WO2020073834A1 - 一种抗人心肌肌钙蛋白i的抗体及其应用 - Google Patents

一种抗人心肌肌钙蛋白i的抗体及其应用 Download PDF

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WO2020073834A1
WO2020073834A1 PCT/CN2019/108689 CN2019108689W WO2020073834A1 WO 2020073834 A1 WO2020073834 A1 WO 2020073834A1 CN 2019108689 W CN2019108689 W CN 2019108689W WO 2020073834 A1 WO2020073834 A1 WO 2020073834A1
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complementarity determining
determining region
region cdr
binding protein
antibody
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PCT/CN2019/108689
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English (en)
French (fr)
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崔鹏
何志强
孟媛
钟冬梅
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东莞市朋志生物科技有限公司
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Priority to US17/278,345 priority Critical patent/US20220033484A1/en
Priority to KR1020217010376A priority patent/KR20210055754A/ko
Priority to JP2021518970A priority patent/JP7307158B2/ja
Priority to EP19871448.7A priority patent/EP3842454A4/en
Publication of WO2020073834A1 publication Critical patent/WO2020073834A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
    • G01N2470/04—Sandwich assay format
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Definitions

  • the present disclosure relates to the fields of biotechnology and medical technology, in particular to an antibody against human cardiac troponin I and its application.
  • WHO World Health Organization
  • myocardial enzyme activity is more sensitive and specific than phosphocreatine kinase (CK), phosphocreatine kinase isoenzyme (CK-MB), and lactate dehydrogenation. Enzyme and aspartate aminotransferase and other biomarkers.
  • Cardiac troponin I (cTnI) exists only in the myocardium and is a marker of cardiomyocytes.
  • cTnI in normal people's blood is generally less than 0.3 ⁇ g / L.
  • ELISA enzyme-linked immunosorbent assay
  • chemiluminescence chemiluminescence
  • colloidal gold etc.
  • the present disclosure relates to a novel isolated binding protein containing cardiac troponin I (cTnI) antigen binding domain, and studies on the preparation and application of the binding protein.
  • cTnI cardiac troponin I
  • the antigen binding domain includes at least one complementarity determining region selected from the following amino acid sequence, or has at least 80% sequence identity with the complementarity determining region of the following amino acid sequence and has K D ⁇ 9.96 ⁇ 10 -8 with cTnI Affinity of mol / L;
  • the complementarity determining region CDR-VH1 is G-Y-X1-F-T-X2-Y-V-X3-H, where,
  • X1 is S or T
  • X2 is I or L
  • X3 is V, L or I;
  • the complementarity determining region CDR-VH2 is Y-I-X1-P-Y-X2-D-G-T-X3-Y-N-E-K, where,
  • X1 is Q, N or Y, X2 is I or L, X3 is R or K;
  • the complementarity determining region CDR-VH3 is R-X1-G-Y-G-X2-Y-G-L-A, where,
  • X1 is S or T, X2 is Q, N or G;
  • the complementarity determining region CDR-VL1 is S-X1-G-A-X2-T-T-S-X3-Y-A-N, where,
  • X1 is S or T
  • X2 is A or V
  • X3 is Q or N
  • the complementarity determining region CDR-VL2 is G-S-X1-N-R-X2-P, where,
  • X1 is N or Q, X2 is A or V;
  • the complementarity determining region CDR-VL3 is A-X1-V-Y-S-N-X2-W, where,
  • X1 is I or L
  • X2 is Q
  • X2 is Q
  • X2 is Q
  • binding protein has strong activity and has high affinity and sensitivity with human cTnI.
  • X1 is T
  • X3 is K
  • X1 is S
  • X1 is T
  • X2 is A
  • X1 is L.
  • X2 is I and X3 is V.
  • X2 is I and X3 is L.
  • X2 is I and X3 is I.
  • X2 is L and X3 is V.
  • X2 is L and X3 is L.
  • X2 is L and X3 is I.
  • X1 is Q and X2 is I.
  • X1 is Q and X2 is L.
  • X1 is N and X2 is I.
  • X1 is N and X2 is L.
  • X1 is Y and X2 is I.
  • X1 is Y and X2 is L.
  • X2 is Q in the complementarity determining region CDR-VH3.
  • X2 is N in the complementarity determining region CDR-VH3.
  • X2 is G in the complementarity determining region CDR-VH3.
  • X2 is A and X3 is Q.
  • X2 is A and X3 is N.
  • X2 is V and X3 is Q.
  • X2 is V and X3 is N.
  • X1 is N in the complementarity determining region CDR-VL2.
  • X1 is Q in the complementarity determining region CDR-VL2.
  • X2 is Q in the complementarity determining region CDR-VL3.
  • X2 is H in the complementarity determining region CDR-VL3.
  • X2 is N in the complementarity determining region CDR-VL3.
  • the mutation site of each complementarity determining region is selected from any one of the following mutation combinations:
  • the binding protein includes at least 3 CDRs; alternatively, the binding protein includes at least 6 CDRs.
  • the binding protein is a complete antibody comprising variable and constant regions.
  • the binding protein is one of Nanobody, F (ab ') 2, Fab', Fab, Fv, scFv, bispecific antibody, and antibody minimum recognition unit.
  • the binding protein includes light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 whose sequence is as shown in SEQ ID NO: 1-4, and / or Or, the sequence is as shown in SEQ ID NO: 5-8 heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4.
  • the binding protein further comprises antibody constant region sequences.
  • the constant region sequence is selected from any one of the constant region sequences of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
  • the species source of the constant region is cattle, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink , Chicken, duck, goose, turkey, cockfight or human.
  • the constant region is derived from mice
  • the sequence of the light chain constant region is shown in SEQ ID NO: 9;
  • the sequence of the heavy chain constant region is shown in SEQ ID NO: 10.
  • the present disclosure also provides an isolated nucleic acid encoding the aforementioned binding protein.
  • the present disclosure also provides a vector including the above-mentioned nucleic acid.
  • the present disclosure also provides a host cell comprising the above nucleic acid or vector.
  • the present disclosure also provides a method for producing the above binding protein, including the following steps:
  • the above-mentioned host cells are cultured in a culture medium, and the produced binding protein is recovered from the culture medium or from the cultured host cells.
  • the present disclosure also provides the use of the aforementioned binding protein in the preparation of a diagnostic agent or kit for diagnosing acute myocardial infarction, acute coronary syndrome, pulmonary infarction, unstable angina, and myocardial injury.
  • the present disclosure also relates to a method of detecting troponin I antigen in a test sample, which includes:
  • the troponin I antigen is cardiac troponin I antigen.
  • the immune complex in step a), further includes a second antibody, and the second antibody binds to the binding protein.
  • the immune complex further includes a second antibody, and the second antibody binds to the troponin I antigen.
  • the present disclosure also provides a kit including one or more of the aforementioned binding protein, the aforementioned isolated nucleic acid, or the aforementioned carrier.
  • the disclosure also relates to the use of the binding proteins described herein in the diagnosis of diseases associated with cardiac troponin I.
  • the present disclosure also relates to a method of diagnosing diseases associated with cardiac troponin I, including:
  • the presence of the immune complex indicates the presence of a disease associated with cardiac troponin I.
  • the method is based on fluorescent immunoassay, chemiluminescence, colloidal gold immunoassay, radioimmunoassay, and / or enzyme-linked immunoassay.
  • the sample is selected from at least one of whole blood, peripheral blood, serum, plasma, or myocardial tissue.
  • the subject is a mammal, such as a primate, such as a human.
  • the disease associated with cardiac troponin I is a cardiovascular disease.
  • the disease associated with cardiac troponin I is selected from the group consisting of acute myocardial infarction, acute coronary syndrome, pulmonary infarction, unstable angina, myocardial injury, or a combination thereof.
  • amino acid refers to a naturally occurring or non-naturally occurring carboxy alpha-amino acid.
  • amino acid as used in this application may include naturally occurring amino acids and non-naturally occurring amino acids.
  • Naturally occurring amino acids include alanine (three letter code: A1a, single letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), Cysteine (Cys, c), glutamine (G1n, Q), glutamic acid (G1u, E), glycine (G1y, G), histidine (His, H), isoleucine (I1e , I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P) , Serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W
  • Non-naturally occurring amino acids include but are not limited to ⁇ -aminoadipate, aminobutyric acid, citrulline, homocitrulline, homoleucine, homoarginine, hydroxyproline, norleucine, pyridine Alanine, sarcosine and so on.
  • isolated binding protein is a protein that does not bind to naturally bound components due to derived origin or source, which naturally accompany it in its natural state; essentially free from the same species Other proteins; expressed by cells from different species; or not found in nature. Therefore, a protein that is chemically synthesized or synthesized in a cell system that is different from the cell in which it is naturally derived will be “isolated” from its naturally associated components.
  • the protein can also be substantially free of naturally bound components by isolation, for example, using protein purification techniques well known in the art.
  • isolated binding protein including an antigen binding domain generally refers to any protein / protein fragment that includes a CDR region.
  • antibody includes polyclonal antibodies and monoclonal antibodies and antigen compound binding fragments of these antibodies, including Fab, F (ab ') 2, Fd, Fv, scFv, bispecific antibodies and antibody minimum recognition units, and these antibodies And fragments of single-chain derivatives.
  • the type of antibody can be IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.
  • antibody includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, and related synthetic isomeric forms (isoforms).
  • antibody is used interchangeably with "immunoglobulin”.
  • variable region refers to the amino-terminal domain of the heavy or light chain of the antibody.
  • the variable domain of the heavy chain may be referred to as "VH”.
  • variable domain of the light chain may be referred to as "VL”.
  • CDRs complementarity determining regions
  • framework region of the antibody that is, the framework region that constitutes the combination of the light chain and the heavy chain, plays a role of locating and aligning CDRs, which are mainly responsible for binding to the antigen.
  • backbone region means the region of the antibody variable domain that excludes those defined as CDRs.
  • the framework region of each antibody variable domain can be further subdivided into adjacent regions (FR1, FR2, FR3, and FR4) separated by CDRs.
  • variable regions VL / VH of the heavy and light chains can be obtained by connecting the numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the term “purified” or “isolated” in association with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its natural medium or in its natural form.
  • isolated includes polypeptides or nucleic acids taken from the original environment, for example, if it is naturally occurring.
  • an isolated polypeptide generally does not contain at least some proteins or other cellular components that it is usually bound to or usually mixed with or in solution.
  • Isolated polypeptides include the naturally produced polypeptides contained in cell lysates, the polypeptides in purified or partially purified form, recombinant polypeptides, the polypeptides expressed or secreted by cells, and in heterologous host cells or cultures Of the polypeptide.
  • the term isolated or purified indicates that the nucleic acid is not in its natural genomic background (eg, in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).
  • bispecific antibody or “bifunctional antibody” refers to an artificial hybrid binding protein with two different heavy / light chain pairs and two different binding sites. Bispecific binding proteins can be produced by a variety of methods, including fusion of hybridomas or attachment of Fab 'fragments.
