WO2015014228A1 - Glp-1类似物融合蛋白，及其制备方法和用途 - Google Patents

Glp-1类似物融合蛋白，及其制备方法和用途 Download PDF

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WO2015014228A1
WO2015014228A1 PCT/CN2014/082798 CN2014082798W WO2015014228A1 WO 2015014228 A1 WO2015014228 A1 WO 2015014228A1 CN 2014082798 W CN2014082798 W CN 2014082798W WO 2015014228 A1 WO2015014228 A1 WO 2015014228A1
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glp
fusion protein
analogue
hsa
analog
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PCT/CN2014/082798
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English (en)
French (fr)
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黄岩山
杨志愉
徐正学
裘霁宛
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江苏泰康生物医药有限公司
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Priority to US14/909,143 priority Critical patent/US20160280762A1/en
Priority to ES14831257T priority patent/ES2713378T3/es
Priority to JP2016530329A priority patent/JP6519929B2/ja
Priority to EP14831257.2A priority patent/EP3029072B1/en
Publication of WO2015014228A1 publication Critical patent/WO2015014228A1/zh
Priority to US16/596,430 priority patent/US20200109182A1/en

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    • 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
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • GLP-1 analog fusion protein preparation method and use thereof
  • the present invention relates to a novel class of GLP-1 analog fusion proteins and methods for their preparation of such fusion proteins.
  • GLP-1 analog fusion proteins are useful in the treatment of diabetes and a variety of related diseases or disorders. Background technique
  • Glucagon-like peptide-1 (GLP-1) and its analogs such as Exendin-4 are widely used to study type 2 diabetes, since the GLP-1 polypeptide is in vivo by the protease dipeptidyl peptidase IV (DPP- IV) rapid inactivation, short half-life in plasma, making it difficult to be widely used clinically; Exendin-4 is not sensitive to enzymatic degradation of DPP-IV, its stability is increased, but molecular weight is lower (4187 61D), the half-life in the body is short, so it must be injected twice a day, which hinders clinical use. Many efforts are currently being made to solve this technical problem, such as sustained release microspheres, PEG modification, fatty acid chain modification, and albumin fusion. Among them, albumin fusion technology greatly enhances its half-life in vivo by blending with human albumin while maintaining the biological and therapeutic functions of the protein of interest.
  • DPP- IV protease dipeptidyl peptidase IV
  • GLP-1 preparations and their derivatives are practical for the treatment of diabetes, but because diabetic patients must be administered long-term after diagnosis, and receive treatment for life, the safety, economy and ease of use of such preparations are convenient. Sexual requirements are extremely urgent.
  • existing GLP-1/HSA fusion formulations have significant drawbacks.
  • the albumin has a large molecular weight compared to the GLP-1 molecule, and thus the GLP-1/HSA fusion protein has substantially no biological activity due to steric hindrance after fusion.
  • Albugon is a new GLP-1/HSA fusion protein designed by Laurie L. Baggio et al., which inserts an additional GLP-1 molecule as a spacer between them, but its biological activity only retains about 1%. The decrease in biological activity leads to the clinical dose of the large towel H (Laue L. Baggio, Qingling Huang, Theodore J. Brown, and Daniel J. Drucker, DIABETES Vol. 53: 2492-2500 (2004)).
  • GLP-1 analogue exenatide (Byetta®) is clinically administered at a dose of only 5-10 micrograms per day, 1_2 times per day, but the effective dose of Albugon is 4 mg/day, and the moles of synthetic moles are increased. 22 times.
  • a large increase in clinical doses raises two questions: 1. Increase the potential for immunogenicity. It is limited to the injection administration capacity problem, the dose increase will inevitably lead to an increase in the concentration of the pharmaceutical preparation, such as the GLP-1 analog preparation
  • Byetta single administration is only 5-10 micrograms (50 microliters), the concentration is only 0. 25 mg / In milliliters, the clinical single dose of Albugon reaches 30 mg/person and the concentration of the preparation is as high as 30-50 mg/ml.
  • High concentrations of protein preparations are prone to increase protein aggregates during transport and storage. Previous studies have shown that increased therapeutic protein aggregates increase immunogenicity (Anne S. De Groot and David W. Scott, Trends Immunol Vol. 28
  • Recombinant protein multimers initiate B cell and T cell immunity by cross-linking B cell receptors to activate B cell proliferation (Rosenberg, AS Effects of protein aggregates : an immunologic perspective. AAPS J. 8: 501 - 507 (2006) ). Moreover, recombinant protein multimers are also more susceptible to phagocytosis by antigen-presenting cells (APCs), thereby accelerating the drive of dendritic cells (dendritic The maturation of cel l , DC) stimulates multiple immune responses. (Anne S. De Groot and David W. Scott, Trends Immunol Vol. 28 No. 11: 482-490).
  • APCs antigen-presenting cells
  • GLP-1/HSA fusion protein preparations need to be prepared by extremely complex bioengineering techniques, the cost per unit of protein High, the large increase in the dose will make the price of the drug unacceptable to diabetic patients.
  • the object of the present invention is to overcome the deficiencies in the prior art and to design and prepare a novel GLP-1 analogue fusion protein consisting of three regions: GLP-1 analogue-linker-human serum white Protein (HSA). Compared to existing products, this fusion protein has the following outstanding advantages:
  • the GLP-1 analog-containing compound prepared by the present invention has a low production cost, a higher biological activity, and a better in vitro and in vivo stability, and thus is expected to be a better class of diabetes therapeutic drugs.
  • the invention discloses a novel GLP-1 analog fusion protein having the structure: GLP-1 analogue-linker peptide-human serum albumin (HSA).
  • GLP-1 analogue-linker peptide-human serum albumin HSA
  • the GLP-1 analogue fusion protein of the present invention wherein the first region of the structure is a GLP-1 analogue, the sequence of which is shown as SEQ ID NO: 1 : HGEGTFTSDVSSYLEEQAAKEFIAWLVK, or at least 85%, 90% thereof, 95%, or 99% homology; further, 2, 3 GLP-1 or analogs thereof may be included; further, the first region may also be a homologue similar to GLP-1. Exendin - 4.
  • Native GLP-1 is processed in vivo and the mature peptide cleaves the first 6 amino acids from the molecule. Therefore, the first amino acid of GLP-1 is designated as No. 7 according to the art. All amino acids in the polypeptide are numbered consecutively as shown in SEQ ID NO: 1. For example, the 7th position is histidine and the 8th position is glycine.
  • Non-conserved positions in the GLP-1 sequence can be replaced by other amino acids without altering their activity, such as Gly8 ⁇ Ala Ser or Cys; Glu9 ⁇ Asp, Gly, Ser, Cys, Thr, Asp, Gin, Tyr, Ala, Val, He, Leu, Met or Phe; GlylO ⁇ Ser, Cys, Thr, Asp, Glu, Tyr , Ala, Val, Ile Leu Met or Phe; Aspl5 ⁇ Glu; Val l6 ⁇ Leu or Tyr; Serl8 ⁇ Lys ; Glu21 ⁇ Asp ; Gly22 ⁇ Glu or Ser; Glu23 ⁇ Arg ; Ala24 ⁇ Arg ; Lys26 ⁇ Gly, Ser, Cys, Thr, Asp, Glu, Tyr, Ala, Val, lie, Leu, Met, Phe, Arg; Lys34 ⁇ Gly, Ser, Cys, Thr, Asp, Glu, Tyr, Ala, Val, lie, Leu, Met, Phe,
  • Any combination of one or more of Xaa selected from G A S means that Xaa at different positions may be selected from three amino acid residues of G A S, and Xaa at different positions may be identical or inconsistent.
  • linked peptide sequence is selected from the group consisting of:
  • the GLP-1 analog fusion protein, the third region is human serum albumin (HSA). Its sequence is as shown in SEQ ID NO: 2, or at least 85%, 90%, 95%, 99% homology to it. Non-conserved positions in the HSA sequence can be replaced by other amino acids without changing their activity, such as Cys34 ⁇ S er ; Leu407 ⁇ Ala Leu408 ⁇ Val Arg408 ⁇ Val Val409 ⁇ Ala Arg410 ⁇ Ala Lys413 ⁇ Gln; Arg410 ⁇ Ala (PI idge Etc., International Patent W02011051489)
  • the amino acid sequence of the GLP-1 analog fusion protein is selected from the group consisting of SEQ ID NOs: 3-5.
  • a polynucleotide encoding the GLP-1 analog fusion protein is disclosed.
  • the GLP-1 analog fusion protein has the nucleotide coding sequence of SEQ ID NO: 10, and the corresponding protein sequence is SEQ ID NO: 5.
  • the GLP-1 analog fusion protein of the present invention; the nucleotide coding sequence thereof may also be SEQ ID NO: 8, the corresponding protein sequence is SEQ ID NO: 3; or the corresponding protein of SEQ ID NO: 9.
  • the sequence is SEQ ID NO: 4.
  • Nucleotide sequences encoding fusion proteins comprising a GLP-1 analog can be prepared by any suitable technique well known to those skilled in the art. These include, but are not limited to, recombinant DNA techniques, chemical synthesis, and the like; nucleotide sequences having a GLP-1 amino acid sequence can also be first synthesized and then inserted, replaced, or otherwise by site-directed mutagenesis, directed mutagenesis, or by other techniques well known in the art. The sequence is knocked out to obtain the desired nucleotide sequence.
  • nucleotide sequence encoding the carrier protein can be prepared by any suitable technique well known to those skilled in the art.
  • the nucleotide sequence of the carrier protein is a nucleotide sequence encoding human serum albumin or at least 95% of the sequence is identical thereto.
  • a method for producing the aforementioned fusion protein comprising: constructing an expression vector containing a fusion protein gene sequence, and then transforming an expression vector containing the fusion protein gene sequence into a host cell to induce expression,
  • the fusion protein is isolated and isolated from the expression product.
  • An expression vector containing a fusion protein gene sequence can be constructed by first synthesizing a nucleotide sequence encoding a GLP-1 analog, and then merging the nucleotide sequence with a nucleotide sequence encoding human serum albumin to construct an appropriate expression. Obtained in the carrier.
  • Gene sequences expressing a fusion protein of a GLP-1 analog can be obtained by a table well known to those skilled in the art.
