CA2126091C - Novel biologically active polypeptides, preparation thereof and pharmaceutical composition containing said polypeptides - Google Patents
Novel biologically active polypeptides, preparation thereof and pharmaceutical composition containing said polypeptides Download PDFInfo
- Publication number
- CA2126091C CA2126091C CA002126091A CA2126091A CA2126091C CA 2126091 C CA2126091 C CA 2126091C CA 002126091 A CA002126091 A CA 002126091A CA 2126091 A CA2126091 A CA 2126091A CA 2126091 C CA2126091 C CA 2126091C
- Authority
- CA
- Canada
- Prior art keywords
- albumin fusion
- recombinant
- leu
- fusion polypeptide
- therapeutically active
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
- B65B—MACHINES, APPARATUS OR DEVICES FOR, OR METHODS OF, PACKAGING ARTICLES OR MATERIALS; UNPACKING
- B65B69/00—Unpacking of articles or materials, not otherwise provided for
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
- A61P7/06—Antianaemics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
-
- 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)
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
-
- 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
Abstract
Novel biologically active polypeptides, preparation thereof and pharmaceutical compositions containing said polypeptides.
Description
I
NOVEL BIOLOGICALLY ACTIVE POLYPEPTIDES, PREPARATION
THEREOF AND PHARMACEUTICAL COMPOSITION CONTAINING
SAID POLYPEPTIDES
The present invention relates to new biologically active polypeptides, their preparation and pharmaceutical compositions containing them.
More particularly, the present invention relates to essentially recombinant polypeptides composed of an active part derived from a natural or artificial polypeptide having a therapeutic activity and coupled to an albumin or to a variant of albumin. It is understood that the therapeutic activity of the polypeptides of the invention can be either direct (treatment of diseases), or indirect (and for example capable of being used in the prevention of diseases, in the design of vaccines, in medical imaging techniques and the like).
It is understood in the following text that the albumin variants designate any protein with a high plasma half-life which is obtained by modification (mutation, deletion and/or addition), by genetic engineering techniques, of a gene encoding 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. Albumin being highly polymorphic, numerous natural variants have been identified and classified Weitkamp L. R. et al., [Ann. Hum. Genet. 37 (1973) 219].
The aim of the present invention is to prepare artificial proteins which are biologically active and can be used pharmaceutically. Indeed, numerous polypeptides possessing one or more potential therapeutic activities cannot be exploited pharmaceutically. This may have various reasons, such as especially their low stability in vivo, their complex or fragile structure, the difficulty of producing them on an industrially acceptable scale and the like. Likewise, some polypeptides do not give the expected results in vivo because of problems of administration, of packaging, of pharmacokinetics and the like.
NOVEL BIOLOGICALLY ACTIVE POLYPEPTIDES, PREPARATION
THEREOF AND PHARMACEUTICAL COMPOSITION CONTAINING
SAID POLYPEPTIDES
The present invention relates to new biologically active polypeptides, their preparation and pharmaceutical compositions containing them.
More particularly, the present invention relates to essentially recombinant polypeptides composed of an active part derived from a natural or artificial polypeptide having a therapeutic activity and coupled to an albumin or to a variant of albumin. It is understood that the therapeutic activity of the polypeptides of the invention can be either direct (treatment of diseases), or indirect (and for example capable of being used in the prevention of diseases, in the design of vaccines, in medical imaging techniques and the like).
It is understood in the following text that the albumin variants designate any protein with a high plasma half-life which is obtained by modification (mutation, deletion and/or addition), by genetic engineering techniques, of a gene encoding 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. Albumin being highly polymorphic, numerous natural variants have been identified and classified Weitkamp L. R. et al., [Ann. Hum. Genet. 37 (1973) 219].
The aim of the present invention is to prepare artificial proteins which are biologically active and can be used pharmaceutically. Indeed, numerous polypeptides possessing one or more potential therapeutic activities cannot be exploited pharmaceutically. This may have various reasons, such as especially their low stability in vivo, their complex or fragile structure, the difficulty of producing them on an industrially acceptable scale and the like. Likewise, some polypeptides do not give the expected results in vivo because of problems of administration, of packaging, of pharmacokinetics and the like.
The present invention makes it possible to overcome these disadvantages. The present invention indeed provides new molecules which permit an optimal therapeutic exploitation of the biological properties of these polypeptides. The present invention results especially from the demonstration that it is possible to couple genetically any active structure derived from a biologically active polypeptide to another protein structure consisting of albumin, without impairing the said biological properties thereof. It also results from the demonstration by the Applicant that human serum albumin makes it possible efficiently to present the active structure to its sites for interaction, and that it provides a high plasma stability for the polypeptide of the invention. The polypeptides of the invention thus make it possible to maintain, in the body, a given biological activity for a prolonged period. They thus make it possible to reduce the administered doses and, in some cases, to potentiate the therapeutic effect, for example by reducing the side effects following a higher administration. The polypeptides of the invention make it possible, in addition, to generate and to use structures derived from biologically active polypeptides which are very small and therefore very specific for a desired effect. It is understood that the peptides having a biological activity, which are of therapeutic interest, may also correspond to non-natural peptide sequences isolated for example from random peptide libraries.
The polypeptides of the invention possess, moreover, a particularly advantageous distribution in the body, which modifies their pharmacokinetic properties and favours the development of their biological activity and their use. In addition, they also have the advantage of being weakly or non-immunogenic for the organism in which they are used. Finally, the polypeptides of the invention can be expressed (and preferentially secreted) by recombinant organisms, at levels permitting their industrial exploitation.
One subject of the present invention therefore relates to polypeptides containing an active part derived from a polypeptide having a therapeutic activity, coupled to an albumir.i or a variant of albumin.
The polypeptides of the invention possess, moreover, a particularly advantageous distribution in the body, which modifies their pharmacokinetic properties and favours the development of their biological activity and their use. In addition, they also have the advantage of being weakly or non-immunogenic for the organism in which they are used. Finally, the polypeptides of the invention can be expressed (and preferentially secreted) by recombinant organisms, at levels permitting their industrial exploitation.
One subject of the present invention therefore relates to polypeptides containing an active part derived from a polypeptide having a therapeutic activity, coupled to an albumir.i or a variant of albumin.
In a specific embodiment, the peptides possessing a therapeutic activity are not of human origin. For example, there may be mentioned peptides, or their derivatives, possessing properties which are potentially useful in the pathologies of the blood and interstitial compartments, such as hirudin, trigramine, antistatine, tick anticoagulant peptides (TAP), arietin, applagin and the like.
