WO2020125136A1 - 一种抗人n末端脑钠肽前体的重组抗体 - Google Patents

一种抗人n末端脑钠肽前体的重组抗体 Download PDF

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WO2020125136A1
WO2020125136A1 PCT/CN2019/109792 CN2019109792W WO2020125136A1 WO 2020125136 A1 WO2020125136 A1 WO 2020125136A1 CN 2019109792 W CN2019109792 W CN 2019109792W WO 2020125136 A1 WO2020125136 A1 WO 2020125136A1
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complementarity determining
determining region
region cdr
binding protein
antibody
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PCT/CN2019/109792
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English (en)
French (fr)
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孟媛
钟冬梅
叶庆妮
梁碧
游辉
马秋燕
李蔚芝
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东莞市朋志生物科技有限公司
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Priority to EP19900628.9A priority Critical patent/EP3901170A4/en
Priority to JP2021526431A priority patent/JP7339338B2/ja
Priority to US17/293,044 priority patent/US20220025008A1/en
Priority to KR1020217014265A priority patent/KR20210114922A/ko
Publication of WO2020125136A1 publication Critical patent/WO2020125136A1/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
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
    • 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
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • 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
    • G01N2333/575—Hormones
    • G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
    • 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 N-terminal brain natriuretic peptide precursor.
  • BNP Brain Natriuretic Peptide
  • proBNP When myocardial cells are stimulated, proBNP is cleaved into 76 amino acids by biological enzymes, without biological
  • the active N-terminal pro-B-type natriuretic peptide (NT-proBNP) and the active B-type natriuretic peptide (BNP) containing 32 amino acids are of the same origin and released into the blood circulation by equimolar secretion.
  • NT-proBNP N-terminal brain natriuretic peptide precursor
  • BNP index concentrations will increase abnormally.
  • NT-proBNP has better biological stability than BNP and a longer half-life ( 120min), the concentration is relatively stable, the effective detection time is long, the blood content is about 16-20 times higher than that of BNP, so the detection is relatively easy, and the stability of plasma samples in vitro is long (>48h), which is a diagnosis of heart failure and The best myocardial marker for evaluating cardiac function.
  • NT-proBNP The content of NT-proBNP in the blood of normal people is generally less than 0.3ng/mL.
  • NT-proBNP When heart function is impaired and myocardium dilates, NT-proBNP will be quickly synthesized and released into human blood in large amounts.
  • accurate, sensitive, efficient and stable determination of the amount of NT-proBNP in the blood can provide treatment for cardiogenic and non-cardiac heart failure in early cardiac dysfunction, heart failure, dyspnea and Prognostic monitoring and the classification of acute coronary syndrome provide a fast and accurate basis for early diagnosis.
  • the methods used to detect the content of NT-proBNP mainly include gold calibration test, fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA) and magnetic particle chemiluminescence method (CMIA), but these measurement methods need to be targeted to NT-proBNP
  • ELISA enzyme-linked immunosorbent assay
  • CMIA magnetic particle chemiluminescence method
  • the present disclosure relates to a novel isolated binding protein containing an N-terminal brain natriuretic peptide precursor (NT-proBNP) antigen binding domain, and studies on the preparation, application, etc. of the binding protein.
  • NT-proBNP N-terminal brain natriuretic peptide precursor
  • the antigen binding domain includes at least one complementarity determining region selected from the following amino acid sequence: OR; at least 80% sequence identity with the complementarity determining region of the following amino acid sequence and K D ⁇ 2.26 with NT-proBNP ⁇ 10 -8 affinity;
  • the complementarity determining region CDR-VH1 is G-X1-S-X2-T-T-Y-Y-X3-D, where,
  • X1 is P or F
  • X2 is I, V or L
  • X3 is I, V or L;
  • the complementarity determining region CDR-VH2 is M-T-K-D-X1-N-A-V-H-X2-P-T-X3-R-S, where,
  • X1 is G or A, X2 is Q or N, X3 is I, V or L;
  • the complementarity determining region CDR-VH3 is V-X1-G-X2-I-D-X3-G, where,
  • X1 is K or R
  • X2 is I, V or L
  • X3 is F or W
  • the complementarity determining region CDR-VL1 is G-S-S-D-X1-V-G-X2-G-D-Y-X3-N, where,
  • X1 is Q or N
  • X2 is F or P
  • X3 is I, V or L;
  • the complementarity determining region CDR-VL2 is I-F-X1-A-X2-S-R-X3-R-G, where,
  • X1 is A or G
  • X2 is T
  • X3 is I, V or L;
  • the complementarity determining region CDR-VL3 is G-S-X1-N-S-R-X2-Y-V-X3-G, where,
  • X1 is P, A or G, X2 is GG or N, and X3 is W or F.
  • binding protein has strong activity and has a high affinity with human NT-proBNP.
  • X1 is F
  • X1 is G
  • X1 is R
  • X2 is F
  • X1 is G
  • X3 is F.
  • X2 is 1 in the complementarity determining region CDR-VH1.
  • X2 is V in the complementarity determining region CDR-VH1.
  • X2 is L in the complementarity determining region CDR-VH1.
  • X3 is I in the complementarity determining region CDR-VH1.
  • X3 is V in the complementarity determining region CDR-VH1.
  • X3 is L in the complementarity determining region CDR-VH1.
  • X2 is Q in the complementarity determining region CDR-VH2.
  • X2 is N in the complementarity determining region CDR-VH2.
  • X3 is I in the complementarity determining region CDR-VH2.
  • X3 is V in the complementarity determining region CDR-VH2.
  • X3 is L in the complementarity determining region CDR-VH2.
  • X2 is I in the complementarity determining region CDR-VH3.
  • X2 is V in the complementarity determining region CDR-VH3.
  • X2 is L in the complementarity determining region CDR-VH3.