  • Systemic expression including but not limited to bacteria transformed with vectors such as recombinant phage, plasmids, yeast transformed with a yeast expression vector, filamentous fungi transformed with a fungal vector, insect cells infected with a viral vector, animal cells, and the like.
  • the expression system is expressed in a secreted form of Pichia pastoris Pichia pas tor is), P. pastoris has high level expression, low cost, and protein having a eukaryotic expression system The advantages of processing, folding, and post-translational modification.
  • cells can be cultured in shake flasks in the laboratory or fermented in fermenters (including continuous, batch, fed-batch, and solid state fermentation).
  • the fusion protein secreted into the culture medium can be purified by methods well known to those skilled in the art, including, but not limited to, ultrafiltration, ammonium sulfate precipitation, acetone precipitation, and ion exchange chromatography, hydrophobic chromatography, reversed phase chromatography, molecular sieve chromatography. Wait.
  • the inventors purified the fusion protein to homogeneity by means of a three-step chromatography using a combination affinity chromatography, hydrophobic chromatography and ion exchange chromatography.
  • the use of the GLP-1 analogue fusion protein for the manufacture of a medicament for the treatment of diabetes and related diseases is disclosed.
  • a pharmaceutical composition comprising the aforementioned GLP-1 analogue fusion protein and at least one pharmaceutically acceptable carrier or excipient is disclosed.
  • the main use of the pharmaceutical composition is in the treatment of diabetes and related diseases.
  • the related diseases include: type 2 diabetes, type I diabetes, obesity, severe cardiovascular events and other serious complications in patients with type 2 diabetes (Madsbad S, Kielgast U, Asmar M, et al. Diabetes Obes Metab. 2011 May; 13 (5): 394-407; Issa CM, Azar ST. Curr Diab Rep, 2012 Oct; 12 (5): 560-567; Neff LM, Kushner RF. Diabetes Metab Syndr Obes, 2010 Jul 20 ; 3 : 263- 273; Sivertsen J, Rosenmeier J, Hoist JJ, et al. Nat Rev Cardiol, 2012 Jan 31 ; 9 (4): 209-222 ).
  • Therapeutic inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, saccharides or derivatives thereof, amino acids or derivatives thereof, surfactants, vegetable oils, waxes, fats, polyhydroxy compounds, for example Polyethylene glycol, alcohols, glycerin, various preservatives, antioxidants, stabilizers, salts, buffers, water, and the like may also be added, which are used to help stabilize the formulation or to help improve the formulation as needed. Activity or its bioavailability.
  • compositions of this invention may be formulated by techniques well known to those skilled in the art, including liquids or gels, lyophilized or otherwise, to produce a storage stable drug suitable for administration to humans or animals.
  • a method of treating diabetes and a related disease thereof comprises administering a GLP-1 analogue fusion protein of the invention to a subject.
  • the aforementioned fusion protein for the treatment of patients including non-insulin dependent and insulin dependent diabetes, obesity and various other diseases can be referred to existing GLP-1 drug preparations such as Byetta® (GLP-1 similar peptide) 5-1 ⁇ / ⁇ Albugon® (GLP-1/HSA fusion protein), Dulaglutide® ((GLP-1/Fc fusion protein)), etc., used in a dose range of 0.05-1 mg / kg.
  • GLP-1 drug preparations such as Byetta® (GLP-1 similar peptide) 5-1 ⁇ / ⁇ Albugon® (GLP-1/HSA fusion protein), Dulaglutide® ((GLP-1/Fc fusion protein)), etc.
  • the proteins of the invention may be administered alone, or in various combinations, and in combination with other therapeutic agents.
  • the present invention uses the following abbreviations:
  • GLP-1 (glucagon like protein_l) ⁇ glucagon-like moon _1; HSA (human serum albumin) human serum albumin.
  • FIG. 1 Electrophoresis pattern of GLP-1 analogue fusion protein expression in different structures. Lanes 1-9 are the expression results of the fusion protein of sequence 1-9 shown in Table 1, respectively.
  • GLP-1-E3-HSA blood glucose level of rhesus monkeys after graded glucose infusion 1 day after subcutaneous injection
  • B blood glucose level of rhesus monkeys after graded glucose infusion 4 days after subcutaneous injection of GLP-1-E3-HSA
  • C Insulin levels in rhesus monkeys after fractional glucose infusion 1 day after subcutaneous injection of GLP-1-E3-HSA
  • D Insulin levels in rhesus monkeys after graded glucose infusion 4 days after subcutaneous injection of GLP-1-E3-HSA
  • Figure 3 Drug-time curve after single administration of macaque
  • GLP-1 analog nucleotide coding sequence (SEQ ID NO: 6): CTTGGCTGGTGAAA
  • the one-line part is the (GLP-1 analog) 2 gene sequence, and the rest is the HSA N-terminal partial coding sequence.
  • the one-line part is the GLP-1 analog _ (Gly 4 Ser) 3 gene sequence, and the rest is the HSA N-terminal partial coding sequence.
  • the one-line part is the GLP-1 analog _ (Gly 4 Ser) 4 gene sequence, and the rest is the HSA N-terminal partial coding sequence.
  • the one-line part is the GLP-1 analog-E1 gene sequence, and the rest is the HSA N-terminal partial coding sequence.
  • the one-line part is the GLP-1 analog-E2 gene sequence, and the rest is the HSA N-terminal partial coding sequence.
  • the rest is the HSA N-terminal partial coding sequence.
  • the one-line part is the GLP-1 analog-E3 gene sequence, and the rest is the HSA N-terminal partial coding sequence.
  • the one-line part is the GLP-1 analog-E4 gene sequence, and the rest is the HSA N-terminal partial coding sequence.
  • the one-line part is the GLP-1 analog-E5 gene sequence, and the rest is the HSA N-terminal partial coding sequence.
  • the one-line part is the GLP-1 analog-E6 gene sequence, and the rest is the HSA N-terminal partial coding sequence.
  • GLP-1/P1 SEQ ID NO: 26
  • HSA/Pl SEQ ID NO: 27 : 5 ' -GATGCACACAAGAGTGAGG-3 '
  • HSA/P2C SEQ ID NO: 28 5 ' - TTAGCGGCCGCTTATAAGCCTAAGGCAGCTTG-3, (Not I restriction site)
  • Human Serum Albumin/HSA/ALB Gene cDNA Clone/ORF Clone gene (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was used as a template, HSA/P1 and HSA/P2 were used as primers to amplify HSA fragments.
  • the PCR system was: template 0. 5 ⁇ 1, 25 ⁇ 1/1 of HSA/Pl and HSA/P2 primers each 1 ⁇ 1, 2mmol/L dNTP 4 ⁇ 1, 5xPS reaction buffer 10 ⁇ 1, PrimeStar DNA polymerase 2. 5 U, ddH20 supplement volume to 50 ⁇ 1.
  • the PCR conditions were: denaturation at 98 ° C for 10 minutes, 68 ° C for 1 minute and 48 seconds, and after 25 cycles, incubation at 4 ° C.
  • the PCR product was subjected to agarose gel electrophoresis to recover a band having a molecular weight of about 1750 bp.
  • the (GLP-1 analog) 2 gene fragment was mixed equimolarly with the PCR product of HSA as a template, GLP-1/P1 and HSA/P2 were primers, and amplified (GLP-1 analog) 2 _HSA, and the PCR system was: 0. 5 ⁇ 1 template, 25 ⁇ 1/1 GLP-1/P1 and HSA/P2 primers each 1 ⁇ 1, 2mmol/L dNTP 4 ⁇ 1, 5xPS reaction buffer 10 ⁇ 1, PrimeStar DNA polymerase 2. 5 U, ddH20 supplement volume to 50 ⁇ 1. PCR conditions: 98 ° C for 10 seconds, 68 ° C for 2 minutes and 30 seconds, after 4 cycles of incubation at 4 ° C. The PCR product was subjected to agarose gel electrophoresis to recover a band having a molecular weight of about 1950 bp.
  • the equimolar mixture of the GLP-1 analogue-(Gly 4 Ser) 3 gene fragment and the PCR product of HSA is used as a template, and the PCR system and PCR conditions are the same as 3. 1.
  • the PCR product is recovered by agarose gel electrophoresis to recover a molecular weight of about 1930 bp. Strips.
  • the equimolar mixture of the GLP-1 analogue-(Gly 4 Ser) 4 gene fragment and the PCR product of HSA is a template, and the PCR system and the PCR conditions are the same as 3. 1.
  • the PCR product is recovered by agarose gel electrophoresis to recover a molecular weight of about 1950 bp. Strips.
  • the equimolar mixture of the GLP-1 analogue-E1 gene fragment and the PCR product of HSA was used as a template, and the PCR system and PCR conditions were the same as 3. 3.
  • the PCR product was subjected to agarose gel electrophoresis to recover a band having a molecular weight of about 1930 bp.
  • the equimolar mixture of the GLP-1 analogue-E2 gene fragment and the PCR product of HSA is a template, and the PCR system and the PCR conditions are the same as 3. 1.
  • the PCR product is subjected to agarose gel electrophoresis to recover a strip having a molecular weight of about 1960 bp. Belt.
  • the equimolar mixture of the GLP-1 analogue-E3 gene fragment and the PCR product of HSA was used as a template, the PCR system and the PCR conditions were the same as 3.1, and the PCR product was subjected to agarose gel electrophoresis to recover a band having a molecular weight of about 1940 bp.
  • the equimolar mixture of the GLP-1 analogue-E4 gene fragment and the PCR product of HSA was used as a template, and the PCR system and PCR conditions were the same as 3.1.
  • the PCR product was subjected to agarose gel electrophoresis to recover a band having a molecular weight of about 1960 bp.
  • the equimolar mixture of the GLP-1 analog-E5 gene fragment and the PCR product of HSA was used as a template, and the PCR system and PCR conditions were the same as 3.1.
  • the PCR product was subjected to agarose gel electrophoresis to recover a band having a molecular weight of about 1930 bp.
  • the equimolar mixture of the GLP-1 analogue-E6 gene fragment and the PCR product of HSA was used as a template, and the PCR system and PCR conditions were the same as 3.1.
  • the PCR product was subjected to agarose gel electrophoresis to recover a band having a molecular weight of about 1970 bp.
