector was used. Infected T cells. Twenty-four hours after virus infection of cells, the cells were centrifuged to change the medium, and the culture was continued in the above-mentioned culture system.
  • CAR-T cells containing Strep-Tag II tag were collected, washed with DPBS and counted, and the cells were aliquoted into EP tubes, about 3 ⁇ 10 5 cells per tube. After the supernatant was collected by centrifugation, 100 ⁇ L/well of hybridoma supernatant was added, and incubated at room temperature for 15 minutes. After washing twice, add 5 ⁇ L of labeled PE-labeled rat anti-mouse IgG (BD, Cat#: 550083) to each tube, incubate at room temperature for 15 minutes, and then wash the cells twice with DPBS for flow cytometry FACS analysis.
  • PE-labeled rat anti-mouse IgG BD, Cat#: 550083
  • cDNA was generated by RT-PCR, and then the heavy and light chains were amplified by PCR (RT-PCR kit was purchased from Quanjin Gold, Cat#: AE311-03, GXL high-fidelity DNA polymerase was used for PCR, purchased from TaKaRa , Cat#: R050A, see the product manual for specific operation). Then, the PCR product was cleaned with a cleaning kit (AXYGEN, Cat#: 155. For the specific operation, see the product manual), followed by sequencing, and the sequencing was handed over to Beijing Ruibo Xingke Co., Ltd.
  • the amino acid sequences of the light chain variable region and the heavy chain variable region are shown in Tables 1 and 2, and the Sequence Listing.
  • the screened antibody clone numbers are 8A882 and 8F8D1, respectively.
  • mice Five Balb/c mice aged 6-8 weeks were ordered, and 1 mL of paraffin (purchased from Jiangxi Yipusheng Pharmaceutical) was injected after one week of feeding, and 1 ⁇ 10 6 monoclonal hybridoma cells were injected into each mouse the following week. After one week, the mice were observed to have obvious abdominal distension, and the mice were punctured and drawn every 48 hours. 3-4 mL of ascites was collected from each mouse, and a total of 20 mL of ascites was collected.
  • paraffin purchased from Jiangxi Yipusheng Pharmaceutical
  • the antibody was purified by affinity purification method, and the gravity column was equilibrated with Binding Buffer (pH 7.0) and washed with 2-5 times the column volume. Pass the filtered ascites through a column (ProteinA/G column, ordered by Thermo Fisher, Cat#: 89930); rinse the column with 5-10 column volumes of Binding Buffer, and then elute the target protein with 0.1M glycine at pH 3.5, and then use Tris-HCl pH 9.0 was adjusted to neutrality. Finally, BCA kit (Thermo; Cat#: 23227, see the product manual for specific operations) was used to determine the antibody concentration. The purity of the antibody was identified by SDS-PAGE, and Figure 2 shows that the antibody has a higher purity.
  • the purified high-purity antibody was identified by FACS.
  • the CAR-T cells containing the Strep-Tag II tag (manufactured by our company) prepared in Example 2.2 were collected, washed with FACS buffer and counted. Aliquot cells into EP tubes at approximately 3 x 105 cells per tube. After centrifuging to remove the supernatant, add 8A882 antibody or 8F8D1 antibody or positive control antibody at a final concentration of 1 ⁇ g/mL, and incubate at room temperature for 15 minutes. After 2 washes, 5 ⁇ L of PE-labeled rat anti-mouse IgG was added to each tube, and incubated at room temperature for 15 minutes. Cells were then washed 2 times before FACS analysis.
  • the experimental results show that under the condition of the same amount of detection cells (CAR-T), after treatment with the 8F8D1 and 8A882 antibodies of the present invention, the expression rates of CAR-T cells containing the Strep-Tag II tag are 48.07% and 36.41%, respectively. , much higher than the 27.63% expression rate of the positive control antibody treatment.
  • the above results demonstrate that the antibody of the present invention can specifically recognize the Strep-Tag II short peptide, and the antibody of the present invention has a higher affinity for the Strep-Tag II short peptide than the commercial positive control antibody (Fig. 3).
  • the negative control was no Strep-Tag II tag antibody but only negative control IgG was added, and the positive control was a commercial anti-Strep-Tag II tag antibody (Abeam; Cat#: ab184224).
  • the antibodies of the present invention are labeled with two different fluorescences, FITC and PE, respectively, for the purpose of multi-site detection and analysis.
  • Antibody fluorescent labeling was done by Beijing Sizhengbai Biotechnology Co., Ltd.
  • the antibody affinity detection of the present invention adopts the dynamic equilibrium assay method (saturated concentration method).
  • saturated concentration method saturated concentration method
  • the antibody is serially diluted, and the concentration of the antigen-antibody complex is detected.
  • concentration of the antigen-antibody complex accounts for half of the total antigen concentration
  • the corresponding concentration value (EC50) of the antibody is the concentration of the antibody relative to the antigen. KD value.
  • Strep-Tag II-tagged CAR-T cells were collected, washed with DPBS and counted. Aliquot into several EP tubes, approximately 3 x 105 cells per tube. After centrifugation to remove the supernatant, serial dilutions of 10 concentrations of 8A882 or 8F8D1 and a positive control antibody (commercial Strep-Tag II antibody, Abcam; Cat#: ab184224) were added and incubated at room temperature for 15 minutes. After washing the cells twice with DPBS, PE-labeled rat anti-mouse IgG was added, and incubated at room temperature for 15 minutes. After washing the cells twice, FACS analysis and GraphPad Prism software were used to calculate the EC50 value.
  • the EC50 values of 8A882 and 8F8D1 and the positive control antibody obtained by software analysis were 0.4 nM, 0.2 nM and 0.55 nM, respectively (Fig. 4). Compared with the positive control antibody, the two strain antibodies of the present invention have high affinity.
