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WO2022218277A1 - 一种抗fgf21羧基末端的抗体及其应用 - Google Patents

一种抗fgf21羧基末端的抗体及其应用 Download PDF

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WO2022218277A1
WO2022218277A1 PCT/CN2022/086227 CN2022086227W WO2022218277A1 WO 2022218277 A1 WO2022218277 A1 WO 2022218277A1 CN 2022086227 W CN2022086227 W CN 2022086227W WO 2022218277 A1 WO2022218277 A1 WO 2022218277A1
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antibody
seq
amino acid
acid sequence
fgf21
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PCT/CN2022/086227
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English (en)
French (fr)
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刘亮
林树珊
李静
王茜
凌伊
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广东东阳光药业有限公司
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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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • 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]
    • 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
    • G01N2333/475—Assays involving growth factors
    • G01N2333/50—Fibroblast growth factors [FGF]

Definitions

  • the invention relates to the field of biotechnology, in particular to an antibody against the carboxyl terminus of FGF21 and its application.
  • Fibroblast growth factor 21 (FGF21) consists of 181 amino acids, including a beta clover-like core domain and random amino- and carboxy-terminal structures.
  • Administration of recombinant FGF21 reduced plasma glucose and insulin levels, decreased liver and circulating triglyceride and cholesterol levels, and improved insulin sensitivity, energy expenditure, hepatic steatosis, and obesity in a series of animal models of insulin resistance (Xie T, Leung P S. Fibroblast growth factor 21: a regulator of metabolic disease and health span[J]. American Journal of Physiology-Endocrinology and Metabolism, 2017, 313(3):E292-E302.). FGF21 has emerged as the most promising therapeutic agent for the treatment of human type 2 diabetes and related metabolic syndrome.
  • FGF21 has been reported to lose about 90% of its activity after losing 3 amino acids (YAS, tyrosine-alanine-serine) at the carboxy terminus (Yie J, Hecht R, Patel J, et al. FGF21N ⁇ and C ⁇ termini play different roles in receptor interaction and activation[J]. FEBS letters, 2009, 583(1):19-24.). Therefore, it is very important to establish a biological sample analysis method that can distinguish the intact molecule of FGF21 at the carboxy terminus and metabolites with 3 or more amino acids missing from the carboxy terminus to accurately characterize the pharmacokinetics of FGF21 or FGF21-containing fusion protein drugs in vivo.
  • YAS tyrosine-alanine-serine
  • the protein/polypeptide biological sample analysis method is mainly based on immunoassay technology, which is essentially the binding reaction of antigen and antibody. Therefore, the specificity of the antibody used in the analysis method will determine the specificity of the method.
  • immunoassay technology which is essentially the binding reaction of antigen and antibody. Therefore, the specificity of the antibody used in the analysis method will determine the specificity of the method.
  • antigen-binding sites For example, anti-FGF21 antibodies are only known to bind to FGF21, but the specific binding sites and which amino acids bind to are not clear. Therefore, it is very necessary to develop a monoclonal antibody with a clear antigen-binding site that binds only to the 3 amino acids of the carboxyl terminal of FGF21 molecule.
  • the object of the present invention is to provide an antibody against the carboxyl terminus of FGF21 and its application.
  • the antigen binding site of the antibody is clear and can specifically bind to the carboxyl terminus of fibroblast growth factor 21; when FGF21 lacks three amino acids at the carboxyl terminus ( YAS), the antibody of the present invention does not bind to it or has a very low binding ability; only when FGF21 or a fusion protein comprising FGF21 is complete at the carboxyl terminus (including YAS), the antibody of the present invention can specifically bind to it.
  • the present invention provides an anti-FGF21 carboxyl-terminal antibody comprising a light chain variable region containing the amino acid sequences of LCDR1, LCDR2 and LCDR3 and a heavy chain variable region containing HCDR1, HCDR2 and HCDR3; wherein , the amino acid sequence of the LCDR1 is RSSKSLLHSNGITYLY (SEQ ID NO: 1), the amino acid sequence of the LCDR2 is QMSSLAS (SEQ ID NO: 2) or QMSNLAS (SEQ ID NO: 3), and the amino acid sequence of the LCDR3 is AQTLELPT (SEQ ID NO: 4), the amino acid sequence of HCDR1 is GYTFTNY (SEQ ID NO: 5), the amino acid sequence of HCDR2 is NTYTGK (SEQ ID NO: 6), and the amino acid sequence of HCDR3 is NYYDYDVAY (SEQ ID NO: 7 ) or NYYDYDIAY (SEQ ID NO: 8).
  • SEQ ID NO: 1 is RSSKSLLHSNGITYLY (
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 9 or SEQ ID NO: 10.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 11 or SEQ ID NO: 12.
  • amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 9
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 11
  • amino acid sequence of the chain variable region is shown in SEQ ID NO: 10
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 12.
  • the antibody is a full-length antibody, Fab fragment, F(ab) 2 fragment, double-chain Fv fragment or single-chain Fv fragment (scFv).
  • the antibody is a monoclonal antibody.
  • the antibody also includes a light chain constant region selected from kappa or lambda subtypes.
  • the light chain constant region is of kappa subtype.
  • the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14.
  • the antibody also includes a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 subtypes.
  • the heavy chain constant region is of IgG1 subtype.
  • the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
  • the second aspect of the present invention provides a nucleic acid molecule encoding the anti-FGF21 carboxyl-terminal antibody or its antigen-binding portion of the present invention.
  • the third aspect of the present invention provides a vector comprising the nucleic acid molecule of the present invention.
