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
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • 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
  • C07K14/745—Blood coagulation or fibrinolysis factors
  • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• • 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
  • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

• ANTITHROMBOTIC POLYPEPTIDES ANTAGONISTS OF THE VWF BINDING TO PLATELETS AND / OR THE SUBENDOTHELIUM.
• the present invention relates to new antithrombotic polypeptides, their preparation and pharmaceutical compositions containing them. More particularly, the present invention relates to new polypeptides comprising a part derived from the structure of von Willebrand factor (vWF) and intrinsically capable of binding to blood platelets and / or to the subendothelium.
• vWF von Willebrand factor
• VWF is a glycosylated protein of 2813 amino acids comprising a signal sequence of 22 residues, a "pro" region of 741 residues and a mature protein of 2050 amino acids organized in several repeated structures [Titani K. et al., Biochemistry 25 ( 1986) 3171-3184; Verweij CL. et al., EMBO J. 5 (1986) 1839-1847].
• This complex glycoprotein is present in vivo, either stored in specialized vesicles of endothelial cells or platelets, or in circulating form in the blood plasma after secretion and proteolytic maturation during the secretion process.
• vWF The circulating forms of vWF are present in the form of multimers of high molecular weight (up to 20,000 kDa) and the protomer of which is a dimer of approximately 450 kDa.
• the vWF gene has been cloned and sequenced by several teams and mapped to the short arm of chromosome 12 [Sadler J.E. et al., Proc. Natl. Acad. Sci. £ 2 (1985) 6394-6398; Verweij CL. et al., EMBO J. 50 (1986) 1839-1847; Shelton-Inloes B.B. et al., Biochemistry 2 £ (1986) 3164-3171; Bonthron D. et al., Nucleic Acids Res. XZ (1986) 7125-7127; Ginsburg D. et al., Science 22 £ (1985) 1401-1406].
• VWF is involved in the genesis of arterial thrombi by a complex and poorly understood interaction between certain components of the subendothelium on the one hand and blood platelets on the other (and in particular platelet GP1b receptors).
• An important point is that the circulating plasma vWF does not spontaneously bind the GPlb receptors of platelets, and it is likely that its interaction with the subendothelium is necessary to unmask its site (s) of interaction with platelets, for example following a conformational change in the vWF.
• vWF fibrino-cellular thrombus
• platelet GPlb The interaction between the vWF thus activated and platelet GPlb leads to the activation of blood platelets which then acquire the ability to aggregate and generate a fibrino-cellular thrombus in the presence of certain adhesive proteins (fibrinogen, thrombospondin, vWF etc.).
• vWF constitutes a pharmacological target of choice for the production of antithrombotic agents.
• many difficulties must be overcome to be able to exploit this molecule on the pharmacological level: the inability of circulating vWF to bind platelets, the ignorance of the respective contribution of the different adhesive functions of vWF (subendothelium and platelets) in its thrombogenic activity, the difficulty of producing at sufficiently high levels sufficiently pure and homogeneous products to be able to be used as therapeutic agents, the large size of the vWF and its complexity, the dynamics of its tertiary structure, etc. Some fragments of the vWF have were obtained by proteolytic digestion and studied pharmacologically.
• the present invention provides new molecules intrinsically capable of at least partially antagonizing platelet activation.
• the molecules of the invention comprise an adhesive part derived from the structure of vWF and a part allowing its functional presentation and ensuring the stability and in vivo distribution of the molecule.
• the Applicant has indeed shown that it is possible to genetically couple vWF to a structure of protein nature, and to produce such molecules at satisfactory levels.
• the molecules of the invention also make it possible to generate and use small structures derived from vWF and therefore very specific for a desired effect (for example antagonists of the sole vWF-GPlb interaction).
• the Applicant has also shown that such coupling promotes the presentation of this structure at its link site (s).
• the polypeptides of the invention therefore make it possible to exhibit, within a stable structure, structures derived from vWF capable of at least partially antagonizing the binding of vWF to platelets, and therefore of inhibiting platelet activation .
• the polypeptides of the invention also make it possible to expose, within a stable structure, structures derived from vWF capable of at least partially antagonizing the binding of vWF to the subendothelium.
• An object of the present invention therefore relates to molecules comprising an adhesive part derived from the structure of vWF capable of at least partially antagonizing the binding of vWF to platelets and / or to the subendothelium, and a part of a protein nature allowing its stabilization. and its presentation in vivo.
• the adhesive part consists of all or part of the peptide sequence comprised between residues 445-733 of vWF or a variant thereof.