  • X3 is F in the complementarity determining region CDR-VH3.
  • X3 is W in the complementarity determining region CDR-VH3.
  • X1 is Q in the complementarity determining region CDR-VL1.
  • X1 is N in the complementarity determining region CDR-VL1.
  • X3 is I in the complementarity determining region CDR-V L1.
  • X3 is V in the complementarity determining region CDR-V L1.
  • X3 is L in the complementarity determining region CDR-V L1.
  • X2 is T in the complementarity determining region CDR-VL2.
  • X2 is Y.
  • X2 is S in the complementarity determining region CDR-VL2.
  • X3 is I in the complementarity determining region CDR-VL2.
  • X3 is V in the complementarity determining region CDR-VL2.
  • X3 is L in the complementarity determining region CDR-VL2.
  • X1 is P in the complementarity determining region CDR-VL3.
  • X1 is A in the complementarity determining region CDR-VL3.
  • X1 is G in the complementarity determining region CDR-VL3.
  • X2 is GG.
  • 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 (for example, 3 light chain CDRs or 3 heavy chain 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 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, dairy 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 sheep
  • 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 that encodes the 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 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 for diagnosing heart failure and evaluating cardiac function.
  • the present disclosure also relates to a method for detecting NT-proBNP in a test sample, which includes:
  • 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 NT-proBNP antigen;
  • the present disclosure also relates to a kit including the binding protein as described above.
  • the present disclosure also relates to the use of the binding proteins described herein in the diagnosis of diseases associated with NT-proBNP.
  • the present disclosure also relates to a method for diagnosing NT-proBNP-related diseases or evaluating cardiac function, including:
  • the presence of the immune complex indicates the presence of NT-proBNP-related diseases or the level of cardiac function.
  • 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, preferably a primate, and more preferably a human.
  • the disease associated with NT-proBNP is heart disease.
  • the disease associated with NT-proBNP is selected from the group consisting of heart failure, cardiac insufficiency, cardiogenic dyspnea, pulmonary dyspnea, acute coronary syndrome, or a combination thereof group.
  • the heart failure is cardiogenic heart failure or non-cardiac heart failure.
  • FIG. 1 is an electrophoresis diagram of monoclonal antibodies against human NT-proBNP in an embodiment of the present disclosure.
  • 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 chain or heavy chain variable region (VL or VH) is composed of three framework regions called “complementarity determining regions" or "CDRs" and 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 a region other than those regions of the antibody variable domain that are defined as CDRs.
  • Each antibody variable domain framework region 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 following 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. Therefore, the term “isolated” includes polypeptides or nucleic acids taken from the original environment, for example, if it is naturally occurring. For example, 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); Needleman's algorithm; or Meyers' algorithm. In one or more embodiments, 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. In one or more embodiments, 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.
  • BLAST Basic Local Alignment Search Tool
  • Needleman's algorithm or Meyers' algorithm.
  • 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
  • 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 includes at least one complementarity determining region selected from the following amino acid sequence: OR; and the complementarity determining region with the following amino acid sequence has at least 80% sequence identity and K D ⁇ 2.26 ⁇ 10 -8 affinity with NT-proBNP;
  • the complementarity determining region CDR-VH1 is G-X1-S-X2-T-T-Y-Y-X3-D, where,
  • X1 is P or F
  • X2 is I, V or L
  • X3 is I, V or L;
  • the complementarity determining region CDR-VH2 is M-T-K-D-X1-N-A-V-H-X2-P-T-X3-R-S, where,
  • X1 is G or A, X2 is Q or N, X3 is I, V or L;
  • the complementarity determining region CDR-VH3 is V-X1-G-X2-I-D-X3-G, where,
  • X1 is K or R
  • X2 is I, V or L
  • X3 is F or W
  • the complementarity determining region CDR-VL1 is G-S-S-D-X1-V-G-X2-G-D-Y-X3-N, where,
  • X1 is Q or N
  • X2 is F or P
  • X3 is I, V or L;
  • the complementarity determining region CDR-VL2 is I-F-X1-A-X2-S-R-X3-R-G, where,
  • X1 is A or G
  • X2 is T
  • X3 is I, V or L;
  • the complementarity determining region CDR-VL3 is G-S-X1-N-S-R-X2-Y-V-X3-G, where,
  • X1 is P, A or G, X2 is GG or N, and X3 is W or F.
  • N-terminal brain natriuretic peptide precursor NT-proBNP
  • N-terminal pro-B-type natriuretic peptide refer to a precursor of brain natriuretic peptide or B-type natriuretic peptide in digestion Thereafter, for example, after digestion with an endonuclease, the biologically inactive N-terminal fragment is produced.
  • the antigen binding domain and the complementarity determining region of the amino acid sequence described below have 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 K D ⁇ 2.26 ⁇ 10 -8 mol/L with NT-proBNP, K D value can also be selected 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, 7 ⁇ 10 -9 mol
  • KD is less than or equal to 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, 7 ⁇ 10 -9 mol/L, 8 ⁇ 10 -9 mol/L, 9 ⁇ 10 -9 mol/L, 1 ⁇ 10 -10 mol/L, 3 ⁇ 10 -10 mol/L, 5 ⁇ 10 -10 mol/L, 7 ⁇ 10 -10 mol/L, 9 ⁇ 10 -10 mol/L, 1 ⁇ 10 -8 mol/L;
  • the affinity is measured according to the method in the present specification.
  • X1 is F
  • X1 is G
  • X1 is R
  • X2 is F
  • X1 is G
  • X3 is F.
  • X2 is 1 in the complementarity determining region CDR-VH1.
  • X2 is V in the complementarity determining region CDR-VH1.
  • X2 is L in the complementarity determining region CDR-VH1.
  • X3 is I in the complementarity determining region CDR-VH1.