  • sequence identity refers to the similarity between at least two different sequences. This percentage identity can be determined by standard algorithms, such as Basic Local Alignment Search Tool (BLAST); Algorithms such as Needleman, etc .; or algorithms such as Meyers.
  • BLAST Basic Local Alignment Search Tool
  • Algorithms such as Needleman, etc .
  • Meyers or algorithms such as Meyers.
  • a set of parameters may be a Blosum 62 scoring matrix and a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of Meyers and Miller ((1989) CABIOS 4: 11-17), which has been incorporated Into the ALIGN program (version 2.0), use the PAM120 weight residue table, gap length penalty of 12, and gap penalty of 4. Percent identity is usually calculated by comparing sequences of similar length.
  • affinity refers to the strength of binding of an antigen binding domain of a binding protein or antibody to an antigen or antigen epitope. Affinity can be measured by KD value, the smaller the KD value, the greater the affinity.
  • the present disclosure provides an isolated binding protein comprising an antigen binding domain, the antigen binding domain comprising at least one complementarity determining region selected from the amino acid sequence described below, or having at least 80 % Sequence identity and affinity with cTnI for K D ⁇ 9.96 ⁇ 10 -8 mol / L;
  • the complementarity determining region CDR-VH1 is G-Y-X1-F-T-X2-Y-V-X3-H, where,
  • X1 is S or T
  • X2 is I or L
  • X3 is V, L or I;
  • the complementarity determining region CDR-VH2 is Y-I-X1-P-Y-X2-D-G-T-X3-Y-N-E-K, where,
  • X1 is Q, N or Y, X2 is I or L, X3 is R or K;
  • the complementarity determining region CDR-VH3 is R-X1-G-Y-G-X2-Y-G-L-A, where,
  • X1 is S or T, X2 is Q, N or G;
  • the complementarity determining region CDR-VL1 is S-X1-G-A-X2-T-T-S-X3-Y-A-N, where,
  • X1 is S or T
  • X2 is A or V
  • X3 is Q or N
  • the complementarity determining region CDR-VL2 is G-S-X1-N-R-X2-P, where,
  • X1 is N or Q, X2 is A or V;
  • the complementarity determining region CDR-VL3 is A-X1-V-Y-S-N-X2-W, where,
  • X1 is I or L
  • X2 is Q
  • X2 is Q
  • X2 is Q
  • the binding specificity and affinity of antibodies are mainly determined by CDR sequences. According to mature and well-known existing technologies, the amino acid sequence of non-CDR regions can be easily changed to obtain changes with similar biological activities. body. Therefore, the present disclosure also includes "functional derivatives" of the binding protein. “Functional derivative” refers to a variant of amino acid substitution. A functional derivative retains detectable binding protein activity, preferably the activity of an antibody capable of binding cTnI. "Functional derivatives" may include "variants” and “fragments” because they have the same CDR sequences as the binding proteins described in this disclosure, and therefore have similar biological activities.
  • the complementarity determining region of the antigen binding domain and the following amino acid sequence has at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, Or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, Or at least 97%, or at least 98%, or at least 99% sequence identity and having KD ⁇ 9.96 ⁇ 10 -8 mol / L with cardiac troponin I, KD value can also be selected 2.26 ⁇ 10 -8 mol / L , 6.76 ⁇ 10 -8 mol / L , 3.54 ⁇ 10 -8 mol / L, 9.41 ⁇ 10 -9 mol / L, 7.95 ⁇ 10 -9 mol / L, 5.41 ⁇ 10 -9 mol / L, 4.20 ⁇ 10 - 9 mol / L
  • the affinity is measured according to the method in the present specification.
  • X1 is T
  • X3 is K
  • X1 is S
  • X1 is T
  • X2 is A
  • X1 is L.
  • X2 is I and X3 is V.
  • X2 is I and X3 is L.
  • X2 is I and X3 is I.
  • X2 is L and X3 is V.
  • X2 is L and X3 is L.
  • X2 is L and X3 is I.
  • X1 is Q and X2 is I.
  • X1 is Q and X2 is L.
  • X1 is N and X2 is I.
  • X1 is N and X2 is L.
  • X1 is Y and X2 is I.
  • X1 is Y and X2 is L.
  • X2 is Q in the complementarity determining region CDR-VH3.
  • X2 is N in the complementarity determining region CDR-VH3.
  • X2 is G in the complementarity determining region CDR-VH3.
  • X2 is A and X3 is Q.
  • X2 is A and X3 is N.
  • X2 is V and X3 is Q.
  • X2 is V and X3 is N.
  • X1 is N in the complementarity determining region CDR-VL2.
  • X1 is Q in the complementarity determining region CDR-VL2.
  • X2 is Q in the complementarity determining region CDR-VL3.
  • X2 is H in the complementarity determining region CDR-VL3.
  • X2 is N in the complementarity determining region CDR-VL3.
  • the mutation site of each complementarity determining region is selected from any one of the following mutation combinations:
  • X1 appearing in the six CDR regions of the binding protein described in the present disclosure each independently of each other represents the amino acid defined in the present disclosure; appearing in the six of the binding protein described in the present disclosure X2 in each CDR region independently represents the amino acids defined in the present disclosure; X3 appearing in the six CDR regions of the binding protein described in the present disclosure each independently represents the amino acids defined in the present disclosure.
  • the binding protein includes at least 3 CDRs; alternatively, the binding protein includes at least 6 CDRs.
  • the binding protein is a complete antibody comprising variable and constant regions.
  • the binding protein is one of Nanobody, F (ab ') 2, Fab', Fab, Fv, scFv, bispecific antibody, and antibody minimum recognition unit.
  • the binding protein includes light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 whose sequence is as shown in SEQ ID NO: 1-4, and / or Or, the sequence is as shown in SEQ ID NO: 5-8 heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4.
  • the binding protein further comprises antibody constant region sequences.
  • the constant region sequence is selected from any one of the constant region sequences of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
  • the species source of the constant region is cattle, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink , Chicken, duck, goose, turkey, cockfight or human.
  • the constant region is derived from mice
  • the sequence of the light chain constant region is shown in SEQ ID NO: 9;
  • the sequence of the heavy chain constant region is shown in SEQ ID NO: 10.
  • the present disclosure also provides an isolated nucleic acid encoding the aforementioned binding protein.
  • nucleic acid contains variants of its conservative substitutions (eg substitution of degenerate codons) and complementary sequences.
  • nucleic acid and polynucleotide are synonymous and include genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides.
  • the present disclosure also provides a vector including the above-mentioned nucleic acid.
  • the nucleic acid sequence is operably linked to at least one regulatory sequence.
  • "Operably linked” means that the coding sequence is linked to the regulatory sequence in a manner that allows expression of the coding sequence.
  • Regulatory sequences are selected to direct the expression of the protein of interest in a suitable host cell, and include promoters, enhancers, and other expression control elements.
  • a vector may refer to a molecule or agent that contains the nucleic acid of the present disclosure or a fragment thereof, is capable of carrying genetic information, and can deliver genetic information to cells.
  • Typical vectors include plasmids, viruses, bacteriophages, cosmids, and mini-chromosomes.
  • the vector can be a cloning vector (ie a vector used to transfer genetic information into a cell, the cell can be propagated and the cell can be selected with or without the genetic information) or an expression vector (ie contains the necessary genetic elements Thereby allowing the genetic information of the vector to be expressed in the cell).
  • a cloning vector may contain a selection marker and an origin of replication that matches the cell type specified by the cloning vector, while an expression vector contains the regulatory elements necessary to affect expression in the designated target cell.
  • the nucleic acid or fragment thereof of the present disclosure can be inserted into a suitable vector to form a cloning vector or expression vector carrying the nucleic acid fragment of the present disclosure.
  • This new carrier is also part of this disclosure.
  • the vector may include plasmids, bacteriophages, cosmids, mini-chromosomes or viruses, as well as naked DNA that is only transiently expressed in specific cells.
  • the cloning vectors and expression vectors of the present disclosure can replicate spontaneously, and therefore can provide high copy numbers for high-level expression or high-level replication purposes for subsequent cloning.
  • the expression vector may include a promoter for driving expression of the nucleic acid fragment of the present disclosure, optionally a nucleic acid sequence encoding a signal peptide that secretes or integrates the peptide expression product on the membrane, a nucleic acid fragment of the present disclosure, and optional Nucleic acid sequence encoding a terminator.
  • the expression vector When the expression vector is manipulated in a production strain or cell line, the vector may or may not be integrated into the genome of the host cell when introduced into the host cell.
  • the vector usually carries a replication site and a marker sequence that can provide phenotypic selection in the transformed cell.
  • the expression vector of the present disclosure is used to transform host cells. Such transformed cells are also part of the present disclosure and can be cultured cells or cell lines used to propagate the nucleic acid fragments and vectors of the present disclosure, or to recombinantly prepare the polypeptides of the present disclosure.
  • the transformed cells of the present disclosure include microorganisms such as bacteria (eg, E. coli, Bacillus, etc.). Host cells also include cells from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells, preferably cells from mammals, such as CHO cells.
  • the transformed cell can replicate the nucleic acid fragment of the present disclosure.
  • the expression product may be exported to the culture medium or carried on the surface of the transformed cell.
  • the present disclosure also provides a method for producing the above binding protein, including the following steps:
  • the above-mentioned host cells are cultured in a culture medium, and the produced binding protein is recovered from the culture medium or from the cultured host cells.
  • the method may be, for example, transfecting a host cell with a nucleic acid vector encoding at least a portion of the binding protein, and culturing the host cell under suitable conditions to express the binding protein.
  • the host cell can also be transfected with one or more expression vectors, which can contain DNA encoding at least a portion of the binding protein, alone or in combination.
  • the binding protein can be separated from the culture medium or cell lysate using conventional techniques for protein and peptide purification, which include ammonium sulfate precipitation, chromatography (eg ion exchange, gel filtration, affinity chromatography, etc.) and / or Electrophoresis.
  • Suitable vectors containing coding and regulatory sequences of interest can be performed using standard ligation and restriction techniques known in the art.
  • the isolated plasmid, DNA sequence or synthetic oligonucleotide is cut, tailed and religated in the desired form.
  • Mutations can be introduced into the coding sequence by any method to produce the variants of the present disclosure, and these mutations can include deletions or insertions or substitutions and the like.
  • the present disclosure also provides antibodies that can react with epitopes of cTnI, including monoclonal and polyclonal antibodies.
  • the antibody may contain the entire binding protein, or a fragment or derivative thereof.
  • Preferred antibodies contain all or part of the binding protein.
  • the present disclosure also provides the use of the aforementioned binding protein in the preparation of a diagnostic agent or kit for diagnosing acute myocardial infarction, acute coronary syndrome, pulmonary infarction, unstable angina, and myocardial injury.
  • the present disclosure also relates to a method of detecting troponin I antigen in a test sample, which includes:
  • the binding protein may be labeled with an indicator that shows signal intensity, so that the complex is easily detected.
  • the troponin I antigen is cardiac troponin I antigen.