• the peptide sequence of vWF having been published, the numbering of the residues of the adhesive part of the molecules of the invention refers to the numbering of the sequence of vWF published by Titani et al. [Biochemistry 2 £ (1986) 3171-3184]. It is understood that this function can be redundant within the molecules of the present invention. Part of this sequence of vWF (residues Thr470 to Val713) is indicated in Figure 1, in which it is coupled at the C-terminal of human serum albumin.
• variant designates any molecule obtained by modification of the sequence capable of at least partially antagonizing the binding of vWF to platelets and / or to the subendothelium.
• modification one must understand any mutation, substitution, deletion, addition or modification obtained, for example, by means of genetic engineering techniques.
• variants can be generated for different purposes, such as in particular that of increasing the affinity of the molecule for its site (s) of fixation, that of improving its production levels, that of reducing its susceptibility to proteases, that of increasing its therapeutic effectiveness or of reducing its side effects, or that of imparting new properties to it pharmacokinetics or biology such as in particular adhesive functions expressed in an intrinsically non-cryptic manner.
• polypeptides of the invention are those in which the adhesive part has: (a) the peptide sequence comprised between residues 445-733 of vWF, or,
• (c) a structure derived from structures (a) or (b) by structural modifications (mutation, substitution addition and / or deletion of one or more residues) and capable of at least partially antagonizing the binding of vWF to GPlb and or at the subendothelium, or,
• an unnatural peptide sequence for example isolated from peptide libraries and capable of at least partially antagonizing the binding of vWF to GPlb and / or to the subendothelium.
• type (b) mention may be made more particularly of those which have retained the capacity to antagonize the interaction between vWF and platelet GPlb, such as for example the peptides G10 or D5 described by Mori et al. [J. Biol. Chem. 2 £ 3_ (1988) 17901-17904], or the peptides which have retained the capacity to bind collagen [Pareti FI et al., J. Biol. Chem.
• Structures of type (c) include, for example, molecules in which certain N- or O-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, or alternatively point and / or multiple mutants relating to at least one residue involved in HB-type pathologies associated with vWF such as residues Arg543, Arg545, Trp550, Val553 or Arg578 for example.
• molecules obtained from (a) or (b) by deletion of regions having little or no involvement in the interaction with the binding sites considered and molecules comprising, with respect to (a) or (b ) additional residues, such as for example an N-terminal methionine and / or a signal secretion sequence and or a polypeptide adapter allowing junction to the stabilizing structure.
• polypeptides of the invention comprising the stabilizing structure coupled: - to a peptide of PI type, the minimum version of which corresponds to the peptide
• - a peptide of type X * defined as any molecular variant of peptides of type X and XD, or, to any combination of these peptides, and among others: - peptides of type P1-P2;
• the adhesive portion of the molecules' of the invention can be coupled, either directly or via a linking peptide to the protein stabilizing structure.
• it can constitute the N-terminal end as the C-terminal end of the molecule.
• the adhesive part constitutes the C-terminal part of the chimera.
• the stabilizing structure of the polypeptides of the invention is a polypeptide having a high plasma half-life.
• it may be a protein such as albumin, an apolipoprotein, an immunoglobulin or a transferin, etc. It may also be peptides derived from such proteins by structural modifications, or peptides artificially or semi-artificially synthesized, 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 polypeptide of the invention is used.
• the stabilizing structure is an albumin or a variant of albumin and for example human serum albumin (S AH).
• 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. Since albumin is very polymorphic, many natural variants have been identified and listed [Weitkamp L.R. et al., Ann. Hmm. Broom. 27 (1973) 219]. For example, the chimeras between the said adhesive function (s) and mature SAH have pharmacokinetic properties and antithrombotic activities which are particularly useful in therapy.
• Another subject of the invention relates to a process for the preparation of the chimeric molecules described above. More specifically, this method consists in causing a eukaryotic or prokaryotic cellular host to express a nucleotide sequence coding for the desired polypeptide, then in collecting the polypeptide produced.
• yeasts examples include animal cells, yeasts, or fungi.
• yeasts mention may be made of yeasts of the genus Saccharomyces. Kl ⁇ yveromyces. Pichia. Schwanniomyces. 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. or Streptomyces.
• 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 reading phase the sequences coding for each of the functional parts of the polypeptide. These can be isolated by the techniques of a person skilled in the art, and for example directly from cellular messenger RNAs (mRNA), or by recloning from a complementary DNA library (cDNA) carried out at 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.
• mRNA 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 tryptophan operons (Ptrp) or lactose (Plac) -
• this control region can be modified, for example by in vitro mutagenesis, by the 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 nucleotide sequence coding for a polypeptide of the invention is preceded by a "leader" sequence (or signal sequence) directing the nascent polypeptide in the pathways secretion from the host used.
