HK1068915A - Novel biologically active polypeptides, preparation thereof and pharmaceutical composition containing said polypeptides - Google Patents

Abstract

Novel biologically active polypeptides, preparation thereof and pharmaceutical compositions containing said polypeptides.

Description

The present invention relates to novel biologically active polypeptides, their preparation and pharmaceutical compositions containing them.

In particular, the present invention concerns recombinant polypeptides consisting essentially of an active part derived from a polypeptide, natural or artificial, having therapeutic activity, and coupled to an albumin or albumin variant. It is understood that the therapeutic activity of the polypeptides of the invention may be either direct (treatment of diseases), or indirect (and for example usable in disease prevention, in vaccine design, in medical imaging techniques, etc.).

It is understood that albumin variants are 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 human serum albumin, as well as any macromolecule with a high plasma half-life obtained by in vitro modification of the protein encoded by such genes.

The purpose of the present invention is to develop biologically active and pharmaceutically usable artificial proteins. Many polypeptides with one or more potential therapeutic activities cannot be used pharmaceutically. This may be for various reasons, such as their low in vivo stability, complex or fragile structure, difficulty in producing them on an industrially acceptable scale, etc. Similarly, some polypeptides do not give the expected results in vivo due to problems with administration, conditioning, pharmacokinetics, etc.

The present invention provides new molecules which allow the therapeutic exploitation of the biological properties of these polypeptides to be optimized. The present invention is based on the demonstration that it is possible to genetically couple any active structure derived from a biologically active polypeptide to another protein structure consisting of albumin without altering the said biological properties. It is also based on the demonstration by the applicant that the human serum albumin effectively presents the active structure at its sites of interaction and ensures high plasma stability of the invention's polypeptide.The polypeptides of the invention thus enable the maintenance of a given biological activity in the body for a prolonged period of time. They thus reduce the doses administered and in some cases potentiate the therapeutic effect, for example by reducing the side effects following a larger dose. The polypeptides of the invention further enable the generation and use of structures derived from very small biologically active polypeptides and therefore very specific to a desired effect. It is understood that peptides with biological activity of therapeutic interest may also correspond to unnatural peptide sequences, for example isolated from random peptide banks.The polypeptides of the invention also have a particularly advantageous distribution in the body, which modifies their pharmacokinetic properties and promotes the development of their biological activity and use. In addition, they also have the advantage of being weakly or non-immunogenic to the organism in which they are used. Finally, the polypeptides of the invention can be expressed (and preferably secreted) by recombinant organisms at levels that allow their industrial exploitation.

Therefore, one of the objects of the present invention concerns polypeptides with an active part derived from a polypeptide with therapeutic activity, coupled to an albumin or albumin variant.

In one particular mode of production, peptides with therapeutic activity are not of human origin, for example peptides or their derivatives with potentially useful properties in blood and interstitial compartment pathologies, such as hirudin, trigramine, antistatin, tick anticoagulant peptides (TAP), arietine, applagin, etc.

In particular, in the molecules of the invention, the polypeptide with therapeutic activity is a human polypeptide or a molecular variant. For example, it may be all or part of an enzyme, an enzyme inhibitor, an antigen, an antibody, a hormone, a factor involved in the control of clotting, an interferon, a cytokine [interleukins, but also their natural antagonist variants of their attachment to the SISx receptor], small secreted cytokines and for example macrophage inflammatory proteins (MIPs), etc., a growth and differentiation factor [e.g. growth factors and TGFs], etc.blood cell differentiation factors (erythropoietin, M-CSF, G-CSF, GM-CSF, etc.), insulin and growth factors similar to insulin (IGFs), or cell permeability factors (VPF/VEGF, etc.), a factor involved in bone tissue generation/resorption (e.g. OIF and osteospontine), a factor involved in cell motility or migration (and for example autocrine motility factor (AMF), migration stimulating factor (MSF), or ster factor/ hepatocyte growth factor), a bactericidal or antifungal factor, e.g. a chemical platelet agent (P4),or monocyte chemoreceptor peptides (MCP/MCAF) or neutrophils (NCAF), etc. ... , a cytostatic factor (and for example proteins that bind to galactosides), a plasma adhesive molecule (and for example von Willebrand factor, fibrinogen etc.) or interstitial (laminine, tenascin, vitronectin, etc.) or extracellular matrices, or any peptide sequence antagonistic or agonist of molecular and/or intercellular interactions involved in pathologies of the circulatory compartments and interstitials and for example formation of arterial and vein thrombuses, osteostatic tumours, angiogenesis, inflammatory cholesterolactasis, autoimmune diseases, pathologies of the bone marrow, etc.

The active part of the polypeptides of the invention may be, for example, the polypeptide having a therapeutic activity entirely, or a derived structure thereof, or a non-natural polypeptide isolated from a peptide bank. For the purposes of this invention, derived structure means any polypeptide obtained by modification and retaining a therapeutic activity. Modification shall mean any mutation, substitution, deletion, addition or modification of a genetic and/or chemical nature. Such derivatives may be generated for different purposes, such as increasing the affinity of the molecule for binding sites, improving its levels, increasing its resistance to proteases, increasing its effectiveness in the production of new pharmaceutical or therapeutic products or reducing its biological or therapeutic properties, or for the prevention of disease.

Polypeptides of the invention are particularly advantageous if the active ingredient is: (a) the entire peptide structure or (b) a structure derived from (a) by structural modification (mutation, substitution, addition and/or deletion of one or more residues) and having therapeutic activity.

Structures of type (b) include in particular molecules in which some N- or O-glycosylation sites have been modified or removed, molecules in which one or more residues have been replaced, or molecules in which all cysteine residues have been replaced, molecules obtained from (a) by deletion of regions with little or no interaction with the relevant binding sites, or with undesirable activity, and molecules with additional residues relative to (a), such as an N-terminal and/or a secretory signal and/or a junction peptide.

The active part of the molecules of the invention may be coupled either directly or via an artificial peptide to albumin. Furthermore, it may constitute the N-terminal as well 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. It is also understood that the biologically active part may be redundant within the chimera.

Another subject of the invention concerns a process for preparing the chimeric molecules described above, namely, by having a nucleotide sequence coding for the desired polypeptide expressed by a eukaryotic or prokaryotic cell host and then harvesting the resulting polypeptide.

