WO2020073831A1 - 一种抗人心肌肌钙蛋白i的重组抗体 - Google Patents

一种抗人心肌肌钙蛋白i的重组抗体 Download PDF

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Publication number
WO2020073831A1
WO2020073831A1 PCT/CN2019/108681 CN2019108681W WO2020073831A1 WO 2020073831 A1 WO2020073831 A1 WO 2020073831A1 CN 2019108681 W CN2019108681 W CN 2019108681W WO 2020073831 A1 WO2020073831 A1 WO 2020073831A1
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complementarity determining
determining region
region cdr
binding protein
antibody
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PCT/CN2019/108681
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English (en)
French (fr)
Inventor
崔鹏
何志强
孟媛
韩日才
钟冬梅
范凌云
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东莞市朋志生物科技有限公司
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Priority to EP19872158.1A priority Critical patent/EP3872092A4/en
Priority to JP2021542251A priority patent/JP7439108B2/ja
Priority to KR1020217009161A priority patent/KR20210049894A/ko
Publication of WO2020073831A1 publication Critical patent/WO2020073831A1/zh

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    • 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
    • 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
    • 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
    • C07K2317/50—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
    • 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
    • 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
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure

Definitions

  • the present disclosure relates to the technical field of immunology, in particular, to a recombinant antibody against human cardiac troponin I.
  • 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 ⁇ with cardiac troponin I 3.33 ⁇ 10 -8 mol / L affinity;
  • the complementarity determining region CDR-VH1 is G-F-T-X1-S-N-Y-A-M-S, where,
  • X1 is Y or F
  • the complementarity determining region CDR-VH2 is I-S-X1-G-G-S-Y-S-X2-Y-P-D-S-V-K, where,
  • X1 is T or S, X2 is Y or F;
  • the complementarity determining region CDR-VH3 is A-R-X1-D-N-Y-X2-G-S-X3-F-M-D-Y, where,
  • X1 is K or R
  • X2 is Y
  • X3 is T or S
  • the complementarity determining region CDR-VL1 is R-A-S-Q-X1-I-T-X2-Y-L-N, where,
  • X1 is D or E, X2 is Q or N;
  • the complementarity determining region CDR-VL2 is I-Y-Y-X1-S-R-X2-H-S-G-V-S, where,
  • X1 is S or T, X2 is I or L;
  • the complementarity determining region CDR-VL3 is Q-Q-G-X1-T-X2-P-L-T, where,
  • X1 is N or H, and X2 is I or L.
  • binding protein has strong activity and has a high affinity with human cTnI protein.
  • X1 is S
  • X1 is R
  • X1 is T.
  • X1 is Y in the complementarity determining region CDR-VH1.
  • X1 is F in the complementarity determining region CDR-VH1.
  • X2 is Y in the complementarity determining region CDR-VH2.
  • X2 is F in the complementarity determining region CDR-VH2.
  • X2 is Y and X3 is T.
  • X2 is P and X3 is T.
  • X2 is F and X3 is T.
  • X2 is Y and X3 is S.
  • X2 is P and X3 is S.
  • X2 is F and X3 is S.
  • X1 is D in the complementarity determining region CDR-VL1.
  • X1 is E in the complementarity determining region CDR-VL1.
  • X2 is 1 in the complementarity determining region CDR-VL2.
  • X2 is L in the complementarity determining region CDR-VL2.
  • X1 is N and X2 is I.
  • X1 is H and X2 is I.
  • X1 is N and X2 is L.
  • X1 is H and X2 is L.
  • 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 (for example, 3 CDRs for the heavy chain, or 3 CDRs for the light chain); or, the binding protein includes at least 6 CDRs.
  • the binding protein is a complete antibody comprising variable and constant regions.
  • the binding protein is a "functional fragment" of an antibody, such as Nanobody, F (ab ') 2 , Fab', Fab, Fv, scFv, bispecific antibody, and antibody minimum recognition unit One of them.
  • 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 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 murine
  • 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 relates to an isolated nucleic acid molecule, which is DNA or RNA, which encodes a binding protein as described above.
  • the present disclosure also relates to a vector comprising the nucleic acid molecule as described above.
  • the present disclosure also relates to a host cell transformed with the vector as described above.
  • the host cell may be a eukaryotic cell, such as a mammalian cell.
  • the present disclosure also relates to a method of producing the binding protein as described above, the method comprising the following steps:
  • the host cells as described above are cultured in the culture medium and under appropriate culture conditions, and the binding protein thus produced is recovered from the culture medium or from the cultured host cells.