  • Example 6 Using antibodies to detect CAR-T cells in peripheral blood of tumor-bearing mice
  • mice A total of 6 6-8 week old NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., China) were injected with 1.0 ⁇ 10 6 Nalm-6-LAE cells (ATCC, USA) from the tail vein of each mouse, 5 After a few days, the mice were analyzed by luciferase in vivo imaging (Lumina II small animal in vivo imaging system, PerkinElmer, USA) to confirm that the mouse leukemia model was successfully made, and each mouse was injected with CD19 CAR-T cells from the tail vein (refer to the implementation of The method in Example 2.2 was prepared by our company, 2 ⁇ 10 6 cells/mouse).
  • luciferase in vivo imaging Lumina II small animal in vivo imaging system, PerkinElmer, USA

Abstract

本发明公开了一种特异性结合Strep-TagII标签的抗体，进一步公开了本发明抗体的氨基酸序列、克隆或表达载体、宿主细胞、以及用于表达或分离抗体的方法，还公开了包含本发明抗体的组合物。本发明还公开了使用本发明抗体检测或纯化表达Strep-Tag II标签的生物样品的方法。

Description

一种特异性结合Strep-Tag II标签的抗体及其应用 技术领域
本发明涉及一种特异性结合Strep-Tag II标签的抗体，并公开了该抗体的氨基酸序列、克隆或表达载体、宿主细胞、生产抗体的方法以及使用该抗体纯化或检测表达Strep-Tag II标签的生物样品的方法。
背景技术
随着肿瘤免疫治疗的发展，CAR-T(Chimeric antigen receptor-T cell，嵌合抗原受体T细胞)和TCR-T(T cell receptor-T cell，工程化T细胞受体T细胞)等细胞免疫治疗策略受到极大关注。CAR-T或TCR-T在体内的扩增水平是指征其疗效的一个重要指标，因此，研发快速准确检测细胞的试剂或方法，将有助于有效监测其在体内外的扩增效率。
目前对于CAR-T或TCR-T细胞进行检测的方法有两种：使用定量实时聚合酶联反应(Quantitative real time PCR，qPCR)检测T细胞中的CAR或TCR的基因表达；以及检测T细胞中的CAR蛋白或TCR蛋白表达。其中，对于检测T细胞中的CAR蛋白表达，常用特异性结合CAR蛋白中scFv(Single-chain variable fragment)片段的抗体对T细胞进行流式细胞检测，或用结合Fab片段的抗体或protein L等检测CAR蛋白中的scFv片段。对于检测T细胞中的TCR蛋白的表达，常用特异针对某TCR的HLA和多肽的复合物对T细胞进行流式检测。但这些方法存在明显的局限性：qPCR检测基因拷贝数方法不能准确反映表达CAR的T细胞，因为对于整合了CAR基因但未正常表达CAR蛋白的细胞，使用qPCR检测方法会造成检测的假阳性；而对于CAR蛋白阳性的T细胞的检测方法，Fab抗体的检测背景值较高，容易造成假阳性；protein L只适用于检测带有κ轻链的scFv的CAR，而针对CAR蛋白中scFv片段的抗体，以及针对TCR的HLA和多肽复合物，则需要针对不同的CAR蛋白或TCR筛选出不同的抗体或复合物，并建立相关检测方法，耗时长，效率低，成本 高。如果能开发出一种准确、灵敏且通用的抗体检测CAR-T，将大大提高基因转导的细胞治疗药物的检测效率，降低检测成本。
发明内容
本发明公开了一种特异性结合Strep-Tag II标签的分离的抗体，其包括：重链可变区(以下缩写为VH)和轻链可变区(以下缩写为VL)，其中所述VH包括：包含SEQ ID NO:1的氨基酸序列的VH-CDR1，包含SEQ ID NO:2的氨基酸序列的VH-CDR2和包含SEQ ID NO:3或SEQ ID NO:4的氨基酸序列的VH-CDR3；所述VL包括：包含SEQ ID NO:5或SEQ ID NO:6的氨基酸序列的VL-CDR1，包含SEQ ID NO:7或SEQ ID NO:8的氨基酸序列的VL-CDR2和包含SEQ ID NO:9或SEQ ID NO:10的氨基酸序列的VL-CDR3。
在一些实施方案中，所述VH包括：由SEQ ID NO:1所示的氨基酸序列组成的VH-CDR1，由SEQ ID NO:2所示的氨基酸序列组成的VH-CDR2和由SEQ ID NO:3或SEQ ID NO:4所示的氨基酸序列组成的VH-CDR3；所述VL包括：由SEQ ID NO:5或SEQ ID NO:6所示的氨基酸序列组成的VL-CDR1，由SEQ ID NO:7或SEQ ID NO:8所示的氨基酸序列组成的VL-CDR2和由SEQ ID NO:9或SEQ ID NO:10所示的氨基酸序列组成的VL-CDR3。
在一些实施方案中，所述VH包括：包含SEQ ID NO:1的氨基酸序列的VH-CDR1，包含SEQ ID NO:2的氨基酸序列的VH-CDR2和包含SEQ ID NO:3的氨基酸序列的VH-CDR3。