Eukaryotic hosts for use in the present invention include animal cells, yeasts, or fungi. In particular, yeasts include yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula. Animal cells include COS, CHO, C127, etc. Fungi that may be used in the present invention include, in particular, Aspergillus ssp. or Trichoderma ssp. As prokaryotic hosts, it is preferable to use bacteria such as Escherichia coli, or those of the genera Corynebacterium, Bacillus, or Streptomyces.

The nucleotide sequences used in the present invention can be prepared in a variety of ways. Generally, they are obtained by assembling the sequences coding for each functional part of the polypeptide in the reading phase. These can be isolated by state-of-the-art techniques, e.g. directly from cellular messenger RNAs (mRNAs), or recloned from a complementary DNA bank (cDNA), or they can be completely synthetic nucleotide sequences. It is further understood that nucleotide sequences can also be subsequently modified, e.g. by genetic engineering techniques, to obtain derivatives or sequence variants.

In the invention process, the nucleotide sequence may be part of an expression cassette comprising a transcription initiation region (promoter region) that allows the expression of the nucleotide sequence under its control in the host cells and encodes for the polypeptides of the invention. This region may come from promoter regions of genes that are regulated in the host cell used, the expression being highly constitutive or regulated. In the case of yeasts, it may be the promoter of the phosphoglycerate kinase (PGK) gene, the glycophosphate-3-nophosphate polyhydrogenase (GPD), the lactase receptor (LAQP4), or a substitute for the in vitro expression of genes (e.g. SLAP), or a region that can be used for the control of the expression of mutations or defects (e.g. in the control of gene expression, the control of gene expression, the control of gene expression, the control of gene expression, or the control of gene expression, etc.).

Err1:Expecting ',' delimiter: line 1 column 525 (char 524)

In addition to the expression cassette, one or more markers for recombinant host selection may be added, such as the URA3 gene of S. cerevisiae yeast, or genes conferring resistance to antibiotics such as geneticin (G418) or any other toxic compound such as certain metal ions.

The set of the expression cassette and the selection marker can be introduced directly into the host cells concerned or be pre-inserted into a functional self-replicating vector. In the first case, homologous sequences to regions in the genome of the host cells are preferentially added to this set; these sequences are then positioned on either side of the expression cassette and the selection gene in such a way as to increase the frequency of integration of the set into the host genome by targeting sequence integration by homologous recombination. In the case where the expression cassette is inserted into a replicative system, a replication system preferred for KluK platelet-derived plasmid or plasmid-derived PK1 or PK2 is the preferred replication system for all types of plasmid.

In addition, expression plasmids can be shuttle vectors between a bacterial host such as Escherichia coli and the chosen host cell. In this case, a replication origin 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 allows these sequences to be removed by cutting and binding in vitro of the vector before transformation of the host cells, which can result in an increase in the number of copies and increased stability of the expression plasmids in said hosts. For example, such restriction sites may correspond to sequences such as 5GCNCCNCCN-3G or 5GNCCN-3G (GNGG-NNGG) where these are extremely rare and not in the range of expression.

After constructing such vectors or expression cassette, these are introduced into the selected host cells according to the classical techniques described in the literature. In this regard, any method that allows the introduction of foreign DNA into a cell can be used. These may include transformation, electroporation, conjugation, or any other technique known to man. As an example for yeast-type hosts, the different strains of Kluyveromyces using the classical techniques described in the literature were transformed into whole cells in the presence of lithium acetate and polyethylene glycol, according to the technique described by J. Ito et al. 153 [J. Bacteriol. (1983) 163]. The transformation technique described by Duret et al. [C. Gen. Letters F. 7 and K. Gen. K. 7] is also described in the following protocols.

Err1:Expecting ',' delimiter: line 1 column 262 (char 261)

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The present invention also relates to nucleotide sequences coding for the chimeric polypeptides described above, as well as recombinant cells, eukaryotic or prokaryotic, including such sequences.

The present invention also concerns the pharmaceutical application of polypeptides according to the present invention. In particular, the invention concerns any pharmaceutical composition comprising one or more polypeptides or nucleotide sequences as described above. Nucleotide sequences can be used in gene therapy.

The present invention will be described more fully by the following examples, which are to be considered as illustrative and not limitative.