  • the present disclosure also relates to the use of the binding protein as described above in the preparation of a diagnostic agent or kit for diagnosing acute myocardial infarction, acute coronary syndrome, pulmonary infarction, unstable angina, and myocardial damage.
  • the present disclosure also relates to a method for 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 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 relates to a kit including the binding protein as described above.
  • the troponin I antigen is cardiac troponin I antigen.
  • 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.
  • Isolated binding protein comprising an antigen binding domain generally refers to any protein / protein fragment comprising 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”.
  • the "variable region” or “variable domain” of an antibody 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”.
  • the variable domain of the light chain may be referred to as "VL”. These domains are usually the most variable part of an antibody and contain antigen binding sites.
  • the light or heavy chain variable region (VL or VH) is composed of three hypervariable regions called “complementarity determining regions" or "CDRs" and a framework region separating the three complementarity determining regions.
  • the framework and CDR ranges have been precisely defined, for example, in Kabat (see “Sequences of Proteins of Immunological Interests", E. Kabat, etc., US Department of Health and Human Services (USDepartment of Health) and Human Services), (1983)) and Chothia.
  • the framework region of an antibody serves to locate and align CDRs, which are mainly responsible for binding to antigens.
  • backbone region means the region of the antibody variable domain that excludes those defined as CDRs.
  • Each antibody variable domain framework 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 occurring 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 relates to an isolated binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises at least one complementarity determining region selected from the following amino acid sequence; or; the complementarity determining region with the following amino acid sequence has at least 80% sequence identity and affinity with cardiac troponin I K D ⁇ 3.33 ⁇ 10 -8 mol / L;
  • the complementarity determining region CDR-VH1 is A-S-G-F-X1-F-X2-T-F-A-M-S, where,
  • the complementarity determining region CDR-VH1 is G-F-T-X1-S-N-Y-A-M-S, where,
  • X1 is Y or F
  • the complementarity determining region CDR-VH2 is I-S-X1-G-G-S-Y-S-X2-Y-P-D-S-V-K, where,
  • X1 is T or S, X2 is Y or F;
  • the complementarity determining region CDR-VH3 is A-R-X1-D-N-Y-X2-G-S-X3-F-M-D-Y, where,
  • X1 is K or R
  • X2 is Y
  • X3 is T or S
  • the complementarity determining region CDR-VL1 is R-A-S-Q-X1-I-T-X2-Y-L-N, where,
  • X1 is D or E, X2 is Q or N;
  • the complementarity determining region CDR-VL2 is I-Y-Y-X1-S-R-X2-H-S-G-V-S, where,
  • X1 is S or T, X2 is I or L;
  • the complementarity determining region CDR-VL3 is Q-Q-G-X1-T-X2-P-L-T, where,
  • X1 is N or H, and X2 is I or L.
  • 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 K D ⁇ 3.33 ⁇ 10 -8 mol / L with cardiac troponin I, such as 1 ⁇ 10 -9 mol / L, 2 ⁇ 10 -9 mol / L, 3 ⁇ 10 -9 mol / L, 4 ⁇ 10 -9 mol / L, 4.5 ⁇ 10 -9 mol / L, 5 ⁇ 10 -9 mol / L, 6 ⁇ 10 -9 mol / L, 6 ⁇ 10 -9 mol / L L L L L
  • the affinity is measured according to the method in the present specification.
  • X1 is S
  • X1 is R
  • X1 is T.
  • X1 is Y in the complementarity determining region CDR-VH1.
  • X1 is F in the complementarity determining region CDR-VH1.
  • X2 is Y in the complementarity determining region CDR-VH2.
  • X2 is F in the complementarity determining region CDR-VH2.
  • X2 is Y and X3 is T.
  • X2 is P and X3 is T.
  • X2 is F and X3 is T.
  • X2 is Y and X3 is S.
  • X2 is P and X3 is S.
  • X2 is F and X3 is S.
  • X1 is D in the complementarity determining region CDR-VL1.
  • X1 is E in the complementarity determining region CDR-VL1.
  • X2 is 1 in the complementarity determining region CDR-VL2.
  • X2 is L in the complementarity determining region CDR-VL2.
  • X1 is N and X2 is I.
  • X1 is H and X2 is I.
  • X1 is N and X2 is L.
  • X1 is H and X2 is L.
  • 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 (for example, 3 CDRs for the heavy chain, or 3 CDRs for the light chain); or, the binding protein includes at least 6 CDRs.
  • the binding protein is a complete antibody comprising variable and constant regions.
  • the binding protein is a "functional fragment" of an antibody, such as Nanobody, F (ab ') 2 , Fab', Fab, Fv, scFv, bispecific antibody, and antibody minimum recognition unit One of them.