• This “leader” sequence can be the natural signal sequence of the vWF 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 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, either directly into the host cells considered, or 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. It is also possible to position restriction sites surrounding the bacterial and unique sequences on the expression vector: this makes it possible to remove these sequences by cleavage and in vitro religation of the truncated vector before transformation of the host cells, which may result in an increase in the number of copies and in an increased stability of the expression plasmids in said hosts.
• restriction sites can correspond to sequences such as 5'-GGCCNNNNNGGCC-3 '(SfiD or 5'- GCGGCCGC-3' (Notl) since these sites are extremely rare and generally absent from a vector of expression.
• 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. JL ⁇ (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 1 £ 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 antithrombotic 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 organisms ("G_enerally Recognized As S_afe").
• Preferred yeasts are preferentially industrial strains of the genus Kluyveromyces capable of stably replicating said plasmids derived from the plasmid pKDl and into which a selection marker has been inserted 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 for the prevention or treatment of thromboses.
• Figure 1 Nucleotide sequence of a Hindi ⁇ restriction fragment coding for a chimeric protein of the S type AH-vWF.
• the black arrows indicate the end of the "pre” and “pro” regions of HSA.
• the Mstll and Pstl restriction sites are underlined.
• the amino acid numbering (right column) corresponds to the mature chimeric protein SAH-vWF470-> 713 (829 residues); the Thr470-Val713 fragment of the vWF of this particular chimera is numbered from residues Thr586 to Val829.
• the Thr470, Leu494, Asp498, Pro502, Tyr508, Leu694, Pro704, and Pro708 residues of mature vWF are underlined.
• Figure 2 Diagram of chimeras of type S AH-vWF (A), of type vWF-SAH (B) or vWF-S AH-vWF (C).
• M / LP methionine residue initiating translation, possibly followed by a signal secretion sequence
• SAH mature human serum albumin or a variant thereof
• vWF fragment (s) of vWF having a property of binding to platelets and / or to the subendothelium, or one (or more) variants obtained by genetic engineering techniques.
• the black arrow indicates the N-terminus of the mature protein.
• FIG. 3 A, 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 transcriptional terminator
• SAH transcriptional terminator
• IR inverted repeat sequences of the plasmid pKD1
• Ap r and Km r respectively designate the genes for resistance to ampicillin (E. coli) and to G418 (yeasts).
• B genetic characteristics and parentage of the main expression plasmids of the hybrids between SAH and vWF exemplified in the present invention.
• the plasmids of the first column are pUC type plasmids comprising a HindIII restriction fragment corresponding to translational fusions between all of the SAH and a fragment or a molecular variant of vWF.
• the expression plasmids correspond to the cloning in the productive orientation of these HindIII fragments into the HindIII site of the plasmid pYG105 ( LAC4).
• Figure 6 Characterization of the material secreted by K. lactis ' transformed by the plasmids pKan707 (control plasmid, lane 2), pYG1206 (expression plasmid of a chimera of the SAH-P2 type, lane 3), pYG1214 (plasmid expression of a chimera of the SAH-P1 type, lane 4) and pYG1223 (plasmid of expression of a chimera of the SAH-P1-XD-P2 type, lane 5); molecular weight standard (lane 1).
• the deposits correspond to 50 ⁇ l of supernatant from a stationary culture after growth in YPD medium, migration into a gel containing 8.5% acrylamide and staining with coomassie blue.
• Figure 9 Assay of the antagonistic activity in vitro of the agglutination of human platelets attached to paraformaldehyde: IC50 of the hybrids SAH-vWF694- 708, [SAH-vWF470-713 C471G, C474G] and [SAH-vWF470-704 C471G, C474G] relative to the standard RG12986.
• the determination of the dose-dependent inhibition of platelet agglutination is carried out with stirring at 37 ° C., using a PAP-4 aggregameter, in the presence of human vWF, botrocetin (8.2 mg ml) and the product to be test at different dilutions. The concentration of the product making it possible to inhibit control agglutination by half (absence of product) is then determined (IC50).
• the pBR322, pUC and phage plasmids of the M13 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 ligase.
• phage T4 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. 13. (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.
• the amino acid numbering of the vWF is that of Titani et al. [Biochemistry 25 (1986) 3171-3184].
• 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 (lâe-POZYA, X74, galU, galK, 3_rA r ), or E. coli TG1 Qâ ⁇ , proA.B. supE. tM, h ⁇ dD5 / PîraD36, proA + B + . iaçW, laçZ, M15).
• the yeast strains used belong to budding yeasts and more particularly to yeasts of the genus Kluyveromyces.
