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EP-3901170-B1 - RECOMBINANT ANTIBODY OF ANTI-HUMAN N-TERMINAL BRAIN NATRIURETIC PEPTIDE PRECURSOR

EP3901170B1EP 3901170 B1EP3901170 B1EP 3901170B1EP-3901170-B1

Inventors

  • MENG, Yuan
  • ZHONG, Dongmei
  • YE, Qingni
  • LIANG, Bi
  • YOU, Hui
  • MA, Qiuyan
  • LI, WEIZHI

Dates

Publication Date
20260506
Application Date
20191001

Claims (14)

  1. An isolated binding protein comprising an N-terminal pro-brain natriuretic peptide antigen-binding domain, wherein the antigen-binding domain comprises six complementarity determining regions (CDR) of the following amino acid sequences; the complementarity determining region CDR-VH1 is G-X1-S-X2-T-T-Y-Y-X3-D, wherein X1 is F, X2 is I, V or L, and 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, wherein X1 is G, X2 is Q or N, and X3 is I, V or L; the complementarity determining region CDR-VH3 is V-X1-G-X2-I-D-X3-G, wherein X1 is R, X2 is I, V or L, and 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, wherein X1 is Q or N, X2 is F, and X3 is I, V or L; the complementarity determining region CDR-VL2 is I-F-X1-A-X2-S-R-X3-R-G, wherein X1 is G, X2 is T, Y or S, and X3 is I, V or L; the complementarity determining region CDR-VL3 is G-S-X1-N-S-R-X2-Y-V-X3-G, wherein X1 is P, A or G, X2 is GG or N, and X3 is F; the six complementarity determining regions have any one of the following mutation combinations: Site CDR-VH1 X2/X3 CDR-VH2 X2/X3 CDR-VH3 X2/X3 CDR-VL1 X1/X3 CDR-VL2 X2/X3 CDR-VL3 X1/X2 Mutation combination 1 I/I Q/I I/W Q/V T/I P/GG Mutation combination 2 I/L N/I I/F Q/L T/L P/N Mutation combination 3 I/V Q/L L/W Q/I T/V A/GG Mutation combination 4 L/I N/L L/F N/V Y/I A/N Mutation combination 5 L/L Q/V V/W N/L Y/L G/GG Mutation combination 6 L/V N/V V/F N/I Y/V G/N Mutation combination 7 V/I N/V I/W N/I S/I P/N Mutation combination 8 V/L Q/V I/F N/L S/L G/N Mutation combination 9 V/V N/L L/W N/V S/V G/GG Mutation combination 10 I/I Q/L L/F Q/I S/I A/GG Mutation combination 11 I/L N/I V/W Q/L S/L A/N Mutation combination 12 I/V Q/I V/F Q/V S/V P/GG Mutation combination 13 L/I Q/I I/W Q/V Y/I P/GG Mutation combination 14 L/L N/I I/F Q/L Y/L P/N Mutation combination 15 L/V Q/L L/W Q/I Y/V A/GG Mutation combination 16 V/I N/L L/F N/V T/I A/N Mutation combination 17 V/L Q/V V/W N/L T/L G/GG Mutation combination 18 V/V N/V V/F N/I T/V G/N Mutation combination 19 I/I N/V I/W N/I T/I P/N Mutation combination 20 I/L Q/V I/F N/L T/L G/N Mutation combination 21 I/V N/L L/W N/V T/V G/GG Mutation combination 22 L/I Q/L L/F Q/I Y/I A/GG Mutation combination 23 L/L N/I V/W Q/L Y/L A/N Mutation combination 24 L/V Q/I V/F Q/V Y/V P/GG Mutation combination 25 V/I Q/I I/W Q/V S/I P/GG Mutation combination 26 V/L N/I I/F Q/L S/L P/N Mutation combination 27 V/V Q/L L/W Q/V S/V A/GG Mutation combination 28 I/I N/L L/F N/V S/I A/N Mutation combination 29 I/L Q/V V/W N/L S/L G/GG Mutation combination 30 I/V N/V V/F N/I S/V G/N Mutation combination 31 L/I N/V I/W N/I T/I P/N Mutation combination 32 L/L Q/V I/F N/L T/L G/N Mutation combination 33 L/V N/L L/W N/V T/V G/GG Mutation combination 34 V/I Q/L L/F Q/I Y/I A/GG Mutation combination 35 V/L N/I V/W Q/L Y/L A/N Mutation combination 36 V/V Q/I V/F Q/V Y/V P/GG Mutation combination 37 I/I Q/I I/W Q/V T/I P/GG Mutation combination 38 I/L N/I I/F Q/L T/L A/N Mutation combination 39 I/V Q/L L/W Q/I T/V G/GG Mutation combination 40 L/I N/L L/F N/V Y/I G/N Mutation combination 41 L/L Q/V V/W N/L Y/L P/N Mutation combination 42 V/I N/V I/W N/I Y/V G/N Mutation combination 43 V/I N/V I/W N/I S/I G/GG Mutation combination 44 V/L Q/V I/F N/L S/L A/GG Mutation combination 45 V/V N/L L/W N/V S/V A/N Mutation combination 46 I/I Q/L L/F Q/I S/I P/GG Mutation combination 47 I/L N/I V/W Q/L S/L P/GG Mutation combination 48 I/V Q/I V/F Q/V S/V P/N Mutation combination 49 L/I Q/I I/W Q/V T/I P/GG Mutation combination 50 L/L N/I I/F Q/L T/L P/N Mutation combination 51 L/V Q/L L/W Q/I T/V A/GG Mutation combination 52 V/I N/L L/F N/V Y/I A/N Mutation combination 53 V/L Q/V V/W N/L Y/L G/GG Mutation combination 54 V/V N/V V/F N/I Y/V G/N the binding protein comprises light chain framework regions FR-L1, FR-L2, FR -L3 and FR-L4 with sequences correspondingly shown in SEQ ID NO: 1-4, and, heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 with sequences correspondingly shown in SEQ ID NO: 5-8.