  • the "functional fragment” described in the present disclosure particularly refers to an antibody fragment having the same specificity as cTnI as the parent antibody. In addition to the above functional fragments, it also includes any fragments whose half-life has been increased.
  • antibody fragments of the present disclosure can obtain the above-mentioned functions by methods such as enzymatic digestion (including pepsin or papain) and / or methods of chemically reducing the cleavage of disulfide bonds according to the contents described in the description of the present disclosure. Fragment.
  • Antibody fragments can also be obtained by peptide synthesis by recombinant genetics techniques that are also known to those skilled in the art or by, for example, automatic peptide synthesizers such as those sold by Applied BioSystems.
  • 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 species source of the skeleton region may be human to constitute a humanized antibody.
  • 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 murine
  • 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 relates to an isolated nucleic acid molecule, which is DNA or RNA, which encodes a binding protein as described above.
  • the present disclosure also relates to a vector comprising the nucleic acid molecule as described above.
  • the present disclosure further includes at least one nuclear construct encoding the nucleic acid molecule as described above, such as a plasmid, and further an expression plasmid, and a method for constructing the vector will be introduced in an embodiment of the present application.
  • the present disclosure also relates to a host cell transformed with the vector as described above.
  • the host cell may be a eukaryotic cell, such as a mammalian cell.
  • the host cell is a CHO cell.
  • the present disclosure also relates to a method of producing the binding protein as described above, the method comprising the following steps:
  • the host cells as described above are cultured in the culture medium and under appropriate culture conditions, and the binding protein thus produced is recovered from the culture medium or from the cultured host cells.
  • the present disclosure also relates to the use of the binding protein as described above in the preparation of a diagnostic agent or kit for diagnosing acute myocardial infarction, acute coronary syndrome, pulmonary infarction, unstable angina, and myocardial damage.
  • the present disclosure also relates to a method for 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 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, 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.
  • the present disclosure also relates to a kit including the binding protein as described above.
  • the troponin I antigen is cardiac troponin I antigen.
  • 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.
  • This embodiment provides an exemplary method for preparing a recombinant antibody against human cardiac troponin I.
  • restriction enzyme and Prime DNA polymerase in this example were purchased from Takara;
  • MagExtractor-RNA extraction kit was purchased from TOYOBO; BD SMART TM RACE cDNA Amplification Kit was purchased from Takara;
  • the pMD-18T vector was purchased from Takara;
  • the plasmid extraction kit was purchased from Tiangen Company;
  • 5’RACE upstream primers for amplification of heavy and light chains 5’RACE upstream primers for amplification of heavy and light chains:
  • NUP Nested Universal Primer A
  • RNA from the hybridoma cell line secreting Anti-cTnI 8C4 monoclonal antibody, and use the SMARTER TM RACE cDNA Amplification Kit and the SMARTER II A oligonucleotide and 5'-CDS primer in the first strand for the first strand cDNA synthesis, the first-strand cDNA product obtained as a template for PCR amplification.
  • the light chain genes were amplified with Universal Primer A Mixture (UPM), Nested Universal Primer A (NUP) and mkR primers, and the heavy chain genes were amplified with Universal Primer A Mixture (UPM), Nested Universal Primer A (NUP) and mHR primers Perform amplification.
  • UPM Universal Primer A Mixture
  • NUP Nested Universal Primer A
  • NUP Nested Universal Primer A
  • mHR primers Perform amplification.
  • the primer pair of the light chain amplifies the target band of about 0.8KB
  • the primer pair of the heavy chain amplifies the target band of about 1.4KB.
  • the product was purified and recovered by agarose gel electrophoresis. The product was added with rTaq DNA polymerase and then inserted into pMD-18T vector to transform into DH5 ⁇ competent cells. After the growth of bacteria, the heavy chain and light chain genes were cloned respectively. Four clones were sent to Invitrogen for sequencing.
  • VL gene sequence is 342bp, which belongs to the VkII gene family, and there is a 57bp leader peptide sequence in front of it; in the gene fragment amplified by the heavy chain primer pair, the VH gene sequence is 357bp, which belongs to the VH1 gene family, which has 57bp in front Leader sequence.
  • the vector is a constructed recombinant antibody eukaryotic expression vector.
  • This expression vector has been introduced into polyclonal restriction sites such as HindIII, BamHI, EcoRI, etc., and is named pcDNA3.4A expression vector, subsequently referred to as 3.4A expression vector;
  • the VL and VH gene-specific primers of the CTNI 8C4 antibody were designed with HindIII, EcoRI cleavage sites and protective bases at both ends.
