WO2021057978A1 - 抗vhh域抗体及其用途 - Google Patents

抗vhh域抗体及其用途 Download PDF

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WO2021057978A1
WO2021057978A1 PCT/CN2020/118150 CN2020118150W WO2021057978A1 WO 2021057978 A1 WO2021057978 A1 WO 2021057978A1 CN 2020118150 W CN2020118150 W CN 2020118150W WO 2021057978 A1 WO2021057978 A1 WO 2021057978A1
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seq
antibody
antigen
chain variable
variable region
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French (fr)
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覃喜建
孙立伟
王婉仪
宋光伟
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Nanjing Jinsirui Science and Technology Biology Corp
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Nanjing Jinsirui Science and Technology Biology Corp
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Priority to US17/753,021 priority Critical patent/US12590171B2/en
Priority to CN202080067484.5A priority patent/CN114829401A/zh
Priority to EP20867925.8A priority patent/EP4036116A4/en
Publication of WO2021057978A1 publication Critical patent/WO2021057978A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • 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
    • G01N33/6854—Immunoglobulins
    • 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
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention relates to a group of anti-VHH domain antibodies.
  • the invention also relates to methods for preparing and obtaining the antibody.
  • the present invention relates to the application of the antibody in the development, screening and purification of Nanobodies.
  • the invention also relates to the application of the antibody in the field of immunotherapy
  • Camel-derived Nanobody also known as single-domain antibody, heavy chain variable region antibody (variable domain of heavy chain of HCAb, VHH), it only includes the variable region fragment of camel-derived heavy chain antibody , The size is only about 15kDa, can bind to antigens with high affinity and high specificity, and has very important applications in the fields of antibody drug development and immune cell therapy.
  • Ablynx has successfully developed and marketed the first therapeutic nanobody drug caplacizumab based on nanobody development technology for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) [https://www.ablynx.com/rd -portfolio/clinical-programmes/caplacizumab/].
  • aTTP acquired thrombotic thrombocytopenic purpura
  • Nanjing Legend's LCAR-B38M is the first cell therapy therapy in China to pass the cFDA clinical application, and it is also the first project to achieve dual approvals in both China and the United States.
  • the project has shown amazing treatment in the current public data. The effect is of great significance in the development of China's immune cell therapy.
  • the project adopted a unique nanobody design chimeric antigen receptor in the CAR design, avoiding the shortcomings of poor stability and low affinity of scFv in conventional technical routes.
  • nanobodies are becoming more and more popular among researchers.
  • nanobodies are of great significance in the field of antibody drug development and immune cell therapy, there is currently a lack of an antibody that recognizes camel-derived nanobodies in order to better develop camel-derived nanobodies or optimize the identification of CART cells in immune cell therapy. Sorting and magnetic separation.
  • the present invention has developed a set of antibodies directed against camel-derived nanobodies with high affinity, high specificity and high functionality, which effectively solves the above-mentioned problems and meets the needs of various application fields.
  • the present invention provides an antibody or antigen-binding fragment thereof, which specifically binds to a VHH domain.
  • the VHH domain is the VHH domain of a camel-derived antibody.
  • the camel-derived antibody is a single-domain antibody or heavy-derived antibody derived from Camelus dromedarius, Camelus bactrianus, Vicugna pacos or Lama glama. Chain antibody.
  • the present invention provides an antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof disclosed in the present invention contains a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein (A) the heavy chain variable region includes a heavy chain complementary
  • the determining regions are HCDR1, HCDR2 and HCDR3,
  • the HCDR1 sequence is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15
  • the HCDR2 sequence is selected from the amino acid sequence shown in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or with the above sequence
  • the LCDR2 sequence is selected from SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 of the amino acid sequence or the above sequence has at least 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the LCDR3 sequence is selected from SEQ ID NO: 76, 77, 78, 79, 80 , 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96 % ⁇ 97% ⁇ 98% ⁇ 9 9% or 100% identical amino acid sequence
  • the light chain complementarity determining region LCDR contains one
  • the antibody or antigen-binding fragment thereof disclosed in the present invention contains (1) SEQ ID NO: 1 HCDR1, SEQ ID NO: 16 HCDR2, SEQ ID NO: 31 HCDR3, SEQ ID LCDR1 shown in NO: 46, LCDR1 shown in SEQ ID NO: 61, and LCDR3 shown in SEQ ID NO: 76; (2) HCDR1 shown in SEQ ID NO: 2 HCDR1, SEQ ID NO: 17 HCDR2, SEQ ID NO: HCDR3 shown in 32, LCDR1 shown in SEQ ID NO: 47, LCDR2 shown in SEQ ID NO: 62, and LCDR3 shown in SEQ ID NO: 77; (3) HCDR1 shown in SEQ ID NO: 3, and LCDR1 shown in SEQ ID NO: 18.
  • the antibody or antigen-binding fragment thereof disclosed in the present invention comprises a heavy chain variable region (HCVR) whose amino acid sequence is selected from SEQ ID NO: 91, 92, 93, 94, 95, 96, 97, 98 , 99, 100, 101, 102, 103, 104, or 105 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or 100% identical amino acid sequence.
  • HCVR heavy chain variable region
  • the antibody or antigen-binding fragment thereof disclosed in the present invention comprises a light chain variable region (LCVR) whose amino acid sequence is selected from SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113 , 114, 115, 116, 117, 118, 119 or 120 amino acid sequence or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Amino acid sequence with 99% or 100% identity.
  • LCVR light chain variable region
  • the antibody or antigen-binding fragment thereof disclosed in the present invention contains (1) the heavy chain variable region shown in SEQ ID NO: 91 and the light chain variable region shown in SEQ ID NO: 106; (2) The heavy chain variable region shown in SEQ ID NO: 92 and the light chain variable region shown in SEQ ID NO: 107; (3) the heavy chain variable region shown in SEQ ID NO: 93 and the light chain variable region shown in SEQ ID NO: 108 Chain variable region; (4) the heavy chain variable region shown in SEQ ID NO: 94 and the light chain variable region shown in SEQ ID NO: 109; (5) the heavy chain variable region shown in SEQ ID NO: 95 and The light chain variable region shown in SEQ ID NO: 110; (6) the heavy chain variable region shown in SEQ ID NO: 96 and the light chain variable region shown in SEQ ID NO: 111; (7) SEQ ID NO: 97 The heavy chain variable region shown in SEQ ID NO: 112 and the light chain variable region shown in SEQ ID NO: 112; (8) The heavy chain variable region shown in
  • the antibody or antigen-binding fragment thereof of the invention specifically binds to the VHH domain.
  • the VHH domain is the VHH domain of a camel-derived antibody. That is to say, the antibody or antigen-binding fragment thereof specified by the above sequence has good binding ability of camel-derived single domain antibody or camel-derived heavy chain antibody.
  • the camelid-derived antibody is a single domain antibody or heavy chain antibody derived from a dromedary, bactrian camel, llama or vicuna.
  • the antibody or antigen-binding fragment thereof of the present invention binds at the framework region of the VHH domain. In one embodiment of the above aspects, the antibody or antigen-binding fragment thereof of the invention binds at a conformational epitope of the VHH domain. In one embodiment of the above aspects, the antibody or antigen-binding fragment thereof of the present invention binds at a conformational epitope in the framework region of the VHH domain.
  • the antibody or antigen-binding fragment of the present invention specifically binds to the VHH domain shown in any one of SEQ ID NO: 241 and 246-255, or is combined with any one of SEQ ID NO: 241 and 246-255 Has at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least A VHH domain with 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity, or the consensus sequence of SEQ ID NO: 241 and 246-255.
  • the antibody or antigen-binding fragment of the present invention specifically binds to the framework region of the VHH domain shown in any one of SEQ ID NO: 241 and 246-255, or with SEQ ID NO: 241 and 246-
  • the framework region of any one of the VHH domains shown in 255 has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% , At least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity framework region, or SEQ ID NO: 241 and 246-255 consensus framework sequence.
  • the antibody or antigen-binding fragment thereof of the present invention specifically binds to the VHH domain with a K D of 10 nM to 1 pM, for example, 1 nM to 10 pM, for example, 1 pM to 10 pM, for example, 1 pM to 5 pM, for example
  • the antibody or antigen-binding fragment thereof of the present invention binds to the VHH domain at an epitope comprising one or more positions selected from the group consisting of: positions 3-4, 6- according to Kabat numbering. 10, 13, 15, 17-18, 20-22, 25-26, 36, 38, 41, 46, 48, 66-67, 69-70, 72, 75, 79-80, 82C, 85-88, 90, 92-93, 105-107, 109 and 111-112; or with SEQ ID NO: 241 position 3-4, 6-10, 13, 15, 17-18, 20-22, 25 according to sequential numbering -26, 36, 38, 41, 46, 48, 66-67, 69-70, 72, 75, 79-80, 85, 88-91, 93, 95-96, 107-109, 111 and 113-114 The corresponding position (see Figure 11).
  • the antibody or antigen-binding fragment thereof of the present invention binds to the VHH domain at an epitope comprising one or more positions selected from the group consisting of: positions 3-10, 13, 13 according to Kabat numbering. 15, 17-22, 25-26, 36-43, 45-46, 48-49, 66-70, 72-73, 75-82, 82B-88, 90-94 and 103-113; or in accordance with consecutive SEQ ID NO: 248 and/or 255 positions 3-10, 13, 15, 17-22, 25-26, 36-43, 45-46, 48-49, 66-70, 72-73, 75 -82, 84-91, 93-97 and 105-115 corresponding positions.
  • the antibody or antigen-binding fragment thereof of the present invention binds to the VHH domain at an epitope comprising one or more positions selected from the group consisting of: positions 3-10, 13, 13 according to Kabat numbering. 15, 17-22, 25-26, 36-38, 40-41, 43, 45-46, 48-49, 66-70, 72-73, 75-77, 79-82, 82C-88, 90- 94, 103-109 and 111-113; or with SEQ ID NO: 248 and/or 255 positions 3-10, 13, 15, 17-22, 25-26, 36-38, 40-41 according to sequential numbering , 43, 45-46, 48-49, 66-70, 72-73, 75-77, 79-82, 85-91, 93-97, 105-111 and 113-115 corresponding positions.
  • the antibody or antigen-binding fragment thereof of the present invention binds to the VHH domain at an epitope comprising one or more positions selected from the group consisting of: positions 39, 42, 78, and positions according to Kabat numbering. 82B and 110; or the positions corresponding to positions 39, 42, 78, 84, and 112 of SEQ ID NO: 248 and/or 255 according to sequential numbering.
  • the present invention encompasses an antibody or antigen-binding fragment thereof that binds to the same epitope as the aforementioned antibody or antigen-binding fragment thereof. In another aspect, the present invention encompasses an antibody or antigen-binding fragment thereof that competes for binding with the aforementioned antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof of the invention is a naked antibody or antigen-binding fragment thereof.
  • the present invention provides one or more polynucleotides, which encode the antibody or antigen-binding fragment thereof of the present invention.
  • the invention provides one or more vectors comprising the polynucleotide of the invention.
  • the vector is selected from cloning vectors and expression vectors.
  • the present invention provides a host cell comprising the polynucleotide or vector of the present invention.
  • the host cell is selected from prokaryotic cells, yeast cells, insect cells and mammalian cells.
  • the present invention provides a method for producing an antibody or antigen-binding fragment thereof, which comprises culturing a host cell of the present invention under conditions suitable for antibody production to express the antibody or antigen-binding fragment thereof. In one embodiment, the method further comprises recovering the antibody or antigen-binding fragment thereof.
  • the present invention provides a conjugate comprising the antibody of the present invention or an antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof is coupled to fluorescein, biotin, enzyme, agarose resin, magnetic beads or biochip.
  • the present invention provides a kit comprising a container containing the antibody or antigen-binding fragment or conjugate thereof of the present invention.
  • the present invention provides a method for detecting a VHH domain, which comprises adding the antibody or antigen-binding fragment or conjugate thereof of the present invention to a sample known or suspected of containing a VHH domain, and detecting the antibody or The antigen-binding fragment or the complex formed between the conjugate and the VHH domain.
  • the present invention provides a method for isolating VHH domains, which comprises adding the antibody or antigen-binding fragment or conjugate of the present invention to a sample known or suspected to contain VHH domains, and isolating the antibody or its The antigen-binding fragment or the complex formed between the conjugate and the VHH domain.
  • the VHH domain is in a camel-derived antibody.
  • the camelid-derived antibody is a single domain antibody or heavy chain antibody derived from a dromedary, bactrian camel, llama or vicuna.
  • the VHH domain is in a chimeric antigen receptor.
  • the chimeric antigen receptor is on an immune cell.
  • the immune cells are selected from PBMC, T cells, NK cells or macrophages.
  • the method is performed using FACS or MCS.
  • Figure 1 shows the schematic diagram and amino acid sequence of camel-derived Nanobody VHH-His used in animal immunization.
  • Figure 2 shows the ELISA titer detection of fusion animal serum.
  • Figure 3 shows the ELISA titer detection of supernatant from parent clones.
  • Figure 4 shows the ELISA titer detection of subclonal strain supernatant.
  • FIGS 5a and 5b show the PAGE purity detection of 15 strains of purified antibodies.
  • Figures 6a and 6b show the EC50 affinity detection of 15 strains of this antibody and 10 different camel-derived Nanobodies.
  • Figure 7 shows the amino acid sequence alignment of the framework regions of camel-derived Nanobodies.
  • Figure 8 shows the affinity detection of clone R166.C5.
  • Figure 9 shows the flow cytometry of CART cells.
  • Figure 10 shows the flow cytometry of magnetically separated CART cells.
  • Figure 11 shows the Kabat numbering and residue frequency of camel-derived Nanobodies used in animal immunization.
  • Figure 12 shows the FACS image of five strains of PE-labeled purified antibodies for flow cytometric detection of camel PBMC cells.
  • Figure 13 shows the SDS-PAGE images of the antibody coupled magnetic beads before and after the camel serum heavy chain antibody purification.
  • anti-VHH domain antibodies refer to antibodies capable of binding to VHH domains with sufficient affinity.
  • the degree of anti-VHH domain antibody binding irrespective of the binding of the anti-VHH domain antibody is less than about 10% of the binding of the antibody to the VHH domain according to, for example, measurement by radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • an antibody binding VHH domains having ⁇ 1 ⁇ M, ⁇ 100nM, ⁇ 10nM, ⁇ 1nM, ⁇ 0.1nM, ⁇ 0.01nM, or 0.001 nM e.g.
  • anti-VHH domain antibodies bind to VHH domain epitopes that are conserved among VHH domains from different species.
  • HCAb heavy chain antibody
  • HCAb refers to a functional antibody that contains a heavy chain but lacks the light chain commonly found in 4-chain antibodies.
  • Camelids such as Camelus dromedarius, Camelus bactrianus, Vicugna pacos, or Lama glama
  • HCAb Light chain antibody
  • single domain antibody refers to a single antigen binding polypeptide with three complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • the sdAb alone can bind to the antigen without pairing with the corresponding CDR-containing polypeptide.
  • single domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains are referred to herein as "VHH" (heavy chain variable domains of heavy chain antibodies).
  • VHH heavy chain variable domains of heavy chain antibodies.
  • Camelid sdAb is the smallest known antigen-binding antibody fragment.
  • the basic VHH has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and where CDR1 to CDR3 refer to complementarity determining region 1. To 3.
  • acceptor human framework refers to a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below The framework of the amino acid sequence.
  • An acceptor human framework "derived” from a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less , Or 2 or less.
  • the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.
  • Bind refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). Unless otherwise indicated, as used herein, "binding affinity” refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X to its partner Y can usually be expressed by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
  • an “affinity mature” antibody refers to an antibody that has one or more changes in one or more hypervariable regions (HVR). Compared with a parent antibody that does not possess such changes, such changes result in the antibody's anti-antigen Improved affinity.
  • HVR hypervariable regions
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments, as long as they exhibit The desired antigen-binding activity can be obtained.
  • Antibody fragment refers to a molecule that is different from an intact antibody, which contains a part of an intact antibody and binds to the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (such as scFv); and multispecific antibody fragments Sex antibody.
  • the antigen-binding fragment may comprise the variable domain of the heavy chain and/or the variable domain of the light chain of an antibody.
  • the "antibody that binds to the same epitope as the reference antibody” refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and on the contrary, the reference antibody binds to the antibody in a competition assay. The binding of the antigen is blocked by 50% or more.
  • An exemplary competition assay is provided herein.
  • chimeric antibody refers to an antibody in which a part of the heavy and/or light chain is derived from a specific source or species, and the remaining part of the heavy and/or light chain is derived from a different source or species.
  • the "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • FR Framework or "FR” refers to variable domain residues other than hypervariable region (HVR) residues.
  • HVR hypervariable region
  • the FR of a variable domain consists of 4 FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
  • full-length antibody “whole antibody”, and “whole antibody” are used interchangeably herein and refer to a structure that is substantially similar to the structure of a natural antibody or has a heavy chain containing an Fc region as defined herein Of antibodies.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived therefrom regardless of the number of passages.
  • the offspring may not be exactly the same as the parent cell in nucleic acid content, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the original transformed cell.
  • Human antibody refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by human or human cells or derived from a non-human source using human antibody repertoires or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.
  • Human consensus framework refers to a framework representing the most frequently present amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
  • a selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the sequence subgroup is the subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3.
  • the subgroup is as Kabat et al., see subgroup ⁇ I above.
  • for VH the subgroup is as Kabat et al., see subgroup III above.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues derived from non-human HVR and amino acid residues derived from human FR.
  • a humanized antibody will comprise at least one, and usually two, substantially the entire variable domain, wherein all or substantially all of the HVR (e.g., CDR) corresponds to those of the non-human antibody, and all or substantially All FRs above correspond to those of human antibodies.
  • a humanized antibody may comprise at least a part of the constant region of an antibody derived from a human antibody.
  • Antibodies, such as "humanized forms" of non-human antibodies refer to antibodies that have undergone humanization.
  • HVR hypervariable region
  • CDR complementarity determining region
  • Hypervariable loop structurally defined loop
  • an antibody contains 6 HVRs: three in VH (H1, H2, H3), and three in VL (L1, L2, L3).
  • Exemplary HVRs in this article include:
  • (d) A combination of (a), (b), and/or (c), including HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56( L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3), and 94-102(H3).
  • HVR residues and other residues in the variable domain are numbered herein according to Kabat et al., see above.
  • Immunoconjugate refers to an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent, such as a conjugate comprising the antibody of the present invention.
  • isolated nucleic acid encoding an anti-VHH domain antibody refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or different vectors, and present in host cells One or more positions in such nucleic acid molecules.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, each antibody constituting the population is the same and/or binds the same epitope, except for example containing naturally occurring mutations or In addition to possible variant antibodies that occur during the production of monoclonal antibody preparations, such variants are generally present in extremely small amounts. Unlike polyclonal antibody preparations which usually contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the properties of an antibody obtained from a group of substantially homogeneous antibodies, and should not be interpreted as requiring the production of antibodies by any specific method.
  • a variety of techniques can be used to generate monoclonal antibodies to be used in accordance with the present invention, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and the use of transgenic animals containing all or part of human immunoglobulin loci Methods, such methods and other exemplary methods for generating monoclonal antibodies are described herein.
  • naked antibody refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radioactive label. Naked antibodies may be present in pharmaceutical formulations.
  • “Native antibodies” refer to naturally occurring immunoglobulin molecules with different structures.
  • a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light chains and two identical heavy chains bonded by disulfide. From N to C-terminus, each heavy chain has a variable region (VH), also called variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also called variable light domain or light chain variable domain, followed by a constant light (CL) domain.
  • antibody light chains can be classified into one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ).
  • the "percent (%) amino acid sequence identity" of the reference polypeptide sequence is defined as when the sequence is aligned and gaps are introduced when necessary to obtain the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity, The percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence.
  • the comparison for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the technical scope of 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 the appropriate parameters for aligning sequences, including any algorithm required to obtain maximum alignment over the entire length of the sequence being compared.
  • the% amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted to the US Copyright Office (US Copyright Office, Washington D.C., 20559) together with user documentation, where it is registered under the US copyright registration number TXU510087.
  • the public can obtain the ALIGN-2 program from Genentech, Inc., South San Francisco, California, or can compile it from the source code.
  • the ALIGN2 program should be compiled to be used on the UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.
  • the% amino acid sequence identity of a given amino acid sequence A relative to (to), with (with), or against (against) a given amino acid sequence B (or can be expressed as A given amino acid sequence that has or contains a certain% amino acid sequence identity with respect to, with, or for a given amino acid sequence B.
  • A is calculated as follows: score X/Y times 100 where X is determined by the sequence alignment program ALIGN-2 The number of amino acid residues that are scored as identical matches in the A and B alignment of this program, and where Y is the total number of amino acid residues in B.
  • variable region refers to the domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen.
  • the heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, where each domain contains 4 conserved framework regions (FR) and 3 hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th edition, W.H. Freeman and Co., p. 91 (2007)).
  • FR conserved framework regions
  • HVR hypervariable regions
  • VH or VL domain from the antigen-binding antibody can be used to screen a library of complementary VL or VH domains to isolate antibodies that bind a specific antigen, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • vector refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it is linked.
  • the term includes a vector that is a self-replicating nucleic acid structure and a vector incorporated into the genome of a host cell into which it is introduced.
  • Certain vectors can direct the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • “Expression” generally refers to the process by which information (eg, genetically encoded and/or epigenetic) is transformed into a structure that exists and operates in a cell. Therefore, as used herein, “expression” can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modification (e.g., post-translational modification of a polypeptide).
  • Transcribed polynucleotides, translated polypeptides, or fragments of polynucleotide and/or polypeptide modifications should also be considered expressed, regardless of whether they are derived from transcription generated by alternative splicing Or degraded transcript, or derived from post-translational processing of the polypeptide (e.g., by proteolysis).
  • "Expressed genes” include genes that are transcribed into polynucleotides (such as mRNA) and then translated into polypeptides, as well as genes that are transcribed into RNA but not translated into polypeptides (such as transfer and ribosomal RNA).
  • the present invention provides a group of anti-VHH domain antibodies or antigen-binding fragments thereof, which specifically recognize the conformational epitopes of the camel-derived VHH domain framework region.
  • the present invention found that the antibody has high affinity, high specificity and high functionality.
  • the present invention provides a preparation method of the antibody and its application in the field of camel-derived nano antibody development and immune cell therapy.
  • the antibodies and methods disclosed in the present invention are used to detect Nanobodies or heavy chain antibodies derived from dromedary camels. In other embodiments, the antibodies and methods disclosed in the present invention are used to detect Nanobodies or heavy chain antibodies derived from Bactrian camels. In still other embodiments, the antibodies and methods disclosed in the present invention are used to detect nanobodies or heavy chain antibodies derived from llamas. In a preferred embodiment, the antibodies and methods disclosed in the present invention are used to detect nano antibodies or heavy chain antibodies derived from llamas.
  • the antibodies and methods disclosed in the present invention are used for the isolation of camelid-derived heavy chain antibody-specific PBMC cells, and the antibodies are also suitable for the isolation of other types of cells.
  • the antibody is used for binding analysis of Nanobodies.
  • the antibody is coupled to a solid-phase carrier medium for affinity purification of VHH antibodies.
  • the antibodies disclosed in the present invention are used for flow cytometric identification of a type of CART cells.
  • the antibody is used for flow cytometric sorting of CART cells.
  • the antibody is used for MACS separation and purification of CART cells.
  • the present invention provides anti-VHH domain antibodies or antigen-binding fragments thereof.
  • the antibody or antigen-binding fragment thereof of the invention specifically binds to the VHH domain.
  • the VHH domain is the VHH domain of a camel-derived antibody.
  • the camelid-derived antibody is a single domain antibody or heavy chain antibody derived from a dromedary, bactrian camel, llama or vicuna.
  • the antibody or antigen-binding fragment thereof of the present invention binds at the framework region of the VHH domain. In one embodiment, the antibody or antigen-binding fragment thereof of the invention binds at a conformational epitope of the VHH domain. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention binds at a conformational epitope in the framework region of the VHH domain.
  • the antibody or antigen-binding fragment thereof of the present invention specifically binds to the VHH domain shown in any one of SEQ ID NOs: 241 and 246-255, or has at least 35 in combination with any one of SEQ ID NO: 241 and 246-255. %, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, A VHH domain with at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity, or the consensus sequence of SEQ ID NO: 241 and 246-255.
  • the antibody or antigen-binding fragment of the present invention specifically binds to the framework region of the VHH domain shown in any one of SEQ ID NO: 241 and 246-255, or is combined with any one of SEQ ID NO: 241 and 246-255
  • the framework region of the shown VHH domain has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% %, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity framework region, or the consensus framework sequence of SEQ ID NO: 241 and 246-255.
  • the present invention provides an anti-VHH domain antibody comprising at least one, two, three, four, five, or six HVRs selected from the following: (a) comprising SEQ ID NO : HVR-H1 with the amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; (b) comprising SEQ ID NO: 16, 17, HVR-H2 of the amino acid sequence of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; (c) contains SEQ ID NO: 31, 32, 33, 34, 35 , 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid sequence of HVR-H3; (d) comprising SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, HVR-L1 of the amino acid sequence of 53, 54, 55, 56, 57, 58, 59 or 60; (e) includes SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 ,
  • the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the following: (a) comprising SEQ ID NO: 1, 2, 3, 4, HVR-H1 with the amino acid sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; (b) contains SEQ ID NO: 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, or 30 amino acid sequence HVR-H2; and (c) comprising SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39 , 40, 41, 42, 43, 44, or 45 amino acid sequence HVR-H3.
  • the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the following: (a) comprising SEQ ID NO: 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid sequence HVR-L1; (b) includes SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 amino acid sequence of HVR-L2; and (c) comprising SEQ ID NO: 76, 77, 78, 79, 80, 81, 82, 83, HVR-L3 with an amino acid sequence of 84, 85, 86, 87, 88, 89, or 90.
  • the antibody of the present invention comprises (a) a VH domain, which comprises at least one, at least two, or all three VH HVR sequences selected from the following: (i) comprising SEQ ID NO: 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid sequence of HVR-H1; (ii) comprising SEQ ID NO: 16, 17, 18, 19, HVR-H2 of the amino acid sequence of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; and (iii) comprising SEQ ID NO: 31, 32, 33, 34, 35, 36, HVR-H3 of the amino acid sequence of 37, 38, 39, 40, 41, 42, 43, 44, or 45; and (b) VL domain comprising at least one, at least two, or all three selected from the following VL HVR sequence: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59
  • the anti-VHH domain antibody comprises an amino acid sequence with SEQ ID NO: 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 that has an amino acid sequence of at least 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the heavy chain variable domain (VH) sequence.
  • VH heavy chain variable domain
  • the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions relative to the reference sequence (E.g.
  • the anti-VHH domain antibody containing this sequence retains the ability to bind to the VHH domain.
  • SEQ ID NO: 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 are substituted, inserted and/or deleted A total of 1 to 10 amino acids.
  • the substitution, insertion, or deletion occurs in a region other than the HVR (ie, in the FR).
  • the anti-VHH domain antibody comprises the VH sequence in SEQ ID NO: 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 or 105, including the Post-translational modification of the sequence.
  • an anti-VHH domain antibody comprising SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or
  • the amino acid sequence of 120 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the light chain variable domain (VL ).
  • VL light chain variable domain
  • a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions relative to the reference sequence (E.g.
  • the anti-VHH domain antibody containing this sequence retains the ability to bind to the VHH domain.
  • SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 are substituted, inserted and/or deleted A total of 1 to 10 amino acids.
  • the substitution, insertion, or deletion occurs in a region other than the HVR (ie, in the FR).
  • the anti-VHH domain antibody comprises the VL sequence in SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120, including the Post-translational modification of the sequence.
  • an anti-VHH domain antibody comprising the VH in any of the embodiments provided above and the VL in any of the embodiments provided above.
  • the anti-VHH domain antibody is humanized.
  • the anti-VHH domain antibody comprises the HVR of any of the above embodiments, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.
  • the present invention provides antibodies that bind to the same epitope as the anti-VHH domain antibodies provided herein.
  • antibodies that bind to the same epitope as the anti-VHH domain antibodies provided herein are provided.
  • the anti-VHH domain antibody is a monoclonal antibody, including a chimeric antibody, a humanized antibody or a human antibody.
  • the anti-VHH domain antibody is an antibody fragment, such as Fab, Fab', Fab'-SH, Fv, single chain variable fragment (scFv), and (Fab') 2 fragments.
  • the antibody is a full-length antibody, such as a complete IgG class antibody or IgG1 isotype or other antibody class or isotype.
  • an anti-VHH domain antibody may incorporate any of the features described in sections 1-8 below, singly or in combination:
  • the antibodies provided herein have a dissociation constant (Kd) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g., 10 -8 M Or less, such as 10 -8 M to 10 -12 M, such as 10 -9 M to 10 -12 M, such as 10 -10 M to 10 -12 M, such as 10 -11 M to 10 -12 M).
  • Kd dissociation constant
  • Kd is measured by a radiolabeled antigen binding assay (RIA).
  • the RIA is performed with the antibody of interest in Fab format and its antigen.
  • the solution binding affinity of the Fab to the antigen is measured by equilibrating the Fab with the minimum concentration of (125 I) labeled antigen in the presence of a titration series of unlabeled antigen, and then capturing the bound antigen with an anti-Fab antibody coating plate (see For example, Chen et al., J. Mol. Biol. 293:865-881 (1999)).
  • VHH domain Multi-well plates (Thermo Scientific) were coated with 5 ⁇ g/ml capture anti-Fab antibody (Cappel Labs) in 50mM sodium carbonate (pH 9.6) overnight, followed by 2% (w/v) bovine serum albumin in PBS at room temperature (Approximately 23°C) Close for 2-5 hours.
  • a non-adsorbed plate (Nunc#269620), 100pM or 26pM [125I]-antigen and serially diluted Fab of interest (for example, with Presta et al., Cancer Res. 57:4593-4599 (1997) anti-VEGF antibody, Fab -12 evaluation is consistent) mixed.
  • the Fab of interest is then incubated overnight; however, the incubation can continue for a longer period of time (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (for example, 1 hour). Then remove the solution and use 0.1% polysorbate 20 in PBS Wash the plate 8 times. After the plate is dry, add 150 ⁇ l/well of scintillation fluid (VHH domain ROSCINT-20 TM ; Packard), and then count the plate on a TOPCOUNTTM gamma counter (Packard) for 10 minutes. The concentration of each Fab that gives less than or equal to 20% of the maximum binding is selected for the competitive binding assay.
  • Kd is used Measured by surface plasmon resonance measurement.
  • CM5 chip at 25°C in about 10 response units (RU) or (BIAcore, Inc., Piscataway, NJ) performs the assay.
  • EDC N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
  • NHS N-hydroxysuccinimide
  • the antigen was diluted with 10 mM sodium acetate pH 4.8 to 5 ⁇ g/ml (about 0.2 ⁇ M), and then injected at a flow rate of 5 ⁇ l/min to obtain about 10 response units (RU) of coupled protein. After the antigen was injected, 1M ethanolamine was injected to block unreacted groups. For kinetic measurement, two-fold serial dilutions of Fab (0.78 nM to 0.78 nM to PBST) containing 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant were injected at 25°C at a flow rate of about 25 ⁇ l/min. 500nM).
  • the binding rate can be measured using fluorescence quenching technology, that is, according to a spectrometer such as a spectrophotometer equipped with a cut-off device (Aviv Instruments ) Or 8000 series SLM-AMINCO TM spectrophotometer (ThermoSpectronic) with stirring cuvette measurement, in the presence of increasing concentration of antigen, measure 20nM anti-antigen antibody (Fab format) in PBS pH7.2 at 25
  • the antibodies provided herein are antibody fragments.
  • Antibody fragments include but are not limited to Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other fragments described below.
  • Fab fragment antigen binding protein
  • Fab' fragment antigen binding protein
  • Fab'-SH fragment antigen binding protein
  • F(ab')2 fragment antigen binding protein
  • scFv fragments see, for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185 ; And U.S. Patent Nos.
  • Diabodies are antibody fragments with two antigen binding sites, which can be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Tribodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).
  • Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody.
  • the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516 B1).
  • Antibody fragments can be produced by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and the production of recombinant host cells (e.g., E. coli or phage), as described herein.
  • recombinant host cells e.g., E. coli or phage
  • the antibodies provided herein are chimeric antibodies.
  • Certain chimeric antibodies are described in, for example, U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).
  • a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or a non-human primate, such as a monkey) and a human constant region.
  • a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from the class or subclass of the parent antibody. Chimeric antibodies include their antigen-binding fragments.
  • the chimeric antibody is a humanized antibody.
  • non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibodies.
  • a humanized antibody contains one or more variable domains, where HVR, such as CDR (or part thereof) is derived from a non-human antibody, and FR (or part thereof) is derived from a human antibody sequence.
  • HVR such as CDR (or part thereof) is derived from a non-human antibody
  • FR or part thereof
  • the humanized antibody will also contain at least a portion of the human constant region.
  • some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody derived from HVR residues), for example, to restore or improve antibody specificity or affinity.
  • Human framework regions that can be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, for example, Sims et al., J. Immunol. 151: 2296 (1993)); Framework regions derived from the consensus sequence of human antibodies of a specific subgroup of light or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al., J Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front.Biosci.
  • the antibodies provided herein are human antibodies.
  • a variety of techniques known in the art can be used to generate human antibodies.
  • human antibodies are described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).
  • Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to generate intact human antibodies or intact antibodies with human variable regions in response to an antigenic challenge.
  • Such animals usually contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, or it exists extrachromosomally or is randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated.
  • Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Pages 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006).
  • Human antibodies can also be generated by the Fv clone variable domain sequence selected from a human-derived phage display library. Then, such variable domain sequences can be combined with the desired human constant domains. The techniques for selecting human antibodies from antibody libraries are described below.
  • the antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies having the desired activity or activities.
  • various methods for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics are known in the art. Such methods are reviewed in, for example, Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., Human Press, Totowa, NJ, 2001), and further described in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol.
  • the complete sets of VH and VL genes are cloned by polymerase chain reaction (PCR) and randomly recombined in a phage library, and then the phage library can be screened for antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994).
  • Phages usually display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies against the immunogen without the need to construct hybridomas.
  • the natural repertoire can be cloned (for example from humans) to provide a single source of antibodies against a large number of non-self and self-antigens without any immunity, such as by Griffiths et al., EMBO J, 12: 725-734 ( 1993) described.
  • an unimmunized library by cloning the unrearranged V gene segment from stem cells, using PCR primers containing random sequences to encode the highly variable CDR3 region and rearrangement in vitro, as shown by Hoogenboom and Winter , J. Mol. Biol., 227:381-388 (1992).
  • Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publication No. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007 /0292936 and 2009/0002360.
  • the antibodies or antibody fragments isolated from the human antibody library are considered to be human antibodies or human antibody fragments herein.
  • the antibodies provided herein are multispecific antibodies, such as bispecific antibodies.
  • Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for the VHH domain and the other is for any other antigen.
  • bispecific antibodies can bind to two different epitopes of the VHH domain. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing VHH domains. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.
  • Techniques for generating multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 /08829, and Traunecker et al., EMBO J.10:3655 (1991)), and "section-entry-acupuncture” engineering (see, for example, U.S. Patent No. 5,731,168). It can also be used to generate the engineered electrostatic manipulation effect of antibody Fc-heterodimer molecules (WO2009/089004A1); cross-linking two or more antibodies or fragments (see, for example, U.S. Patent No.
  • engineered antibodies with three or more functional antigen binding sites including “octopus antibodies” (see, for example, US 2006/0025576A1).
  • the antibody or fragment herein also includes a "dual acting FAb” or “DAF” containing an antigen binding site that binds to a VHH domain and another different antigen (see, for example, US 2008/0069820).
  • amino acid sequence variants of the antibodies provided herein are encompassed.
  • Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be performed to obtain the final construct, as long as the final construct possesses the desired characteristics, for example, antigen binding.
  • antibody variants with one or more amino acid substitutions are provided.
  • Sites of interest for alternative mutagenesis include HVR and FR.
  • Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions”. More substantial changes are provided in Table 1 under the heading of "Exemplary Substitutions" and are described further below with reference to amino acid side chain categories.
  • Amino acid substitutions can be introduced into the antibody of interest, and the product screened for the desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
  • amino acids can be grouped as follows:
  • Non-conservative substitutions would require replacing members of one of these categories with members of another category.
  • substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody).
  • a parent antibody e.g., a humanized or human antibody.
  • the resulting variants selected for further research will have certain biological changes (e.g. improvement) (e.g. increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain the parent antibody Some of the biological characteristics.
  • An exemplary alternative variant is an affinity matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein.
  • one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activities (such as binding affinity).
  • HVR Changes (e.g., substitutions) can be made to HVR, for example to improve antibody affinity.
  • HVR can be "hot spots", that is, residues encoded by codons that undergo mutations at a high frequency during the somatic cell maturation process (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or Such changes are made to residues in contact with the antigen, wherein the resulting variant VH or VL is tested for binding affinity.
  • Affinity maturation through the construction and reselection of secondary libraries has been described in, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O’Brien et al.
  • affinity maturation diversity is introduced as a variable gene of maturation selection by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, create a secondary library. Then, the library is screened to identify any antibody variants with the desired affinity.
  • Another method of introducing diversity involves an HVR-directed method, in which several HVR residues (eg, 4-6 residues at a time) are randomized. Alanine scanning mutagenesis or modeling can be used, for example, to specifically identify HVR residues involved in antigen binding. In particular, CDR-H3 and CDR-L3 are often targeted.
  • substitutions, insertions, or deletions can occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind antigen.
  • conservative changes e.g., conservative substitutions, as provided herein
  • HVR that do not substantially reduce binding affinity.
  • such changes can be outside of the antigen contact residues in the HVR.
  • each HVR is unchanged or contains no more than 1, 2, or 3 amino acid substitutions.
  • alanine scanning mutagenesis One method that can be used to identify residues or regions in antibodies that can be targeted for mutagenesis is called “alanine scanning mutagenesis", as described by Cunningham and Wells (1989) Science, 244:1081-1085.
  • residues or groups of target residues for example, charged residues such as arg, asp, his, lys, and glu
  • neutral or negatively charged amino acids for example, alanine Acid or polyalanine
  • Further substitutions can be introduced at amino acid positions that indicate functional sensitivity to the initial substitution.
  • the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen.
  • contact residues and neighboring residues can be targeted or eliminated.
  • the variants can be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include fusions of amino and/or carboxyl ends of polypeptides with a length ranging from 1 residue to 100 or more residues, and in-sequence insertions of single or multiple amino acid residues.
  • terminal insertions include antibodies with N-terminal methionyl residues.
  • Other insertional variants of the antibody molecule include fusions of the N- or C-terminus of the antibody with an enzyme (for example, for ADEPT) or a polypeptide that extends the serum half-life of the antibody.
  • the antibodies provided herein can be further modified to contain additional non-proteinaceous modules known in the art and readily available.
  • Modules suitable for antibody derivatization include, but are not limited to, water-soluble polymers.
  • water-soluble polymers include but are not limited to polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1 ,3-Dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene Pyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerin), polyvinyl alcohol and mixtures thereof.
  • PEG polyethylene glycol
  • polyethylene glycol propionaldehyde Due to its stability in water, polyethylene glycol propionaldehyde may have advantages in production.
  • the polymer can be of any molecular weight and can be branched or unbranched.
  • the number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally speaking, the number and/or type of polymers used for derivatization can be determined based on the following considerations, including but not limited to the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used for treatment under specified conditions, etc. .
  • nucleic acids encoding an anti-VHH domain antibody described herein are provided.
  • nucleic acids may encode the amino acid sequence comprising the VL of the antibody and/or the amino acid sequence of the VH of the antibody (e.g., the light and/or heavy chain of the antibody).
  • vectors e.g., expression vectors
  • a host cell comprising such nucleic acid is provided.
  • the host cell comprises (e.g., has been transformed with the following vector): (1) a vector comprising a nucleic acid encoding the amino acid sequence of the VL comprising the antibody and the amino acid sequence of the VH comprising the antibody, or (2) A first vector and a second vector, the first vector comprising a nucleic acid encoding the amino acid sequence of the VL comprising the antibody, and the second vector comprising the nucleic acid encoding the amino acid sequence of the VH comprising the antibody.
  • the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells).
  • a method of producing an anti-VHH domain antibody comprising culturing a host cell containing a nucleic acid encoding the antibody in a medium under conditions suitable for expression of the antibody, as provided above, and optionalally, the antibody is recovered from the host cell (or host cell culture medium).
  • the nucleic acid encoding the antibody (for example, as described above) is isolated and inserted into one or more vectors for further cloning and/or expression in host cells.
  • Such nucleic acids can be easily separated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding genes encoding the heavy and light chains of antibodies).
  • Host cells suitable for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein.
  • antibodies can be produced in bacteria (such as E. coli), especially when glycosylation and Fc effector functions are not required.
  • bacteria such as E. coli
  • For the expression of antibody fragments and polypeptides in bacteria see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (Edited by BKCLo, Humana Press, Totowa, NJ, 2003), Page 245-254, which describes the expression of antibody fragments in E. coli.).
  • the antibody can be separated from the bacterial cell mass in the soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable for cloning or expression hosts for antibody-encoding vectors, including their glycosylation pathways that have been "humanized”, resulting in partial or fully human glycosylation.
  • Style antibodies to fungal and yeast strains See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
  • Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include insect cells. Many baculovirus strains have been identified that can be used with insect cells, especially for transfecting Spodoptera frugiperda cells.
  • plant cell culture as a host. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES TM technology for generating antibodies in transgenic plants).
  • mammalian cell lines suitable for growth in suspension may be useful.
  • useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 or 293 cells, as described in, for example, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, as described in, for example, Mather, Biol. Reprod.
  • anti-VHH domain antibodies provided herein can be identified, screened, or characterized by their physical/chemical properties and/or biological activities by a variety of assays known in the art.
  • the antibody of the present invention is tested for its antigen binding activity, for example, by a known method such as ELISA, Western blot, and the like.
  • competition assays can be used to identify antibodies that compete with any of the anti-VHH domain antibodies described herein for binding to the VHH domain.
  • such competitive antibodies bind to the same epitope (e.g., linear or conformational epitope) as bound by any of the anti-VHH domain antibodies described herein. See Morris (1996) “Epitope Mapping Protocols", Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ) for detailed exemplary methods for locating epitopes bound by antibodies.
  • a first labeled antibody (which binds to a VHH domain, such as any of the anti-VHH domain antibodies described herein) and a second unlabeled antibody (which are to be tested to compete with the first antibody) are included in an exemplary competition assay.
  • a first labeled antibody (which binds to a VHH domain, such as any of the anti-VHH domain antibodies described herein) and a second unlabeled antibody (which are to be tested to compete with the first antibody) are included in an exemplary competition assay.
  • the second antibody may be present in the supernatant of the hybridoma.
  • the immobilized VHH domain was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody.
  • the excess unbound antibody is removed, and the amount of label associated with the immobilized VHH domain is measured. If the amount of the label associated with the immobilized VHH domain in the test sample is substantially reduced compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to the VHH domain. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
  • any of the anti-VHH domain antibodies provided herein can be used to detect the presence of VHH domains in biological samples.
  • the term “detection” encompasses quantitative or qualitative detection.
  • an anti-VHH domain antibody for use in a detection method is provided.
  • a method of detecting the presence of a VHH domain in a biological sample is provided.
  • the method includes contacting a biological sample with an anti-VHH domain antibody under conditions that allow the anti-VHH domain antibody to bind to the VHH domain, as described herein, and detecting whether the anti-VHH domain antibody is in contact with the VHH domain. Complexes are formed between domains.
  • Such methods can be in vitro or in vivo methods.
  • Labels include, but are not limited to, directly detectable labels or modules (such as fluorescence, chromogenic, electronic compaction, chemiluminescence, and radioactive labels), and modules that are detected indirectly via enzyme reactions or molecular interactions, such as enzymes or ligands, for example. body.
  • Exemplary labels include but are not limited to the radioisotopes 32 P, 14 C, 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dan Acyl, umbelliferone, luciferase, for example, firefly luciferase and bacterial luciferase (US Patent No.
  • luciferin 2,3-dihydrophthalazinedione, horseradish Oxidase (HRP), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate deoxidase Hydrogenase, heterocyclic oxidase such as uricase and xanthine oxidase (which are coupled with enzymes that use hydrogen peroxide dye precursors such as HRP), lactoperoxidase, or microperoxidase, biotin /Avidin, spin marker, phage marker, stable free radical, etc.
  • HRP horseradish Oxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase ⁇ -galactosidase
  • glucoamylase
  • Example 1 Obtaining antibody hybridoma cell lines
  • the antigen used for animal immunization is the recombinantly expressed His tag camel-derived nanobody VHH-His (amino acid sequence is shown in Figure 1 or SEQ ID NO: 241).
  • This Nanobody AS718 is an antigen used for animal immunization and has a C-terminal His tag. Containing a total of 121 amino acids, the CDR domain and His tag are shown in Figure 1 underlined.
  • Figure 2 In the indirect ELISA mode, the immunogen AS718VHH-His is used to coat the ELISA test plate, and the animal serum is diluted at the initial ratio of 1:1,000, and then the ELISA detection is carried out with a 2-fold serial dilution to 1:512,000, and the OD450 is read.
  • S/N stands for Signal-to-Noise, which is the signal-to-noise ratio.
  • the effective serum titer criterion is 2.1 times the blank background value.
  • the serum dilution ratio is measured at 1:64,000. If the value is greater than 1.0, fusion is possible.
  • the detection value of animal #6119 serum was 1.323 when the dilution ratio was 1:64,000, and the detection value was 0.488 when the serum dilution ratio was 1:512,000; the detection value of animal #563 serum was 1.637 when the dilution ratio was 1:64,000, and the serum was diluted When the ratio is 1:512,000, the detection value is 0.528. Since the detection value of the two animals at the serum dilution ratio of 1:512,000 is greater than 2.1 times the blank background value, the effective serum titer of the two animals is greater than 1:512,000, and the detection value of 1:64,000 is greater than 1.0, which is consistent with the animal Convergence standards.
  • the rabbit spleen is extracted and homogenized to produce a single-cell suspension, and a single-cell suspension of myeloma cells is prepared at the same time.
  • the ELISA plate was coated with 0.5 ⁇ g/ml VHH-His or His protein in 100 ⁇ l/well of PBS overnight at 4°C; the plate was washed with PBS-T (0.05% Tween20), and 200 ⁇ l/well containing Block with 1% BSA in PBST at 37°C for 0.5 hours; aspirate the blocking solution, add 100 ⁇ l of hybridoma cell culture supernatant to each well, and then incubate at room temperature for 1 hour; after the incubation, discard the supernatant, and The plate was washed three times with PBST and incubated with 100 ⁇ l/well of goat anti-rabbit IgG (GenScript Goat Anti-Rabbit IgG Antibody (H&L)[HRP],pAb;CAT#:A00098) at 37°C for 0.5 Hours; wash the plate five times with PBST, then add TMB color development solution and incubate in the dark at room temperature for 15 minutes.
  • H&L goat anti-rabbit
  • a detection value greater than 0.5 was judged as positive, and a detection value less than 0.2 was judged as negative.
  • the results showed that the detection values of positive serum and VHH-His, positive serum and His-tag protein, Anti-His antibody and VHH-His, Anti-His antibody and His-tag protein binding detection values were 2.953, 0.747, 2.906, 2.858, all greater than 0.5 If it is judged as positive, the positive control is valid, and the detection value of the binding between the positive serum and the His-tag protein is 0.747, which means that antibodies against the His-tag protein are still produced in the fusion animal.
  • the ELISA reaction values of 15 parent clone supernatant stocks and immunogen AS718VHH-His were 2.758, 3.060, 2.908, 2.883, 2.893, 2.556, 2.913, 2.655, 2.561, 2.356, 3.059, 2.130, 2.083, 2.454, 2.428, respectively If it is greater than 0.5, the supernatant of 15 maternal clones is determined to be positive for VHH-His; the ELISA reaction values of 15 maternal clone supernatant stock and His tag protein are 0.074, 0.130, 0.091, 0.102, 0.082, 0.100, 0.121, respectively.
  • the ELISA test data of the subclonal strain is shown in Figure 4 (the subclonal strain supernatant ELISA titer test): Pass In the indirect ELISA mode, the immunogen AS718VHH-His is used to coat the ELISA test plate, and the positive animal serum diluted 1:1,000 is used as a positive control. The OD450 reading is used to detect the titers of the cell supernatants of 15 parent clones after subcloning. . The cell supernatant stock solution is used in the first well, and it is diluted to 1:81 with a 3-fold ratio in turn. When the cell supernatant dilution ratio is 1:9, the detection value is greater than 1.0, and antibody production can be carried out.
  • Example 2 Sequencing of the variable region of the antibody and recombinant production of the antibody
  • TRIzol (Ambion, CAT#: 15596-026) to extract total RNA from 3 ⁇ 10 6 -5 ⁇ 10 6 hybridoma cells, and use antibody subtype-specific primers and universal primers (Takara PrimeScript TM 1st Strand cDNA Synthesis Kit , CAT#:6110A) reverse transcribed it into cDNA. Subsequently, the rabbit immunoglobulin heavy chain and light chain variable region fragments were amplified by RACE PCR, and the resulting PCR fragments were subcloned into the pMD18-T vector system (Takara, CAT#: 6011), and vector-specific primers were used Sequence the insert.
  • nucleotide/protein sequences of the heavy and light chain variable regions of 15 monoclonal antibodies were obtained, including clone R166.C5, clone R166.G3, clone R166.F2, clone R166.G8, clone R166.H9, clone R166.D1, clone R166.G10, clone R166.H10, clone R166.F9, clone R166.G2, clone R166.E5, clone R166.E3, clone R166.H8, clone R166.E7, and clone R166.A5 .
  • Antibodies are produced by recombinant expression, taking a preferred clone as an example.
  • the light chain variable region + constant region (amino acid sequence is shown in SEQ ID NO: 244, and the nucleotide sequence is shown in SEQ ID NO: 245) and heavy chain variable region + constant region (amino acid sequence is shown in SEQ ID NO: 242, the nucleotide sequence is shown in SEQ ID NO: 243) DNA fragments, inserted into the pTT5 expression vector to form expression plasmids; the above plasmids were co-transfected into CHO-3E7 cells, and After culturing in a shake flask at 37°C for 6 days, collect the supernatant for antibody purification; use 0.2M NaOH to depyrogenate the tubing and protein A column, and then use a buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0) on the column Rebalance; Dilute the harvested cell culture supernatant with 2 ⁇ the above buffer 1:1 and filter sterilize.
  • the gel was stained with eStain2.0 (GenScript) and the molecular size and purity were estimated by comparing the stained band with the Protein Ladder (GenScript).
  • the purity identification results of 15 antibodies are shown in Figures 5a and 5b, under non-reducing conditions
  • the two heavy chains and two light chains of the antibody still maintain the tetravalent structure through disulfide bonds, and only one band of interest is shown in the electrophoretic staining results.
  • the displayed molecular weight is slightly lower than the actual molecular weight, which is comparable to the 116kDa marker.
  • the strips are of comparable size. Under reducing conditions, the presence of the reducing agent destroys the disulfide bond structure in the antibody, and the heavy chain of the antibody is separated from the light chain.
  • Example 3 Binding of the antibody to the recombinant camelid nanobody
  • the indirect ELISA method was used to evaluate the binding ability of the purified antibodies of 15 camel-derived Nanobodies to 10 different camel-derived Nanobodies VHH (amino acid sequence shown in SEQ ID NO: 246-255).
  • the ELISA plate was coated overnight at 4°C with 100 ⁇ l/well of PBS 0.5 ⁇ g/ml in 10 different VHHs; the plate was washed with PBS-T (0.05% Tween), and 200 ⁇ l/well was used.
  • PBST containing 1% BSA was blocked at 37°C for 0.5 hours; aspirate the blocking solution, add 100 ⁇ l of 1 ⁇ g/ml purified antibody to the first well, and dilute it in a 3-fold gradient, a total of 11 test concentration gradients, incubate at room temperature for 1 hour; Wash the plate three times with PBST, and incubate with 100 ⁇ l/well of goat anti-rabbit IgG (GenScript Goat Anti-Rabbit IgG Antibody (H&L)[HRP],pAb;CAT#:A00098) at 37°C. 0.5 hour; wash the plate five times with PBST, then add TMB color development solution and incubate in the dark at room temperature for 15 minutes.
  • H&L GenScript Goat Anti-Rabbit IgG Antibody
  • the reaction was terminated by adding 50 ⁇ l of 1M HCl stop solution.
  • antibodies of the same camel-derived Nanobody have different affinities with different camel-derived Nanobodies; on the other hand, antibodies of different camel-derived Nanobodies have different affinities from the same camel-derived Nanobody. But overall, these 15 purified antibodies can all be recognized with 10 different camel-derived Nanobodies.
  • the EC50 calculated according to the ELISA concentration gradient experiment shows that these antibodies all show a relatively high affinity for the antigen.
  • the aforementioned 10 camel-derived Nanobodies and AS718 Nanobodies have 74.7% (AS154, AS325), 77.0% (AS587), 79.3% (AS675), 80.5% (AS200, AS519), 81.6% (AS656), 82.8 % (AS588, AS988), 83.9% (AS673) of the framework region sequence identity.
  • the above 10 camel-derived Nanobodies and AS718 Nanobodies have 62.3% (AS154), 62.9% (AS200), 63.6% (AS325), 64.8% (AS656), 66.1% ( AS675, AS673), 66.4% (AS587, AS988), 68.0% (AS588), 69.9% (AS519) sequence identity.
  • the identity of all 11 Nanobodies is 37.7%.
  • a Biacore 8K instrument (GE healthcare) was used to determine the affinity level of the antibody clone R166.C5 with multiple camel-derived nanobodies through SPR experimental technology.
  • CM5 chip pretreatment equilibration buffer HBS 10 ⁇ l/min after 5 minutes, treat the chip 10 ⁇ l/min with NHS/EDC activator for 7 minutes, then add 10 ⁇ l of coupled anti-His antibody (GenScript, CAT#:A00186) /min react for 7 minutes, and finally block with 10 ⁇ l/min aminoethanol.
  • Capture of camel-derived nanoantibodies After equilibrating with 10 ⁇ l/min equilibration buffer HBS for 5 minutes, add 10 strains of 20 ⁇ l/ml VHH-His antibody, and fully react at 30 ⁇ l/min for 1 minute; use 200nM antibody concentration and 30 ⁇ l in the mobile phase The affinity level of the antibody was determined at a flow rate of 1/min.
  • the immunogen AS718 was used as a positive control for camel-derived nanoantibodies.
  • the results showed that the affinity between the immunogen AS718 and the cloned antibody reached 3.47 pmol.
  • 6 strains of AS325, AS656, AS673, AS588, AS988 and AS519 all reached the pmol level with affinities of 4.04pmol, 1.34pmol, 4.36pmol, 7.09pmol, 7.08pmol and 1.66pmol, respectively.
  • the one camel-derived Nanobody with the lowest affinity with the cloned antibody is AS587, and its affinity is also up to 2.2 nmol.
  • a fluorescently-labeled camelid-derived nanobody antibody is used to isolate heavy chain antibody-specific PBMC by flow cytometry.
  • the PBMC cells of non-immunized camels are used in the present invention.
  • PBMC cells for analysis, wash the cells once with 500 ⁇ l FACS buffer (PBS, 1% BSA), centrifuge at 1000 rpm for 5 minutes and discard the supernatant; choose 200 ⁇ l FACS buffer to resuspend the PBMC cells and add 2 ⁇ g algae Antibodies R166.C5, R166.H8, R166.H9, R166.E7, and R166.G8 of camel-derived nanobodies labeled with fluorescein (PE), in the dark, after incubating for 15 minutes in an ice bath, use 500 ⁇ l FACS Wash the cells once with buffer, centrifuge at 1000rpm for 5 minutes and discard the supernatant; fix the cells with 2% polymethanol solution for 30 minutes, wash the cells with 500 ⁇ l FACS buffer once, centrifuge at 1000rpm for 5 minutes and discard the supernatant; use 200 ⁇ l FACS buffer Resuspend the cells and perform flow sorting of heavy chain antibody-specific PBMC cells.
  • FACS buffer PBS, 1% B
  • the antibody of the present invention is used to purify heavy chain antibodies in the serum of a llama or llama, and the serum of a non-immune alpaca is used in the present invention.
  • the purified antibodies (including the serum supernatant before magnetic bead purification, the eluate after magnetic bead purification and the eluted sample after purification) were passed through the BioRad electrophoresis system with 10% precast gel (GenScript, CAT#: M00665) and passed SDS- PAGE to analyze.
  • Use the gel L1 Protein Staining System (GenScript) stains and estimates the molecular size and purity by comparing the stained band with the Protein Ladder.
  • the molecular weights of the labeled Camelidae IgG2 and IgG3 antibodies are 46KD and 43KD, respectively.
  • the obtained magnetic beads can specifically purify heavy chain antibodies. After purification, the overall purity of heavy chain antibodies IgG2 and IgG3 are respectively They are 77.4%, 78.3%, 71.6%, 80.1%, 81.1% and 73.8%.
  • the antibody of the present invention can be used for flow cytometric identification of CART cells.
  • the CART cells that meet the above conditions are compared with the initial ratio (0%, 10%, 20%, 40%, 80%, 95%) T cells were mixed to prepare cell samples to be tested; 5 ⁇ 10 5 cells were selected for each sample, and the cells were washed once with 500 ⁇ l FACS buffer (PBS, 1% BSA), centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded; 400 ⁇ l FACS buffer was selected Resuspend the cells in the solution and add 4.5 ⁇ g of biotin-labeled camel-derived nanobody antibody clone R166.A5.
  • FACS buffer PBS, 1% BSA
  • the CART cells and the initial T cells were mixed at different fixed ratios of 0%, 10%, 20%, 40%, 80% and 95%, and the antibody of the clone R166.A5 labeled with biotin and the PE labeled Streptavidin was used for flow cytometric identification.
  • the results showed that the proportion of CAR-positive cells were 0.01%, 9.72%, 20.9%, 39.3%, 80.5% and 93.7%, and the deviation from the premix ratio was less than ⁇ 1.5%
  • the data show that the antibody can be well applied to CART cell detection using camel-derived nanoantibodies to construct chimeric receptors. This antibody can well recognize CAR-positive T cells.
  • the antibody of the present invention can be used for magnetic separation of CART cells.
  • the number of CART cells to be separated is 1 ⁇ 10 7 ; centrifuge at 1000 rpm for 10 minutes and discard the supernatant, using 100 ⁇ l PBE buffer (PBS, pH 7.2, 0.5% BSA, 2 mM EDTA) fully Resuspend the cells; add 2 ⁇ g of biotin-labeled camel-derived nanobody antibody clone R166.A5, mix thoroughly, and incubate at 4°C for 10 minutes; wash the cells once with 2ml PBE buffer, and fully reconstitute with 100 ⁇ l PBE buffer Suspended cells; add 20 ⁇ l anti-biotin magnetic beads (Miltenyi), mix well, incubate at 4°C for 10 minutes, use a magnetic stand for cell separation, perform flow cytometry on the cells obtained above to verify the magnetic separation results, shown in Figure 10, the use of naive T cells CAR negative cells as a control, 10% CAR positive T cells and naive T cells were mixed as a