More particularly, in the molecules of the invention, the polypeptide having a therapeutic activity is a polypeptide of human origin or a molecular variant. For example, this may be all or part of an enzyme, an enzyme inhibitor, an antigen, an antibody, a hormone, a factor involved in the control of coagulation, an interferon, a cytokine [the interleukins, but also their variants which are natural antagonists of their binding to the receptor(s), the SIS (small induced secreted) type cytokines and for example the macrophage inflammatory proteins (MIPs), and the like], of a growth factor and/or of differentiation [and for example the transformant growth factors (TGFs), the blood cell differentiation factors (erythropoietin, M-CSF, G-CSF, GM-CSF and the like), insulin and the growth factors resembling it (IGFs), or alternatively cell permeability factors (VPF/VEGF), and the like], of a factor involved in the genesis/resorption of bone tissues (OIF and osteospontin for example), of a factor involved in cellular motility or migration [and for example autocrine motility factor (AMF), migration stimulating factor (MSF), or alternatively the scatter factor (scatter factor/hepatocyte growth factor)], of a bactericidal or antifungal factor, of a chemotactic factor and for example platelet factor 4 (PF4), or alternatively the monocyte chemoattracting peptides (MCP/MCAF) or neutrophil chemoattracting peptides (NCAF), and the like, of a cytostatic factor (and for example the proteins which bind to galactosides), of a plasma (and for example von Willebrand factor, fibrinogen and the like) or interstitial (laminin, tenascin, vitronectin and the like) adhesive molecule or extracellular matrices, or alternatively any peptide sequence which is an antagonist or agonist of molecular and/or intercellular interactions involved in the pathologies of the circulatory and interstitial compartments and for example the formation of arterial and venous thrombi, cancerous metastases, tumour angiogenesis, inflammatory shock, autoimmune diseases, bone and osteoarticular pathologies and the like.
The active part of the polypeptides of the invention may consist for example of the polypeptide having a whole therapeutic activity, or of a structure derived therefrom, or alternatively of a non-natural polypeptide isolated from a peptide library. For the purposes of the present invention, a derived structure is understood to mean any polypeptide obtained by modification and preserving a therapeutic activity. Modification should be understood to mean any mutation, substitution, deletion, addition or modification of genetic and/or chemical nature.
Such derivatives may be generated for various reasons, such as especially that of increasing the affinity of the molecule for its binding sites, that of improving its levels of production, that of increasing its resistance to proteases, that of increasing its therapeutic efficacy or alternatively of reducing its side effects, or that of conferring on it new biological properties. As an example, the chimeric polypeptides of the invention possess pharmacokinetic properties and a biological activity which can be used for the prevention or treatment of diseases.
Particularly advantageous polypeptides of the invention are those in which the active part has:
(a) the whole peptide structure or, (b) a structure derived from (a) by structural modification (mutation, substitution addition and/or deletion of one or more residues) and possessing a therapeutic activity.
Among the structures of the (b) type, there may be mentioned more particularly the molecules in which certain N- or 0-glycosylation sites have been modified or suppressed, the molecules in which one or more residues have been substituted, or the molecules in which all the cystein residues have been substituted. There may also be mentioned molecules obtained from (a) by deletion of regions not involved or not highly involved in the interaction with the binding sites considered, or expressing an undesirable activity, and molecules containing, compared to (a), additional residues such as for example an N-terminal methionine and/or a signal for secretion and/or a joining peptide.
The active part of the molecules of the invention can be coupled either directly or via an artificial peptide to albumin. Furthermore, it may constitute the N-terminal end 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 repetitive within the chimera. A schematic representation of the molecules of the invention is given in FIG. 1.
Another subject of the invention relates to a process for preparing the chimeric molecules described above. More specifically, this process consists in causing a eukaryotic or prokaryotic cellular host to express a nucleotide sequence encoding the desired polypeptide, and then in harvesting the polypeptide produced.
Among the eukaryotic hosts which can be used within the framework of the present invention, there may be mentioned animal cells, yeasts or fungi. In particular, as regards yeasts, there may be mentioned yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula. As regards animal cells, there may be mentioned COS, CHO and C127 cells and the like. Among the fungi capable of being used in the present invention, there may be mentioned more particularly Aspergillus spp, or Trichoderma spp. As prokaryotic hosts, the use of bacteria such as Escherichia coli, or belonging to the genera Corynebacterium, Bacillus, or Streptomyces is preferred.
The nucleotide sequences which can be used within the framework of the present invention can be prepared in various ways. Generally, they are obtained by assembling, in reading phase, the sequences encoding each of the functional parts of the polypeptide. The latter may be isolated by the techniques of persons skilled in the art, and for example directly from cellular messenger RNAs (mRNAs), or by recloning from a complementary DNA (cDNA) library, or alternatively they may be completely synthetic nucleotide sequences. It is understood, furthermore, that the nucleotide sequences may also be subsequently modified, for example by the techniques of genetic engineering, in order to obtain derivatives or variants of the said sequences.
More preferably, in the process of the invention, the nucleotide sequence is part of an expression cassette comprising a region for initiation of transcription (promoter region) permitting, in the host cells, the expression of the nucleotide sequence placed under its control and encoding the polypeptides of the invention. This region may come from promoter regions of genes which are highly expressed in the host cell used, the expression being constitutive or regulatable. As regards yeasts, it may be the promoter of the gene for phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GPD), lactase (LAC4), enolases (ENO), alcohol dehydrogenases (ADH), and the like. As regards bacteria, it may be the promoter of the right-hand or left-hand genes from the lambda bacteriophage (PL, PR), or alternatively the promoters of the genes for the tryptophan (Pt,r) or lactose (Plaj operons. In addition, this control region can be modified, for example by in vitro mutagenesis, by the introduction of additional control elements or of synthetic sequences, or by deletions or substitutions of the original control elements. The expression cassette may also comprise a region for termination of transcription which is functional in the host envisaged, positioned immediately downstream of the nucleotide sequence encoding a polypeptide of the invention.
In a preferred mode, 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 product of expression of the said sequence into the culture medium. It is indeed particularly advantageous to be able to obtain, by the recombinant route, nlolecules directly in the culture medium. In this case, the nucleotide sequence encoding a polypeptide of the invention is preceded by a "leader" sequence (or signal sequence) directing the nascent polypeptide in the secretory pathways of the host used. This "leader" sequence may be the natural signal sequence of the biologically active polypeptide in the case where the latter is a naturally secreted protein, or that of the stabilizing structure, but it may also be any other functional "leader" sequence, or an artificial "leader" sequence.
The choice of one or the other of these sequences is especially guided by the host used.
Examples of functional signal sequences include those of the genes for the sexual pheromones or the "killer" toxins of yeasts.
In addition to the expression cassette, one or several markers which make it possible to select the recombinant host may be added, such as for example the URA3 gene from the yeast S. cerevisiae, or genes conferring the resistance to antibiotics such as geneticin (G418) or to any other toxic compound such as certain metal ions.