  • the primers are as follows:
  • C8C4-HR 5 ’> CATCGAATTCTTATCATTTACCAGGAGAGTGGGAGA ⁇ 3’;
  • C8C4-LF 5 ’> CATCAAGCTTGCCGCCACCATGAAGTTGCCTGTTAGGCTGTTGG ⁇ 3’;
  • C8C4-LR 5 ’> CAGCGAATTCTTACTAACACTCATTCCTGTTGAAGC ⁇ 3’;
  • the 0.75KB light chain gene fragment and 1.42kb 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.
  • Example 1 The antibody obtained in Example 1 (having light and heavy chains whose sequences are shown in SEQ ID NO: 11 and 12) was analyzed, and the complementarity determining region (WT) of the heavy chain:
  • CDR-VH1 is G-F-T-Y (X1) -S-N-Y-A-M-S;
  • CDR-VH2 is I-S-T (X1) -G-G-S-Y-S-Y (X2) -Y-P-D-S-V-K;
  • CDR-VH3 is A-R-K (X1) -D-N-Y-Y (X2) -G-S-T (X3) -F-M-D-Y;
  • CDR-VL1 is R-A-S-Q-D (X1) -I-T-Q (X2) -Y-L-N;
  • CDR-VL2 is I-Y-Y-S (X1) -S-R-I (X2) -H-S-G-V-S;
  • CDR-VL3 is Q-Q-G-N (X1) -T-I (X2) -P-L-T;
  • X1, X2, X3 are mutation sites.
  • the inventors made the above mutations in the CDR sites in WT to obtain antibodies with better activity.
  • mutation 1 has the best activity effect, so mutation 1 is used as the backbone sequence to screen 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 10ug / ml, and the CTNI quality control product recombinant protein (antigen produced by the company) was diluted with PBST: 769.2nmol / ml, 384.6nmol / ml, 192.3nmol / ml, 96.2 nmol / ml, 48.1nmol / ml, 24nmol / ml, 12nmol / ml, 0nmol / ml;
  • 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。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≤3.33×10 -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的重组抗体
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201811180362.8、名称为“一种抗人心肌肌钙蛋白I的重组抗体”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
本公开涉及免疫技术领域，具体而言，涉及一种抗人心肌肌钙蛋白I的重组抗体。
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％的序列同一性且与心肌肌钙蛋白I具有K D≤3.33×10 -8mol/L的亲和力；
互补决定区CDR-VH1为G-F-T-X1-S-N-Y-A-M-S，其中，
X1是Y或F；
互补决定区CDR-VH2为I-S-X1-G-G-S-Y-S-X2-Y-P-D-S-V-K，其中，
X1是T或S、X2是Y或F；
互补决定区CDR-VH3为A-R-X1-D-N-Y-X2-G-S-X3-F-M-D-Y，其中，
X1是K或R，X2是Y、P或F，X3是T或S；
互补决定区CDR-VL1为R-A-S-Q-X1-I-T-X2-Y-L-N，其中，
X1是D或E，X2是Q或N；
互补决定区CDR-VL2为I-Y-Y-X1-S-R-X2-H-S-G-V-S，其中，
X1是S或T，X2是I或L；
互补决定区CDR-VL3为Q-Q-G-X1-T-X2-P-L-T，其中，
X1是N或H，X2是I或L。
一个重要优点在于，所述结合蛋白活性强，与人cTnI蛋白具有很高的亲和力。
在一种或多种实施方式中：
所述互补决定区CDR-VH2中，X1是S；
所述互补决定区CDR-VH3中，X1是R；
所述互补决定区CDR-VL1中，X2是N；
所述互补决定区CDR-VL2中，X1是T。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是Y。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是F。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是Y。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是F。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是Y，X3是T。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是P，X3是T。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是F，X3是T。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是Y，X3是S。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是P，X3是S。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是F，X3是S。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是D。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是E。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VL3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-VL3中，X1是H，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VL3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-VL3中，X1是H，X2是L。