  • the immune complex further includes a second antibody, and the second antibody binds to the binding protein
  • the binding protein forms a paired antibody with the second antibody in the form of a first antibody for binding to different epitopes of cTnI;
  • the second antibody may be labeled with an indicator that shows signal intensity, so that the complex is easily detected.
  • the immune complex further includes a second antibody that binds to the troponin I antigen
  • the binding protein serves as an antigen of the second antibody
  • the second antibody may be labeled with an indicator that shows signal intensity, so that the complex is easily detected.
  • the indicator showing the signal intensity includes fluorescent substances, quantum dots, digoxin-labeled probes, biotin, radioisotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electrons Any one of dense substance, chemiluminescent marker, ultrasound contrast agent, photosensitizer, colloidal gold or enzyme.
  • the fluorescent substance includes Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY -FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ′, 5′-dichloro-2 ′, 7′-dimethoxyfluorescein, 5-carboxy-2 ′, 4 ′ , 5 ′, 7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,
  • the radioisotope includes 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y , 89 Zr, 94 mTc, 94 Tc, 99 mTc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re , 51 Mn, 52 mMn, 55 Co, 72 As, 75 Br, 76 Br, 82 mRb and 83 Sr.
  • the enzyme includes any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.
  • the fluorescent microspheres are: polystyrene fluorescent microspheres, with rare earth fluorescent ion europium wrapped inside.
  • kits including one or more of the above-mentioned binding protein, the above-mentioned isolated nucleic acid or the above-mentioned carrier.
  • the kit further includes a label for labeling the binding protein.
  • the present disclosure provides a kit for determining the presence of, for example, troponin I in a subject infected with cardiomyocytes, the kit comprising at least one binding protein provided by the present disclosure, related buffers Reagents, reagents required to react the liquid sample with the binding protein, and reagents used to determine whether there is a positive or negative binding reaction between troponin I and the binding protein.
  • the kit may, for example, use a labeled binding protein as an antibody, where the label may be any suitable label, such as a colloidal gold label.
  • the disclosure also relates to the use of the binding proteins described herein in the diagnosis of diseases associated with cardiac troponin I.
  • cardiac troponin I-related disease refers to a disease that uses cardiac troponin I, including its protein or encoding nucleic acid, as a marker.
  • a disease associated with cardiac troponin I may refer to a disease characterized by an increase in the level of cardiac troponin I in blood.
  • a cardiac troponin I-related disease may refer to a disease characterized by a decrease in cardiac troponin I levels in cardiac muscle tissue or cardiomyocytes.
  • the present disclosure also relates to a method of diagnosing diseases associated with cardiac troponin I, including:
  • the presence of the immune complex indicates the presence of a disease associated with cardiac troponin I.
  • the method is based on fluorescent immunoassay, chemiluminescence, colloidal gold immunoassay, radioimmunoassay, and / or enzyme-linked immunoassay.
  • the sample is selected from at least one of whole blood, peripheral blood, serum, plasma, or myocardial tissue.
  • the subject is a mammal, such as a primate, such as a human.
  • the disease associated with cardiac troponin I is a cardiovascular disease.
  • the disease associated with cardiac troponin I is selected from the group consisting of acute myocardial infarction, acute coronary syndrome, pulmonary infarction, unstable angina, myocardial injury, or a combination thereof.
  • the restriction enzyme and Prime DNA polymerase were purchased from Takara.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO.
  • SMARTERTM RACE cDNA Amplification Kit kit was purchased from Takara.
  • the pMD-18T vector was purchased from Takara Corporation.
  • the plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were completed by Invitrogen.
  • the hybridoma cell line secreting the Anti-cTnI-21C5 monoclonal antibody is our existing hybridoma cell line, and it is ready for recovery.
  • UPM Universal Primer A Mixture
  • NUP Nested Universal Primer A
  • MIgG-CKR 5’-CGCCTAACACTCATTCCTGTTGAAGC-3 ’;
  • MIgG-CHR 5'-CCGCTCATTTACCCGGAGACCG-3 '.
  • RNA from the hybridoma cell line secreting Anti-cTnI 21C5 monoclonal antibody and use the SMARTERTM RACE cDNA Amplification Kit Kit and the SMARTER II Oligonucleotide and 5'-CDS primers in the kit to perform first-strand cDNA After synthesis, the obtained first-strand cDNA product is used as a template for PCR amplification.
  • Light chain genes were amplified with Universal Primer A Mixture (UPM), Nested Universal Primer A (NUP) and MIgG-CKR primers, and Heavy Chain Genes were amplified with Universal Primer A Mixture (UPM), Nested Universal Primer A (NUP) and MIgG-CHR primers are used for amplification.
  • the primer pair of the light chain amplifies the target band of about 0.7KB
  • the primer pair of the heavy chain amplifies the target band of about 1.4KB.
  • VL gene sequence is 357bp, which belongs to the VkII gene family, with a 57bp leader peptide sequence in front
  • VH gene sequence is 366bp, which belongs to the VH1 gene family, which has 57bp in front Leader sequence.
  • the vector is a constructed recombinant antibody eukaryotic expression vector.
  • the expression vector has been introduced into polyclonal cleavage sites such as HindIII, BamHI, EcoRI, etc., and is named pcDNA 3.4A expression vector, subsequently referred to as 3.4A expression vector; according to the above pMD-18T
  • the results of the sequencing of the antibody genes were designed.
  • Anti-cTnI 21C5 antibody heavy and light chain gene-specific primers were designed with HindIII and EcoRI cleavage sites and protective bases at both ends. The primers are as follows:
  • cTnI-21C5-HF 5’-CCCAAGCTTGCCGCCACCATGAGTGTGCTCACTCAGGTCCTGGGGT-3 ’;
  • cTnI-21C5-HR 5’-GGGGAATTCTCATTTACCCGGAGACCGGGAGATGGTCTTC-3 ’;
  • cTnI-21C5-LF 5’-CCCAAGCTTGCCGCCACCATGAAGTCACAGACCCAGGTCTTCGTA-3 ’;
  • cTnI-21C5-LR 5'-CCCGAATTCTCAACACTCATTCCTGTTGAAGCTCTTGACGATG-3 '.
  • the 0.73KB light chain gene fragment and the 1.45KB heavy chain gene fragment were amplified by PCR amplification.
  • the heavy chain and light chain gene fragments were digested with HindIII / EcoRI double digestion, and the 3.4A vector was digested with HindIII / EcoRI double digestion. After the fragments and vector were purified and recovered, the heavy chain gene and light chain gene were connected to the 3.4A expression vector, respectively. Recombinant expression plasmids for heavy and light chains were obtained.
  • Recombinant antibody expression plasmids are stably transfected, and stable cell lines are screened under pressure:
  • the cells were recovered, they were first cultured in 125ml shake flasks, the inoculation volume was 30ml, the culture medium was 100% Dynamis medium, and placed in a shaker with a rotation speed of 120r / min, a temperature of 37 ° C, and carbon dioxide of 8%. Incubate for 72h, and inoculate expansion at an inoculation density of 500,000 cells / ml. The expansion volume is calculated according to production requirements. The medium is 100% Dynamis medium. After every 72h expansion. When the cell volume meets the production needs, strictly control the seeding density to be around 500,000 cells / ml for production.
  • Shake flask parameters speed 120r / min, temperature 37 ° C, carbon dioxide 8%.
  • Feeding feed feeding in the shake flask until 72h starts feeding every day, HyCloneTM Cell Booster Feed 7a feeds 3% of the initial culture volume every day, Feed 7b feeds the volume daily as 1 / 1000th of the initial culture volume Make up to the 12th day (feeding on the 12th day).
  • Glucose was supplemented with 3g / L on the sixth day. Sample collection on the 13th day.
  • Use proteinA affinity chromatography column for affinity purification After purification, 500 mg of recombinant antibody was obtained, and 4 ⁇ g of the purified antibody was subjected to reducing SDS-PAGE.
  • the antibody of sample 1 obtained in Example 1 (having the light chain and heavy chain shown in SEQ ID NO: 20 and 18) has the ability to bind cTnI protein, but the affinity and antibody activity are not ideal, so the applicant passed The light chain CDR and heavy chain CDR of this antibody were mutated.
  • the complementary determination region (WT) of the heavy chain After analysis, the complementary determination region (WT) of the heavy chain:
  • CDR-VH1 is G-Y-S (X1) -F-T-L (X2) -Y-V-V (X3) -H;
  • CDR-VH2 is Y-I-Q (X1) -P-Y-L (X2) -D-G-T-R (X3) -Y-N-E-K;
  • CDR-VH3 is R-T (X1) -G-Y-G-G (X2) -Y-G-L-A;
  • CDR-VL1 is S-S (X1) -G-A-A (X2) -T-T-S-Q (X3) -Y-A-N;
  • CDR-VL2 is G-S-N (X1) -N-R-V (X2) -P;
  • CDR-VL3 is A-I (X1) -V-Y-S-N-H (X2) -W;
  • X1, X2, X3 are mutation sites.
  • the coating solution was diluted with goat anti-mouse IgG 1 ⁇ g / ml for microplate coating, 100 ⁇ L per well, overnight at 4 °C; the next day, the washing solution was washed twice and patted dry; adding blocking solution (20% BSA + 80% PBS), 120 ⁇ L per well, 37 ° C, 1h, pat dry; add diluted cTnI monoclonal antibody, 100 ⁇ L / well, 37 ° C, 60min; shake off the liquid in the plate, pat dry, add Sealed with 20% murine negative blood, 120 ⁇ L per well, 37 °C, 1h; shake off the liquid in the plate, pat dry, add diluted cTnI antigen (0.15 ⁇ g / ml), 100 ⁇ L per well, 37 °C, 40min; wash with washing solution 5 Time, pat dry; add another cTnI monoclonal antibody (1: 5K) labeled with HRP, 100 ⁇ L per well, 37 ° C.
  • mutation 1 has the best activity effect, so mutation 1 is used as the backbone sequence to screen for mutation sites with better titers (to ensure that the activity of the antibody obtained by screening is similar to mutation 1, and the antibody activity is ⁇ 10%), part of the results as follows.
  • the purified antibody was diluted with PBST to 10 ⁇ g / ml, and the cTnI quality control recombinant protein (recombinant antigen produced by the company) was diluted with PBST: 769.2 nmol / ml, 384.6 nmol / ml, 192.3 nmol / ml 96.2nmol / ml, 48.1nmol / ml, 24nmol / ml, 12nmol / ml, 0nmol / ml;
  • the WT mutant sequence also has a certain affinity.
  • the present disclosure provides isolated binding proteins that include an antigen binding domain that binds to cardiac troponin I, including specific heavy chain CDRs and light chain CDRs.
  • the binding protein can specifically recognize and bind cardiac troponin I protein, and has high sensitivity and specificity.
  • the binding protein has high affinity with human cTnI protein.