  • X3 is V in the complementarity determining region CDR-VH1.
  • X3 is L in the complementarity determining region CDR-VH1.
  • X2 is Q in the complementarity determining region CDR-VH2.
  • X2 is N in the complementarity determining region CDR-VH2.
  • X3 is I in the complementarity determining region CDR-VH2.
  • X3 is V in the complementarity determining region CDR-VH2.
  • X3 is L in the complementarity determining region CDR-VH2.
  • X2 is I in the complementarity determining region CDR-VH3.
  • X2 is V in the complementarity determining region CDR-VH3.
  • X2 is L in the complementarity determining region CDR-VH3.
  • X3 is F in the complementarity determining region CDR-VH3.
  • X3 is W in the complementarity determining region CDR-VH3.
  • X1 is Q in the complementarity determining region CDR-VL1.
  • X1 is N in the complementarity determining region CDR-VL1.
  • X3 is I in the complementarity determining region CDR-V L1.
  • X3 is V in the complementarity determining region CDR-V L1.
  • X3 is L in the complementarity determining region CDR-V L1.
  • X2 is T in the complementarity determining region CDR-VL2.
  • X2 is Y.
  • X2 is S in the complementarity determining region CDR-VL2.
  • X3 is I in the complementarity determining region CDR-VL2.
  • X3 is V in the complementarity determining region CDR-VL2.
  • X3 is L in the complementarity determining region CDR-VL2.
  • X1 is P in the complementarity determining region CDR-VL3.
  • X1 is A in the complementarity determining region CDR-VL3.
  • X1 is G in the complementarity determining region CDR-VL3.
  • X2 is GG.
  • 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 represent the amino acids 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 acid 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 acid defined in the present disclosure.
  • the binding protein includes at least 3 CDRs (eg, 3 light chain CDRs or 3 heavy chain 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 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 NT-proBNP as the parent antibody. In addition to the above functional fragments, it also includes any fragments whose half-life has been increased.
  • Antibody fragments can also be obtained by peptide synthesis through recombinant genetics techniques that are also known to those skilled in the art or through, 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, dairy 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 sheep
  • 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 a 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 for diagnosing heart failure and evaluating cardiac function.
  • the cancer includes acute lymphoblastic leukemia, acute myelogenous leukemia, cholangiocarcinoma, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon Rectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, kidney cancer, ovarian cancer , Pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer and urinary bladder cancer.
  • the present disclosure also relates to a method for detecting NT-proBNP in a test sample, which includes:
  • the binding protein may be labeled with an indicator that shows signal intensity to make the complex easy to detect.
  • 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 NT-proBNP;
  • the binding protein forms a paired antibody with the second antibody in the form of a first antibody for binding to different epitopes of NT-proBNP;
  • the second antibody may be labeled with an indicator showing signal intensity, so that the complex is easily detected.
  • the immune complex further includes a second antibody, and the second antibody binds to the NT-proBNP 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 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-1,3-diazo
  • 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 present disclosure also relates to the use of the binding proteins described herein in the diagnosis of diseases associated with NT-proBNP.
  • NT-proBNP refers to a disease in which NT-proBNP, including its protein or encoding nucleic acid, is used as a marker.
  • a disease associated with NT-proBNP may refer to a disease characterized by an increase in NT-proBNP levels in blood, such as whole blood, plasma, or serum.
  • the present disclosure also relates to a method for diagnosing NT-proBNP-related diseases or evaluating cardiac function, including:
  • the presence of the immune complex indicates the presence of NT-proBNP-related diseases or the level of cardiac function.
  • 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, preferably a primate, and more preferably a human.
  • the disease associated with NT-proBNP is heart disease.
  • the disease associated with NT-proBNP is selected from the group consisting of heart failure, cardiac insufficiency, cardiogenic dyspnea, pulmonary dyspnea, acute coronary syndrome, or a combination thereof group.
  • the heart failure is cardiogenic heart failure or non-cardiac heart failure.
  • This example provides an exemplary method for preparing a recombinant antibody against human NT-proBNP.
  • restriction enzyme and Prime DNA polymerase were purchased from Takara;
  • 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;
  • the anti-NT-proBNP 8B9 monoclonal antibody is secreted as an existing hybridoma cell line, and is used for recovery.
  • NUP Nested Universal Primer A
  • RNA from the hybridoma cell line secreting Anti-NT-proBNP 8B9 monoclonal antibody and use the SMARTERTM RACE cDNA Amplification Kit kit and the SMARTER II oligonucleotide and 5'-CDS primers in the kit for the first Strand cDNA synthesis, the first strand cDNA product obtained as a PCR amplification template.
  • Light chain genes are amplified with Universal Primer A Mixture (UPM), Nested Universal Primer A (NUP) and mkR
  • Heavy chain genes are amplified with Universal Primer A Mixture (UPM), Nested Universal Primer A (NUP) and mHR increase.
  • 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 402bp, which belongs to the VkII gene family, and there is a 60bp leader peptide sequence in front of it; in the gene fragment amplified by the heavy chain primer pair, the VH gene sequence is 402bp, 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 pcDNA3.4A expression vector, subsequently referred to as 3.4A expression vector; according to the above pMD-18T
  • anti-NT-proBNP 8B9 antibody VL and VH gene-specific primers were designed with HindIII and EcoRI cleavage sites and protective bases at both ends.
  • the primers are as follows:
  • the 0.7KB light chain gene fragment and the 1.4kb 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.
  • S21 recombinant antibody expression plasmid was transiently transfected into CHO cells to determine the activity of the expression plasmid
  • plasmid Dilute the plasmid to 400ng/ml with ultrapure water, adjust the CHO cells to 1.43 ⁇ 10 7 cells/ml in a centrifuge tube, mix 100ul of plasmid with 700ul of cells, transfer it to an electric rotating cup, and transfer it to electricity. Samples are counted on days 3, 5, and 7. Sample collection and detection on the 7th day.