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 PCTCN2019108681-appb-000001
Figure PCTCN2019108681-appb-000002
在一种或多种实施方式中，所述结合蛋白中包括至少3个CDR(例如重链的3个CDR，或者轻链的3个CDR)；或者，所述结合蛋白包括至少6个CDR。
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，所述结合蛋白为抗体的“功能片段”，例如纳米抗体、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。
本公开还涉及一种分离的核酸分子，所述核酸分子为DNA或RNA，其编码如上所述的结合蛋白。
本公开还涉及一种载体，其包含如上所述的核酸分子。
本公开还涉及一种宿主细胞，其被如上所述的载体转化。
所述宿主细胞可以为真核细胞，比如哺乳动物细胞。
本公开还涉及一种生产如上所述的结合蛋白的方法，所述方法包括如下步骤：
在培养基中和合适的培养条件下培养如上所述的宿主细胞，从培养基中或从所培养的宿主细胞中回收如此产生的结合蛋白。
本公开还涉及如上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。
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，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。
根据本公开的一方面，本公开还涉及一种试剂盒，其包括如上所述的结合蛋白。
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。
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。
名词定义
“包含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”此用语包括天然发生的抗体以及非天然发生的抗体，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(VL或VH)由三个称为“互补决定区”或“CDR”的高变区以及将三个互补决定区间隔开的骨架区构成。骨架区和CDR的范围已被精确定义，例如在Kabat(参见《免疫重要的蛋白质的序列》(Sequences of Proteins of Immunological Interest)，E.Kabat等，美国卫生与人类服务部(U.S.Department of Health and Human Services)，(1983))和Chothia中。抗体的骨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构架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存在的所述多肽，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。
本公开的示例性实施方案
本公开涉及一种包含抗原结合结构域的分离的结合蛋白，其中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％的序列同一性且与心肌肌钙蛋白I具有K D≤3.33×10 -8mol/L的亲和力；
互补决定区CDR-VH1为A-S-G-F-X1-F-X2-T-F-A-M-S，其中，
互补决定区CDR-VH1为G-F-T-X1-S-N-Y-A-M-S，其中，
X1是Y或F；
互补决定区CDR-VH2为I-S-X1-G-G-S-Y-S-X2-Y-P-D-S-V-K，其中，
X1是T或S、X2是Y或F；
互补决定区CDR-VH3为A-R-X1-D-N-Y-X2-G-S-X3-F-M-D-Y，其中，
X1是K或R，X2是Y、P或F，X3是T或S；
互补决定区CDR-VL1为R-A-S-Q-X1-I-T-X2-Y-L-N，其中，
X1是D或E，X2是Q或N；
互补决定区CDR-VL2为I-Y-Y-X1-S-R-X2-H-S-G-V-S，其中，
X1是S或T，X2是I或L；
互补决定区CDR-VL3为Q-Q-G-X1-T-X2-P-L-T，其中，
X1是N或H，X2是I或L。
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K D≤3.33×10 -8mol/L，例如1×10 -9mol/L、2×10 -9mol/L、3×10 -9mol/L、4×10 -9mol/L、4.5×10 -9mol/L、5×10 -9mol/L、6×10 -9mol/L、7×10 -9mol/L、8×10 -9mol/L、9×10 -9mol/L、1×10 -10mol/L、3×10 -10mol/L、5×10 -10mol/L、7×10 -10mol/L、或9×10 -10mol/L的亲和力；或者8.30×10 -10mol/L≤K D≤3.33×10 -8mol/L，或KD小于或等于1×10 -9mol/L、2×10 -9mol/L、3×10 -9mol/L、4×10 -9mol/L、4.5×10 -9mol/L、5×10 -9mol/L、6×10 -9mol/L、7×10 -9mol/L、8×10 -9mol/L、9×10 -9mol/L、1×10 -10mol/L、3×10 -10mol/L、5×10 -10mol/L、7×10 -10mol/L、或9×10 -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-VH2中，X1是S；
所述互补决定区CDR-VH3中，X1是R；
所述互补决定区CDR-VL1中，X2是N；
所述互补决定区CDR-VL2中，X1是T。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是Y。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是F。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是Y。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是F。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是Y，X3是T。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是P，X3是T。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是F，X3是T。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是Y，X3是S。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是P，X3是S。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是F，X3是S。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是D。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是E。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VL3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-VL3中，X1是H，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VL3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-VL3中，X1是H，X2是L。