• 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 Baam (Netherlands) where it was registered under the number CBS 579.88.
• CBS Centraalbureau voor Schimmelkulturen
• a bacterial strain (E. coli) transformed with the plasmid pET-8c52K was deposited on April 17, 1990 with the American Type Culture Collection under the number ATCC 68306.
• 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.
• the vWF cDNA fragment encoding residues 445 to 733 of human vWF has several crucial determinants of the interaction between vWF and platelets on the one hand, and certain elements of the basement membrane and the subendothelial tissue of somewhere else. Amplification of these genetic determinants can be carried out, for example from a human cell line expressing vWF, and for example from a line of endothelial cells of human umbilical cord veins [Verweij CL. et al., Nucleic Acids Res. 1_3_ (1985) 4699-4717], or also from human platelet RNA, for example according to the protocol described by Ware et al. [Proc Natl. Acad. Sci.
• RNAs are purified using the guanidium thiocyanate extraction technique initially described by Cathala et al. [DNA 4 (1983) 329-335] and used as a template for the synthesis of complementary DNA (cDNA) including the part of the vWF to be amplified.
• cDNA complementary DNA
• the synthesis of the non-coding strand is carried out using the kit distributed by Amersham and an oligodeoxynucleotide complementary to the nucleotide sequence of the mRNA coding for contiguous residues located at the C-terminal of the part to be amplified.
• the resulting solution is then subjected to 30 cycles of enzymatic amplification by the PCR technique, using as initiates the preceding oligodeoxynucleotide and an oligodeoxynucleotide identical to the nucleotide sequence coding for contiguous residues located at the N-terminal of the part of the vWF to be amplified.
• the amplified fragments are then cloned into vectors of the M13 type with a view to their verification by sequencing using either the universal primers located on either side of the cloning multisite, or oligodeoxynucleotides specific for the amplified region of the vWF gene, of which the sequence of several isomorphs is known [Sadler JE et al., Proc Natl. Acad. Sci. S2 (1985) 6394-6398; Verweij CL. et al., EMBO J. 5 (1986) 1839-1847; Shelton-Inloes BB et al., Biochemistry 25. (1986) 3164-3171; Bonthron D.
• the plasmid pET-8c52K is particularly useful because it contains a fragment of the cDNA of vWF coding for residues 445 to 733 of human vWF and includes in particular the peptides G10 and D5 antagonists of the interaction between vWF and GPlb [Mori H. et al ., J. Biol. Chem. 263 (1988) 17901-17904].
• the fragment of vWF present in the plasmid p5E is identical to the fragment of the vWF of the plasmid pET-8c52K with the exception that the cysteine residues at positions 459, 462, 464, 471 and 474 were mutated into glycine residues by site-directed mutagenesis.
• the plasmid p7E is identical to the plasmid p5E except that the cysteine residues at positions 509 and 695 have also been mutated into glycine residues by site-directed mutagenesis.
• EXAMPLE 2 CONSTRUCTION OF A MSTII-HINDIII RESTRICTION FRAGMENT INCLUDING A VWF BINDING SITE TO BLOOD PLATES
• the peptide sequence thus amplified comprises a restriction fragment MstlI-HindHI including the residues Thr470 to Val713 of vWF and whose peptide sequence is identical to the corresponding sequence described by Titani et al. [Biochemistry 25_ (1986) 3171-3184].
• the plasmid pYG1220 comprises this restriction fragment MstlI-HindlII preceded by the HindlII-Mstll fragment of the plasmid pYG404 (cf. Example 4 and Figure 3B).
• Residue 705 of natural vWF is O-glycosylated and is located inside the peptide D5 defined by residues Leu694 to Pro708 of vWF [Mon H. et al., J. Biol. Chem. 26_2 (1988) 17901-17904], Furthermore, it is known that treatment of natural vWF with a neuraminidase, the function of which is to release terminal sialic acids from glycosylations of mammalian cells, makes it possible to expose the binding sites from vWF to platelet GPlb in the absence of platelet agglutination cofactors such as botrocetin for example.
• a fragment MstlI-HindlII including the residues Thr470 to Pro704 of vWF is therefore generated in a similar manner to the previous example: the fragments resulting from the PCR amplification of the plasmid p5E with the oligodeoxynucleotides 5'-CCCGG- GATCCCTTAGGCTTAACCGGTGAAGCCGGC-3 '(Sq2149 , the BamHI and Mstll sites are underlined) and 5'-CCATGGATCCAAGCTTAAGGAGGAGGGGCTTCA- GGGGCAAGGTC-3 '(Sq2622, the BamHI and HindIII sites are underlined) are first cloned into a vector of type pUC in the form of a restriction fragment BamHI.