  • reaction OD was still greater than 1.0 after the cell supernatant was diluted 1000 times, and the reaction OD was less than 0.1 without the cell supernatant, indicating that the antibody produced after the plasmid transient was active against recombinant NT protein.
  • Buffer 50ul DNA 100ug/tube, 10ul of PuvI enzyme, make up to 500ul with sterile water, digest at 37°C in water bath overnight; first use equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1 , followeded by extraction with chloroform (aqueous phase) in sequence; 0.1 volume (aqueous phase) of 3M sodium acetate and 2 volumes of ethanol precipitated on ice, 70% ethanol rinse the precipitate, remove the organic solvent, after the ethanol has evaporated completely with a proper amount of The bacteria water was reconstituted, and finally the concentration was measured.
  • S23 recombinant antibody expression plasmid was stably transfected, and stable cell lines were screened under pressure
  • 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.
  • Example 1 The antibody obtained in Example 1 was analyzed to have a light chain with the sequence shown in SEQ ID NO: 11 and a heavy chain with 12 shown.
  • the complementary determination region (WT) of the heavy chain After analysis, the complementary determination region (WT) of the heavy chain:
  • CDR-VH1 is G-P(X1)-S-I(X2)-T-T-Y-Y-I(X3)-D;
  • CDR-VH2 is M-T-K-D-A(X1)-N-A-V-H-Q(X2)-P-T-I(X3)-R-S;
  • CDR-VH3 is V-K(X1)-G-I(X2)-I-D-W(X3)-G;
  • CDR-VL1 is G-S-S-D-Q(X1)-V-G-P(X2)-G-D-Y-V(X3)-N;
  • CDR-VL2 is I-F-A(X1)-A-T(X2)-S-R-I(X3)-R-G;
  • CDR-VL3 is G-S-P(X1)-N-S-R-GG(X2)-Y-V-W(X3)-G;
  • X1, X2, and X3 are all sites to be mutated.
  • the inventors made the above mutations in the CDR sites in WT to obtain antibodies with better activity.
  • NT antigen Dilute the recombinant NT antigen to 1ug/ml in the coating solution for microplate coating, 100uL per well at 4°C overnight; the next day, wash the solution twice and pat dry; add blocking solution (20% BSA+80% PBS) , 120uL per well, 37°C, 1h, pat dry; add diluted NT monoclonal antibody, 100uL/well, 37°C, 30min (partial supernatant 1h); wash solution 5 times, pat dry; add rabbit anti-sheep -HRP, 100uL per well, 37°C, 30min; wash solution 5 times, pat dry; add solution A (50uL/well), add solution B (50uL/well), 10min; add stop solution , 50uL/well; read the OD value at 450nm (reference 630nm) on the microplate reader.
  • 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.
  • Enzyme-free indirect method is used to do data in the same way as activity identification, and the package is made into four gradients 3ug/ml, 1.5ug/ml, 0.75ug/ml, 0.375ug/ml; the antibody is diluted 2-fold to 0.977ng from 1000ng/ml /ml loading. The OD values corresponding to different antibody concentrations at different coating concentrations are obtained.
  • the applicant verified the above antibody in Table 4 with another internal antibody (antibody paired with the original WT sequence antibody) by paired antibody experiment verification.
  • the double antibody sandwich method paired experiment verified that the specificity maintained the original high level, but Due to the increased activity and affinity of the mutant antibody, a higher sensitivity is exhibited.
  • the mutation 1 antibody and the WT antibody were used as coating antibodies, and they were used in conjunction with another NT-oroBNP antibody as a label.
  • the performance difference was compared in the chemiluminescence evaluation table. The specific performance is shown in the table below:
  • the binding protein described in the present disclosure has strong activity and has a high affinity with human NT-proBNP.
  • the binding protein described in the present disclosure can be used to diagnose diseases related to NT-proBNP, such as heart failure, and can be used to evaluate cardiac function, with high sensitivity and specificity.

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Abstract

提供一种包含NT-proBNP抗原结合结构域的分离的结合蛋白及其制备方法，所述抗原结合结构域包括选自限定的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NT-proBNP具有K D≤2.26×10 -8的亲和力，可应用于NT-proBNP蛋白的检测领域。

Description

一种抗人N末端脑钠肽前体的重组抗体