The unit formed by the expression cassette and by the selectable marker can be introduced directly into the considered host cells, or previously inserted in a functional self-replicating vector. In the first case, sequences homologous to regions present in the genome of the host cells are preferably added to this unit; the said sequences then being positioned on each side of the expression cassette and of the selectable gene so as to increase the frequency of integration of the unit into the genome of the host by targeting the integration of the sequences by homologous recombination. In the case where the expression cassette is inserted in a replicative system, a preferred replication system for yeasts of the genus Kluyveromyces is derived from the plasmid pKDI originally isolated from K.
drosophilarum; a preferred replication system for yeasts of the genus Saccharomyces is derived from the 2 plasmid from S. cerevisiae. Furthermore, this expression plasmid may contain all or part of the said replication systems, or may combine elements derived both from the plasmid pKDI and the 2 plasmid.
In addition, the expression plasmids may be shuttle vectors between a bacterial host such as Escherichia coli and the chosen host cell. In this case, a replication origin and a selectable 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 suppress these sequences by cutting and religation in vitro 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 the said hosts. For example, such restriction sites may correspond to sequences such as 5'-GGCCNNNNNGGCC-3' (Sfil) or 5'-GCGGCCGC-3' (Notl) in so far as these sites are extremely rare and generally absent from an expression vector.
After construction of such vectors or expression cassette, the latter are introduced into the host cells selected according to the conventional techniques described in the literature. In this respect, any method permitting the introduction of a foreign DNA into a cell can be used. This may be especially transformation, electroporation, conjugation, or any other technique known to persons skilled in the art. As an example of yeast-type hosts, the various strains of Kluyveromyces used were transformed by treating the 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.
Genet. 18 (1990) 7] using ethylene glycol and dimethyl sulphoxide was also used.
It is also possible to transform the yeasts by electroporation, according to the method described by Karube et al. [FEBS Letters 182 (1985) 90]. An alternative procedure is also described in detail in the examples below.
After selection of the transformed cells, the cells expressing the said polypeptides are inoculated and the recovery of the said polypeptides can be carried out, either during the cell growth for the "continuous" processes, or at the end of growth for the "batch" cultures. The polypeptides which are the subject of the present invention are then purified from the culture supernatant for their molecular, pharmacokinetic and biological characterization.
A preferred expression system for the polypeptides of the invention consists in using yeasts of the genus Kluyveromyces as host cell, transformed by certain vectors derived from the extrachromosomal replicon pKD 1 originally isolated from K. marxianus var. drosophilarum. These yeasts, and in particular K.
lactis and K. fragilis are generally capable of stably replicating the said vectors and possess, in addition, the advantage of being included in the list of G.R.A.S.
("Generally Recognized As Safe") organisms. Favoured yeasts are preferably industrial yeasts of the genus Kluyveromyces which are capable of stably replicating the said plasmids derived from the plasmid pKDI and in which has been inserted a selectable marker as well as an expression cassette permitting the secretion, at high levels, of the polypeptides of the invention.
The present invention also relates to the nucleotide sequences encoding the chimeric polypeptides described above, as well as the eukaryotic or prokaryotic recombinant cells comprising such sequences.
The present invention also relates to the application, as medicinal products, of the polypeptides according to the present invention. More particularly, the subject of the invention is any pharmaceutical composition comprising one or more polypeptides or nucleotide sequences as described above. The nucleotide sequences can indeed be used in gene therapy.
The present invention will be more fully described with the aid of the following examples, which should be considered as illustrative and non-limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
The representations of the plasmids indicated in the following figures are not plotted to scale and only the restriction sites important for the understanding of the clonings carried out have been indicated.
Figure 1: Schematic representation of the chimera of the HSA-PEPTIDE type (A), a chimera of the PEPTIDE-HSA type (B) and a chimera of the PEPTIDE-HSA-PEPTIDE type (C). Abbreviations used: M/LP, translational initiator methionine residue, optionally followed by a signal sequence for secretion;
HSA, mature albumin or one of its molecular variants; PEP, peptide of natural or artificial origin possessing a given therapeutic property. The PEP sequence may be present several times in the molecules of type A, B and C. The black arrow indicates the N-terminal end of the mature protein.
Figure 2: Examples of the nucleotide sequence of a HindIll restriction fragment encoding a chimeric protein of the prepro-HSA-PEPTIDE type. The black arrows indicate the end of the "pre" and "pro" regions of HSA. The MstII
restriction site is underlined and the codon specifying the termination of translation is in bold characters.
Figure 3: Restriction map for the plasmid pYG105 and generic strategy for construction of the plasmids for expression of the chimeric proteins of the present invention. Abbreviations used: P, transcriptional promoter; T, transcriptional terminator; IR, inverted repeat sequences of the plasmid pKDI;
LP, signal sequence for secretion; Apr and Kmr designate the genes for resistance to ampicillin (E. coli) and to G418 (yeasts), respectively.
Figure 4: Examples of nucleotide sequences of MstII-HindIII
restriction fragments derived from the von Willebrand factor. Representation of the structure of the Mstll-Hindlll fragment of the plasmid pYG1248 (panel A).
Representation of the structure of the Mstll-Hindlll fragment of the plasmid pYG1214 (panel B). Representation of the Mstll-Hindlll fragment of the plasmid pYG1206 (panel C); in this particular chimera, the Leu694 residue of the vWF
is also the last residue (Leu585) of the HSA. Representation of the Mstll-Hindlll fragment of the plasmid pYG1223 (panel D). The numbering of the amino acids corresponds to the numbering of the mature vWF according to Titani et al.
[Biochemistry 25 (1986) 3171-3184]. The Mstll and HindIII restriction sites are underlined and the translation termination codon is in bold characters. FIG.
4E is a nucleotide sequence (SEQ ID NO:3) of the Mstll-Hindlll restriction fragment of the plasmid pYG1248. The numbering of the amino acids (right-hand column) corresponds to the mature chimeric protein HSA-vWF470-*713 (829 residues).
The Thr470, Leu494, Asp498, Pro502, Tyr508, Leu694, Pro704 and Pro708 residues of the mature vWF are underlined.
Figure 5: The characterization of the material secreted after 4 days of culture (Erlenmeyers) of the strain CBS 293.91 transformed with the plasmids pYG1248 (plasmid for expression of a chimera of the HSA-vWF Thr470->Va1713) and pKan707 (control plasmid). In this experiment, the polypeptides for panels A, B and C were run on the same gel (8.5% SDS-PAGE) and then treated separately.
A: the results of Coomassie blue staining of a molecular weight standard (lane 2); of a supernatant equivalent to 50 l of the culture transformed with the plasmid pKan707 in YPL medium (lane 1); the plasmid pYG 1248 in YPD medium (lane 3) and the plasmid pYG 1248 in YPL medium (lane 4).