• the sequence of the MstlI-HindlII fragment thus generated corresponds to the corresponding sequence given in FIG. 1 except that the TAA codon specifying the translational stop is located immediately downstream of the Pro704 residue of the vWF and that the residues 471 and 474 are glycine residues and not cysteine residues.
• the plasmid pYG1310 comprises this restriction fragment MstlI-HindlII preceded by the HindIII-MstlI fragment of the plasmid p YG404 (cf. Example 4 and Figure 3B).
• the peptide sequence present in the plasmid pYG1310 still has the threonine or serine residues at positions 485, 492, 493 and 500 which are naturally O-glycosylated in the native molecule of human vWF, located at immediate proximity to the G10 peptide defined by Mori et al. [J. Biol. Chem. 263 (1988) 17901-17904].
• the amplified fragments are first cut by the enzymes Kpn1 and BamHI to be cloned in a vector of pUC type cut by the same enzymes. A particular clone is isolated which corresponds to the expected sequence verified by sequencing.
• This Kpnl-BamHI fragment therefore comprises an MstlI-HindIII fragment including the residues Leu494 to Pro704 of human vWF.
• the plasmid pYG1373 comprises this restriction fragment MstlI-HindlH preceded by the HindIII-MstlI fragment of the plasmid pYG404 (cf. Example 4 and Figure 3B).
• the peptide sequence present after PCR amplification in the plasmid pYG1373 still has the threonine residue at position 500 which is naturally O-glycosylated in the native molecule of human vWF.
• CAAGCTTAAGGAGGAGGGGCTTCAGGGGCAAGGTC-3 '(Sq2622, BamHI and HindIII sites are underlined) generates a fragment including residues Tyr508 to Pro704 of vWF.
• the amplified fragments are first cut by the enzymes Kpn1 and BamHI to be cloned in a vector of the pUC type cut by the same enzymes. A particular clone is isolated which corresponds to the expected sequence verified by sequencing.
• This Kpnl-BamHI fragment therefore comprises an MstlI-HindIII fragment including the residues Tyr508 to Pro704 of human vWF.
• the plasmid pYG1309 comprises this restriction fragment MstlI-HindlII preceded by the Hindi ⁇ -Mstll fragment of the plasmid pYG404 (cf. Example 4 and Figure 3B).
• the peptide sequence corresponding to residues Pro502 to Pro704 of human vWF is generated from the preceding plasmid by insertion of the oligcxieoxynucleotides 5'-TTAGGGT ACCACCTTTGCATGACTTCTACTGCA-3 ' (Sq2751) and 5'-GTAGAAGTCATGCAAAGGTGGTAACCC-3 '(Sq2752) which by pairing can be cloned between the Mstll and PstI sites of the plasmid obtained after PCR amplification according to Example E.2.I.4., Which allows generate a MstlI-HindlII restriction fragment including residues Pro502 to Pro704 of human vWF.
• the plasmid pYG1350 comprises this restriction fragment MstlI-HindlII preceded by the HindIII-MstlI fragment of the plasmid pYG404 (cf. Example 4 and Figure 3B).
• the vWF binding site is a peptide including residues Thr470 to Asp498 of mature vWF.
• This sequence includes the peptide G10 (Cys474-Pro488) described by Mori et al. [J. Biol. Chem. 2 £ 2 (1988) 17901-17904] and capable of antagonizing the interaction of human vWF with the GPlb of human platelets.
• the sequence including the peptide G10 is first generated in the form of a restriction fragment MstlI-HindlII. for example by means of the PCR amplification technique, or even directly using synthetic oligodeoxynucleotides.
• the PCR amplification products of the plasmid ⁇ ET-8c52K with the oligodeoxynucleotides Sql969 and 5'-CCCG- GGATCCAAGCTTAGTCCTCCACATACAG-3 '(Sql970, the BamHI and HindIII sites are underlined) are first cut by the enzyme BamHI then clones in the BamHI site of a vector of the pUC type. A particular clone is isolated which corresponds to the expected sequence verified by sequencing.
• This BamHI fragment therefore comprises an MstlI-HindlII fragment including the residues Thr470 to Asp498 of human vWF.
• the plasmid pYG1210 comprises this restriction fragment MstlI-HindlII preceded by the HindIII-MstlI fragment of the plasmid pYG404 (cf. Example 4 and Figure 3B).
• vWF peptide of type P2.
• the binding site of vWF to GPlb is directly designed using synthetic oligodeoxynucleotides, for example the oligodeoxynucleotides 5'-TTAGGCCTCTGTGACCTTGCCCCTG-
• oligodeoxynucleotides form by pairing a MstlI-BglII restriction fragment including the MstlI-HindlII fragment corresponding to the D5 peptide defined by residues Leu694 to Pro708 of the vWF [Mori H. et al., J. Biol. Chem. 263 (1988) 17901-17904].
