Classifications

• • 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
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • 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
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/53—Colony-stimulating factor [CSF]
  • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
• • 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

Definitions

• the present invention relates to new polypeptides having activity of stimulating colonies of human granulocytes, their preparation and pharmaceutical compositions containing them.
• the present invention relates in particular to chimeric polypeptides composed of a biologically active part consisting of all or part of G-CSF or of a variant of G-CSF, and of an essentially protein stabilizing structure conferring on it new biological properties.
• Human G-CSF is a secreted polypeptide of 174 amino acids, having a molecular weight of approximately 18 kD. It was initially isolated from a cancer cell line (EP 169,566), and its gene was cloned, sequenced, and expressed in different cell hosts by genetic engineering techniques (EP 215,126, EP 220,520). An mRNA potentially coding for a form of G-CSF having 177 amino acids has also been demonstrated [Nagata S. et al., EMBO J. 5 (1986) 575-581]. G-CSF has the ability to stimulate the differentiation and proliferation of bone marrow progenitor cells into granulocytes.
• G-CSF is degraded quickly by the body once administered. This is all the more sensitive since G-CSF is generally used at low doses. In addition, the use of larger doses could not improve the capacities therapeutic effects of this molecule and can induce undesirable side effects. These elimination and degradation phenomena in vivo therefore constitute for the moment an obstacle to the exploitation of the biological activity of G-CSF as a pharmaceutical agent.
• the present invention overcomes these drawbacks.
• the present invention provides indeed new molecules allowing an optimal exploitation on the therapeutic level of the biological properties of G-CSF. The Applicant has in fact demonstrated that the optimal activity of G-CSF manifests itself when G-CSF is present at low dose and for an extended time.
• the applicant has now produced molecules capable of maintaining G-CSF activity in the body for a sufficiently long time.
• the Applicant has shown that it is possible to express, in cellular hosts at high levels, genetic fusions generating chimeras exhibiting new pharmacokinetic properties and the desirable biological properties of G-CSF.
• the hybrid polypeptides of the invention retain their affinity for the G-CSF receptors, and are sufficiently functional to lead to cell proliferation and differentiation.
• the molecules of the invention also have a distribution and pharmacokinetic properties which are particularly advantageous in the organism and allow the therapeutic development of their biological activity.
• An object of the present invention therefore relates to recombinant polypeptides comprising an active part consisting of all or part of G-CSF, or of a variant of G-CSF, and an essentially protein stabilizing structure.
• variant of G-CSF designates any molecule obtained by modification of the sequence between the residues Thr586 and Pro759 of the sequence presented in FIG. 1, retaining G-CSF activity, that is to say ie the ability to stimulate the differentiation of target cells and the formation of granulocyte colonies.
• This sequence corresponds to that of mature G-CSF described by Nagata et al. [EMBO J. £ (1986) 575-581].
• Such variants can be generated for different purposes, such as in particular that of increasing the affinity of the molecule for the G-CSF receptor (s), that of improving its production levels, that of increasing its resistance to proteases, that of increasing its therapeutic efficacy or reducing its side effects, or that of giving it new pharmacokinetic and / or biological properties.
• polypeptides include, for example, molecules in which certain glycosylation sites have been modified or deleted, as well as molecules in which one, more, or even all of the cysteine residues have been substituted. It also includes molecules obtained from (a) or (b) by deletion of regions having little or no activity, or occurring in an undesirable activity, and molecules comprising, with respect to (a) or ( b) additional residues, such as for example an N-terminal methionine or a secretion signal.
• the chimeric polypeptides of the invention comprise an active part of type (a).
• the active part of the molecules of the invention can be coupled to the protein stabilizing structure, either directly or through a junction peptide. In addition, it can constitute the N-terminal end as the C-terminal end of the molecule. Preferably, in the molecules of the invention, the active part constitutes the C-terminal part of the chimera.
• the stabilizing structure of the polypeptides of the invention is essentially protein.
• this structure is a polypeptide having a high plasma half-life.
• it may be an albumin, an apolipoprotein, an immunoglobulin or even a transfemin. They may also be peptides derived from such proteins by structural modifications, or peptides synthesized artificially or semi-artificially, and having a high plasma half-life.
