WO2018178796A1 - Procédé pour rendre des polypeptides sensibles à la protéase kex1 à l'aide d'une souche de levure - Google Patents

Procédé pour rendre des polypeptides sensibles à la protéase kex1 à l'aide d'une souche de levure Download PDF

Info

Publication number
WO2018178796A1
WO2018178796A1 PCT/IB2018/051788 IB2018051788W WO2018178796A1 WO 2018178796 A1 WO2018178796 A1 WO 2018178796A1 IB 2018051788 W IB2018051788 W IB 2018051788W WO 2018178796 A1 WO2018178796 A1 WO 2018178796A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
carboxypeptidase
precursor
amino acid
yeast
Prior art date
Application number
PCT/IB2018/051788
Other languages
English (en)
Inventor
Rajamannar Thennati
Sanjay Kumar Singh
Nishith Chaturvedi
Nitin Bhimrao NAGE
Ranjit Sudhakar RANBHOR
Mn RAVISHANKARA
Nandkumar Govind BHAGAT
Original Assignee
Sun Pharmaceutical Industries Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sun Pharmaceutical Industries Limited filed Critical Sun Pharmaceutical Industries Limited
Publication of WO2018178796A1 publication Critical patent/WO2018178796A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts

Definitions

  • the present invention relates to novel methods and compositions for producing KEX1 protease-sensitive heterologous polypeptides in yeast.
  • yeast have been the focus of genetic engineers as hosts for production of heterologous proteins. As hosts, they offer several advantages such as rapid growth, genetic background, established fermentation technology and simple media components, a post- translational modification pattern as in eucaryotes, and the ability to secrete the heterologous polypeptides. Thus, yeast may be regarded as an ideal host for producing large quantities of recombinant biopharmaceuticals.
  • yeast expression systems are available like Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia, etc. Saccharomyces cerevisiae, a particularly well characterized eucaryotic model organism, has become an attractive host cell for producing recombinant heterologous polypeptides.
  • yeast produces several proteases which lead to proteolytic degradation of the expression product.
  • a number of endogenous yeast proteases degrade yeast heterologous protein degradation including KEX1, PEP4 and YPS3.
  • Killer expression-defective protein 1 also called pheromone-processing carboxypeptidase KEXl, is a yeast protease involved in the processing of C-terminal lysine and arginine residues from the endogenous precursors of Kl, K2 and K28 killer toxins and alpha-factor (mating pheromone) (Cooper et ah, 1989).
  • KEXl Killer expression-defective protein 1
  • KEXl pheromone-processing carboxypeptidase
  • yeast protease involved in the processing of C-terminal lysine and arginine residues from the endogenous precursors of Kl, K2 and K28 killer toxins and alpha-factor (mating pheromone) (Cooper et ah, 1989).
  • expression of a heterologous polypeptide in yeast with a C-terminal lysine, arginine or glycine residue also results in C- terminal
  • the present invention pertains to methods and compositions of matter for making KEXl protease- sensitive heterologous polypeptides in yeast strains.
  • the present invention offers a solution to the problem faced by the art without having to knock out or even disable or down-regulate endogenous yeast protease genes. Rather, it entails transformation of yeast with heterologous nucleic acid that encodes a precursor of a heterologous, biologically active polypeptide that is not susceptible to cleavage of C-terminal amino acid residues by KEXl.
  • the precursor heterologous polypeptide includes at least one additional C-terminal amino acid which is not sensitive to cleavage by endogenous yeast proteases, particularly KEXl.
  • the precursor heterologous polypeptide is also designed to facilitate efficient post-translational cleavage of the at least one additional C-terminal amino acid residue by carboxypeptidase B, which results in formation of the mature, heterologous polypeptide.
  • a first aspect of the present invention is directed to a method of producing a heterologous polypeptide in yeast, wherein the polypeptide is sensitive to KEX1 degradation.
  • the method comprises transforming a KEX1 -functional yeast cell with a heterologous polynucleotide that comprises a nucleic acid having a sequence that encodes a precursor of the heterologous polypeptide which in its mature form has a C-terminal amino acid that is susceptible to degradation by KEX1 (e.g., glycine, arginine and lysine).
  • KEX1 e.g., glycine, arginine and lysine
  • the nucleic acid comprises (or consists of) at its 3 ' end, an oligonucleotide (also referred to herein as a fragment or tail) containing (or consisting of) one or more codons wherein the 3 ' codon in the oligonucleotide encodes an amino acid that is resistant to degradation by KEX1 but which is susceptible to degradation by carboxypeptidase B (which in some embodiments is a tyrosine (TYR) residue), and wherein each amino acid encoded by the 3 ' oligonucleotide fragment is susceptible to cleavage by carboxypeptidase B.
  • the yeast cell is cultured under conditions suitable for expression of the nucleic acid, thus producing the precursor polypeptide.
  • the precursor polypeptide expression product is biologically inactive, it is contacted with carboxypeptidase B whereby the at least one C- terminal amino acid residue is cleaved, resulting in the mature form of the heterologous polypeptide.