• Plasmid pYG1204 contains this fragment of MstlI-HindlII restriction preceded by the HindIII-MstlI fragment of the plasmid pYG404 (cf. Example 4 and Figure 3B).
• Useful variants of the plasmid pET-8c52K are deleted by site-directed mutagenesis between the peptides G10 and D5, for example binding sites to collagen, and / or to heparin, and / or to botrocetin, and / or to sulfatides and or ristocetin.
• An example is the plasmid pMMB9 deleted by site-directed mutagenesis between residues Cys509 and Ile662.
• PCR amplification of this plasmid with the oligodeoxynucleotides Sql969 and Sq2029 generates a restriction fragment Mstll-HindIII including residues Thr470 to Tyr508 and Arg663 to Val713 and in particular the peptides G10 and D5 of vWF and deleted in particular from its binding site to collagen located between residues Glu542 and Met622 [Roth GJ. et al. Biochemistry 2g (1986) 8357-8361].
• the plasmid pYG1217 comprises this restriction fragment Mst ⁇ -HindHI preceded by the HindIII-MstlI fragment of the plasmid pYG404 (cf. Example 4 and Figure 3B).
• the use of the combined techniques of site-directed mutagenesis and PCR amplification makes it possible to generate at will variants of the restriction fragment MstlI-HindlII of FIG. 1 but deleted from one or more binding sites. sulfatides and / or botrocetin and or heparin and / or collagen.
• PCR amplification products of plasmids p5E and p7E with the oligodeoxynucleotides Sq2149 (5'-CCCGGGATCCCTTAGGCTTAACCGGTG- AAGCCGGC-3 ', the BamHI and Mstll sites are underlined) and Sq2029 are first cloned into a vector of type pUC under the shape of a restriction fragment
• the sequence of the MstlI-HindIII fragment thus generated corresponds to the corresponding sequence given in FIG. 1 with the exception that residues 471 and 474 of vWF are glycine residues and not cysteine residues.
• the plasmid pYG1271 comprises this restriction fragment Mst ⁇ -HindlII preceded by the HindIII-MstlI fragment of the plasmid pYG404 (cf. Example 4 and Figure 3B).
• Plasmid pYG1269 is generated similarly except that the plasmid p7E is used as a template during the PCR amplification by the oligodeoxynucleotides Sq2149 and Sq2029. E.2.5.2. Conformational alteration by introduction of type IIB mutations Other particularly useful mutations relate to at least one residue involved in type IIB pathologies associated with vWF (increase in the intrinsic affinity of vWF for GPlb), such as the residues Arg543, Arg545 , Trp550, Val551, Val553, Pro574 or Arg578 for example.
• the plasmids pYG1359 (R543W) and pYG1360 (P574L) comprise these restriction fragments MstlI-HindlII preceded by the HindIII-MstlI fragment of the plasmid pYG404 (cf. Example 4 and Figure 3B).
• Mutagenesis using the oligodeoxynucleotide Sq2851 also introduces the SalI sites. EcoRV and MluI at positions Val538, Ile546 and Val551, respectively. These restriction sites are not present in the corresponding natural sequence of human vWF and are therefore particularly useful for easily introducing any desirable mutation between residues Val538 and Val551.
• the oligodeoxynucleotides 5'-ATCCCAGAAGTGCGTA-3 '(Sq3017, the codon specifying the type IIB mutant Cys550 is underlined) and 5'-CGCGTACGCACTTCTGGGAT-3' (Sq3018) form by matching a restriction fragment EcoRV-MluI which can be cloned into the plasmid pYG1359 cut by the enzymes EcoRV and MluI. which generates the plasmid pYG1374 comprising the mutations R543W and W550C ( Figure 3B).
• EXAMPLE 3 CONSTRUCTION OF AN MSTII / HINDIII RESTRICTION FRAGMENT INCLUDING A VWF LINK SITE TO THE SUBENDOTHELIUM
• the binding sites of vWF to the components of the subendothelial tissue and of collagen in particular are generated by PCR amplification of the plasmid pET-8c52K.
• Molecular variants of types XD (cf. E.2.4.) Or X * (cf. E.2.5.) Can also be generated according to the same strategy and which comprise any desirable combination between the sites for binding of vWF to sulfatides and / or botrocetin and / or heparin and / or collagen and / or any residue responsible for a modification of the affinity of vWF for GPlb (type II pathologies associated with vWF).