  • the vectors include plasmids, bacteriophages, plant cell viruses, mammalian cell viruses or retroviruses.
  • the fourth aspect of the present invention provides a host cell containing the nucleic acid molecule of the present invention, or the vector of the present invention.
  • the host cells include prokaryotic cells, yeast cells, insect cells or mammalian cells.
  • the host cells are mammalian cells, such as HEK293 cells.
  • the fifth aspect of the present invention provides a detection kit comprising the antibody of the present invention, the nucleic acid molecule of the present invention, or the vector of the present invention.
  • the sixth aspect of the present invention provides the use of the antibody of the present invention in immunodetection for diagnostic or non-diagnostic purposes; or the use of the antibody of the present invention for immunodetection for diagnostic or non-diagnostic purposes.
  • the immunoassay includes: detecting the integrity of the carboxyl terminus of FGF21.
  • the antigen-binding site of the antibody provided by the present invention is clear and can specifically bind to the complete carboxyl terminus of FGF21; when FGF21 lacks the three amino acids (YAS) at the carboxyl terminus, the antibody of the present invention does not bind to it or has a very low binding ability;
  • the antibody of the present invention can specifically bind to FGF21 or a fusion protein comprising FGF21 in the condition that the carboxyl terminus is intact (including YAS). Based on this, the present invention provides a biological sample analysis method that can distinguish the intact molecule of FGF21 at the carboxyl terminus and metabolites whose carboxyl terminus is missing 3 or more amino acids.
  • Fig. 1 is the purified electrophoresis diagram of six monoclonal antibodies obtained after the second subcloning screening; wherein, lane M is the molecular weight marker, lane 1 is non-reducing electrophoresis, and lane 2 is reducing electrophoresis;
  • Fig. 2 is the SDS-PAGE detection result of MM21T antibody; wherein, lane M is molecular weight marker, lane 1 is reducing electrophoresis, and lane 2 is non-reducing electrophoresis;
  • Figure 3 is the detection result of MM24T antibody SDS-PAGE; wherein, lane M is molecular weight marker, lane 1 is reducing electrophoresis, and lane 2 is non-reducing electrophoresis.
  • antibody refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • antibody includes not only intact (ie, full-length) antibodies, but also antigen-binding fragments thereof (eg, Fab, Fab, F(ab) 2 , Fv), variants thereof, fusions comprising antibody portions Proteins, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g. bispecific antibodies) and any other immunoglobulin molecule containing an antigen recognition site of the desired specificity modified configurations of antibodies, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • full or full-length antibodies contain two heavy chains and two light chains.
  • Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • Each light chain contains a light chain variable region (VL) and a light chain constant region (CL).
  • a full-length antibody can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or a subclass of the above), but the antibody need not belong to any particular class.
  • Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the constant domain of the heavy chain.
  • immunoglobulins there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 .
  • binding refers to a non-random binding reaction between two molecules, eg, binding of an antibody to an epitope.
  • antigen binding portion refers to a portion or region of an intact antibody molecule responsible for binding an antigen.
  • the antigen binding portion may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both.
  • VH and VL typically contains three complementarity determining regions CDR1, CDR2 and CDR3
  • CDRs complementarity determining regions
  • CDR1, CDR2 and CDR3 are the regions in the variable region that have the greatest impact on the affinity and specificity of antibodies.
  • the middle CDR amino acid sequence of the variable region amino acid sequence can be analyzed in a variety of ways, for example as determined herein from Chothia using the online software Abysis (http://www.abysis.org/ ).
  • the polypeptide A for immunization was coupled separately for animal immunization.
  • the animal serum with high titer for immunization polypeptide A and low titer for screening polypeptide B was screened.
  • the specific steps include: coating the ELISA plate with polypeptide A for immunization, polypeptide B for screening (SinoA8627), protein A (SEQ ID NO: 22) and protein B (SEQ ID NO: 23), and using HRP to label goat anti-microplate Mouse IgG Fc antibody (ICL company, product number: GGPC-90P) was used as the detection antibody, and the serum titer was determined by indirect ELISA method.
  • the mouse numbered SBI180064-2#C was selected for booster immunization by injecting the polypeptide SinoA8624; 3 days later, the spleen was taken for fusion, and the fused cells were suspended in HAT medium, according to 6 ⁇ 10 4 per well Each cell was seeded in a 96-well cell culture plate, that is, a total of 30 96-well cell culture plates were seeded. On the 6th day and the 8th day after fusion, the medium in the culture plate was discarded, fresh HAT medium was added, and the hybridoma cell culture supernatant was taken on the 10th day after fusion for primary clone screening (the results are shown in Table 6). ).
  • the positive cells obtained by screening were counted, and then seeded into a 96-well cell culture plate at 0.75 cells/well, and 0.5 cell culture plates were seeded for each positive cell. After 7 days of culture, the cell state was observed under a microscope, and the wells containing only a single cell cluster were selected and marked, and the culture supernatant was taken for ELISA screening.
  • the screening results are shown in Table 7 and Table 8 (S: signal value; S-B: background subtraction post signal value).
  • the positive cells obtained by screening were subjected to the second limiting dilution according to the above steps until stable positive monoclonal cells were obtained. After indirect ELISA screening, the supernatants of the 6 hybridoma cell strains in Table 8 were obtained, which could satisfy the binding to SinoA8624 and protein A, but did not bind to SinoA8627 and protein B.