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Abstract

本发明提供一组抗VHH域抗体及其用途。本发明进一步提供了上述抗体在纳米抗体开发、筛选及纯化中的应用，及上述抗体在免疫治疗领域中的应用。

Description

抗VHH域抗体及其用途 技术领域
本发明涉及一组抗VHH域抗体。本发明还涉及该抗体的制备和获得方法。同时，本发明涉及该抗体在纳米抗体开发、筛选及纯化中的应用。本发明还涉及该抗体在免疫治疗领域中的应用
背景技术
骆驼源纳米抗体(Nanobody)又称单域抗体(Single-domain antibody)、重链可变区抗体(variable domain of heavy chain of HCAb,VHH)，它只包括骆驼源重链抗体的可变区片段，大小只有15kDa左右，可以高亲和力，高特异性的与抗原结合，在抗体药物开发及免疫细胞治疗等领域具有非常重要的应用。
1993年，Hamers-Casterman等人首次发现骆驼科动物中存在缺失轻链的重链抗体(heavy-chain antibodies,HCAbs)[C.Hamers,et al.,Naturally occurring antibodies devoid of light chains.Nature,1993.Vol 363:p 466-468]，这种抗体只包含重链可变区和CH2与CH3恒定区。通过分子克隆手段表达的VHH可变区具有非常好的稳定性和亲和力，是目前已知的最小的抗体单元。目前Ablynx公司基于纳米抗体开发技术已经成功研发并上市了第一款治疗性纳米抗体药物caplacizumab，用于治疗获得性血栓性血小板减少性紫癜(aTTP)[https://www.ablynx.com/rd-portfolio/clinical-programmes/caplacizumab/]。
同时，骆驼源纳米抗体在免疫细胞治疗领域也有非常重要的应用。南京传奇公司的LCAR-B38M是中国第一个通过cFDA临床申报的细胞治疗疗法，同时也是第一个实现中美双报双批的项目，该项目在目前公开的数据中展现出了惊人的治疗效果，在中国免疫细胞疗法的发展历程中具有重要的意义。该项目在CAR的设计上就采用了独特的纳米抗体设计嵌合抗原受体，避免了常规技术路线中scFv的稳定性差，亲和力低的缺点。伴随着免疫细胞治疗技术的发展，纳米抗体也越来越受到研究者的青睐。
虽然纳米抗体在抗体药物开发和免疫细胞治疗领域具有重要的意义，但是目前缺乏一种识别骆驼源纳米抗体的抗体，以便更好的开发骆驼源纳米抗体或优化免疫细胞治疗中CART细胞的鉴定，分选和磁分离。本发明开发了一组高亲和力，高特异性，高功能性的针对骆驼源纳米抗体的抗体，行之有效的解决了上述问题，满足了各个应用领域的需求。
发明概述
在一个方面，本发明提供一种抗体或其抗原结合片段，其特异性结合VHH域。在一个实施方案中，所述VHH域是骆驼源抗体的VHH域。在一个实施方案中，所述骆驼源抗体是单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Vicugna pacos)或小羊驼(Lama glama)来源的单域抗体或重链抗体。
在另一个方面，本发明提供一种抗体或其抗原结合片段。在一些实施方案中，本发明公开的抗体或其抗原结合片段含有重链可变区(HCVR)和轻链可变区(LCVR)，其中(A)所述重链可变区包括重链互补决定区HCDR1、HCDR2和HCDR3，(a)所述HCDR1序列选自SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14或15所示的氨基酸序列或与上述序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同 一性的氨基酸序列，(b)所述HCDR2序列选自SEQ ID NO:16、17、18、19、20、21、22、23、24、25、26、27、28、29或30所示的氨基酸序列或与上述序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，且(c)所述HCDR3序列选自SEQ ID NO:31、32、33、34、35、36、37、38、39、40、41、42、43、44或45所示的氨基酸序列或与上述序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，(d)重链互补决定区HCDR含有一处或多处不超过3个氨基酸取代，缺失或插入的(a)、(b)和(c)的氨基酸序列；且(B)所述轻链可变区包括轻链互补决定区LCDR1、LCDR2和LCDR3，(e)所述LCDR1序列选自SEQ ID NO:46、47、48、49、50、51、52、53、54、55、56、57、58、59或60所示的氨基酸序列或与上述序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，(f)所述LCDR2序列选自SEQ ID NO:61、62、63、64、65、66、67、68、69、70、71、72、73、74或75所示的氨基酸序列或与上述序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，(g)所述LCDR3序列选自SEQ ID NO:76、77、78、79、80、81、82、83、84、85、86、87、88、89或90所示的氨基酸序列或与上述序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，(h)轻链互补决定区LCDR含有一处或多处不超过3个氨基酸取代，缺失或插入的(e)、(f)和(g)的氨基酸序列。
在进一步的实施方案中，本发明公开的抗体或其抗原结合片段含有(1)SEQ ID NO:1所示HCDR1、SEQ ID NO:16所示HCDR2、SEQ ID NO:31所示HCDR3、SEQ ID NO:46所示LCDR1、SEQ ID NO:61所示LCDR2和SEQ ID NO:76所示LCDR3；(2)SEQ ID NO:2所示HCDR1、SEQ ID NO:17所示HCDR2、SEQ ID NO:32所示HCDR3、SEQ ID NO:47所示LCDR1、SEQ ID NO:62所示LCDR2和SEQ ID NO:77所示LCDR3；(3)SEQ ID NO:3所示HCDR1、SEQ ID NO:18所示HCDR2、SEQ ID NO:33所示HCDR3、SEQ ID NO:48所示LCDR1、SEQ ID NO:63所示LCDR2和SEQ ID NO:78所示LCDR3；(4)SEQ ID NO:4所示HCDR1、SEQ ID NO:19所示HCDR2、SEQ ID NO:34所示HCDR3、SEQ ID NO:49所示LCDR1、SEQ ID NO:64所示LCDR2和SEQ ID NO:79所示LCDR3；(5)SEQ ID NO:5所示HCDR1、SEQ ID NO:20所示HCDR2、SEQ ID NO:35所示HCDR3、SEQ ID NO:50所示LCDR1、SEQ ID NO:65所示LCDR2和SEQ ID NO:80所示LCDR3；(6)SEQ ID NO:6所示HCDR1、SEQ ID NO:21所示HCDR2、SEQ ID NO:36所示HCDR3、SEQ ID NO:51所示LCDR1、SEQ ID NO:66所示LCDR2和SEQ ID NO:81所示LCDR3；(7)SEQ ID NO:7所示HCDR1、SEQ ID NO:22所示HCDR2、SEQ ID NO:37所示HCDR3、SEQ ID NO:52所示LCDR1、SEQ ID NO:67所示LCDR2和SEQ ID NO:82所示LCDR3；(8)SEQ ID NO:8所示HCDR1、SEQ ID NO:23所示HCDR2、SEQ ID NO:38所示HCDR3、SEQ ID NO:53所示LCDR1、SEQ ID NO:68所示LCDR2和SEQ ID NO:83所示LCDR3；(9)SEQ ID NO:9所示HCDR1、SEQ ID NO:24所示HCDR2、SEQ ID NO:39所示HCDR3、SEQ ID NO:54所示LCDR1、SEQ ID NO:69所示LCDR2和SEQ ID NO:84所示LCDR3；(10)SEQ ID NO:10所示HCDR1、SEQ ID NO:25所示HCDR2、SEQ ID NO:40所示HCDR3、SEQ ID NO:55所示LCDR1、SEQ ID NO:70所示LCDR2和SEQ ID NO:85所示LCDR3；(11)SEQ ID NO:11所示HCDR1、SEQ ID NO:26所示HCDR2、SEQ ID NO:41所示HCDR3、SEQ ID NO:56所示LCDR1、SEQ ID NO:71所示LCDR2和SEQ ID NO:86所示LCDR3；(12)SEQ  ID NO:12所示HCDR1、SEQ ID NO:27所示HCDR2、SEQ ID NO:42所示HCDR3、SEQ ID NO:57所示LCDR1、SEQ ID NO:72所示LCDR2和SEQ ID NO:87所示LCDR3；(13)SEQ ID NO:13所示HCDR1、SEQ ID NO:28所示HCDR2、SEQ ID NO:43所示HCDR3、SEQ ID NO:58所示LCDR1、SEQ ID NO:73所示LCDR2和SEQ ID NO:88所示LCDR3；(14)SEQ ID NO:14所示HCDR1、SEQ ID NO:29所示HCDR2、SEQ ID NO:44所示HCDR3、SEQ ID NO:59所示LCDR1、SEQ ID NO:74所示LCDR2和SEQ ID NO:89所示LCDR3；或(15)SEQ ID NO:15所示HCDR1、SEQ ID NO:30所示HCDR2、SEQ ID NO:45所示HCDR3、SEQ ID NO:60所示LCDR1、SEQ ID NO:75所示LCDR2和SEQ ID NO:90所示LCDR3。
在一些实施方案中，本发明公开的抗体或其抗原结合片段包含重链可变区(HCVR)，其氨基酸序列选自SEQ ID NO:91、92、93、94、95、96、97、98、99、100、101、102、103、104或105所示的氨基酸序列或与上述序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，本发明公开的抗体或其抗原结合片段包含轻链可变区(LCVR)，其氨基酸序列选自SEQ ID NO:106、107、108、109、110、111、112、113、114、115、116、117、118、119或120的氨基酸序列或与上述序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在进一步的实施方案中，本发明公开的抗体或其抗原结合片段含有(1)SEQ ID NO:91所示重链可变区和SEQ ID NO:106所示轻链可变区；(2)SEQ ID NO:92所示重链可变区和SEQ ID NO:107所示轻链可变区；(3)SEQ ID NO:93所示重链可变区和SEQ ID NO:108所示轻链可变区；(4)SEQ ID NO:94所示重链可变区和SEQ ID NO:109所示轻链可变区；(5)SEQ ID NO:95所示重链可变区和SEQ ID NO:110所示轻链可变区；(6)SEQ ID NO:96所示重链可变区和SEQ ID NO:111所示轻链可变区；(7)SEQ ID NO:97所示重链可变区和SEQ ID NO:112所示轻链可变区；(8)SEQ ID NO:98所示重链可变区和SEQ ID NO:113所示轻链可变区；(9)SEQ ID NO:99所示重链可变区和SEQ ID NO:114所示轻链可变区；(10)SEQ ID NO:100所示重链可变区和SEQ ID NO:115所示轻链可变区；(11)SEQ ID NO:101所示重链可变区和SEQ ID NO:116所示轻链可变区；(12)SEQ ID NO:102所示重链可变区和SEQ ID NO:117所示轻链可变区；(13)SEQ ID NO:103所示重链可变区和SEQ ID NO:118所示轻链可变区；(14)SEQ ID NO:104所示重链可变区和SEQ ID NO:119所示轻链可变区；或(15)SEQ ID NO:105所示重链可变区和SEQ ID NO:120所示轻链可变区。
在任何上述实施方案中，本发明的抗体或其抗原结合片段特异性结合VHH域。在一个实施方案中，所述VHH域是骆驼源抗体的VHH域。也就是说，上述序列所规定的抗体或其抗原结合片段具有良好的骆驼源单域抗体或骆驼源重链抗体结合能力。在一个实施方案中，所述骆驼源抗体是单峰驼、双峰驼、大羊驼或小羊驼来源的单域抗体或重链抗体。
在上述方面的一个实施方案中，本发明的抗体或其抗原结合片段在VHH域的框架区处结合。在上述方面的一个实施方案中，本发明的抗体或其抗原结合片段在VHH域的构象表位处结合。在上述方面的一个实施方案中，本发明的抗体或其抗原结合片段在VHH域的框架区构象表位处结合。
在上述方面的一个实施方案中，本发明的抗体或其抗原结合片段特异性结合SEQ ID NO：241和246-255任一所示VHH域，或与SEQ ID NO：241和246-255任一具有至少35％、至 少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％氨基酸序列同一性的VHH域，或SEQ ID NO：241和246-255共有序列。在上述方面的一个实施方案中，本发明的抗体或其抗原结合片段特异性结合SEQ ID NO：241和246-255任一所示VHH域的框架区，或与SEQ ID NO：241和246-255任一所示VHH域的框架区具有至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％氨基酸序列同一性的框架区，或SEQ ID NO：241和246-255共有框架序列。
在上述方面的一个实施方案中，本发明的抗体或其抗原结合片段以K D为10nM至1pM，例如1nM至10pM，例如1pM至10pM，例如1pM至5pM的结合亲和力特异性结合VHH域，例如SEQ ID NO：241和246-255任一所示VHH域，或与SEQ ID NO：241和246-255任一具有至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％氨基酸序列同一性的VHH域，或SEQ ID NO：241和246-255共有序列，或者包含SEQ ID NO：241和246-255任一所示所示VHH域的框架区，或与SEQ ID NO：241和246-255任一所示所示VHH域的框架区具有至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％氨基酸序列同一性的框架区的VHH域，或SEQ ID NO：241和246-255共有框架序列。在一个实施方案中，结合亲和力是通过表面等离振子共振(SPR)技术测定的。
在上述方面的一个实施方案中，本发明的抗体或其抗原结合片段在包含一个或多个选自下组的位置的表位处结合VHH域：依照Kabat编号方式的位置3-4、6-10、13、15、17-18、20-22、25-26、36、38、41、46、48、66-67、69-70、72、75、79-80、82C、85-88、90、92-93、105-107、109和111-112；或与依照连续编号方式的SEQ ID NO：241位置3-4、6-10、13、15、17-18、20-22、25-26、36、38、41、46、48、66-67、69-70、72、75、79-80、85、88-91、93、95-96、107-109、111和113-114对应的位置(见图11)。
在上述方面的一个实施方案中，本发明的抗体或其抗原结合片段在包含一个或多个选自下组的位置的表位处结合VHH域：依照Kabat编号方式的位置3-10、13、15、17-22、25-26、36-43、45-46、48-49、66-70、72-73、75-82、82B-88、90-94和103-113；或与依照连续编号方式的SEQ ID NO：248和/或255位置3-10、13、15、17-22、25-26、36-43、45-46、48-49、66-70、72-73、75-82、84-91、93-97和105-115对应的位置。
在上述方面的一个实施方案中，本发明的抗体或其抗原结合片段在包含一个或多个选自下组的位置的表位处结合VHH域：依照Kabat编号方式的位置3-10、13、15、17-22、25-26、36-38、40-41、43、45-46、48-49、66-70、72-73、75-77、79-82、82C-88、90-94、103-109和111-113；或与依照连续编号方式的SEQ ID NO：248和/或255位置3-10、13、15、17-22、25-26、36-38、40-41、43、45-46、48-49、66-70、72-73、75-77、79-82、85-91、93-97、105-111和113-115对应的位置。
在上述方面的一个实施方案中，本发明的抗体或其抗原结合片段在包含一个或多个选自下组的位置的表位处结合VHH域：依照Kabat编号方式的位置39、42、78、82B和110； 或与依照连续编号方式的SEQ ID NO：248和/或255位置39、42、78、84和112对应的位置。
在另一个方面，本发明涵盖一种抗体或其抗原结合片段，其与前述抗体或其抗原结合片段结合相同表位。在另一个方面，本发明涵盖一种抗体或其抗原结合片段，其与前述抗体或其抗原结合片段竞争结合。
在一个方面，本发明的抗体或其抗原结合片段是裸抗体或其抗原结合片段。
在一个方面，本发明提供一种或多种多核苷酸，其编码本发明的抗体或其抗原结合片段。在一个方面，本发明提供一种或多种载体，其包含本发明的多核苷酸。在一个实施方案中，所述载体选自克隆载体和表达载体。在一个方面，本发明提供一种宿主细胞，其包含本发明的多核苷酸或载体。在一个实施方案中，所述宿主细胞选自原核细胞、酵母细胞、昆虫细胞和哺乳动物细胞。在一个方面，本发明提供一种生成抗体或其抗原结合片段的方法，其包括在适合抗体生成的条件下培养本发明的宿主细胞使得所述抗体或其抗原结合片段表达。在一个实施方案中，所述方法还包括回收所述抗体或其抗原结合片段。
在一个方面，本发明提供一种偶联物，其包含本发明的抗体或其抗原结合片段。在一个实施方案中，所述抗体或其抗原结合片段与荧光素、生物素、酶、琼脂糖树脂、磁珠或生物芯片偶联。
在一个方面，本发明提供一种试剂盒，其包括包含本发明的抗体或其抗原结合片段、或偶联物的容器。
在一个方面，本发明提供一种检测VHH域的方法，其包括将如本发明的抗体或其抗原结合片段或偶联物添加至已知或怀疑含有VHH域的样品，并检测所述抗体或其抗原结合片段或所述偶联物与所述VHH域之间形成的复合物。
在一个方面，本发明提供一种分离VHH域的方法，其包括将本发明的抗体或其抗原结合片段或偶联物添加至已知或怀疑含有VHH域的样品，并分离所述抗体或其抗原结合片段或所述偶联物与所述VHH域之间形成的复合物。
在一个实施方案中，所述VHH域在骆驼源抗体中。在一个实施方案中，所述骆驼源抗体是单峰驼、双峰驼、大羊驼或小羊驼来源的单域抗体或重链抗体。在一个实施方案中，所述VHH域在嵌合抗原受体中。在一个实施方案中，所述嵌合抗原受体在免疫细胞上。在一个实施方案中，所述免疫细胞选自PBMC、T细胞、NK细胞或巨噬细胞。在一个实施方案中，所述方法利用FACS或MCS进行。
附图说明
图1显示动物免疫采用的骆驼源纳米抗体VHH-His的示意图和氨基酸序列。
图2显示融合动物血清ELISA效价检测。
图3显示母克隆定株上清ELISA效价检测。
图4显示亚克隆定株上清ELISA效价检测。
图5a、5b显示15株纯化抗体PAGE纯度检测。
图6a、6b显示15株该抗体与10种不同骆驼源纳米抗体EC50亲和力检测。
图7显示骆驼源纳米抗体的框架区氨基酸序列比对。
图8显示克隆R166.C5亲和力检测。
图9显示CART细胞流式检测。
图10显示磁分离CART细胞流式检测。
图11显示动物免疫采用的骆驼源纳米抗体的Kabat编号方式及残基发生频率。
图12显示五株PE标记的该纯化抗体流式分离检测骆驼PBMC细胞的FACS图。
图13显示该抗体偶联磁珠在骆驼血清重链抗体纯化前后的SDS-PAGE图。
发明详述
I.定义
如本说明书和所附权利要求书中使用的，单数形式“一个”，“一种”和“该/所述”包括复数提及物，除非上下文明确另有规定。如此，例如，提及“一个/种分子”任选包括两个/种或更多个/种此类分子的组合，诸如此类。
如本文中使用的，术语“约”指技术领域技术人员容易知道的相应数值的常规误差范围。本文中提及“约”某个数值或参数包括(并描述)涉及该数值或参数本身的实施方案。
理解的是，本文中描述的本发明的方面和实施方案包括“包含”，“由……组成”，和“基本上由……组成”的方面和实施方案。
术语“抗VHH域抗体”，“结合VHH域的抗体”和“特异性结合VHH域的抗体”指能够以足够亲和力结合VHH域的抗体。在一个实施方案中，根据例如通过放射免疫测定法(RIA)的测量，抗VHH域抗体结合无关的，非VHH域的蛋白质的程度小于该抗体对VHH域的结合的约10％。在某些实施方案中，结合VHH域的抗体具有≤1μM，≤100nM，≤10nM，≤1nM，≤0.1nM，≤0.01nM，或≤0.001nM(例如10 -8M或更少，例如10 -8M到10 -13M，例如10 -9M到10 -13M)的解离常数(Kd)。在某些实施方案中，抗VHH域抗体结合在来自不同物种的VHH域中保守的VHH域表位。
术语“重链抗体”或“HCAb”是指包含重链，但缺乏通常见于4链抗体中的轻链的功能性抗体。已知骆驼科动物(诸如单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Vicugna pacos)或小羊驼(Lama glama))会产生HCAb。
术语“单域抗体”或“sdAb”是指具有三个互补决定区(CDR)的单一抗原结合多肽。单独sdAb能够在不与相应含CDR多肽配对下结合抗原。在一些情况下，单域抗体从骆驼科动物HCAb工程化，并且它们的重链可变域在本文中称为“VHH”(重链抗体的重链可变域)。骆驼科动物sdAb是一种最小已知抗原结合抗体片段。基本VHH从N末端至C末端具有以下结构：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，其中FR1至FR4分别是指框架区1至4，并且其中CDR1至CDR3是指互补决定区1至3。
出于本文中的目的，“受体人框架”指包含自人免疫球蛋白框架或如下文定义的人共有框架衍生的轻链可变域(VL)框架或重链可变域(VH)框架的氨基酸序列的框架。自人免疫球蛋白框架或人共有框架“衍生”的受体人框架可以包含其相同的氨基酸序列，或者它可以含有氨基酸序列变化。在一些实施方案中，氨基酸变化的数目是10或更少，9或更少，8或更少，7或更少，6或更少，5或更少，4或更少，3或更少，或2或更少。在一些实施方案中，VL受体人框架与VL人免疫球蛋白框架序列或人共有框架序列在序列上相同。
“亲和力”指分子(例如抗体)的单一结合位点与其结合配偶体(例如抗原)之间全部非共价相互作用总和的强度。除非另有指示，如本文中使用的，“结合亲和力”指反映结合对的成员(例如抗体和抗原)之间1:1相互作用的内在结合亲和力。分子X对其配偶体Y的亲和力通常可以用解离常数(Kd)来表述。亲和力可以通过本领域知道的常用方法来测量，包括本文中所描述的方法。下文描述了用于测量结合亲和力的具体的说明性和例示性的实施方案。
“亲和力成熟的”抗体指在一个或多个高变区(HVR)中具有一处或多处改变的抗体，与不拥有此类改变的亲本抗体相比，此类改变导致该抗体对抗原的亲和力改善。
本文中的术语“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)，和抗体片段，只要它们展现出期望的抗原结合活性。