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 PCTCN2019108681-appb-000003
Figure PCTCN2019108681-appb-000004
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CDR(例如重链的3个CDR，或者轻链的3个CDR)；或者，所述结合蛋白包括至少6个CDR。
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，所述结合蛋白为抗体的“功能片段”，例如纳米抗体、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。
scFv(sc＝单链)，双特异抗体(diabodies)。
本公开所述的“功能片段”特别地指对于cTnI具有与母体抗体相同特异性的抗体片段。除上述功能片段外，还包括半衰期已增加的任何片段。
这些功能片段通常具有与其来源抗体相同的结合特异性。本领域技术人员根据本公开说明中记载的内容推断，本公开的抗体片段可以通过比如酶消化的方法(包括胃蛋白酶或木瓜蛋白酶)和/或通过化学还原分裂二硫键的方法获得上述的功能片段。
抗体片段还可以通过也是本领域技术人员所知的重组遗传学技术或通过例如自动肽合成仪，比如Applied BioSystems等销售的自动肽合成仪，通过肽合成获得。
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。
需要说明的是，除本申请上述公开的氨基酸序列外，骨架区的种属来源可以为人，以构成人源化抗体。
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。
本公开还涉及一种分离的核酸分子，所述核酸分子为DNA或RNA，其编码如上所述的结合蛋白。
本公开还涉及一种载体，其包含如上所述的核酸分子。
本公开进一步包含至少一种编码如上所述的核酸分子的核构建体，例如质粒，进一步为表达质粒，在本申请的一个实施例中会介绍该载体的构建方法。
本公开还涉及一种宿主细胞，其被如上所述的载体转化。
所述宿主细胞可以为真核细胞，比如哺乳动物细胞。
在一种或多种实施方式中，所述宿主细胞为CHO细胞。
本公开还涉及一种生产如上所述的结合蛋白的方法，所述方法包括如下步骤：
在培养基中和合适的培养条件下培养如上所述的宿主细胞，从培养基中或从所培养的宿主细胞中回收如此产生的结合蛋白。
本公开还涉及如上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。
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，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。
根据本公开的一方面，本公开还涉及一种试剂盒，其包括如上所述的结合蛋白。
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。
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。
下面将结合实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本公开，而不应视为限制本公开的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
本实施例提供了一种抗人心肌肌钙蛋白I的重组抗体的示例性制备方法。
S10，构建表达质粒：
其中，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；BD SMART TM 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；
S11，引物的设计与合成：
扩增重链和轻链的5’RACE上游引物：
扩增重链和轻链5’RACE引物：
SMARTER II A寡核苷酸：
5’>AAGCAGTGGTATCAACGCAGAGTACXXXXX<3’；
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’
扩增轻链基因下游引物：
mkR：
5’>TTTTCCTTTTGAATTCCTAACACTCATTCCTGTTGAAGC<3’。
扩增重链基因的下游引物：
mHR：
5’>TTTTCCTTTTGAATTCTCATTTACCAGGAGAGTGGGAGA<3’。
S12，抗体可变区基因克隆及测序：
从分泌Anti-cTnI 8C4单克隆抗体的杂交瘤细胞株中提取中RNA，用SMARTER TM 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mkR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mHR引物进行扩增。其中轻链的引物对扩增出0.8KB左右的目的条带，重链的引物对扩增出1.4KB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反应后插入到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。
S13，CTNI 8C4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342bp，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 357bp，属于VH1基因家族，其前方有57bp的前导肽序列。
S14，重组抗体表达质粒的构建：
pcDNA TM 3.4
Figure PCTCN2019108681-appb-000005
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pcDNA3.4A表达载体，后续简称3.4A表达载体；根据上述pMD-18T中抗体可变区基因测序结果，设计CTNI 8C4抗体的VL和VH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：
C8C4-HF：
5’>CAGCAAGCTTGCCGCCACCATGGAATGCAGCTGTGTCATGCTCTTCTTC<3’；