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Abstract

本发明公开了一种包含心肌肌钙蛋白I（cTnI）抗原结合结构域的分离的结合蛋白，以及该结合蛋白的制备、应用等。所述抗原结合结构域包括选自本文限定的氨基酸序列的至少一个互补决定区：或；与所述氨基酸序列的互补决定区具有至少80％的序列同一性且与cTnI具有K D≤9.96×10 -8mol/L的亲和力。所述结合蛋白可用于cTnI蛋白的检测领域。

Description

一种抗人心肌肌钙蛋白I的抗体及其应用
相关申请的交叉引用
本申请要求于2018年10月10日提交中国专利局的申请号为201811183078.6、名称为“一种抗人心肌肌钙蛋白I的抗体及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及生物技术和医学技术领域，尤其是涉及一种抗人心肌肌钙蛋白I的抗体及其应用。
背景技术
20世纪80年代前，世界卫生组织(WHO)一直将心肌酶谱活性作为急性心肌梗死(AMI)的诊断标准之一。20世纪80年代末，科研人员发现，肌钙蛋白(troponin，Tn)的敏感性和特异性高于磷酸肌酸激酶(CK)、磷酸肌酸激酶同工酶(CK-MB)、乳酸脱氢酶和天冬氨酸氨基转移酶等生物标志物。心肌肌钙蛋白I(cTnI)仅存于心肌，是心肌细胞的标志物，其异常改变可影响心脏的舒缩功能，并可用于诊断心肌坏死，判断心肌损伤等，成为心肌细胞损伤敏感性和特异性最强的标志物之一，是公认的快速诊断AMI和急性冠脉综合征(acute coronary syndromes，ACS)以及协助ACS危险分层和反映其预后的主要生化标志。
正常人血液中cTnI含量一般低于0.3μg/L。当心肌细胞胞膜完整性因缺血或缺氧等受到破坏时，游离的cTnI可迅速透过细胞膜进入血流。因此，在发病初期快速、灵敏且准确的测定人血中的cTnI及其变化趋势对急性心肌梗死的诊断、急性冠状动脉综合征的危险分层、监测各种因素导致的心肌损伤等有着重要的临床意义。临床上用于检测cTnI水平的方法有酶联免疫吸附法(ELISA)，化学发光，胶体金等，不同方法都有各自的优缺点，但是都需要针对于cTnI的特异性单克隆抗体。
现有的cTnI抗体由于活性低、亲和力差，无法很好地应用于cTnI蛋白的检测中，因此本领域对于有效且特异性结合检测cTnI并对其进行检测的抗体存在着强烈需求。
发明内容
本公开涉及一种新颖的包含心肌肌钙蛋白I(cTnI)抗原结合结构域的分离的结合蛋白，并对该结合蛋白的制备、应用等方面进行研究。
所述抗原结合结构域包括选自下述氨基酸序列的至少一个互补决定区，或与下述氨基酸序列的互补决定区具有至少80％的序列同一性且与cTnI具有K D＜9.96×10 -8mol/L的亲和力；
互补决定区CDR-VH1为G-Y-X1-F-T-X2-Y-V-X3-H，其中，
X1是S或T，X2是I或L，X3是V、L或I；
互补决定区CDR-VH2为Y-I-X1-P-Y-X2-D-G-T-X3-Y-N-E-K，其中，
X1是Q、N或Y，X2是I或L，X3是R或K；
互补决定区CDR-VH3为R-X1-G-Y-G-X2-Y-G-L-A，其中，
X1是S或T，X2是Q、N或G；
互补决定区CDR-VL1为S-X1-G-A-X2-T-T-S-X3-Y-A-N，其中，
X1是S或T，X2是A或V，X3是Q或N；
互补决定区CDR-VL2为G-S-X1-N-R-X2-P，其中，
X1是N或Q，X2是A或V；
互补决定区CDR-VL3为A-X1-V-Y-S-N-X2-W，其中，
X1是I或L，X2是Q、H或N。
一个重要优点在于，所述结合蛋白活性强，与人cTnI具有很高的亲和力和灵敏度。
在一种或多种实施方式中，
所述互补决定区CDR-VH1中，X1是T；
所述互补决定区CDR-VH2中，X3是K；
所述互补决定区CDR-VH3中，X1是S；
所述互补决定区CDR-VL1中，X1是T；
所述互补决定区CDR-VL2中，X2是A；
所述互补决定区CDR-VL3中，X1是L。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是I，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是I，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是I，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是L，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是L，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是L，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是Q，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是Q，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是N，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是N，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是Y，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是Y，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是Q。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是N。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是G。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是A，X3是Q。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是A，X3是N。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是V，X3是Q。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是V，X3是N。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X1是N。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X1是Q。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X2是Q。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X2是H。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X2是N。
在一种或多种实施方式中，各互补决定区的突变位点选自下述突变组合中的任一种：
Figure PCTCN2019108689-appb-000001
Figure PCTCN2019108689-appb-000002
Figure PCTCN2019108689-appb-000003
在一种或多种实施方式中，所述结合蛋白中包括至少3个CDRs；或者，所述结合蛋白包括至少6个CDRs。
在一种或多种实施方式中，所述结合蛋白为包含可变区和恒定区的完整抗体。
在一种或多种实施方式中，所述结合蛋白为纳米抗体、F(ab’)2、Fab’、Fab、Fv、scFv、双特异抗体和抗体最小识别单位中的一种。
在一种或多种实施方式中，所述结合蛋白包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4，和/或，序列依次如SEQ ID NO:5-8所示的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4。
在一种或多种实施方式中，所述结合蛋白还包含抗体恒定区序列。
在一种或多种实施方式中，所述恒定区序列选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任何其中之一恒定区的序列。
在一种或多种实施方式中，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在一种或多种实施方式中，所述恒定区来源于小鼠；
轻链恒定区序列如SEQ ID NO:9所示；
重链恒定区序列如SEQ ID NO:10所示。
本公开还提供了一种分离的核酸，所述核酸编码上述的结合蛋白。
本公开还提供了一种载体，所述载体包含上述的核酸。
本公开还提供了一种宿主细胞，包含上述核酸或载体。
本公开还提供了一种生产上述结合蛋白的方法，包括如下步骤：
在培养基中培养上述的宿主细胞，从培养基中或从所培养的宿主细胞中回收产生的结合蛋白。
本公开还提供了上述的结合蛋白在制备用于诊断急性心肌梗死、急性冠状动脉综合征、肺梗、不稳定性心绞痛、心肌损伤的诊断剂或试剂盒中的应用。
根据本公开的一方面，本公开还涉及一种检测测试样品中的肌钙蛋白I抗原的方法，其包括：
a)在足以发生抗体/抗原结合反应的条件下，使所述测试样品中的肌钙蛋白I抗原与权利要求如上所述的结合蛋白接触以形成免疫复合物；和
b)检测所述免疫复合物的存在，所述复合物的存在指示所述测试样品中所述肌钙蛋白I抗原的存在。
在一种或多种实施方式中，所述肌钙蛋白I抗原为心肌肌钙蛋白I抗原。
在一种或多种实施方式中，在步骤a)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述结合蛋白结合。
在一种或多种实施方式中，在步骤a)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述肌钙蛋白I抗原结合。
本公开还提供了一种试剂盒，所述试剂盒包括上述的结合蛋白、上述的分离的核酸或上述的载体中的一种或多种。
本公开还涉及的本文所述结合蛋白，在诊断与心肌肌钙蛋白I相关的疾病的应用。
本公开还涉及一种诊断与心肌肌钙蛋白I相关的疾病的方法，包括：
A)在足以发生结合反应的条件下，使来自受试者的样品与本公开所述的结合蛋白接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物，
其中，所述免疫复合物的存在指示与心肌肌钙蛋白I相关的疾病的存在。
在一种或多种实施方式中，所述方法基于荧光免疫技术、化学发光技术、胶体金免疫技术、放射免疫分析和/或酶联免疫技术。
在一种或多种实施方式中，所述样品选自全血，外周血，血清，血浆或心肌组织中的至少一种。
在一种或多种实施方式中，所述受试者为哺乳动物，例如为灵长类动物，例如为人类。
在一种或多种实施方式中，所述与心肌肌钙蛋白I相关的疾病为心血管疾病。
在一种或多种实施方式中，所述与心肌肌钙蛋白I相关的疾病选自由急性心肌梗死、急性冠状动脉综合征、肺梗、不稳定性心绞痛、心肌损伤或其组合组成的组。
具体实施方式
本公开可通过后续对于本公开一些实施方案描述以及其中所包括的实施例的详细内容而更容易被了解。
在进一步叙述本公开之前，应明了本公开不会被局限于所述特定实施方案中，因为这些实施方案必然是多样的。亦应明了本说明书中所使用的用语仅是为了阐述特定实施方案，而非作为限制，因为本公开的范围将会被仅仅界定在所附的权利要求中。
除非本文另有定义，连同本公开使用的科学和技术术语应具有本领域普通技术人员通常理解的含义。术语的含义和范围应当清晰，然而，在任何潜在不明确性的情况下，本文提供的定义优先于任何字典或外来定义。在本申请中，除非另有说明，“或”的使用意味着“和/或”。此外，术语“包括”及其他形式的使用是非限制性的。