  2. The isolated binding protein comprising an N-terminal pro-brain natriuretic peptide antigen-binding domain according to claim 1, wherein the binding protein is one of F(ab') 2 , Fab', Fab, Fv, scFv, and a bispecific antibody .
  3. The isolated binding protein comprising an N-terminal pro-brain natriuretic peptide antigen-binding domain according to any one of claims 1 to 2, wherein the binding protein further comprises a constant region sequence of antibody; preferably, the constant region sequence is a sequence of any one constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD; preferably, the constant region is derived from species consisting of cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human; preferably, the constant region is derived from the sheep; a light chain constant region sequence is shown in SEQ ID NO: 9; a heavy chain constant region sequence is shown in SEQ ID NO: 10; preferably the binding protein is labeled by an indicator which shows signal intensity.
  4. An isolated nucleic acid molecule, the nucleic acid molecule being DNA or RNA, which encodes the binding protein according to any one of claims 1-3.
  5. A vector, comprising the nucleic acid according to claim 4.
  6. A host cell transformed with the vector according to claim 5.
  7. A method for producing the binding protein according to any one of claims 1-3, the method comprising the steps of: culturing the host cell according to claim 6 in a culture medium and under suitable culture conditions, recovering such produced binding protein from the culture medium or from the cultured host cell.
  8. Use of the binding protein according to any one of claims 1-3 in the in vitro/ex vivo diagnosis of heart failure and evaluation of a cardiac function, and in vitro/ex vivo diagnosis of a disease related to NT-proBNP, wherein the disease related to NT-proBNP is selected from heart failure, cardiac insufficiency, cardiogenic dyspnea, pulmonary dyspnea, acute coronary syndrome, or a combination thereof; wherein, the heart failure is cardiogenic heart failure or non-cardiac heart failure.
  9. A method for detecting NT-proBNP in a test sample, the method comprising: a) contacting the NT-proBNP antigen in the test sample with the binding protein according to any one of claims 1-3 to form an immune complex under conditions sufficient for taking an antibody/antigen binding reaction; and b) detecting a presence of the immune complex, which indicates a presence of the NT-proBNP in the test sample; preferably, in step a), the immune complex further comprises a second antibody that binds to the binding protein; preferably, in step a), the immune complex further comprises a second antibody that binds to the NT-proBNP; preferably, the presence of the immune complex indicates the presence of a disease related to NT-proBNP or indicates the level of the cardiac function.
  10. The method according to claim 9, wherein the method is based on fluorescence immunoassay, chemiluminescence, colloidal gold immunoassay, radioimmunoassay, and/or enzyme-linked immunoassay.
  11. The method according to claim 9 or 10, wherein the test sample is selected from at least one of whole blood, peripheral blood, serum, plasma or myocardial tissue.
  12. The method according to any one of claims 9-11, wherein a subject which the test sample is derived from is mammal, preferably primate, more preferably human.
  13. The method according to any one of claims 9-12, wherein the disease related to NT-proBNP is a cardiac disease; further, the disease related to NT-proBNP is selected from heart failure, cardiac insufficiency, cardiogenic dyspnea, pulmonary dyspnea, acute coronary syndrome, or a combination thereof; further, the heart failure is cardiogenic heart failure or non-cardiac heart failure.
  14. A kit, comprising the binding protein as defined in any one of claims 1-3.