“抗体片段”指与完整抗体不同的分子，其包含完整抗体的一部分且结合完整抗体结合的抗原。抗体片段的例子包括但不限于Fv，Fab，Fab’，Fab’-SH，F(ab’)2；双抗体；线性抗体；单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。例如，抗原结合片段可以包含抗体的重链可变域和/或轻链可变域。
与参照抗体“结合相同表位的抗体”指在竞争测定法中将参照抗体对其抗原的结合阻断50％或更多的抗体，且相反，参照抗体在竞争测定法中将该抗体对其抗原的结合阻断50％或更多。本文中提供了例示性的竞争测定法。
术语“嵌合抗体”指其中的重和/或轻链的一部分自特定的来源或物种衍生，而重和/或轻链的剩余部分自不同来源或物种衍生的抗体。
抗体的“类”指其重链拥有的恒定域或恒定区的类型。抗体有5大类：IgA，IgD，IgE，IgG，和IgM，并且这些中的几种可以进一步分成亚类(同种型)，例如，IgG1，IgG2，IgG3，IgG4，IgA1，和IgA2。与不同类免疫球蛋白对应的重链恒定域分别称作α，δ，ε，γ，和μ。
“框架”或“FR”指除高变区(HVR)残基外的可变域残基。一般地，可变域的FR由4个FR域组成：FR1，FR2，FR3，和FR4。因而，HVR和FR序列在VH(或VL)中一般以如下的顺序出现：FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4。
术语“全长抗体”，“完整抗体”，和“全抗体”在本文中可互换使用，指与天然抗体结构具有基本上类似的结构或者具有含有如本文中所限定的Fc区的重链的抗体。
术语“宿主细胞”，“宿主细胞系”，和“宿主细胞培养物”可互换使用，并且指已经导入外源核酸的细胞，包括此类细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞及自其衍生的后代而不考虑传代的次数。后代在核酸内容物上可以与亲本细胞不完全相同，而是可以含有突变。本文中包括具有与在初始转化细胞中筛选或选择的相同功能或生物学活性的突变体后代。
“人抗体”指拥有与由人或人细胞生成的或利用人抗体全集或其它人抗体编码序列自非人来源衍生的抗体的氨基酸序列对应的氨基酸序列的抗体。人抗体的此定义明确排除包含非人抗原结合残基的人源化抗体。
“人共有框架”指代表人免疫球蛋白VL或VH框架序列选集中最常存在的氨基酸残基的框架。通常，人免疫球蛋白VL或VH序列选集来自可变域序列亚组。通常，序列亚组是如Kabat等，Sequences of Proteins of Immunological Interest,第五版,NIH Publication 91-3242,Bethesda MD(1991),第1-3卷中的亚组。在一个实施方案中，对于VL，亚组是如Kabat等，见上文中的亚组κI。在一个实施方案中，对于VH，亚组是如Kabat等，见上文中的亚组III。
“人源化”抗体指包含来自非人HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在某些实施方案中，人源化抗体会包含至少一个，通常两个基本上整个可变域，其中所有或基本上所有HVR(例如，CDR)对应于非人抗体的那些，且所有或基本上所有FR对应于人 抗体的那些。任选地，人源化抗体可以至少包含自人抗体衍生的抗体恒定区的一部分。抗体，例如非人抗体的“人源化形式”指已经经历人源化的抗体。
如本文中使用的，术语“高变区”或“HVR”指抗体可变域中在序列上高变的(“互补决定区”或“CDR”)和/或形成结构上限定的环(“高变环”)和/或含有抗原接触残基(“抗原接触”)的每个区。一般地，抗体包含6个HVR：三个在VH中(H1，H2，H3)，且三个在VL中(L1，L2，L3)。本文中的例示性HVR包括：
(a)高变环，存在于氨基酸残基26-32(L1)，50-52(L2)，91-96(L3)，26-32(H1)，53-55(H2)，和96-101(H3)(Chothia and Lesk,J.Mol.Biol.196:901-917(1987))；
(b)CDR，存在于氨基酸残基24-34(L1)，50-56(L2)，89-97(L3)，31-35b(H1)，50-65(H2)，和95-102(H3)(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD(1991))；
(c)抗原接触，存在于氨基酸残基27c-36(L1)，46-55(L2)，89-96(L3)，30-35b(H1)，47-58(H2)，和93-101(H3)(MacCallum et al.J.Mol.Biol.262:732-745(1996))；和
(d)(a)，(b)，和/或(c)的组合，包括HVR氨基酸残基46-56(L2)，47-56(L2)，48-56(L2)，49-56(L2)，26-35(H1)，26-35b(H1)，49-65(H2)，93-102(H3)，和94-102(H3)。
除非另有说明，HVR残基及可变域中的其它残基(例如FR残基)在本文中是依照Kabat等，见上文编号的。
“免疫缀合物”指与一种或多种异源分子，包括但不限于细胞毒剂缀合的抗体，例如包含本发明的抗体的偶联物。
“编码抗VHH域抗体的分离的核酸”指编码抗体重和轻链(或其片段)的一种或多种核酸分子，包括单一载体或不同载体中的此类核酸分子，和存在于宿主细胞中的一个或多个位置的此类核酸分子。
如本文中使用的，术语“单克隆抗体”指从一群基本上同质的抗体获得的抗体，即构成群体的各个抗体是相同的和/或结合相同表位，除了例如含有天然存在的突变或在单克隆抗体制备物的生成期间发生的可能的变体抗体外，此类变体一般以极小量存在。与通常包含针对不同决定簇(表位)的不同抗体的多克隆抗体制备物不同，单克隆抗体制备物的每种单克隆抗体针对抗原上的单一决定簇。如此，修饰语“单克隆”指示抗体自一群基本上同质的抗体获得的特性，而不应解释为要求通过任何特定方法来生成抗体。例如，可以通过多种技术来生成要依照本发明使用的单克隆抗体，包括但不限于杂交瘤方法，重组DNA方法，噬菌体展示方法，和利用含有所有或部分人免疫球蛋白基因座的转基因动物的方法，本文中描述了用于生成单克隆抗体的此类方法和其它例示性方法。
“裸抗体”指未与异源模块(例如细胞毒性模块)或放射性标记物缀合的抗体。裸抗体可以存在于药学配制剂中。
“天然抗体”指具有不同结构的天然存在的免疫球蛋白分子。例如，天然IgG抗体是约150,000道尔顿的异四聚糖蛋白，由二硫化物键合的两条相同轻链和两条相同重链构成。从N至C端，每条重链具有一个可变区(VH)，又称作可变重域或重链可变域，接着是三个恒定域(CH1，CH2，和CH3)。类似地，从N至C端，每条轻链具有一个可变区(VL)，又称作可变轻域或轻链可变域，接着是一个恒定轻(CL)域。
根据其恒定域氨基酸序列，抗体轻链可归入两种型中的一种，称作卡帕(κ)和拉姆达(λ)。
关于参照多肽序列的“百分比(％)氨基酸序列同一性”定义为比对序列并在必要时引入缺口以获取最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分时，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式进行，例如使用公众可得到的计算机软件，诸如BLAST，BLAST-2，ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以决定用于比对序列的合适参数，包括对所比较序列全长获得最大对比所需的任何算法。然而，为了本发明的目的，％氨基酸序列同一性值是使用序列比较计算机程序ALIGN-2产生的。ALIGN-2序列比较计算机程序由Genentech,Inc.编写，并且源代码已经连同用户文档一起提交给美国版权局(US Copyright Office,Washington D.C.,20559)，其中其以美国版权注册号TXU510087注册。公众自Genentech,Inc.,South San Francisco,California可获得ALIGN-2程序，或者可以从源代码编译。ALIGN2程序应当编译成在UNIX操作系统，包括数码UNIX V4.0D上使用。所有序列比较参数由ALIGN-2程序设定且不变。
在采用ALIGN-2来比较氨基酸序列的情况中，给定氨基酸序列A相对于(to)，与(with)，或针对(against)给定氨基酸序列B的％氨基酸序列同一性(或者可表述为具有或包含相对于，与，或针对给定氨基酸序列B的某一％氨基酸序列同一性的给定氨基酸序列A)如下计算：分数X/Y乘100其中X是由序列比对程序ALIGN-2在该程序的A和B比对中评分为相同匹配的氨基酸残基数，且其中Y是B中的氨基酸残基总数。应当领会，若氨基酸序列A的长度与氨基酸序列B的长度不相等，则A相对于B的％氨基酸序列同一性将不等于B相对于A的％氨基酸序列同一性。除非另有明确说明，本文中所使用的所有％氨基酸序列同一性值都是依照上一段所述，使用ALIGN-2计算机程序获得的。
术语“可变区”或“可变域”指抗体重或轻链中牵涉抗体结合抗原的域。天然抗体的重链和轻链可变域(分别为VH和VL)一般具有类似的结构，其中每个域包含4个保守的框架区(FR)和3个高变区(HVR)。(见例如Kindt等,Kuby Immunology,第6版,W.H.Freeman and Co.,第91页(2007))。单个VH或VL域可以足以赋予抗原结合特异性。此外，可以分别使用来自结合抗原的抗体的VH或VL域筛选互补VL或VH域的文库来分离结合特定抗原的抗体。见例如，Portolano等,J.Immunol.150:880-887(1993)；Clarkson等,Nature 352:624-628(1991)。
如本文中使用的，术语“载体”指能够增殖与其连接的另一种核酸的核酸分子。该术语包括作为自身复制型核酸结构的载体及并入接受其导入的宿主细胞的基因组中的载体。某些载体能够指导与其可操作连接的核酸的表达。此类载体在本文中称为“表达载体”。
“表达”一般指信息(例如基因编码的和/或表观遗传的)转化成细胞中存在并运行的结构的过程。因此，如本文中使用的，“表达”可以指转录成多核苷酸，翻译成多肽，或甚至多核苷酸和/或多肽修饰(例如多肽的翻译后修饰)。转录的多核苷酸的，翻译的多肽的，或多核苷酸和/或多肽修饰(例如多肽的翻译后修饰)的片段也应视为表达的，无论它们是源自通过可变剪接生成的转录物或经过降解的转录物，或者是源自多肽的翻译后加工(例如通过蛋白质水解)。“表达的基因”包括转录成多核苷酸(如mRNA)，然后翻译成多肽的基因，还有转录成RNA但不翻译成多肽的基因(例如转运和核糖体RNA)。
II.组合物和使用方法
本发明提供一组抗VHH域抗体或其抗原结合片段，其特异性识别骆驼源VHH域框架区的构象表位。本发明发现该抗体具有高亲和力，高特异性，高功能性。同时本发明提供了该抗体的制备方法和其在骆驼源纳米抗体开发及免疫细胞治疗领域的应用。
在一些实施方案中，本发明所公开的抗体和方法用于检测来源于单峰驼的纳米抗体或重链抗体。在另一些实施方案中，本发明所公开的抗体和方法用于检测来源于双峰驼的纳米抗体或重链抗体。在还有一些实施方案中，本发明所公开的抗体和方法用于检测来源于大羊驼的纳米抗体或重链抗体。在优选的实施方案中，本发明所公开的抗体和方法用于检测来源于小羊驼纳米抗体或重链抗体。
在一个实施方案中，本发明公开的抗体和方法用于骆驼源重链抗体特异性PBMC细胞的分离，该抗体同样适用于其他类型细胞的分离。在另一个实施方案中，该抗体用于纳米抗体的结合分析。在一个优选方案中，该抗体通过偶联固相载体介质，用于VHH类抗体的亲和纯化。
在一个实施方案中，本发明所公开的抗体用于一类CART细胞的流式鉴定。在另一个实施方案中，该抗体用于CART细胞的流式细胞分选。在一个优选的实施方案中，该抗体用于CART细胞的MACS分离纯化。
A.例示性抗VHH域抗体
在一个方面，本发明提供抗VHH域抗体或其抗原结合片段。
在一个实施方案中，本发明的抗体或其抗原结合片段特异性结合VHH域。在一个实施方案中，所述VHH域是骆驼源抗体的VHH域。在一个实施方案中，所述骆驼源抗体是单峰驼、双峰驼、大羊驼或小羊驼来源的单域抗体或重链抗体。
在一个实施方案中，本发明的抗体或其抗原结合片段在VHH域的框架区处结合。在一个实施方案中，本发明的抗体或其抗原结合片段在VHH域的构象表位处结合。在一个实施方案中，本发明的抗体或其抗原结合片段在VHH域的框架区构象表位处结合。
在一个实施方案中，本发明的抗体或其抗原结合片段特异性结合SEQ ID NO：241和246-255任一所示VHH域，或与SEQ ID NO：241和246-255任一具有至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％氨基酸序列同一性的VHH域，或SEQ ID NO：241和246-255共有序列。在一个实施方案中，本发明的抗体或其抗原结合片段特异性结合SEQ ID NO：241和246-255任一所示VHH域的框架区，或与SEQ ID NO：241和246-255任一所示VHH域的框架区具有至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％氨基酸序列同一性的框架区，或SEQ ID NO：241和246-255共有框架序列。
在另一个方面，本发明提供一种抗VHH域抗体，其包含至少一种，两种，三种，四种，五种，或六种选自下述的HVR：(a)包含SEQ ID NO：1、2、3、4、5、6、7、8、9、10、11、12、13、14或15的氨基酸序列的HVR-H1；(b)包含SEQ ID NO：16、17、18、19、20、21、22、23、24、25、26、27、28、29或30的氨基酸序列的HVR-H2；(c)包含SEQ ID NO：31、32、33、34、35、36、37、38、39、40、41、42、43、44或45的氨基酸序列的HVR-H3；(d)包含SEQ ID NO：46、47、48、49、50、51、52、53、54、55、56、57、58、59或60的氨基酸序列的HVR-L1；(e)包含SEQ ID NO：61、62、63、64、65、66、67、68、69、70、 71、72、73、74或75的氨基酸序列的HVR-L2；和(f)包含SEQ ID NO：76、77、78、79、80、81、82、83、84、85、86、87、88、89或90的氨基酸序列的HVR-L3。
在一个方面，本发明提供一种抗体，其包含至少一种，至少两种，或所有三种选自下述的VH HVR序列：(a)包含SEQ ID NO：1、2、3、4、5、6、7、8、9、10、11、12、13、14或15的氨基酸序列的HVR-H1；(b)包含SEQ ID NO：16、17、18、19、20、21、22、23、24、25、26、27、28、29或30的氨基酸序列的HVR-H2；和(c)包含SEQ ID NO：31、32、33、34、35、36、37、38、39、40、41、42、43、44或45的氨基酸序列的HVR-H3。
在另一个方面，本发明提供一种抗体，其包含至少一种，至少两种，或所有三种选自下述的VL HVR序列：(a)包含SEQ ID NO：46、47、48、49、50、51、52、53、54、55、56、57、58、59或60的氨基酸序列的HVR-L1；(b)包含SEQ ID NO：61、62、63、64、65、66、67、68、69、70、71、72、73、74或75的氨基酸序列的HVR-L2；和(c)包含SEQ ID NO：76、77、78、79、80、81、82、83、84、85、86、87、88、89或90的氨基酸序列的HVR-L3。
在另一个方面，本发明的抗体包含(a)VH域，其包含至少一种，至少两种，或所有三种选自下述的VH HVR序列：(i)包含SEQ ID NO：1、2、3、4、5、6、7、8、9、10、11、12、13、14或15的氨基酸序列的HVR-H1；(ii)包含SEQ ID NO：16、17、18、19、20、21、22、23、24、25、26、27、28、29或30的氨基酸序列的HVR-H2；和(iii)包含SEQ ID NO：31、32、33、34、35、36、37、38、39、40、41、42、43、44或45的氨基酸序列的HVR-H3；和(b)VL域，其包含至少一种，至少两种，或所有三种选自下述的VL HVR序列：(i)包含SEQ ID NO：46、47、48、49、50、51、52、53、54、55、56、57、58、59或60的氨基酸序列的HVR-L1；(ii)包含SEQ ID NO：61、62、63、64、65、66、67、68、69、70、71、72、73、74或75的氨基酸序列的HVR-L2；和(iii)包含SEQ ID NO：76、77、78、79、80、81、82、83、84、85、86、87、88、89或90的氨基酸序列的HVR-L3的氨基酸序列的HVR-L3。
在另一个方面，抗VHH域抗体包含与SEQ ID NO：91、92、93、94、95、96、97、98、99、100、101、102、103、104或105的氨基酸序列具有至少90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，或100％序列同一性的重链可变域(VH)序列。在某些实施方案中，具有至少90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％同一性的VH序列相对于参照序列含有替代(例如保守替代)，插入，或删除，但是包含该序列的抗VHH域抗体保留结合VHH域的能力。在某些实施方案中，在SEQ ID NO：91、92、93、94、95、96、97、98、99、100、101、102、103、104或105中替代，插入和/或删除了总共1至10个氨基酸。在某些实施方案中，替代，插入，或删除发生在HVR以外的区域中(即在FR中)。任选地，该抗VHH域抗体包含SEQ ID NO：91、92、93、94、95、96、97、98、99、100、101、102、103、104或105中的VH序列，包括该序列的翻译后修饰。
在另一个方面，提供一种抗VHH域抗体，其中该抗体包含与SEQ ID NO：106、107、108、109、110、111、112、113、114、115、116、117、118、119或120的氨基酸序列具有至少90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，或100％序列同一性的轻链可变域(VL)。在某些实施方案中，具有至少90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％同一性的VL序列相对于参照序列含有替代(例如保守替代)，插入，或删除，但是包含该序列的抗VHH域抗体保留结合VHH域的能力。在某些实施方 案中，在SEQ ID NO：106、107、108、109、110、111、112、113、114、115、116、117、118、119或120中替代，插入和/或删除了总共1至10个氨基酸。在某些实施方案中，替代，插入，或删除发生在HVR以外的区域中(即在FR中)。任选地，该抗VHH域抗体包含SEQ ID NO：106、107、108、109、110、111、112、113、114、115、116、117、118、119或120中的VL序列，包括该序列的翻译后修饰。
在另一个方面，提供一种抗VHH域抗体，其中该抗体包含上文提供的任何实施方案中的VH和上文提供的任何实施方案中的VL。
在任何上述实施方案中，抗VHH域抗体是人源化的。在一个实施方案中，抗VHH域抗体包含任何上述实施方案的HVR，且进一步包含受体人框架，例如人免疫球蛋白框架或人共有框架。
在又一个方面，本发明提供与本文中提供的抗VHH域抗体结合相同表位的抗体。例如，在某些实施方案中，提供与本文中提供的抗VHH域抗体结合相同表位的抗体。
在本发明的又一个方面，依照任何上述实施方案的抗VHH域抗体是单克隆抗体，包括嵌合抗体，人源化抗体或人抗体。在一个实施方案中，抗VHH域抗体是抗体片段，例如Fab，Fab’，Fab’-SH，Fv，单链可变片段(scFv)，和(Fab’) 2片段。在另一个实施方案中，该抗体是全长抗体，例如完整IgG类抗体或IgG1同种型或其它抗体类或同种型。
在又一个方面，依照任何上述实施方案的抗VHH域抗体可单一地或组合地并入下文1-8节中描述的任何特征：
1.抗体亲和力
在某些实施方案中，本文中提供的抗体具有≤1μM，≤100nM，≤10nM，≤1nM，≤0.1nM，≤0.01nM，或≤0.001nM的解离常数(Kd)(例如10 -8M或更少，例如10 -8M至10 -12M，例如10 -9M至10 -12M，例如10 -10M至10 -12M，例如10 -11M至10 -12M)。
在一个实施方案中，Kd是通过放射性标记抗原结合测定法(RIA)来测量的。在一个实施方案中，用Fab型式的感兴趣抗体及其抗原实施RIA。例如，通过在存在未标记抗原的滴定系列的情况中用最小浓度的( 125I)标记抗原平衡Fab，然后用抗Fab抗体包被板捕捉结合的抗原来测量Fab对抗原的溶液结合亲和力(见例如Chen等,J.Mol.Biol.293:865-881(1999))。为了建立测定法的条件，将VHH域
Figure PCTCN2020118150-appb-000001
多孔板(Thermo Scientific)用50mM碳酸钠(pH 9.6)中的5μg/ml捕捉用抗Fab抗体(Cappel Labs)包被过夜，随后用PBS中的2％(w/v)牛血清清蛋白于室温(约23℃)封闭2-5小时。在非吸附板(Nunc#269620)中，将100pM或26pM[125I]-抗原与连续稀释的感兴趣Fab(例如与Presta等,Cancer Res.57:4593-4599(1997)中抗VEGF抗体，Fab-12的评估一致)混合。然后将感兴趣的Fab温育过夜；然而，温育可持续更长时间(例如约65小时)以确保达到平衡。此后，将混合物转移至捕捉板，于室温温育(例如1小时)。然后除去溶液，并用PBS中的0.1％聚山梨酯20
Figure PCTCN2020118150-appb-000002
Figure PCTCN2020118150-appb-000003
洗板8次。平板干燥后，加入150μl/孔闪烁液(VHH域ROSCINT-20 TM；Packard)，然后在TOPCOUNT TM伽马计数器(Packard)上对平板计数10分钟。选择各Fab给出小于或等于最大结合之20％的浓度用于竞争性结合测定法。
依照另一个实施方案，Kd是使用
Figure PCTCN2020118150-appb-000004
表面等离振子共振测定法测量的。例如，于25℃使用固定化抗原CM5芯片在约10个响应单位(RU)使用
Figure PCTCN2020118150-appb-000005
或
Figure PCTCN2020118150-appb-000006
(BIAcore,Inc.,Piscataway,NJ)实施测定法。在一个实施方案中，依照供应商的用法说明书用盐酸N-乙基-N’-(3-二甲氨基丙基)-碳二亚胺(EDC)和N-羟基琥珀酰亚胺 (NHS)活化羧甲基化右旋糖苷生物传感器芯片(CM5,BIACORE,Inc.)。将抗原用10mM乙酸钠pH 4.8稀释至5μg/ml(约0.2μM)，然后以5μl/分钟的流速注射以获得约10个响应单位(RU)的偶联蛋白质。注入抗原后，注入1M乙醇胺以封闭未反应基团。为了进行动力学测量，于25℃以约25μl/分钟的流速注入在含0.05％聚山梨酯20(TWEEN-20 TM)表面活性剂的PBS(PBST)中两倍连续稀释的Fab(0.78nM至500nM)。使用简单一对一朗格缪尔(Langmuir)结合模型(
Figure PCTCN2020118150-appb-000007