List of characters

The plasmid representations shown in the following Figures are not scaled and only restriction sites important for understanding cloning have been reported. Figure 1 : Schematic of chimeras of the SAH-PEPTIDE (A), PEPTIDE-SAH (B) or PEPTIDE-SAH-PEPTIDE (C) type. Abbreviations used: M/LP, methionine residue initiating translation, possibly followed by a sequence of secretion signals; SAH, mature albumin or one of its molecular variants; PEP, peptide of natural or artificial origin with a given therapeutic property. The PEP sequence may be present several times in type A, B or C molecules.Err1:Expecting ',' delimiter: line 1 column 308 (char 307)Secretion signal sequence; Apr and Kmr refer to ampicillin resistance genes (E. coli and G418 (yeast) respectively).Figure 4 : Examples of nucleotide sequences of MstII-HindIII restriction fragments derived from von Willebrand factor; Structure representation of MstII-HindIII fragments of the plasmids pYG1248 (panel A), pYG1214 (panel B), pYG1206 (panel C, in this particular site the vWF residue Leu694 is also the last residue (Leu585) of the SAH) and pYG1223 (panel D) the amino acid numbering corresponding to the VF; the alchemical termination of the Hindus and alchemical termination of Titanium is highlighted.E-line: nucleotide sequence of the MstII-HindIII restriction fragment of the pYG1248 plasmid.The amino acid numbering (right column) corresponds to the mature chimeric protein SAH-vWF470->713 (829 residues).The residues Thr470, Leu494, Asp498, Pro502, Tyr508, Leu694, Pro704, and Pro708 of the mature vWF are highlighted.Figure 5: Characterisation of the material secreted after 4 days of culture (erlenmeyers) of the CBS29 strain 293.91 transformed by the pYG1248 plasmids (expression medium of a chimeric type of the HVWF470->173Va) and pI770 (pm).In this experiment, the results of the ADS and BPA-S are migrated to the same control panels, the results of which were obtained on the same BPA-S (Canadian Throma) and CPA-S (Canadian Throma) plasmids.5%) and then treated separately. A, co-mass blue staining; molecular weight standard (track 2); surfeit equivalent to 50 μl of culture transformed by pKan707 plasmids in YPL medium (track 1), or pYG1248 in YPD medium (track 3) or YPL medium (track 4).B, immunological characterization of material secreted after use of mouse antibodies directed against human vWF: same legend as in A except that biotinilled molecular weight standards were used.C, immunological characterization of material secreted after use of rabbit antibodies against human albumin: surfeit equivalent to culture of plasmids directed by pKan707 plasmids in YPL 1),Err1:Expecting ',' delimiter: line 1 column 373 (char 372)Figure 7: Characterisation of material secreted by K. lactis transformed by plasmids pKan707 (control plasmid, track 2), pYG1206 (track 3), pYG1214 (track 4) and pYG1223 (track 5); molecular weight standard (track 1). The deposits correspond to 50 μl of overflowed from a stationary culture after growth in YPD, migration in an 8.5% acrylamide gel and co-mass blue colouration.Figure 8 : Nucleotide sequence of the MstII-HindIII restriction fragment of the plasmid pYG1341 (SAH-UK>135). The limit of EGF-UK (UK1-UK>1346) is shown in the pYG131 (SAH-UK>1354) plasmids restriction fragment. The residual number of the protein is the same as the mature amino acid MstII-HindIII (7H131-HG40).Figure 9: Secretion of the SAH-UK1-46 and SAH-UK1-135 chimeras by the CBS 293.91 strain transformed by the plasmids pYG1343 (SAH-UK1-46) and pYG1345 (SAH-UK1-135), respectively, after 4 days of growth (mean YPL+G418). Deposits (equivalent to 50 μl of culture) are migrated to PAGE-SDS gel at 8.5% and coloured in co-mass blue: surrogate of a clone transformed by the plasmids pKan707 (track 1), pYG1343 (track 3) or pYG1345 (track 4); the standard molecular weight sites (track 2).Figure 10: Nucleotide sequence of the plasmid fragment restriction of the SCSI-HY-III (SCSI-HY-III) SCSI-HF12 (SCSI-HF74), the restraint site of the GCSI-HY-I1 is underlined.Err1:Expecting ',' delimiter: line 1 column 306 (char 305)Figure 12: Characterization of the material secreted after 4 days of culture (erlenmeyers) of CBS 293.91 strain transformed by the plasmids pYG1266 (plasmids expressing a chimera of type SAH-G.CSF) and pKan707 (control plasmids). A, co-mass blue staining; molecular weight standard (track 2); 100 μl survival of the culture transformed by the pKan707 plasmids in YPL (track 1), or pYG1266 in YPD (track 3) or YPL (track 4).B, immunological characterization of the material secreted after use of primary antibodies directed against human G-CSF: same legend as in A.C, immunological characterisation of the material secreted after use of primary antibodies directed against human albumin: same legend as in A.Figure 13 : characterisation of the material secreted after 4 days of culture (erlenmeyers in YPD medium) of the CBS 293.91 strain transformed by the plasmids pYG1267 (SAH-G.CSF chimera), pYG1303 (G.CSF-Gly4-SAH chimera) and pYG1352 (SAH-Gly4-G.CSF chimera) after migration on SDS-PAGE 8.5% gel. A, co-mass blue colouring; 100 μl survival of the culture transformed by the plasmids pYG1303 (track 1), pYG1267 (track 2) or pYG1352 (track 3); molecular weight standard (track 4).B, immunological characterisation of the material secreted after use of primary antibodies directed against human G-CSF: same legend as in A.Figure 14 : Nucleotide sequence of the MstII-HindIII restriction fragment of the plasmid pYG1382 (SAH-Fv). The VH (124 residues) and VL (107 residues) domains of the Fv' fragment are separated by the synthetic linker (GGGGS) x3. The amino acid numbering corresponds to the mature chimeric protein SAH-Fv' (831 residues).Figure 15 : Secretion of the SAH-Fv' chimeric by the strain 293.91 transformed by the CBS plasmid pYG1383 (LAC4) after 4 days of growth into ermeyleners at 28°C in YPD (track 2), YPL (track 3) or standard molecular weight medium (track 1).The results of the study were published in the journal Nature. A,: co-mass blue gel staining.B,: immunological characterisation of secreted material after use of primary antibodies directed against HAS.Figure 16: Dose of in vitro antagonistic activity of formaldehyde-bound human platelet agglutination: CI50 of the hybrids SAH-vWF694-708, [SAH-vWF470-713 C471G, C474G] and [SAH-vWF470-704 C471G, C474G] relative to the RG12986 standard.Determination of dose-dependent inhibition of platelet agglutination is performed according to the method described by C.Technology et al. [B/Fio 10/Cio 62] using a prior test using 37° optical variations in the presence of aggregation in human transmission.The concentration of the product which allows half-inhibition of control agglutination (in the absence of product) is then determined (CI50).Figure 17: Activity on cell proliferation in vitro in the NFS60 mouse lineage.Radioactivity (3H-thymidine) incorporated into cell nuclei after 6 hours of incubation is represented in cpm; the amount of product indicated in abscissa is expressed in molarity (arbitrary units).Figure 18: Activity on granulopoiesis in vivo in rats.The number of neutrophils (mean 7) is represented in cpm as a function of animal time.The products tested were the chimeric SAH-G.CSF (pYG1266, 4 or 40 mg/rat/day), the reference G-CSF (10 mg/rat/day), the recombinant SAH purified from Kluyveromyces lactis supernatant (SAH, 30 mg/rat/day, see EP 361 991), or the physiological serum.

Examples The Commission has also adopted a proposal for a regulation on the use of genetically modified organisms in animal cloning.

Err1:Expecting ',' delimiter: line 1 column 639 (char 638)

Restriction enzymes were supplied by New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Amersham and are used as recommended by suppliers.

Plasmids of the pBR322 type, pUC and phages of the M13 series are of commercial origin (Bethesda Research Laboratories).

For ligatures, DNA fragments are separated by size by electrophoresis into agarose or acrylamide gels, extracted with phenol or a phenol/chloroform mixture, precipitated with ethanol and then incubated in the presence of T4 phage DNA ligase (Biolabs) as recommended by the supplier.

The 5' prominent ends are filled with the Klenow fragment of E. coli DNA Polymerase I (Biolabs) as specified by the supplier. The 3' prominent ends are destroyed in the presence of T4 phage DNA Polymerase (Biolabs) as recommended by the manufacturer. The 5' prominent ends are destroyed by a S1 nuclease-controlled treatment.

In vitro directed mutagenesis by synthetic oligodeoxynucleotides is performed using the method developed by Taylor et al. [Nucleic Acids Res. 13 (1985) 8749-8764] using the kit distributed by Amersham.