一般地，连同本文描述的细胞和组织培养、分子生物学、免疫学、微生物学、遗传学以及蛋白和核酸化学和杂交使用的命名法和其技术是本领域众所周知和通常使用的那些。除非另有说明，本公开的方法和技术一般根据本领域众所周知，且如各种一般和更具体的参考文献中所述的常规方法来进行，所述参考文献在本说明书自始至终引用和讨论。酶促反应和纯化技术根据制造商的说明书、如本领域通常实现的或如本文所述来进行。连同本文描述的分析化学、合成有机化学以及医学和药物化学使用的命名法、以及其实验室程序和技术是本领域众所周知和通常使用的那些。
为了本公开可以更容易地理解，选择的术语在下文定义。
术语“氨基酸”表示天然存在或非天然存在的羧基α-氨基酸。术语“氨基酸”用在本申请中可以包括天然存在的氨基酸和非天然存在的氨基酸。天然存在的氨基酸包括丙氨酸(三字母密码：A1a，单字母密码：A)，精氨酸(Arg，R)，天冬酰胺(Asn，N)，天冬氨酸(Asp，D)，半胱氨酸(Cys，c)，谷氨酰胺(G1n，Q)，谷氨酸(G1u，E)，甘氨酸(G1y，G)，组氨酸(His，H)，异亮氨酸(I1e，I)，亮氨酸(Leu，L)，赖氨酸(Lys，K)，甲硫氨酸(Met，M)，苯丙氨酸(Phe，F)，脯氨酸(Pro，P)，丝氨酸(Ser，S)，苏氨酸(Thr，T)，色氨酸(Trp，W)，酪氨酸(Tyr，Y)，和缬氨酸(Va1，V)。非天然存在的氨基酸包括但不限于α-氨基己二酸，氨基丁酸，瓜氨酸，高瓜氨酸，高亮氨酸，高精氨酸，羟基脯氨酸，正亮氨酸，吡啶基丙氨酸，肌氨酸等等。
术语“分离的结合蛋白”是这样的蛋白，其由于衍生起源或来源不与天然结合的组分结合，所述天然结合的组分在其天然状态下与其伴随；基本上不含来自相同物种的其他蛋白；由来自不同物种的细胞表达；或在自然界中不存在。因此，化学合成或在不同于其天然起源的细胞的细胞***中合成的蛋白将是与其天然结合的组分“分离的”。还可以通过分离，例如使用本领域众所周知的蛋白纯化技术，使得蛋白基本上不含天然结合的组分。
术语“包括抗原结合结构域的分离的结合蛋白”泛指包含CDR区的一切蛋白/蛋白片段。“抗体”此用语包括多克隆抗体及单克隆抗体以及这些抗体的抗原化合物结合片段，包括Fab、F(ab’)2、Fd、Fv、scFv、双特异抗体和抗体最小识别单位，以及这些抗体和片段的单链衍生物。抗体的类型可以选择IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD。 此外，“抗体”此用语包括天然存在的抗体以及非天然存在的抗体，包括例如嵌合型(chimeric)、双功能型(bifunctional)和人源化(humanized)抗体，以及相关的合成异构形式(isoforms)。“抗体”此用语可和“免疫球蛋白”互换使用。
抗体的“可变区”或“可变结构域”是指抗体的重链或轻链的氨基端结构域。重链的可变结构域可以被称为“VH”。轻链的可变结构域可以被称为“VL”。这些结构域通常是抗体的最可变的部分，并含有抗原结合位点。轻链或重链可变区由被三个称为“互补决定区”或“CDR”的高变区以及将其分隔开的骨架区(framework region)构成。抗体的骨架区，即构成要件轻链和重链的组合的构架区，起到定位和对齐CDR的作用，所述CDR主要负责与抗原的结合。
当在本文中使用时，“骨架区”、“构架区”或“FR”意味着抗体可变结构域的排除被定义为CDR的那些区域之外的区域。每个抗体可变结构域骨架区可以被进一步细分成被CDR分隔开的毗邻区域(FR1、FR2、FR3和FR4)。
通常情况下，重链和轻链的可变区VL/VH可由以下编号的CDR与FR按如下组合排列连接获得：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
当在本文中使用时，与多肽或核酸相关联的术语“纯化的”或“分离的”是指多肽或核酸不是处于其天然介质中或天然形式下。因此，术语“分离的”包括从其原始环境，例如如果它是天然存在的，从天然环境取出的多肽或核酸。例如，分离的多肽通常不含通常与其结合或通常与其混合或在溶液中的至少某些蛋白质或其他细胞组分。分离的多肽包括细胞裂解物中包含的天然生产的所述多肽，纯化或部分纯化形式的所述多肽，重组多肽，被细胞表达或分泌的所述多肽，以及在异源宿主细胞或培养物中的所述多肽。与核酸相关联，术语分离的或纯化的指示例如所述核酸不在其天然的基因组背景中(例如在载体中，作为表达盒，连接到启动子，或人工引入到异源宿主细胞中)。
如本文所用，术语“双特异性抗体”或“双功能性抗体”指具有两对不同重/轻链和两个不同结合位点的人造杂合结合蛋白。双特异性结合蛋白可通过多种方法来生成，包括融合杂交瘤或连接Fab’片段。
如本文所用，术语“序列同一性”指至少两种不同序列之间的相似性。此百分比同一性可通过标准算法来确定，例如基本局部比对搜索工具(Basic Local Alignment Search Tool，BLAST)；Needleman等的算法；或Meyers等的算法。在一种或多种实施方式中，一组参数可以是Blosum 62评分矩阵及缺口罚分12、缺口延伸罚分4、和移码缺口罚分5。在一种或多种实施方式中，两种氨基酸或核苷酸序列之间的百分比同一性还可以使用Meyers和Miller((1989)CABIOS 4：11-17)的算法来确定，该算法已经掺入ALIGN程序(2.0版)，使用PAM120权重残基表、缺口长度罚分12、和缺口罚分4。百分比同一性通常通过比较相似长度的序列来计算。
如本文所用，术语“亲和力”是指结合蛋白或抗体的抗原结合结构域与抗原或抗原表位的结合强度。亲和力可以用KD值来衡量，KD值越小代表亲和力越大。
本公开提供的一种包含抗原结合结构域的分离的结合蛋白，所述抗原结合结构域包括选自下述氨基酸序列的至少一个互补决定区，或与下述氨基酸序列的互补决定区具有至少80％的序列同一性且与cTnI具有K D≤9.96×10 -8mol/L的亲和力；
互补决定区CDR-VH1为G-Y-X1-F-T-X2-Y-V-X3-H，其中，
X1是S或T，X2是I或L，X3是V、L或I；
互补决定区CDR-VH2为Y-I-X1-P-Y-X2-D-G-T-X3-Y-N-E-K，其中，
X1是Q、N或Y，X2是I或L，X3是R或K；
互补决定区CDR-VH3为R-X1-G-Y-G-X2-Y-G-L-A，其中，
X1是S或T，X2是Q、N或G；
互补决定区CDR-VL1为S-X1-G-A-X2-T-T-S-X3-Y-A-N，其中，
X1是S或T，X2是A或V，X3是Q或N；
互补决定区CDR-VL2为G-S-X1-N-R-X2-P，其中，
X1是N或Q，X2是A或V；
互补决定区CDR-VL3为A-X1-V-Y-S-N-X2-W，其中，
X1是I或L，X2是Q、H或N。
本领域公知，抗体的结合特异性及亲合力均主要由CDR序列决定，根据成熟、公知的现有各项技术可轻易地将非CDR区域的氨基酸序列改变而获得具有相类似的生物活性的变体。因此，本公开也包括该结合蛋白的“功能性衍生物”。“功能性衍生物”是指氨基酸替换的变体，一个功能性衍生物保留有可检测的结合蛋白活性，优选为能结合cTnI的抗体的活性。“功能性衍生物”可以包含“变体”和“片段”，因其具有与本公开所述的结合蛋白完全相同的CDR序列，因此具有相类似的生物活性。
在一种或多种实施方式中，所述抗原结合结构域与下述氨基酸序列的互补决定区具有至少50％，或至少55％，或至少60％，或至少65％，或至少70％，或至少75％，或至少80％，或至少85％，或至少90％，或至少91％，或至少92％，或至少93％，或至少94％，或至少95％，或至少96％，或至少97％，或至少98％，或至少99％的序列同一性且与心肌肌钙蛋白I具有KD≤9.96×10 -8mol/L，KD值也可以选择2.26×10 -8mol/L、6.76×10 -8mol/L、3.54×10 -8mol/L、9.41×10 -9mol/L、7.95×10 -9mol/L、5.41×10 -9mol/L、4.20×10 -9mol/L、1.08×10 -9mol/L、8.66×10 -10mol/L、6.97×10 -10mol/L、3.99×10 -10mol/L、1.06×10 -10mol/L，或者1.06×10 -10mol/L≤KD≤9.96×10 -8mol/L，或者1.06×10 -10mol/L≤KD≤9.41×10 -9mol/L；或KD小于或等于2.26×10 -8mol/L、6.76×10 -8mol/L、3.54×10 -8mol/L、9.41×10 -9mol/L、7.95×10 -9mol/L、5.41×10 -9mol/L、4.20×10 -9mol/L、1.08×10 -9mol/L、8.66×10 -10mol/L、6.97×10 -10mol/L、3.99×10 -10mol/L或1.06×10 -10mol/L。
其中，亲和力按照本公开说明书中的方法测定。
在一种或多种实施方式中，
所述互补决定区CDR-VH1中，X1是T；
所述互补决定区CDR-VH2中，X3是K；
所述互补决定区CDR-VH3中，X1是S；
所述互补决定区CDR-VL1中，X1是T；
所述互补决定区CDR-VL2中，X2是A；
所述互补决定区CDR-VL3中，X1是L。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是I，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是I，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是I，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是L，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是L，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是L，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是Q，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是Q，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是N，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是N，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是Y，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是Y，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是Q。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是N。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是G。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是A，X3是Q。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是A，X3是N。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是V，X3是Q。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是V，X3是N。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X1是N。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X1是Q。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X2是Q。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X2是H。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X2是N。
在一种或多种实施方式中，各互补决定区的突变位点选自下述突变组合中的任一种：
Figure PCTCN2019108689-appb-000004