  • Sample loading and equilibration The feed liquid is loaded onto the Protein A affinity chromatography column. After the sample is loaded, rinse with equilibration buffer until the baseline is stable;
  • Example 3 Take the monoclonal antibody purified in Example 3 as the coating antibody, use HRP-labeled mouse anti-human IgG4 Fc antibody (Southern Biotech, product number 9200-05) as the detection antibody, and use double-antibody sandwich ELISA to detect protein A and Protein B, the specific steps include:
  • Coating Coat the purified mouse monoclonal antibody at 2 ⁇ g/mL, 100 ⁇ L/well, overnight at 4°C;
  • Sample preparation Dilute protein A and protein B to 5ng/mL and 10ng/mL respectively, add 100 ⁇ L/well to the well plate, add sample diluent to blank control, 100 ⁇ L/well, mix well, and react at room temperature for 2h;
  • HRP-labeled mouse anti-human IgG4 Fc monoclonal antibody (Southern biotech, Cat. No. 9200-05) at a dilution of 1:2000, 100 ⁇ L/well, and incubate at room temperature for 1 h after sealing;
  • Termination 50 ⁇ L of stop solution was added to each well, and the OD value was immediately measured at a wavelength of 450 nm.
  • the detection results are shown in Table 9 (S: signal value; S-B: signal value after background subtraction).
  • MM21H, MM22H, MM23H, MM24H, MM25H, MM26H can be paired with the detection antibody to detect protein A without interference from protein B.
  • MM21H and MM24H with higher response and lower background were selected as candidate monoclonal antibody strains.
  • the candidate monoclonal antibody strains MM21H and MM24H screened in Example 4 were entrusted to Beijing Sino Biological Inc. to detect and sequence the antibody subtypes of the hybridoma cell lines.
  • the cultured supernatants of MM21H and MM24H hybridoma cells were respectively taken for subtype detection using a subtype detection kit (Southern Biotech). According to the ELISA test results shown in Table 10, the subtypes of MM21H and MM24H mouse monoclonal antibodies were both IgG1 /kappa.
  • the light chain variable region of MM21T contains the following three complementarity determining regions:
  • CDR-L1 its amino acid sequence is RSSKSLLHSNGITYLY (SEQ ID NO: 1), and its coding nucleotide sequence is SEQ ID NO: 24;
  • CDR-L2 its amino acid sequence is QMSSLAS (SEQ ID NO: 2), and its coding nucleotide sequence is SEQ ID NO: 25;
  • CDR-L3 its amino acid sequence is AQTLELPT (SEQ ID NO: 4), and its coding nucleotide sequence is SEQ ID NO: 26.
  • amino acid sequence of the light chain of MM21T is SEQ ID NO: 27; wherein, positions 1-19 are signal peptides, positions 20-130 are light chain variable regions, and positions 131-237 are light chain constants Area.
  • the nucleotide sequence encoding the light chain of MM21T is SEQ ID NO:28.
  • the heavy chain variable region of MM21T contains the following three complementarity determining regions:
  • CDR-H1 its amino acid sequence is GYTFTNY (SEQ ID NO: 5), and its coding nucleotide sequence is SEQ ID NO: 29;
  • CDR-H2 its amino acid sequence is NTYTGK (SEQ ID NO: 6), and its coding nucleotide sequence is SEQ ID NO: 30;
  • CDR-H3 its amino acid sequence is NYYDYDVAY (SEQ ID NO: 7), and its coding nucleotide sequence is SEQ ID NO: 31.
  • amino acid sequence of the heavy chain of MM21T is SEQ ID NO: 32; wherein, positions 1-19 are the signal peptide, positions 20-137 are the variable region of the heavy chain, and positions 138-461 are the constant heavy chain Area.
  • the nucleotide sequence encoding the heavy chain of MM21T is SEQ ID NO:33.
  • the light chain variable region of MM24T contains the following three complementarity determining regions:
  • CDR-L1' its amino acid sequence is RSSKSLLHSNGITYLY (SEQ ID NO: 1), and its encoding nucleotide sequence is SEQ ID NO: 24;
  • CDR-L2' its amino acid sequence is QMSNLAS (SEQ ID NO: 3), and its coding nucleotide sequence is SEQ ID NO: 34;
  • CDR-L3' its amino acid sequence is AQTLELPT (SEQ ID NO: 4), and its encoding nucleotide sequence is SEQ ID NO: 26.
  • amino acid sequence of the light chain of MM24T is SEQ ID NO: 35; wherein, positions 1-19 are signal peptides, positions 20-130 are light chain variable regions, and positions 131-237 are light chain constant regions .
  • the nucleotide sequence encoding the light chain of MM24T is SEQ ID NO:36.
  • the heavy chain variable region of MM24T contains the following three complementarity determining regions:
  • CDR-H1' its amino acid sequence is GYTFTNY (SEQ ID NO: 5), and its encoding nucleotide sequence is SEQ ID NO: 29;
  • CDR-H2' its amino acid sequence is NTYTGK (SEQ ID NO: 6), and its coding nucleotide sequence is SEQ ID NO: 37;
  • CDR-H3' its amino acid sequence is NYYDYDIAY (SEQ ID NO: 8), and its encoding nucleotide sequence is SEQ ID NO: 38.
  • amino acid sequence of the heavy chain of MM24T is SEQ ID NO: 39; wherein, positions 1-19 are the signal peptide, positions 20-137 are the variable region of the heavy chain, and positions 138-461 are the constant heavy chain Area.
  • the nucleotide sequence encoding the heavy chain of MM24T is SEQ ID NO:40.