  • a second aspect of the present invention is directed to a culture of yeast cells in a suitable medium, wherein the yeast cells are KEX1 -functional yeast cells and comprise the heterologous polynucleotide.
  • the yeast cells further comprise a heterologous polynucleotide comprising a nucleic acid having a sequence that encodes a carboxypeptidase B.
  • the medium comprises the carboxypeptidase B.
  • a third aspect of the present invention is directed to a composition
  • a composition comprising the precursor heterologous polypeptide and a carboxypeptidase B.
  • the composition further includes the fermentation broth or medium used for cultivation of the yeast.
  • the composition further includes (new) medium.
  • the composition is substantially free of yeast cells.
  • aspects of the present invention are directed to the precursors of the heterologous polypeptides, per se.
  • the precursor polypeptides have been surprisingly and unexpectedly found to be biologically active.
  • GLP-1 (7-37)-TYR or GLP-1 analogue 2357/05.
  • related aspects of the invention pertain to methods of making GLP-1 (7-37)-TYR or GLP-1 analogue (2357/05), which do not require the post-translational step of cleaving the C-terminal TYR residue, and methods of using this polypeptide to treat Parkinson's disease or metabolic disorders such as type II diabetes and obesity.
  • Still further aspects of the present invention are directed to genetic constructs, including the heterologous polynucleotides, per se, expression cassettes containing the polynucleotides, vectors containing the expression cassettes, and yeast cells transformed with the vectors.
  • Figure 1 is a schematic diagram of a plasmid vector useful is the present invention wherein P_Gall is galactose inducible promoter; GOI is the gene of interest; M_URA3 is the URA3 gene encodes orotidine-5' phosphate decarboxylase, an enzyme that is required for the biosynthesis of uracil; Ori_lum is an origin of replication which allow Saccharomyces cerevisiae cells to maintain 20-50 copies of the vector; M_Ampicillin-r is selection marker for E. coli; Ori_pUC is the origin of replication for E. coli.
  • Figure 2 is a graph that shows percent cleavage of C-terminal amino acid residue of pentapeptide 5P by carboxypeptidase B over the course of a 16-hour incubation.
  • Figure 3 is a graph that shows is a graph that shows percent cleavage of C-terminal amino acid residue of pentapeptide 6PM by carboxypeptidase B over the course of a 16-hour incubation.
  • Figure 4 is a graph that shows is a graph that shows percent cleavage of C-terminal amino acid residue of pentapeptide 6PY by carboxypeptidase B over the course of a 16 -hour incubation.
  • Figure 5 is a graph that shows is a graph that shows percent cleavage of C-terminal amino acid residue of pentapeptide 6PR by carboxypeptidase B over the course of a 16 -hour incubation.
  • Figure 6 is a graph that shows is a graph that shows percent cleavage of C-terminal amino acid residue of pentapeptide 6PL by carboxypeptidase B over the course of a 16-hour incubation.
  • Figure 7 is a graph that shows is a graph that shows percent cleavage of C-terminal amino acid residue of pentapeptide 6PK by carboxypeptidase B over the course of a 16 -hour incubation.
  • Figure 8 is a graph that shows percent cleavage of C-terminal amino acid residue of various polypeptides produced in yeast, by carboxypeptidase B over the course of a 6-7-hour incubation.
  • Figure 9 is a graph that shows percent conversion of GLP-1 analogues (sequence id 1), 1532/2357/03 (sequence id 2) and 1532/2357/04 (sequence id 3) by carboxypeptidase B over the course of a 6-hour incubation.
  • Figure 10 is a graph that shows percent conversion of GLP-1 analogue and a precursor thereof, GLP-1 analogue-TYR, by carboxypeptidase B over the course of a 6-hour incubation.
  • Figure 11 is a graph that shows change in blood glucose in blood collected from rats over time at various doses of liraglutide, GLP-1 analogue (2357/05) and Victoza®.
  • Figures 12A and B are graphs showing concentration of GLP-1 analogue (2357/05) and Victoza®, respectively, in blood collected from rats injected with 0.5 mg/kg, as a function of time.
  • Figures 13A and B are graphs showing concentration of GLP-1 analogue (2357/05) and Victoza®, respectively, in blood collected from rats injected with 1.0 mg/kg, as a function of time.
  • KEX1 or KEXlp refers to a serine carboxypeptidase, which is endogenous to yeast, that catalyzes the C-terminal cleavage of lysine, arginine and glycine residues.
  • functional KEX1 gene, it is meant the endogenous, unmodified KEX1 gene or a disabled or down-regulated form thereof that still possesses non-negligible enzymatic activity (as compared to a KEX1 "knock out”).
  • a precursor polypeptide is a polypeptide that includes at least one additional amino acid at its C-terminus of a mature, biologically active polypeptide, wherein the C-terminal amino acid of the precursor is not sensitive to KEX1 protease but wherein the at least one amino acid is sensitive to cleavage by carboxypeptidase B.
  • enzymatic cleavage refers to the hydrolysis or breakage of the polypeptide chains at specific sites within the polypeptide or at the terminal thereof into smaller peptides or amino acids, which are catalysed by enzymes called proteases.