  • the expression vector plasmid PPIC9 was first digested with XhoI and Notl. The specific conditions were as follows: Expression vector plasmid pPIC9, 10 ⁇ 1 ; Xhol 1 ⁇ 1, Not I 1 ⁇ 1, 10X digestion buffer ( ⁇ ) 4 ⁇ 1 (purchased from Takara); dd3 ⁇ 40 24 ⁇ 1, total volume 40 ⁇ 1. (GLP-1 analogue) 2 The _HSA fragment was subjected to the same double digestion. The reaction was carried out for 2 hours in a 37 ° C constant temperature water bath, and the linearized plasmid DNA and the (GLP-1 analog) 2 -HSA gene fragment were recovered by agarose gel electrophoresis.
  • the recovered vector and gene fragment were ligated into a fusion protein expression plasmid (GLP-1 analog) 2 -HSA/pPIC9.
  • the ligation system is generally 10 ⁇ l, the molar ratio of the vector to the gene fragment is 1:2-10, 10XT4 DNA ligase buffer 1 ⁇ l, ⁇ 4 DNA ligase 1 ⁇ l, and sterile water is added to the total volume ⁇ .
  • the ligation reaction was carried out in a constant temperature water bath at 16 ° C for 1 hour.
  • the ligation product was transformed into E. coli 7 7 competent cells, and the transformed clones were identified by PCR using universal primers 5' A0X1 and 3' A0X1 as primers.
  • the correct cloned bacterial solution was sent to Kingsray Biotech Co., Ltd., using universal primer 5' A0X1 and 3' A0X1 were sequenced. It was verified by sequencing to meet expectations.
  • the specific conditions of the linearized plasmid were as follows: Expression vector plasmid 60 ⁇ l, Sai l 2. 5 ⁇ 1 , 10 ⁇ buffer ( ⁇ ) 20 ⁇ l, dd 0 was added to a total volume of 200 ⁇ l. The reaction was carried out in a constant temperature water bath at 37 °C for 3 hours.
  • the specific method for preparation of competent cells is as follows: First, the preparation of the cells is carried out, and a single colony of the yeast is picked, inoculated into a 50 ml flask containing 5 ml of YPD medium, and cultured at 30 ° C, 250 rpm overnight.
  • the culture was then inoculated into a 250 ml flask containing 50 ml of YPD medium, 30 ° C, 250 rpm overnight, to 0 D600 reached 1-1.
  • the cell culture was pre-cooled for 10 min on ice, centrifuged at 1500 g for 5 min at 4 ° C, the supernatant was discarded, and the cell pellet was resuspended with 40 ml of pre-cooled sterile water. After centrifugation, the pellet was resuspended in 25 ml of ice-cold sterile water.
  • the pellet was resuspended with 5 ml of ice-cold 1 M sorbitol solution, centrifuged again, and the pellet was resuspended with 80 ⁇ l of ice-cold 1 M sorbitol solution.
  • the specific method of electroporation is as follows: The ⁇ linearized plasmid was mixed with 80 ⁇ l of the above competent cells, and transferred to a 0.2 cm ice pre-cooled electrotransformation cup. The electric conversion cup was ice bathed for 5 minutes, and then an electric shock was applied with a voltage of 1500V.
  • Example 3 Preparation of GLP-1 Analog Fusion Protein
  • the strains expressing each of the GLP-1 analog fusion proteins obtained in Example 2 were inoculated into YPD medium, and cultured at 30 ° C, 220 to 280 rpm, to a wet weight of about 50 g. /L, 10% inoculum into the tank (Biostat C10, Sartorius), 30 ° C, ⁇ ⁇ 5.0, 30% saturation, dissolved oxygen for 20 h, continuous flow of methanol to induce, control dissolved oxygen 40% saturation, induced After 4 h, the temperature was lowered to 22 ° C. After 50 h, the induction was terminated, and the fermentation supernatant was collected by centrifugation at 10,000 g for 15 min.
  • the enzyme substrate (Bright-GloTM Luciferase Assay System, Promega, E2620) was reacted for 2 minutes, transferred to a costar 96-well all-white microplate, and the fluorescence was measured on a multi-plate reader (SpectraMax M5 system, Molecular Device). Value, the dose response curve is plotted against the fluorescence value to determine EC 5 . value.
  • the relative activity of each fusion protein was calculated based on the activity of (GLP-1 analog) 2 _HSA of 100%. The results are shown in Table 1.
  • Gly 4 Ser acts as a linker peptide, and its in vitro activity is substantially similar to that of the GLP-1 analog as a linker peptide; and when a sequence according to claim 1 is inserted between the GLP-1 analog and the HSA ( Sequence E1-E6), the in vitro activity of the fusion protein was increased by about 7-10 fold.
  • GLP-1 analogue fusion protein stock solution Take a highly purified GLP-1 analogue fusion protein stock solution, add appropriate amount of sodium chloride, disodium hydrogen phosphate and sodium dihydrogen phosphate, adjust pH 7.4 with sodium hydroxide or hydrochloric acid, and add water for injection to make GLP per ml. -1 analog protein 5.
  • Omg sodium chloride 9 mg, phosphate 20 ⁇ mol. Filtered with 0.22 ⁇ m PVDF or PES filter, aseptically packaged in a vial in a 100-stage environment, stored in a stability chamber at a storage temperature of 25 ° C, at 0, 1, 3 months Samples were stored in a -70 degree refrigerator for inspection. All samples to be analyzed were combined to detect SDS-PAGE purity and cell biological activity.
  • the SDS-PAGE purity detection method was as described in Example 1, and the sample to be tested was loaded at 10 ug.
  • the in vitro activity assay method was as described in Example 4, with 100% activity at 0 months for each sample.
  • the sample before the activity measurement was separated by a Superdex 75 10/30 molecular sieve column (GE Healthcare) to remove the degradation fragments with a molecular weight of less than 1000OOa to avoid interference with the measurement.
  • the results are shown in Table 2.
  • the GLP-1 analogue was inserted as a linker peptide between the GLP-1 analogue and HSA, the activity retention rate was the worst and the fusion protein was the most unstable.
  • Table 2 0 1 3 0 1 3
  • GLP-1 analogue _ (Gly 4 Ser) 3 -HSA 97. 3 84. 3 67. 3 100 79. 0 47. 2
  • GLP-1 analogue _ (Gly 4 Ser) 4 -HSA 98. 0 89. 4 66. 0 100 85. 5 45. 3
  • the purified high-concentration GLP-1 analogue fusion protein stock was added to monkey serum at a volume ratio of 1:25, sterilized by filtration, and packaged into a vial aseptically, and incubated at 37 °C. On day 0, 15 days, 30 days, samples were stored in a -70 degree refrigerator for testing. All the samples to be analyzed were combined, and the fusion protein concentration was determined by sandwich ELISA using Anti-GLP- ⁇ monoclonal antibody (Antibodyshop) as a capture antibody and Goat ant i -Human Albumin-HRP (Bethyl Laboratories) as a detection antibody.
  • Anti-GLP- ⁇ monoclonal antibody Antibodyshop
  • Goat ant i -Human Albumin-HRP Bethyl Laboratories
  • the capture antibody binds to the GLP-1 analog portion of the fusion protein and the detection antibody binds to the albumin portion, the measured fusion protein concentration is positively correlated with the undegraded portion content.
  • the results are shown in Table 3. After 30 days, most of the monkey serum (GLP-1 analogue) 2- HSA had degraded, while the other samples retained nearly 40%.
  • mice male and female, overnight fasting for 18 hours, subcutaneous injection of 1. 0 mg / kg HSA (control), (GLP-1 analogue) 2 -HSA, GLP-1 analogue-E3-HSA and GLP-1 analogue-E6-HSA.
  • IPGTT glucose tolerance test
  • the blood glucose level of the (GLP-1 analogue) 2 -HSA, GLP-1 analogue-E3-HSA and GLP-1 analogue-E6-HSA group was significantly lower than that of the blood glucose curve.
  • area (AUC._ 12. "") were significantly lower than the control group (results Table 4).
  • Rhesus monkeys were given a subcutaneous injection of 0.5 mg/kg (GLP-1 analogue) 2 _HSA or GLP-1 analogue-E3-HSA, followed by a graded intravenous glucose test after 24 h and 96 h: intravenous glucose Solution (20% dextrose solution, 200 mg/ml) 10 mg/kg/min (3.0 ml/kg/h) for 20 min, followed by glucose solution 25 mg/kg/min (7.5 ml/kg) /h) lasts 20 minutes. Blood was collected at 0, 10, 20, 30, and 40 min after glucose injection to measure blood glucose and insulin.
  • the blood glucose was measured using a YSI 2700 biochemical analyzer, and the insulin assay was performed using an enzyme-linked immunosorbent assay (Insulin ELISA kit, DRG International, Inc.). There was no significant difference in blood glucose between the two groups at each time point after the drug (see Figure 2). There was a significant difference between the groups at 10, 30, 40 min after 4 days of administration (P ⁇ 0.05 or P ⁇ 0.01). The insulin between the groups was significant at the Id and 4d 20 and 40 min time points. Difference (P ⁇ 0.01).
  • ELISA Enzyme-linked immunosorbent assay
  • human serum albumin (final concentration of 60 ⁇ M) was added as an antagonist in the serum samples to be tested under similar measurement conditions to further analyze the antibody specificity produced (see Table 6). The results showed that both antibodies were produced after multiple administrations, and the highest titer reached 1: 6400, and the antibody trends and titers produced by the two were basically the same. Furthermore, human serum albumin (HSA) was added to the serum for antagonistic analysis. The results showed that the serum titer decreased significantly in the presence of HSA, indicating that the antibodies produced were basically antagonized by HSA. Therefore, it is indicated that most of the antibodies produced by repeated injection of the fusion protein in monkeys are directed against the human serum albumin portion of the fusion protein, but not the antibody against the GLP-1 analog portion.