C8C4-HR:5’>CATCGAATTCTTATCATTTACCAGGAGAGTGGGAGA<3’；
C8C4-LF:5’>CATCAAGCTTGCCGCCACCATGAAGTTGCCTGTTAGGCTGTTGG<3’；
C8C4-LR:5’>CAGCGAATTCTTACTAACACTCATTCCTGTTGAAGC<3’；
通过PCR扩增方法扩出0.75KB的轻链基因片段和1.42kb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。
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，100ul质粒与700ul细胞混合，转入电转杯，电转，转入10ml含CD CHO AGT培养基中，37度摇床中培养(8％CO 2、震幅150)；每天取样检测细胞活率，当细胞活率低于50％，离心细胞培养上清。
用proteinA亲和层析柱进行亲和纯化。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28KD的轻链(序列如SEQ ID NO:11所示)，另一条为50KD的重链(序列如SEQ ID NO:12所示)。
实施例2
实施例1得到的抗体(具有序列如SEQ ID NO:11以及12所示的轻链和重链)经分析，重链的互补决定区(WT)：
CDR-VH1为G-F-T-Y(X1)-S-N-Y-A-M-S；
CDR-VH2为I-S-T(X1)-G-G-S-Y-S-Y(X2)-Y-P-D-S-V-K；
CDR-VH3为A-R-K(X1)-D-N-Y-Y(X2)-G-S-T(X3)-F-M-D-Y；
轻链的互补决定区：
CDR-VL1为R-A-S-Q-D(X1)-I-T-Q(X2)-Y-L-N；
CDR-VL2为I-Y-Y-S(X1)-S-R-I(X2)-H-S-G-V-S；
CDR-VL3为Q-Q-G-N(X1)-T-I(X2)-P-L-T；
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
位点 CDR-VH2 CDR-VH3 CDR-VL1 CDR-VL2
  X1 X1 X2 X1
WT T K Q S
突变1 S R N T
突变2 T R Q T
突变3 S K N S
突变4 S K Q S
突变5 T R Q S
发明人将WT中的CDR位点进行上述突变，以获得活性更好的抗体。
包被液稀释CTNI质控品重组抗原到1ug/ml进行微孔板包被，每孔100uL，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120uL，37℃，1h，拍干；加入稀释后的cTnI单克隆抗体，100uL/孔，37℃,30min；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100uL，37℃,30min；洗涤液清洗5次，拍干；加入显色液A液(50uL/孔)，加入显色液B液(50uL/孔)，10min；加入终止液，50uL/孔；酶标仪上450nm(参考630nm)处读OD值。
表2抗体活性分析数据
Figure PCTCN2019108681-appb-000006
从表2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 PCTCN2019108681-appb-000007
Figure PCTCN2019108681-appb-000008
亲和力分析
利用AMC传感器，纯化出来的抗体用PBST稀释到10ug/ml，CTNI质控品重组蛋白(公司自产抗原)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。(K D表示平衡解亲常数既亲和力；Kon表示结合速率；kdis表示解离速率。)
表4亲和力分析数据
位点 K D(M) K ON(1/Ms) kdis(1/s)
突变1 7.04E-09 8.28E+04 5.83E-04
突变1-1 3.47E-09 3.58E+04 1.24E-04
突变1-2 2.43E-09 5.32E+04 1.29E-04
突变1-3 4.58E-09 1.25E+04 5.73E-05
突变1-4 7.10E-09 7.46E+04 5.30E-04
突变1-5 8.30E-10 9.72E+05 8.07E-04
突变1-6 9.81E-09 2.80E+04 2.75E-04
突变1-7 4.71E-09 3.94E+04 1.86E-04
突变1-8 6.81E-09 9.80E+04 6.67E-04
突变1-9 8.60E-09 2.14E+04 1.84E-04
突变1-10 5.05E-09 6.85E+04 3.46E-04
突变1-11 4.40E-09 1.08E+04 4.75E-05
突变1-12 1.52E-09 8.86E+04 1.35E-04
突变1-13 4.12E-09 8.60E+04 3.54E-04
突变1-14 7.76E-09 6.42E+04 4.98E-04
突变1-15 3.80E-09 3.81E+04 1.45E-04
突变1-16 9.72E-10 8.57E+05 8.33E-04
突变1-17 9.22E-09 3.64E+04 3.36E-04
突变1-18 4.40E-09 5.75E+04 2.53E-04
突变1-19 8.19E-09 9.43E+04 7.72E-04
突变1-20 2.87E-09 5.98E+04 1.72E-04
突变1-21 9.55E-09 6.62E+04 6.32E-04
突变1-22 9.07E-09 3.44E+04 3.12E-04
突变1-23 4.31E-09 7.79E+04 3.36E-04
突变1-24 5.37E-09 4.61E+04 2.48E-04
突变1-25 5.36E-09 6.79E+04 3.64E-04
突变1-26 1.38E-09 2.52E+04 3.48E-05
突变1-27 6.30E-09 7.96E+04 5.01E-04
突变1-28 3.68E-09 1.17E+04 4.31E-05
突变1-29 7.59E-09 2.60E+04 1.97E-04
突变1-30 5.76E-09 2.83E+04 1.63E-04
突变1-31 8.73E-09 8.52E+04 7.44E-04
突变1-32 8.38E-09 3.17E+04 2.66E-04
突变1-33 9.79E-10 5.32E+05 5.21E-04
突变1-34 8.44E-09 3.02E+04 2.55E-04
突变1-35 5.25E-09 3.07E+04 1.61E-04
突变1-36 9.27E-09 8.35E+04 7.74E-04
突变1-37 4.20E-09 7.10E+04 2.99E-04
突变1-38 7.74E-09 7.37E+04 5.70E-04
突变1-39 6.25E-09 8.14E+04 5.09E-04
突变1-40 3.12E-09 8.39E+04 2.62E-04
突变1-41 9.24E-09 8.38E+04 7.74E-04
突变1-42 3.09E-09 3.52E+04 1.09E-04
突变1-43 5.37E-09 4.61E+04 2.48E-04