Figure PCTCN2019108689-appb-000005
在一种或多种实施方式中，出现在本公开所述的结合蛋白的六个CDR区中的X1各自彼此独立地代表本公开所限定的氨基酸；出现在本公开所述的结合蛋白的六个CDR区中的X2各自彼此独立地代表本公开所限定的氨基酸；出现在本公开所述的结合蛋白的六个CDR区中的X3各自彼此独立地代表本公开所限定的氨基酸。
在一种或多种实施方式中，所述结合蛋白中包括至少3个CDRs；或者，所述结合蛋白包括至少6个CDRs。
在一种或多种实施方式中，所述结合蛋白为包含可变区和恒定区的完整抗体。
在一种或多种实施方式中，所述结合蛋白为纳米抗体、F(ab’)2、Fab’、Fab、Fv、scFv、双特异抗体和抗体最小识别单位中的一种。
在一种或多种实施方式中，所述结合蛋白包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4，和/或，序列依次如SEQ ID NO:5-8所示的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4。
在一种或多种实施方式中，所述结合蛋白还包含抗体恒定区序列。
在一种或多种实施方式中，所述恒定区序列选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任何其中之一恒定区的序列。
在一种或多种实施方式中，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在一种或多种实施方式中，所述恒定区来源于小鼠；
轻链恒定区序列如SEQ ID NO:9所示；
重链恒定区序列如SEQ ID NO:10所示。
本公开还提供了一种分离的核酸，所述核酸编码上述的结合蛋白。
在本文中，核酸包含其保守置换的变体(例如简并密码子的置换)和互补序列。术语“核酸”和“多核苷酸”是同义的，包含基因、cDNA分子、mRNA分子以及它们的片段 例如寡核苷酸。
本公开还提供了一种载体，所述载体包含上述的核酸。
其中的核酸序列与至少一种调节序列可操作连接。“可操作连接”指的是编码序列以允许编码序列的表达的方式与调节序列连接。调节序列选择用来在合适的宿主细胞中指导目的蛋白质的表达，包含启动子、增强子和其它的表达调控元件。
在本文中，载体可以指包含本公开的核酸或其片段的、能够携带遗传信息并且可以将遗传信息递送到细胞中的分子或试剂。典型的载体包括质粒、病毒、噬菌体、黏粒和微型染色体。载体可以是克隆载体(即用于将遗传信息转移到细胞中的载体，可以繁殖所述细胞并且可以选择存在或不存在所述遗传信息的所述细胞)或表达载体(即包含必要的遗传元件从而允许所述载体的遗传信息在细胞中表达的载体)。因此，克隆载体可以包含选择标记，以及与所述克隆载体所指定的细胞类型相匹配的复制起点，而表达载体则包含对于影响指定靶细胞中的表达必要的调节元件。
本公开的核酸或其片段可以***到合适的载体中以形成携带本公开核酸片段的克隆载体或表达载体。这种新载体也是本公开的一部分。所述载体可以包括质粒、噬菌体、黏粒、微型染色体或病毒，也包括只在特定细胞中瞬时表达的裸DNA。本公开克隆载体和表达载体能够自发的复制，因此能够为用于随后克隆的高水平表达或高水平复制目的提供高拷贝数。表达载体可以包括用于驱动本公开的核酸片段表达的启动子，可选的编码使所述肽表达产物分泌或整合到膜上的信号肽的核酸序列，本公开的核酸片段，以及可选的编码终止子的核酸序列。当在生产菌株或细胞系中操作表达载体时，载体引入到宿主细胞中时可以整合到宿主细胞的基因组中，也可以不能被整合到宿主细胞基因组中。载体通常携带复制位点，以及能够在转化细胞中提供表型选择的标记序列。
本公开的表达载体用于转化宿主细胞。这种转化细胞也是本公开的一部分，可以是用于增殖本公开的核酸片段和载体、或用于重组制备本公开的多肽的培养细胞或细胞系。本公开的转化细胞包括微生物如细菌(如大肠杆菌、芽抱杆菌等)。宿主细胞也包括来自多细胞生物如真菌、昆虫细胞、植物细胞或哺乳动物细胞，优选来自哺乳动物的细胞，例如CHO细胞。所述转化细胞能够复制本公开的核酸片段。当重组制备本公开的肽组合时，所述表达产物可以输出到培养基中或携带在所述转化细胞的表面。
本公开还提供了一种生产上述结合蛋白的方法，包括如下步骤：
在培养基中培养上述的宿主细胞，从培养基中或从所培养的宿主细胞中回收产生的结合蛋白。
所述方法可以是例如，用编码至少一部分结合蛋白的核酸载体转染宿主细胞，在合适的条件下培养该宿主细胞使其表达该结合蛋白。宿主细胞也可以用一个或多个表达载体转染，该表达载体可以单独或结合地包含编码至少一部分结合蛋白的DNA。利用常规的纯化蛋白质和肽的技术可从培养基或细胞裂解物中分离结合蛋白，所述技术包括硫酸铵沉淀，层析(如离子交换，凝胶过滤，亲合层析等)和/或电泳。
构建合适的含有目的编码和调控序列的载体可以使用本领域公知的标准连接和限制技术进行。将分离的质粒、DNA序列或合成的寡核苷酸按需要的形式切割、加尾和再连接。可以用任何方法向编码序列中引入突变以产生本公开的变体，这些突变可以包含缺失或***或置换等。
本公开也提供抗体，能与cTnI的表位发生反应，包含单克隆的和多克隆的抗体。该抗体可以含有完整的结合蛋白，或其片段或衍生物。优选的抗体含有全部或部分的结合蛋白。
本公开还提供了上述的结合蛋白在制备用于诊断急性心肌梗死、急性冠状动脉综合征、肺梗、不稳定性心绞痛、心肌损伤的诊断剂或试剂盒中的应用。
根据本公开的一方面，本公开还涉及一种检测测试样品中的肌钙蛋白I抗原的方法，其包括：
a)在足以发生抗体/抗原结合反应的条件下，使所述测试样品中的肌钙蛋白I抗原与权 利要求如上所述的结合蛋白接触以形成免疫复合物；和
b)检测所述免疫复合物的存在，所述复合物的存在指示所述测试样品中所述肌钙蛋白I抗原的存在；
在此实施方式中，所述结合蛋白可以标记由显示信号强度的指示剂，以使得所述复合物容易被检测。
在一种或多种实施方式中，所述肌钙蛋白I抗原为心肌肌钙蛋白I抗原。
在一种或多种实施方式中，在步骤a)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述结合蛋白结合；
在此实施方式中，所述结合蛋白以第一抗体的形式与所述第二抗体形成配对抗体，用于结合cTnI的不同抗原表位；
所述的第二抗体可以标记由显示信号强度的指示剂，以使得所述复合物容易被检测。
在一种或多种实施方式中，在步骤a)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述肌钙蛋白I抗原结合；
在此实施方式中，所述结合蛋白作为所述第二抗体的抗原，所述的第二抗体可以标记由显示信号强度的指示剂，以使得所述复合物容易被检测。
在一种或多种实施方式中，所述显示信号强度的指示剂包括荧光物质、量子点、地高辛标记探针、生物素、放射性同位素、放射性造影剂、顺磁离子荧光微球、电子致密物质、化学发光标记物、超声造影剂、光敏剂、胶体金或酶中的任一种。
在一种或多种实施方式中，所述荧光物质包括Alexa 350、Alexa 405、Alexa 430、Alexa 488、Alexa 555、Alexa 647、AMCA、氨基吖啶、BODIPY 630/650、BODIPY 650/665、BODIPY-FL、BODIPY-R6G、BODIPY-TMR、BODIPY-TRX、5-羧基-4′，5′-二氯-2′，7′-二甲氧基荧光素、5-羧基-2′，4′，5′，7′-四氯荧光素、5-羧基荧光素、5-羧基罗丹明、6-羧基罗丹明、6-羧基四甲基罗丹明、Cascade Blue、Cy2、Cy3、Cy5、Cy7、6-FAM、丹磺酰氯、荧光素、HEX、6-JOE、NBD(7-硝基苯并-2-氧杂-1，3-二唑)、Oregon Green 488、Oregon Green 500、Oregon Green514、Pacific Blue、邻苯二甲酸、对苯二甲酸、间苯二甲酸、甲酚固紫、甲酚蓝紫、亮甲酚蓝、对氨基苯甲酸、赤藓红、酞菁、偶氮甲碱、花青、黄嘌呤、琥珀酰荧光素、稀土金属穴状化合物、三双吡啶基二胺铕、铕穴状化合物或螯合物、二胺、双花青苷、La Jolla蓝染料、别藻蓝蛋白、allococyanin B、藻蓝蛋白C、藻蓝蛋白R、硫胺、藻红青蛋白、藻红蛋白R、REG、罗丹明绿、罗丹明异硫氰酸酯、罗丹明红、ROX、TAMRA、TET、TRIT(四甲基罗丹明异硫醇)、四甲基罗丹明和德克萨斯红中的任一种。
在一种或多种实施方式中，所述放射性同位素包括 110In、 111In、 177Lu、 18F、 52Fe、 62Cu、 64Cu、 67Cu、 67Ga、 68Ga、 86Y、 90Y、 89Zr、 94mTc、 94Tc、 99mTc、 120I、 123I、 124I、 125I、 131I、 154-158Gd、 32P、 11C、 13N、 15O、 186Re、 188Re、 51Mn、 52mMn、 55Co、 72As、 75Br、 76Br、 82mRb和 83Sr中的任一种。
在一种或多种实施方式中，所述酶包括辣根过氧化酶、碱性磷酸酶和葡萄糖氧化酶中的任一种。
在一种或多种实施方式中，所述荧光微球为：聚苯乙烯荧光微球，内部包裹有稀土荧光离子铕。
进一步地，本公开还提供了一种试剂盒，所述试剂盒包括上述的结合蛋白、上述的分离的核酸或上述的载体中的一种或多种。
优选地，所述试剂盒还包括用于标记所述结合蛋白的标记物。
如在一些实施方案中，本公开提供用于确定例如心肌细胞感染的受试者中的肌钙蛋白I存在的试剂盒，所述试剂盒包含至少一种本公开提供的结合蛋白，相关的缓冲剂，用于使液体样品与所述结合蛋白反应所需的试剂，以及用于确定肌钙蛋白I和结合蛋白之间存在阳性或阴性结合反应的试剂。为了确定肌钙蛋白I的存在，所述试剂盒可以例如利用带有标记的结合蛋白作为抗体，其中所述标记可以是任何合适的标记，如胶体金标记。
本公开还涉及的本文所述结合蛋白，在诊断与心肌肌钙蛋白I相关的疾病的应用。
如本文所用术语“心肌肌钙蛋白I相关的疾病”是指以心肌肌钙蛋白I，包括其蛋白质或编码核酸，作为标志物的疾病。特别地，在本公开的一种或多种实施方式中，心肌肌钙蛋白I相关的疾病可以指以血液中心肌肌钙蛋白I水平的升高作为特征的疾病。在本公开的一种或多种实施方式中，心肌肌钙蛋白I相关的疾病可以指以心肌组织或心肌细胞中心肌肌钙蛋白I水平降低作为特征的疾病。
本公开还涉及一种诊断与心肌肌钙蛋白I相关的疾病的方法，包括：
A)在足以发生结合反应的条件下，使来自受试者的样品与本公开所述的结合蛋白接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物，
其中，所述免疫复合物的存在指示与心肌肌钙蛋白I相关的疾病的存在。
在一种或多种实施方式中，所述方法基于荧光免疫技术、化学发光技术、胶体金免疫技术、放射免疫分析和/或酶联免疫技术。
在一种或多种实施方式中，所述样品选自全血，外周血，血清，血浆或心肌组织中的至少一种。
在一种或多种实施方式中，所述受试者为哺乳动物，例如为灵长类动物，例如为人类。
在一种或多种实施方式中，所述与心肌肌钙蛋白I相关的疾病为心血管疾病。
在一种或多种实施方式中，所述与心肌肌钙蛋白I相关的疾病选自由急性心肌梗死、急性冠状动脉综合征、肺梗、不稳定性心绞痛、心肌损伤或其组合组成的组。
下文提供了一些实例用于示例性说明本公开，而不是限制本公开的范围。
实施例1
本实施例中限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。SMARTERTM RACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由Invitrogen公司完成。分泌Anti-cTnI-21C5单克隆抗体的杂交瘤细胞株为我司已有的杂交瘤细胞株，复苏备用。
1、引物
扩增重链和轻链5’RACE引物：
SMARTER II A寡核苷酸：
Figure PCTCN2019108689-appb-000006
5'-RACE CDS引物(5'-CDS)：5'-(T) 25VN-3’(N＝A,C,G,orT；V＝A,G,orC)；
通用引物A混合物(UPM)：5’-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3’；
巢式通用引物A(NUP)：5’-AAGCAGTGGTATCAACGCAGAGT-3’；