在一些实施方案中，所述VH包括：包含SEQ ID NO:1的氨基酸序列的VH-CDR1，包含SEQ ID NO:2的氨基酸序列的VH-CDR2和包含SEQ ID NO:4的氨基酸序列的VH-CDR3。
在一些实施方案中，所述VL包括：包含SEQ ID NO:5的氨基酸序列的VL-CDR1，包含SEQ ID NO:7的氨基酸序列的VL-CDR2和包含SEQ ID NO:9的氨基酸序列的VL-CDR3。
在一些实施方案中，所述VL包括：包含SEQ ID NO:6的氨基酸序列的VL-CDR1，包含SEQ ID NO:8的氨基酸序列的VL-CDR2和包含 SEQ ID NO:10的氨基酸序列的VL-CDR3。
在一些实施方案中，所述抗体包括VH和VL，其中所述VH包括：包含SEQ ID NO:1的氨基酸序列的VH-CDR1，包含SEQ ID NO:2的氨基酸序列的VH-CDR2和包含SEQ ID NO:3的氨基酸序列的VH-CDR3；并且所述VL包括：包含SEQ ID NO:5的氨基酸序列的VL-CDR1，包含SEQ ID NO:7的氨基酸序列的VL-CDR2和包含SEQ ID NO:9的氨基酸序列的VL-CDR3。
在一些实施方案中，所述抗体包括VH和VL，其中所述VH包括：包含SEQ ID NO:1的氨基酸序列的VH-CDR1，包含SEQ ID NO:2的氨基酸序列的VH-CDR2和包含SEQ ID NO:4的氨基酸序列的VH-CDR3；并且所述VL包括：包含SEQ ID NO:6的氨基酸序列的VL-CDR1，包含SEQ ID NO:8的氨基酸序列的VL-CDR2和包含SEQ ID NO:10的氨基酸序列的VL-CDR3。
在一些实施方案中，所述抗体包括VH和VL，所述VH包括：由SEQ ID NO:1所示的氨基酸序列组成的VH-CDR1，由SEQ ID NO:2所示的氨基酸序列组成的VH-CDR2和由SEQ ID NO:3所示的氨基酸序列组成的VH-CDR3；并且所述VL包括：由SEQ ID NO:5所示的氨基酸序列组成的VL-CDR1，由SEQ ID NO:7所示的氨基酸序列组成的VL-CDR2和由SEQ ID NO:9所示的氨基酸序列组成的VL-CDR3；或
所述VH包括：由SEQ ID NO:1所示的氨基酸序列组成的VH-CDR1，由SEQ ID NO:2所示的氨基酸序列组成的VH-CDR2和由SEQ ID NO:4所示的氨基酸序列组成的VH-CDR3；并且所述VL包括：由SEQ ID NO:6所示的氨基酸序列组成的VL-CDR1，由SEQ ID NO:8所示的氨基酸序列组成的VL-CDR2和由SEQ ID NO:10所示的氨基酸序列组成的VL-CDR3。
在本发明中，所述VH和所述VL中各CDR由Kabat编号系统确定。
在一些实施方案中，所述VH包含SEQ ID NO:23或24的氨基酸序列、或基本上由其组成或由其组成，或所述VH包含与SEQ ID NO:23或24具有85％以上、优选90％以上、更优选95％以上或进一步优选99％以上同一性的氨基酸序列、或基本上由其组成或由其组成。
在一些实施方案中，所述VL包含SEQ ID NO:25或26的氨基酸序 列、或基本上由其组成或由其组成，或所述VL包含与SEQ ID NO:25或26具有85％以上、优选90％以上、更优选95％以上或进一步优选99％以上同一性的氨基酸序列、或基本上由其组成或由其组成。
在一些实施方案中，所述抗体的VH或VL进一步包括一个以上的框架区(以下缩写为FWR)。
在一些实施方案中，所述VH包括一个、两个、三个或四个FWR，每个FWR包含一个氨基酸序列，所述氨基酸序列选自由SEQ ID NO:11、12、13、14、15和16所示的序列，与其具有至少90％、优选至少95％、更优选至少99％同一性的氨基酸序列，与SEQ ID NO:11，12，13，14，15和16中任一个的氨基酸序列相比具有一个以上的氨基酸置换、插入或缺失的氨基酸序列组成的组中的氨基酸序列。在一些具体实施方案中，前述VH包括四个FWR，其中FWR1包含SEQ ID NO:11所示的氨基酸序列，FWR2包含SEQ ID NO:12或SEQ ID NO:13所示的氨基酸序列，FWR3包含SEQ ID NO:14或SEQ ID NO:15所示的氨基酸序列，FWR4包含SEQ ID NO:16所示的氨基酸序列。
在一些实施方案中，所述VL包括一个、两个、三个或四个FWR，每个FWR包含一个氨基酸序列，所述氨基酸序列选自由SEQ ID NO:17、18、19、20、21和22所示的序列，与其至少90％、至少95％、至少99％同一性的氨基酸序列，与SEQ ID NO:17，18，19，20，21和22中任一个的氨基酸序列相比具有一个以上氨基酸置换、插入或缺失的氨基酸序列组成的组中的氨基酸序列。在一些具体实施方案中，前述VL包括四个FWR，其中FWR1包含SEQ ID NO:17或SEQ ID NO:18所示的氨基酸序列，FWR2包含SEQ ID NO:19所示的氨基酸序列，FWR3包含SEQ ID NO:20或SEQ ID NO:21所示的氨基酸序列，FWR4包含SEQ ID NO:22所示的氨基酸序列。
在一些实施方案中，所述VH包含SEQ ID NO:23的氨基酸序列、或基本上由其组成或由其组成，并且所述VL包含SEQ ID NO:25的氨基酸序列、或基本上由其组成或由其组成。
在一些实施方案中，所述VH包含SEQ ID NO:24的氨基酸序列、或基本上由其组成或由其组成，并且所述VL包含SEQ ID NO:26的氨基酸 序列、或基本上由其组成或由其组成。
在一些实施方案中，所述抗体的重链包含SEQ ID NO:27或29的氨基酸序列、或基本上由其组成或由其组成，或所述抗体的重链包含与SEQ ID NO:27或29具有至少85％、优选至少90％、更优选至少95％或进一步优选至少99％同一性的氨基酸序列、或基本上由其组成或由其组成。