• the stabilizing structure used is more preferably a weakly or non-immunogenic polypeptide for the organism in which the polypeptides of the invention are used.
• the stabilizing structure is an albumin or a variant of albumin and for example human serum albumin (S AH).
• the albumin variants designate any protein with a high plasma half-life obtained by modification (mutation, deletion and / or addition) by genetic engineering techniques of a gene coding for a given isomorph of serum- human albumin, as well as any macromolecule with a high plasma half-life obtained by in vitro modification of the protein encoded by such genes.
• albumin is very polymorphic, many natural variants have already been identified, and more than 30 different genetic types have been identified [Weitkamp L.R. et al., Ann. Hmm. Broom. 21 (1973) 219]. More preferably, the stabilizing structure is a mature albumin.
• polypeptides of the invention comprising, in the N-terminal -> C-terminal direction, (i) the sequence of mature SAH directly coupled to the sequence of mature G-CSF (cf. Figure 1), or (ii) the sequence of mature G-CSF coupled via a peptide binding to the sequence of mature HSA.
• Another subject of the invention relates to a process for the preparation of the chimeric molecules described above. More precisely, this method consists in causing a eukaryotic oti prokaryotic cellular host to express a nucleotide sequence coding for the desired polypeptide, then in harvesting the produced polypeptide.
• eukaryotic hosts which can be used in the context of the present invention, mention may be made of animal cells, yeasts, or fungi.
• yeasts mention may be made of yeasts of the genus Saccharomyces. Kluyveromyces. Pichia. Sc warmiomyces. or Hansenula.
• animal cells mention may be made of COS, CHO, C127 cells, etc.
• fungi capable of being used in the present invention there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp.
• prokaryotic hosts it is preferred to use bacteria such as Escherichia coli. or belonging to the genera Corynebacterium. Bacillus.
• nucleotide sequences which can be used in the context of the present invention can be prepared in different ways. Generally, they are obtained by assembling in the reading phase the sequences coding for each of the functional parts of the polypeptide. These can be isolated by techniques skilled in the art, and for example directly from cellular messenger RNAs (mRNAs), or by recloning from a complementary DNA library (cDNA) isolated from from producer cells, or it can be completely synthetic nucleotide sequences. It is further understood that the nucleotide sequences can also be subsequently modified, for example by genetic engineering techniques, to obtain derivatives or variants of said sequences.
• mRNAs messenger RNAs
• cDNA complementary DNA library
• the nucleotide sequence is part of an expression cassette comprising a region for initiating transcription (promoter region) allowing, in host cells, the expression of the nucleotide sequence placed under its control and coding for the polypeptides of the invention.
• This region can come from promoter regions of genes strongly expressed in the host cell used, the expression being constitutive or regulable. In the case of yeasts, it may be the promoter of the phosphoglycerate kinase (PGK) gene.
• PGK phosphoglycerate kinase
• GPD glyceraldehyde-3-phosphate dehydrogenase
• lactase lactase (LAC4). enolases (ENO). alcohol dehydrogenases (ADH). etc.
• bacteria can be the promoter of the right or left genes of bacteriophage lambda (PL, PR), OR promoters of the genes of the operons tryptophan (Ptrp) or lactose (PJac) -
• this control region can be modified, for example by in vitro mutagenesis, by introduction of additional control elements or synthetic sequences, or by deletions or substitutions of the original control elements.
• the expression cassette can also comprise a transcription termination region functional in the envisaged host, positioned immediately downstream of the nucleotide sequence coding for a polypeptide of the invention.
• the polypeptides of the invention result from the expression in a eukaryotic or prokaryotic host of a nucleotide sequence and from the secretion of the expression product of said sequence in the culture medium. It is in fact particularly advantageous to be able to obtain molecules by recombinant route directly in the culture medium.
• the sequence The nucleotide coding for a polypeptide of the invention is preceded by a "leader" sequence (or signal sequence) directing the nascent polypeptide in the secretory pathways of the host used.
• This "leader” sequence can be the natural signal sequence of G-CSF or of the stabilizing structure in the case where this is a naturally secreted protein, but it can also be any other functional "leader” sequence, or of an artificial leader sequence. The choice of one or the other of these sequences is in particular guided by the host used. Examples of functional signal sequences include those of genes for sex pheromones or yeast "killer” toxins.