MIgG-CKR：5’-CGCCTAACACTCATTCCTGTTGAAGC-3’；
MIgG-CHR：5’-CCGCTCATTTACCCGGAGACCG-3’。
2、抗体可变区基因克隆及测序
从分泌Anti-cTnI 21C5单克隆抗体的杂交瘤细胞株中提取中RNA，用SMARTERTM RACE cDNA Amplification Kit试剂盒及试剂盒中的SMARTER II A寡核苷酸和5'-CDS引物进行第一链cDNA合成，获得的第一链cDNA产物作为PCR扩增模板。轻链基因以通用引物A混合物(UPM)、巢式通用引物A(NUP)和MIgG-CKR引物进行扩增，重链基因以通用引物A混合物(UPM)、巢式通用引物A(NUP)和MIgG-CHR引物进行扩增。其中轻链的引物对扩增出0.7KB左右的目的条带，重链的引物对扩增出1.4 KB左右的目的条带。用琼脂糖凝胶电泳纯化回收，产物用rTaq DNA聚合酶进行加A反应后***到pMD-18T载体中，转化到DH5α感受态细胞中，长出菌落后分别取重链及轻链基因克隆各4个克隆送Invitrogen公司进行测序。
3、Anti-cTnI 21C5抗体可变区基因的序列分析
将上述测序得到的基因序列放在IMGT抗体数据库中进行分析，并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的，其中轻链扩增出的基因片段中，VL基因序列为357bp，属于VkII基因家族，其前方有57bp的前导肽序列；重链引物对扩增出的基因片段中，VH基因序列为366bp，属于VH1基因家族，其前方有57bp的前导肽序列。
4、重组抗体表达质粒的构建
pcDNA TM3.4
Figure PCTCN2019108689-appb-000007
载体为构建的重组抗体真核表达载体，该表达载体已经引入HindIII、BamHI、EcoRI等多克隆酶切位点，并命名为pcDNA 3.4A表达载体，后续简称3.4A表达载体；根据上述pMD-18T中抗体基因测序结果，设计Anti-cTnI 21C5抗体的重链和轻链基因特异性引物，两端分别带有HindIII、EcoRI酶切位点和保护碱基，引物如下：
cTnI-21C5-HF：5’-CCCAAGCTTGCCGCCACCATGAGTGTGCTCACTCAGGTCCTGGGGT-3’；
cTnI-21C5-HR：5’-GGGGAATTCTCATTTACCCGGAGACCGGGAGATGGTCTTC-3’；
cTnI-21C5-LF：5’-CCCAAGCTTGCCGCCACCATGAAGTCACAGACCCAGGTCTTCGTA-3’；
cTnI-21C5-LR：5’-CCCGAATTCTCAACACTCATTCCTGTTGAAGCTCTTGACGATG-3’。
通过PCR扩增方法扩出0.73KB的轻链基因片段和1.45KB的重链基因片段。重链和轻链基因片段分别采用HindIII/EcoRI双酶切，3.4A载体采用HindIII/EcoRI双酶切，将片段和载体纯化回收后重链基因和轻链基因分别连接3.4A表达载体中，分别得到重链和轻链的重组表达质粒。
5、筛选稳定细胞株
5.1质粒用超纯水稀释至400ng/ml，调节CHO细胞1.43×10 7cells/ml于离心管中，100μl质粒与700μl细胞混合，转入电转杯，电转，第3、5、7天取样计数，第7天收样检测。
包被液稀释相应抗原到指定浓度，每孔100μL，4℃过夜；次日，洗涤液清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120μL，37℃，1h，拍干；加入稀释后的细胞上清，100μL/孔，37℃,30min(部分上清1h)；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100μL，37℃,30min；洗涤液清洗5次，拍干；加入显色液A液(50μL/孔)，加入显色液B液(50μL/孔)，10min；加入终止液，50μL/孔；酶标仪上450nm(参考630nm)处读OD值。以标准品浓度和OD值作标准曲线，计算细胞上清中抗体含量。
5.2重组抗体表达质粒线性化
准备下述试剂：Buffer 50μl、DNA 100μg/管、PuvⅠ酶10μl、无菌水补至500μl，37℃水浴酶切过夜；先用等体积酚/氯仿/异戊醇(下层)25:24:1，再用氯仿(水相)依次进行抽提；0.1倍体积(水相)3M醋酸钠和2倍体积乙醇冰上沉淀，70％乙醇漂洗沉淀，去除有机溶剂，待乙醇挥发完全用适量的灭菌水进行复融，最后进行浓度的测定。
重组抗体表达质粒稳定转染，加压筛选稳定细胞株：
质粒用超纯水稀释至400ng/ml，调节CHO细胞1.43×10 7cells/ml于离心管中，100μl质粒与700μl细胞混合，转入电转杯，电转，次日计数；25μmol/L MSX 96孔加压培养约25天。
显微镜下观察标记长有细胞的克隆孔,并记录汇合度；取培养上清，送样检测；挑选抗体浓度、相对浓度高的细胞株转24孔，3天左右转6孔；3天后保种批培，调整细胞密度0.5×10 6cells/ml，2.2ml进行批培养，细胞密度0.3×10 6cells/ml，2ml进行保种；7天6孔批培上清送样检测，挑选抗体浓度及细胞直径较小的细胞株转TPP保种传代。
6、生产重组抗体
6.1细胞扩培
细胞复苏之后先在125ml规格的摇瓶中培养，接种体积为30ml，培养基为100％Dynamis培养基，放置于转速120r/min，温度为37℃，二氧化碳为8％的摇床中。培养72h，以50万cells/ml接种密度接种扩培，扩培体积根据生产需求进行计算，培养基为100％Dynamis培养基。之后每72h扩培一次。当细胞量满足生产需求时，严格控制接种密度为50万cells/ml左右进行生产。
6.2摇瓶生产及纯化
摇瓶参数：转速120r/min，温度为37℃，二氧化碳为8％。流加补料：在摇瓶中培养至72h时开始每天补料，HyCloneTM Cell BoostTM Feed 7a每天流加初始培养体积的3％，Feed 7b每天流加量为初始培养体积的千分之一，一直补到第12天(第12天补料)。葡萄糖在第六天补加3g/L。第13天收样。用proteinA亲和层析柱进行亲和纯化。纯化后得到500mg重组抗体，取4μg纯化的抗体进行还原性SDS-PAGE。在还原性SDS-PAGE后显示两条带，1条为23KD左右的轻链(序列如SEQ ID NO:11所示)，另一条为50KD左右的重链(序列如SEQ ID NO:12所示)。
实施例2
实施例1得到的样品1的抗体(具有序列如SEQ ID NO:20以及18所示的轻链和重链)虽然具备结合cTnI蛋白的能力，但亲和力和抗体活性均不够理想，因而申请人通过对该抗体的轻链CDR及重链CDR进行突变。
经分析，重链的互补决定区(WT)：
CDR-VH1为G-Y-S(X1)-F-T-L(X2)-Y-V-V(X3)-H；
CDR-VH2为Y-I-Q(X1)-P-Y-L(X2)-D-G-T-R(X3)-Y-N-E-K；
CDR-VH3为R-T(X1)-G-Y-G-G(X2)-Y-G-L-A；
轻链的互补决定区：
CDR-VL1为S-S(X1)-G-A-A(X2)-T-T-S-Q(X3)-Y-A-N；
CDR-VL2为G-S-N(X1)-N-R-V(X2)-P；
CDR-VL3为A-I(X1)-V-Y-S-N-H(X2)-W；
其中，X1、X2、X3均为突变位点。
表1与抗体活性有关的突变位点
Figure PCTCN2019108689-appb-000008
在突变后对抗体活性进行检测，包被液稀释羊抗鼠IgG 1μg/ml进行微孔板包被，每孔100μL，4℃过夜；次日，洗涤液清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120μL，37℃，1h，拍干；加入稀释后的cTnI单克隆抗体，100μL/孔，37℃，60min；甩掉板内液体，拍干，加入20％鼠阴性血封闭，每孔120μL，37℃，1h；甩掉板内液体，拍干，加入稀释的cTnI抗原(0.15μg/ml)，每孔100μL，37℃，40min；洗涤液清洗5次，拍干；加入标记HRP的另一株cTnI单克隆抗体(1:5K)，每孔100μL，37℃,30min；加入显色液A液(50μL/孔)，加入显色液B液(50μL/孔)，10min；加入终止液，50μL/孔；酶标仪上450nm(参考630nm)处读OD值。
部分结果如下：
表2抗体活性分析数据
浓度(ng/ml) WT 突变1 突变2 突变3 突变4 突变5
37.037 2.162 2.326 2.095 1.578 0.821 0.478
12.346 1.968 2.243 1.942 0.824 0.119 0.110
4.115 1.187 1.888 1.512 0.347 - -
1.372 0.612 0.940 0.421 0.150 - -
0.457 0.365 0.524 0.211 - - -
0.000 0.130 0.114 0.117 - - -
“-”代表无活性。
从上表可知，突变1的活性效果最佳，因而以突变1作为骨架序列筛选效价较好的突变位点(保证筛选得到的抗体活性与突变1相近，抗体活性±10％)，部分结果如下。
表3与抗体亲和力有关的突变位点
Figure PCTCN2019108689-appb-000009
Figure PCTCN2019108689-appb-000010
亲和力分析
利用AMC传感器，纯化出来的抗体用PBST稀释到10μg/ml，cTnI质控品重组蛋白(公司自产重组抗原)用PBST进行梯度稀释：769.2nmol/ml、384.6nmol/ml、192.3nmol/ml、96.2nmol/ml、48.1nmol/ml、24nmol/ml、12nmol/ml、0nmol/ml；
运行流程：缓冲液1(PBST)中平衡60s，抗体溶液中固化抗体300s，缓冲液2(PBST)中孵育180s，抗原溶液中结合420s，缓冲液2中解离1200s，用10mM pH 1.69 GLY溶液及缓冲液3进行传感器再生，输出数据。KD表示平衡解亲常数即亲和力；Kon表示结合速率；Kdis表示解离速率。
表4亲和力分析数据
组别 KD(M) Kon(1/Ms) Kdis(1/S)
突变1 4.20E-09 5.10E+04 2.14E-04
突变1-1 8.27E-09 8.61E+04 7.12E-04
突变1-2 7.64E-09 7.99E+04 6.10E-04
突变1-3 3.99E-10 2.39E+04 9.54E-06
突变1-4 5.03E-09 4.62E+04 2.32E-04
突变1-5 1.20E-09 3.68E+04 4.40E-05
突变1-6 6.97E-10 7.66E+04 5.34E-05
突变1-7 2.77E-09 6.36E+04 1.76E-04
突变1-8 9.31E-09 2.62E+04 2.44E-04
突变1-9 5.43E-09 4.42E+04 2.40E-04
突变1-10 7.31E-10 5.63E+04 4.12E-05
突变1-11 4.23E-09 3.35E+04 1.42E-04
突变1-12 7.69E-09 7.92E+04 6.09E-04
突变1-13 1.39E-10 2.73E+04 3.79E-06
突变1-14 8.66E-10 2.55E+04 2.21E-05
突变1-15 7.59E-10 6.19E+04 4.70E-05
突变1-16 6.48E-09 3.43E+04 2.22E-04
突变1-17 7.37E-09 3.21E+04 2.37E-04
突变1-18 9.31E-09 6.30E+04 5.86E-04
突变1-19 4.81E-09 5.70E+04 2.74E-04
突变1-20 7.74E-09 7.00E+04 5.42E-04
突变1-21 1.47E-09 3.88E+04 5.71E-05
突变1-22 1.06E-10 2.70E+04 2.86E-06
突变1-23 8.49E-09 4.16E+04 3.53E-04
突变1-24 6.30E-10 5.74E+04 3.62E-05
突变1-25 6.60E-09 8.03E+04 5.30E-04
突变1-26 4.65E-10 3.98E+04 1.85E-05