B: the results of immunological characterization of the secreted material after using mouse antibodies directed against human vWF. The lanes are the same as described for FIG. 5A except that biotinylated molecular weight standards were used (lane 2).
C: the results of immunological characterization of the secreted material after using rabbit antibodies directed against human albumin: supernatant equivalent to 50 l of the culture transformed with the plasmid pKan707 in YPL
medium (lane 1), the plasmid pYG1248 in YPD medium (lane 2) the plasmid pYG1248 in YPL medium (lane 3).
Figure 6: The kinetic analysis of secretion of a chimera of the invention by the strain CBS 293.91 transformed with the plasmid pYG1206 (HSA-vWF Leu694-Pro708).
A: Coomassie blue staining was employed. Lane 1 is the molecular weight standard, lane 2 is the supernatant equivalent to 2.5 l of a "Fed Batch"
culture in YPD medium after 24 hours of growth; lane 3 is the supernatant of the same culture after 40 hours; and lane 4 is the supernatant of the same culture after 46 hours of growth.
B: immunological characterization of the secreted material after using mouse antibodies directed against the human vWF. The lanes are the same as in A
except that biotinylated molecular weight standards were used.
Figure 7: Characterization of the material secreted by K. lactis transformed with the plasmids pKan707 (control plasmid, lane 2), pYG 1206 (lane 3), pYG1214 (lane 4) and pYG1223 (lane 5); molecular weight standard (lane 1).
The deposits correspond to 50 l of supernatant from a stationary culture after growing in YPD medium, running on an 8.5% acrylamide gel and staining with Coomassie blue.
Figure 8: Nucleotide sequence of the MstI1-Hindlll restriction fragment of the plasmid pYG1341 (HSA-UK1--),135). The limit of the EGF-like domain (UK1-46) present in the MstII-Hindlll restriction fragment of the plasmid pYG1340 is indicated. The numbering of the amino acids corresponds to the mature chimeric protein SAU-UK1- 135 (720 residues).
Figure 9: Secretion of the HSA-UK1-46 and HSA-UK1-135 chimeras by the strain CBS 293.91 respectively transformed with the plasmids pYG1343 (HSA-UKI-46) and pYG1345 (HSA-UK1-135), after 4 days of growth (YPL+G418 medium). The deposits (equivalent to 50 l of culture) are run on an 8.5% PAGE-SDS gel and stained with Coomassie blue: supernatant from a clone transformed with the plasmids pKan707 (lane 1), pYG1343 (lane 3) or pYG1345 (lane 4); molecular weight standard (lane 2).
Figure 10: Nucleotide sequence of the MstII-Hindlll restriction fragment of the plasmid pYG1259 (HSA-G.CSF). The limit of the G-CSF part (174 residues) is indicated. The Apal and Sstl (Sstl) restriction sites are underlined. The numbering of the amino acids corresponds to the mature chimeric protein HSA-G.CSF (759 residues).
Figure 11: The nucleotide sequence of the HindIII restriction fragment of the plasmid pYG1301 (chimera G.CSF-Gly4 -HSA). The black arrows indicate the end of the "pre" and "pro" regions of HSA. The Apal, Sstl (SacI) and MstIl restriction sites are underlined. The G.CSF (174 residues) and HSA (585 residues) domains are separated by the synthetic linker GGGG. The numbering of the amino acids corresponds to the mature chimeric protein G.CSF-Gly4-SAH (763 residues).
The nucleotide sequence between the translation termination codon and the HindIIl site comes from the HSA complementary DNA (cDNA) as described in Patent Application EP 361 991.
Figure 12: The characterization of the material secreted after 4 days of culture (erlenmeyers) of the strain CBS 293.91 transformed with the plasmids pYG1266 (plasmid for expression of a chimera of the HSA-G.CSF type) and pKan707 (control plasmid). In this experiment, the polypeptides for panels A, B
and C were run on the same gel (8.5% SDS-PAGE) and then treated separately.
A: coomassie blue staining of a molecular weight standard (lane 2);
supernatant equivalent to 100 l of culture transformed with the plasmid pKan707 in YPL medium (lane 1); the plasmid pYG1266 in YPD medium (lane 3) and the plasmid pYG 1266 in YPL medium (lane 4).
B: immunological characterization of the material secreted after using primary antibodies directed against human G-CSF. The lanes are as described above for A.
C: immunological characterization of the material secreted after using primary antibodies directed against human albumin. The lanes are as described above for A.
Figure 13: Characterization of the material secreted after 4 days of culture (erlenmeyers in YPD medium) of the strain CBS 293.91 transformed with the plasmids pYG1267 (chimera HSA-G.CSF), pYG1303 (chimera G.CSF-Gly4-HSA) and pYG1352 (chimera HSA-G1y4-G.CSF) after running on an 8.5% SDS-PAGE gel.
A: coomassie blue staining of a supernatant equivalent to 100 l of the culture transformed with the plasmid pYG1303 (lane 1), the plasmid pYG1267 (lane 2), and the plasmid pYG1352 (lane 3). Lane 4 is the molecular weight standard.
B: immunological characterization of the material secreted after using primary antibodies directed against the human G-CSF: same legend as in A.
Figure 14: Nucleotide sequence of the MstII-HindIIl restriction fragment of the plasmid pYG1382 (HSA-Fv'). The VH (124 residues) and VL (107 residues) domains of the Fv' fragment are separated by the synthetic linker (GGGGS)x3. The numbering of the amino acids corresponds to the mature chimeric protein HSA-Fv' (831 residues).
Figure 15: Characterization of the secretion of the chimera HSA-Fv' by the strain CBS 293.91 transformed with the plasmid pYG1383 (LAC4) after 4 days of growth in erlenmeyers at 28 C. in YPD medium (lane 2), and in YPL medium (lane 3). Lane 1 shows the molecular weight standard. The deposits, equivalent to 200 1 of culture (precipitation with ethanol), are run on a PAGE-SDS gel (8.5%).
A: coomassie blue staining of the gel.
B: immunological characterization of the material secreted after using primary antibodies directed against HSA.
Figure 16: Assay of the in vitro antagonistic activity of the agglutination of human platelets fixed with formaldehyde: IC50 of the hybrids HSA-vWF694-708, [HSA-vWF470-713 C471G, C474G] and [HSA-vWF470-704 C471G, C474G] compared with the standard RG12986. The determination of the dose-dependent inhibition of the platelet agglutination is carried out according to the method described by C. Prior et al. [Bio/Technology (1992) 10 66] using an aggregameter recording the variations in optical transmission, with stirring, at 37 C. in the presence of human vWF, botrocetin (8.2 mg/ml) of the test product at various dilutions. The concentration of the product which makes it possible to inhibit the control agglutination (in the absence of product) by half is then determined (IC50).