  • HSA human serum albumin
  • ND I ND 1: 1600 1: 1600 NDNDNDND Injection antibody titer ⁇ 1:100 is defined as wood detection (ND)
  • ND wood detection

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Abstract

提供了一种GLP-1类似物融合蛋白及其制备方法和用途，所述融合蛋白由三个区组成：GLP-1类似物-连接肽-HSA，该融合蛋白生产成本低、生物活性高、体内外稳定性好，可用于治疗糖尿病、肥胖和肠易激综合症等。

Description

GLP-1类似物融合蛋白， 及其制备方法和用途 技术领域
本发明涉及一类新型 GLP-1类似物融合蛋白，及其制备这种融合蛋白的方法。 该类 GLP-1类似物融合蛋白用于治疗糖尿病及相关的多种疾病或紊乱。 背景技术
胰高血糖素样肽 -1 (GLP-1 ) 及其类似物例如 Exendin-4被广泛的用来研究治 疗 2型糖尿病，由于 GLP-1多肽在体内被蛋白酶二肽基肽酶 IV (DPP-IV)所迅速失活， 在血浆中的半衰期很短， 使其难以在临床上被广泛使用； 而 Exendin-4对 DPP-IV 的酶降解不敏感， 其稳定性增加， 但分子量较低（4187. 61D) ， 在体内半衰期短， 所以每天必须注射两次， 这阻碍了在临床上使用。 目前许多努力用来解决这个技 术难题， 例如缓释微球、 PEG修饰， 脂肪酸链修饰， 白蛋白融合。 其中白蛋白融合 技术在保持目的蛋白的生物学和治疗功能的同时， 通过与人白蛋白融合大大提高 了其在体内半衰期。
尽管 GLP-1类制剂及其衍生物用于治疗糖尿病是现实可行的. 但是因为糖尿 病患者一经诊断后必须长期持续给药， 终生接受治疗， 因此对于该类制剂的安全 性、经济性和使用便利性的要求都是极其迫切的。但是， 现有的 GLP-1/HSA融合类 制剂还存在很大弊端。
首先， 与 GLP-1分子相比， 白蛋白分子量巨大， 因此两者融合后因为空间位阻 原因导致 GLP-1/HSA融合蛋白基本不具有生物学活性。 Albugon是 Laurie L. Baggio 等设计了一种新的 GLP-1/HSA融合蛋白， 就是在其两者间插入一个额外的 GLP-1分 子作为间隔物， 但其生物学活性仅仅保留了约 1%; 生物学活性的下降导致临床剂 量的大巾 H增力口 ( Laurie L. Baggio, Qingl ing Huang, Theodore J. Brown, and Daniel J. Drucker, DIABETES Vol. 53： 2492-2500 ( 2004) ) 。 例如 GLP-1类似 物埃塞那肽 (Byetta®),临床每次给药剂量仅为 5_10微克，每天 1_2次，但是 Albugon 临床有效给药剂量达到 4毫克 /天， 折合成摩尔数增加了近 22倍。 临床剂量的大幅 增加会带来两个问题： 1.增加潜在的免疫原性风险。 限于注射给药容量问题， 剂 量增大势必导致药物制剂浓度的增加， 如 GLP-1类似物制剂 Byetta单次给药仅为 5-10微克（50微升）， 浓度仅为 0. 25毫克 /毫升， 而临床中 Albugon单次剂量达到 30 毫克 /人，制剂浓度高达 30-50毫克 /毫升。高浓度的蛋白制剂在运输和贮存期间容 易造成蛋白质聚体增加。 已有的研究表明， 治疗用蛋白聚体增加， 会增加免疫原 性 (Anne S. De Groot and David W. Scott, Trends Immunol Vol. 28
No. 11 : 482-490)。 重组蛋白多聚体会通过交联 B细胞受体， 激活 B细胞增生从而启 动 B细胞禾口 T细胞免疫 ( Rosenberg, A. S. Effects of protein aggregates : an immunologic perspective. AAPS J. 8： 501 - 507 (2006) ) 。 而且， 重组蛋白多 聚体也更容易被抗原递呈细胞（APC)所吞噬，从而加快驱使树突细胞（dendritic cel l ， DC)的成熟从而激发多种免疫反应。（Anne S. De Groot and David W. Scott, Trends Immunol Vol. 28 No. 11 : 482-490)。 因此， GLP-1/HSA融合蛋白制剂剂量的 显著增加必然会导致抗体生成的风险加大； 2. GLP-1/HSA融合蛋白类制剂需要通 过极其复杂的生物工程技术制备， 其单位蛋白量成本高昂， 给药剂量的大幅增加 将使得药物价格无法被糖尿病患者所承受。
其次，由于 GLP-1序列大部分呈无规则卷曲构象，极易受蛋白酶攻击而被降解， 因此额外加入的第二个 GLP-1使得 Albugon更易受到蛋白酶攻击而不稳定。 这个不 稳定在两个方面都体现了弊端： 1.在进行重组表达 Albugon时，无论是生产成本低 廉的酵母表达系统还是高昂的哺乳动物细胞表达系统， 分泌在培养上清中的 GLP-1/HSA融合蛋白易受蛋白酶降解，既导致表达量下降，也会产生很多不均一酶 解产物而使得最终产物不均一； 2. Albugon注射进入体内后在体内循环时也易受 蛋白酶降解而失效。
此外， 限于制品稳定性问题， 目前该类制品都需要低温保存和运输， 因此对 于糖尿病人外出旅行携带极不方便。 发明内容
本发明的目的是克服现有技术中的缺陷， 设计并制备了一种新型的 GLP-1类 似物融合蛋白， 该融合蛋白由三个区域组成： GLP-1 类似物-连接肽 -人血清白蛋 白 （HSA) 。 与现有产品相比， 该融合蛋白具有如下突出优点：
1.更好的热稳定性，可以在室温条件下长期保存而活性不下降，便于病人随身 携带使用
2.更好的耐蛋白酶稳定性， 在发酵上清及体内稳定性是现有该类融合蛋白的 3 倍以上， 易于工业化制备。
3.更高的生物学活性， 其生物学活性是现有该类融合蛋白的 10倍以上。
应用本发明制备的包含 GLP-1类似物的化合物具有很低的生产成本， 更高的 生物学活性， 更优的体内外稳定性， 从而有望成为一类更佳的糖尿病治疗药物。
本发明一方面公开了一种新型的 GLP-1类似物融合蛋白，该融合蛋白结构为： GLP-1类似物-连接肽-人血清白蛋白 （HSA) 。
本发明所述的 GLP-1类似物融合蛋白，其结构中第一个区域为 GLP-1类似物， 其序列如 SEQ ID NO: 1 ： HGEGTFTSDVSSYLEEQAAKEFIAWLVK所示， 或者至少与其 保持 85%， 90%, 95%, 或 99%的同源性； 进一步的， 也可包含 2个， 3个 GLP-1或 其类似物重复序列； 进一步的， 第一区域也可以是与 GLP-1 功能相似的同系物 Exendin - 4。
天然 GLP-1在体内经过加工， 成熟肽从该分子切割前 6个氨基酸。 因此， 按 照本领域习惯将 GLP-1的第一个氨基酸指定为 7号。 如 SEQ ID NO: 1中所示对该 多肽中的所有氨基酸连续编号。例如， 第 7位为组氨酸， 第 8位为甘氨酸。 GLP-1 序列中的非保守位置可以被其他氨基酸所替换而不改变其活性， 如 Gly8→Ala Ser或 Cys; Glu9→Asp, Gly, Ser, Cys, Thr, Asp, Gin, Tyr, Ala, Val , He, Leu, Met或 Phe; GlylO→Ser, Cys, Thr, Asp, Glu, Tyr, Ala, Val , Ile Leu Met或 Phe; Aspl5→Glu; Val l6→Leu或 Tyr; Serl8→Lys; Glu21→Asp; Gly22→Glu或 Ser; Glu23→Arg; Ala24→Arg; Lys26→Gly, Ser, Cys, Thr, Asp, Glu, Tyr, Ala, Val , lie, Leu, Met, Phe, Arg; Lys34→Gly, Ser, Cys, Thr, Asp, Glu, Tyr, Ala, Val , lie, Leu, Met, Phe, Arg; Arg36→Gly, Ser, Cys, Thr, Asp, Glu, Tyr, Ala, Val , He, Leu, Met, Phe, lys GLP- 1序列 C末端 亦可以缺失 1 2个或 3个氨基酸。 (Wolfgang Glaesner等，美国专利 US7452966) 所述的 GLP-1类似物融合蛋白， 其结构中第二个区域为一个长度不超过 26个 氨基酸的连接肽， 具有如下通式： （Xaa) x- (Pro) y- (Xaa) z, 其中 Xaa选自 G A S中的一种或几种的任意组合， x y z均为整数， 且 x z ^3, 26^x+y+z ^ l4, 10^y^3, l y/ (X+Z) 0. 13。 连接肽 N端与第一区域 C末端以肽键相连， C端 与 HSA的 N端以肽键相连。
Xaa选自 G A S中的一种或几种的任意组合是指： 不同位置的 Xaa均可从 G A S这三种氨基酸残基中任选， 不同位置的 Xaa可以一致也可以不一致。
进一步的， 所述连接肽序列选自：
a) GGGSSPPPGGGGSS ( SEQ ID NO: 11 )
b) GGGSSGGGSSPPPAGGGSSGGGSS ( SEQ ID NO: 12)
c) GGGAPPPPPPPPPPSSGGG ( SEQ ID NO: 13)
d) AGGGAAGGGSSGGGPPPPPGGGGS ( SEQ ID NO: 14)
e) GGSSGAPPPPGGGGS ( SEQ ID NO: 15)
f) GGGSSGAPPPSGGGGSGGGGSGGGGS ( SEQ ID NO: 16)