Evaluation Software version 3.2)通过同时拟合结合和解离传感图计算结合速率(kon)和解离速率(koff)。平衡解离常数(Kd)以比率koff/kon计算。见例如Chen等,J.Mol.Biol.293:865-881(1999)。如果根据上文表面等离振子共振测定法，结合速率超过106M-1S-1，那么结合速率可使用荧光淬灭技术来测定，即根据分光计诸如配备了断流装置的分光光度计(Aviv Instruments)或 8000系列SLM-AMINCO TM分光光度计(ThermoSpectronic)中用搅拌比色杯的测量，在存在浓度渐增的抗原的情况中，测量PBS pH7.2中20nM抗抗原抗体(Fab形式)于25℃的荧光发射强度(激发＝295nm；发射＝340nm，16nm带通)的升高或降低。
2.抗体片段
在某些实施方案中，本文中提供的抗体是抗体片段。抗体片段包括但不限于Fab，Fab’，Fab’-SH，F(ab’)2，Fv，和scFv片段，及下文所描述的其它片段。关于某些抗体片段的综述，见Hudson等,Nat.Med.9:129-134(2003)。关于scFv片段的综述，见例如Pluckthün,于The Pharmacology of Monoclonal Antibodies,第113卷,Rosenburg和Moore编,(Springer-Verlag,New York),第269-315页(1994)；还可见WO 93/16185；及美国专利No.5,571,894和5,587,458。关于包含补救受体结合表位残基，并且具有延长的体内半衰期的Fab和F(ab’)2片段的讨论，见美国专利No.5,869,046。
双抗体是具有两个抗原结合位点的抗体片段，其可以是二价的或双特异性的。见例如EP 404,097；WO 1993/01161；Hudson等,Nat.Med.9:129-134(2003)；及Hollinger等,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)。三抗体和四抗体也记载于Hudson等,Nat.Med.9:129-134(2003)。
单域抗体是包含抗体的整个或部分重链可变域或整个或部分轻链可变域的抗体片段。在某些实施方案中，单域抗体是人单域抗体(Domantis,Inc.,Waltham,MA；见例如美国专利No.6,248,516B1)。
可以通过多种技术，包括但不限于对完整抗体的蛋白水解消化及重组宿主细胞(例如大肠杆菌或噬菌体)的生成来生成抗体片段，如本文中所描述的。
3.嵌合抗体和人源化抗体
在某些实施方案中，本文中提供的抗体是嵌合抗体。某些嵌合抗体记载于例如美国专利No.4,816,567；及Morrison等,Proc.Natl.Acad.Sci.USA,81:6851-6855(1984))。在一个例子中，嵌合抗体包含非人可变区(例如，自小鼠，大鼠，仓鼠，家兔，或非人灵长类，诸如猴衍生的可变区)和人恒定区。在又一个例子中，嵌合抗体是“类转换的”抗体，其中类或亚类已经自亲本抗体的类或亚类改变。嵌合抗体包括其抗原结合片段。
在某些实施方案中，嵌合抗体是人源化抗体。通常，将非人抗体人源化以降低对人的免疫原性，同时保留亲本非人抗体的特异性和亲和力。一般地，人源化抗体包含一个或多个可变域，其中HVR，例如CDR(或其部分)自非人抗体衍生，而FR(或其部分)自人抗体序列衍生。任选地，人源化抗体还会至少包含人恒定区的一部分。在一些实施方案中，将人源 化抗体中的一些FR残基用来自非人抗体(例如衍生HVR残基的抗体)的相应残基替代，例如以恢复或改善抗体特异性或亲和力。
人源化抗体及其生成方法综述于例如Almagro和Fransson,Front.Biosci.13:1619-1633(2008)，并且进一步记载于例如Riechmann等,Nature 332:323-329(1988)；Queen等,Proc.Nat’l Acad.Sci.USA 86:10029-10033(1989)；美国专利No.5,821,337,7,527,791,6,982,321和7,087,409；Kashmiri等,Methods 36:25-34(2005)(描述了特异性决定区(SDR)嫁接)；Padlan,Mol.Immunol.28:489-498(1991)(描述了“重修表面”)；Dall’Acqua等,Methods 36:43-60(2005)(描述了“FR改组”)；及Osbourn等,Methods 36:61-68(2005)和Klimka等,Br.J.Cancer,83:252-260(2000)(描述了FR改组的“引导选择”方法)。
可以用于人源化的人框架区包括但不限于：使用“最佳拟合(best-fit)”方法选择的框架区(见例如Sims等,J.Immunol.151:2296(1993))；自轻或重链可变区的特定亚组的人抗体的共有序列衍生的框架区(见例如Carter等,Proc.Natl.Acad.Sci.USA,89:4285(1992)；及Presta等,J.Immunol.,151:2623(1993))；人成熟的(体细胞突变的)框架区或人种系框架区(见例如Almagro和Fransson,Front.Biosci.13:1619-1633(2008))；和通过筛选FR文库衍生的框架区(见例如Baca等,J.Biol.Chem.272:10678-10684(1997)及Rosok等,J.Biol.Chem.271:22611-22618(1996))。
4.人抗体
在某些实施方案中，本文中提供的抗体是人抗体。可以使用本领域中已知的多种技术来生成人抗体。一般地，人抗体记载于van Dijk和van de Winkel,Curr.Opin.Pharmacol.5:368-74(2001)及Lonberg,Curr.Opin.Immunol.20:450-459(2008)。
可以通过对转基因动物施用免疫原来制备人抗体，所述转基因动物已经修饰为响应抗原性攻击而生成完整人抗体或具有人可变区的完整抗体。此类动物通常含有所有或部分人免疫球蛋白基因座，其替换内源免疫球蛋白基因座，或者其在染色体外存在或随机整合入动物的染色体中。在此类转基因小鼠中，一般已经将内源免疫球蛋白基因座灭活。关于自转基因动物获得人抗体的方法的综述，见Lonberg,Nat.Biotech.23:1117-1125(2005)。还可见例如美国专利No.6,075,181和6,150,584，其描述了XENOMOUSE TM技术；美国专利No.5,770,429，其描述了
Figure PCTCN2020118150-appb-000008
技术；美国专利No.7,041,870，其描述了K-M
Figure PCTCN2020118150-appb-000009
技术，和美国专利申请公开文本No.US 2007/0061900，其描述了
Figure PCTCN2020118150-appb-000010
技术)。可以例如通过与不同人恒定区组合进一步修饰来自由此类动物生成的完整抗体的人可变区。
也可以通过基于杂交瘤的方法生成人抗体。已经描述了用于生成人单克隆抗体的人骨髓瘤和小鼠-人异骨髓瘤细胞系(见例如Kozbor J.Immunol.,133:3001(1984)；Brodeur等,Monoclonal Antibody Production Techniques and Applications,第51-63页(Marcel Dekker,Inc.,New York,1987)；及Boerner等,J.Immunol.,147:86(1991))。经由人B细胞杂交瘤技术生成的人抗体也记载于Li等,Proc.Natl.Acad.Sci.USA,103:3557-3562(2006)。其它方法包括那些例如记载于美国专利No.7,189,826(其描述了自杂交瘤细胞系生成单克隆人IgM抗体)和Ni,Xiandai Mianyixue,26(4):265-268(2006)(其描述了人-人杂交瘤)的。人杂交瘤技术(Trioma技术)也记载于Vollmers和Brandlein,Histology and Histopathology,20(3):927-937(2005)及Vollmers和Brandlein,Methods and Findings in Experimental and Clinical Pharmacology,27(3):185-91(2005)。
也可以通过分离自人衍生的噬菌体展示文库选择的Fv克隆可变域序列生成人抗体。然后，可以将此类可变域序列与期望的人恒定域组合。下文描述了自抗体文库选择人抗体的技术。
5.文库衍生的抗体
可以通过对组合文库筛选具有期望的一种或多种活性的抗体来分离本发明的抗体。例如，用于生成噬菌体展示文库并对此类文库筛选拥有期望结合特征的抗体的多种方法是本领域中已知的。此类方法综述于例如Hoogenboom等,于Methods in Molecular Biology 178:1-37(O’Brien等编,Human Press,Totowa,NJ,2001)，并且进一步记载于例如McCafferty等,Nature348:552-554；Clackson等,Nature 352:624-628(1991)；Marks等,J.Mol.Biol.222:581-597(1992)；Marks和Bradbury,于Methods in Molecular Biology 248:161-175(Lo编,Human Press,Totowa,NJ,2003)；Sidhu等,J.Mol.Biol.338(2):299-310(2004)；Lee等,J.Mol.Biol.340(5):1073-1093(2004)；Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004)；及Lee等,J.Immunol.Methods 284(1-2):119-132(2004)。
在某些噬菌体展示方法中，将VH和VL基因的全集分别通过聚合酶链式反应(PCR)克隆，并在噬菌体文库中随机重组，然后可以对所述噬菌体文库筛选抗原结合噬菌体，如记载于Winter等,Ann.Rev.Immunol.,12:433-455(1994)的。噬菌体通常以单链Fv(scFv)片段或以Fab片段展示抗体片段。来自经免疫的来源的文库提供针对免疫原的高亲和力抗体，而不需要构建杂交瘤。或者，可以(例如自人)克隆天然全集以在没有任何免疫的情况中提供针对一大批非自身和还有自身抗原的抗体的单一来源，如由Griffiths等,EMBO J,12:725-734(1993)描述的。最后，也可以通过自干细胞克隆未重排的V基因区段，并使用含有随机序列的PCR引物编码高度可变的CDR3区并在体外实现重排来合成生成未免疫文库，如由Hoogenboom和Winter,J.Mol.Biol.,227:381-388(1992)所描述的。描述人抗体噬菌体文库的专利公开文本包括例如：美国专利No.5,750,373，和美国专利公开文本No.2005/0079574,2005/0119455,2005/0266000,2007/0117126,2007/0160598,2007/0237764,2007/0292936和2009/0002360。
认为自人抗体文库分离的抗体或抗体片段是本文中的人抗体或人抗体片段。
6.多特异性抗体
在某些实施方案中，本文中提供的抗体是多特异性抗体，例如双特异性抗体。多特异性抗体是对至少两个不同位点具有结合特异性的单克隆抗体。在某些实施方案中，结合特异性之一针对VHH域，而另一种针对任何其它抗原。在某些实施方案中，双特异性抗体可以结合VHH域的两个不同表位。也可以使用双特异性抗体来将细胞毒剂定位于表达VHH域的细胞。双特异性抗体可以以全长抗体或抗体片段制备。
用于生成多特异性抗体的技术包括但不限于具有不同特异性的两对免疫球蛋白重链-轻链对的重组共表达(见Milstein和Cuello,Nature 305:537(1983))，WO 93/08829，和Traunecker等,EMBO J.10:3655(1991))，和“节-入-穴”工程化(见例如美国专利No.5,731,168)。也可以通过用于生成抗体Fc-异二聚体分子的工程化静电操纵效应(WO2009/089004A1)；交联两个或更多个抗体或片段(见例如美国专利No.4,676,980,及Brennan等,Science,229:81(1985))；使用亮氨酸拉链来生成双特异性抗体(见例如Kostelny等,J.Immunol.,148(5):1547-1553(1992))；使用用于生成双特异性抗体片段的“双抗体”技术(见例如Hollinger等,Proc.Natl.Acad.Sci.USA,90:6444-6448(1993))；及使用单链Fv(sFv)二 聚体(见例如Gruber等,J.Immunol.,152:5368(1994))；及如例如Tutt等,J.Immunol.147:60(1991)中所描述的，制备三特异性抗体来生成多特异性抗体。
本文中还包括具有三个或更多个功能性抗原结合位点的工程化改造抗体，包括“章鱼抗体”(见例如US 2006/0025576A1)。
本文中的抗体或片段还包括包含结合VHH域及另一种不同抗原的抗原结合位点的“双重作用FAb”或“DAF”(见例如US 2008/0069820)。
7.抗体变体
在某些实施方案中，涵盖本文中提供的抗体的氨基酸序列变体。例如，可以期望改善抗体的结合亲和力和/或其它生物学特性。可以通过将合适的修饰引入编码抗体的核苷酸序列中，或者通过肽合成来制备抗体的氨基酸序列变体。此类修饰包括例如对抗体的氨基酸序列内的残基的删除，和/或插入和/或替代。可以进行删除，插入，和替代的任何组合以得到最终的构建体，只要最终的构建体拥有期望的特征，例如，抗原结合。
在某些实施方案中，提供了具有一处或多处氨基酸替代的抗体变体。替代诱变感兴趣的位点包括HVR和FR。保守替代在表1中在“优选的替代”的标题下显示。更实质的变化在表1中在“例示性替代”的标题下提供，并且如下文参照氨基酸侧链类别进一步描述的。可以将氨基酸替代引入感兴趣的抗体中，并且对产物筛选期望的活性，例如保留/改善的抗原结合，降低的免疫原性，或改善的ADCC或CDC。
表1：氨基酸替代
初始残基 例示性替代 优选的替代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp,Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；正亮氨酸 Leu
Leu(L) 正亮氨酸；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸 Leu
依照共同的侧链特性，氨基酸可以如下分组：
(1)疏水性的：正亮氨酸,Met,Ala,Val,Leu,Ile；
(2)中性，亲水性的：Cys,Ser,Thr,Asn,Gln；
(3)酸性的：Asp,Glu；
(4)碱性的：His,Lys,Arg；
(5)影响链取向的残基：Gly,Pro；
(6)芳香族的：Trp,Tyr,Phe。
非保守替代会需要用这些类别之一的成员替换另一个类别的。
一类替代变体牵涉替代亲本抗体(例如人源化或人抗体)的一个或多个高变区残基。一般地，为进一步研究选择的所得变体相对于亲本抗体会具有某些生物学特性的改变(例如改善)(例如升高的亲和力，降低的免疫原性)和/或会基本上保留亲本抗体的某些生物学特性。例示性的替代变体是亲和力成熟的抗体，其可以例如使用基于噬菌体展示的亲和力成熟技术诸如本文中所描述的那些技术来方便地生成。简言之，将一个或多个HVR残基突变，并将变体抗体在噬菌体上展示，并对其筛选特定的生物学活性(例如结合亲和力)。
可以对HVR做出变化(例如，替代)，例如以改善抗体亲和力。可以对HVR“热点”，即由在体细胞成熟过程期间以高频率经历突变的密码子编码的残基(见例如Chowdhury,Methods Mol.Biol.207:179-196(2008))，和/或接触抗原的残基做出此类变化，其中对所得的变体VH或VL测试结合亲和力。通过次级文库的构建和再选择进行的亲和力成熟已经记载于例如Hoogenboom等,于Methods in Molecular Biology 178:1-37(O’Brien等编,Human Press,Totowa,NJ,(2001))。在亲和力成熟的一些实施方案中，通过多种方法(例如，易错PCR，链改组，或寡核苷酸指导的诱变)任一将多样性引入为成熟选择的可变基因。然后，创建次级文库。然后，筛选文库以鉴定具有期望的亲和力的任何抗体变体。另一种引入多样性的方法牵涉HVR指导的方法，其中将几个HVR残基(例如，一次4-6个残基)随机化。可以例如使用丙氨酸扫描诱变或建模来特异性鉴定牵涉抗原结合的HVR残基。特别地，经常靶向CDR-H3和CDR-L3。
在某些实施方案中，可以在一个或多个HVR内发生替代，插入，或删除，只要此类变化不实质性降低抗体结合抗原的能力。例如，可以对HVR做出保守变化(例如，保守替代，如本文中提供的)，其不实质性降低结合亲和力。例如，此类变化可以在HVR中的抗原接触残基以外。在上文提供的变体VH和VL序列的某些实施方案中，每个HVR是未改变的，或者含有不超过1，2或3处氨基酸替代。
一种可用于鉴定抗体中可以作为诱变靶位的残基或区域的方法称作“丙氨酸扫描诱变”，如由Cunningham和Wells(1989)Science,244:1081-1085所描述的。在此方法中，将残基或靶残基的组(例如，带电荷的残基诸如arg，asp，his，lys，和glu)鉴定，并用中性或带负电荷的氨基酸(例如，丙氨酸或多丙氨酸)替换以测定抗体与抗原的相互作用是否受到影响。可以在对初始替代表明功能敏感性的氨基酸位置引入进一步的替代。或者/另外，利用抗原-抗体复合物的晶体结构来鉴定抗体与抗原间的接触点。作为替代的候选，可以靶向或消除此类接触残基和邻近残基。可以筛选变体以确定它们是否含有期望的特性。
氨基酸序列插入包括长度范围为1个残基至含有100或更多个残基的多肽的氨基和/或羧基端融合，及单个或多个氨基酸残基的序列内插入。末端插入的例子包括具有N端甲硫 氨酰基残基的抗体。抗体分子的其它插入变体包括抗体的N或C端与酶(例如对于ADEPT)或延长抗体的血清半衰期的多肽的融合物。
8.抗体衍生物
在某些实施方案中，可以进一步修饰本文中提供的抗体以含有本领域知道的且易于获得的额外非蛋白质性质模块。适合于抗体衍生化的模块包括但不限于水溶性聚合物。水溶性聚合物的非限制性例子包括但不限于聚乙二醇(PEG)，乙二醇/丙二醇共聚物，羧甲基纤维素，右旋糖苷，聚乙烯醇，聚乙烯吡咯烷酮，聚-1,3-二氧戊环，聚-1,3,6-三口恶烷，乙烯/马来酸酐共聚物，聚氨基酸(均聚物或随机共聚物)，和右旋糖苷或聚(n-乙烯吡咯烷酮)聚乙二醇，丙二醇均聚物，环氧丙烷/环氧乙烷共聚物，聚氧乙烯化多元醇(例如甘油)，聚乙烯醇及其混合物。由于其在水中的稳定性，聚乙二醇丙醛在生产中可能具有优势。聚合物可以是任何分子量，而且可以是分支的或不分支的。附着到抗体上的聚合物数目可以变化，而且如果附着了超过一个聚合物，那么它们可以是相同或不同的分子。一般而言，可根据下列考虑来确定用于衍生化的聚合物的数目和/或类型，包括但不限于抗体要改进的具体特性或功能，抗体衍生物是否将用于指定条件下的治疗等。
B.重组方法和组合物
可以使用重组方法和组合物来生成抗体，例如，如记载于美国专利No.4,816,567的。在一个实施方案中，提供了编码本文中所描述的抗VHH域抗体的分离的核酸。此类核酸可以编码包含抗体VL的氨基酸序列和/或包含抗体VH的氨基酸序列(例如，抗体的轻和/或重链)。在又一个实施方案中，提供了包含此类核酸的一种或多种载体(例如，表达载体)。在又一个实施方案中，提供了包含此类核酸的宿主细胞。在一个此类实施方案中，宿主细胞包含(例如，已经用下列载体转化)：(1)包含核酸的载体，所述核酸编码包含抗体的VL的氨基酸序列和包含抗体的VH的氨基酸序列，或(2)第一载体和第二载体，所述第一载体包含编码包含抗体的VL的氨基酸序列的核酸，所述第二载体包含编码包含抗体的VH的氨基酸序列的核酸。在一个实施方案中，宿主细胞是真核的，例如中国仓鼠卵巢(CHO)细胞或淋巴样细胞(例如，Y0，NS0，Sp20细胞)。在一个实施方案中，提供了一种生成抗VHH域抗体的方法，其中该方法包括在适合于表达抗体的条件下在培养基中培养包含编码抗体的核酸的宿主细胞，如上文提供的，并且任选地，自宿主细胞(或宿主细胞培养基)回收抗体。
对于抗VHH域抗体的重组生成，将编码抗体的核酸(例如如上文所描述的)分离，并插入一种或多种载体中，以在宿主细胞中进一步克隆和/或表达。可以使用常规规程将此类核酸容易地分离并测序(例如，通过使用寡核苷酸探针来进行，所述寡核苷酸探针能够特异性结合编码抗体的重和轻链的基因)。
适合于克隆或表达抗体编码载体的宿主细胞包括本文中所描述的原核或真核细胞。例如，可以在细菌(诸如大肠杆菌)中生成抗体，特别是在不需要糖基化和Fc效应器功能时。对于抗体片段和多肽在细菌中的表达，见例如美国专利No.5,648,237,5,789,199和5,840,523(还可见Charlton,Methods in Molecular Biology,第248卷(B.K.C.Lo编,Humana Press,Totowa,NJ,2003),第245-254页，其描述了抗体片段在大肠杆菌(E.coli.)中的表达)。表达后，可以将抗体在可溶性级分中自细菌细胞团糊分离，并可以进一步纯化。
在原核生物外，真核微生物诸如丝状真菌或酵母是适合于抗体编码载体的克隆或表达宿主，包括其糖基化途径已经“人源化”，导致生成具有部分或完全人的糖基化样式的抗体的真 菌和酵母菌株。见Gerngross,Nat.Biotech.22:1409-1414(2004),及Li等,Nat.Biotech.24:210-215(2006)。
适合于表达糖基化抗体的宿主细胞也自多细胞生物体(无脊椎动物和脊椎动物)衍生。无脊椎动物细胞的例子包括昆虫细胞。已经鉴定出许多杆状病毒株，其可以与昆虫细胞一起使用，特别是用于转染草地夜蛾(Spodoptera frugiperda)细胞。
也可以利用植物细胞培养物作为宿主。见例如美国专利No.5,959,177,6,040,498,6,420,548,7,125,978和6,417,429(其描述了用于在转基因植物中生成抗体的PLANTIBODIES TM技术)。
也可以使用脊椎动物细胞作为宿主。例如，适合于在悬浮液中生长的哺乳动物细胞系可以是有用的。有用的哺乳动物宿主细胞系的其它例子是经SV40转化的猴肾CV1系(COS-7)；人胚肾系(293或293细胞，如记载于例如Graham等,J.Gen Virol.36:59(1977)的)；幼年仓鼠肾细胞(BHK)；小鼠塞托利(sertoli)细胞(TM4细胞，如记载于例如Mather,Biol.Reprod.23:243-251(1980)的)；猴肾细胞(CV1)；非洲绿猴肾细胞(VERO-76)；人宫颈癌细胞(HELA)；犬肾细胞(MDCK；牛鼠(buffalo rat)肝细胞(BRL 3A)；人肺细胞(W138)；人肝细胞(Hep G2)；小鼠乳房肿瘤(MMT 060562)；TRI细胞，如记载于例如Mather等,Annals N.Y.Acad.Sci.383:44-68(1982)的；MRC 5细胞；和FS4细胞。其它有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞(Urlaub等,Proc.Natl.Acad.Sci.USA 77:4216(1980))；和骨髓瘤细胞系诸如Y0，NS0和Sp2/0。关于适合于抗体生成的某些哺乳动物宿主细胞系的综述，见例如Yazaki和Wu,Methods in Molecular Biology,第248卷(B.K.C.Lo编,Humana Press,Totowa,NJ),第255-268页(2003)。
C.测定法
可以通过本领域中已知的多种测定法对本文中提供的抗VHH域抗体鉴定，筛选，或表征其物理/化学特性和/或生物学活性。
一方面，对本发明的抗体测试其抗原结合活性，例如通过已知的方法诸如ELISA，Western印迹，等来进行。