Figure 17: Activity on the in vitro cellular proliferation of the murine line NFS60. The radioactivity (3H-thymidine) incorporated into the cellular nuclei after 6 hours of incubation is represented on the y-axis (cpm); the quantity of product indicated on the x-axis is expressed in molarity (arbitrary units).
Figure 18: Activity on granulopoiesis in vivo in rats. The number of neutrophils (average for 7 animals) is indicated on the y-axis as a function of time.
The products tested are the chimera HSA-G.CSF (pYG1266), 4 or 40 mg/rat/day), the reference G-CSF (10 mg/rat/day), the recombinant HSA purified from Kluyveromyces lactis supernatant (HSA, 30 mg/rat/day, cf. EP 361 991), or physiological saline.
EXAMPLES
GENERAL CLONING TECHNIQUES
The methods conventionally used in molecular biology, such as the preparative extractions of plasmid DNA, the centrifugation of plasmid DNA in caesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, extractions of proteins with phenol or phenol-chloroform, DNA precipitation in saline medium with ethanol or isopropanol, transformation in Escherichia coli, and the like are well known to persons skilled in the art and are widely described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F. M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987].
The restriction enzymes were provided by New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Amersham and are used according to the recommendations of the suppliers.
The pBR322 and pUC type plasmids and the phages of the M13 series are of commercial origin (Bethesda Research Laboratories).
For the ligations, the DNA fragments are separated according to their size by electrophoresis on agarose or acrylamide gels, extracted with phenol or with a phenol/chloroform mixture, precipitated with ethanol and then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the recommendations of the manufacturer.
The filling of the protruding 5' ends is carried out by the Klenow fragment of DNA polymerase I of E. coli (Biolabs) according to the specifications of the supplier. The destruction of the protruding 3' ends is carried out in the presence of phage T4 DNA polymerase (Biolabs) used according to the recommendations of the manufacturer. The destruction of the protruding 5' ends is carried out by a controlled treatment with S 1 nuclease.
Site-directed mutagenesis in vitro with 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.
The enzymatic amplification of DNA fragments by the so-called PCR technique Polymerase-catalyzed Chain Reaction, [Saiki R. K. et al., Science 230 (1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth. Enzym. 155 (1987) 335-350] is carried out using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the specifications of the manufacturer.
The verification of the nucleotide sequences is carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 5467] using the kit distributed by Amersham.
The transformations of K. lactis with DNA from the plasmids for expression of the proteins of the present invention are carried out by any technique known to persons skilled in the art, and of which an example is given in the text.
Except where otherwise stated, the bacterial strains used are E. coli MC1060 (lacIPOZYA, X74, galU, galK, strA`), or E. coli TG1 (lac, proA,B, supE, thi, hsdD5/FtraD36, proA+ B+, IacI9, lacZ, M15).
The yeast strains used belong to the budding yeasts and more particularly to yeasts of the genus Kluyveromyces. The K. lactis MW98-8C (a, uraA, arg, lys, K, pKD1 ) and K. lactis CBS 293.91 strain were particularly used;
a sample of the MW98-8C strain was deposited on 16 Sep. 1988 at Centraalbureau voor Schimmelkulturen (CBS) at Baarn (the Netherlands) where it was registered under the number CBS 579.88.
A bacterial strain (E. coli) transformed with the plasmid pET-8c52K
was deposited on 17 Apr. 1990 with the American Type Culture Collection under the number ATCC 68306.
The yeast strains transformed with the expression plasmids encoding the proteins of the present invention are cultured in erlenmeyers or in 21 pilot fermenters (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.
EXAMPLE 1: COUPLING AT THE C-TERMINUS OF HSA
The plasmid pYG404 is described in Patent Application EP 361 991.
This plasmid contains a HindIII restriction fragment encoding the prepro-HSA
gene preceded by the 21 nucleotides naturally present immediately upstream of the initiator ATG for translation of the PGK gene of S. cerevisiae. The nucleotide sequence of this restriction fragment is included in that of FIG. 2. The Mstll site localized in the coding sequence, three residues from the codon specifying the end of translation is particularly useful as site for cloning a biologically active peptide which it is desired to couple in translational phase at the C-terminus of HSA.
In a specific embodiment, it is useful to use peptides whose sequence is encoded by an MstIl-Hindlll restriction fragment of the type: 5'-CCTTAGGCTTA [3xN]P
TAAGCTT-3', the sequence encoding the biologically active peptide (p residues) is [3xN]P). The ligation of this fragment to the HindIll-MstII restriction fragment corresponding to the entire gene encoding HSA, with the exception of the three C-terminal-most amino acids (leucine-glycine-leucine residues) generates a HindIII
restriction fragment containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro"
export region of HSA. In another embodiment, the biologically active peptide may be present more than once in the chimera.
EXAMPLE 2: COUPLING AT THE N-TERMINUS OF HSA
In a specific embodiment, the combined techniques of site-directed mutagenesis and PCR amplification make it possible to construct hybrid genes encoding a chimeric protein resulting from the translational coupling between a signal peptide (and for example the prepro region of HSA), a sequence including the biologically active peptide and the mature form of HSA or one of its molecular variants. These hybrid genes are preferably bordered in 5' of the translational initiator ATG and in 3' of the translational stop codon by HindIII restriction sites and encode chimeric proteins of the PEPTIDE-HSA type (FIG. 1, panel B). In a still more specific embodiment, the biologically active peptide may be present more than once in the chimera.
EXAMPLE 3: COUPLING AT THE N- AND C-TERMINUS OF HSA
The combined techniques of site-directed mutagenesis and PCR
amplification described in Examples 1 and 2 make it possible to construct hybrid genes encoding a chimeric protein resulting from the translational coupling between the mature form of HSA, or one of its molecular variants, and a biologically active peptide coupled to the N- and C-terminal ends of HSA.
These hybrid genes are preferably bordered in 5' of the translational initiator ATG
and in 3' of the translational stop codon by Hindlll restriction sites and encode chimeric proteins of the PEPTIDE-HSA-PEPTIDE type (FIG. 1, panel C), immediately preceded by the "prepro" export region of HSA. In a still more specific embodiment, the biologically active peptide may be present more than once in the chimera.