Err1:Expecting ',' delimiter: line 1 column 298 (char 297)

The verification of nucleotide sequences is carried out by the method developed by Sanger et al. [Prof. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.

The transformations of K. lactis with DNA from the protein expression plasmids of the present invention are performed by any technique known to man, an example of which is given in the text.

Unless otherwise specified, the bacterial strains used are E. coli MC1060 (lacIPOZYA, X74, galU, galK, StrAr), or E. coli TG1 (lac, proA,B, supE, thi, hsdD5 / FtraD36, proA+B+, lacIq, lacZ, M15).

The strains of yeast used are budding yeast and in particular yeast of the genus Kluyveromyces. The strains K. lactis MW98-8C (a, uraA, arg, lys, K+, pKD1°) and K. lactis CBS 293.91 were particularly used; a sample of the strain MW98-8C was deposited on 16 September 1988 at the Central Bureau for Schimmelkulturen (CBS) in Baam (Netherlands) where it was registered under the number CBS 579.88.

A bacterial strain (E. coli) transformed with the plasmid pET-8c52K was filed on 17 April 1990 with the American Type Culture Collection under number ATCC 68306.

Yeast strains transformed by expression plasmids coding for the proteins of the present invention are grown in 21 erlenmeyers or pilot fermenters (SETRIC, France) at 28°C in a rich medium (YPD: 1% yeast extract, 2% Bactopeptone, 2% glucose; or YPL: 1% yeast extract, 2% Bactopeptone, 2% lactose) under constant agitation.

The test shall be carried out in accordance with the requirements of the test procedure.

The plasmid pYG404 is described in patent application EP 361 991 This plasmid contains a HindIII restriction fragment coding for the prepro-SAH gene preceded by 21 naturally occurring nucleotides immediately upstream of the translation initiator ATG of the S. cerevisiae PGK gene. The nucleotide sequence of this restriction fragment is included in Figure 2. The MstII site located in the coding sequence, with three codon residues specifying the end of translation, is particularly useful as a cloning site for a biologically active peptide to be coupled in the C-ninterral phase translation of SAH.Err1:Expecting ',' delimiter: line 1 column 730 (char 729)In another embodiment, the biologically active peptide may be present more than once in the chimera.

The following is the list of the types of vehicles which are to be used in the production of the vehicle:

In a particular embodiment, the combined techniques of directed mutagenesis and PCR amplification allow the construction of hybrid genes coding for a chimeric protein resulting from the translational coupling between a signal peptide (and for example the prepro region of the SAH), a sequence including the biologically active peptide and the mature form of the SAH or one of its molecular variants. These hybrid genes are preferably bounded at 5' of the translation initiator ATG and 3' of the end restriction codon by translation sites HindIII and code for chimeric proteins of the PEPT-SAH type (Figure 1, panel BIDE).

EXAMPLE 3: coupling at the N- and C-terminal of the shaft

Err1:Expecting ',' delimiter: line 1 column 722 (char 721)

The following is a list of the substances that are used in the preparation of the test chemical:

Err1:Expecting ',' delimiter: line 1 column 788 (char 787)The plasmid pYG105 corresponds to the plasmid pKan707 described in patent application EP 361 991 in which the unique HindIII restriction site located in the gene for resistance to genetics (G418) was destroyed by directed mutagenesis while retaining an unchanged protein (oligodeoxynucleotide 5'-GAAATGCATAAGCTCTCCCATTCACTT-3). The SalI-SacI fragment encoding for URA3 of the promoted plasmid was then replaced by a SalI-SacI restriction fragment containing a cassette of expression of the LAC4 lactase constituent gene (KCG4 and the SIG-H1 form of the SalI-CGIII gene).The pYG105 plasmid is mitotically very stable in Kluyveromyces yeasts and a restriction map is given in Figure 3. The pYG105 and pYG106 plasmids differ only in the nature of the transcription promoter encoded by the SalI-SacIII fragment.

EXAMPLE 5 - The processing of yeast

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 treatment of whole cells with lithium acetate (Ito H. et al., J. Bacteriol. 153 (1983) 163-168), adapted as follows:The cells are then incubated at 30°C for 1 hour under moderate agitation. 0.1 ml aliquots of the resulting suspension of competent cells are incubated at 30°C for 1 hour in the presence of DNA and at a final concentration of 35% polyethylene glycol (PEG4000, Sigma). After a 5 minute heat shock at 42°C, the cells are washed twice, resuspended in 0,2 ml sterile water and incubated for 16 hours at 28°C in 2 ml YPD to allow phenotypic expression of the resistance gene G4184 expressed by the control medium Pk1 (cf.EP 361 991); 200 μl of the cell suspension is then spread on selective YPD boxes (G418, 200 μg/ml).

Example 6: Secretion of chimeras

After selection on a G418-rich medium, the recombinant clones are tested for their ability to secrete the mature form of the chimeric proteins. A few clones corresponding to the CBS 293.91 or MW98-8C strain transformed by the chimeric expression plasmids between the SAH and the biologically active part are incubated in YPD or YPL at 28°C. The cell surfactants are recovered by centrifugation when the cells reach the stationary growth phase, and then possibly concentrated 10 times by precipitation for 30 minutes at -20°C in a final concentration of 60% ethanol, tested after electrophoresis in SDS-PAGE gel at 8.Err1:Expecting ',' delimiter: line 1 column 592 (char 591)according to the manufacturer's recommendations.