另一方面，可使用竞争测定法来鉴定与本文中描述的任何抗VHH域抗体竞争对VHH域的结合的抗体。在某些实施方案中，此类竞争性抗体结合与本文中描述的任何抗VHH域抗体所结合表位相同的表位(例如线性或构象表位)。用于定位抗体所结合表位的详细例示性方法见Morris(1996)“Epitope Mapping Protocols”,Methods in Molecular Biology vol.66(Humana Press,Totowa,NJ)。
在一种例示性竞争测定法中，在包含第一经标记抗体(其结合VHH域，例如本文中描述的任何抗VHH域抗体)和第二未标记抗体(其要测试与第一抗体竞争对VHH域的结合的能力)的溶液中温育固定化VHH域。第二抗体可存在于杂交瘤上清液中。作为对照，在包含第一经标记抗体但不包含第二未标记抗体的溶液中温育固定化VHH域。在允许第一抗体结合VHH域的条件下温育后，除去过量的未结合抗体，并测量与固定化VHH域联合的标记物的量。如果测试样品中与固定化VHH域联合的标记物的量与对照样品相比实质性降低，那么这指示第二抗体与第一抗体竞争对VHH域的结合。参见Harlow and Lane(1988)Antibodies:A Laboratory Manual ch.14(Cold Spring Harbor Laboratory,Cold Spring Harbor,NY)。
D.用于检测的方法和组合物
在某些实施方案中，本文中提供的任何抗VHH域抗体可用于检测生物学样品中VHH域的存在。如本文中使用的，术语“检测”涵盖定量或定性检测。
在一个实施方案中，提供了在检测方法中使用的抗VHH域抗体。在又一方面，提供了检测生物学样品中VHH域的存在的方法。在某些实施方案中，该方法包括在容许抗VHH域抗体结合VHH域的条件下使生物学样品与抗VHH域抗体接触，如本文中所描述的，并检测是否在抗VHH域抗体与VHH域间形成复合物。此类方法可以是体外或体内方法。
在某些实施方案中，提供了经标记的抗VHH域抗体。标记物包括但不限于直接检测的标记物或模块(诸如荧光，发色，电子致密，化学发光，和放射性标记物)，及例如经由酶反应或分子相互作用间接检测的模块，诸如酶或配体。例示性的标记物包括但不限于放射性同位素 32P， 14C， 125I， 3H，和 131I，荧光团诸如稀土螯合物或荧光素及其衍生物，罗丹明及其衍生物，丹酰，伞形酮，萤光素酶，例如，萤火虫萤光素酶和细菌萤光素酶(美国专利No.4,737,456)，萤光素，2,3-二氢酞嗪二酮，辣根过氧化物酶(HRP)，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶菌酶，糖类氧化酶，例如，葡萄糖氧化酶，半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，杂环氧化酶诸如尿酸酶和黄嘌呤氧化酶(其与采用过氧化氢氧化染料前体的酶诸如HRP偶联)，乳过氧化物酶，或微过氧化物酶，生物素/亲合素，自旋标记物，噬菌体标记物，稳定的自由基，等等。
序列的说明
Figure PCTCN2020118150-appb-000011
Figure PCTCN2020118150-appb-000012
Figure PCTCN2020118150-appb-000013
Figure PCTCN2020118150-appb-000014
Figure PCTCN2020118150-appb-000015
Figure PCTCN2020118150-appb-000016
Figure PCTCN2020118150-appb-000017
Figure PCTCN2020118150-appb-000018
Figure PCTCN2020118150-appb-000019
Figure PCTCN2020118150-appb-000020
Figure PCTCN2020118150-appb-000021
Figure PCTCN2020118150-appb-000022
Figure PCTCN2020118150-appb-000023
Figure PCTCN2020118150-appb-000024
Figure PCTCN2020118150-appb-000025
Figure PCTCN2020118150-appb-000026
实施例
实施例1：抗体的杂交瘤细胞株获得
1)动物免疫。动物免疫的抗原采用重组表达的His标签骆驼源纳米抗体VHH-His(氨基酸序列如图1或SEQ ID NO：241所示)，该纳米抗体AS718为动物免疫采用的抗原，具有C端His标签，共计含有121个氨基酸，CDR结构域和His标签通过下划线在图1中示出。
用含有200μg VHH-His及200μl弗氏完全佐剂(Sigma-Aldrich,CAT#:F5881)的1:1乳液皮下免疫8只新西兰兔；每2周皮下注射含有200μg VHH-His及弗氏不完全佐剂(Sigma-Aldrich,CAT#:F5506)的1:1乳液4次，从而对新西兰兔进行加强免疫；骨髓瘤融合前4天，ELISA检测血清抗体滴度(如图2所示)，检测动物的血清效价，结果显示效价1:64,000检测值均大于1.0，符合标准的2只兔接受了400μg VHH-His(不含佐剂)的腹腔和静脉加强免疫。
图2通过间接ELISA的模式，采用免疫原AS718VHH-His包被ELISA检测板，将动物血清按1:1,000的初始比例稀释，之后采用2倍比例梯度稀释至1:512,000进行ELISA检测，通过OD450读数测定两只待融合动物的血清效价，其中S/N代表Signal-to-Noise即为信噪比，血清有效效价判定标准是空白背景值的2.1倍，血清稀释比在1:64,000时检测值大于1.0则可以进行融合。结果显示动物#6119的血清在稀释比1:64,000时检测值为1.323，血清稀释比1:512,000时检测值为0.488；动物#563的血清在稀释比1:64,000时检测值为1.637，血清稀释比1:512,000时检测值为0.528。由于两只动物在血清稀释比1:512,000时检测值均大于空白背景值的2.1倍，所以两只动物的血清有效效价均大于1:512,000，同时1:64,000检测值均大于1.0，符合动物融合标准。
2)杂交瘤融合和筛选
提取兔脾脏并进行均质化以产生单细胞悬液，同时准备骨髓瘤细胞单细胞悬液。使用电融合将脾细胞与1.0×10 8个脾细胞与1.2×10 5个骨髓瘤细胞进行融合；将融合的细胞重悬于100ml含杂交瘤细胞选择剂胸腺核苷嘧啶，次黄嘌呤和氨基蝶呤的DMEM/10％FBS培养基中，并用移液器以100μl的体积移至150×96孔板中；将板在37℃下在6％CO 2中孵育。9天孵育后，使用间接ELISA用于评估上清液中抗体对于VHH-His的结合能力，说明VHH-His的抗体的存在情况。
将ELISA板用100μl/孔的PBS中0.5μg/ml的VHH-His或His蛋白在4℃下包被过夜；用PBS-T(0.05％Tween20)洗涤板，并将其用200μl/孔的含1％BSA的PBST在37℃封闭0.5小时；吸弃封闭液，向每个孔加入100μl杂交瘤细胞培养上清液，然后在室温下孵育1小时；孵育结束后，弃去上清液，将板用PBST洗涤三次，并用100μl/孔的缀和辣根过氧化物酶的山羊抗兔IgG(GenScript Goat Anti-Rabbit IgG Antibody(H&L)[HRP],pAb；CAT#:A00098)37℃孵育0.5小时；将板用PBST洗涤五次，然后加入TMB显色液并在室温下在黑暗中孵育15分钟。通过加入50μl的1M HCl终止液终止反应。使用酶标仪在450nm下读板。图3通过间接ELISA的模式，采用免疫原AS718VHH-His包被ELISA检测板，通过OD450读数检测15株母克隆上清原液的效价，同时检测它们与His标签蛋白的交叉反应，排除非特异的结合。实验中采用阳性血清作为免疫原AS718VHH-His的阳性对照，采用Anti-His抗体作为反筛检测原His标签蛋白的阳性对照，检测值大于0.5判定为阳性，检测值小于0.2判定为阴性。结果显示：阳性血清与VHH-His，阳性血清与His标签蛋白，Anti-His抗体与VHH-His，Anti-His抗体与His标签蛋白结合的检测值分别为2.953，0.747， 2.906，2.858均大于0.5，判定为阳性即阳性对照有效，其中阳性血清与His标签蛋白结合检测值为0.747代表融合动物体内依然产生了针对His标签蛋白的抗体。15株母克隆上清原液与免疫原AS718VHH-His的ELISA反应检测值分别为2.758，3.060，2.908，2.883，2.893，2.556，2.913，2.655，2.561，2.356，3.059，2.130，2.083，2.454，2.428均大于0.5，判定15株母克隆上清对VHH-His反应为阳性；15株母克隆上清原液与His标签蛋白的ELISA反应检测值分别为0.074，0.130，0.091，0.102，0.082，0.100，0.121，0.083，0.091，0.114，0.123，0.085，0.112，0.082，0.079均小于0.2，判定15株母克隆上清对His标签蛋白反应为阴性，即与非相关的His标签蛋白没有交叉反应。
3)杂交瘤亚克隆，采用有限稀释法进行亚克隆
使用血球细胞计数器并在含杂交瘤细胞选择剂胸腺核苷嘧啶、次黄嘌呤和氨基蝶呤的DMEM/10％FBS培养基中对细胞进行系列稀释来确定细胞数量，直至细胞密度达到5-15个细胞/ml；对于每个杂交瘤，将200μl的细胞溶液用移液器移至96孔中，密度为1-3个细胞/孔；将培养物在37℃下在5％CO 2中培养1周后，对上清液进行上述ELISA结合测试，来评估针对VHH的抗体存在情况，亚克隆定株的ELISA检测数据如图4所示(亚克隆定株上清ELISA效价检测)：通过间接ELISA的模式，采用免疫原AS718VHH-His包被ELISA检测板，以1：1,000稀释的阳性动物血清作为阳性对照，通过OD450读数检测15株母克隆通过亚克隆定株后细胞上清的效价。首孔采用细胞上清原液，并依次采用3倍倍比稀释至1:81，细胞上清稀释比在1:9时检测值大于1.0则可以进行抗体生产。结果显示15株克隆的细胞上清在稀释比1:9时检测值为2.460，1.954，1.945，2.143，2.243，2.047，2.064，2.268，2.197，1.528，2.377，1.530，2.485，1.929，1.475均大于1.0，符合杂交瘤细胞克隆定株标准，可以继续进行抗体生产。
实施例2：该抗体的可变区测序及抗体重组生产
使用TRIzol(Ambion,CAT#:15596-026)从3×10 6–5×10 6个杂交瘤细胞提取总RNA，并利用抗体亚型特异性引物和通用引物(Takara PrimeScript TM 1st Strand cDNA Synthesis Kit，CAT#:6110A)将其逆转录为cDNA。随后通过RACE PCR扩增兔免疫球蛋白重链和轻链可变区域片段，并将所得的PCR片段亚克隆至pMD18-T载体系统(Takara,CAT#:6011)中，并使用载体特异性引物对插入片段进行测序。最终获得了15株单克隆抗体的重链和轻链可变区域核苷酸/蛋白序列，包括clone R166.C5,clone R166.G3,clone R166.F2,clone R166.G8,clone R166.H9,clone R166.D1,clone R166.G10,clone R166.H10,clone R166.F9,clone R166.G2,clone R166.E5,clone R166.E3,clone R166.H8,clone R166.E7,和clone R166.A5。
抗体均采用重组表达的方式进行生产，以一株优选的克隆为例。
分别合成编码轻链可变区+恒定区(氨基酸序列如SEQ ID NO：244所示，核苷酸序列如SEQ ID NO：245所示)与重链可变区+恒定区(氨基酸序列如SEQ ID NO：242所示，核苷酸序列如SEQ ID NO：243所示)的DNA片段，将其分别插入pTT5表达载体中，形成表达质粒；将上述质粒共转染CHO-3E7细胞，并于37℃摇瓶中培养6天后，收集上清用于抗体纯化；管道和蛋白A柱用0.2M NaOH去热原，随后将柱用含有0.05M Tris和1.5M NaCl(pH8.0)的缓冲液重新平衡；将收获的细胞培养物上清液，使用2×上述缓冲液1:1稀释并过滤除菌。将过滤的上清液和蛋白A柱室温孵育2小时，采用1×上述缓冲液洗涤柱后，使用无菌0.1M柠檬酸钠(pH3.5)洗脱IgG，收集了洗脱液并用九分之一体积的无菌1M Tris-HCl(pH9.0)中和；在无菌条件下，将所述产品缓冲液交换为PBS(pH7.4)以除去任 何的洗脱缓冲液并浓缩所述样品。浓缩之后，使用1.43的消光系数Ec(0.1％)通过OD280nm对抗体进行定量；纯化的抗体通过BioRad电泳系统用10％预制胶(GenScript)通过SDS-PAGE来分析。将所述凝胶用eStain2.0(GenScript)染色并通过比较染色带与Protein Ladder(GenScript)来估计分子大小和纯度，15株抗体纯度鉴定结果如图5a、5b所示，在非还原条件下，抗体的两条重链和两条轻链仍通过二硫键保持四价体结构，在电泳染色结果中只呈现出一条目的条带，显示的分子量大小略低于实际分子量，与116kDa的marker条带大小相当。而在还原条件下，由于还原剂的存在破坏了抗体中的二硫键结构，抗体的重链与轻链分离，电泳时呈现出独立的电泳条带，大小分别是55kDa和25kDa。结果显示所有15株抗体纯度均大于90％，符合抗体生产纯化的标准，可用于后续实验研究。
实施例3：该抗体对重组骆驼纳米抗体的结合
采用间接ELISA的方法评估纯化后的15株骆驼源纳米抗体的抗体对于10株不同骆驼源纳米抗体VHH(氨基酸序列分别如SEQ ID NO：246-255所示)的结合能力。
分别用100μl/孔的PBS中0.5μg/ml的10种不同VHH将ELISA板的在4℃下包被过夜；用PBS-T(0.05％吐温)洗涤板，并将其用200μl/孔的含1％BSA的PBST在37℃封闭0.5小时；吸弃封闭液，将首孔加入1μg/ml的纯化抗体100μl，并按照3倍梯度稀释，共计11个测试浓度梯度，在室温孵育1小时；将板用PBST洗涤三次，并用100μl/孔的缀和辣根过氧化物酶的山羊抗兔IgG(GenScript Goat Anti-Rabbit IgG Antibody(H&L)[HRP],pAb；CAT#:A00098)37℃孵育0.5小时；将板用PBST洗涤五次，然后加入TMB显色液并在室温下在黑暗中孵育15分钟。通过加入50μl的1M HCl终止液终止反应。使用酶标仪在450nm下读板；得到15株克隆对于VHH-His的结合能力(如图6a、6b所示)，结果显示15株骆驼源纳米抗体的抗体与不同的骆驼源纳米抗体的亲和力有差异，一方面，同一骆驼源纳米抗体的抗体与不同的骆驼源纳米抗体亲和力不同；另一方面，不同的骆驼源纳米抗体的抗体与同一骆驼源纳米抗体亲和力不同。但是整体上，这15株纯化抗体全部可以与10种不同骆驼源纳米抗体识别。根据ELISA浓度梯度实验计算得到的EC50表明这些抗体对于抗原都表现出相当高的亲和力。
将上述10种不同的骆驼源纳米抗体序列和实施例1中用于免疫动物的AS718纳米抗体序列通过Kabat分析得到框架区FR1,FR2,FR3和FR4的序列，再利用BioEdit软件进行序列一致性分析，结果显示FR1区域序列一致性为53.3％，FR2区域序列一致性为35.7％，FR3区域序列一致性为50％，FR4区域序列一致性为54.5％，所有框架区的序列一致性为49.4％，见图7。而且，上述10种骆驼源纳米抗体与AS718纳米抗体分别具有74.7％(AS154、AS325)、77.0％(AS587)、79.3％(AS675)、80.5％(AS200、AS519)、81.6％(AS656)、82.8％(AS588、AS988)、83.9％(AS673)的框架区序列一致性。
类似地，在VHH域全长上，上述10种骆驼源纳米抗体与AS718纳米抗体分别具有62.3％(AS154)、62.9％(AS200)、63.6％(AS325)、64.8％(AS656)、66.1％(AS675、AS673)、66.4％(AS587、AS988)、68.0％(AS588)、69.9％(AS519)的序列一致性。全部11种纳米抗体的一致性为37.7％。
实施例4：抗体与多株骆驼源纳米抗体亲和力的测定
在一个例示性的实施方案中，利用Biacore 8K仪器(GE healthcare)，通过SPR实验技术测定了该抗体克隆R166.C5与多株骆驼源纳米抗体的亲和力水平。
CM5芯片预处理：平衡缓冲液HBS 10μl/min平衡5分钟后，采用NHS/EDC活化剂处理芯片10μl/min，7分钟，再加入偶联的anti-His抗体(GenScript,CAT#:A00186)10μl/min反应7分钟，最后用10μl/min的氨基乙醇封闭。骆驼源纳米抗体的捕获：平衡缓冲液HBS 10μl/min平衡5分钟后，分别加入10株20μl/ml的VHH-His抗体，30μl/min充分反应1分钟；流动相中以200nM的抗体浓度和30μl/min的流速测定该抗体的亲和力水平，其中采用免疫原AS718作为骆驼源纳米抗体的阳性对照，结果显示免疫原AS718与该克隆抗体的亲和力达到3.47pmol水平，同时在检测的10株骆驼源纳米抗体中有6株AS325,AS656,AS673,AS588,AS988和AS519与该抗体的亲和力均达到pmol水平，亲和力分别是4.04pmol,1.34pmol,4.36pmol,7.09pmol,7.08pmol和1.66pmol。其中与该克隆抗体亲和力最低的1株骆驼源纳米抗体是AS587，亲和力也达到2.2nmol，这些数据表明克隆R166.C5骆驼源纳米抗体的抗体与10株不同的骆驼源纳米抗体均具有较高的亲和力(如图8所示)。
实施例5：抗体在骆驼源重链抗体特异性PBMC分离中的应用
在一个例示性的实施方案中，利用荧光标记的骆驼源纳米抗体的抗体，通过流式细胞术分离重链抗体特异性的PBMC。本发明中使用的是非免疫骆驼的PBMC细胞。
选取5×10 6个PBMC细胞用于分析，采用500μl FACS缓冲液(PBS,1％BSA)洗涤细胞一次，1000rpm离心5分钟并弃上清；选取200μl FACS缓冲液重悬PBMC细胞并加入2μg藻红蛋白荧光素(PE)标记的骆驼源纳米抗体的抗体R166.C5、R166.H8、R166.H9、R166.E7以及R166.G8，避光条件下，冰浴孵育15分钟后，采用500μl FACS缓冲液洗涤细胞一次，1000rpm离心5分钟并弃上清；采用2％多聚甲醇溶液固定细胞30分钟，采用500μl FACS缓冲液洗涤细胞一次，1000rpm离心5分钟并弃上清；采用200μl FACS缓冲液重悬细胞并进行重链抗体特异性PBMC细胞的流式分选。FACS检测结果如图12所示，5株PE标记的不同克隆抗体FACS分选得到的重链抗体特异性的PBMC细胞比例分别为22.83％、23.58％、24.24％、23.33％和17.24％，与理论值(约20％～30％)相符，显示出该5株抗体均具有良好的重链抗体特异性。
实施例6：抗体在骆驼源重链抗体纯化中的应用
在一个例示性的实施方案中，利用本发明的抗体纯化小羊驼或大羊驼血清中的重链抗体，本发明中使用的是非免疫羊驼的血清。
将骆驼源纳米抗体的抗体(示例性的抗VHH抗体)偶联到NHS活化好的磁珠上，采取如下质量体积比例(抗体:磁珠＝3:1；4:1；5:1；6:1；7:1；8:1)进行偶联，得到VHH纳米抗体纯化介质。选取1ml羊驼的血清，加入4ml PBS稀释血清充分混匀后，加入1ml磁珠纯化介质并置于4℃环境中孵育1小时；采用磁力架吸附磁珠后弃上清，采用PBST洗涤磁珠3次；使用无菌0.1M柠檬酸钠(pH3.5)洗脱重链抗体，收集了洗脱液并用九分之一体积的无菌1M Tris-HCl(pH9.0)中和；在无菌条件下，将所述产品缓冲液交换为PBS(pH7.4)以除去任何的洗脱缓冲液并浓缩所述样品。浓缩之后，使用1.43的消光系数Ec(0.1％)通过OD280nm对抗体进行定量。
纯化的抗体(包括磁珠纯化前的血清上清、磁珠纯化后的洗脱液以及纯化后的洗脱样品)通过BioRad电泳系统用10％预制胶(GenScript，CAT#:M00665)通过SDS-PAGE来分析。将所述凝胶用
Figure PCTCN2020118150-appb-000027
L1 Protein Staining System(GenScript)染色并通过比较染色带与Protein Ladder来估计分子大小和纯度。如图13所示，标出的驼科IgG2和IgG3抗体的分子 量分别为46KD和43KD，所获得的磁珠均可特异性的纯化重链抗体，纯化后重链抗体IgG2和IgG3总体的纯度分别为77.4％、78.3％、71.6％、80.1％、81.1％和73.8％。
实施例7：抗体在CART细胞治疗中的应用-CART细胞的流式细胞鉴定
当CART细胞治疗中利用骆驼源的纳米抗体构建嵌合受体时，本发明所述的抗体可用于CART细胞的流式细胞鉴定。
将符合上述条件的CART细胞以如下比例(0％,10％,20％,40％,80％,95％)与初始
Figure PCTCN2020118150-appb-000028
T细胞混合，制备待检测细胞样品；每个样品选取5×10 5个细胞，采用500μl FACS缓冲液(PBS,1％BSA)洗涤细胞一次，1000rpm离心5分钟并弃上清；选取400μl FACS缓冲液重悬细胞并加入4.5μg生物素标记的骆驼源纳米抗体的抗体克隆R166.A5，冰浴孵育45分钟后，采用500μl FACS缓冲液洗涤细胞一次，1000rpm离心5分钟并弃上清；选取500μl FACS缓冲液重悬细胞并加入0.2μg PE标记的链霉亲合素(Biolegend,CAT#:740452)，室温孵育15分钟后，采用500μl FACS缓冲液洗涤细胞一次，1000rpm离心5分钟并弃上清；采用2％多聚甲醇溶液固定细胞30分钟，采用500μl FACS缓冲液洗涤细胞一次，1000rpm离心5分钟并弃上清；采用200μl FACS缓冲液重悬细胞并进行流式细胞分析，分析结果如图9所示，将CART细胞与初始T细胞按不同的固定比例0％,10％,20％,40％,80％和95％混合，通过生物素标记的克隆R166.A5的抗体及PE标记的链霉亲合素进行进行流式细胞染色鉴定，结果显示CAR阳性的细胞比例分别是0.01％,9.72％,20.9％,39.3％,80.5％和93.7％，与预混比例的偏差小于±1.5％，数据表明该抗体可以很好应用于利用骆驼源的纳米抗体构建嵌合受体的CART细胞检测。该抗体可以很好的识别CAR阳性的T细胞。
实施例8：抗体在CART细胞治疗中的应用-CART细胞的磁分离
当CART细胞治疗中利用骆驼源的纳米抗体构建嵌合受体时，本发明所述的抗体可用于CART细胞的磁分离。
细胞计数，在一次例示性的实施方案中，待分离CART细胞个数为1x10 7；1000rpm离心10分钟并弃上清，采用100μl PBE缓冲液(PBS,pH 7.2,0.5％BSA,2mM EDTA)充分重悬细胞；加入2μg生物素标记的骆驼源纳米抗体的抗体克隆R166.A5，充分混匀后，4℃环境中孵育10分钟；采用2ml PBE缓冲液洗涤细胞一次，并用100μl PBE缓冲液充分重悬细胞；加入20μl抗生物素磁珠(Miltenyi)，充分混匀后，4℃环境中孵育10分钟，采用磁力架进行细胞分离，将上述得到的细胞进行流式细胞分析，以验证磁分离的效果，如图10所示，采用初始T细胞作为CAR阴性的细胞对照，以10％CAR阳性T细胞与初始T细胞混合作为阳性待分离样品，分别取用1x10 7个细胞，通过生物素标记的克隆R166.A5的抗体和抗生物素的磁珠进行CART细胞的磁分离，通过流式细胞检测CART细胞磁分离的效果。结果显示CAR阴性的细胞在磁分离前后检测到CAR阳性的比例均小于0.5％，属于背景噪音信号；而CAR阳性的细胞经过磁分离后，CAR阳性细胞的比例从预混的10％提升至分离后的88.6％，数据表明该抗体可以很好的应用于CART细胞的磁分离。结果显示该抗体可以很好的应用于CART细胞的磁分离。