所述的 GLP-1类似物融合蛋白， 第三个区域为人血清白蛋白 （HSA)。 其序列 如 SEQ ID NO: 2 所示， 或者至少与其保持 85% 90%, 95%, 99%的同源性。 HSA 序列中的非保守位置可以被其他氨基酸所替换而不改变其活性， 如 Cys34→Ser ; Leu407→Ala Leu408→Val Arg408→Val Val409→Ala Arg410→Ala Lys413→Gln; Arg410→Ala (PI idge等， 国际专利 W02011051489)
SEQ ID NO: 2 ADDKETCFAEEGKKLVAASQAALGL
在本案的较佳案例中，所述的 GLP-1类似物融合蛋白的氨基酸序列选自 SEQ ID NO: 3—5。
SEQ ID NO:
Figure imgf000005_0001
GL
c) SEQ ID NO: 5： AASQAALGL
本发明第二方面， 公开了一种多核苷酸， 其编码所述 GLP-1类似物融合蛋白。 在本案的较佳实例中， 所述的 GLP-1类似物融合蛋白， 其核苷酸编码序列为 SEQ ID NO: 10， 其对应的蛋白序列为 SEQ ID NO: 5。 本发明的 GLP-1类似物融 合蛋白； 其核苷酸编码序列还可为 SEQ ID NO: 8， 其对应的蛋白序列为 SEQ ID NO: 3； 或者为 SEQ ID NO: 9， 其对应的蛋白序列为 SEQ ID NO: 4。
-1类似物融合蛋白核苷酸编码序列
Figure imgf000006_0001
SEQ ID NO: 9. GLP-1类似物融合蛋白核苷酸编码序列
Figure imgf000007_0001
GGCTTATAA
SEQ ID NO: 10. GLP-1类似物融合蛋白核苷酸编码序列
GCTGCAAGTCAAGCTGCCTTAGGCTTATAA
编码含有 GLP-1类似物融合蛋白的核苷酸序列可以通过本领域技术人员熟知 的任何适当的技术制备。 包括但不限于重组 DNA技术、 化学合成等方法； 也可以 先合成具有 GLP-1氨基酸序列的核苷酸序列， 然后通过定点突变、 定向诱变或以 其它本领域熟知的技术来插入、 替换、 剔除序列以获得所需的核苷酸序列。
编码载体蛋白的核苷酸序列可以通过本领域技术人员熟知的任何适当的技术 制备。 在本发明的一个具体实施例中， 载体蛋白的核苷酸序列为编码人血清白蛋 白的核苷酸序列或至少 95 %的序列与其一致。
编码 GLP-1类似物核苷酸序列和编码载体蛋白的核苷酸序列之间的融合技术 见于本领域的一般描述，如《分子克隆实验指南》（J.萨姆布鲁克等，科学出版社， 1995 )
本发明第三方面， 公开了一种制备前述融合蛋白的方法， 该方法包括： 构建 含有融合蛋白基因序列的表达载体， 然后将含有融合蛋白基因序列的表达载体转 化至宿主细胞中诱导表达， 从表达产物中分离获得所述的融合蛋白。
构建含有融合蛋白基因序列的表达载体， 可通过先合成编码 GLP-1类似物的 核苷酸序列， 而后将此核苷酸序列与编码人血清白蛋白的核苷酸序列融合并构建 至合适表达载体中获得。
有表达 GLP-1类似物融合蛋白的基因序列可以通过本领域技术人员熟知的表 达系统表达， 包括但不限于用重组噬菌体、 质粒等载体转化的细菌， 用酵母表达 载体转化的酵母， 用真菌载体转化的丝状真菌、 用病毒载体感染的昆虫细胞、 动 物细胞等等。 在本发明的一个具体实施例中， 表达系统选用巴斯德毕赤酵母 Pichia pas tor is) 分泌形式表达， 巴斯德毕赤酵母具有高水平表达、 成本低、 以及具有真核表达系统的蛋白质加工、折叠、翻译后修饰的优点。在具体生产中， 细胞可以在实验室中通过摇瓶培养， 或者通过发酵罐发酵培养（包括连续、分批、 补料分批和固体状态发酵）。
分泌到培养基的融合蛋白可以通过本领域技术人员熟知的方法纯化， 包括担 不限于超滤、硫酸铵沉淀、 丙酮沉淀、 以及离子交换层析、 疏水层析、 反相层析、 分子筛层析等。 在本发明的一个具体实施例中， 发明者采用联合亲和层析、 疏水 层析和离子交换层析的三步层析手段使融合蛋白纯化到均一。
本发明第四方面， 公开了所述 GLP-1类似物融合蛋白在制备治疗糖尿病及相 关疾病药物上的用途。
本发明第五方面， 公开了一种药物组合物， 含有前述 GLP-1类似物融合蛋白 及至少一种药学可接受的载体或赋形剂。
所述药物组合物的主要用途为治疗糖尿病及相关疾病。 所述相关疾病包括： Π型糖尿病、 I型糖尿病、 肥胖、 II型糖尿病患者严重心血管事件和其他严重并 发症等 (Madsbad S, Kielgast U， Asmar M， et al. Diabetes Obes Metab. 2011 May； 13 (5)： 394-407； Issa CM, Azar ST. Curr Diab Rep, 2012 Oct； 12 (5)： 560-567； Neff LM， Kushner RF. Diabetes Metab Syndr Obes, 2010 Jul 20 ; 3 : 263-273； Sivertsen J, Rosenmeier J, Hoist JJ, et al. Nat Rev Cardiol, 2012 Jan 31 ; 9 (4) : 209-222 ) 。
本领域枝术人员已知的治疗惰性的无机或有机的载体包括 (但不限于)糖类或 其衍生物、 氨基酸类或其衍生物、 表面活性剂， 植物油、 蜡、 脂肪、 多羟基化合 物例如聚乙二醇、 醇类、 甘油， 各种防腐剂、 抗氧化剂、 稳定剂、 盐、 缓冲液， 水、 诸如此类也可加入其中， 这些物质根据需要用于帮助配方的稳定性或有助于 提高活性或它的生物有效性。
本发明的药物组合物可以用本领域技术人员所熟知的技术配制， 包括液体或 凝胶体， 冻干的或其他形式， 以产生储存稳定的适合对人或动物给药的药物。
本发明第六方面， 公开了一种治疗糖尿病及其相关疾病的方法， 为向对象施 用本发明的 GLP-1类似物融合蛋白。
前述融合蛋白用于治疗包括患有非胰岛素依赖型和胰岛素依赖型糖尿病、 肥 胖及多种其他疾病患者的方法可参考现有的 GLP-1 类药物制剂， 如 Byetta® (GLP-1类似肽）， Albugon® (GLP-1/HSA融合蛋白）， Dulaglutide® ( (GLP- 1/Fc 融合蛋白） ） 等， 其使用剂量范围为 0. 05-1毫克 /公斤。
本发明的蛋白质可以单独给药， 或以各种组合给药， 以及与其它治疗药剂一 起结合形式给药。 本发明使用以下缩写：
GLP-1 (glucagon like protein_l)姨高血糖素样月太 _1 ; HSA (human serum albumin) 人血清白蛋白。 附图说明
图 1 不同结构 GLP-1类似物融合蛋白表达电泳图， 泳道 1-9分别为表 1所述 1-9 号序列融合蛋白表达结果。
图 2正常恒河猴单次皮下注射 GLP-1类似物融合蛋白药效动力学试验
A: GLP-1-E3-HSA 皮下注射 1天后进行分级葡萄糖输注时恒河猴的血糖水平 B : GLP-1-E3-HSA皮下注射 4天后进行分级葡萄糖输注时恒河猴的血糖水平 C: GLP-1-E3-HSA皮下注射 1天后进行分级葡萄糖输注时恒河猴的胰岛素水平 D : GLP-1-E3-HSA皮下注射 4天后进行分级葡萄糖输注时恒河猴的胰岛素水平 图 3猕猴单次给药后药物 -时间曲线图
序列说明：
SEQ ID NO: 1. GLP-1类似物氨基酸序列
SEQ ID NO: 2. HSA氨基酸序列
SEQ ID NO: 3. GLP-1类似物融合蛋白氨基酸序列
SEQ ID NO: 4. GLP-1类似物融合蛋白氨基酸序列
SEQ ID NO: 5. GLP-1类似物融合蛋白氨基酸序列
SEQ ID NO: 6. GLP-1类似物核苷酸编码序列
SEQ ID NO: 7. HSA核苷酸编码序列
SEQ ID NO: 8. GLP-1类似物融合蛋白核苷酸编码序列
SEQ ID NO: 9. GLP-1类似物融合蛋白核苷酸编码序列
SEQ ID NO: 10. GLP-1类似物融合蛋白核苷酸编码序歹
具体实施方式
以下通过特定的具体实例说明本发明的实施方式， 本领域技术人员可由本说 明书所揭露的内容轻易地了解本发明的其他优点与功效。 本发明还可以通过另外 不同的具体实施方式加以实施或应用， 本说明书中的各项细节也可以基于不同观 点与应用， 在没有背离本发明的精神下进行各种修饰或改变。
除非另外说明， 本发明中所公开的实验方法、检测方法、制备方法均采用本 技术领域常规的分子生物学、 生物化学、 染色质结构和分析、 分析化学、 细胞培 养、 重组 DNA技术及相关领域的常规技术。 实施例 1 重组融合蛋白表达质粒构建
GLP-1类似物核苷酸编码序列 （SEQ ID NO: 6)： CTTGGCTGGTGAAA
1.1 3' 端带 HSA融合片段的 (GLP-1类似物 )2基因片段:
人工合成如下寡核苷酸序列 （SEQ ID NO: 17)：
CTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGATGCACACAAGAGTGAGG
其中，单划线部分为 (GLP-1类似物 )2基因序列，其余部分为 HSA N端部分编码 序列。
1.2 3' 端带 HSA融合片段的 GLP-1类似物 -( Gly4Ser) 3基因片段
人工合成如下寡核苷酸序列 （SEQ ID NO: 18)：
ACAAGAGTGAGG
其中，单划线部分为 GLP-1类似物 _( Gly4Ser)3基因序列，其余部分为 HSA N 端部分编码序列。
1.3 3' 端带 HSA融合片段的 GLP-1类似物 -( Gly4Ser) 4基因片段
人工合成如下寡核苷酸序列 （SEQ ID NO: 19)：
GGGGTTCAGATGCACACAAGAGTGAGG
其中，单划线部分为 GLP-1类似物 _( Gly4Ser)4基因序列，其余部分为 HSA N 端部分编码序列。
1.4 3' 端带 HSA融合片段的 GLP-1类似物 -E1基因片段
Figure imgf000011_0001
AGAGTGAGG
其中，单划线部分为 GLP-1类似物 -E1基因序列，其余部分为 HSA N端部分编 码序列。
1.5 3' 端带 HSA融合片段的 GLP-1类似物 -E2基因片段