EXAMPLE 4: EXPRESSION PLASMIDS
The chimeric proteins of the preceding examples can be expressed in yeasts using functional, regulatable or constitutive promoters such as, for example, those present in the plasmids pYG105 (LAC4 promoter of Kluyveromyces lactis), pYG106 (PGK promoter of Saccharomyces cerevisiae), pYG536 (PHO5 promoter of S. cerevisiae), or hybrid promoters such as those described in Patent Application EP 361 991. The plasmids pYG105 and pYG106 are particularly useful here because they permit the expression of the genes encoded by the HindIII
restriction fragments as described in the preceding examples and cloned into the HindIII site and in the productive orientation (defined as the orientation which places the "prepro" region of albumin proximally relative to the promoter for transcription), using promoters which are functional in K. lactis, regulatable (pYG105) or constitutive (pYG106). The plasmid pYG105 corresponds to the plasmid pKan707 described in Patent Application EP 361 991 in which the HindIII
restriction site which is unique and localized in the gene for resistance to geneticin (G418) has been destroyed by site-directed mutagenesis while preserving an unchanged protein (oligodeoxynucleotide 5'-GAAA-TGCATAAGCTCTTGCCATTCTCACCG-3'). The Sall-Sacl fragment encoding the URA3 gene of the mutated plasmid was then replaced with a Sall-SacI
restriction fragment containing an expression cassette consisting of the LAC4 promoter of K. lactis (in the form of a SalI-HindIII fragment) and the terminator of the PGK gene of S. cerevisiae (in the form of a HindIII-Sac1 fragment). The plasmid pYG105 is mitotically very stable in the Kluyveromyces yeasts and a restriction map thereof is given in FIG. 3. The plasmids pYG105 and pYG106 differ from each other only in the nature of the promoter for transcription encoded by the SalI-HindI1I fragment.
EXAMPLE 5: TRANSFORMATION OF THE YEASTS
The transformation of the yeasts belonging to the genus Kluyveromyces, and in particular the strains MW98-8C and CBS 293.91 of K.
lactis is carried out for example by the technique for treating whole cells with lithium acetate Ito H. et al., [J. Bacteriol. 153 (1983) 163-168], adapted as follows.
The growth of the cells is carried out at 28 C. in 50 ml of YPD medium, with stirring and up to an optical density of 600 nm (OD600) of between 0.6 and 0.8; the cells are harvested by centrifugation at low speed, washed in a sterile solution of TE
(10 mM Tris HCl pH 7.4; 1 mM EDTA), resuspended in 3-4 ml of lithium acetate (0.1M in TE) in order to obtain a cellular density of about 2 x 108 cells/ml, and then incubated at 30 C. for 1 hour with moderate stirring. Aliquots of 0.1 ml 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 heat shock of 5 minutes at 42 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 in order to permit the phenotypic expression of the gene for resistance to G418 expressed under the control of the Pkl promoter (cf. EP 361 991); 200 l of the cellular suspension are then plated 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.
EXAMPLE 6:SECRETION OF THE CHIMERAS
After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the mature form of the chimeric proteins. Few clones, corresponding to the strain CBS 293.91 or MW98-8C transformed by the plasmids for expression of the chimeras between HSA and the biologically active part, are incubated in YPD or YPL medium at 28 C. The cellular supernatants are recovered by centrifugation when the cells reach the stationary growth phase, optionally concentrated 10 times by precipitation for minutes at -20 C. in a final concentration of 60% ethanol, and then tested after electrophoresis on an 8.5% SDS-PAGE gel, either directly by staining the gel with coomassie blue, or after immunoblotting using primary antibodies directed against the biologically active part or a rabbit polyclonal serum directed against HSA.
During the experiments for immunological detection, the nitrocellulose filter is first incubated in the presence of specific primary antibodies, washed several times, incubated in the presence of goat antibodies directed against the primary antibodies, and then incubated in the presence of an avidin-peroxidase complex using the "ABC kit" distributed by Vectastain (Biosys S. A., Compiegne, France). The immunological reaction is then revealed by the addition of 3,3'-diamino benzidine tetrahydrochloride (Prolabo) in the presence of hydrogen peroxide, according to the recommendations of the manufacturer.
EXAMPLE 7: CHIMERAS DERIVED FROM THE VON WILLEBRAND
FACTOR
E.7.1. Fragments Antagonizing the Binding of vWF to the Platelets E.7.1.1. Thr470-Va1713 Residues of vWF
The plasmid pET-8c52K contains a fragment of the vWF cDNA encoding residues 445 to 733 of human vWF and therefore includes several crucial determinants of the interaction between vWF and the platelets on the one hand, and certain elements of the basal membrane and the sub-endothelial tissue on the other, and especially the peptides G10 and D5 which antagonize the interaction between vWF and GPIb 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]. The amplification of these genetic determinants can be carried out using the plasmid pET-8c52K, for example by the PCR amplification technique, using as primer oligodeoxynucleotides encoding contiguous residues localized on either side of the sequence to be amplified.
The amplified fragments are then cloned into vectors of the M 13 type for their verification by sequencing using either the universal primers situated on either side of the multiple cloning site, or oligodeoxynucleotides specific for the amplified region of the vWF gene of which the sequence of several isomorphs is known Sadler J. E. et al., [Proc. Natl. Acad. Sci. 82 (1985) 6394-6398]; Verweij C.
L. et al., [EMBO J. 5 (1986) 1839-1847]; Shelton-Inloes B. B. et al., [Biochemistry (1986) 3164-3171]; Bonthron D. et al., [Nucleic Acids Res. 17 (1986) 7125-7127].
Thus, the PCR amplification of the plasmid pET-8c52K with the oligodeoxynucleotides 5'-CCCGGGATCCCTTAGGCTTAACCTGTGAAGCCTG
C-3' (Sq1969, the MstII site is underlined) and 5'-CCCGGGATCCAAGCTTA-GACTTGTGCCATGTCG-3' (Sq2029, the Hindlll site is underlined) generates an MstII-HindI1l restriction fragment including the Thr470 to Va1713 residues of vWF
(FIG. 4, panel E). The ligation of this fragment to the HindI1I-MstII
restriction fragment corresponding to the entire gene encoding HSA, with the exception of the three C-terminal most amino acids (cf. FIG. 2) generates a Hindlll restriction fragment containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro" export region of HSA. This restriction fragment is cloned in the productive orientation and into the Hindlll site of the plasmid pYG105, which generates the expression plasmid pYG 1248 (HSA-vWF470-713).
E.7.1.2. Molecular Variants:
In another embodiment, the binding site of vWF is a peptide including the Thr470 to Asp498 residues of the mature vWF. This sequence including 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 the GPIb of the human platelets. The sequence corresponding to the peptide G10 is first included in an MstII-Hindlll restriction fragment (FIG. 4, panel B), for example by PCR amplification of the plasmid pET-8c52K with the oligodeoxynucleotides Sq1969 and 5'-CCCGGGATCCAAGCTTAGTCCTCCACATACAG-3' (Sq1970, the HindIII site is underlined), which generates an MstII-HindI11 restriction fragment including the peptide G 10, and whose sequence is: 5'-CCTTAGGCTTAACCTGTGAAGCCTGCCAGGAGCCGGGAGGCCTGGT-GGTGCCTCCCACAGATGCCCCGGTGAGCCCCACCACTCTGTA-TGTGGAGGACTAAGCTT-3' (the sequence encoding the peptide G10 is in bold characters). The ligation of this fragment to the Hindlll-Mstll restriction fragment corresponding to the entire gene encoding HSA, with the exception of the three C-terminal most amino acids (cf. FIG. 2) generates a HindIII restriction fragment containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro" export region of HSA.