Claims (35)

  1. 一种抗体或其抗原结合片段，其特异性结合VHH域。
  2. 一种抗体或其抗原结合片段，其中该抗体含有重链可变区和轻链可变区，其中(A)所述重链可变区包括重链互补决定区(HCDR)1、HCDR2和HCDR3，(a)所述HCDR1序列选自SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14或15所示的氨基酸序列或与上述序列具有至少90％同一性的氨基酸序列，(b)所述HCDR2序列选自SEQ ID NO:16、17、18、19、20、21、22、23、24、25、26、27、28、29或30所示的氨基酸序列或与上述序列具有至少90％同一性的氨基酸序列，且(c)所述HCDR3序列选自SEQ ID NO:31、32、33、34、35、36、37、38、39、40、41、42、43、44或45所示的氨基酸序列或与上述序列具有至少90％同一性的氨基酸序列；且(B)所述轻链可变区包括轻链互补决定区(LCDR)1、LCDR2和LCDR3，(d)所述LCDR1序列选自SEQ ID NO:46、47、48、49、50、51、52、53、54、55、56、57、58、59或60所示的氨基酸序列或与上述序列具有至少90％同一性的氨基酸序列，(e)所述LCDR2序列选自SEQ ID NO:61、62、63、64、65、66、67、68、69、70、71、72、73、74或75所示的氨基酸序列或与上述序列具有至少90％同一性的氨基酸序列，且(f)所述LCDR3序列选自SEQ ID NO:76、77、78、79、80、81、82、83、84、85、86、87、88、89或90所示的氨基酸序列或与上述序列具有至少90％同一性的氨基酸序列。
  3. 如权利要求2所述的抗体或其抗原结合片段，其中该抗体含有
    (1)SEQ ID NO:1所示HCDR1、SEQ ID NO:16所示HCDR2、SEQ ID NO:31所示HCDR3、SEQ ID NO:46所示LCDR1、SEQ ID NO:61所示LCDR2和SEQ ID NO:76所示LCDR3；
    (2)SEQ ID NO:2所示HCDR1、SEQ ID NO:17所示HCDR2、SEQ ID NO:32所示HCDR3、SEQ ID NO:47所示LCDR1、SEQ ID NO:62所示LCDR2和SEQ ID NO:77所示LCDR3；
    (3)SEQ ID NO:3所示HCDR1、SEQ ID NO:18所示HCDR2、SEQ ID NO:33所示HCDR3、SEQ ID NO:48所示LCDR1、SEQ ID NO:63所示LCDR2和SEQ ID NO:78所示LCDR3；
    (4)SEQ ID NO:4所示HCDR1、SEQ ID NO:19所示HCDR2、SEQ ID NO:34所示HCDR3、SEQ ID NO:49所示LCDR1、SEQ ID NO:64所示LCDR2和SEQ ID NO:79所示LCDR3；
    (5)SEQ ID NO:5所示HCDR1、SEQ ID NO:20所示HCDR2、SEQ ID NO:35所示HCDR3、SEQ ID NO:50所示LCDR1、SEQ ID NO:65所示LCDR2和SEQ ID NO:80所示LCDR3；
    (6)SEQ ID NO:6所示HCDR1、SEQ ID NO:21所示HCDR2、SEQ ID NO:36所示HCDR3、SEQ ID NO:51所示LCDR1、SEQ ID NO:66所示LCDR2和SEQ ID NO:81所示LCDR3；
    (7)SEQ ID NO:7所示HCDR1、SEQ ID NO:22所示HCDR2、SEQ ID NO:37所示HCDR3、SEQ ID NO:52所示LCDR1、SEQ ID NO:67所示LCDR2和SEQ ID NO:82所示LCDR3；
    (8)SEQ ID NO:8所示HCDR1、SEQ ID NO:23所示HCDR2、SEQ ID NO:38所示HCDR3、SEQ ID NO:53所示LCDR1、SEQ ID NO:68所示LCDR2和SEQ ID NO:83所示LCDR3；
    (9)SEQ ID NO:9所示HCDR1、SEQ ID NO:24所示HCDR2、SEQ ID NO:39所示HCDR3、SEQ ID NO:54所示LCDR1、SEQ ID NO:69所示LCDR2和SEQ ID NO:84所示LCDR3；
    (10)SEQ ID NO:10所示HCDR1、SEQ ID NO:25所示HCDR2、SEQ ID NO:40所示HCDR3、SEQ ID NO:55所示LCDR1、SEQ ID NO:70所示LCDR2和SEQ ID NO:85所示LCDR3；
    (11)SEQ ID NO:11所示HCDR1、SEQ ID NO:26所示HCDR2、SEQ ID NO:41所示HCDR3、SEQ ID NO:56所示LCDR1、SEQ ID NO:71所示LCDR2和SEQ ID NO:86所示LCDR3；
    (12)SEQ ID NO:12所示HCDR1、SEQ ID NO:27所示HCDR2、SEQ ID NO:42所示HCDR3、SEQ ID NO:57所示LCDR1、SEQ ID NO:72所示LCDR2和SEQ ID NO:87所示LCDR3；
    (13)SEQ ID NO:13所示HCDR1、SEQ ID NO:28所示HCDR2、SEQ ID NO:43所示HCDR3、SEQ ID NO:58所示LCDR1、SEQ ID NO:73所示LCDR2和SEQ ID NO:88所示LCDR3；
    (14)SEQ ID NO:14所示HCDR1、SEQ ID NO:29所示HCDR2、SEQ ID NO:44所示HCDR3、SEQ ID NO:59所示LCDR1、SEQ ID NO:74所示LCDR2和SEQ ID NO:89所示LCDR3；或
    (15)SEQ ID NO:15所示HCDR1、SEQ ID NO:30所示HCDR2、SEQ ID NO:45所示HCDR3、SEQ ID NO:60所示LCDR1、SEQ ID NO:75所示LCDR2和SEQ ID NO:90所示LCDR3。
  4. 如权利要求2所述的抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列选自SEQ ID NO:91、92、93、94、95、96、97、98、99、100、101、102、103、104或105所示的氨基酸序列或与上述序列具有至少90％同一性的氨基酸序列。
  5. 如权利要求2或4所述的抗体或其抗原结合片段，其中所述轻链可变区的氨基酸序列选自SEQ ID NO:106、107、108、109、110、111、112、113、114、115、116、117、118、119或120所示的氨基酸序列或与上述序列具有至少90％同一性的氨基酸序列。
  6. 如权利要求2所述的抗体或其抗原结合片段，其中该抗体含有
    (1)SEQ ID NO:91所示重链可变区和SEQ ID NO:106所示轻链可变区；
    (2)SEQ ID NO:92所示重链可变区和SEQ ID NO:107所示轻链可变区；
    (3)SEQ ID NO:93所示重链可变区和SEQ ID NO:108所示轻链可变区；
    (4)SEQ ID NO:94所示重链可变区和SEQ ID NO:109所示轻链可变区；
    (5)SEQ ID NO:95所示重链可变区和SEQ ID NO:110所示轻链可变区；
    (6)SEQ ID NO:96所示重链可变区和SEQ ID NO:111所示轻链可变区；
    (7)SEQ ID NO:97所示重链可变区和SEQ ID NO:112所示轻链可变区；
    (8)SEQ ID NO:98所示重链可变区和SEQ ID NO:113所示轻链可变区；
    (9)SEQ ID NO:99所示重链可变区和SEQ ID NO:114所示轻链可变区；
    (10)SEQ ID NO:100所示重链可变区和SEQ ID NO:115所示轻链可变区；
    (11)SEQ ID NO:101所示重链可变区和SEQ ID NO:116所示轻链可变区；
    (12)SEQ ID NO:102所示重链可变区和SEQ ID NO:117所示轻链可变区；
    (13)SEQ ID NO:103所示重链可变区和SEQ ID NO:118所示轻链可变区；
    (14)SEQ ID NO:104所示重链可变区和SEQ ID NO:119所示轻链可变区；或
    (15)SEQ ID NO:105所示重链可变区和SEQ ID NO:120所示轻链可变区。
  7. 如权利要求2到6中任一项所述的抗体或其抗原结合片段，其特异性结合VHH域。
  8. 如权利要求1或7所述的抗体或其抗原结合片段，其以K D为10nM至1pM的结合亲和力特异性结合VHH域。
  9. 如权利要求1、7和8中任一项所述的抗体或其抗原结合片段，其中所述VHH域是骆驼源抗体的VHH域。
  10. 如权利要求9所述的抗体或其抗原结合片段，其中所述骆驼源抗体是单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Vicugna pacos)或小羊驼(Lama glama)来源的单域抗体或重链抗体。
  11. 如权利要求1和7到10中任一项所述的抗体或其抗原结合片段，其特异性结合SEQ ID NO：241所示VHH域或与SEQ ID NO：241具有至少60％氨基酸序列同一性的VHH域。
  12. 如权利要求1和7到11中任一项所述的抗体或其抗原结合片段，其在VHH域的框架区处结合。
  13. 如权利要求1和7到11中任一项所述的抗体或其抗原结合片段，其在VHH域的构象表位处结合。
  14. 如权利要求1和7到11任中一项所述的抗体或其抗原结合片段，其在VHH域的框架区构象表位处结合。
  15. 如权利要求1和7到11中任一项所述的抗体或其抗原结合片段，其特异性结合SEQ ID NO：241所示VHH域的框架区或与SEQ ID NO：241所示VHH域的框架区具有至少70％氨基酸序列同一性的框架区。
  16. 如权利要求1到15中任一项所述的抗体或其抗原结合片段，其选自Fab、F(ab’) 2、scFv、嵌合抗体、人源化抗体、双抗体和多特异性抗体。
  17. 一种抗体或其抗原结合片段，其与如权利要求1到16中任一项所述的抗体或其抗原结合片段结合相同表位。
  18. 一种抗体或其抗原结合片段，其与如权利要求1到16中任一项所述的抗体或其抗原结合片段竞争结合。
  19. 一种或多种多核苷酸，其编码如权利要求1到18中任一项所述的抗体或其抗原结合片段。
  20. 一种或多种载体，其包含如权利要求19所述的多核苷酸。
  21. 如权利要求20所述的载体，其选自克隆载体和表达载体。
  22. 一种宿主细胞，其包含如权利要求19所述的多核苷酸或如权利要求20或21所述的载体。
  23. 如权利要求22所述的宿主细胞，其选自原核细胞、酵母细胞、昆虫细胞或哺乳动物细胞。
  24. 一种生成抗体或其抗原结合片段的方法，其包括在适合抗体生成的条件下培养权利要求22或23的宿主细胞使得所述抗体或其抗原结合片段表达。
  25. 如权利要求24所述的方法，其还包括回收所述抗体或其抗原结合片段。
  26. 一种偶联物，其包含如权利要求1到18中任一项所述的抗体或其抗原结合片段。
  27. 如权利要求26所述的偶联物，其中所述抗体或其抗原结合片段与荧光素、生物素、酶、琼脂糖树脂、磁珠或生物芯片偶联。
  28. 一种检测VHH域的方法，其包括将如权利要求1到18中任一项所述的抗体或其抗原结合片段或如权利要求26或27所述的偶联物添加至已知或怀疑含有VHH域的样品，并检测所述抗体或其抗原结合片段或所述偶联物与所述VHH域之间形成的复合物。
  29. 一种分离VHH域的方法，其包括将如权利要求1到18中任一项所述的抗体或其抗原结合片段或如权利要求26或27所述的偶联物添加至已知或怀疑含有VHH域的样品，并分离所述抗体或其抗原结合片段或所述偶联物与所述VHH域之间形成的复合物。
  30. 如权利要求28或29所述的方法，其中所述VHH域在骆驼源抗体中。
  31. 如权利要求28或29所述的方法，其中所述VHH域在嵌合抗原受体中。
  32. 如权利要求31所述的方法，其中所述嵌合抗原受体在免疫细胞上。
  33. 如权利要求32所述的方法，其中所述免疫细胞选自PBMC、T细胞、NK细胞或巨噬细胞。
  34. 如权利要求28到33中任一项所述的方法，其中所述方法利用FACS或MCS进行。
  35. 一种试剂盒，其包括包含权利要求1到18中任一项所述的抗体或其抗原结合片段和/或如权利要求26或27所述的偶联物的容器。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12583925B2 (en) 2022-12-12 2026-03-24 LaNova Medicines Limited Bispecific antibodies targeting PD1 and VEGF