在一些实施方案中，所述抗体的轻链包含SEQ ID NO:28或30的氨基酸序列、或基本上由其组成或由其组成，或所述抗体的轻链包含与SEQ ID NO:28或30具有至少85％、优选至少90％、更优选至少95％或进一步优选至少99％同一性的氨基酸序列、或基本上由其组成或由其组成。
在一些实施方案中，所述抗体的重链包含SEQ ID NO:27的氨基酸序列、或基本上由其组成或由其组成，并且所述抗体的轻链包含SEQ ID NO:28的氨基酸序列、或基本上由其组成或由其组成。
在一些实施方案中，所述抗体的重链包含SEQ ID NO:29的氨基酸序列、或基本上由其组成或由其组成，并且所述抗体的轻链包含SEQ ID NO:30的氨基酸序列、或基本上由其组成或由其组成。
在一些实施方案中，前述抗体进一步包括重链恒定区和轻链恒定区的至少一个。所述重链恒定区选自小鼠IgG1、小鼠IgG2a、小鼠IgG2b或小鼠IgG3，所述轻链恒定区选自κ链或者λ链。优选所述重链恒定区为小鼠IgG1，且所述轻链恒定区为小鼠抗体的κ链。
在一些实施方案中，前述抗体是全长抗体、Fab、Fab’、F(ab’)2、Fv、scFv、单克隆抗体、双特异性抗体或多特异性抗体。
所述示例性抗体的序列如表1和序列表所示。
表1示例性抗体氨基酸序列
Figure PCTCN2021136113-appb-000001
Figure PCTCN2021136113-appb-000002
Figure PCTCN2021136113-appb-000003
所述示例性抗体的CDR序列和FWR序列如表2和表3所示。
表2示例性抗体的CDR序列
Figure PCTCN2021136113-appb-000004
表3示例性抗体的FWR序列
Figure PCTCN2021136113-appb-000005
本发明还公开了一种分离的核酸分子，该核酸分子编码如前所述的抗体。
本发明还公开了一种克隆载体或表达载体，该表达载体包含如前所述的核酸分子。
本发明还公开了一种宿主细胞，该宿主细胞包含一个以上如前所述的克隆载体或表达载体。
本发明还公开了一种用于生产如前所述的抗体的方法，包括培养如前所述的宿主细胞，并且分离所述抗体。
本发明还公开了一种组合物或试剂盒，包含如前所述的抗体，以及一种以上药学可接受的赋形剂、稀释剂或载体。
本发明还公开了一种偶联物，包含连接至可检测标记的如前所述的抗体。本领域技术人员可以理解任何可检测的标记均可用于构建本发明的偶联物。示例性的可检测标记包括但不限于荧光标签、酶底物标签、放射性同位素、地高辛素、生物素或亲和素、用于检测的DNA分子或金。其中，荧光标签包括但不限于荧光素、罗丹明、丹西尔、藻红蛋白或德州红。酶底物标签包括但不限于辣根过氧化物酶、碱性磷酸酶、糖化酶、溶菌酶、糖氧化酶或β-D-半乳糖苷酶。放射性同位素包括但不限于 123I、 124I、 125I、 131I、 35S、 3H、 111In、 112In、 14C、 64Cu、 67Cu、 86Y、 88Y、 90Y、 177Lu、 211At、 186Re、 188Re、 153Sm、 212Bi、 32P、其它镧系元素。所述标签还可以是一种发光标记或一种发色基团。
本发明还公开了一种组合物或试剂盒，包含如前所述的偶联物和一种以上药学可接受的赋形剂、稀释剂或载体。
本发明还公开了如前所述的抗体在制备用于在样品中检测含有Strep-Tag II标签的融合多肽的试剂或试剂盒中的用途。
本发明还公开了一种检测样品中含有Strep-Tag II标签的融合多肽的方法，包括(i)在允许抗体和所述融合多肽发生相互作用的条件下，体外使含有所述融合多肽的样品接触本发明公开的分离的抗体，和(ii)检测所述抗体和融合多肽之间的复合物的形成。其中检测所述复合物的方法包括但不限于流式、蛋白质印迹、免疫组化、免疫荧光中的至少一种。
在一些具体实施方式中，含有Strep-Tag II标签的样品是含有Strep-Tag II标签的CAR-T细胞。
本发明还公开了一种纯化样品中含有Strep-Tag II标签的融合多肽的 方法，包括(i)在允许所述抗体和所述融合多肽发生相互作用的条件下，体外使含有所述融合多肽的样品接触本发明公开的的分离的抗体，和(ii)纯化抗体和融合多肽之间的复合物。其中纯化方法包括但不限于亲和层析、免疫沉淀等。
在一些实施方案中，所述样品是包括血液、尿液、唾液、淋巴液、脑脊液、骨髓、组织器官、细胞中至少一种的生物样品。
在一个具体实施例中，所述血液包括血清、血浆和全血中的至少一种。
在一些实施方案中，所述样品包含嵌合抗原受体细胞或工程化细胞受体细胞。
在一些实施方案中，所述样品是嵌合抗原受体细胞或工程化细胞受体细胞。
发明的有益效果
本发明抗体的亲和力更强，可更特异地结合嵌合抗原受体细胞或工程化细胞受体细胞中的Strep-Tag II标签，有效监测嵌合抗原受体细胞或工程化细胞受体细胞在体内外的扩增效率。
附图说明
图1示出小鼠经过四次免疫后血清的效价曲线。
图2示出抗体8F8D1的聚丙烯酰胺凝胶电泳图谱。泳道M为分子量标准(300KD以内)；泳道1为抗体8F8D1的重链(约55KD)和轻链(约24KD)。
图3示出抗体8F8D1和8A882的流式检测图。
图4示出抗体8F8D1和8A882的亲和力检测图。
图5示出使用抗体8F8D1监测小鼠体内CAR-T细胞的代谢曲线图。
具体实施方式
下面通过具体实施方式对本发明作进一步的说明。除非另有限定，本文中所使用的学术语具有与本领域普通技术人员通常理解相同的含义。