This restriction fragment is cloned in the productive orientation into the HindIIl site of the plasmid pYG105, which generates the expression plasmid pYG1214.
In another embodiment, the site for binding of vWF to GPlb is directly designed with the aid of synthetic oligodeoxynucleotides, and for example the oligodeoxynucleotides 5'-TTAGGCCTCTGTGACCTTGCCCCTGA-AG-CCCCTCCTCCTACTCTGCCCCCCTAAGCTTA-3' (SEQ ID NO:26) and 5'-GATCTAAG-CTTAGGGGGGCAGAGTAGGAGGAGGGGCTTCAGGG-GCAAGGTCACAGAGGCC-3' (SEQ ID NO:27). These oligodeoxynucleotides form, by pairing, a Mstll-Bg1Il restriction fragment including the MstII-Hindlll fragment (FIG. 4, panel C) corresponding to the peptide D5 defined by the Leu694 to Pro708 residues of vWF. The ligation of the MstII-HindIII fragment to the HindI1l-MstII restriction fragment corresponding to the entire gene encoding HSA
with the exception of the three C-terminal most amino acids (cf. FIG. 2) generates a Hindlll restriction fragment containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro" export region of HSA. This restriction fragment is cloned in the productive orientation into the Hindlll site of the plasmid pYG 105, which generates the expression plasmid pYG1206.
Useful variants of the plasmid pET-8c52K are deleted by site-directed mutagenesis between the peptides GIO and G5, for example sites for binding to collagen, and/or to heparin, and/or to botrocetin, and/or to sulphatides and/or to ristocetin. One example is the plasmid pMMB9 deleted by site-directed mutagenesis between the residues Cys509 and I1e662. The PCR amplification of this plasmid with the oligodeoxynucleotides Sq1969 and Sq2029 generates an Mstll-HindIIl restriction fragment (FIG. 4, panel D) including the Thr470 to Tyr508 and Arg663 to Va1713 residues and in particular the peptides G10 and D5 of vWF and deleted in particular of its site for binding to collagen localized between the residues G1u542 and Met622 Roth G. J. et al., [Biochemistry 25 (1986) 8357-8361]. The ligation of this fragment to the HindIII-Mst11 restriction fragment corresponding to the entire gene encoding HSA, with the exception of the three C-terminal most amino acids (cf. FIG. 2) generates a Hindlll restriction fragment containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro" export region of HSA.
This restriction fragment is cloned in the productive orientation into the Hindlll site of the plasmid pYG105, which generates the expression plasmid pYG1223.
In other embodiments, the use of combined techniques of site-directed mutagenesis and PCR amplification makes it possible to generate at will variants of the MstII-HindIIl restriction fragment of panel A of FIG. 4 but deleted of one or more sites for binding to sulphatides and/or to botrocetin and/or to heparin and/or to collagen, and/or substituted by any residue involved in the vWF-associated emergence of IIB type pathologies.
In other useful variants of the plasmid pET-8c52K, mutations are introduced, for example by site-directed mutagenesis, in order to replace or suppress all or part of the set of cysteines present at positions 471, 474, 509 and 695 of the human vWF. Specific examples are the plasmids p5E and p7E in which the cysteins present at positions 471 and 474, on the one hand, and at positions 471, 474, 509 and 695, on the other hand, have been respectively replaced by glycine residues. The PCR amplification of these plasmids with the oligodeoxynucleotides Sq2149 (5'-CCCGGGATCCCTTAGGCTTAACCGGTGAAGCCGGC-3' (SEQ ID
NO:28), the MstII site is underlined) and Sq2029 makes it possible to generate MstII-HindIlI restriction fragments including the Thr470 to Va1713 residues of the natural vWF with the exception that at least the cystein residues at positions and 474 were mutated to glycine residues. The ligation of these fragments to the HindIII-MstII restriction fragment corresponding to the entire gene encoding HSA
with the exception of the three C-terminal most amino acids (cf. FIG. 2) generates a HindIIl restriction fragment containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro" export region of HSA. These restriction fragments are cloned in the productive orientation into the HindIII site of the plasmid pYG105, which generates the expression plasmids pYG1283 (chimera HSA-vWF470-713, C471G, C474G) and pYG1279 (chimera HSA-vWF470-713, C471G, C474G, C509G, C695G).
Other particularly useful mutations affect at least one residue involved in vWF-associated type IIB pathologies (increase in the intrinsic affinity of vWF
for GP1b), such as the residues Arg543, Arg545, Trp550, Va1551, Va1553, Pro574 or Arg578 for example. The genetic recombination techniques in vitro also make it possible to introduce at will one or more additional residues into the sequence of vWF and for example a supernumerary methionine between positions Asp539 and G1u542.
E.7.2. Fragments Antagonizing the Binding of vWF to the Sub-Endothelium In a specific embodiment, the sites for binding of vWF to the components of the sub-endothelial tissue, and for example collagen, are generated by PCR
amplification of the plasmid pET-8c52K, for example with the oligodeoxynucleotides Sq2258 (5'-GGATCCTTAGGGCT-GTGCAGCAGGCTACTGGACCTGGTC-3', the Mstll site is underlined) and Sq2259 (5'-GAATTCAAGCTTAACAGAGGTAGCTAA-CGATCTCGTCCC-3', the Hindlll site is underlined), which generates an MstII-HindIIl restriction fragment encoding the Cys509 to Cys695 residues of the natural vWF. Deletion molecular variants or modified variants are also generated which contain any desired combination between the sites for binding of vWF to the sulphatides and/or to botrocetin and/or to heparin and/or to collagen and/or any residue responsible for a modification of the affinity of vWF for GPIb (vWF-associated type II
pathologies). In another embodiment, the domain capable of binding to collagen may also come from the vWF fragment which is between the residues 911 and 1114 and described by Pareti et al. [J. Biol. Chem. (1987) 262: 13835-13841].
The ligation of these fragments to the HindIIl-MstII restriction fragment corresponding to the entire gene encoding HSA with the exception of the three C-terminal most amino acids (cf. FIG. 2) generates Hindlll restriction fragments containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro" export region of HSA. These restriction fragments are cloned in the productive orientation into the Hindlll site of the plasmid pYG105, which generates the corresponding expression plasmids, and for example the plasmid pYG1277 (HSA-vWF509-695).