Example 7: The Chimes of the Factor by Willebrand E.7.1. Fragments which antagonize the binding of vWF to platelets. E.7.1.1 Residue of Thr470-Va1713 from the vWF

The plasmid pET-8c52K contains a fragment of vWF cDNA encoding for human vWF residues 445 to 733 and thus includes several crucial determinants of the interaction between vWF and platelets on the one hand, and some elements of the basement membrane and sub-endothelial tissue on the other hand, including the G10 and D5 antagonist peptides of the interaction between vWF and GP1b [Mori H. et al., J. Biol. Chem. 263 (1988) 17901-17904]. This peptide sequence is identical to the corresponding sequence described by Titani et al. [Biochemistry 25 (1986) 3171-3184].For example, by PCR amplification, using oligodeoxynucleotides coding for contiguous residues located on one side and on the other of the amplified sequence as the precursor. The amplified fragments are then cloned into M13-type vectors for sequencing verification using either the universal precursors on both sides of the cloning multisite or specific oligodeoxynucleotides from the amplified region of the vWF gene whose sequence of several isomorphs is known (Sadler J.E. et al., Proc. Natl. Acad. 82 (1985) 6394-6398; Verij C.E. and al., EMBO J.L. 5 (1986); 1839-1847; Shelton B.Inlowe).Thus, PCR amplification of the pET-8c52K plasmid with the 5'-CCCGGGATCCCTTAGGCTTAACCTGTGAAGCCTGC-3' (Sq1969, MstII site is underlined) and 5'-CCCGGGATCCAAGCTTAGACTTGTG SACCATGTCG-3' (Sq2029, SACTIII site is underlined) oligodeoxynucleotides generates a MstII-HindIII restriction fragment including the Thr470 to Val713 residues of the vWFIDE (Figure 4, EEP panel). This protein fragment binds with the HindIII-MstIII restriction fragment corresponding to the gene coding for the three-type of the HFII-HindIII restriction (see Figure 1, EEP panel).Err1:Expecting ',' delimiter: line 1 column 106 (char 105)

The following shall be used for the calculation of the maximum level of the test substance:

In another embodiment, the vWF binding site is a peptide including the Thr470 to Asp498 residues of the mature vWF. This sequence includes the peptide G10 (Cys474-Pro488) described by Mori et al. [J. Biol. Chem. 263 (1988) 17901-17904] and capable of antagonizing the interaction of human vWF with human platelet GP1b. The corresponding sequence for the G10 peptide is first included in a restriction fragment MstII-HindIII (Figure 4, panel B), e.g. by amplification of the PCR plasmid pET-8c52K with the oligododexyloproteins SQ1969 and 5'-CCGGATCCAAGCCTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAErr1:Expecting ',' delimiter: line 1 column 643 (char 642)

Err1:Expecting ',' delimiter: line 1 column 989 (char 988)

Useful variants of the pET-8c52K plasmid are deleted by directed mutagenesis between peptides G10 and D5, e.g. from binding sites to collagen, and/or heparin, and/or botocetin, and/or sulfatides and/or ristocetin. An example is the plasmid pMMB9 deleted by directed mutagenesis between residues Cys509 and Ile662. PCR amplification of this plasmid with oligodeoxynucleotides Sq1969 and Sq2029 generates a MstII-HindIII restriction fragment (Figure 4, panel D) including residues Thr470 at Tyr850 and Argothene at Val663137 and in particular the peptides GF10 and DW5 and its localized deletion site between collagen and GG226R5 [R].Err1:Expecting ',' delimiter: line 1 column 485 (char 484)

In other embodiments, the use of combined directed mutagenesis and PCR amplification techniques allows the arbitrary generation of variants of the MstII-HindIII restriction fragment of panel A of Figure 4 but deleted from one or more sulfatide and/or butocetin and/or heparin and/or collagen binding sites and/or replaced by any residue involved in the emergence of vWF-associated type IIB pathologies.

In other useful variants of the plasmid pET-8c52K mutations are introduced, for example by directed mutagenesis, to replace or remove all or part of the cysteines at positions 471, 474, 509 and 695 of the human vWF. Particular examples are the plasmids p5E and p7E in which the cysteines at positions 471 and 474 on the one hand and 471, 474, 509 and 695 on the other hand have been replaced by glycine residues respectively. PCR amplification of these plasmids with the oligodeoxycysteine oligocycloids Sq2149 (5CCGGATCCCCCTTACTAGAGGTAG, the site is highlighted SGCGC-320), allows the generation of fragments of cysteine residues at positions 471 and 474 except for the natural cysteine residues at positions 477 and MQGV413 in the Valgulin.Err1:Expecting ',' delimiter: line 1 column 443 (char 442)

Other particularly useful mutations involve at least one residue involved in vWF-associated type IIB pathologies (increased intrinsic affinity of vWF for GP1b), such as Arg543, Arg545, Trp550, Val551, Val553, Pro574, or Arg578 residues.

E.7.2. Fragments antagonistic to the attachment of vWF to the sub-endothelium.

In a particular embodiment, the binding sites of the vWF to components of the sub-endothelial tissue, and for example collagen, are generated by PCR amplification of the plasmid pET-8c52K, for example with oligodeoxynucleotides Sq2258 (5'-GGATCCTTAGGGCTG-TGCAGCAGGCTACTTAGGACTTGGTC-3', the MstII site is underlined) and Sq2259 (5'GAATTCAAGCTTAACAGAACTAGTGGTCCC-3', the Hindouin site is underlined), which generates a MstII-HGGIII restriction fragment coding for residues Cys950 to Cys695 GPWV. Molecular or gene-modified variants are also associated with the genetic modification of any desirable combination of vWF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/WF/W/WF/WF/WF/WF/WF/WF/WF/WF/W/W/WF/WF/WF/W/WF/WF/W/WF/W/W/W/WF/W/W/WF/W/W/WF/W/W/W/W/W/WErr1:Expecting ',' delimiter: line 1 column 676 (char 675)These restriction fragments are clones in the productive orientation at the HindIII site of the plasmid pYG105, which generates the corresponding expression plasmids, and for example the plasmid pYG1277 (SAH-vWF509-695).

E.7.3. Purification and molecular characterisation of chimeras between SAH and vWF.

The chimeras present in the culture surfactants corresponding to the transformed CBS 293.91 strain, e.g. by the expression plasmids in examples E.7.1 and E.7.2, are first characterised by specific antibodies to the SAH part and the vWF part. The results in Figures 5 to 7 show that K. lactis yeast is capable of secreting chimeric proteins between the SAH and a fragment of the vWF, and that these chimeras are immunologically reactive. It may also be desirable to purify some of these chimeras.The resulting concentrate is then dialysed against a solution of Tris HCl (50 mM pH 8) and then purified on a column. For example, the concentrate corresponding to the culture surfactant of the CBS strain 293.91 transformed by the plasmid pYG1206 is purified by Blue-Trisacryl affinity chromatography (IBF). A purification by ion exchange chromatography may also be used. For example, in the case of the SAH-Ultra-vWF4707-713, the resulting concentration is dialysed against a solution of Tris HCl (50 mM pH),then deposited in 20 ml fractions on a column (5 ml) of a cation exchanger (S Fast Flow, Pharmacia) balanced in the same buffer. The column is then washed several times with Tris HCl solution (50 mM pH 8) and the chimeric protein is then elicited from the column by a gradient (0 to 1 M) of NaCl. The fractions containing the chimeric protein are then collected and dialysed against a solution of Tris HCl 50 mM (pH 8) and then deposited on column S Fast Flow. After elusion, the fractions containing the protein are collected,Err1:Expecting ',' delimiter: line 1 column 422 (char 421)C474G]. behaves under these conditions as a protein with an apparent molecular weight of 95 kDa demonstrating its monomeric character.