本文所用的术语“抗原”是指引起免疫应答的分子，该免疫应答可涉 及抗体产生，或特异性免疫活性细胞的活化。本领域技术人员均可理解任何大分子包括所有的蛋白质或肽均可用作抗原。例如本发明中的免疫抗原可以为Strep-Tag II，其氨基酸序列为NWSHPQFEK，亦可为偶联血蓝素(Keyhole Limpet Hemocyanin，KLH)的Strep-Tag II(NWSHPQFEK-KLH)。抗原可被产生、合成或可源自生物学样品，这种生物学样品包括但不限于组织样品、肿瘤样品、细胞或生物学流体。本文使用的术语“检测抗原”是指用于测定抗体效价的抗原，例如偶联鸡卵白蛋白(Ovalbumin，OVA)的Strep-Tag II(NWSHPQFEK-OVA)，但本领域技术人员均可理解检测抗原并不局限于NWSHPQFEK-OVA，包含NWSHPQFEK或者其部分的抗原也可作为检测抗原使用，因此该具体检测抗原的使用并不应认为是对抗体制备方法的限制。
本文使用的术语“肽”和“多肽”是指由肽键共价连接的氨基酸残基组成的化合物。本文使用的“融合多肽”指的是连接了Strep-Tag II标签的多肽，例如在嵌合抗原受体的胞外结构域scFv中重链可变区或轻链可变区的N端或C端连接Strep-Tag II标签。多肽包括天然肽、重组肽、合成肽或其组合。
本文所用的术语“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。所述抗体包括但不限于Fab、Fab’、F(ab’)2、Fv、scFv、di-scFv、tri-scFv、Fd和其它保留抗原结合功能的抗体片段；还可以是二聚体结构Diabody或三聚体结构Triabody。抗原结合片段通常包括抗体轻链可变区(VL)和抗体重链可变区(VH)，VH和VL区可以进一步细分成：称为互补决定区(CDR)的高变区，以及穿插分布的称为框架区(FWR)的更保守区域。本发明公开的抗体和抗原结合片段的CDR由Kabat编号所定义或识别。
所述全长抗体是指包含至少两条重链和两条轻链并通过二硫键相互连接的蛋白质。每条重链由重链可变区和重链恒定区组成，每条轻链由轻链可变区和轻链恒定区组成。本发明具体实施方案中所述重链恒定区可以选自小鼠来源的IgG1、IgG2a、IgG2b或IgG3，所述轻链恒定区选 自κ链或者λ链。
本文使用的术语“嵌合抗原受体”或“CAR”是指被工程化以在免疫效应细胞上表达并能特异性地结合抗原的人工受体。“嵌合抗原受体细胞”是指细胞上表达能特异性地结合抗原的人工受体的免疫细胞，其中免疫细胞包括淋巴细胞、自然杀伤细胞、树突状细胞、单核/巨噬细胞、粒细胞、肥大细胞等，可以被用于使用过继细胞转移的疗法。本发明所用的术语“工程化细胞受体”或“TCR”，又称工程化T细胞受体，是通过体外基因工程化改造TCR的肽链构成的异元二聚体，使之更有效地识别由MHC递呈的肿瘤胞内抗原多肽，进而杀伤和治疗肿瘤。本发明中的融合多肽包括融合了Strep-Tag II标签的CAR或TCR。
本文使用的“分离的”是指从自然状态改变或移出。例如，天然存在于活动物中的核酸或肽不是“分离的”，但是部分或完全与它的自然状态的共存物质分开的相同的核酸或肽是“分离的”。分离的核酸或蛋白质可以以基本上纯化的形式存在，或可以存在于非自然环境，例如，宿主细胞中。“分离的”抗体是已鉴定并与其天然环境的组分分开和/或从其回收的抗体。在一些实施方案中，将抗体纯化至大于95％或99％纯度，如通过电泳(例如SDS-PAGE、等电聚焦IEF、毛细管电泳)或色谱法(例如离子交换或反相HPLC)所测定。本文使用的宿主细胞可以是含有克隆载体或表达载体的任何原核细胞或真核细胞，包括已经进行基因工程改造以在宿主细胞的染色体或基因组中含有克隆化基因的那些原核细胞或真核细胞。具有经修饰的免疫系统的特殊转基因动物也可用于制备抗体。
本文陈述的数值极限或范围包括端点，具体包括在数值极限或范围内的所有值和子范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
实施例：
实施例1抗原、小鼠免疫及杂交瘤制备
1.抗原
免疫抗原是偶联血蓝素(Keyhole Limpet Hemocyanin，KLH)的Strep-Tag II(NWSHPQFEK-KLH)；检测抗原是偶联鸡卵白蛋白(Ovalbumin， OVA)的Strep-Tag II(NWSHPQFEK-OVA)，均购自杭州中肽生化有限公司。
2.免疫
将免疫抗原蛋白(偶联KLH的Strep-Tag II(NWSHPQFEK-KLH)多肽)溶解于生理盐水中，浓度为0.5mg/mL。将100μL的上述抗原蛋白溶液和等体积的弗氏完全佐剂(Sigma；Cat#：1001646446)混匀，经过腹部皮下多点注射免疫Balb/c小鼠(雌性，6-8周，购自北京维通利华实验动物技术有限公司)的方式使其产生免疫应答，之后将上述抗原蛋白溶液用生理盐水稀释至浓度为0.25mg/mL，分别在第15天、第29天、第43天将100uL稀释后的抗原蛋白溶液和弗氏不完全佐剂(Sigma；Cat#:1002036152)按体积比1:1混合，经过腹部皮下多点注射免疫Balb/c小鼠进行加强免疫。每次每只小鼠注射200μL体积，共计抗原免疫4次。在第22天、36天和50天分别进行断尾采血测定血清效价。小鼠血清效价通过使用NWSHPQFEK-OVA多肽进行酶联免疫吸附测定(ELISA)检测，方法如下：在4℃下，将1μg/mL偶联OVA的Strep-Tag II(NWSHPQFEK-OVA)添加到96孔板(购自康宁公司)中过夜，清洗酶标板并用含0.05％Tween-20的磷酸盐缓冲液(PBST)洗涤5次。室温下，用1％牛血清白蛋白将平板封闭2小时，并用PBST清洗5次。后将小鼠血清从1:1000进行4倍梯度稀释至1:102400加入酶标板，室温孵育1小时后用PBST洗涤5次。加入100μL/孔的标记HRP的山羊抗鼠二抗(Invitrogen，Cat#：31430)，并在洗涤后室温孵育1小时。然后在平板中加入50μL/孔的3,3',5,5'-四甲基联苯胺(TMB)，在室温下孵育15分钟，然后停止反应并读取结果。
根据免疫小鼠血清效价的检测结果显示了5只小鼠的效价结果(图1)，编号#4小鼠的效价相较于其它小鼠偏高。挑选#4小鼠进行杂交瘤融合，融合前3天，以50μg免疫抗原腹腔注射小鼠进行冲击。