  • Carboxypeptidase B which catalyzes the hydrolysis of C-terminal tyrosine, lysine, valine, leucine, isoleucine, asparagine, glutamine and arginine residues from the polypeptides.
  • gene disruption or “gene knock-out” refers to the replacement of the functional gene by an inactive gene or deletion of the functional gene. This can be achieved by homologous recombination and/or antisense technology and/or lambda red recombinase and/or CRISPRE and/or zinc finger nuclease technology, and/or regulation of the gene using antisense technology.
  • heterologous expression refers to the expression of a nucleic acid in a yeast host cell which naturally does not have the nucleic acid, i.e., the nucleic acid is non- native to the yeast.
  • transformation is a process by which exogenous DNA or nucleic acid is introduced into cells. Such methods are described, for example, in U.S. Patent Nos. 4,845,075 and 4,599,311.
  • the heterologous polynucleotide containing the nucleic acid encoding the precursor polypeptide is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression of the nucleic acid.
  • the vector is then introduced into the yeast host through standard techniques. Generally, it will be necessary to select for transformed host cells.
  • the nucleic acid having the sequence encoding the precursor polypeptide may be operably linked to a DNA sequence encoding a promoter functional in the yeast, a leader sequence and/or other DNA sequences that are necessary for the precursor heterologous polypeptide to be expressed in and secreted from the yeast.
  • Suitable promoters for S. cerevisiae include the MFal promoter, galactose inducible promoters such as GAL1, GAL7 and GAL 10 promoters, glycolytic enzyme promoters including TPI and PGK promoters, TRP1 promoter, CYCI promoter, CUP1 promoter, PH05 promoter, ADH1 promoter, and HSP promoter.
  • a suitable promoter in the genus Pichia is the AOX1 (methanol utilization) promoter.
  • the DNA constructs that are used for providing secretory expression of the desired polypeptide comprise a DNA sequence that includes a leader sequence linked to the polypeptide by a yeast processing signal.
  • the leader sequence contains a signal peptide ("pre- sequence”) for protein translocation across the endoplasmic reticulum and optionally contains an additional sequence (“pro-sequence”), which may or may not be cleaved within yeast cells before the polypeptide is released into the surrounding medium.
  • useful leaders are the signal peptide of mouse a-amylase, S. cerevisiae MF1, YAP3, BAR1, HSP150 and S. kluyveri MFa signal peptides and prepro-sequences of S. cerevisiae MFal, YAP3, PRC, HSP150, and S. kluyveri MFa and synthetic leader sequences described in WO 92/11378, WO 90/10075 and WO 95/34666.
  • the transcription terminal signal may include the 3' flanking sequence of a eukaryotic gene which contains proper signal for transcription termination and polyadenylation.
  • Suitable 3' flanking sequences may, e.g., be those of the gene naturally linked to the expression control sequence used, i.e., corresponding to the promoter.
  • Yeast plasmid vectors that may be useful in the practice of the present invention include the POT (Kjeldsen et al, Gene. 7010-112 (1996)) and Yepl3, Yep24 (Rose et al, Methods Enzymol. 85:234-279 (1990)), and pG plasmids (Schena et al, Methods Enzymol. 94:289-398 (1991)).
  • the exogenous nucleic acid is inserted into an yeast integration plasmid vector, such as pJJ215, pJJ250, pJJ236, pJJ248, pJJ242 (Jones & Prakash, Yeast 6: 363 (1990)) or pDP6 (Fleig et al, Gene 46:231 (1986)), in proper orientation and correct reading frame for expression, which is flanked with homologous sequences of any non-essential yeast genes, transposon sequence or ribosomal genes. Flanking sequences may include genes used as a selective marker.
  • the DNA is then integrated into host chromosome(s) by homologous recombination occurred in the flanking sequences, by using standard techniques show in Rothstein (Method in Enzymol. 794:281- 301 (1991)) and Cregg et al., (Bio/Technol. 77:905-910 (1993)).
  • the yeast host cell into which the nucleic acid vector is introduced may be any yeast cell which is capable of expressing the polypeptide.
  • Representative examples include Saccharomyces spp. Or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae or Saccharomyces kluyveri.
  • Further examples of suitable yeast cells are strains of Kluyveromyces, such as K. lactis, Hansemula, e.g., H. polymorpha, or Pichia, e.g., P. pastoris (cf. Gleeson, et al, J. Gen. Microbiol. 732:3459-3465 (1986); U.S. Patent 4,882,279).
  • the KEX1 gene may be unmodified or down-regulated.
  • the yeast is modified in that one of more of the endogenous protease genes e.g., YAP3, STE13, PRBl, PEP4, YPS3, YPSl, MKC7, YPS5, YPS6, and YPS7, are down-regulated or knocked out.
  • Methods for transforming yeast cells with heterologous nucleic acid (DNA) and producing heterologous polypeptides therefrom are well known in the art, as described, e.g., in U.S. Patent Nos. 4,599,311, 4,931,373, 4,870,008, 5,037,743 and 4,845,075.
  • Methods for the transformation of S. cerevisiae include the spheroplast transformation, lithium acetate transformation, and electroporation, cf. Methods in Enzymol. 194 (1991).