• the domain capable of binding to collagen can also come from the vWF fragment comprised between residues 911 and 1114 and described by Pareti et al. [J. Biol. Chem. (1987) 2 £ 2: 13835-13841].
• 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 in upstream of the translation initiating ATG of the PGK gene of S. cerevisiae.
• This fragment comprises a HindIII-MstlI restriction fragment corresponding to the entire gene coding for SAH with the exception of the three most C-terminal amino acids (leucine-glycine-leucine residues).
• HindIII restriction fragments including composite genes coding for chimeric proteins in which a fragment of vWF endowed with particular properties is positioned in the translational phase of C-terminal reading of the SAH molecule.
• Such composite genes are exemplified in the Table of Figure 3B.
• These hybrid genes are preferably bordered 5 'to the translation initiating ATG and 3' to the translation end codon by HindIII restriction sites, which makes it possible to generate expression plasmids for these chimeric proteins, for example according to the strategy detailed in the following example.
• the chimeric proteins of the preceding 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 Kluyveromyces lactis).
• pYG106 PGK promoter of Saccharomyces cerevisiae
• pYG536 PHO5 promoter of S. cerevisiae
• hybrid promoters such as those described in patent application EP 361 991.
• the plasmids pYG105 and pYG106 are particularly useful here because they allow the expression of the genes encoded by the HindIII restriction fragments of Examples E.4. and E.5. from functional promoters in K. lactis.
• the plasmid pYG105 corresponds to the plasmid pKan707 described in the Patent application EP 361 991 in which the unique HindIII restriction site located in the geneticin resistance gene (G418) was destroyed by site-directed mutagenesis while retaining an unchanged protein (oligodeoxynucleotide 5'-GAAATGCATAAGCTCTTGCCATTCTCACCG-3 ').
• the Sall-Sacl fragment coding for the URA3 gene of the mutated plasmid was then replaced by a Sall-Sacl restriction fragment comprising an expression cassette consisting of the LAC4 promoter from K.
• lactis in the form of a SalI-HindHI fragment
• terminator of the PGK gene of S. cerevisiae in the form of a HindJJI-Sacl fragment
• the plasmid pYG105 is mitotically very stable in Kluyveromyces yeasts and a restriction map is given in FIG. 3.
• the plasmids pYG105 and pYG106 differ from each other only in the nature of the transcription promoter encoded by the SalI-HindIII fragment.
• 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 cells are washed twice, resuspended in 0.2 ml of sterile water and incubated for 16 hours at 28 ° C in 2 ml of YPD medium to allow the phenotypic expression of the G418 resistance gene expressed under control the promoter P ⁇ 1 (cf. EP 361 991); 200 ⁇ l of the cell suspension are then spread on selective YPD dishes (G418, 200 ⁇ g / ml). The dishes are incubated at 28 ° C and the transformants appear after 2 to 3 days of cell growth.
• the cell supernatants are recovered by centrifugation when the cells reach the stationary growth phase, possibly concentrated 10 times by precipitation for 30 minutes at -20 °. C in a final concentration of 60% ethanol, then tested after electrophoresis in SDS-PAGE gel at 8.5%, either directly by staining the gel with coomassie blue, or after immunoblotting using, as primary antibodies, antibodies to mice directed against vWF or a polyclonal rabbit serum directed against HSA.
• the nitrocellulose filter is first incubated in the presence of specific primary antibodies, washed several times, incubated in the presence of goat anti-mouse antibodies (immunoblot anti-vWF) or anti-rabbit (immunoblot anti-HSA), then incubated in the presence of an avidin-peroxidase complex using the "ABC kit" distributed by Vectastain (Biosys SA, Compiègne, France).
• the immunological reaction is then revealed by the addition of 3,3-diamino benzidine tetrachlorydrate (Prolabo) in the presence of hydrogen peroxide, according to the manufacturer's recommendations.
• the results of FIGS. 4 to 8 demonstrate that the yeast K.
• lactis is capable of secreting chimeric proteins between SAH and a fragment of vWF, and that these chimeras are recognized by antibodies specific for SAH or vWF.
• EXAMPLE 9 PURIFICATION AND MOLECULAR CHARACTERIZATION OF THE SECRET PRODUCTS
• the chimeras present in the culture supernatants corresponding to the strain CBS 293.91 transformed, for example by the expression plasmids according to example 6, are first characterized using antibodies specific for the HSA part and the vWF part.
• the results of FIGS. 4 to 8 demonstrate that the yeast K. lactis is capable of secreting chimeric proteins between SAH and a fragment of vWF, and that these chimeras are immunologically reactive. It may also be desirable to purify some of these chimeras.