EXAMPLE 8 - Chimes from the Urokinases E.8.1. construction work

A MstII-HindIII restriction fragment including human urokinase ATF is shown in Figure 8. The binding of the pYG404 plasmid's HindIII-MstII fragment to this MstII-HindIII fragment allows the generation of the pYG1341 plasmid fragment, respectively, which codes for a gene-producing protein in which the SAH molecule is restrictively coupled to the PATF (UKSA-H131-UKSA-H135). Similarly, the MstII-HindIII restriction fragment contains the pYG40 plasmid (UKSA-H131-UKSA-H134-UKSA-H134-PYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY

E.8.2.Secretion of hybrids

After selection on G418-rich media, recombinant clones are tested for their ability to secrete the mature form of the SAH-UK chimeric proteins. A few clones corresponding to the K. lactis CBS 293.91 strain transformed by the expression plasmids in example E.9.1. are incubated in a selective complete liquid medium at 28°C. The cell survivors are then tested after electrophoresis in 8.5% acrylamide gel, either directly by staining the gel to co-mass blue, or after primary serum using a polyclonal rabbit serum against albumin Klutein or against human protein kinase. The results of Figure 9 show that SAH-UK461-H1H1H1 and SAH-UK135 hybrids are particularly well directed by immunity.

E.8.3.Purification of chimeras between SAH and urokinase.

After centrifugation of a culture of the CBS 293.91 strain transformed by expression plasmids in example E.8.1, the culture surfactant was passed through a 0.22 mm filter (Millipore) and then concentrated by ultrafiltration (Amicon) using a membrane with a discrimination threshold of 30 kDa. The resulting concentrate was then adjusted to 50 mM Tris HCl from a stock solution of Tris HCl 1M (pH 7) and then deposited in 20 ml fractions on a column (3 ml) of an anion exchanger (D-Zephyr, Sepracorlu) balanced in the buffer. The chimeric protein (SAH-UK1->46>SAH-UK1->135) is then collected in a column or gradient of the same color by a dialysis of the Tris HCl 1M (HCl) and its reactions against the same chemical, and the fractions are then displaced on the same column and the HCl (D-Zephyr) are then collected in the same column and the reactions against the dialysis of the dialysis of the protein (HCl) and their reactions against the HCl are then balanced.

EXAMPLE 9 - Chymers derived from G-CSF E.9.1 Construction work The test shall be carried out in accordance with the requirements of Annex I to this Regulation.

A restriction fragment MstII-HindIII including the mature form of human G-CSF is generated, for example, according to the following strategy: a restriction fragment KpnI-HindIII is first obtained by the PCR enzyme amplification technique using the oligodeoxynucleotides Sq2291 (5'-CAAGGATCCAAGCTTCAGGGCTCGCGCGCGCGTAG-3', the HindIII site is underlined) and Sq2292 (5'-CGGGGTACCTAACTACCCT SAGCCCTGCCAGC-3', the KpnGGI site is underlined) as a precursor on the BBG13 plasmid serving as a restriction nucleus. The BBG13 plasmid is the same gene. This sequence of amino acids (174 for the BCSF125F) is generated by the human plasmid, allowing the fusion of the PHG-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HINY-HIN-HINY-HINY-HIN-HINY-HIN-HIN-HIN-HINY-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-HIN-

A MstII-HindIII restriction fragment is for example generated by substitution of the MstII-ApaI fragment of the plasmid pYG1255 by the oligodeoxynucleotides Sq2742 (5'-TTAGGCTTAGGTGGGCGGTACCCCTGCC-3', codons coding for glycine residues from this particular linker are highlighted) and Sq2741 (5CAGGGGTACCGACC'-CACCTAAGCC-3') which form by pairing a MstII-ApaI fragment. The plasmid thus generates a MstII-ApaI restriction fragment, the sequence of which is identical to the MstII-ApaI fragment in Figure 10.

The binding of the HindIII-MstII fragment of the pYG404 plasmid with the MstII-HindIII fragment of the pYG1255 plasmid allows the generation of the HindIII fragment of the pYG1259 plasmid, which encodes for a chimeric protein in which the mature G-CSF B form is positioned by genetic coupling at the C-terminal translational phase of the SAH molecule (SAH-G.CSF).

A HindIII restriction fragment identical to the MstII-ApaI fragment can also be easily generated, which codes for a chimeric protein in which the B form of the mature G-CSF is positioned by genetic coupling in the C-terminal translational phase of the SAH molecule and a particular peptide linker.

The HindIII restriction fragment of the pYG1259 plasmid is cloned in the productive orientation and at the HindIII restriction site of the pYG105 expression plasmid, generating the pYG1266 expression plasmid (SAH-G.CSF).In another example, cloning of the HindIII restriction fragment of the pYG1259 plasmid in the productive orientation and at the HindIII site of the pYG106 plasmid generates the pYG1267 plasmid.The pYG1266 and pYG1267 plasmids are isogenous to each other except for the SalI-HindIII restriction fragment coding for the promoter of LAC.lactis (pYG1266) or the promoter of S.plasmiae (pYG1267).

In another example, cloning in the productive direction of the HindIII restriction fragment of the pYG1336 plasmid (SAH-Gly4-G.CSF chimera) at the HindIII site of the pYG105 (LAC4) and pYG106 (PGK) plasmids generates the pYG1351 and pYG1352 expression plasmids, respectively.

The N-terminal coupling of the SAH.