Figure imgf000011_0002
CTTCAGGTGGAGGCTCTTCAGATGCACACAAGAGTGAGG
其中，单划线部分为 GLP-1类似物 -E2基因序列，其余部分为 HSA N端部分编 码序列。 1.6 3' 端带 HSA融合片段的 GLP-1类似物 -E3基因片段
Figure imgf000012_0001
GCGGTGATGCACACAAGAGTGAGG
其中，单划线部分为 GLP-1类似物 -E3基因序列，其余部分为 HSA N端部分编 码序列。
1.7 3' 端带 HSA融合片段的 GLP-1类似物 -E4基因片段
Figure imgf000012_0002
CACCAGGAGGCGGGGGTTCAGATGCACACAAGAGTGAGG
其中，单划线部分为 GLP-1类似物 -E4基因序列，其余部分为 HSA N端部分编 码序列。
1.8 3' 端带 HSA融合片段的 GLP-1类似物 -E5基因片段
Figure imgf000012_0003
ACAAGAGTGAGG
其中，单划线部分为 GLP-1类似物 -E5基因序列，其余部分为 HSA N端部分编 码序列。
1.9 3' 端带 HSA融合片段的 GLP-1类似物 -E6基因片段
Figure imgf000012_0004
GTGGAGGTTCCGGAGGCGGGGGTTCAGATGCACACAAGAGTGAGG
其中，单划线部分为 GLP-1类似物 -E6基因序列，其余部分为 HSA N端部分编 码序列。
El: GGGSSPPPGGGGSS (SEQ ID NO: 11)
E2: GGGSSGGGSSPPPAGGGSSGGGSS (SEQ ID NO: 12)
E3: GGGAPPPPPPPPPPSSGGG (SEQ ID NO: 13)
E4: AGGGAAGGGSSGGGPPPPPGGGGS (SEQ ID NO: 14)
E5: GGSSGAPPPPGGGGS (SEQ ID NO: 15)
E6: GGGSSGAPPPSGGGGSGGGGSGGGGS (SEQ ID NO: 16)
注： (Xaa) x- (Pro)y- (Xaa)z, 其中 Xaa为 G， A， S中的一种或几种的任意组合， X, z^3, 26^x+y+z^l4, 10^y^3, l^y/(x+z) ^O.13, 。 连接肽 N端与第一区 C末 端以肽键相连， C端与 HSA的 N端以肽键相连。
增强域 X Y Z X+Y+Z Y/X+Z
E1 5 3 6 14 0. 272727
E2 10 3 11 24 0. 142857
E3 4 10 7 21 0. 909091
E4 14 5 5 24 0. 263158
E5 6 4 5 15 0. 363636
E6 7 3 16 26 0. 130435
2. HSA基因的扩增
Figure imgf000013_0001
GCTTATAA 引物设计：
GLP-1/P1 ( SEQ ID NO : 26 ) ：
5 ' - TCTCTCGAGAAAAGACACGGCGAAGGGACCTTTACCAGTG- 3， ( Xhol酶切位点）
HSA/Pl ( SEQ ID NO : 27 ): 5 ' -GATGCACACAAGAGTGAGG- 3 '
HSA/P2C SEQ ID NO : 28)： 5 ' - TTAGCGGCCGCTTATAAGCCTAAGGCAGCTTG- 3， ( Not I 酶切位点）
以 Human Serum Albumin/HSA/ALB Gene cDNA Clone/ORF Clone基因 （北京 义翘神州生物技术有限公司）为模板， HSA/P1与 HSA/P2为引物， 扩增 HSA片断， PCR体系为： 模板 0. 5μ1， 25μπιο1/1的 HSA/Pl与 HSA/P2引物各 1μ1， 2mmol/L 的 dNTP 4μ1， 5xPS反应缓冲液 10μ1， PrimeStar DNA聚合酶 2. 5 U， ddH20补充 体积至 50μ1。
PCR条件为： 98°C变性 10分钟， 68°C 1分 48秒， 25个循环后， 4°C保温。 PCR 产物用琼脂糖凝胶电泳切胶回收分子量约 1750bp的条带。
3. 融合基因的扩增
3. 1 (GLP-1类似物 ) 2_HSA融合基因的扩增
将 (GLP-1类似物 ) 2基因片段与 HSA的 PCR产物等摩尔混合作为模板， GLP-1/P1 和 HSA/P2为引物，扩增（GLP-1类似物）2_HSA， PCR体系为： 0. 5μ1模板， 25μπιο1/1 的 GLP-1/P1和 HSA/P2引物各 1μ1， 2mmol/L的 dNTP 4μ1， 5xPS反应缓冲液 10μ1， PrimeStar DNA聚合酶 2. 5 U， ddH20补充体积至 50μ1。 PCR条件： 98 °C 10秒， 68 °C 2分 30秒， 25个循环后 4°C保温。 PCR产物用琼脂糖凝胶电泳切胶回收分子 量约 1950bp的条带。
3. 2 GLP-1类似物 _ ( Gly4Ser) 3-HSA融合基因的扩增
GLP-1类似物 - ( Gly4Ser) 3基因片段与 HSA的 PCR产物的等摩尔混合物为模板， PCR体系及 PCR条件同 3. 1，PCR产物用琼脂糖凝胶电泳切胶回收分子量约 1930bp 的条带。
3. 3 GLP-1类似物 _ ( Gly4Ser) 4-HSA融合基因的扩增
GLP-1类似物 - ( Gly4Ser) 4基因片段与 HSA的 PCR产物的等摩尔混合物为模板， PCR体系及 PCR条件同 3. 1，PCR产物用琼脂糖凝胶电泳切胶回收分子量约 1950bp 的条带。
3. 4 GLP-1类似物 -E1-HSA融合基因的扩增
GLP-1类似物 -E1基因片段与 HSA的 PCR产物的等摩尔混合物为模板， PCR体 系及 PCR条件同 3. 1， PCR产物用琼脂糖凝胶电泳切胶回收分子量约 1930bp的条 带。
3. 5 GLP-1类似物 -E2-HSA融合基因的扩增
GLP-1类似物 -E2基因片段与 HSA的 PCR产物的等摩尔混合物为模板， PCR体 系及 PCR条件同 3. 1， PCR产物用琼脂糖凝胶电泳切胶回收分子量约 1960bp的条 带。
3.6 GLP-1类似物 -E3-HSA融合基因的扩增
GLP-1类似物 -E3基因片段与 HSA的 PCR产物的等摩尔混合物为模板， PCR体 系及 PCR条件同 3.1， PCR产物用琼脂糖凝胶电泳切胶回收分子量约 1940bp的条 带。
3.7 GLP-1类似物 -E4-HSA融合基因的扩增
GLP-1类似物 -E4基因片段与 HSA的 PCR产物的等摩尔混合物为模板， PCR体 系及 PCR条件同 3.1， PCR产物用琼脂糖凝胶电泳切胶回收分子量约 1960bp的条 带。
3.8 GLP-1类似物 -E5-HSA融合基因的扩增
GLP-1类似物 -E5基因片段与 HSA的 PCR产物的等摩尔混合物为模板， PCR体 系及 PCR条件同 3.1， PCR产物用琼脂糖凝胶电泳切胶回收分子量约 1930bp的条 带。
3.9 GLP-1类似物 -E6-HSA融合基因的扩增
GLP-1类似物 -E6基因片段与 HSA的 PCR产物的等摩尔混合物为模板， PCR体 系及 PCR条件同 3.1， PCR产物用琼脂糖凝胶电泳切胶回收分子量约 1970bp的条 带。
4. 融合蛋白表达质粒的构建
4.1 (GLP-1类似物）2_HSA表达质粒的构建
先对表达载体质粒 PPIC9进行 XhoI、 Notl双酶切。 具体条件如下： 表达载体 质粒 pPIC9， 10μ1; Xhol 1μ1， Not I 1μ1， 10X酶切缓冲液（Η) 4μ1 (购自 Takara 公司）； dd¾0 24μ1， 总体积 40μ1。 （GLP-1类似物) 2_HSA片段做同样的双酶切。 37°C恒温水浴箱内反应 2 小时， 通过琼脂糖凝胶电泳回收线性化的质粒 DNA和 (GLP-1类似物 )2-HSA基因片段。将回收的载体和基因片段连接构建成融合蛋白表 达质粒 (GLP-1类似物 )2-HSA/pPIC9。连接体系一般为 10μ1， 载体和基因片段的摩 尔比为 1:2-10， 10XT4 DNA ligase缓冲液 1μ1， Τ4 DNA ligase 1μ1， 加无菌水 至总体积 ΙΟμΙ。连接反应于 16°C恒温水浴锅内反应 1小时。连接产物转化 E. coli 7 7 感受态细胞，转化克隆斑用通用引物 5' A0X1和 3' A0X1为引物 PCR鉴定， 将鉴定正确的克隆的菌液送金斯瑞生物技术公司， 使用通用引物 5' A0X1 和 3' A0X1进行测序。 经测序验证符合预期。
4.2 GLP-1类似物 _( Gly4Ser)3-HSA表达质粒的构建
除融合蛋白基因片段采用 GLP-1类似物 _( Gly4Ser)3-HSA融合基因替代外， 其余均同 4.1， 测序验证符合预期。
4.3 GLP-1类似物 _( Gly4Ser)4-HSA表达质粒的构建
除融合蛋白基因片段采用 GLP-1类似物 _( Gly4Ser)4-HSA融合基因替代外， 其余均同 4.1， 测序验证符合预期。 4. 4 GLP-1类似物 -El-HSA表达质粒的构建
除融合蛋白基因片段采用 GLP-1类似物- -E1-HSA融合基因替代外， 其余均同 4. 1， 测序验证符合预期。
4. 5 GLP-1类似物 -E2-HSA表达质粒的构建
除融合蛋白基因片段采用 GLP-1类似物- ■E2-HSA融合基因替代外， 其余均同 4. 1， 测序验证符合预期。
4. 6 GLP-1类似物 -E3-HSA表达质粒的构建
除融合蛋白基因片段采用 GLP-1类似物- •E3-HSA融合基因替代外， 其余均同 4. 1， 测序验证符合预期。
4. 7 GLP-1类似物 -E4-HSA表达质粒的构建
除融合蛋白基因片段采用 GLP-1类似物- •E4-HSA融合基因替代外， 其余均同 4. 1， 测序验证符合预期。
4. 8 GLP-1类似物 -E5-HSA表达质粒的构建