3.融合
通过聚乙二醇融合法，提取#4小鼠的脾脏，研碎后用杜氏磷酸缓冲液(DPBS)重悬，以提取小鼠脾脏B细胞单细胞，500g离心5分钟，用DPBS重悬沉淀后，500g再离心5分钟洗涤细胞，将脾细胞与小鼠骨 髓瘤细胞系sp2/0(购自北欧生物科技(北京)有限公司)按照3:1(细胞数量比)的比例混合，加入1mL聚乙二醇(罗氏；Cat#：10783641001)，将融合后的细胞用HAT培养基(Gibco；Cat#：21060-017)重悬，铺在96孔细胞培养板，在二氧化碳培养箱中37℃培养7天后用ELISA和流式细胞术进行杂交瘤的初筛。筛选出来的杂交瘤细胞在二氧化碳培养箱中37℃培养过夜后，将融合细胞以有限稀释法接种到96孔板中。
实施例2阳性杂交瘤细胞的筛选
通过ELISA试验和流式细胞术检测杂交瘤培养上清液中抗Strep-Tag II抗体的存在，最终筛到既可以无限增殖又可以分泌抗体的杂交瘤细胞。具体如下：
1.ELISA筛选
在4℃下，将1μg/mL偶联OVA的Strep-Tag II(NWSHPQFEK-OVA)添加到96孔板(购自康宁公司)中过夜，然后丢弃上清并用含0.05％Tween-20的磷酸盐缓冲液(PBST)洗涤5次。室温下，用1％牛血清白蛋白将平板封闭2小时，并用PBST清洗5次。加入100μL/孔杂交瘤上清液，室温孵育1小时，然后用PBST洗涤5次。加入100μL/孔的标记HRP的羊抗鼠二抗(Invitrogen，Cat#：31430)，并在洗涤后室温孵育1小时。然后在平板中加入50μL/孔的3,3',5,5'-四甲基联苯胺(TMB)，在室温下孵育15分钟，然后停止反应并读取结果。ELISA筛选挑取阳性杂交瘤，96孔培养板中转到24孔培养板扩大培养，5天后对24孔培养板孔中的上清进行复筛，复筛采用流式细胞术方法(FACS)分析。
2.流式细胞术筛选
编码靶向CD19的CAR慢病毒转移质粒的构建和慢病毒制备：1)将编码Strep-Tag II和靶向CD19的scFv的嵌合基因通过基因合成的方法合成(北京博迈德基因技术有限公司)。2)以已有的靶向CD19的CAR质粒为模板(靶向CD19的CAR核苷酸序列参见专利CN 105177031B中SEQ ID NO:13)，利用PCR克隆出CAR分子中含有CD8α铰链区、CD8α跨膜区、4-1BB(对应于NP_001552.2)的胞内区、以及CD3ζ(对应于NP_000725.1)胞内区的核酸片段。3)以步骤1)获得的嵌合基因和 步骤2)获得的核酸片段为模板，通过PCR克隆出编码Strep-Tag II和靶向CD19 CAR的完整核酸片段。4)通过限制性酶切和连接的方法，将步骤3)获得的完整核酸片段插入慢病毒载体pLenti6.3/V5(Thermo Fisher，Waltham，MA，USA)，获得载带Strep-Tag II和靶向CD19 CAR基因的慢病毒转移质粒。5)将慢病毒包装质粒pLP/VSVG,pLP1/MDK,pLP2/RSK(Thermo Fisher，Waltham，MA，USA)与步骤4)获得的转移质粒，用Lipofectamine 3000(Thermo Fisher，Waltham，MA，USA)转染至HEK293T细胞，48小时后收集培养基，300g离心去除细胞碎片后，用超速离心机25000rpm离心3小时。将沉淀用1mL生理盐水溶解，即为所需的慢病毒载体。
制备包含Strep-Tag II标签的CAR-T细胞：使用CD3/CD28 Dynabeads(Thermo Fisher)从健康志愿者的外周血单个核细胞(妙通(上海)生物科技有限公司，中国)中分离T细胞，将分离得到的T细胞(此时的T细胞与CD3/CD28 Dynabeads连在一起)在含IL-2(500IU/ml)的新鲜X-VIVO 15培养体系中培养48小时后，用上述慢病毒载体感染T细胞。病毒感染细胞24小时后，细胞离心换液，并于上述培养体系中继续培养。细胞培养4天后，收集培养体系中的所有细胞，并用磁力架去除培养体系中的Dynabeads，T细胞离心并计数，用流式细胞仪(NovoCyte 2060R，ACEA Biosciences，San Diego，CA，USA)检测CAR-T细胞含量。
收集上述包含Strep-Tag II标签的CAR-T细胞，用DPBS清洗细胞并计数，将细胞分装到EP管中，每管约3×10 5个细胞。离心取上清后加入100μL/孔杂交瘤上清液，在室温孵育15分钟。洗涤2次后每管加入5μL标记PE标记的大鼠抗小鼠IgG(BD，Cat#：550083)，在室温孵育15分钟，然后用DPBS洗涤细胞2次后进行流式细胞术FACS方法分析。
3.阳性亚克隆
通过有限稀释法亚克隆阳性杂交瘤克隆至96孔板，并通过FACS筛选方法(同上述步骤)鉴定和挑选阳性单克隆。将得到的单克隆细胞提取RNA，反转录为DNA后，用抗体重链和轻链基因特异结合的引物进行PCR，将PCR产物进行测序。
实施例3测序
挑选阳性单克隆，采用TRIZOL法提取总RNA。RT-PCR产生cDNA，随后重链和轻链分别用PCR扩增(RT-PCR试剂盒购自全式金，Cat#：AE311-03，PCR使用的是GXL高保真DNA聚合酶，购自TaKaRa，Cat#：R050A，具体操作见产品说明书)。然后将PCR产物用清洁试剂盒(AXYGEN，Cat#：155。具体操作见产品说明书)清洁后进行测序，测序交予北京睿博兴科有限公司。轻链可变区和重链可变区的氨基酸序列参见表1和表2，以及序列表。筛选的抗体克隆号分别是8A882和8F8D1。
实施例4腹水制备和抗体纯化
1.腹水制备
订购5只6～8周的Balb/c小鼠，饲养一周后注射1mL石蜡(购自江西益普生药业)，次周每只小鼠注射1×10 6的单克隆杂交瘤细胞。待一周后小鼠观察到有明显的腹胀，每隔48小时对小鼠穿刺抽液。每只小鼠收集3～4mL腹水，共收集到20mL腹水。
2.抗体纯化