突变1-27 3.53E-10 2.07E+04 7.31E-06
突变1-28 1.76E-09 8.96E+04 1.58E-04
突变1-29 3.80E-09 5.45E+04 2.07E-04
突变1-30 3.71E-09 5.95E+04 2.20E-04
突变1-31 9.41E-09 3.61E+04 3.40E-04
突变1-32 7.65E-10 6.93E+04 5.30E-05
突变1-33 7.95E-09 4.17E+04 3.31E-04
突变1-34 3.41E-09 5.80E+04 1.98E-04
突变1-35 8.32E-10 5.18E+04 4.31E-05
突变1-36 3.29E-09 3.60E+04 1.18E-04
突变1-37 5.21E-09 3.99E+04 2.08E-04
突变1-38 2.54E-10 6.77E+04 1.72E-05
突变1-39 1.08E-09 4.36E+04 4.72E-05
突变1-40 1.85E-09 2.19E+04 4.05E-05
突变1-41 7.89E-09 8.20E+04 6.47E-04
突变1-42 8.21E-10 8.28E+04 6.80E-05
突变1-43 4.64E-10 7.29E+04 3.38E-05
突变1-44 6.56E-09 3.13E+04 2.05E-04
突变1-45 7.41E-09 3.86E+04 2.86E-04
突变1-46 5.41E-09 4.01E+04 2.17E-04
突变1-47 3.05E-09 6.41E+04 1.96E-04
突变1-48 5.53E-10 7.82E+04 4.32E-05
突变1-49 1.51E-09 8.84E+04 1.34E-04
突变1-50 4.61E-09 5.05E+04 2.33E-04
突变1-51 7.30E-09 3.05E+04 2.23E-04
突变1-52 6.01E-09 8.18E+04 4.92E-04
突变1-53 8.38E-09 6.48E+04 5.43E-04
突变1-54 7.78E-09 4.13E+04 3.21E-04
突变1-55 4.31E-09 5.54E+04 2.39E-04
从表4可以看出，表3中列出的突变位点对抗体的亲和力影响不大。
为验证上述结果，以WT作为骨架序列重复上述实验，进行突变位点的亲和力验证，部分结果如下。
表5以WT为骨架进行的突变
Figure PCTCN2019108689-appb-000011
表6亲和力分析数据
位点 KD(M) Kon(1/Ms) Kdis(1/S)
WT 7.09E-08 4.80E+04 3.40E-03
WT 1-3 5.17E-08 8.90E+03 4.60E-04
WT 1-8 8.03E-08 6.09E+04 4.89E-03
WT 1-22 9.96E-08 8.70E+03 8.67E-04
WT 1-35 2.17E-08 4.20E+04 9.11E-04
WT 1-45 6.76E-08 4.83E+04 3.27E-03
WT 1-48 4.26E-08 1.36E+04 5.79E-04
WT 1-51 3.54E-08 1.49E+04 5.27E-04
从表5和表6分析，在保证具有抗体活性的前提下，WT突变序列也有一定的亲和力。
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。
工业实用性
本公开提供的包括与心肌肌钙蛋白I结合的抗原结合结构域的分离的结合蛋白，包括特定的重链CDR和轻链CDR。该结合蛋白能够特异性地识别并结合心肌肌钙蛋白I蛋白， 具有较高的灵敏度和特异性，特别地，该结合蛋白与人cTnI蛋白具有很高的亲和力。从而实现对心肌肌钙蛋白I相关疾病的检测和诊断。

Claims (18)

  1. 一种包含抗原结合结构域的分离的结合蛋白，其特征在于，所述抗原结合结构域包括选自下述氨基酸序列的至少一个互补决定区，或与下述氨基酸序列的互补决定区具有至少80％的序列同一性且与cTnI具有K D≤9.96×10 -8mol/L的亲和力；
    互补决定区CDR-VH1为G-Y-X1-F-T-X2-Y-V-X3-H，其中，
    X1是S或T，X2是I或L，X3是V、L或I；
    互补决定区CDR-VH2为Y-I-X1-P-Y-X2-D-G-T-X3-Y-N-E-K，其中，
    X1是Q、N或Y，X2是I或L，X3是R或K；
    互补决定区CDR-VH3为R-X1-G-Y-G-X2-Y-G-L-A，其中，
    X1是S或T，X2是Q、N或G；
    互补决定区CDR-VL1为S-X1-G-A-X2-T-T-S-X3-Y-A-N，其中，
    X1是S或T，X2是A或V，X3是Q或N；
    互补决定区CDR-VL2为G-S-X1-N-R-X2-P，其中，
    X1是N或Q，X2是A或V；
    互补决定区CDR-VL3为A-X1-V-Y-S-N-X2-W，其中，
    X1是I或L，X2是Q、H或N。
  2. 根据权利要求1所述的结合蛋白，其特征在于，所述互补决定区CDR-VH1中，X1是T；
    所述互补决定区CDR-VH2中，X3是K；
    所述互补决定区CDR-VH3中，X1是S；
    所述互补决定区CDR-VL1中，X1是T；
    所述互补决定区CDR-VL2中，X2是A；
    所述互补决定区CDR-VL3中，X1是L；
    进一步地，所述互补决定区CDR-VH1中，X2是I，X3是V；
    进一步地，所述互补决定区CDR-VH1中，X2是I，X3是L；
    进一步地，所述互补决定区CDR-VH1中，X2是I，X3是I；
    进一步地，所述互补决定区CDR-VH1中，X2是L，X3是V。
    进一步地，所述互补决定区CDR-VH1中，X2是L，X3是L；
    进一步地，所述互补决定区CDR-VH1中，X2是L，X3是I；
    进一步地，所述互补决定区CDR-VH2中，X1是Q，X2是I；
    进一步地，所述互补决定区CDR-VH2中，X1是Q，X2是L；
    进一步地，所述互补决定区CDR-VH2中，X1是N，X2是I；
    进一步地，所述互补决定区CDR-VH2中，X1是N，X2是L；
    进一步地，所述互补决定区CDR-VH2中，X1是Y，X2是I；
    进一步地，所述互补决定区CDR-VH2中，X1是Y，X2是L；
    进一步地，所述互补决定区CDR-VH3中，X2是Q；
    进一步地，所述互补决定区CDR-VH3中，X2是N；
    进一步地，所述互补决定区CDR-VH3中，X2是G；
    进一步地，所述互补决定区CDR-VL1中，X2是A，X3是Q；
    进一步地，所述互补决定区CDR-VL1中，X2是A，X3是N；
    进一步地，所述互补决定区CDR-VL1中，X2是V，X3是Q；
    进一步地，所述互补决定区CDR-VL1中，X2是V，X3是N；
    进一步地，所述互补决定区CDR-VL2中，X1是N；
    进一步地，所述互补决定区CDR-VL2中，X1是Q；
    进一步地，所述互补决定区CDR-VL3中，X2是Q；
    进一步地，所述互补决定区CDR-VL3中，X2是H；
    进一步地，所述互补决定区CDR-VL3中，X2是N；
    优选地，各互补决定区的突变位点选自下述突变组合中的任一种：
    Figure PCTCN2019108689-appb-100001
    Figure PCTCN2019108689-appb-100002
  3. 根据权利要求1或2所述的结合蛋白，其特征在于，所述结合蛋白中包括至少3个CDRs；或者，所述结合蛋白包括至少6个CDRs；
    进一步地，所述结合蛋白为纳米抗体、F(ab’)2、Fab’、Fab、Fv、scFv、双特异抗体和抗体最小识别单位中的一种；
    进一步地，所述结合蛋白包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4，和/或，序列依次如SEQ ID NO:5-8所示的重链骨架 区FR-H1、FR-H2、FR-H3及FR-H4。
  4. 根据权利要求1或2所述的结合蛋白，其特征在于，所述结合蛋白还包含抗体恒定区序列；
    进一步地，所述恒定区序列选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任何其中之一恒定区的序列；
    进一步地，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人；
    进一步地，所述恒定区来源于小鼠；
    轻链恒定区序列如SEQ ID NO:9所示；
    重链恒定区序列如SEQ ID NO:10所示。
  5. 一种分离的核酸，其特征在于，所述核酸编码权利要求1-4任一项所述的结合蛋白。
  6. 一种载体，其特征在于，其包含权利要求5所述的核酸。
  7. 一种宿主细胞，其特征在于，所述宿主细胞包括权利要求5所述的核酸或权利要求6所述的载体。
  8. 一种生产权利要求1-4任一项所述的结合蛋白的方法，其特征在于，包括如下步骤：
    在培养基中培养权利要求7所述的宿主细胞，从培养基中或从所培养的宿主细胞中回收产生的结合蛋白。
  9. 权利要求1-4任一项所述的结合蛋白在制备用于诊断急性心肌梗死、急性冠状动脉综合征、肺梗、不稳定性心绞痛、心肌损伤的诊断剂或试剂盒中的应用。
  10. 一种试剂盒，其特征在于，所述试剂盒包括权利要求1-4任一项所述的结合蛋白、权利要求5所述的分离的核酸或权利要求6所述的载体中的一种或多种；
    优选地，所述试剂盒还包括用于标记所述结合蛋白的标记物。
  11. 一种检测测试样品中的肌钙蛋白I抗原的方法，其包括：
    a)在足以发生抗体/抗原结合反应的条件下，使所述测试样品中的肌钙蛋白I抗原与权利要求3所述的结合蛋白接触以形成免疫复合物；和
    b)检测所述免疫复合物的存在，所述复合物的存在指示所述测试样品中所述肌钙蛋白I抗原的存在；
    优选的，所述免疫复合物中还包括第二抗体，所述第二抗体与所述结合蛋白结合；
    优选的，在步骤a)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述 肌钙蛋白I抗原结合；
    优选的，所述肌钙蛋白I抗原为心肌肌钙蛋白I抗原。
  12. 如权利要求1～4任一项所述的结合蛋白，在诊断与心肌肌钙蛋白I相关的疾病的应用。
  13. 一种诊断与心肌肌钙蛋白I相关的疾病的方法，包括：
    A)在足以发生结合反应的条件下，使来自受试者的样品与权利要求1～4中任一项所述的结合蛋白接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物，
    其中，所述免疫复合物的存在指示与心肌肌钙蛋白I相关的疾病的存在。
  14. 根据权利要求13所述的方法，其中所述方法基于荧光免疫技术、化学发光技术、胶体金免疫技术、放射免疫分析和/或酶联免疫技术。
  15. 根据权利要求13或14所述的方法，其中所述样品选自全血，外周血，血清，血浆或心肌组织中的至少一种。
  16. 根据权利要求13～15中任一项所述的方法，其中所述受试者为哺乳动物，优选地为灵长类动物，更优选地为人类。
  17. 根据权利要求12所述的应用或根据权利要求13～16中任一项所述的方法，其中所述与心肌肌钙蛋白I相关的疾病为心血管疾病。
  18. 根据权利要求12所述的应用或根据权利要求13～16中任一项所述的方法，其中所述与心肌肌钙蛋白I相关的疾病选自由急性心肌梗死、急性冠状动脉综合征、肺梗、不稳定性心绞痛、心肌损伤或其组合组成的组。
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JP2021518970A JP7307158B2 (ja) 2018-10-10 2019-09-27 抗ヒト心筋型トロポニンｉ抗体及びその応用
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