除融合蛋白基因片段采用 GLP-1类似物- •E5-HSA融合基因替代外， 其余均同 4. 1， 测序验证符合预期。
4. 9 GLP-1类似物 -E6-HSA表达质粒的构建
除融合蛋白基因片段采用 GLP-1类似物- •E6-HSA融合基因替代外， 其余均同 4. 1， 测序验证符合预期。 实施例 2融合蛋白表达工程菌的构建
分 别 挑 选 含 有 （GLP-1 类 似 物 ）2-HSA/pPIC9 、 GLP-1 类 似 物 - (Gly4Ser) 3- HSA/pPIC9、 GLP- 1 类似物-（ Gly4Ser) 4- HSA/pPIC9、 GLP- 1 类似物 -El-HSA/pPIC9、GLP-l类似物 _E2-HSA/pPIC9、GLP_l类似物 _E3-HSA/pPIC9、GLP_l 类似物 _E4-HSA/pPIC9、GLP-l类似物 _E5-HSA/pPIC9、GLP_l类似物 _E6_HSA/pPIC9 表达载体质粒的细菌克隆，分别提取各表达载体质粒，然后分别用 Sai l线性化各 质粒， 通过琼脂糖凝胶电泳回收线性化的各质粒 DNA， 最后用电转化的方法分别 把各线性化质粒转入毕赤酵母 GS115 感受态细胞中。 电击完后马上加入 1ml 1M 山梨醇溶液将菌体混匀， 转至 1. 5ml离心管中， 30 °C静置 1. 5小时后将菌体悬液 涂布于 RDB选择性平板上， 每 300μ1涂布一块平板。将平板置于 30 °C培养， 直至 单菌落出现， 将获得的阳性菌落转至新鲜的 RDB平板上培养 24h后， 分别挑取在 RDB平板上长出对应各 GLP-1类似物融合蛋白的单菌落接于 10ml BMGY培养基中， 30 V , 250rpm培养 24小时， 静置沉淀后弃上清， 用 10ml BMMY ( 2%甲醇） 重悬， 30 V , 250rpm诱导 48小时， 离心取上清进行 10%SDS_PAGE电泳检测融合蛋白的 表达， 如果条带大小符合预期， 将融合蛋白 N端测序， 测序结果符合预期的单菌 落即为各 GLP-1类似物融合蛋白的表达工程菌株。
线性化质粒具体条件如下： 表达载体质粒 60μ1， Sai l 2. 5μ1 , 10χ缓冲液（Η) 20μ1， dd 0补至总体积 200μ1。 37 °C恒温水浴箱内反应 3小时， 感受态细胞制备具体方法如下： 先进行菌体的准备， 挑取酵母单菌落， 接种 至含有 5ml YPD培养基的 50ml三角瓶中， 30°C、 250rpm培养过夜。 然后取 30μ1 的培养物接种至含有 50ml YPD培养基的 250ml三角瓶中， 30°C、 250rpm培养过 夜， 至 0D600达到 1-1. 5。 再将细胞培养物于冰上预冷 lOmin后， 于 4°C， 1500g 离心 5min，弃上清，用 40ml的预冷的无菌水将菌体沉淀重悬。离心后，再用 25ml 的冰预冷的无菌水将菌体沉淀重悬。再离心，用 5ml的冰预冷的 1M的山梨醇溶液 将菌体沉淀重悬， 再离心， 用 80μ1的冰预冷的 1M山梨醇溶液将菌体沉淀重悬。
电转化具体方法如下： 将 ΙΟμΙ线性化质粒与 80μ1的上述感受态菌体混匀， 转至 0. 2cm冰预冷的电转化杯中。将电转化杯冰浴 5min， 然后用 1500V电压进行 电击。 实施例 3 GLP-1类似物融合蛋白的制备
参考毕赤酵母表达手册 （A Manual of Methods for Expression of
Recombinant Proteins in Pichia pas tori s. Invitrogen Corporation) 将实施 例 2获得的表达各 GLP-1类似物融合蛋白的菌种接种于 YPD培养基， 30°C 220〜 280rpm 振荡培养至菌体湿重约 50g/L， 按 10 %接种量进罐 （Biostat C10, Sartorius) , 30°C， ρΗ5. 0, 30%饱和度溶氧培养 20h后连续流加甲醇开始诱导， 控制溶氧 40 %饱和度，诱导 4h后将温度降低到 22°C， 50h后结束诱导，用 10000g 离心 15min收集发酵上清液。
纯化采用 BLUE亲和、 PHE疏水、 DEAE离子交换和凝胶排阻四步层析。 首先将 发酵上清液用 20 mM pH 7. 0磷酸钠溶液稀释 3倍， 过 Blue Sepharose Fast Flow (XK 50/20, GE healthcare)亲和层析柱， 用 PBS平衡后， 再用 2M NaCl , 20 mM pH 6. 5磷酸钠溶液洗脱目的蛋白。 向所收蛋白溶液中加入 （NH4) 2S04， 使终浓度达到 0. 5M, 过 PHE Sepharose Fast Flow (XK 50/20, GE healthcare)层析柱， 经 0. 6M (NH4) 2S04溶液平衡后， 用 5 mM pH 6. 5 磷酸钠缓冲液洗脱蛋白。 将收到的蛋白再 用 5mM pH 6. 5 磷酸钠缓冲液稀释 2倍后过离子交换层析， 采用 DEAE Sepharose Fast Flow (XK 50/20, GE healthcare)层析柱， 直接用 PBS洗脱目的蛋白。 最后 通过 S印 hadex G25 coarse (XK 50/60， GE healthcare)凝胶层析柱脱盐置换到 5mM pH 6. 5 磷酸钠缓冲液中。 将表达上清和纯化的融合蛋白分别用非还原 SDS-PAGE分析。 如图 1所示， 不同结构的 GLP-1类似物融合蛋白在表达时的稳定 性就有较大差异， 其中 (GLP-1类似物 ) 2_HSA稳定性最差。 实施例 4体外活性试验
根据文献( Ί—. j.oka.rri k G, Negu lescu ?A, napp ΊΈ, Mere [_., Burres N, Feng
L, Whitney Roemer K, Tsi en RY. Sci ence. 279 (5347) : 84-8. ( 1998 ))， 构建带有人 GLP-1受体和 CRE-Luc报告基因的 HEK-293细胞， 以 50000细胞 /孔 /200 μ 1含 10%FBS的 DMEM培养基接种到 Costar 96孔细胞培养板中。 接种后第 二天， 吸掉培养液， 将含 500 μ Μ ΙΒΜΧ、 梯度稀释的 GLP-1类似物融合蛋白的无 血清 DMEM培养液 50 μ 1加入各孔， 孵育 5-6小时后， 加入 50 μ 1荧光素酶底物 (Bright-Glo™ Luciferase Assay System, Promega, E2620 ), 反应 2分钟， 转 移到 costar 96 孔全白微孔板中， 在多功能酶标仪（SpectraMax M5 system, Molecular Device)上测定荧光值， 根据荧光值描绘剂量反应曲线， 确定 EC5。值。 以（GLP-1类似物 ) 2_HSA的活性为 100%计，计算各融合蛋白的相对活性。结果见表 1， Gly4Ser作为连接肽， 其体外活性与 GLP-1类似物作为连接肽基本相似； 而当 GLP-1类似物与 HSA之间插入一段如权利要求 1所述的序列后 （序列 E1-E6 ) , 融 合蛋白的体外活性提高了 7-10倍左右。
¾1
Figure imgf000018_0001
实施例 5 体外稳定性分析
取高度纯化的 GLP-1类似物融合蛋白原液， 加入适量的氯化钠、 磷酸氢二钠 和磷酸二氢钠， 用氢氧化钠或盐酸调 PH7. 4， 再加注射用水使每毫升含 GLP-1类 似物蛋白 5. Omg, 氯化钠 9mg， 磷酸盐 20 μ mol。 用 0. 22 μ m的 PVDF或 PES滤膜 过滤除菌， 100 级环境下无菌封装于西林瓶， 样品存放于稳定性试验箱， 保存温 度 25°C， 在 0， 1， 3个月时取样保存于 -70度冰箱待检。 合并所有待分析样品检 测 SDS-PAGE纯度和细胞生物学活性。 SDS-PAGE纯度检测方法如实施例 1所述， 待检样品的上样量为 10ug。 另上自身对照 lug， 0. 5ug, 0. 2ug, 0. lug, 0. 05ug, 经光密度扫描， 获得标准曲线， 计算各杂蛋白含量百分比， 最终算得融合蛋白纯 度。 体外活性测定方法如实施例 4所述，以各样品 0个月时活性为 100%计。 活性 测定前样品用 Superdex 75 10/30 分子筛柱（GE Healthcare)分离去除分子量小 于 lOOOODa的降解片段， 避免其对测活的干扰。 结果见表 2， GLP-1类似物与 HSA 之间插入 GLP-1类似物作为连接肽时， 活性保留率最差， 融合蛋白最不稳定。 表 2
Figure imgf000018_0002
0 1 3 0 1 3
1 (GLP-1类似物）2-HSA 97. 1 65. 3 34. 3 100 45. 4 13. 5
2 GLP-1类似物 _ ( Gly4Ser) 3-HSA 97. 3 84. 3 67. 3 100 79. 0 47. 2
3 GLP-1类似物 _ ( Gly4Ser) 4-HSA 98. 0 89. 4 66. 0 100 85. 5 45. 3
4 GLP-1类似物 -El-HSA 97. 5 93. 4 77. 9 100 97. 6 60. 6
5 GLP-1类似物 - E2- HSA 98. 3 94. 3 72. 8 100 93. 4 57. 9
6 GLP-1类似物 -E3- HSA 97. 9 95. 1 77. 3 100 91. 7 49. 8
7 GLP-1类似物 -E4- HSA 97. 5 94. 8 75. 2 100 96. 2 55. 6
8 GLP-1类似物 -E5- HSA 98. 4 95. 7 81. 2 100 93. 3 47. 2
9 GLP-1类似物 -E6- HSA 98. 2 96. 7 80. 1 100 91. 7 53. 9 实施例 6 血清稳定性分析