  • Transformed yeast cells may be selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g., leucine.
  • Host cells that have been transformed by the exogenous DNA invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression and optimally secretion of the precursor heterologous polypeptides.
  • the nucleic acid (DNA) sequence encoding the desired mature heterologous peptide may be of genomic or cDNA origin, for instance be obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (see, for example, Sambrook, J, Fritsch, E F and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989).
  • the DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in U.S. Patent 4,683,202 or Saiki et al., Science 239:487-491 (1988).
  • the nucleic acid encoding the precursor heterologous polypeptide can then be prepared by joining, e.g., ligating, the oligonucleotide tail to the 3' end of the nucleic acid prepared above.
  • the DNA sequence encoding the heterologous precursor polypeptide may also be prepared synthetically by established standard methods, e.g., the phosphoamidite method described by Beaucage et al., Tetrahedron Letters 22:1859-1869 (1981), or the method described by Matthes et al, EMBO Journal 3:801-805 (1984).
  • Heterologous polypeptides of interest especially biologically active polypeptides having C-terminal lysine, arginine and glycine residues (and their corresponding DNA sequences), which can be advantageously produced in accordance with the present methods, are known in the art.
  • Representative polypeptides include pancreatic polypeptide and its analogs, amylin and amylin analogs, PP and analogs, PYY and analogs, oxytocin, vasopressin, incretins such as GLP- 1 and its derivatives and analogues (e.g.
  • GLP- 1 agonists/incretin mimetics such as liraglutide (marketed by Novo Nordisk under the tradename Victoza®), Lixisenatixe (C-terminal Lys), Albiglutide (C-terminal Lys), Dulaglutide (C-terminal Gly) and Semaglutide (C-terminal Gly)), secretin, calcitonin, gastrin, NPY, FMRF amide, GRHR, CRF, neurokinin A, gastrin releasing peptide, insulin and analogs, and alpha-MSH.
  • liraglutide marketed by Novo Nordisk under the tradename Victoza®
  • Lixisenatixe C-terminal Lys
  • Albiglutide C-terminal Lys
  • Dulaglutide C-terminal Gly
  • Semaglutide C-terminal Gly
  • the nucleic acids encoding these polypeptides may be modified by adding to the mature coding sequence a 3 ' oligonucleotide that encodes at least one amino acid that is not susceptible to degradation by KEX1 but which is susceptible to carboxypeptidase B.
  • the 3 ' tail may encode more than one amino acid provided that that C-terminal amino acid of the precursor polypeptide is an amino acid residue that is not susceptible to degradation by KEX1 but is susceptible to degradation by carboxypeptidase B.
  • the 3 ' codon encodes tyrosine.
  • the present inventors have surprisingly and unexpectedly discovered that the carboxypeptidase B cleaves the C-terminal tyrosine residues of polypeptides.
  • the 3 ' oligonucleotide fragment may encode a C-terminal oligopeptide having, for example, 1- 10 amino acid residues and in some embodiments, 1-5 amino acid residues and in some other embodiments, 1-3 amino acid residues and in some other embodiments, 1 amino acid residue.
  • the 3 ' oligonucleotide may thus include codons for amino acids valine, leucine, isoleucine, asparagine, glutamine, lysine, arginine and tyrosine, arranged in appropriate order such that it is not susceptible to degradation by KEX1 (i.e., the C-terminal amino acid is not cleavable by KEX1), and that upon contact with carboxypeptidase B, the mature, biologically active heterologous polypeptide is produced (i.e., each of the at least one amino acids is cleavable by carboxypeptidase B).
  • Representative additional embodiments of the at least one C-terminal amino acid may have the following amino acid sequences:
  • a representative example of a polynucleotide encoding a precursor polypeptide of the present invention contains a 3 ' trinucleotide tail encoding tyrosine, is as follows:
  • Carboxypeptidase B is a pancreatic, zinc-containing exopeptidase (EC 3.4.17.2) which as of the time the present invention was made, was known to cleave (via hydrolysis) peptide linkages at basic amino acids, such as lysine, arginine and ornithine, at the C-termini of the polypeptides. Applicant' s discovery thus makes possible the novel methods and compositions which utilize C-terminal tyrosine residue(s) as the substrate for carboxypeptidase B.
  • the carboxypeptidase may be any suitable natural or recombinant carboxypeptidase such as carboxypeptidase B or a mutated variant thereof.
  • the yeast cell may also be genetically modified (e.g. , transformed with) with nucleic acid encoding the carboxypeptidase gene (sequences of which are known in the art e.g. , U.S. Patent Application Pubs 2008/0311619 Al and 2005/0142633 Al).
  • the DNA encoding carboxypeptidase B may be associated with a leader sequence to allow for secretion of the enzyme from the yeast cell into the culture medium/milieu (or fermentation broth).
  • the precursor polypeptide is secreted from or otherwise isolated from the yeast cell (e.g. , via centrifugation) and then added to a suitable medium along with the carboxypeptidase B.