相关申请的交叉引用
本申请要求于2018年12月19日提交中国专利局的申请号为201811557468.5、名称为“一种抗人N末端脑钠肽前体的重组抗体”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N末端脑钠肽前体的重组抗体。
背景技术
1988年日本学者Sudoh首次从猪脑内分离得到一种具有强有力的利尿、扩血管和降压作用的多肽，将其命名为脑钠肽(Brain Natriuretic Peptide，BNP)。BNP分布在心脏含量最高，但心肌细胞首先合成的是含有108个氨基酸的proBNP(BNP前体)，当心肌细胞受到刺激后，proBNP在内切酶的作用下裂解为含有76个氨基酸、无生物活性的N末端B型利钠肽原(NT-proBNP)和含有32个氨基酸、具有活性的B型利钠肽(BNP)，两者来源相同并且等摩尔分泌释放进入血循环。
当心脏容量负荷增加或心脏功能受损时，N末端脑钠肽前体(NT-proBNP)与BNP的指标浓度会异常升高，其中NT-proBNP相对BNP生物稳定性较好，半衰期较长(120min)，浓度相对较稳定，有效检测时间长，血液中含量相对比BNP高约16～20倍，因此检测相对容易，并且血浆标本在体外的稳定性长(>48h)，是诊断心力衰竭和评价心脏功能的最佳心肌标志物。
正常的人血液中NT-proBNP含量一般低于0.3ng/mL。当心脏功能受损，心肌扩张时，NT-proBNP会快速合成并大量分泌释放进入到人体血液中。在发现一些相关早期病症的时候，准确、灵敏、高效稳定地测定血液中NT-proBNP的量，能给早期心功能不全、心衰、呼吸困难的心源性及非心源性心衰治疗及预后监测、急性冠状动脉综合征的分级等方面提供快速、准确的早期诊断依据。目前用于检NT-proBNP含量的方法主要有金标定性试验、荧光免疫法、酶联免疫吸附试验(ELISA)和磁微粒化学发光法(CMIA)，但是这些测量方法都需要针对于NT-proBNP的特异性单克隆抗体，目前国内用于检测NT-proBNP的单克隆抗体灵敏度、特异性上都不够理想。
发明内容
本公开涉及一种新颖的包含N末端脑钠肽前体(NT-proBNP)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。
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NT-proBNP具有K D≤2.26×10 -8的亲和力；
互补决定区CDR-VH1为G-X1-S-X2-T-T-Y-Y-X3-D，其中，
X1是P或F，X2是I、V或L，X3是I、V或L；
互补决定区CDR-VH2为M-T-K-D-X1-N-A-V-H-X2-P-T-X3-R-S，其中，
X1是G或A，X2是Q或N，X3是I、V或L；
互补决定区CDR-VH3为V-X1-G-X2-I-D-X3-G，其中，
X1是K或R，X2是I、V或L，X3是F或W；
互补决定区CDR-VL1为G-S-S-D-X1-V-G-X2-G-D-Y-X3-N，其中，
X1是Q或N，X2是F或P，X3是I、V或L；
互补决定区CDR-VL2为I-F-X1-A-X2-S-R-X3-R-G，其中，
X1是A或G，X2是T、Y或S，X3是I、V或L；
互补决定区CDR-VL3为G-S-X1-N-S-R-X2-Y-V-X3-G，其中，
X1是P、A或G，X2是GG或N，X3是W或F。
一个重要优点在于，所述结合蛋白活性强，与人NT-proBNP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-VH1中，X1是F；
所述互补决定区CDR-VH2中，X1是G；
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F；
所述互补决定区CDR-VL2中，X1是G；
所述互补决定区CDR-VL3中，X3是F。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2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。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是Q。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是N。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X3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I。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X3是F。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X3是W。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是Q。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是N。
在一种或多种实施方式中，所述互补决定区CDR-V L1中，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-V L1中，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-V L1中，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是T。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是Y。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是S。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X3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P。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是A。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是G。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X2是GG。
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 PCTCN2019109792-appb-000001
Figure PCTCN2019109792-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CDRs(例如3个轻链CDR或3个重链 CDR)；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，所述恒定区来源于羊；
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，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。
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。
根据本公开的一方面，本公开还涉及如上所述的结合蛋白在制备用于诊断心力衰竭和评价心脏功能的诊断剂中的应用。
根据本公开的一方面，本公开还涉及一种检测测试样品中NT-proBNP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NT-proBNP抗原与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NT-proBNP的存在。
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NT-proBNP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。
本公开还涉及本文所述的结合蛋白，在诊断与NT-proBNP相关的疾病的应用。
本公开还涉及一种诊断与NT-proBNP相关的疾病或评价心脏功能的方法，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NT-proBNP相关的疾病的存在或指示心脏功能水平。
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，优选地为灵长类动物，更优选地为人类。
在一种或多种实施方式中，所述与NT-proBNP相关的疾病为心脏疾病。
在一种或多种实施方式中，所述与NT-proBNP相关的疾病选自由心力衰竭、心功能不全、心源性呼吸困难、肺源性呼吸困难、急性冠状动脉综合征或其组合组成的组。
在一种或多种实施方式中，所述心力衰竭是心源性心力衰竭或非心源性心力衰竭。
附图说明
为了更清楚地说明本公开具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本公开的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本公开一个实施例中抗人NT-proBNP的单克隆抗体电泳图。