采用亲和纯化法纯化抗体，用Binding Buffer(pH7.0)平衡重力柱，2-5倍柱体积冲洗。将过滤的腹水过柱(ProteinA/G柱，Thermo Fisher订购，Cat#：89930)；用5-10倍柱体积的Binding Buffer冲洗柱子，再用pH 3.5的0.1M甘氨酸洗脱目的蛋白，后用pH 9.0的Tris-HCl调节至中性。最后用BCA试剂盒(Thermo；Cat#：23227，具体操作见产品说明书)测定抗体浓度。通过SDS-PAGE鉴定抗体纯度，图2显示抗体有较高的纯度。
实施例5抗体的验证和亲和力的检测
1.流式细胞术验证
将纯化出的高纯度抗体进行FACS鉴定。收集实施例2.2中制备的含有Strep-Tag II标签的CAR-T细胞(本公司制备)，用FACS缓冲液清洗细胞并计数。将细胞分装到EP管中，每管约3×10 5个细胞。离心去 上清后加入终浓度为1μg/mL的8A882抗体或8F8D1抗体或阳性对照抗体，在室温孵育15分钟。洗涤2次后每管加入5μL PE标记的大鼠抗小鼠IgG，在室温孵育15分钟。然后洗涤细胞2次后进行FACS分析。实验结果显示，在检测细胞(CAR-T)相同量的情况下，使用本发明的8F8D1和8A882抗体处理后，含有Strep-Tag II标签的CAR-T细胞的表达率分别为48.07％和36.41％，远远高于阳性对照抗体处理的27.63％的表达率。上述结果证明本发明抗体可以特异性识别Strep-Tag II短肽且相比商用的阳性对照抗体，本发明的抗体对Strep-Tag II短肽的亲和力更高(图3)。阴性对照为未加入Strep-Tag II标签抗体而仅加入阴性对照IgG，阳性对照为商用抗Strep-Tag II标签抗体(Abcam；Cat#：ab184224)。
2.抗体荧光标记
将本发明抗体分别用FITC和PE两种不同荧光标记，目的为了多位点检测分析。抗体荧光标记由北京四正柏生物科技有限公司完成。
3.荧光标记抗体亲和力检测
本发明抗体亲和力检测采用动态平衡测定法(饱和浓度法)。少量抗原存在的情况下，将抗体进行梯度稀释，检测抗原抗体复合物的浓度，当抗原抗体复合物的浓度占总抗原浓度的一半时，抗体对应的浓度值(EC50)即抗体相对于抗原的KD值。
收集含有Strep-Tag II标签的CAR-T细胞，用DPBS清洗细胞并计数。分装到数个EP管中，每管约3×10 5个细胞。离心去上清后加入连续梯度稀释10个浓度的8A882或8F8D1以及阳性对照抗体(商用的Strep-Tag II抗体，Abcam；Cat#：ab184224)，在室温孵育15分钟。用DPBS洗涤细胞2次后再加入PE标记的大鼠抗小鼠IgG，室温孵育15分钟，洗涤细胞2次后进行FACS分析以及GraphPad Prism软件进行EC50值的计算。通过软件分析得到8A882和8F8D1以及阳性对照抗体的EC50值分别是0.4nM、0.2nM、0.55nM(图4)。与阳性对照抗体相比，本发明的两株抗体具有高度亲和力。
实施例6利用抗体检测荷瘤小鼠外周血中的CAR-T细胞
6-8周龄NCG小鼠(江苏集萃药康生物科技有限公司，中国)共6 只，每只小鼠从尾静脉注射1.0×10 6个Nalm-6-LAE细胞(ATCC，USA)，5天后对小鼠进行荧光素酶活体成像(Lumina II小动物活体成像系统，PerkinElmer，USA)分析，确定小鼠白血病模型制作成功后，每只小鼠从尾静脉注射CD19 CAR-T细胞(参照实施例2.2中方法由本公司制备，2×10 6个细胞/只)，小鼠于CAR-T细胞注射后第2、4、8、12、21、28天，分别从眼眶取100μL外周血进行CAR-T检测。具体操作为，将外周血分为2等份，每份50μL，每份加入3μL PE标记的8F8D1抗体，混匀后室温避光孵育20分钟；向样品中加入溶血剂，室温避光孵育15分钟，用DPBS洗涤两次后，再分别用100μL DPBS重悬，用FACS分析CAR-T比例，并计算每100μL外周血中CAR-T细胞数，绘制CAR-T在每只小鼠体内的代谢曲线(图5)。
数据显示，使用PE标记的8F8D1抗体可以有效检测小鼠体内CAR-T细胞的分布情况。

Claims (15)

  1. 一种特异性结合Strep-Tag II标签的分离的抗体，其包括：重链可变区VH和轻链可变区VL，其中所述VH包括：包含SEQ ID NO:1的VH-CDR1、包含SEQ ID NO:2的VH-CDR2、包含SEQ ID NO:3或SEQ ID NO:4的VH-CDR3；所述VL包括：包含SEQ ID NO:5或SEQ ID NO:6的VL-CDR1、包含SEQ ID NO:7或SEQ ID NO:8的VL-CDR2、包含SEQ ID NO:9或SEQ ID NO:10的VL-CDR3。
  2. 如权利要求1所述抗体，其中所述VH包括：包含SEQ ID NO:1的VH-CDR1、包含SEQ ID NO:2的VH-CDR2、包含SEQ ID NO:3的VH-CDR3；并且所述VL包括：包含SEQ ID NO:5的VL-CDR1、包含SEQ ID NO:7的VL-CDR2、包含SEQ ID NO:9的VL-CDR3；或
    所述VH包括：包含SEQ ID NO:1的VH-CDR1、包含SEQ ID NO:2的VH-CDR2、包含SEQ ID NO:4的VH-CDR3；并且所述VL包括：包含SEQ ID NO:6的VL-CDR1、包含SEQ ID NO:8的VL-CDR2、包含SEQ ID NO:10的VL-CDR3。
  3. 如权利要求1所述的抗体，其中所述VH包含SEQ ID NO:23或24的氨基酸序列，或包含与SEQ ID NO:23或24具有至少85％、优选至少90％、更优选至少95％或进一步优选至少99％同一性的氨基酸序列；所述VL包含SEQ ID NO:25或26的氨基酸序列，或包含与SEQ ID NO:25或26具有至少85％、优选至少90％、更优选至少95％或进一步优选至少99％同一性的氨基酸序列。