• one or more markers making it possible to select the recombinant host can be added, such as for example the URA3 gene from the yeast S. cerevisiae. or genes conferring resistance to antibiotics such as geneticin (G418) or to any other toxic compound such as certain metal ions.
• the assembly constituted by the expression cassette and by the selection marker can be introduced directly into the host cells considered, or be inserted beforehand into a functional self-replicating vector.
• sequences homologous to regions present in the genome of the host cells are preferably added to this set; said sequences then being positioned on each side of the expression cassette and of the selection gene so as to increase the frequency of integration of the assembly into the host genome by targeting the integration of the sequences by homologous recombination.
• a preferred replication system for yeasts of the genus Kluyveromyces is derived from the plasmid pKD1 initially isolated from K.
• drosophilarum a preferred replication system for yeasts of the genus Saccharomyces is derived from the plasmid 2 ⁇ of S. cerevisiae.
• this expression plasmid may contain all or part of said replication systems, or may combine elements derived from the plasmid pKDl as well as from the plasmid 2 ⁇ .
• the expression plasmids can be shuttle vectors between a bacterial host such as Escherichia coli and the chosen host cell. In this case, an origin of replication and a selection marker functioning in the bacterial host are required.
• restriction sites can correspond to sequences such as 5'-GGCCNNNNNGGCC-3 '(î ⁇ fil) or 5'- GCGGCCGC-3' (Notl) since these sites are extremely rare and generally absent from an expression vector.
• any method allowing the introduction of foreign DNA into a cell can be used. It may especially be transformation, electroporation, conjugation, or any other technique known to those skilled in the art.
• yeast-type hosts the different Kluyveromyces strains used were transformed by treating whole cells in the presence of lithium acetate and polyethylene glycol, according to the technique described by Ito et al. [J. Bacteriol. 153 (1983) 163].
• the transformation technique described by Durrens et al. [Curr. Broom. 2_ ⁇ (1990) 7] using ethylene glycol and dimethyl sulfoxide was also used. It is also possible to transform yeasts by electroporation, according to the method described by Karube et al. [FEBS Letters ⁇ _2 (1985) 901.
• An alternative protocol is also described in detail in the examples which follow.
• the cells expressing said polypeptides are inoculated and the recovery of said polypeptides can be made, either during cell growth for the "continuous” methods, or at the end of growth for the "batch” cultures ( “batch”).
• the polypeptides which are the subject of the present invention are then purified from the culture supernatant for their molecular, pharmacokinetic and biological characterization.
• a preferred expression system for the polypeptides of the invention consists in the use of yeasts of the genus Kluyveromyces as host cell, transformed by certain vectors derived from the extrachromosomal replicon pKD1 initially isolated from K. marxianus var. drosophilarum. These yeasts, and in particular K. lactis and
• K. fragilis are generally capable of replicating said vectors stably and also have the advantage of being included in the list of GRAS organizations (".Qenerally Recognized As _ £ afe").
• Preferred yeasts are preferably industrial strains of the genus Kluyveromyces capable of stable replication of said plasmids derived from the plasmid pKDl and into which has been inserted a selection marker as well as an expression cassette allowing the secretion at high levels of the polypeptides of the invention.
• the present invention also relates to the nucleotide sequences coding for the chimeric polypeptides described above, as well as the recombinant, eukaryotic or prokaryotic cells, comprising such sequences.
• the present invention also relates to the application as a medicament of the polypeptides according to the present invention.
• the subject of the invention is any pharmaceutical composition comprising one or more polypeptides as described above. More particularly, these compositions can be used in all pathological situations in which the number and / or the activity of the granulocytes must be stimulated. In particular, they can be used for the prevention or treatment of leukopenia or certain leukemias, or in the case of grafts or anti-cancer treatment, to strengthen or restore the immune system.
• Figure 1 Nucleotide sequence of the HindIII restriction fragment of the plasmid pYG1259 (chimera prepro-SAH-G.CSF). The black arrows indicate the end of the "pre” and “pro” regions of HSA. MstlI restriction sites. Apal and SstI (Sacl) are underlined. The peptide sequence of G-CSF is in italics (Thr586-> Pro759, the numbering of the amino acids corresponds to the mature chimeric protein).