• the culture is then centrifuged (10,000 g, 30 min), the 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 dialyzed against a solution of Tris HCl (50 mM pH 8) and then purified on a column.
• the concentrate corresponding to the culture supernatant of the CBS 293.91 strain transformed with the plasmid pYG1206 is purified by affinity chromatography on Blue-Trisacryl (IBF). Purification by ion exchange chromatography can also be used.
• the concentrate obtained after ultrafiltration is dialyzed against a solution of Tris HCl (50 mM pH 8), then deposited in 20 ml fractions on a column (5 ml) exchanging cations (S Fast Flow, Pharmacia) balanced in the same buffer.
• the column is then washed several times with the Tris HCl solution (50 mM pH 8) and the chimeric protein is then eluted from the column by a gradient (0 to 1 M) of NaCl.
• the fractions containing the chimeric protein are then combined and dialyzed against a 50 mM Tris HCl solution (pH 8) and then redeposited on a S Fast Flow column.
• EXAMPLE 10 ANTAGONIST ACTIVITY OF GENETIC HYBRIDS BETWEEN SAH AND VWF FOR PLATELET AGGLUTINATION
• the antagonistic activity of the products is determined by measuring the dose-dependent inhibition of the agglutination of human platelets attached to paraformaldehyde according to the method described by Prior et al. [Bio / Technology (1992) 10: 66]. The measurements are made in an aggregameter (PAP-4, Bio Data, Horsham, PA, USA) which records the variations over time of the optical transmission with stirring at 37 ° C in the presence of vWF, botrocetin (8.2 mg / ml) and of the product to be tested at different dilutions (concentrations). For each measurement, 400 ml (8x10?
• Platelets of a suspension of human platelets stabilized with paraformaldehyde (0.5%, then resuspended in [NaCl (137 mM); MgCl2 (1 mM); NaH2PO4 (0.36 mM) ; NaHC ⁇ 3 (10 mM); KC1 (2.7 mM); glucose (5.6 mM); SAH (3.5 mg / ml); HEPES buffer (10 mM, pH 7.35)] are preincubated at 37 ° C in the cylindrical tank (8.75 x 50 mm, Wellcome Distriwell, 159 rue Nationale, Paris) of the aggregameter for 4 min, then 30 ml of the solution of the product to be tested are added at different dilutions in the formulation vehicle nonpyrogenic [mannitol (50 g / 1); citric acid (192 mg / 1); L-lysine monochlorhydrate (182.6 mg / 1); NaCl (88 mg / 1); pH adjusted to 3.5 by addition of NaOH (
• q ue i is incubated at 37 ° C for 1 min before adding 12.5 ml of botrocetin solution [purified from lyophilized venom of Bothrops jararaca (Sigma) according to the protocol described by Sugimoto et al., Biochemistry (1991) 266: 18172].
• the recording of the transmission reading as a function of time is then carried out for 2 min with stirring using a magnetic bar (Wellcome Distriwell) placed in the tank and under a magnetic stirring of 1100 rpm provided by the aggregameter.
• the average variation in optical transmission (n 3 5 for each dilution) over time is therefore a measure of the platelet agglutination due to the presence of vWF and botrocetin, in the absence or in the presence of variable concentrations of the product to be tested. From such recordings, the% inhibition of platelet agglutination due to each concentration of product is then determined and the line is given giving the% inhibition as a function of the inverse of the dilution of product on a log scale. -log. The IC50 (or concentration of product causing 50% inhibition of agglutination) is then determined on this line.
• the Table in Figure 9 compares the IC50s of some of the SAH-vWF chimeras of the present invention and demonstrates that some of them are better antagonists of platelet agglutination than the product RG12986 described by Prior et al. [Bio / Technology (1992) read: 66] and included in the tests as a standard value. Identical tests for the inhibition of the agglutination of human platelets in the presence of pig plasma vWF (Sigma) also make it possible to demonstrate that some of the hybrids of the present invention, and in particular certain variants of type IIB, are very good antagonists of platelet agglutination in the absence of botrocetin-type co-factors.
• botrocetin-independent antagonism of these particular chimeras can also be demonstrated according to the protocol initially described by Ware et al. [Proc. Natl. Acad. Sci. (1991) 88: 2946] by displacement of the monoclonal antibody ⁇ ⁇ -U-IB1 (10 mg / ml), a competitive inhibitor of vWF binding on platelet GPlb [Handa M. et al., (1986 ) J. Biol. Chem. 26J .: 12579] after 30 min of incubation at 22 ° C in the presence of fresh platelets (10 ⁇ platelets / ml).

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• 1992
  • 1992-01-31 FR FR9201066A patent/FR2686901A1/fr active Pending
• 1993
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