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)由被三个称为“互补决定区”或“CDR”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NT-proBNP具有K D≤2.26×10 -8的亲和力；
互补决定区CDR-VH1为G-X1-S-X2-T-T-Y-Y-X3-D，其中，
X1是P或F，X2是I、V或L，X3是I、V或L；
互补决定区CDR-VH2为M-T-K-D-X1-N-A-V-H-X2-P-T-X3-R-S，其中，
X1是G或A，X2是Q或N，X3是I、V或L；
互补决定区CDR-VH3为V-X1-G-X2-I-D-X3-G，其中，
X1是K或R，X2是I、V或L，X3是F或W；
互补决定区CDR-VL1为G-S-S-D-X1-V-G-X2-G-D-Y-X3-N，其中，
X1是Q或N，X2是F或P，X3是I、V或L；
互补决定区CDR-VL2为I-F-X1-A-X2-S-R-X3-R-G，其中，
X1是A或G，X2是T、Y或S，X3是I、V或L；
互补决定区CDR-VL3为G-S-X1-N-S-R-X2-Y-V-X3-G，其中，
X1是P、A或G，X2是GG或N，X3是W或F。
如本文所用术语“N末端脑钠肽前体(NT-proBNP)”和“N末端B型利钠肽原”可以互换使用，是指脑钠肽前体或B型利钠肽在酶切之后，例如通过内切酶酶切之后，产生的无生物活性的N末端片段。
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NT-proBNP具有K D≤2.26×10 -8mol/L，K D值也可以选择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、9×10 -10mol/L或1×10 -8mol/L的亲和力；
或者8.10×10 -10mol/L≤K D≤2.26×10 -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、9×10 -10mol/L或1×10 -8mol/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F；
所述互补决定区CDR-VH2中，X1是G；
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F；
所述互补决定区CDR-VL2中，X1是G；
所述互补决定区CDR-VL3中，X3是F。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2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。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是Q。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是N。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X3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I。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X3是F。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X3是W。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是Q。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是N。
在一种或多种实施方式中，所述互补决定区CDR-V L1中，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-V L1中，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-V L1中，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是T。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是Y。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是S。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X3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P。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是A。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是G。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X2是GG。
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 PCTCN2019109792-appb-000003
Figure PCTCN2019109792-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CDRs(例如3个轻链CDR或3个重链CDR)；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NT-proBNP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，所述恒定区来源于羊；
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。
根据本公开的一方面，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。
根据本公开的一方面，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。
根据本公开的一方面，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。
根据本公开的一方面，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。
根据本公开的一方面，本公开还涉及如上所述的结合蛋白在制备用于诊断心力衰竭和评价心脏功能的诊断剂中的应用。
在一种或多种实施方式中，所述癌症包括急性淋巴母细胞性白血病、急性骨髓源性白血病、胆管癌、乳腺癌、子宫颈癌、慢性淋巴细胞性白血病、慢性骨髓源性白血病、结肠直肠癌、子宫内膜癌、食道癌、胃癌、头颈部癌、霍奇金氏淋巴瘤、肺癌、甲状腺髓样癌、非霍奇金氏淋巴瘤、多发性骨髓瘤、肾癌、卵巢癌、胰腺癌、神经胶质瘤、黑素瘤、肝癌、前列腺癌和尿路膀胱癌。
根据本公开的一方面，本公开还涉及一种检测测试样品中NT-proBNP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NT-proBNP抗原与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NT-proBNP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NT-proBNP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NT-proBNP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NT-proBNP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。
本公开还涉及本文所述的结合蛋白，在诊断与NT-proBNP相关的疾病的应用。
如本文所用术语“与NT-proBNP相关的疾病”是指以NT-proBNP，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，与NT-proBNP相关的疾病可以指以血液，例如全血，血浆或血清中NT-proBNP水平的升高作为特征的疾病。
本公开还涉及一种诊断与NT-proBNP相关的疾病或评价心脏功能的方法，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NT-proBNP相关的疾病的存在或指示心脏功能水平。
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，优选地为灵长类动物，更优选地为人类。
在一种或多种实施方式中，所述与NT-proBNP相关的疾病为心脏疾病。
在一种或多种实施方式中，所述与NT-proBNP相关的疾病选自由心力衰竭、心功能不全、心源性呼吸困难、肺源性呼吸困难、急性冠状动脉综合征或其组合组成的组。
在一种或多种实施方式中，所述心力衰竭是心源性心力衰竭或非心源性心力衰竭。
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NT-proBNP的重组抗体的示例性制备方法。
S1.构建表达质粒：
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；
分泌Anti-NT-proBNP 8B9单克隆抗体为已有的杂交瘤细胞株，复苏备用。