• Figure 2 Schematization of chimeras of the SAH-G.CSF (A) type, of the G.CSF-SAH (B) or G.CSF-SAH-G.CSF (C) type.
• M / LP methionine initiating translation, possibly followed by a signal secretion sequence
• SAH mature human serum albumin or a variant thereof
• G.CSF peptide derived from G-CSF and having identical or modified activity.
• the black arrow indicates the N-terminus of the mature protein.
• Figure 3 Restriction map of the plasmid pYG105 and strategy for the construction of the expression plasmids of the chimeric proteins of the present invention.
• P transcriptional promoter
• T transcriptional terminator
• IR inverted repeat sequences of the plasmid pKD1
• LPSAH »SAH prepro "region
• Ap 1 " and Km r respectively designate the genes for resistance to ampicillin (E. coli) and to G418 (yeasts).
• Figure 5 Nucleotide sequence of the HindIII restriction fragment of the plasmid pYG1301 (chimera G.CSF-Gly4-SAH).
• the black arrows indicate the end of the "pre” and "pro” regions of HSA.
• SstI (Sacl) and MstH are underlined.
• the G.CSF (174 residues) and SAH (585 residues) domains are separated by the synthetic linker GGGG.
• the amino acid numbering corresponds to the mature chimeric protein G.
• the nucleotide sequence between the translation termination codon and the HindIII site comes from the complementary DNA (cDNA) of SAH as described in patent application EP 361 991.
• Figure 7 Activity on in vitro cell proliferation of the murine line NFS60.
• the radioactivity ( ⁇ H-thymidine) incorporated into the cell nuclei after 6 hours of incubation is represented on the ordinate (cpm); the quantity of product indicated on the abscissa is expressed in molarity (arbitrary units).
• Figure 8 Activity on granulopoiesis in vivo in rats. The number of neutrophils (average of 7 animals) is indicated on the ordinate as a function of time.
• the products tested are the SAH-G.CSF chimera (pYG1266, 4 or 40 mg / rat / day), the reference G-CSF (10 mg / rat / day), the recombinant SAH purified from a supernatant of Kluyveromyces lactis ( rHSA, 30 mg / rat / day, see EP 361 991), or physiological saline.
• the plasmids of type ⁇ BR322, pUC and the phages of the Ml 3 series are of commercial origin (Bethesda Research Laboratories).
• the DNA fragments are separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of DNA.
• phage T4 ligase Biolabs
• the filling of the protruding 5 ′ ends is carried out by the fragment of
• Mutagenesis directed in vitro by synthetic oligodeoxynucleotides is carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 2_1 (1985) 8749-8764] using the kit distributed by Amersham.
• Verification of the nucleotide sequences is carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.
• Transformations of K. lactis with the DNA of the protein expression plasmids of the present invention are carried out by any technique known to those skilled in the art, an example of which is given in the text.
• the bacterial strains used are E. coli MC1060 (lIPOsYA, X74, gâiU, £ _1K, _rtrA r ), or E. coli TG1 ⁇ _____, p A, B, ⁇ ugE, M, hsdD5 / FîraD36, prgA + B + ,] açl _açZ, M15).
• the yeast strains used belong to budding yeasts and more particularly to yeasts of the genus Kluyveromyces.
• the strains K. lactis M 98-8C (a, uraA. Sg, _J_-, + , pKDl °) and K. lactis CBS 293.91 were particularly used; a sample of the strain MW98-8C was deposited on September 16, 1988 at the Centraalbureau voor Schimmelkulturen (CBS) in Baarn (Netherlands) where it was registered under the number CBS 579.88.
• CBS Centraalbureau voor Schimmelkulturen
• the yeast strains transformed by the expression plasmids coding for the proteins of the present invention are cultured in Erlenmeyer flasks or in pilot fermenters of 21 (SETRIC, France) at 28 ° C. in rich medium (YPD: 1% yeast extract, 2 % Bactopeptone, 2% glucose; or YPL: 1% yeast extract, 2% Bactopeptone, 2% lactose) with constant stirring.