突变1-44 3.53E-09 4.85E+04 1.71E-04
突变1-45 9.02E-09 2.11E+04 1.90E-04
突变1-46 4.93E-09 5.56E+04 2.74E-04
突变1-47 2.87E-09 3.30E+04 9.47E-05
突变1-48 7.04E-09 8.28E+04 5.83E-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 PCTCN2019108681-appb-000009
表6亲和力分析数据
位点 K D(M) K ON(1/Ms) kdis(1/s)
WT 2.16E-08 7.83E+03 1.69E-04
WT 1-1 3.33E-08 3.84E+03 1.28E-04
WT 1-5 8.35E-09 8.90E+04 7.43E-04
WT 1-10 4.89E-09 2.65E+04 1.30E-04
WT 1-20 9.39E-09 2.41E+04 2.26E-04
从表5和表6分析，在保证具有抗体活性的前提下，上述突变位点也具有一定的亲和力。
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，但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，其中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％的序列同一性且与心肌肌钙蛋白I具有K D≤3.33×10 -8mol/L的亲和力；
    互补决定区CDR-VH1为G-F-T-X1-S-N-Y-A-M-S，其中，
    X1是Y或F；
    互补决定区CDR-VH2为I-S-X1-G-G-S-Y-S-X2-Y-P-D-S-V-K，其中，
    X1是T或S、X2是Y或F；
    互补决定区CDR-VH3为A-R-X1-D-N-Y-X2-G-S-X3-F-M-D-Y，其中，
    X1是K或R，X2是Y、P或F，X3是T或S；
    互补决定区CDR-VL1为R-A-S-Q-X1-I-T-X2-Y-L-N，其中，
    X1是D或E，X2是Q或N；
    互补决定区CDR-VL2为I-Y-Y-X1-S-R-X2-H-S-G-V-S，其中，
    X1是S或T，X2是I或L；
    互补决定区CDR-VL3为Q-Q-G-X1-T-X2-P-L-T，其中，
    X1是N或H，X2是I或L；
    优选的：
    所述互补决定区CDR-VH2中，X1是S；
    所述互补决定区CDR-VH3中，X1是R；
    所述互补决定区CDR-VL1中，X2是N；
    所述互补决定区CDR-VL2中，X1是T；
    优选的，所述互补决定区CDR-VH1中，X1是Y；
    优选的，所述互补决定区CDR-VH1中，X1是F；
    优选的，所述互补决定区CDR-VH2中，X2是Y；
    优选的，所述互补决定区CDR-VH2中，X2是F；
    优选的，所述互补决定区CDR-VH3中，X2是Y，X3是T；
    优选的，所述互补决定区CDR-VH3中，X2是P，X3是T；
    优选的，所述互补决定区CDR-VH3中，X2是F，X3是T；
    优选的，所述互补决定区CDR-VH3中，X2是Y，X3是S；
    优选的，所述互补决定区CDR-VH3中，X2是P，X3是S；
    优选的，所述互补决定区CDR-VH3中，X2是F，X3是S；
    优选的，所述互补决定区CDR-VL1中，X1是D；
    优选的，所述互补决定区CDR-VL1中，X1是E；
    优选的，所述互补决定区CDR-VL2中，X2是I；
    优选的，所述互补决定区CDR-VL2中，X2是L；
    优选的，所述互补决定区CDR-VL3中，X1是N，X2是I；
    优选的，所述互补决定区CDR-VL3中，X1是H，X2是I；
    优选的，所述互补决定区CDR-VL3中，X1是N，X2是L；
    优选的，所述互补决定区CDR-VL3中，X1是H，X2是L；
    优选的，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 PCTCN2019108681-appb-100001
    Figure PCTCN2019108681-appb-100002
  2. 如权利要求1所述包含抗原结合结构域的分离的结合蛋白，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；
    优选的，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；
    优选的，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。
  3. 如权利要求2所述包含抗原结合结构域的分离的结合蛋白，其特征在于，所述结合蛋白还包含抗体恒定区序列；
    优选的，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；
    优选的，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；
    优选的，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。
  4. 一种分离的核酸分子，其特征在于，所述核酸分子为DNA或RNA，其编码权利要求1～3任一项所述的结合蛋白。
  5. 一种载体，其包含权利要求4所述的核酸分子。
  6. 一种宿主细胞，其被权利要求5所述的载体转化。
  7. 一种生产权利要求1～3任一项所述的结合蛋白的方法，其特征在于，包括如下步骤：
    在培养基中和合适的培养条件下培养权利要求6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如此产生的结合蛋白。
  8. 权利要求1～3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。
  9. 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，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；
    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。
  10. 如权利要求9所述的方法，其中所述肌钙蛋白I抗原为心肌肌钙蛋白I抗原。
  11. 一种试剂盒，其包括权利要求1～3任一项所述的结合蛋白。
  12. 如权利要求1～3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1～3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。
PCT/CN2019/108681 2018-10-10 2019-09-27 一种抗人心肌肌钙蛋白i的重组抗体 WO2020073831A1 (zh)

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