  • Subculture HEK293 cells with 293 serum-free CD medium (article number SMM 293-TI), mix the expression plasmids containing the light chain and heavy chain genes of the target antibody constructed in Example 5 with transfection reagent TF1 and add them to HEK293 cells, Add 293 serum-free feed solution (Cat. No.: M293-SUPI-100) on days 1, 3, and 5 after transfection.
  • Protein purification was performed after 7 days of cell culture. After centrifugation of the HEK293 culture liquid, filter it with a filter, take the supernatant, and then use a Protein A affinity chromatography column to purify the collected cell culture liquid, and collect absorption peaks.
  • the obtained protein samples were monitored by SDS-PAGE and UV OD280 methods to monitor the protein expression process and QC detection of the products.
  • the target antibody can be purified from HEK293 culture supernatant using Protein A affinity purification method.
  • the molecular weight of the antibody analyzed by non-reducing gel is in the range of 150-200KDa, which is in line with the characteristics of the molecular weight of the antibody.
  • the band near the main antibody band on the non-reducing gel may be due to the difference in molecular weight and migration mode caused by different degrees of glycosylation.
  • the band at ⁇ 100KDa on the non-reducing gel may be caused by the overexpression of the antibody heavy chain, but The overall proportion is ⁇ 5%.
  • Embodiment 7 Activity detection of MM24T recombinant antibody
  • MM24T recombinant antibody The activity of MM24T recombinant antibody was detected by ELISA method, respectively coated with 0.1, 1, and 5 ⁇ g/mL of protein A and protein B, and the antibody spotting concentration was 1 ⁇ g/mL. It was verified that MM24T could bind to protein A and not bind to protein B, and was able to bind to protein B. The binding ability of MM24H was consistent, and the detection results were shown in Table 11 (two parallel experiments were set, and the results of OD450 in the table were averaged).
  • MM21T recombinant antibody was detected by ELISA method, respectively coated with protein A and protein B of 0.1, 1, and 5 ⁇ g/mL, and the antibody concentration was 1 ⁇ g/mL. It was verified that MM21T could bind to protein A and not bind to protein B.
  • the test results As shown in Table 12 (two parallel experiments were set, and the results of OD450 in the table were averaged).

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Abstract

涉及一种抗FGF21羧基末端的抗体及其应用，该抗体的抗原结合位点清晰，可以特异性结合FGF21的完整羧基端；当FGF21缺少羧基末端三个氨基酸(YAS)时，该抗体与其不结合或结合能力极低；仅当FGF21或包含FGF21的融合蛋白在羧基末端完整(包含YAS)的情况下，该抗体可与之发生特异性结合。基于此，提供了一种可以区别FGF21的羧基末端完整分子和羧基端丢失3个或以上氨基酸的代谢物的生物样品分析方法。

Description

一种抗FGF21羧基末端的抗体及其应用 技术领域