• YPD 1% yeast extract, 2 % Bactopeptone, 2% glucose
• YPL 1% yeast extract, 2% Bactopeptone, 2% lactose
• EXAMPLE 1 CONSTRUCTION OF AN MSTII / HINDIII RESTRICTION FRAGMENT INCLUDING THE MATURE PART OF THE HUMAN G-CSF
• a restriction fragment MstlI-HindIII including the mature form of human G-CSF is generated, for example according to the following strategy: a restriction fragment Kpnl-HindlII is first obtained by the PCR enzymatic amplification technique using oligodeoxynucleotides Sq2291 (5'- CAAGGATCCAA2CJTICAGGGCTGCGCAAGGTGGCGTAG-3 ', the HindIII site is underlined) and Sq2292 (5'-CGGG ⁇ TA £ ⁇ TAGGCTTAACCCCCCTG- GGCCCTGCCAGC-3', the " Kpnl site is primed as the matrix as the plasmid as for the template) Plasmid BBG13 contains the gene coding for form B (174 amino acids) of mature human G-CSF, obtained from British Bio-technology Limited, Oxford, England.
• the enzymatic amplification product of approximately 550 nucleotides is then digested with the restriction enzymes Kpnl and HindIII and cloned into the vector pUC19 cut with the same enzymes, which generates the recombinant plasmid pYG1255.
• This plasmid is the source of a restriction fragment Ms tI-HindIII, the sequence of which is included in that of FIG. 1.
• An MstlI-HindIII restriction fragment coding for the same polypeptide sequence can also be generated by the PCR amplification technique from the corresponding cDNAs, the sequence of which is known [Nagata S. et al., EMBO J. ⁇ (1986) 575-581].
• cDNAs can be isolated by techniques skilled in the art, for example using the kit distributed by Amersham, from a human cell line expressing G-CSF, and for example the CHU-2 cell line. human carcinoma [Nagata et al., Nature 19 (1986) 415-418]. It may also be desirable to insert a peptide linker between the SAH part and G-CSF, for example to allow a better functional presentation of the transducer part.
• a restriction fragment MstlI-HindIII is for example generated by substitution of the fragment MstlI-Apal of FIG.
• the plasmid pYG1336 thus generated therefore comprises an MstlI-HindIII restriction fragment. whose sequence is identical to that of Figure 1 except for the MstlI-Apal fragment.
• EXAMPLE 2 MERGERS IN THE TRANSLATION PHASE BETWEEN THE SAH AND THE HUMAN G-CSF
• the plasmid pYG404 is described in patent application EP 361 991.
• This plasmid comprises a HindIII restriction fragment coding for the prepro-SAH gene preceded by the 21 nucleotides naturally present immediately upstream of the translation initiator ATG of the PGK gene from S. cerevisiae. More particularly, this fragment comprises a HindIII-MstlI restriction fragment corresponding to the entire gene coding for prepro-SAH with the exception of the three most C-terminal amino acids (leucine-glycine-leucine residues).
• HindIII restriction fragment except for the MstlI-Apal fragment can also be easily generated and which codes for a chimeric protein in which form B of mature G-CSF is positioned by genetic coupling in the C-terminal translational phase of the SAH molecule and of a particular peptide linker.
• this linker consists of 4 glycine residues in the HindIII fragment of the plasmid pYG1336 (chimera SAH-GIy4-G.CSF, cf. FIG. 2, panel A).
• hybrid genes coding for a chimeric protein (FIG. 2, panel B) resulting from the translational coupling between a signal peptide (and for example the prepro region of SAH), a sequence including a gene having G-CSF activity, and the mature form of SAH or one of its molecular variants.
• These hybrid genes are preferably bordered 5 'to the translation initiating ATG and 3' to the translation end codon by HindIII restriction sites.
• the residues underlined correspond in this particular chimera to a peptide linker composed of 4 glycine phase residues allows for mutagenic phase translation) mature form of human G-CSF from plasmid BBG13 immediately upstream of the mature form of HSA, which generates intermediate plasmid A.