  4. 如权利要求1所述的抗体，其中所述VH包括一个以上的框架区，每个所述框架区包含一个氨基酸序列，所述氨基酸序列是SEQ ID NOs:11、12、13、14、15和16所示的序列，或与其具有至少90％、优选至少95％、更优选至少99％同一性的氨基酸序列；
    所述VL包括一个以上的框架区，每个所述框架区包含一个氨基酸序列，所述氨基酸序列是SEQ ID NOs:17、18、19、20、21和22所示的序列，或与其具有至少90％、优选至少95％、更优选至少99％同一性的氨 基酸序列。
  5. 如权利要求1所述的抗体，其中
    所述VH包含SEQ ID NO:23的氨基酸序列或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列，并且所述VL包含SEQ ID NO:25的氨基酸序列或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列；或
    所述VH包含SEQ ID NO:24的氨基酸序列或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列，并且所述VL包含SEQ ID NO:26的氨基酸序列或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列。
  6. 如权利要求1所述的抗体，其中所述抗体重链包含SEQ ID NO:27或29的氨基酸序列、或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列；所述抗体轻链包含SEQ ID NO:28或30的氨基酸序列、或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列。
  7. 如权利要求1所述的抗体，其中所述抗体重链包含SEQ ID NO:27或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列，所述抗体轻链包含SEQ ID NO:28或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列；或
    所述抗体重链包含SEQ ID NO:29或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列，所述抗体轻链包含SEQ ID NO:30或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列。
  8. 一种分离的核酸，其编码如权利要求1-7任一项所述的抗体。
  9. 一种克隆载体或表达载体，其包括如权利要求8所述的核酸分子。
  10. 一种宿主细胞，其包括如权利要求8所述的核酸分子。
  11. 一种抗体偶联物，包含连接至标记的如权利要求1-7任一项的抗体，其中，所述标记选自荧光标签、酶底物标签、放射性同位素、地高辛素、生物素或亲和素、用于检测的DNA分子或金，其中优选地，所述荧光标签选自荧光素、罗丹明、丹西尔、藻红蛋白或德州红；优选地，所述酶底物标签选自辣根过氧化物酶、碱性磷酸酶、糖化酶、溶菌酶、糖氧化酶或β-D-半乳糖苷酶；优选地，所述放射性同位素选自 123I、 124I、 125I、 131I、 35S、 3H、 111In、 112In、 14C、 64Cu、 67Cu、 86Y、 88Y、 90Y、 177Lu、 211At、 186Re、 188Re、 153Sm、 212Bi、 32P、其它镧系元素；优选地，所述标记选自发光标记或发色基团。
  12. 一种组合物或试剂盒，其包含如权利要求1至7中任一项所述的抗体或如权利要求11所述的抗体偶联物，以及一种以上药学可接受的赋形剂、稀释剂或载体。
  13. 一种检测样品中含有Strep-Tag II标签的融合多肽的方法，包括(i)在允许抗体和所述融合多肽发生相互作用的条件下，体外使含有所述融合多肽的样品接触如权利要求1至7任一项的分离的抗体，和(ii)检测抗体和样品之间的复合物的形成；优选地，检测所述复合物的方法包括流式、蛋白质印迹、免疫组化、免疫荧光中的至少一种。
  14. 一种纯化样品中含有Strep-Tag II标签的融合多肽的方法，包括(i)在允许抗体和多肽发生相互作用的条件下，体外使样品接触如权利要求1至7任一项的分离的抗体，和(ii)纯化抗体和样品之间形成的复合物；优选地，纯化方法包括亲和层析、免疫沉淀。
  15. 如权利要求13或14所述的方法，其中所述样品是包括血液、尿液、唾液、淋巴液、脑脊液、骨髓、组织器官、细胞中至少一种的生物样品，优选地，所述血液包括血清、血浆和全血中的至少一种，优选所述样品包含嵌合抗原受体细胞或工程化细胞受体细胞。
PCT/CN2021/136113 2020-12-08 2021-12-07 一种特异性结合Strep-Tag II标签的抗体及其应用 WO2022121899A1 (zh)

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EP2871189A1 (en) * 2013-11-07 2015-05-13 Institut Pasteur High-affinity monoclonal anti-strep-tag antibody
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EP2871189A1 (en) * 2013-11-07 2015-05-13 Institut Pasteur High-affinity monoclonal anti-strep-tag antibody
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