本发明涉及生物技术领域，具体涉及一种抗FGF21羧基末端的抗体及其应用。
背景技术
成纤维细胞生长因子21(FGF21)由181个氨基酸组成，包含β三叶草样核心结构域及无规则的氨基端和羧基端结构。在一系列胰岛素抵抗动物模型中，重组FGF21的使用降低了血浆葡萄糖和胰岛素水平，降低了肝脏和循环中的甘油三酯和胆固醇水平，并改善了胰岛素敏感性、能量消耗、肝脂肪变性和肥胖(Xie T,Leung P S.Fibroblast growth factor 21:a regulator of metabolic disease and health span[J].American Journal of Physiology-Endocrinology and Metabolism,2017,313(3):E292-E302.)。FGF21已经成为治疗人类2型糖尿病和相关代谢综合征的最有潜力的治疗剂。
有报道显示，FGF21在羧基末端丢失3个氨基酸(YAS，络氨酸-丙氨酸-丝氨酸)后，其活性下降约90％(Yie J,Hecht R,Patel J,et al.FGF21N‐and C‐termini play different roles in receptor interaction and activation[J].FEBS letters,2009,583(1):19-24.)。因此，建立可以区别FGF21的羧基末端完整分子和羧基端丢失3个或以上氨基酸的代谢物的生物样品分析方法对于准确描述FGF21或包含FGF21的融合蛋白药物在体内的药代动力学特征非常重要。
在生物大分子药物研发的过程中，建立可以区分完整蛋白/多肽分子和其降解的代谢产物的生物样品分析方法是存在一定挑战的。蛋白/多肽的生物样品分析方法主要是基于免疫分析技术，其本质是抗原和抗体的结合反应，因此，分析方法中所使用的抗体的特异性强弱将决定了该方法的特异性情况。尽管目前商业流通的各类抗体非常多，但通常具有不确定的抗原结合位点，比如抗FGF21抗体，仅知道可与FGF21结合，而具体的结合部位，与哪几个氨基酸结合并不清楚。因此，开发一种仅针对FGF21分子羧基末端3个氨基酸结合的、抗原结合位点清晰的单克隆抗体是非常必要的。
发明内容
本发明的目的在于提供一种抗FGF21羧基末端的抗体及其应用，该抗体的抗原结合位点清晰，可以特异性结合成纤维细胞生长因子21的羧基端；当FGF21缺少羧基末端三个氨基酸(YAS)时，本发明的抗体与其不结合或结合能力极低；仅当FGF21或包含FGF21的融合蛋白在羧基末端完整(包含YAS)的情况下，本发明的抗体可与之发生特异性结合。
为此，第一方面，本发明提供一种抗FGF21羧基末端的抗体，其包含含有LCDR1、LCDR2和LCDR3氨基酸序列的轻链可变区以及含有HCDR1、HCDR2和HCDR3的重链可变区；其中，所述LCDR1的氨基酸序列为RSSKSLLHSNGITYLY(SEQ ID NO：1)，所述LCDR2的氨基酸序列为QMSSLAS(SEQ ID NO：2)或QMSNLAS(SEQ ID NO：3)，所述LCDR3的氨基酸序列为AQTLELPT(SEQ ID NO：4)，所述HCDR1的氨基酸序列为GYTFTNY(SEQ ID NO：5)，HCDR2的氨基酸序列为NTYTGK(SEQ ID NO：6)，HCDR3的氨基酸序列为NYYDYDVAY(SEQ ID NO：7)或NYYDYDIAY(SEQ ID NO：8)。
进一步，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：9或者SEQ ID NO：10所示。
进一步，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：11或者SEQ ID NO：12所示。
进一步，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：9所示，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：11所示；或者所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：10所示，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：12所示。
进一步，所述抗体为全长抗体、Fab片段、F(ab) 2片段、双链Fv片段或单链Fv片段(scFv)。
进一步，所述抗体为单克隆抗体。
进一步，所述抗体还包括选自kappa或lambda亚型的轻链恒定区。
进一步，所述轻链恒定区为kappa亚型。
进一步，所述轻链恒定区包括如SEQ ID NO：13或SEQ ID NO：14所示的氨基酸序列。
进一步，所述抗体还包括选自IgG1、IgG2、IgG3、IgG4亚型的重链恒定区。
进一步，所述重链恒定区为IgG1亚型。
进一步，所述重链恒定区包括如SEQ ID NO：15、SEQ ID NO：16或SEQ ID NO：17所示的氨基酸序列。
本发明的第二方面，提供一种核酸分子，其编码本发明所述的抗FGF21羧基末端的抗体或其抗原结合部分。
本发明的第三方面，提供一种载体，其包含本发明所述的核酸分子。
进一步，所述载体包括质粒、噬菌体、植物细胞病毒、哺乳动物细胞病毒或逆转录病毒。
本发明的第四方面，提供一种宿主细胞，其含有本发明所述核酸分子，或包含本发明所述的载体。
进一步，所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞或哺乳动物细胞。
进一步，所述宿主细胞为哺乳动物细胞，例如HEK293细胞。
本发明的第五方面，提供一种检测试剂盒，包含本发明所述的抗体，本发明所述的核酸分子，或本发明所述的载体。本发明的第六方面，提供本发明所述的抗体在诊断或非诊断目的的免疫检测方面的应用；或本发明所述的抗体用于诊断或非诊断目的的免疫检测方面的用途。
进一步，所述免疫检测包括：检测FGF21羧基末端的完整性。
与现有技术相比，本发明的技术方案具有以下优点：
本发明提供的抗体的抗原结合位点清晰，可以特异性结合FGF21的完整羧基端；当FGF21缺少羧基末端三个氨基酸(YAS)时，本发明的抗体与其不结合或结合能力极低；仅当FGF21或包含FGF21的融合蛋白在羧基末端完整(包含YAS)的情况下，本发明的抗体可与之发生特异性结合。基于此，本发明提供了一种可以区别FGF21的羧基末端完整分子和羧基端丢失3个或以上氨基酸的代谢物的生物样品分析方法。