• the use of the oligodeoxynucleotide Sq2338 .5'-CAGGGAGCTGGCAGGGCCCAGGGGGGTTCGACGAAACACACCCCTG- GAATAAGCCGAGCT-GAATAAGCCGAGCT 1 (non-coding strand), the nucleotides complementary to the nucleotides coding for the first N-terminal residues of the mature form of human G-CSF are underlined] allows by directed mutagenesis to couple in the translational phase of reading the prepro region of SAH immediately upstream of the mature form of human G-CSF, which generates the intermediate plasmid re B.
• Plasmid pYG1301 contains this particular HindIII restriction fragment coding for the chimera G.CSF-Gly4-SAH fused immediately downstream of the prepro region of SAH.
• hybrid genes in which a sequence coding for G-CSF activity is coupled to the N- and C- terminal ends of HSA or one of its molecular variants ( Figure 2, panel C). These hybrid genes are preferably bordered 5 'to the translation initiating ATG and 3' to the translation end codon by HindIII restriction sites.
• the chimeric proteins of the previous examples can be expressed in yeasts from functional, regulatable or constitutive promoters, such as, for example, those present in the plasmids pYG105 (LAC4 promoter from KluwerOmyces lactis). pYG106 (PGK promoter of Saccharomyces cerevisiae). pYG536 (PHO5 promoter of S. cerevisiae). or hybrid promoters such as those carried by the plasmids described in patent application EP 361 991.
• functional, regulatable or constitutive promoters such as, for example, those present in the plasmids pYG105 (LAC4 promoter from KluwerOmyces lactis).
• pYG106 PGK promoter of Saccharomyces cerevisiae
• pYG536 PHO5 promoter of S. cerevisiae
• hybrid promoters such as those carried by the plasmids described in patent application EP 361 991.
• the HindIII restriction fragment of the plasmid pYG1259 is cloned in the productive orientation in the HindIII restriction site of the expression plasmid pYG105, which generates the expression plasmid pYG1266 ( Figure 3).
• the plasmid pYG105 corresponds to the plasmid pKan707 described in patent application EP 361 991 in which the HindIII restriction site was destroyed by site-directed mutagenesis (oligodeoxynucleotide Sql053: 5'-GAAATGCATAAGCTC-TTGCCATTCTCACCG-3 ') and whose fragment Sall-Sacl encoding the URA3 gene has been replaced by a Sall-Sacl restriction fragment comprising the promoter LAC4 (in the form of a SalI-HindIII fragment) and the terminator of the PGK gene of S. cerevisiae (in the form of a fragment HindIII-SacI).
• the plasmid pYG105 is mitotically very stable in the absence of geneticin (G418) and makes it possible to express the chimeric protein from the LAC4 promoter from K. lactis. especially when the carbon source is lactose.
• the cloning in the productive orientation of the HindIII restriction fragment of the plasmid pYG1259 into the HindIII site of the plasmid pYG106 generates the expression plasmid pYG1267.
• the plasmids pYG1266 and pYG1267 are isogenic with each other except for the restriction fragment SalI-HindIII coding for the promoter LAC4 from K. lactis (plasmid pYG1266) or the PGK promoter from S. cerevisiae (plasmid pYG1267).
• the cloning in the productive orientation of the HindIII restriction fragment of the plasmid pYG1336 (chimera SAH-GIy4-G.CSF, cf. E.2J.)
• Into the HindIII site of the plasmids pYG105 and pYG106 generates the plasmids d pYG1351 and pYG1352, respectively.
• HindHI from plasmids pYG105 and pYG106 generates the expression plasmids pYG1302 and pYG1303, respectively.
• the transformation of yeasts belonging to the genus Kluyveromyces. and in particular the MW98-8C and CBS 293.91 strains of K. lactis. is carried out for example by the technique of treating whole cells with lithium acetate (Ito H. et al., J. Bacteriol. 153 (1983) 163-168), adapted as follows. The cells are grown at 28 ° C.
• the cell supernatants are then tested after electrophoresis in 8.5% acrylamide gel, either directly by staining the acrylamide gel with coomassia blue (FIG. 4, panel A), or after immunoblotting using primary antibodies as polyclonal antibodies. rabbits specifically directed against human G-CSF, or against HSA.