S11，引物的设计与合成：
扩增重链和轻链的5’RACE上游引物：
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’；
mkR：5’>CCCGAATTCCTAAGAACACTCTGAGGGCTTCACTG<3’；
mHR：5’>CCCGAATTCTCATTTACCCGGAGGCTTAGAGAT<3’。
S12，抗体可变区基因克隆及测序：
从分泌Anti-NT-proBNP 8B9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mkR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1.4KB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，Anti-NT-proBNP 8B9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402bp，属于VkII基因家族，其前方有60bp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402bp，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 TM3.4
Figure PCTCN2019109792-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Anti-NT-proBNP 8B9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：
NT-proBNP 8B9-HF：
5’>CCCAAGCTTGCCACCATGAACCCACTGTGGACCCTCCTCTTT<3’；
NT-proBNP 8B9-HR:
5’>CCCGAATTCTCATTTACCCGGAGGCTTAGAGAT<3’；
NT-proBNP 8B9-LF:
5’>CCCAAGCTTGCCACCATGGCCTGGTCCCCTCTGCTCCTCAC<3’；
NT-proBNP 8B9-LR:
5’>CCCGAATTCCTAAGAACACTCTGAGGGCTTCACTG<3’；
通过PCR扩增方法扩出0.7KB的轻链基因片段和1.4kb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。
S2.稳定细胞株筛选
S21重组抗体表达质粒瞬时转染CHO细胞，确定表达质粒活性
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，第3、5、7天取样计数，第7天收样检测。
包被液稀释重组NT抗原(自产，161213)到指定浓度，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；加入稀释后的细胞上清，100uL/孔，37℃,30min(部分上清1h)；洗涤液清洗5次，拍干；加入兔抗羊-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。结果显示细胞上清稀释1000倍后反应OD仍大于1.0，未加细胞上清孔反应OD小于0.1，表明质粒瞬转后产生的抗体对重组NT蛋白都有活性。
S22重组抗体表达质粒线性化
准备下述试剂：Buffer 50ul、DNA 100ug/管、PuvⅠ酶10ul、无菌水补至500ul，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。
S23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，100ul质粒与700ul细胞混合，转入电转杯，电转，次日计数；25umol/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。
S3.重组抗体生产
S31细胞扩培
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。
S32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。取4μg纯化的抗体进行还原性SDS-PAGE，4μg外来对照抗体作为对照，电泳图如图1所示。在还原性SDS-PAGE后显示两条带，1条Mr为50KD(重链)，另一条Mr为28KD(轻链)。
实施例2
抗体亲和力分析及活性鉴定
实施例1中得到的抗体经分析具有序列如SEQ ID NO:11所示的轻链以及12所示的重链。
经分析，重链的互补决定区(WT)：
CDR-VH1为G-P(X1)-S-I(X2)-T-T-Y-Y-I(X3)-D；
CDR-VH2为M-T-K-D-A(X1)-N-A-V-H-Q(X2)-P-T-I(X3)-R-S；
CDR-VH3为V-K(X1)-G-I(X2)-I-D-W(X3)-G；
轻链的互补决定区：
CDR-VL1为G-S-S-D-Q(X1)-V-G-P(X2)-G-D-Y-V(X3)-N；
CDR-VL2为I-F-A(X1)-A-T(X2)-S-R-I(X3)-R-G；
CDR-VL3为G-S-P(X1)-N-S-R-GG(X2)-Y-V-W(X3)-G；
其中，X1、X2、X3均为待突变位点。表1与抗体活性有关的突变位点
Figure PCTCN2019109792-appb-000006
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。
包被液稀释重组NT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NT单克隆抗体，100uL/孔，37℃,30min(部分上清1h)；洗涤液清洗5次，拍干；加入兔抗羊-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
样品浓度ng/ml WT 突变1 突变2 突变3 突变4
111.11 1.528 1.827 1.772 1.764 1.724
37.04 1.236 1.568 1.526 1.533 1.501
12.35 0.732 1.110 1.031 1.022 0.959
4.12 0.312 0.564 0.523 0.520 0.489
1.37 0.218 0.355 0.353 0.347 0.300
0 0.067 0.121 0.089 0.076 0.115
由上表可知，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 PCTCN2019109792-appb-000007
Figure PCTCN2019109792-appb-000008
亲和力分析
以活性鉴定相同方式酶免间接法做数据，包被做四个梯度3ug/ml、1.5ug/ml、0.75ug/ml、0.375ug/ml；抗体从1000ng/ml开始2倍梯度稀释至0.977ng/ml上样。得出不用包被浓度下不同抗体浓度对应的OD值。在同一包被浓度下，以抗体浓度为横坐标，OD值为纵坐标，对数作图，根据拟合方程计算出50％最大OD值时的抗体浓度；带入公式：K＝(n-1)/(2×(n×Ab`-Ab))计算出亲和力常数的倒数，其中Ab和Ab`分别表示对应包被浓度(Ag、Ag`)下50％最大OD值时的抗体浓度，n＝Ag/Ag`；每两个包被浓度可以组合计算出一个K值，最后可得六个K值，取其平均数值，再求其倒数则为亲和力常数KD。
表4亲和力分析数据
序号 K D(M) 序号 K D(M)
突变1 1.73E-09 突变1-28 2.25E-09
突变1-1 2.51E-09 突变1-29 1.49E-09
突变1-2 2.61E-09 突变1-30 2.66E-09
突变1-3 1.52E-09 突变1-31 1.84E-09
突变1-4 2.07E-09 突变1-32 1.52E-09
突变1-5 6.10E-09 突变1-33 2.60E-09
突变1-6 1.17E-09 突变1-34 3.12E-09
突变1-7 1.53E-09 突变1-35 1.51E-09
突变1-8 2.22E-09 突变1-36 5.64E-09
突变1-9 1.56E-09 突变1-37 1.57E-09
突变1-10 3.20E-09 突变1-38 1.82E-09
突变1-11 1.52E-09 突变1-39 2.45E-09
突变1-12 2.75E-09 突变1-40 3.47E-09
突变1-13 2.09E-09 突变1-41 2.82E-09
突变1-14 8.75E-10 突变1-42 2.22E-09
突变1-15 1.76E-09 突变1-43 2.93E-09
突变1-16 3.27E-09 突变1-44 1.26E-09
突变1-17 3.39E-09 突变1-45 1.53E-09
突变1-18 1.73E-09 突变1-46 3.49E-09
突变1-19 1.51E-09 突变1-47 2.79E-09
突变1-20 2.61E-09 突变1-48 2.02E-09
突变1-21 2.53E-09 突变1-49 6.83E-09
突变1-22 2.07E-09 突变1-50 2.16E-09
突变1-23 8.10E-10 突变1-51 9.44E-10
突变1-24 1.17E-09 突变1-52 2.71E-09
突变1-25 2.16E-09 突变1-53 2.06E-09
突变1-26 1.51E-09    
突变1-27 8.55E-10    