附图说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领 域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。在附图中：
图1经第二次亚克隆后筛选得到的六株单克隆抗体纯化电泳图；其中，泳道M为分子量marker，泳道1为非还原电泳，泳道2为还原电泳；
图2为MM21T抗体的SDS-PAGE检测结果；其中，泳道M为分子量marker，泳道1为还原电泳，泳道2为非还原电泳；
图3为MM24T抗体SDS-PAGE检测结果；其中，泳道M为分子量Marker，泳道1为还原电泳，泳道2为非还原电泳。
具体实施方式
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
除非另外指明，本文的实施例采用本领域常规的分子生物学、微生物学、细胞生物学、生物化学以及免疫学技术。
除非另外指明，本申请中所用的术语具有本领域技术人员通常所理解的含义。
定义
如本文所用术语“抗体”是指能够经由至少一个位于免疫球蛋白分子的可变区中的抗原识别位点特异性结合到标靶的免疫球蛋白分子。本文所使用的“抗体”不仅包括完整的(即全长的)抗体，而且还包括其抗原结合片段(例如Fab、Fab、F(ab) 2、Fv)、其变异体、包含抗体部分的融合蛋白、人源化抗体、嵌合抗体、双抗体、线性抗体、单链抗体、多特异性抗体(例如双特异性抗体)及任何其他包含所需特异性的抗原识别位点的免疫球蛋白分子的修改配置，包括抗体的糖基化变体、抗体的氨基酸序列变体及共价修饰的抗体。
通常，完整或全长的抗体包含两个重链和两个轻链。每个重链含有重链可变区(VH)和重链恒定区(CH)。每个轻链含有轻链可变区(VL)和轻链恒定区(CL)。全长的抗体可以是任何种类的抗体，例如IgD、IgE、IgG、IgA或IgM(或上述的子类)，但抗体不需要属于任何特定的类别。根据重链的恒定 域的抗体氨基酸序列，可以将免疫球蛋白指定为不同的类别。通常，免疫球蛋白有五种主要的类别：IgA、IgD、IgE、IgG及IgM，而且这些类别中有几个可以再被进一步区分成子类，例如IgG1、IgG2、IgG3、IgG4、IgA1及IgA2。
如本文所用术语“特异性结合”是指两个分子之间的非随机结合反应，例如抗体至抗原表位的结合。
如本文所用术语“抗原结合片段或抗原结合部分”是指负责结合抗原的完整抗体分子的一部分或区域。抗原结合部分可以包含重链可变区(VH)、轻链可变区(VL)或上述两者。VH和VL中的每个通常含有三个互补决定区CDR1、CDR2及CDR3
本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。对于给定抗体的可变区氨基酸序列，可以通过多种方式分析可变区氨基酸序列的中CDR氨基酸序列，例如在本文中利用在线软件Abysis根据Chothia确定(http://www.abysis.org/)。
实施例1动物免疫
设计合成4条多肽，其中3条含有FGF21羧基末端的3个氨基酸(YAS)，1条不含有YAS，其序列如表1所示。
表1合成多肽序列
Figure PCTCN2022086227-appb-000001
将免疫用多肽A分别进行偶联备用于动物免疫。
完成三次免疫后取血测定血清效价。免疫动物的实验分组见表2所示。
表2免疫动物信息
Figure PCTCN2022086227-appb-000002
Figure PCTCN2022086227-appb-000003
利用间接酶联免疫分析方法(间接ELISA)，筛选对免疫用多肽A效价高，且对筛选用多肽B效价低的动物血清。具体步骤包括：分别用免疫用多肽A、筛选用多肽B(SinoA8627)、蛋白A(SEQ ID NO：22)和蛋白B(SEQ ID NO：23)包被酶标板，利用HRP标记山羊抗小鼠IgG Fc抗体(ICL公司，货号：GGPC-90P)作为检测抗体，使用间接ELISA法测定血清效价。筛选出与免疫用多肽A和蛋白A反应信号强，且与筛选用多肽B和蛋白B反应信号弱的动物。血清效价检测结果如表3-表5所示(S：信号值；S-B：扣除本底后信号值)。
表3三免后一周小鼠血清ELISA检测结果
Figure PCTCN2022086227-appb-000004
Figure PCTCN2022086227-appb-000005
表4三免后一周小鼠血清ELISA检测结果
Figure PCTCN2022086227-appb-000006
Figure PCTCN2022086227-appb-000007
表5三免后一周小鼠血清ELISA检测结果
Figure PCTCN2022086227-appb-000008
Figure PCTCN2022086227-appb-000009
实施例2融合杂交瘤细胞及筛选
根据表3-5的结果，选择编号为SBI180064-2#C的小鼠进行注射多肽SinoA8624加强免疫；3天后取脾进行融合，将融合后细胞用HAT培养基悬浮，按照每孔6×10 4个细胞接种于96孔细胞培养板，即共接种30块96孔细胞培养板。分别于融合后第6天、第8天，弃去培养板中培养基，添加新鲜HAT培养基，于融合后第10天取杂交瘤细胞培养上清进行主克隆筛选(结果见表6所示)。
表6主克隆首次筛选ELISA检测结果
Figure PCTCN2022086227-appb-000010
将筛选所得阳性的细胞进行细胞计数，然后按照0.75细胞/孔接种到96孔细胞培养板，每个阳性细胞接种0.5块细胞培养板。培养7天后，显微镜下观察细胞状态，挑选并标记仅含有单个细胞团的孔，取培养上清进行ELISA筛选，筛选结果见表7和表8所示(S：信号值；S-B：扣除本底后信号值)。将筛选所得阳性细胞按上述步骤进行第二次有限稀释，直至获得稳定的阳性单克隆细胞。经间接ELISA筛选，获得表8中的6株杂交瘤细胞上清可以满足与SinoA8624及蛋白A结合，且与SinoA8627及蛋白B不结合。
表7第一次亚克隆ELISA检测结果
Figure PCTCN2022086227-appb-000011
Figure PCTCN2022086227-appb-000012
表8第二次亚克隆ELISA检测结果