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK1976886T3 (en) * 2005-12-16 2015-03-02 Amgen Res Munich Gmbh Means and methods for the treatment of tumor diseases
EP3571508B8 (en) * 2017-01-18 2023-05-24 SRI International Detecting cancer stem cells using a glycan biomarker
KR102719065B1 (ko) 2017-06-08 2024-10-17 블랙 벨트 테라퓨틱스 리미티드 Cd38 조정 항체
AU2018316522B2 (en) 2017-08-16 2025-02-20 Black Belt Therapeutics Limited CD38 antibody
SG11202000321PA (en) 2017-08-16 2020-02-27 Black Belt Therapeutics Ltd Cd38 modulating antibody
MX2020003783A (es) 2017-10-02 2021-01-08 Visterra Inc Moleculas de anticuerpo de cd138 y sus usos.
KR20250078626A (ko) * 2018-01-12 2025-06-02 브리스톨-마이어스 스큅 컴퍼니 Tim3에 대한 항체 및 그의 용도
CN111902428B (zh) * 2018-07-03 2023-11-10 上海健信生物医药科技有限公司 一种双特异性抗体及其用途
WO2020243623A1 (en) * 2019-05-31 2020-12-03 The Council Of The Queensland Institute Of Medical Research Activating anti-gal9 binding molecules
CA3142021A1 (en) * 2019-06-17 2020-12-24 Visterra, Inc. Humanized antibody molecules to cd138 and uses thereof
EP3753954A1 (en) * 2019-06-21 2020-12-23 Université de Franche-Comté Anti-cd123 antibodies, anti-cd123 chimeric antigen receptors and anti-cd123 chimeric antigen receptors t cells
US20230056782A1 (en) * 2019-12-30 2023-02-23 Ming-Tain Lai Antibodies, pharmaceutical compositions and uses thereof
ES2867373B2 (es) * 2020-04-17 2023-01-02 Consejo Superior Investigacion Tratamiento terapeutico de leucemias linfoblasticas agudas de celulas t con un anticuerpo monoclonal frente al receptor pre-tcr
WO2021231732A1 (en) * 2020-05-15 2021-11-18 Bristol-Myers Squibb Company Antibodies to garp
US20220106406A1 (en) * 2020-10-01 2022-04-07 University Of Kentucky Research Foundation Polypeptide directed against protein tyrosine phosphatase 4a proteins, and compositions and methods for use thereof
EP4662247A1 (en) * 2023-02-07 2025-12-17 National Research Council of Canada Broadly reactive anti-vhh antibodies

Citations (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US4737456A (en) 1985-05-09 1988-04-12 Syntex (U.S.A.) Inc. Reducing interference in ligand-receptor binding assays
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
EP0404097A2 (de) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Bispezifische und oligospezifische, mono- und oligovalente Rezeptoren, ihre Herstellung und Verwendung
WO1993001161A1 (en) 1991-07-11 1993-01-21 Pfizer Limited Process for preparing sertraline intermediates
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
WO1993016185A2 (en) 1992-02-06 1993-08-19 Creative Biomolecules, Inc. Biosynthetic binding protein for cancer marker
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
US5587458A (en) 1991-10-07 1996-12-24 Aronex Pharmaceuticals, Inc. Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof
US5648237A (en) 1991-09-19 1997-07-15 Genentech, Inc. Expression of functional antibody fragments
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5750373A (en) 1990-12-03 1998-05-12 Genentech, Inc. Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5789199A (en) 1994-11-03 1998-08-04 Genentech, Inc. Process for bacterial production of polypeptides
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US5840523A (en) 1995-03-01 1998-11-24 Genetech, Inc. Methods and compositions for secretion of heterologous polypeptides
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US5959177A (en) 1989-10-27 1999-09-28 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
US6040498A (en) 1998-08-11 2000-03-21 North Caroline State University Genetically engineered duckweed
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6248516B1 (en) 1988-11-11 2001-06-19 Medical Research Council Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors
US6420548B1 (en) 1999-10-04 2002-07-16 Medicago Inc. Method for regulating transcription of foreign genes
US20050079574A1 (en) 2003-01-16 2005-04-14 Genentech, Inc. Synthetic antibody phage libraries
US20050119455A1 (en) 2002-06-03 2005-06-02 Genentech, Inc. Synthetic antibody phage libraries
US20050266000A1 (en) 2004-04-09 2005-12-01 Genentech, Inc. Variable domain library and uses
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
US20060025576A1 (en) 2000-04-11 2006-02-02 Genentech, Inc. Multivalent antibodies and uses therefor
US7041870B2 (en) 2000-11-30 2006-05-09 Medarex, Inc. Transgenic transchromosomal rodents for making human antibodies
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody
US7125978B1 (en) 1999-10-04 2006-10-24 Medicago Inc. Promoter for regulating expression of foreign genes
US7189826B2 (en) 1997-11-24 2007-03-13 Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
US20070061900A1 (en) 2000-10-31 2007-03-15 Murphy Andrew J Methods of modifying eukaryotic cells
US20070117126A1 (en) 1999-12-15 2007-05-24 Genentech, Inc. Shotgun scanning
US20070160598A1 (en) 2005-11-07 2007-07-12 Dennis Mark S Binding polypeptides with diversified and consensus vh/vl hypervariable sequences
US20070237764A1 (en) 2005-12-02 2007-10-11 Genentech, Inc. Binding polypeptides with restricted diversity sequences
US20070292936A1 (en) 2006-05-09 2007-12-20 Genentech, Inc. Binding polypeptides with optimized scaffolds
US20080069820A1 (en) 2006-08-30 2008-03-20 Genentech, Inc. Multispecific antibodies
US20090002360A1 (en) 2007-05-25 2009-01-01 Innolux Display Corp. Liquid crystal display device and method for driving same
US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies
WO2009089004A1 (en) 2008-01-07 2009-07-16 Amgen Inc. Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
CN109843923A (zh) * 2016-10-11 2019-06-04 南京传奇生物科技有限公司 针对ctla-4的单结构域抗体及其变体

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1163599C (zh) * 1997-06-06 2004-08-25 Asat应用科学技术股份公司 抗gpiib/iiia重组抗体
CN108456252B (zh) * 2017-02-20 2019-03-26 南京金斯瑞生物科技有限公司 高特异性的抗兔IgG天然构象单克隆抗体
CN109734814A (zh) * 2019-02-12 2019-05-10 南京卡提医学科技有限公司 具有免疫受体的工程化t细胞治疗癌症的用途

Patent Citations (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4737456A (en) 1985-05-09 1988-04-12 Syntex (U.S.A.) Inc. Reducing interference in ligand-receptor binding assays
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
US6248516B1 (en) 1988-11-11 2001-06-19 Medical Research Council Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors
EP0404097A2 (de) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Bispezifische und oligospezifische, mono- und oligovalente Rezeptoren, ihre Herstellung und Verwendung
US5959177A (en) 1989-10-27 1999-09-28 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
US6417429B1 (en) 1989-10-27 2002-07-09 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5750373A (en) 1990-12-03 1998-05-12 Genentech, Inc. Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1993001161A1 (en) 1991-07-11 1993-01-21 Pfizer Limited Process for preparing sertraline intermediates
US5648237A (en) 1991-09-19 1997-07-15 Genentech, Inc. Expression of functional antibody fragments
US5587458A (en) 1991-10-07 1996-12-24 Aronex Pharmaceuticals, Inc. Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
WO1993016185A2 (en) 1992-02-06 1993-08-19 Creative Biomolecules, Inc. Biosynthetic binding protein for cancer marker
US5789199A (en) 1994-11-03 1998-08-04 Genentech, Inc. Process for bacterial production of polypeptides
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5840523A (en) 1995-03-01 1998-11-24 Genetech, Inc. Methods and compositions for secretion of heterologous polypeptides
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US7189826B2 (en) 1997-11-24 2007-03-13 Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody
US6040498A (en) 1998-08-11 2000-03-21 North Caroline State University Genetically engineered duckweed
US7125978B1 (en) 1999-10-04 2006-10-24 Medicago Inc. Promoter for regulating expression of foreign genes
US6420548B1 (en) 1999-10-04 2002-07-16 Medicago Inc. Method for regulating transcription of foreign genes
US20070117126A1 (en) 1999-12-15 2007-05-24 Genentech, Inc. Shotgun scanning
US20060025576A1 (en) 2000-04-11 2006-02-02 Genentech, Inc. Multivalent antibodies and uses therefor
US20070061900A1 (en) 2000-10-31 2007-03-15 Murphy Andrew J Methods of modifying eukaryotic cells
US7041870B2 (en) 2000-11-30 2006-05-09 Medarex, Inc. Transgenic transchromosomal rodents for making human antibodies
US20050119455A1 (en) 2002-06-03 2005-06-02 Genentech, Inc. Synthetic antibody phage libraries
US20050079574A1 (en) 2003-01-16 2005-04-14 Genentech, Inc. Synthetic antibody phage libraries
US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies
US20050266000A1 (en) 2004-04-09 2005-12-01 Genentech, Inc. Variable domain library and uses
US20070160598A1 (en) 2005-11-07 2007-07-12 Dennis Mark S Binding polypeptides with diversified and consensus vh/vl hypervariable sequences
US20070237764A1 (en) 2005-12-02 2007-10-11 Genentech, Inc. Binding polypeptides with restricted diversity sequences
US20070292936A1 (en) 2006-05-09 2007-12-20 Genentech, Inc. Binding polypeptides with optimized scaffolds
US20080069820A1 (en) 2006-08-30 2008-03-20 Genentech, Inc. Multispecific antibodies
US20090002360A1 (en) 2007-05-25 2009-01-01 Innolux Display Corp. Liquid crystal display device and method for driving same
WO2009089004A1 (en) 2008-01-07 2009-07-16 Amgen Inc. Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
CN109843923A (zh) * 2016-10-11 2019-06-04 南京传奇生物科技有限公司 针对ctla-4的单结构域抗体及其变体

Non-Patent Citations (55)

* Cited by examiner, † Cited by third party
Title
"Kuby Immunology", 2007, W.H. FREEMAN AND CO., pages: 91
ALMAGROFRANSSON, FRONT. BIOSCI., vol. 13, 2008, pages 1619 - 1633
ANONYMOUS: "Accelerating Nanobody Production Using Polyclonal Anti-VHH Antibodiessing Polyclonal Anti-VHH Antibodies", 13 September 2019 (2019-09-13), XP055793842, Retrieved from the Internet <URL:http://www.xsjk.net/guoneikeyan/201909/2360.html> *
ANONYMOUS: "Jackson alpaca family-nanobody VHH newly launched", 24 September 2019 (2019-09-24), XP055793840, Retrieved from the Internet <URL:http://www.seebio.cn/Article/nmktchh_1.html> *
ANONYMOUS: "MonoRabTM Rabbit Anti-Camelid VHH Antibody [iFluor488], MAb", GENSCRIPT, 5 May 2017 (2017-05-05), pages 1 - 4, XP009527052 *
BACA ET AL., J. BIOL. CHEM., vol. 272, 1997, pages 10678 - 10684
BOERNER ET AL., J. IMMUNOL., vol. 147, 1991, pages 60
BRENNAN ET AL., SCIENCE, vol. 229, 1985, pages 81
BRODEUR ET AL.: "Monoclonal Antibody Production Techniques and Applications", 1987, MARCEL DEKKER, INC., pages: 51 - 63
C. HAMERS ET AL.: "Naturally occurring antibodies devoid of light chains", NATURE, vol. 363, 1993, pages 466 - 468
CARTER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 4285
CHEN ET AL., J. MOL. BIOL., vol. 293, 1999, pages 865 - 881
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CHOWDHURY, METHODS MOL. BIOL., vol. 207, 2008, pages 179 - 196
CLACKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 3242
CUNNINGHAMWELLS, SCIENCE, vol. 244, 1989, pages 1081 - 1085
DALL'ACQUA ET AL., METHODS, vol. 36, 2005, pages 61 - 68
FELLOUSE, PROC. NATL. ACAD. SCI. USA, vol. 101, no. 34, 2004, pages 12467 - 12472
GERNGROSS, NAT. BIOTECH., vol. 22, 2004, pages 1409 - 1414
GRAHAM ET AL., J. GEN VIROL., vol. 36, 1977, pages 59
GRIFFITHS ET AL., EMBO J, vol. 12, 1993, pages 725 - 734
GRUBER ET AL., J. IMMUNOL., vol. 152, 1994, pages 5368
HARLOWLANE: "Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY
HOLLINGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
HOOGENBOOMO'BRIEN ET AL.: "Methods in Molecular Biology", vol. 248, 1996, HUMANA PRESS, article "Epitope Mapping Protocols", pages: 255 - 268
HOOGENBOOMWINTER, J. MOL. BIOL., vol. 227, 1992, pages 381 - 388
HUDSON ET AL., NAT. MED., vol. 9, 2003, pages 129 - 134
KLIMKA ET AL., BR. J. CANCER, vol. 83, 2000, pages 252 - 260
KOSTELNY ET AL., J. IMMUNOL., vol. 148, no. 5, 1992, pages 1547 - 1553
KOZBOR, J. IMMUNOL., vol. 133, 1984, pages 3001
LEE ET AL., J. IMMUNOL. METHODS, vol. 284, no. 1-2, 2004, pages 119 - 132
LI ET AL., NAT. BIOTECH., vol. 24, 2006, pages 210 - 215
LI ET AL., PROC. NATL. ACAD. SCI. USA, vol. 103, 2006, pages 3557 - 3562
LONBERG, CURR. OPIN. IMMUNOL., vol. 20, 2008, pages 450 - 459
LONBERG, NAT. BIOTECH., vol. 23, 2005, pages 1117 - 1125
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745
MATHER ET AL., ANNALS N.Y. ACAD. SCI., vol. 383, 1982, pages 44 - 68
MATHER, BIOL. REPROD., vol. 23, 1980, pages 243 - 251
MCCAFFERTY ET AL., NATURE, vol. 305, 1983, pages 537 - 554
MORRISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 - 6855
NI, XIANDAI MIANYIXUE, vol. 26, no. 4, 2006, pages 265 - 268
PADLAN, MOL. IMMUNOL., vol. 28, 1991, pages 489 - 498
PORTOLANO ET AL., J. IMMUNOL., vol. 151, 1993, pages 2623 - 887
PRESTA ET AL., CANCER RES., vol. 57, 1997, pages 4593 - 4599
QUEEN ET AL., PROC. NAT'L ACAD. SCI. USA, vol. 86, 1989, pages 10029 - 10033
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
ROSOK ET AL., J. BIOL. CHEM., vol. 271, 1996, pages 22611 - 22618
See also references of EP4036116A4
SIDHU ET AL., J. MOL. BIOL., vol. 340, no. 5, 2004, pages 1073 - 1093
TRAUNECKER ET AL., EMBO J., vol. 10, 1991, pages 3655
URLAUB ET AL., PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4216
VAN DIJKVAN DE WINKEL, CURR. OPIN. PHARMACOL., vol. 5, 2001, pages 368 - 74
VOLLMERSBRANDLEIN, HISTOLOGY AND HISTOPATHOLOGY, vol. 20, no. 3, 2005, pages 927 - 937
VOLLMERSBRANDLEIN, METHODS AND FINDINGS IN EXPERIMENTAL AND CLINICAL PHARMACOLOGY, vol. 27, no. 3, 2005, pages 185 - 91
WINTER ET AL., ANN. REV. IMMUNOL., vol. 113, 1994, pages 433 - 455

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12583925B2 (en) 2022-12-12 2026-03-24 LaNova Medicines Limited Bispecific antibodies targeting PD1 and VEGF

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