  • the contacting e.g. , incubation
  • the amount of enzyme added to a composition containing the precursor polypeptide produced by the yeast cell ranges from about 0.1 units to about 100 units per mg of the precursor polypeptide.
  • the at least one additional C-terminal amino acids may include one or more amino acids that is susceptible to cleavage by KEX1, as well as being cleavable by carboxypeptidase B.
  • the C-terminal amino acid of the mature heterologous polypeptide is susceptible to cleavage by carboxypeptidase B, e.g., an arginine or lysine residue
  • carboxypeptidase B e.g., an arginine or lysine residue
  • the mature heterologous polypeptide may be isolated from the composition and purified in accordance with standard techniques.
  • cleavage with carboxypeptidase may take place after purification of the precursor polypeptide.
  • compositions containing GLP-1 (7-37)-TYR and Methods of Use
  • the present invention also related to pharmaceutical compositions comprising GLP-1 (7-37)-TYR or analogues of the present invention and a pharmaceutically acceptable vehicle or carrier.
  • GLP-1 (7-37)-TYR it is meant: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser- Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg/Lys-Gly-Arg- Gly-Tyr.
  • GLP-1 analogue (2286/41), it is meant: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg- Gly-Tyr and at position 26 (Lys) a C16 acyl chain via a glutamyl spacer.
  • GLP-1 analogue (2357/05), it is meant: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg- Gly-Tyr and at position 26 (Lys) a C16 acyl chain via a glutamyl spacer.
  • Native GLP-1 (7-37) has a Lys at position 34.
  • Liraglutide differs from native GLP-1 (7-37) in that it has an Arg as position 34, and at position 26 (Lys) a C16 acyl chain via a glutamyl spacer.
  • GLP-1 (7-37)-TYR may be present in a concentration that is effective to treat the metabolic disease, which generally ranges from about 0.1 mg/ml to about 100 mg/ml, and in some embodiments in a concentration from about 0.1 mg/ml to about 50 mg/ml, and in yet other embodiments in a concentration of from about 0.1 mg/ml to about 10 mg/ml.
  • GLP-1 analogue (2357/05) may be present in a concentration that is effective to treat the metabolic disease, which generally ranges from about 0.1 mg/ml to about 100 mg/ml, and in some embodiments in a concentration from about 0.1 mg/ml to about 50 mg/ml, and in yet other embodiments in a concentration of from about 0.1 mg/ml to about 10 mg/ml.
  • the pharmaceutical compositions may include an isotonic agent and/or a buffer. Examples of isotonic agents include sodium chloride, mannitol and glycerol and propylene glycol.
  • the propylene glycol may be present in a concentration that generally ranges from about 1 to about 50 mg/ml, and in some embodiments in a concentration of from about 5 to about 25 mg/ml, and in yet other embodiments in a concentration of from about 8 to about 16 mg/ml.
  • Propylene glycol-containing compositions may be advantageously formulated to have a pH in the range from about 7.0 to about 9.5. and in some embodiments from about 7.0 to about 8.0, and in some other embodiments from 7.2 to about 8.0, and in some other embodiments from about 7.0 to about 8.3, and in yet further embodiments from 7.3 to about 8.3.
  • the composition may further contain a preservative, representative examples of which include phenol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2- phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, and combinations of two or more thereof.
  • the preservative may be present in a concentration that generally ranges from about 0.1 mg/ml to about 50 mg/ml, and in some embodiments from about 0.1 mg/ml to about 25 mg/ml, and in yet other embodiments from about 0.1 mg/ml to about 10 mg/ml.
  • buffers include sodium acetate, sodium phosphate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, and tris(hydroxymethyl)- aminomethan, and combinations of two or more thereof.
  • the pharmaceutical compositions may further contain a surfactant in order to improve the solubility and/or the stability of the GLP- 1 (7-37)-TYR or its analogues.
  • surfactants such as zwitterionic surfactants, cationic surfactants, non-ionic surfactants and polymeric surfactants as well as detergents, that may be suitable for use in the present invention include ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, poloxamers, such as 188 and 407, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene derivatives such as alkylated and alkoxylated derivatives (tweens, e.g.
  • Tween-20 monoglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, glycerol, cholic acid or derivatives thereof, lecithins, alcohols and phospholipids, glycerophospholipids (lecithins, kephalins, phosphatidyl serine), glyceroglycolipids (galactopyransoide), sphingophospholipids (sphingomyelin), and sphingoglycolipids (ceramides, gangliosides), DSS (docusate sodium, CAS registry no [577- 11-7]), docusate calcium, CAS registry no [128-49-4]), docusate potassium, CAS registry no [7491-09-0]), SDS (sodium dodecyl sulfate or sodium lauryl sulfate), dipalmitoyl phosphatidic acid, sodium caprylate, bile acids and salt
  • N-alkyl- N,N-dimethylammonio- 1 -propanesulfonates 3 -cholamido- 1 -propyldimethylammonio- 1 - propanesulfonate, dodecylphosphocholine, myristoyl lysophosphatidylcholine, hen egg lysolecithin), cationic surfactants (quarternary ammonium bases) (e.g.