取纯化后的高浓度 GLP-1类似物融合蛋白原液， 按体积比 1 : 25加入到猴血清 中， 过滤除菌， 无菌封装到西林瓶中， 37°C孵育。 于第 0天， 15天， 30天取样保 存于 -70度冰箱待检。合并所有待分析样品， 以 Anti-GLP-Ι 单抗（Antibodyshop ) 为捕捉抗体， Goat ant i -Human Albumin-HRP (Bethyl Laboratories ) 为检测抗 体通过夹心 ELISA法测定融合蛋白浓度。 因为捕捉抗体结合于融合蛋白 GLP-1类似 物部分， 检测抗体结合于白蛋白部分， 因此测定的融合蛋白浓度与未降解部分含 量正相关。 结果见表 3， 30天后猴血清中（GLP-1类似物 ) 2-HSA大部分已经降解， 而 其他样品保留了将近 40%。
表 3 血清中融合蛋白含量随时间变化情况
Figure imgf000019_0001
注： 以 0天测定的浓度为 100%计。 实施例 7 小鼠腹腔注射葡萄糖耐受试验
32只 KM小鼠， 雌雄各半， 过夜禁食不禁水 18小时， 皮下注射 1. 0 mg/kg的 HSA (对照组）、 (GLP-1 类似物）2-HSA、 GLP-1 类似物 -E3-HSA 和 GLP-1 类似物 -E6-HSA。给药 1 、 8小时后分别进行腹腔注射葡萄糖耐受试验（IPGTT): 腹腔注 射 1. 5 g/kg葡萄糖， 注射葡萄糖前（t=0)及注射后 10、 20、 30、 60、 90、 120 分 钟后取血测定血液中葡萄糖含量 （YSI2700型生化分析仪）。 与对照组 （HSA组） 比较， （GLP-1类似物）2-HSA、 GLP-1类似物 -E3-HSA和 GLP-1类似物 -E6-HSA组小 鼠血糖显著降低，血糖曲线峰下面积（AUC。_12。„„)均显著低于对照组（结果见表 4)。 其中， 给药 1 小时后进行 IPGTT时， 三组间各个时间点血糖水平类似， 血糖曲线 峰下面积 （AUC。― ιη) 也相似， 组间无显著性差异 （P>0. 05); 但在给药 8 小时后 进行 IPGTT时， 10-30min GLP-1类似物 -E3-HSA和 GLP-1类似物 -E6-HSA组的血糖 水平要显著低于（GLP-1 类似物）2_HSA， 血糖 AUC。_12。„in也显著低于（GLP-1 类似 物） 2-HSA组（P〈0. 01 )。 结果表明， （GLP-1类似物） 2_HSA和 GLP-1类似物 -E3-HSA 均能有效降低小鼠空腹血糖，并具有长效特性，但 GLP-1类似物 -E3-HSA和 GLP-1 类似物 -E6-HSA组较 (GLP-1类似物 ) 2_HSA组具有更显著和持续的降糖效果。
KM小鼠单次药后 lh和 8h IPGTT血糖峰下面积 AUC。-
Figure imgf000020_0001
实施例 8正常恒河猴单次皮下注射 GLP-1类似物融合蛋白药效动力学试验
恒河猴单次皮下注射 0. 5 mg/kg 的（GLP-1 类似物）2_HSA 或 GLP-1 类似物 -E3-HSA, 24 h和 96 h后进行分级静脉注射葡萄糖试验：静脉注射葡萄糖溶液（20% 右旋糖溶液， 200 mg/ml ) 10mg/kg/min ( 3. 0 ml/kg/h) 持续 20 min, 随后给予 葡萄糖溶液 25 mg/kg/min ( 7. 5 ml/kg/h) 持续 20 min。 葡萄糖注射后 0、 10、 20、 30、 40 min采集血液测定血糖和胰岛素。 血糖测定使用 YSI2700型生化分析 仪， 胰岛素测定使用酶联免疫吸附方法 （ Insulin ELISA 试剂盒， DRG International, Inc. )。 两组间血糖在药后 Id各时间点无显著差异 （结果见图 2)。， 给药后 4d时 10、 30、 40 min组间有显著差异 （P〈0. 05或 P〈0. 01 ); 组间胰 岛素在药后 Id和 4d的 20、 40 min时间点都具有显著差异 (P〈0. 01 )。 结果表明， GLP-1类似物 -E3-HSA较 (GLP-1类似物 ) 2_HSA能更好促进胰岛素分泌， 降低正常 恒河猴分级静脉注射葡萄糖试验中血糖水平。 实施例 9 食蟹猴单次给药药物代谢动力学研究
食蟹猴单次皮下注射 0. 5mg/kg 的（GLP-1类似物 ) 2_HSA和 GLP-1类似物 -E3-HSA, 分别于药前 ( t=0)、 药后 4、 8、 12、 24、 48、 72、 96、 120、 144、 216 h采集血样并分离血清， -80度冷冻保存后合并检验。 以 Anti-GLP-Ι 单抗
(Antibodyshop) 为捕捉抗体， Goat ant i -Human Albumin-HRP (Bethyl Laboratories)为检测抗体测定血清中的融合蛋白浓度（见图 3)， 并计算药物代 谢动力学参数（见表 5)。研究表明 0. 5mg/kg的 GLP-1类似物 -E3-HSA在食蟹猴体 内的半衰期为 102h (约 4d)， 而（GLP-1类似物）2-HSA半衰期约为 60h ( 2. 5d)。 表 5
Figure imgf000021_0001
实施例 10 食蟹猴重复皮下给药免疫原性
食蟹猴每周皮下注射一次 lmg/kg的（GLP-1类似物 ) 2_HSA和 GLP-1类似物 -E3-HSA, 连续给药 3个月。 给药前（t=0)、 给药后 1、 2、 3个月分别采集血样并 分离血清， -80度冰冷冻保存后合并检验。 使用酶联免疫吸附方法（ELISA)来测 定可能产生的猴抗融合蛋白抗体。 以对应的融合蛋白为包被物， 加入不同稀释度 的待检血清样品， 用 mouse-anti-monkey IgG为检测抗体来测定抗体的滴度。 同 时， 在相似的测定条件下， 待检血清样品中加入人血清白蛋白 （终浓度为 60 μ Μ) 作为拮抗物， 来进一步分析产生的抗体特异性 （见表 6)。 研究结果显示， 多次给 药后两者均有抗体生成， 最高滴度达到 1 : 6400， 而且两者产生的抗体趋势与滴度 基本一致。进一步在血清中加入人血清白蛋白 （HSA)进行拮抗分析， 结果显示存 在 HSA时血清滴度显著下降， 说明所生成的抗体基本均被 HSA所拮抗。 因此说明 猴反复注射融合蛋白后所生成的抗体大部分是针对融合蛋白中的人血清白蛋白部 分， 而没有生成抗 GLP-1类似物部分的抗体。
表 6
Figure imgf000021_0002
6 N. D. I N. D. 1:1600 1:1600 N. D. N. D. N. D. N. D. 注 抗体滴度< 1:100定义为木检出 （N.D. ) 上述实施例仅例示性说明本发明的原理及其功效， 而非用于限制本发明。 任 何熟悉此技术的人士皆可在不违背本发明的精神及范畴下， 对上述实施例进行修 饰或改变。 因此， 举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的 精神与技术思想下所完成的一切等效修饰或改变， 仍应由本发明的权利要求所涵

Claims

权利要求书 、 一种 GLP-1 类似物融合蛋白， 该融合蛋白结构为： GLP-1 类似物-连接肽 -人血清白蛋 白， 所述连接肽的长度不超过 26 个氨基酸， 且通式如下： （Xaa) χ- (Pro) y- (Xaa) z , 其 中 Xaa 选自 G、 A 和 S 中的一种或几种的任意组合， x， y， z 均为整数， 且 x， z ^3 , 26 ^x+y+z ^ l4, 10 ^y^3, l ^y/ (x+z) ^0. 13, 连接肽 N端与 GLP-1类似物 C末端以肽 键相连， 连接肽 C端与人血清白蛋白的 N端以肽键相连。
、 如权利要求 1所述的 GLP-1类似物融合蛋白， 其特征在于， 所述 GLP-1类似物选自以下 任一：
a) 其氨基酸序列为 SEQ ID NO: 1；
b) 其氨基酸序列与 SEQ ID NO: 1保持 85%， 优选 90%， 更优选 95%， 再优选 99%的同源 性；
c) 包含 a)或 b ) 所述的 GLP-1类似物的 2个或 3个重复序列， 或者包含 GLP-1的 2个 或 3个重复序列；
d) 为 Exendin_4。
、 如权利要求 1所述的 GLP-1类似物融合蛋白， 其特征在于， 所述连接肽的氨基酸序列选 自 SEQ ID NO : 11-16中的任一。
、 如权利要求 1所述的 GLP-1类似物融合蛋白， 其特征在于， 所述人血清白蛋白的氨基酸 序列为 SEQ ID NO: 2， 或者至少与 SEQ ID NO: 2保持 85%， 优选 90%， 更优选 95%， 再 优选 99%的同源性。
、 如权利要求 1所述的 GLP-1类似物融合蛋白， 其特征在于， 所述的 GLP-1类似物融合蛋 白的氨基酸序列选自 SEQ ID NO: 3-5。
、 一种多核苷酸， 其编码权利要求 1-5任一权利要求所述 GLP-1类似物融合蛋白。
、 如权利要求 6 所述的多核苷酸， 其特征在于， 所述的多核苷酸的序列选自 SEQ ID NO:
8-10。
、 一种制备权利要求 1-5任一所述的 GLP-1类似物融合蛋白的方法， 该方法包括： 构建含 有所述 GLP-1 类似物融合蛋白的基因序列的表达载体， 然后将所述的表达载体转化至宿 主细胞中诱导表达， 从表达产物中分离获得所述的融合蛋白。
、 如权利要求 8 所述的 GLP-1 类似物融合蛋白的方法， 其特征在于， 所述表达载体为 PPIC9; 所述宿主细胞为巴斯德毕赤酵母。 、 如权利要求 8所述的 GLP-1 类似物融合蛋白的方法， 其特征在于， 所述从表达产物 中分离获得所述的融合蛋白的方法为： 采用联合亲和层析、 疏水层析和离子交换层析的 三步层析方法分离获得所述的融合蛋白。
、 如权利要求 1-5任一所述的 GLP-1类似物融合蛋白在制备治疗糖尿病及相关疾病药 物上的用途。
、 一种用于治疗糖尿病及糖尿病相关疾病的药物组合物， 含有权利要求 1-5 任一所述 的 GLP-1类似物融合蛋白及至少一种药学可接受的载体或赋形剂。
、 一种治疗糖尿病及其相关疾病的方法， 为向对象施用权利要求 1-5任一所述的 GLP- 1类似物融合蛋白。
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