In a particular embodiment, the combined techniques of directed mutagenesis and PCR amplification allow the construction of hybrid genes coding for a chimeric protein resulting from the translational coupling between a signal peptide (and for example the prepro region of the SAH), a sequence including a gene with G-CSF activity, and the mature form of the SAH or one of its molecular variants (see chimera in panel B, Figure 1). These hybrid genes are preferably bounded in 5' of the translation initiator ATG and in 3' of the end-translation codon by HindIII restriction sites.The highlighted residues (optional) correspond in this particular chimera to a peptide linker composed of 4 glycine residues) allows directed mutagenesis to translate the mature form of human G-CSF from the BBG13 plasmid immediately upstream of the mature form of the SAH, generating the intermediate plasmid A. Similarly, the use of oligodeoxynucleotide Sq2338 [5'-CAGGGAGCTCAGCCCAGGTGGTGACGAAACCTGGGGGGGAGGAGGAG-3' (non-coding strand), the complementary nucleotide precursors to the N-terminal residues of the mature form of human GCSF] are directed by mutagenesis to read the readings in the immediate translation of the GHG protein,which generates the intermediate plasmid B. A HindIII fragment coding for a chimeric protein of the PEPTIDE-SAH type (see Figure 1, panel B) is then generated by combining the HindIII-SstI fragment of plasmid B (SAH prepro region junction + mature G-CSF N-terminal fragment) with the SstI-HindIII fragment of plasmid A [mature G-CSF junction-glycine-x4-SAH]. The plasmid pYG1301 contains this particular HindIII restriction fragment coding for the chimeric G.CSF-Gly4-SAH fusion immediately downstream of the preproproject region of the SAH (Figure 11). The cloning of this HindIII fragment restriction in the productive and orientation site in the plasmids pYYG10 (PYYG10K13, pYG30C14) and pYYG10G130 (PYYG10G13) generates the expression of the HindIII restriction in the productive and orientation plasmids pYYG10K1 and pYG30G1 (PYG10K14)The Commission has

E.9.2 Secretion of hybrids

After selection on G418-rich media, recombinant clones are tested for their ability to secrete the mature form of the chimeric proteins between SAH and G-CSF. A few clones corresponding to the K. lactis CBS 293.91 strain transformed by the plasmids pYG1266 or pYG1267 (SAH-G.CSF), pYG1302 or pYG1303 (G.CSF-Gly4-SAH) or pYG1351 or pYG1352 (SAH-Gly4-G.CSF) are incubated in selective liquid at 28°C. Cell surpluses are tested after a complete electroplast in an 8.5% acrylic acid gel, either directly after staining with a blue coagulation gel, or using a serum antibody directed against the human GCSF-cell antibodies as a primary antibody or using a serum antibodies directed against the GCSF.The results in Figure 12 show that the SAH-G.CSF hybrid protein is recognized by both antibodies directed against human albumin (panel C) and human G-CSF (panel B). The results in Figure 13 indicate that the SAH-Gly4-G.CSF chimera (track 3) is particularly well secreted by Kluyveromyces yeast, possibly because the presence of the peptide linker between SAH part and G-CSF part is more favorable to independent folding of these 2 parts during the chimera transit through the secretory pathway.Runway 1)

E.9.3. Purification and molecular characterisation of chimeras between SAH and G-CSF.

After centrifugation of a culture of the CBS 293.91 strain transformed by the expression plasmids in Example E.9.1, the culture surfactant was passed through a 0.22 mm filter (Millipore) and then concentrated by ultrafiltration (Amicon) using a membrane with a discrimination threshold of 30 kDa. The resulting concentrate was then adjusted to 50 mM Tris HCl from a stock solution of Tris HCl 1M (pH 6) and then deposited in 20 ml fractions on a balanced (5 ml) ion exchange column (Q Fast Flow, Pharmacia) in the same buffer.Err1:Expecting ',' delimiter: line 1 column 679 (char 678)

The following is a list of the chemicals that are used in the preparation of the test chemical: E.10.1 Construction work

A Fv' fragment can be constructed by genetic engineering techniques, and which codes for variable fragments of the heavy and light chains of an immunoglobulin (Ig), linked together by a peptide linker [Bird et al., Science (1988) 242: 423; Huston et al., (1988) Prof. Natl. Acad. Sci. 85:5879]. Schematically, the variable regions (about 120 residues) of the heavy and light chains of a given Ig are cloned from the corresponding hybridome messenger, e.g. using the RT-PCR kit distributed by Pharmacia (Mouse ScFv Module).A MstII-HindIII restriction fragment including the Fv' fragment of an immunoglobulin secreted by a mouse hybridome is shown in Figure 14. The binding of the HindIII-MstII fragment of the pYG404 plasmid to this MstII-HindIII fragment allows the generation of the HindIII fragment of the pYG1382 plasmid which encodes for a chimeric protein in which the SAH molecule is genetically coupled to the Fv' fragment of Figure 14 (SAH-Fv chimeric).

E.10.2 Secretion of hybrids

After selection on a G418-rich medium, the recombinant clones are tested for their ability to secrete the mature form of the chimeric SAH-Fv protein. A few clones corresponding to the K. lactis CBS 293.91 strain transformed by the plasmids pYG1383 or pYG1384 (SAH-Fv') are incubated in a selective complete liquid medium at 28°C. The cell surfactants are then tested after electrophoresis in 8.5% acrylamide gel, either directly by staining the co-mass blue gel, or after cloning using primary antibodies such as a serinyl albumin polyclinic antibody against human albumin, directed directly with antibodies directed against human or bioglobin antibodies (Figure 15), or directed against the human or bioglobin antibodies (Figure 15), which is shown to be immunologically recognized as antibodies against the B-albumin antibodies (Figure 15), and is shown to be antibodies against the human or bioglobin antibodies against the B-albumin antibodies (Figure 15), which are directed directly against the human or bioglobin antibodies against the B-albumin antibodies (Figure 15), and are shown to be immunologically recognized as antibodies against the B-albumin antibodies (Figure 15).

The following is a list of the types of chemicals used in the chemical industry: E.11.1. biological activity in vitro E.11.1.1 Chimeras between SAH and vWF.