Description

Cross-Reference to Related Application This application claims the priority of the Chinese patent application with the application No. 201811557468.5, titled "Recombinant Antibody of Anti-human N-terminal Pro-brain Natriuretic Peptide" filed to the China National Intellectual Property Administration on December 19, 2018. Technical Field The present disclosure relates to the field of immune technology, in particular, to a recombinant antibody of anti-human N-terminal pro-brain natriuretic peptide. Background In 1988, Japanese scholar Sudoh first isolated a polypeptide with potent diuretic, vasodilator and hypotensive effects from pig brain, and named it Brain Natriuretic Peptide (BNP). BNP is most distributed in the heart, but cardiomyocytes first synthesize proBNP containing 108 amino acids. When cardiomyocytes are stimulated, proBNP is cleaved into non-biologically active N-terminal pro-B-type natriuretic peptide (NT-proBNP) containing 76 amino acids and active B-type natriuretic peptide (BNP) containing 32 amino acids, both of which are derived from the same source and are secreted equimolarly and released into the blood circulation. When the heart volume load increases or the cardiac function is impaired, the index concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) and BNP will increase abnormally, wherein NT-proBNP has better biological stability, has a longer half-life ( 120 min), has relatively stable concentration, has a long effective detection time, and has about 16-20 times higher content in blood compared with BNP, therefore, it is relatively easy to detect, and the stability of its plasma samples in vitro is long (> 48 h), which is the best myocardial marker for diagnosing heart failure and evaluating cardiac function. The content of NT-proBNP in the blood of normal people is generally less than 0.3 ng/mL. When the heart function is impaired and the myocardium expands, NT-proBNP will be rapidly synthesized and secreted in large quantities into the human blood. When finding some relevant early symptoms, accurate, sensitive, efficient and stable determination of the amount of NT-proBNP in the blood can provide a fast and accurate basis for early diagnosis for early cardiac insufficiency, heart failure, cardiogenic and non-cardiogenic heart failure with dyspnea treatment and prognosis monitoring, and acute coronary syndrome grading, etc. The current methods used to detect the content of NT-proBNP mainly include gold calibration test, fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA) and chemiluminescence microparticle immunoassays (CMIA), but these measurement methods all require a specific monoclonal antibody against NT-proBNP. However, the sensitivity and specificity of the monoclonal antibody currently used to detect NT-proBNP in China are not ideal. The present invention is defined by the appended claims. . Relevant technologies are also known from the following documents: WILKINS MICHAEL D. ET AL: "Quantum dot enabled lateral flow immunoassay for detection of cardiac biomarker NT-proBNP", SENSING AND BIO-SENSING RESEARCH, vol. 21, 1 November 2018 (2018-11-01), pages 46-53.CN 106 916 225 A.KIM HYUNG-YONG ET AL: "Affinity Maturation of Monoclonal Antibodies by Multi-Site-Directed Mutagenesis", 1 January 2014 (2014-01-01).NIELSEN U B ET AL: "Chapter 37: Affinity maturation by chain shuffling and site directed mutagenesis", 1 January 2001 (2001-01-01).WO 2009/066010 A1.Zhang Shiyun ET AL: "Predicting detection limits of enzyme-linked immunosorbent assay (ELISA) and bioanalytical techniques in general", Analyst, vol. 139, no. 2, 1 January 2014 (2014-01-01), pages 439-445. Summary The present disclosure relates to a novel isolated binding protein including a N-terminal pro-brain natriuretic peptide (NT-proBNP) antigen binding domain, and investigates the preparation, use and other aspects of the binding protein. The invention is set out in the appended set of claims 1-14. Wherein the antigen-binding domain includes six complementarity determining regions (CDR) of the following amino acid sequence; the complementarity determining region CDR-VH1 is G-X1-S-X2-T-T-Y-Y-X3-D (SEQ ID NO:13), wherein X1 is F, X2 is I, V or L, and 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 (SEQ ID NO:14), wherein X1 is G, X2 is Q or N, and X3 is I, V or L; the complementarity determining region CDR-VH3 is V-X1-G-X2-I-D-X3-G (SEQ ID NO:15), wherein X1 is R, X2 is I, V or L, and 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 (SEQ ID NO:16), wherein X1 is Q or N, X2 is F or P, and X3 is I, V or L; the complementarity determining region CDR-VL2 is I-F-X1-A-X2-S-R-X3-R-G (SEQ ID NO:17), wherein X1 is G, X2 is T, Y or S, and X3 is I, V or L; the complementarity determining region CDR-VL3 is G-S-X1-N-S-R-X2-Y-V-X3-G (SEQ ID NO:18), wherein X1 is P, X2 is GG or N, and X3 is W or F; the six com