Figure PCTCN2022086227-appb-000013
实施例3单克隆抗体与小量培养与纯化
取表8所记载的六株杂交瘤细胞，分别将1mL杂交瘤细胞转入100mL培养瓶中，定期加入一定量的培养基进行细胞扩增，培养10天。培养液离心过滤取上清，经Protein A亲和层析纯化，具体步骤包括：
收集培养料液6000rpm离心20min后，用滤器过滤，取上清；
a、层析柱预处理：Protein A亲和层析柱用超纯水冲洗，然后用平衡缓冲液平衡；
b、上样与平衡：料液上样Protein A亲和层析柱，上样完毕后，用平衡缓冲液淋洗至基线平稳；
c、洗脱：用Elution3.0洗脱，收集洗脱峰，用2M Tris中和；
d、Protein A亲和层析柱再生；
e、纯化样品用脱盐至适当的缓冲液中；
f、纯化样品无菌过滤后留样，并用SDS-PAGE分析纯度，结果见图1所示。
实施例4与HRP标记的小鼠抗人IgG4 Fc抗体配对检测FGF21-Fc融合蛋白
取实施例3纯化得到的单克隆抗体作为包被抗体，以HRP标记的小鼠抗人IgG4 Fc抗体(Southern Biotech，货号9200-05)为检测抗体，使用双抗夹心法ELISA分别检测蛋白A和蛋白B，具体步骤包括：
(1)包被：包被纯化后的小鼠单克隆抗体2μg/mL，100μL/孔，4℃包被过 夜；
(2)封闭：板内液体甩净拍干，加入2％BSA，300μL/孔，密封后室温孵育1h；
(3)洗板：300μL/孔洗涤液，洗板2次，最后一次拍干；
(4)样品准备：将蛋白A及蛋白B分别稀释至5ng/mL、10ng/mL，100μL/孔加入至孔板中，空白对照加入样品稀释液，100μL/孔，混合均匀，室温反应2h；
(5)洗板：300μL/孔洗涤液，洗板3次，最后一次拍干；
(6)加入二抗：HRP标记的小鼠抗人IgG4 Fc单抗(Southern biotech，货号9200-05)按1：2000稀释使用，100μL/孔，密封后室温孵育1h；
(7)洗板：同步骤5；
(8)显色：将A液和B液按1:1混匀后，每孔加入200μL，室温避光孵育20min；
(9)终止：每孔加入50μL终止液，立即在450nm波长处测量OD值，检测结果如表9所示(S：信号值；S-B：扣除本底后信号值)。
表9 6株单克隆抗体与HRP标记的小鼠抗人IgG4 Fc抗体双抗夹心法ELISA检测结果
Figure PCTCN2022086227-appb-000014
根据表9，MM21H、MM22H、MM23H、MM24H、MM25H、MM26H均可以与检测抗体配对检测蛋白A，且不受蛋白B干扰。选择响应较高且背景较低的MM21H和MM24H作为候选单克隆抗体株。
实施例5单克隆抗体测序
对实施例4筛选获得的候选单克隆抗体株MM21H和MM24H委托北京义翘 神州科技股份有限公司(Sino Biological Inc.)进行杂交瘤细胞株抗体亚型检测和测序。分别取培养的MM21H和MM24H杂交瘤细胞上清采用亚型检测试剂盒(Southern Biotech)进行亚型检测，根据表10所示的ELISA检测结果，MM21H和MM24H鼠源单克隆抗体亚型均为IgG1/kappa。
表10单克隆抗体亚型检测结果
Figure PCTCN2022086227-appb-000015
分别将MM21H和MM24H杂交瘤细胞离心后，弃上清，采用TriPure Isolation Reagent试剂盒(Roche)提取细胞RNA，经反转录获得cDNA，以cDNA为模板，依据测定的亚型采用特异引物(Sino Biological Inc.)进行PrimeSTAR(TAKARA)高保真PCR，将扩增获得的基因片段插入到表达载体pcDNA系列载体上，克隆测序获得基因序列。将重组抗体分别命名为MM21T及MM24T，根据测序结果，MM21T的轻链可变区包含以下三个互补决定区：
CDR-L1，其氨基酸序列为RSSKSLLHSNGITYLY(SEQ ID NO：1)，其编码核苷酸序列为SEQ ID NO：24；
CDR-L2，其氨基酸序列为QMSSLAS(SEQ ID NO：2)，其编码核苷酸序列为SEQ ID NO：25；
CDR-L3，其氨基酸序列为AQTLELPT(SEQ ID NO：4)，其编码核苷酸序列为SEQ ID NO：26。
根据测序结果，MM21T的轻链的氨基酸序列为SEQ ID NO：27；其中，第1-19位为信号肽，第20-130位为轻链可变区，第131-237位为轻链恒定区。
编码MM21T的轻链的核苷酸序列为SEQ ID NO：28。
MM21T的重链可变区包含以下三个互补决定区：
CDR-H1，其氨基酸序列为GYTFTNY(SEQ ID NO：5)，其编码核苷酸序列为SEQ ID NO：29；
CDR-H2，其氨基酸序列为NTYTGK(SEQ ID NO：6)，其编码核苷酸序列为SEQ ID NO：30；
CDR-H3，其氨基酸序列为NYYDYDVAY(SEQ ID NO：7)，其编码核苷 酸序列为SEQ ID NO：31。
根据测序结果，MM21T的重链的氨基酸序列为SEQ ID NO：32；其中，第1-19位为信号肽，第20-137位为重链可变区，第138-461位为重链恒定区。
编码MM21T的重链的核苷酸序列为SEQ ID NO：33。
MM24T的轻链可变区包含以下三个互补决定区：
CDR-L1’，其氨基酸序列为RSSKSLLHSNGITYLY(SEQ ID NO：1)，其编码核苷酸序列为SEQ ID NO：24；
CDR-L2’，其氨基酸序列为QMSNLAS(SEQ ID NO：3)，其编码核苷酸序列为SEQ ID NO：34；
CDR-L3’，其氨基酸序列为AQTLELPT(SEQ ID NO：4)，其编码核苷酸序列为SEQ ID NO：26。
根据测序结果，MM24T的轻链的氨基酸序列为SEQ ID NO：35；其中，第1-19位为信号肽，第20-130位为轻链可变区，第131-237位轻链恒定区。
编码MM24T的轻链的核苷酸序列为SEQ ID NO：36。
MM24T的重链可变区包含以下三个互补决定区：
CDR-H1’，其氨基酸序列为GYTFTNY(SEQ ID NO：5)，其编码核苷酸序列为SEQ ID NO：29；
CDR-H2’，其氨基酸序列为NTYTGK(SEQ ID NO：6)，其编码核苷酸序列为SEQ ID NO：37；
CDR-H3’，其氨基酸序列为NYYDYDIAY(SEQ ID NO：8)，其编码核苷酸序列为SEQ ID NO：38。