  • acylcarnitines and derivatives N a - acylated derivatives of lysine, arginine or histidine, or side-chain acylated derivatives of lysine or arginine, N a -acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, N a -acylated derivative of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, or the surfactant may be selected from the group of imidazoline derivatives.
  • the pharmaceutical compositions may further contain a chelating agent, representative examples of which include salts of ethlenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and combinations of two or more thereof.
  • EDTA ethlenediaminetetraacetic acid
  • the chelating agent may be present in a concentration generally ranging from 0.1 mg/ml to 5 mg/ml, and in some embodiments from 0.1 mg/ml to 2 mg/ml, and in yet other embodiments from 2 mg/ml to 5 mg/ml.
  • the pharmaceutical compositions may further contain a stabilizer, representative examples of which include high molecular weight polymers or low molecular compounds e.g. , polyethylene glycol (e.g.
  • PEG 3350 polyvinylalcohol (PVA), polyvinylpyrrolidone, carboxymethylcellulose, salts (e.g. sodium chloride), L-glycine, L-histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and combinations of two or more thereof.
  • PVA polyvinylalcohol
  • PVP polyvinylpyrrolidone
  • carboxymethylcellulose salts (e.g. sodium chloride)
  • salts e.g. sodium chloride
  • L-glycine L-histidine
  • imidazole imidazole
  • arginine arginine
  • lysine isoleucine
  • aspartic acid tryptophan
  • threonine isoleucine
  • the high molecular weight polymer is present in a concentration from 0.1 mg/ml to 50 mg/ml, and in other embodiments from 5 mg/ml to 10 mg/ml, and in yet other embodiments from 0.1 mg/ml to 5 mg/ml.
  • the low molecular weight polymer is present in a concentration from 0.1 mg/ml to 50 mg/ml, and in other embodiments from 5 mg/ml to 10 mg/ml, and in yet other embodiments from 0.1 mg/ml to 5 mg/ml.
  • the pharmaceutical compositions may also contain zinc.
  • compositions may further contain another active agent, e.g., an antidiabetic (e.g., insulin such as human insulin, and oral hypoglycemic agents) and/or an anti-obesity agent (e.g. , leptin, amphetamine, dexfenfluramine, sibutramine, orlistat).
  • an antidiabetic e.g., insulin such as human insulin, and oral hypoglycemic agents
  • an anti-obesity agent e.g. , leptin, amphetamine, dexfenfluramine, sibutramine, orlistat.
  • compositions of the present invention may be prepared by conventional techniques, e.g., as described in Remington's Pharmaceutical Sciences, 1985.
  • injectable compositions of the present invention can be prepared using the conventional techniques known in the pharmaceutical industry which involves dissolving and mixing the ingredients as appropriate to give the desired end product.
  • a composition for nasal administration may, for example, be prepared as described in European Patent 272097 (to Novo Nordisk A/S) or in WO 93/18785.
  • GLP-1 (7-37)-TYR is provided in the form of a composition suitable for administration by injection.
  • Such a composition can either be an injectable solution ready for use or it can be an amount of a solid composition, e.g.
  • the injectable solution may contain not less than about 2 mg/ml, and in some embodiments not less than about 5 mg/ml, and in other embodiments not less than about 10 mg/ml and in yet other embodiments, not more than about lmg/ml of GLP-1 (7-37)-TYR or its analogue.
  • GLP-1 (7-37)-TYR or its analogue and pharmaceutical compositions containing it may be used to treat metabolic diseases, examples of which include non-insulin dependent diabetes mellitus, insulin dependent diabetes mellitus, obesity (including glycemic control) and insulin resistance.
  • the pharmaceutical compositions of the present invention may in some embodiments, be administered parenterally to patients in need of such a treatment.
  • Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe.
  • parenteral administration can be performed by means of an infusion pump.
  • GLP-1 (7-37)-TYR in a composition which may be a powder or a liquid for administration via a nasal or pulmonary spray.
  • GLP-1 (7-37)-TYR may be formulated for transdermal administration e.g. from a patch, optionally an iontophoretic patch, or transmucosally, e.g. bucally.
  • starting dosages may be about 0.6 mg/day for about one week (to reduce gastrointestinal symptoms), and then about 1.2 mg day, which may be increased to about 1.8 mg/day in the event the lower dose is ineffective.
  • Figs. 2-7 The results are graphically illustrated in Figs. 2-7.
  • the data show that the peptide 6PY, having the C-terminal tyrosine residue (Y), was the most susceptible to degradation by carboxypeptidase B, wherein 100% of the tyrosine residues were cleaved within about 6 hours. See Fig. 4.
  • the data also show that the C-terminal R and K residues, i.e., arginine and lysine, respectively, were also quite susceptible to carboxypeptidase B degradation, although not as susceptible as tyrosine. See, Figs. 5 and 7.
  • proline (P), methionine (M) and leucine (L) were substantially if not totally resistant to carboxypeptidase B degradation. See, Figs. 2, 3 and 6.
  • Example 2 Yeast Host Cells