• the nitrocellulose filter is first incubated in the presence of the specific antibody, washed several times, incubated in the presence of biotinylated anti-rabbit goat antibodies, then incubated in the presence of an avidin complex. -peroxidase using the "ABC kit" distributed by Vectastain (Biosys SA, Compiègne, France).
• the immunological reaction is then revealed by the addition of diamino-3,3 'benzidine tetrachlorydrate (Prolabo) in the presence of hydrogen peroxide, according to the recommendations of the supplier.
• the results of FIG. 4 demonstrate that the hybrid protein SAH-G.CSF is recognized both by antibodies directed against human albumin (panel C) and human G-CSF (panel B).
• the results of FIG. 6 indicate that the SAH-Gly4-G.CSF chimera (lane 3) is particularly well secreted by the yeast Kluyveromyces. possibly because the presence of the peptide linker between the SAH part and the G-CSF part is more favorable for independent folding of these 2 parts during the transit of the chimera in the secretory pathway.
• the N-terminal fusion (G.CSF-Gly4-SAH) is also secreted by the yeast KluyverOmyces
• the culture supernatant is passed through a 0.22 mm filter (Millipore), then concentrated by ultrafiltration (Amicon) using a membrane with a discrimination threshold of 30 kDa.
• the concentrate obtained is then adjusted to 50 mM Tris HC1 from a stock solution of Tris HC1 1M (pH 6), then deposited in 20 ml fractions on a column (5 ml) ion exchange (Q Fast Flow, Pharmacia) balanced in the same buffer.
• the chimeric protein is then eluted from the column by a gradient (0 to 1 M) of NaCl.
• fractions containing the chimeric protein are then combined and dialyzed against a 50 mM Tris HCl solution (pH 6) and redeposited on a Q Fast Flow column (1 ml) equilibrated in the same buffer. After elution from the column, the fractions containing the protein are combined, dialyzed against water and lyophilized before characterization: for example, sequencing (Applied Biosystem) of the protein SAH-G.CSF secreted by the yeast CBS 293.91 gives the expected N-terminal sequence of SAH (Asp-Ala-His ...), demonstrating a correct maturation of the chimera immediately in C-terminal of the doublet of Arg-Arg residues from the "pro" region of the SAH ( Figure 1).
• the chimeras purified according to Example 6 are tested for their capacity to allow in vitro proliferation of the murine IL3-dependent line NFS60, by measuring the incorporation of tritiated thymidine essentially according to the protocol described by Tsuchiya et al. [Proc. Natl. Acad. Sci. (1986) £ 2. 7633]. For each chimera, the measurements are made between 3 and 6 times in a three-point test (three dilutions of the product) in an area where the relationship between the quantity of active product and incorporation of labeled thymidine (Amersham) is linear.
• the stimulation activity of SAH / G-CSF chimeras on granulopoiesis in vivo is tested after subcutaneous injection in rats (Sprague-Dawley / CD, 250-300 g, 8-9 weeks) and compared with that of G -CSF reference expressed from mammalian cells.
• Each product, tested on the basis of 7 animals, is injected subcutaneously in the dorsoscapular region at the rate of 100 ml for 7 consecutive days (D1-D7).
• 500 ml of blood are collected on days 3-6, J2 (before 2nd injection), J5 (before the 5 th injection) and D8, and complete blood cell count is performed.

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• Animal Behavior & Ethology (AREA)
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• Diabetes (AREA)
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• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

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• 1992
  • 1992-01-31 FR FR9201065A patent/FR2686900B1/fr not_active Expired - Fee Related
• 1993
  • 1993-01-28 JP JP5512987A patent/JPH07503844A/ja active Pending
  • 1993-01-28 EP EP93904130A patent/EP0624200A1/fr not_active Withdrawn
  • 1993-01-28 CA CA002125979A patent/CA2125979A1/fr not_active Abandoned
  • 1993-01-28 FI FI943564A patent/FI943564A7/fi not_active Application Discontinuation
  • 1993-01-28 WO PCT/FR1993/000086 patent/WO1993015211A1/fr not_active Application Discontinuation
  • 1993-01-29 US US08/256,938 patent/US5665863A/en not_active Expired - Lifetime
• 1994
  • 1994-08-01 NO NO942858A patent/NO942858L/no unknown

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