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 PCTCN2019109792-appb-000009
表6亲和力分析数据
序号 K D(M) 序号 K D(M)
WT 1 3.02E-09 WT 1-5 2.26E-08
WT 1-1 1.78E-08 WT 1-6 6.14E-09
WT 1-2 3.48E-09 WT 1-7 5.05E-09
WT 1-3 2.53E-09 WT 1-8 4.78E-09
WT 1-4 3.02E-09 WT 1-9 2.47E-09
从表5和表6分析，在保证具有抗体活性的前提下，上述突变位点与其他位点的关联也不大。
实施例3稳定性分析
将抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天，取7天、14天、21天样品进行状态观察，并对21天样品进行活性检测，结果显示三种考核条件下抗体放置21天均未见明显蛋白状态变化，活性也未随考核温度的升高呈下降越势，说明自产抗体稳定。下表为突变1考核21天的酶免活性检测OD结果。
表7稳定性分析数据
样品浓度(ng/ml) 111.11 12.35 0
4℃，21天样品 1.726 0.864 0.047
-80℃，21天样品 1.845 0.976 0.036
37℃，21天样品 1.813 0.843 0.033
进一步地将随机抽取的8个突变抗体进行稳定性检测；将上述抗体在37℃保存72小时，取出后与同批次的4℃保存72小时的抗体在同一检测条件下检测相同的阴、阳性质控样本，检测方法如上述实施例中所采用的抗体活性分析方法，各组抗体的线性均可达99.50％以上，且CV值低于10％，这说明上述抗体均具有优秀的稳定性。
实施例4配对性能评价
申请人将表4中上述抗体与内部另一株抗体(与原WT序列的抗体配对的抗体)做配对抗体实验验证，经双抗夹心法配对实验验证，特异性均维持原有高水平，但由于突变抗体活性和亲和力的升高而表现出了更高的灵敏度。
将突变1抗体与WT抗体作为包被抗体，分别与另一株作为标记的NT-oroBNP抗体配套使用，在化学发光评台比较性能差异，具体性能见下表：
表8配对性能评价表
性能指标 检测范围 灵敏度 线性 检出率
突变1 0-50000pg/ml 5.8pg/ml 0.97689 100％
WT 0-50000pg/ml 6pg/ml 0.95638 99％
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
本公开所述的结合蛋白活性强，与人NT-proBNP具有很高的亲和力。本公开所述的结合蛋白能够用于诊断与NT-proBNP相关的疾病，例如心力衰竭，并且可以用于评价心脏功能，具有高的灵敏度和特异性。

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NT-proBNP具有K D≤2.26×10 -8的亲和力；
    互补决定区CDR-VH1为G-X1-S-X2-T-T-Y-Y-X3-D，其中，
    X1是P或F，X2是I、V或L，X3是I、V或L；
    互补决定区CDR-VH2为M-T-K-D-X1-N-A-V-H-X2-P-T-X3-R-S，其中，
    X1是G或A，X2是Q或N，X3是I、V或L；
    互补决定区CDR-VH3为V-X1-G-X2-I-D-X3-G，其中，
    X1是K或R，X2是I、V或L，X3是F或W；
    互补决定区CDR-VL1为G-S-S-D-X1-V-G-X2-G-D-Y-X3-N，其中，
    X1是Q或N，X2是F或P，X3是I、V或L；
    互补决定区CDR-VL2为I-F-X1-A-X2-S-R-X3-R-G，其中，
    X1是A或G，X2是T、Y或S，X3是I、V或L；
    互补决定区CDR-VL3为G-S-X1-N-S-R-X2-Y-V-X3-G，其中，
    X1是P、A或G，X2是GG或N，X3是W或F；
    优选的：
    所述互补决定区CDR-VH1中，X1是F；
    所述互补决定区CDR-VH2中，X1是G；
    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F；
    所述互补决定区CDR-VL2中，X1是G；
    所述互补决定区CDR-VL3中，X3是F；
    优选的，所述互补决定区CDR-VH1中，X2是I；
    优选的，所述互补决定区CDR-VH1中，X2是V；
    优选的，所述互补决定区CDR-VH1中，X2是L；
    优选的，所述互补决定区CDR-VH1中，X3是I；
    优选的，所述互补决定区CDR-VH1中，X3是V；
    优选的，所述互补决定区CDR-VH1中，X3是L；
    优选的，所述互补决定区CDR-VH2中，X2是Q；
    优选的，所述互补决定区CDR-VH2中，X2是N；
    优选的，所述互补决定区CDR-VH2中，X3是I；
    优选的，所述互补决定区CDR-VH2中，X3是V；
    优选的，所述互补决定区CDR-VH2中，X3是L；
    优选的，所述互补决定区CDR-VH3中，X2是I；
    优选的，所述互补决定区CDR-VH3中，X2是V；
    优选的，所述互补决定区CDR-VH3中，X2是L；
    优选的，所述互补决定区CDR-VH3中，X3是F；
    优选的，所述互补决定区CDR-VH3中，X3是W；
    优选的，所述互补决定区CDR-VL1中，X1是Q；
    优选的，所述互补决定区CDR-VL1中，X1是N；
    优选的，所述互补决定区CDR-VL1中，X3是I；
    优选的，所述互补决定区CDR-VL1中，X3是V；
    优选的，所述互补决定区CDR-VL1中，X3是L；
    优选的，所述互补决定区CDR-VL2中，X2是T；
    优选的，所述互补决定区CDR-VL2中，X2是Y；
    优选的，所述互补决定区CDR-VL2中，X2是S；
    优选的，所述互补决定区CDR-VL2中，X3是I；
    优选的，所述互补决定区CDR-VL2中，X3是V；
    优选的，所述互补决定区CDR-VL2中，X3是L；
    优选的，所述互补决定区CDR-VL3中，X1是P；
    优选的，所述互补决定区CDR-VL3中，X1是A；
    优选的，所述互补决定区CDR-VL3中，X1是G；
    优选的，所述互补决定区CDR-VL3中，X2是GG；
    优选的，所述互补决定区CDR-VL3中，X2是N；
    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 PCTCN2019109792-appb-100001
    Figure PCTCN2019109792-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1或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心力衰竭和评价心脏功能的诊断剂中的应用。
  9. 一种检测测试样品中的NT-proBNP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NT-proBNP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NT-proBNP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NT-proBNP结合。
  10. 一种试剂盒，其包括权利要求1～3任一项所述的结合蛋白。
  11. 如权利要求1～3任一项所述的结合蛋白，在诊断与NT-proBNP相关的疾病的应用。
  12. 一种诊断与NT-proBNP相关的疾病或评价心脏功能的方法，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NT-proBNP相关的疾病的存在或指示心脏功能水平。
  13. 根据权利要求12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。
  14. 根据权利要求12或13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。
  15. 根据权利要求12～14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。
  16. 根据权利要求11所述的应用或根据权利要求12～15中任一项所述的方法，其中所述与NT-proBNP相关的疾病为心脏疾病。
  17. 根据权利要求11所述的应用或根据权利要求12～15中任一项所述的方法，其中所述与NT-proBNP相关的疾病选自由心力衰竭、心功能不全、心源性呼吸困难、肺源性呼吸困难、急性冠状动脉综合征或其组合组成的组。
  18. 根据权利要求11所述的应用或根据权利要求17所述的方法，其中所述心力衰竭是心源性心力衰竭或非心源性心力衰竭。
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CN115261333A (zh) * 2022-09-26 2022-11-01 玛特瑞尔(吉林)生物技术有限公司 NT-proBNP杂交瘤细胞株及单克隆抗体、试纸条及检测试剂盒

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