  • the S. cerevisiae yeast host cells were obtained from American Type Cell Culture collection (ATCC Number 4026510). Overnight cultures of S. cerevisiae grown in nonselective YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 25 °C were inoculated at 1: 100 in YPD medium and grown to an optical density at 600 nm (OS600) of 1.6 for competent cell preparation. The culture was chilled on ice for 15 min and centrifuged at 4000g. The cell pellet was treated with lOOmL of TE buffer containing Lithium Acetate for 45 min at 30°C shaking at about 85 RPM 2.5 mL of 1 M DTT was added and incubated for 15 min at 30°C. Cells were pelleted down and washed with ice cold water followed by 1M sorbitol solution.
  • a pSCOOl vector was used, operably linked to a galactose inducible promoter ( Figure 1).
  • the aliquot of 40 ⁇ of electro- competent cells was mixed with 1 ⁇ g of expression vector in 0.2 cm cuvettes and electroporated using Gene Plaste Xcell (Make: Biorad).
  • Gene Plaste Xcell Make: Biorad
  • cells were plated directly onto selective agar plate (SD media without uracil, Cat No. G067 Make: Himedia) and the plate was incubated at 25°C for 4 days.
  • the selected colonies were transferred to a flask containing 100 ml SD media and expression was induced with 2% galactose at an optical density 10.
  • Precursor polypeptides were purified with series of chromatography steps.
  • the expressed heterologous polypeptides (5P-A, 6P-B, 6P-C, 6P-D, 6P-E, 6P-F) were treated to cleave the C-terminal additional amino acid according to the same protocol described in example 1.
  • FIG. 9 shows percent conversion of GLP-1 analogue (sequence id 1), 1532/2357/03 (sequence id 2) and 1532/2357/04 (sequence id 3) by carboxypeptidase B over the course of a 6-hour incubation.
  • Figure 10 shows percent conversion of GLP-1 analogue and a precursor thereof, GLP-1 analogue-TYR, by carboxypeptidase B over the course of a 6-hour incubation.
  • Seq id 1 GLP-1 analog
  • GLP-1 Glucagon like peptide 1
  • GLP-1 consists of 30 amino acids and it is derived from tissue-specific post-translational processing of the proglucagon gene located on chromosome 17. GLP- 1 further undergoes post-translational amidation at C-terminus. This amidation and the histidine residue at position 7 are very important for its activity. Amidation has also been shown to prolong the survival of GLP- 1 in the blood stream (Vishal, Indian J. Endocrinol. Metab. 17(3 ):413-21 (2013). This example shows that GLP- 1 analogue (2357/05) has increased plasma half-life compared to liraglutide or GLP- 1 (7-37).
  • Group 1 Normal control received Phosphate Buffer pH 8.0
  • Group 3 GLP-1 Analogue (2357/05, 0.5 mg/kg, s.c.)
  • 20% Glucose solution was prepared. Baseline blood glucose levels were measured with glucose strips using Blood Glucose Meter (One TouchTM UltraTM; LIFESCAN, Johnson & Johnson). After 4 hours of drug administration, all the animals were dosed orally with the 20% glucose solution at a dose of 2 gm/kg. Approximately 5 ⁇ ⁇ of blood was collected and blood glucose levels were measured with glucose strips using Blood Glucose Meter (One TouchTM UltraTM; LIFESCAN, Johnson & Johnson) at 20, 40, 60, 90 and 120 minutes post oral glucose administration.
  • Blood Glucose Meter One TouchTM UltraTM; LIFESCAN, Johnson & Johnson
  • Group 1 GLP-1 analogue (2357/05) (0.5 mg/kg, s.c.)
  • Group 2 GLP-1 Analogue (2357/05) (1 mg/kg, s.c.)
  • Figs. 12A and B show that the plasma half-life of GLP-1 analogue (2357/05) is comparatively higher than Victoza® at the 0.5 mg/ml dose. These data are tabulated in Tables I and II, respectively (for the inventive embodiment and Victoza®).
  • Figs. 13A and B show that GLP-1 analogue (2357/05) exhibited a concentration in plasma of greater than 100 mg/ml at 24 hours, which is higher than the plasma concentration of Victoza® at 24 hours. These data are tabulated in (GLP-1 analogue 2357/05) Tables III and IV.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Mycology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne des procédés et des compositions destinés pour rendre des polypeptides hétérologues sensibles à la protéase KEX1, dans une levure, qui utilisent des acides nucléiques qui codent des formes de précurseur de ces polypeptides qui contiennent, au niveau de leur terminaison C, au moins un acide aminé supplémentaire dont chacun est susceptible de se dégrader sous l'action de la carboxypeptidase B, l'acide aminé de terminaison C du précurseur étant résistant à la dégradation par KEX1. Les polypeptides précurseurs qui sont biologiquement inactifs peuvent être ensuite convertis en polypeptides actifs biologiquement, matures, par clivage d'au moins un acide aminé de terminaison C à l'aide de la carboxypeptidase B. Des précurseurs du polypeptide qui sont actifs biologiquement peuvent être formulés et utilisés pour traiter des maladies telles que des désordres métaboliques. L'invention concerne également des formes précurseurs des polypeptides hétérologues, et des acides nucléiques les codant, ainsi que des constructions génétiques les contenant.
PCT/IB2018/051788 2017-03-27 2018-03-18 Procédé pour rendre des polypeptides sensibles à la protéase kex1 à l'aide d'une souche de levure WO2018178796A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN201721010840 2017-03-27
IN201721010840 2017-03-27