The antagonistic activity of the products is determined by measuring the dose-dependent inhibition of paraformaldehyde-bound human platelet agglutination as described by Prior et al. [Bio/Technology (1992) 10: 66]. Measurements are made in an aggregator (PAP-4, Bio Data, Horsham, PA, USA) which records the variations over time in optical transmission under agitation at 37°C in the presence of vWF, botrocetin (8.2 mg/ml) and the product to be tested at different dilutions (concentrations). For each measurement, 400 (8x107 ml) of paraformaldehyde-stabilized human platelet suspension (0.5%, residues in [NaClM37 m; NaClM2 m; NaClM2 (1,4 ml) ]The test chemical is then added to the test chemical solution at various dilutions in the apyrogenic formulation vehicle (mannitol (50 g/l)); citric acid (192 mg/l) ; L-monochlorinated L-lysine (182.6 mg/l).; NaCl (88 pH/l) ; adjusted to 3.5 mg (1M NaOH) by addition of NaOH), test vehicle or control (only).The resulting suspension is then incubated for 1 minute at 37°C and 12.5 ml of human vWF [American Bioproducts, Parsippany, NJ, USA; 11% von Willebrand activity measured according to the PAP-4 (Platelet Aggregation ProfilerR) recommendations for use with formaldehyde-bound platelets (2x105 platelets/ml), human plasma containing 0-100% vWF and ristocetin (10 mg/ml, cf. pp. 36-45: vW ProgramTM) is added to incubate at 37°C for 1 minute before adding 12.5 ml of botrocine solution [purified from Botrophilised Lyophilized Venom of Botrops (Sigmaca Sugara), as described by the protocole and alimoto.The average variation in optical transmission (n35 for each dilution) over time is therefore a measure of platelet agglutination due to the presence of vWF and botrocetin, in the absence or presence of variable concentrations of the test product. From such recordings, the % inhibition of platelet agglutination due to each product concentration is then determined and the % inhibition trace is given as a function of the dilution of the product on the log-inverse scale.The CI50 (or product concentration causing 50% inhibition of agglutination) is then determined on this line. The Table in Figure 16 compares the CI50s of some of the SAH-vWF chimeras of the present invention and shows that some of them are better platelet agglutination antagonists than the product RG12986 described by Prior et al. [Bio/Technology (1992) 10: 66] and included in the tests as a standard value.Botoxine-independent antagonism of these particular chimeras can also be demonstrated by the protocol originally described by Ware et al. [Proc. Natl. Acad. Sci. (1991) 88:2946] by displacement of the monoclonal antibody 125I-LJ-IB1 (10 mg/ml), a competitive inhibitor of the binding of vWF to platelet GbPI [Handa M. et al., (1986) J. Biol. Chem. 261: 12579] after 30 minutes of incubation at 22°C in the presence of frogs (108 ml/ml).

E.11.1.2 Chimeras between SAH and G-CSF.

The purified chimeras are tested for their ability to allow in vitro proliferation of the IL3-dependent mouse lineage NFS60 by measuring the incorporation of thymidine tritiated essentially according to the protocol described by Tsuchiya et al. [Proc. Natl. Acad. Sci. (1986) 83 7633]. For each chimera, measurements are performed between 3 and 6 times in a three-point test (three dilutions of the product) in an area where the ratio of amount of active product to incorporation of labeled thymidine (sham) is linear. In each microtitration plate, the activity of a specific reference product consisting of G-CSF-Amerin recombinant expressed in human mammary cells is also systematically incorporated. The results of Figure 17 show that the chemical activity of Kluf-F (GCSF-H12H6H) in this particular case is approximately 7 times lower than that of the in vitro signal of G-CSF-Amerin (GCSF-H12H6H6H6O).

E.11.2 Biological activity in vivo is not known.

The stimulating activity of the 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 the reference G-CSF expressed from mammalian cells. Each product, tested in 7 animals, is injected subcutaneously into the dorsoscapular region at 100 ml for 7 consecutive days (J1-J7). 500 ml of blood is collected on days J-6, J2 (before the 2nd injection), J5 (before the 5th injection) and J8, and a specific blood test is performed. In this test, the specific activity (neutral reference units/inhibition) of SAH/GCSF is shown to be 7 times that of SAH. G-CSF (GCSF) has a particularly low in vitro chemical activity, so the chemical properties of G-CSF (GCSF) are shown to be identical to those of G-CSF (GCSF) 1712 (F68).

Claims (19)

1. Recombinant polypeptide with an active part derived from a polypeptide with therapeutic activity, genetically coupled to albumin or albumin variant.
2. Polypeptide according to claim 1 characterised by the fact that the polypeptide with therapeutic activity is a polypeptide of human origin.
3. Polypeptide as claimed 2 characterised by the selection of the polypeptide with therapeutic activity from all or part of enzymes, enzyme inhibitors, antigens, antibodies, hormones, clotting factors, interferons, cytokines, growth and/or differentiation factors, factors involved in the generation/resorption of bone tissue, chemotactic factors, factors of motility or cell migration, cytostatic factors, bactericidal or antifungal factors, or plasma, interstitial or extracellular adhesive molecules.
4. Polypeptide according to claims 1 to 3 characterised by the polypeptide having therapeutic activity being chosen from any peptide sequence antagonist or agonist of molecular and/or cellular interactions involved in circulatory and interstitial compartment pathologies.
5. Polypeptide according to claims 1 to 4 characterised by the active part having a structure chosen from: - What?

   (a) the entire peptide structure, or

   (b) a fragment of (a) or a structure derived from (a) by structural modification (mutation, substitution, addition and/or deletion of one or more residues) and retaining therapeutic activity.
6. Polypeptide according to claims 1 to 5 characterised by the active part being coupled to the N-terminal end of the albumin.
7. Polypeptide according to claims 1 to 5 characterised by the active part being coupled to the C-terminal end of the albumin.
8. Polypeptide according to claims 1 to 7 characterised by the presence of the active substance several times.
9. Nucleotide sequence coding for a polypeptide according to any of claims 1 to 8.
10. Err1:Expecting ',' delimiter: line 1 column 140 (char 139)
11. Expression cassette containing a nucleotide sequence according to claim 9 or 10 under control of a transcription initiation region and possibly a transcription termination region.
12. Self-replicating plasmid with an expression cassette as claimed 11.
13. Recombinant eukaryotic or prokaryotic cell into which a nucleotide sequence according to claim 9 or 10 or an expression cassette according to claim 11 or a plasmid according to claim 12 has been inserted.
14. Recombinant cell according to claim 13 characterised as a yeast, animal cell, fungus or bacterium.
15. Recombinant cell according to claim 14 characterised as a yeast.
16. Recombinant cell according to claim 15 characterised as a yeast of the genus Saccharomyces or Kluyveromyces.
17. Process for preparing a polypeptide as defined in one of claims 1 to 8 characterised by culturing a recombinant cell according to one of claims 13 to 16 under expression conditions and recovering the polypeptide produced.
18. Pharmaceutical formulation containing one or more polypeptides according to any of claims 1 to 8.
19. A pharmaceutical composition containing a nucleotide sequence according to any of claims 9 to 11 for use in gene therapy.