根据测序结果，MM24T的重链的氨基酸序列为SEQ ID NO：39；其中，第1-19位为信号肽，第20-137位为重链可变区，第138-461位为重链恒定区。
编码MM24T的重链的核苷酸序列为SEQ ID NO：40。
实施例6重组抗体MM21T及MM24T的表达与纯化
用293无血清CD培养基(货号SMM 293-TI)传代培养HEK293细胞，将实施例5构建得到的含目的抗体轻链和重链基因的表达质粒与转染试剂TF1混合后加入HEK293细胞中，转染后第1、3、5天添加293无血清加料液(货号:M293-SUPI-100)。细胞培养7天后进行蛋白纯化。HEK293培养料液离心后， 用滤器过滤，取上清，随后使用Protein A亲和层析柱对收集的细胞培养液进行纯化，并收集吸收峰。获取的蛋白样本采取SDS-PAGE和UV OD280的方法对蛋白表达过程进行监控以及对产品进行QC检测。
SDS-PAGE的检测结果如图2-图3所示，通过SDS-PAGE分析可知，可使用Protein A亲和纯化法从HEK293培养上清液中纯化得到目标抗体。通过非还原胶分析抗体的分子量在150～200KDa区间，符合抗体分子量的特性。非还原胶上靠近抗体主条带下方条带可能是由于不同程度糖基化造成的分子量和移动方式差异，同时，非还原胶上～100KDa处条带可能是由于抗体重链过量表达造成，但总体占比<5％。
实施例7MM24T重组抗体的活性检测
使用ELISA方法检测MM24T重组抗体的活性，分别包被0.1、1、5μg/mL的蛋白A及蛋白B，抗体点样浓度1μg/mL，验证MM24T能够与蛋白A结合与蛋白B不结合，且与MM24H结合能力情况一致，检测结果如表11所示(设置有两个平行试验，表中OD450的结果取平均值)。
表11 MM24T重组表达抗体活性检测验证
Figure PCTCN2022086227-appb-000016
实施例8 MM21T重组抗体的活性检测
使用ELISA方法检测MM21T重组抗体的活性，分别包被0.1、1、5μg/mL的蛋白A及蛋白B，抗体点样浓度1μg/mL，验证MM21T能够与蛋白A结合与蛋白B不结合，检测结果如表12所示(设置有两个平行试验，表中OD450的结果取平均值)。
表12 MM21T重组表达抗体活性检测验证
Figure PCTCN2022086227-appb-000017
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (13)

  1. 一种抗FGF21羧基末端的抗体，其特征在于，其包含含有LCDR1、LCDR2和LCDR3氨基酸序列的轻链可变区以及含有HCDR1、HCDR2和HCDR3的重链可变区；其中，所述LCDR1的氨基酸序列为SEQ ID NO：1，所述LCDR2的氨基酸序列为SEQ ID NO：2或SEQ ID NO：3，所述LCDR3的氨基酸序列为SEQ ID NO：4，所述HCDR1的氨基酸序列为SEQ ID NO：5，HCDR2的氨基酸序列为SEQ ID NO：6，HCDR3的氨基酸序列为SEQ ID NO：7或SEQ ID NO：8。
  2. 如权利要求1所述的抗体，其特征在于，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：9或者SEQ ID NO：10所示。
  3. 如权利要求1所述的抗体，其特征在于，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：11或者SEQ ID NO：12所示。
  4. 如权利要求1所述的抗体，其特征在于，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：9所示，并且所述抗体的重链可变区的氨基酸序列如SEQ ID NO：11所示；或者所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：10所示，并且所述抗体的重链可变区的氨基酸序列如SEQ ID NO：12所示。
  5. 如权利要求1-4任一项所述的抗体，其特征在于，所述抗体为全长抗体、Fab片段、F(ab) 2片段、双链Fv片段或单链Fv片段；优选地，所述抗体为单克隆抗体。
  6. 如权利要求1-4任一项所述的抗体，其特征在于，所述抗体还包括选自kappa或lambda亚型的轻链恒定区；优选地，所述轻链恒定区为kappa亚型；优选地，所述轻链恒定区包括如SEQ ID NO：13或SEQ ID NO：14所示的氨基酸序列。
  7. 如权利要求1-4任一项所述的抗体，其特征在于，所述抗体还包括选自IgG1、IgG2、IgG3、IgG4亚型的重链恒定区；优选地，所述重链恒定区为IgG1亚型；优选地，所述重链恒定区包括如SEQ ID NO：15、SEQ ID NO：16或SEQ ID NO：17所示的氨基酸序列。
  8. 一种核酸分子，其编码权利要求1-7任一项所述的抗体或其抗原结合部分。
  9. 一种载体，其包含权利要求8所述的核酸分子；
    优选地，所述载体包括质粒、噬菌体、植物细胞病毒、哺乳动物细胞病毒或逆转录病毒。
  10. 一种宿主细胞，其含有权利要求8所述的核酸分子，或包含权利要求9所述的载体；
    优选地，所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞或哺乳动物细胞；
    优选地，所述宿主细胞为哺乳动物细胞；
    优选地，所述宿主细胞为HEK293细胞。
  11. 一种检测试剂盒，包含权利要求1-7任一项所述的抗体，权利要求8所述的核酸分子，或权利要求9所述的载体。
  12. 权利要求1-7任一项所述的抗体在诊断或非诊断目的的免疫检测方面的应用；
    优选地，所述免疫检测包括：检测FGF21羧基末端的完整性。
  13. 权利要求1-7任一项所述的抗体用于诊断或非诊断目的的免疫检测方面的用途；
    优选地，所述免疫检测包括：检测FGF21羧基末端的完整性。
PCT/CN2022/086227 2021-04-13 2022-04-12 一种抗fgf21羧基末端的抗体及其应用 WO2022218277A1 (zh)

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