Publications (1)

Publication Number Publication Date
WO2018178796A1 true WO2018178796A1 (fr) 2018-10-04

Family

ID=63674550

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2018/051788 WO2018178796A1 (fr) 2017-03-27 2018-03-18 Procédé pour rendre des polypeptides sensibles à la protéase kex1 à l'aide d'une souche de levure

Country Status (1)

Country Link
WO (1) WO2018178796A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109735589A (zh) * 2019-01-02 2019-05-10 珠海冀百康生物科技有限公司 一种胰岛素或胰岛素衍生物前体制备方法
WO2019193576A1 (fr) * 2018-04-05 2019-10-10 Sun Pharmaceutical Industries Limited Nouveaux analogues de glp-1

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4929553A (en) * 1987-05-29 1990-05-29 Canadian Patents & Development Ltd. Protease for specific processing of secreted proteins
US5077204A (en) * 1984-06-21 1991-12-31 Chiron Corporation Yeast endopeptidase for basic amino-acid site cleavage, preparation and use
US20130273606A1 (en) * 2007-12-21 2013-10-17 Fujifilm Manufacturing Europe B.V. Secretion yield of a protein of interest by in vivo proteolytic processing of a multimeric precursor

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5077204A (en) * 1984-06-21 1991-12-31 Chiron Corporation Yeast endopeptidase for basic amino-acid site cleavage, preparation and use
US4929553A (en) * 1987-05-29 1990-05-29 Canadian Patents & Development Ltd. Protease for specific processing of secreted proteins
US20130273606A1 (en) * 2007-12-21 2013-10-17 Fujifilm Manufacturing Europe B.V. Secretion yield of a protein of interest by in vivo proteolytic processing of a multimeric precursor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KRESGE N ET AL.: "The Role of Hemoglobin's C-terminal Region: the Work of Eraldo Antonini", JOURNAL OF BIOCHEMISTRY, vol. 284, no. 30, July 2009 (2009-07-01), pages 9 - 10, XP055551764 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019193576A1 (fr) * 2018-04-05 2019-10-10 Sun Pharmaceutical Industries Limited Nouveaux analogues de glp-1
JP2021520346A (ja) * 2018-04-05 2021-08-19 サン ファーマシューティカル インダストリーズ リミテッドＳｕｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｉｎｄｕｓｔｒｉｅｓ Ｌｔｄ． 新規ｇｌｐ−１類似体
US11242373B2 (en) 2018-04-05 2022-02-08 Sun Pharmaceutical Industries Limited GLP-1 analogues
US11447535B2 (en) 2018-04-05 2022-09-20 Sun Pharmaceutical Industries Limited GLP-1 analogues
EP4122954A1 (fr) * 2018-04-05 2023-01-25 Sun Pharmaceutical Industries Limited Nouveaux analogues de glp-1
JP7250814B2 (ja) 2018-04-05 2023-04-03 サン ファーマシューティカル インダストリーズ リミテッド 新規ｇｌｐ－１類似体
US11866477B2 (en) 2018-04-05 2024-01-09 Sun Pharmaceutical Industries Limited GLP-1 analogues
US11873328B2 (en) 2018-04-05 2024-01-16 Sun Pharmaceutical Industries Limited GLP-1 analogues
EP4364751A3 (fr) * 2018-04-05 2024-06-26 Sun Pharmaceutical Industries Limited Nouveaux analogues de glp-1
CN109735589A (zh) * 2019-01-02 2019-05-10 珠海冀百康生物科技有限公司 一种胰岛素或胰岛素衍生物前体制备方法

Similar Documents

Publication Publication Date Title
AU2007298919C1 (en) Protease resistant insulin analogues
JP5027812B2 (ja) トリプシンの変異体によるインスリン前駆体の切断
AU2007298919B2 (en) Protease resistant insulin analogues
US8030273B2 (en) Protracted exendin-4 compounds
US8846618B2 (en) Stable formulation of modified GLP-1
JP5503968B2 (ja) 成熟インスリンポリペプチドの作製方法
JP2643968B2 (ja) Ｋｅｘ２エンドプロテアーゼ及びその製造方法
EP2045265A1 (fr) Peptides à fusion GLP-1, leur production et utilisation
JP5366546B2 (ja) 成熟インスリンポリペプチドの作製方法
US10301612B2 (en) Recombinant elastase proteins and methods of manufacturing and use thereof
EP2004213A1 (fr) Agonistes du peptide glp-1
JP2019531075A (ja) インスリン受容体との結合力が減少された、インスリンアナログ及びその用途
JP5850848B2 (ja) ポリペプチドの製造方法
WO2018178796A1 (fr) Procédé pour rendre des polypeptides sensibles à la protéase kex1 à l'aide d'une souche de levure
EP0802983B1 (fr) Expression d'inhibiteurs de l'activateur de l'urokinase-plasminogene
CA2094512A1 (fr) Polypeptides de fusion
JPH0687788B2 (ja) システイン残基を含有する生理活性ペプチドの製造方法
Hou et al. High-level expression of fusion protein containing 10 tandem repeated GLP-1 analogs in yeast Pichia pastoris and its biological activity in a diabetic rat model
Ozaki et al. Enzymatic inactivation of major circulating forms of atrial and brain natriuretic peptides
AU2013204017B2 (en) Recombinant elastase proteins and methods of manufacturing and use thereof
NO311895B1 (no) Anvendelse av furin eller furinlignende enzym for spalting av et protein, fremgangsmåte for mikrobiologisk fremstilling av etprotein og pattedyrcelle som omfatter DNA som koder for enzymet
Huang et al. Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18778283

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18778283

Country of ref document: EP

Kind code of ref document: A1