E.7.3. Purification and Molecular Characterization of the Chimeras Between HSA and vWF
The chimeras present in the culture supernatants corresponding to the CBS
293.91 strain transformed, for example with the expression plasmids according to Examples E.7.1. and E.7.2., are characterized in a first instance by means of antibodies specific for the HSA part and for the vWF part. The results of FIGS. 5 to 7 demonstrate that the yeast K. lactis is capable of secreting chimeric proteins between HSA 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) and then concentrated by ultrafiltration (Amicon) using a membrane whose discrimination threshold is situated at 30 kDa. The concentrate obtained is then dialysed against a Tris-HCl solution (50 mM pH 8) and then purified on a column. For example, 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). A purification by ion-exchange chromatography can also be used. For example, in the case of the chimera HSA-vWF470-713, the concentrate obtained after ultrafiltration is dialysed against a Tris-HC1 solution (50 mM pH 8), and then loaded in 20 ml fractions onto a cation-exchange column (5 ml) (S Fast Flow, Pharmacia) equilibrated in the same buffer. The column is then washed several times with the Tris-HC1 solution (50 mM pH 8) and the chimeric protein is then eluted from the column by an NaC1 gradient (0 to 1M). The fractions containing the chimeric protein are then pooled and dialysed against a 50 mM Tris-HCI solution (pH 8) and then reloaded onto the S Fast Flow column. After elution of the column, the fractions containing the protein are pooled, dialysed against water and freeze-dried before characterization:
for example, sequencing (Applied Biosystem) of the protein [HSA-vWF470-704 C471G, C474G] secreted by the yeast CBS 293.91 gives the N-terminal sequence expected for HSA (Asp-Ala-His ...), demonstrating a correct maturation of the chimera immediately at the C-terminus of the doublet of residues Arg-Arg of the "pro" region of HSA (FIG. 2). The essentially monomeric character of the chimeric proteins between HSA and vWF is also confirmed by their elution profile on a TSK
3000 column [Toyo Soda Company, equilibrated with a cacodylate solution (pH 7) containing 0.2M Na2SO4]: for example the chimera [HSA-vWF 470-704 C471G, C474G] behaves under the conditions like a protein with an apparent molecular weight of 95 kDa, demonstrating its monomeric character.
EXAMPLE 8: CHIMERAS DERIVED FROM UROKINASE
E.8.1. Constructs A fragment corresponding to the amino-terminal fragment of urokinase (ATF: EGF-like domain + kringle domain) can be obtained from the corresponding messenger RNA of cells of certain human carcinoma, for example using the RT-PCR kit distributed by Pharmacia. An MstI1-HindIII restriction fragment including the ATF of human urokinase is given in FIG. 8. The ligation of the HindI11-MstII
fragment of the plasmid pYG404 to this MstII-HindIII fragment makes it possible to generate the Hindlll fragment of the plasmid pYG1341 which encodes a chimeric protein in which the HSA molecule is genetically coupled to the ATF
(HSA-UK1-*135). Likewise, the plasmid pYG1340 contains a Hindlll fragment encoding a chimera composed of HSA immediately followed by the first 46 residues of human urokinase (HSA-UK1-46, cf. FIG. 8). The cloning in the productive orientation, of the Hindlll restriction fragment of the plasmid pYG
(HSA-UK1-46) into the Hindlll site of the plasmids pYG105 (LAC4) and pYG106 (PGK) generates the expression plasmids pYG1343 and pYG1342 respectively. Likewise, the cloning, in the productive orientation, of the Hindlll restriction fragment of the plasmid pYG1341 (HSA-UK1--->135) into the Hindlll site of the plasmids pYG105 (LAC4) and pYG106 (PGK) generates the expression plasmids pYG1345 and pYG1344 respectively.
E.8.2. Secretion of the Hybrids After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the mature form of the chimeric proteins HSA-UK. A few clones corresponding to the strain K. lactis CBS
293.91, which is transformed with the expression plasmids according to Example E.9.1., are incubated in selective complete liquid medium at 28 C. The cellular supernatants are then tested after electrophoresis on an 8.5% acrylamide gel, either directly by staining of the gel with coomassie blue, or after immunoblotting using as primary antibodies a rabbit polyclonal serum directed against human albumin or against human urokinase. The results of FIG. 9 demonstrate that the hybrid proteins HSA-UK1--46 and HSA-UK1-*135 are particularly well secreted by the yeast Kluyveromyces.
E.8.3 Purification of the Chimeras Between HSA and Urokinase After centrifugation of a culture of the CBS 293.91 strain transformed with the expression plasmids according to Example E.8.1., the culture supernatant is passed through a 0.22 mm filter (Millipore) and then concentrated by ultrafiltration (Amicon) using a membrane whose discrimination threshold is situated at 30 kDa.
The concentrate obtained is then adjusted to 50 mM Tris-HCl starting with a stock solution of 1M Tris-HCi (pH 7), and then loaded in 20 ml fractions onto an anion-exchange column (3 ml) (D-Zephyr, Sepracor) equilibrated in the same buffer.
The chimeric protein (HSA-UK1--+46 or HSA-UK1-135) is then eluted from the column by a gradient (0 to 1M) of NaCI. The fractions containing the chimeric protein are then pooled and dialysed against a 50 mM Tris-HC1 solution (pH 6) and reloaded onto a D-Zephyr column equilibrated in the same buffer. After elution of the column, the fractions containing the protein are pooled, dialysed against water and freeze-dried before characterization of their biological activity and especially with respect to their ability to displace urokinase from its cellular receptor.
EXAMPLE 9: CHIMERAS DERIVED FROM G-CSF
E.9.1. Constructs E.9.1.1. Coupling at the C-terminus of HSA.
An MstII-HindIII restriction fragment including the mature form of human G-CSF is generated, for example according to the following strategy: a Kpnl-HindIII restriction fragment is first obtained by the enzymatic PCR
amplification technique using the oligodeoxynucleotides Sq2291 (5'-CAAGGATCCAAGCTTCAGGGCTGCGCAAGGTGGCGTAG-3', the HindIII
site is underlined) and Sq2292 (5'-CGGGGTACCTTAGGCTTAACCCCCCTG-GGCCCTGCCAGC-3', the KpnI site is underlined) as primer on the plasmid BBG13 serving as template. The plasmid BBG13 contains the gene encoding the B
form (174 amino acids) of mature human G-CSF, which is obtained from British Bio-technology Limited, Oxford, England. The enzymatic amplification product of about 550 nucleotides is then digested with the restriction enzymes KpnI and HindIII and cloned into the vector pUC19 cut with the same enzymes, which generates the recombinant plasmid pYG1255. This plasmid is the source of an MstII-HindlII restriction fragment which makes it possible to fuse G-CSF
immediately downstream of HSA (chimera HSA-G.CSF) and whose nucleotide sequence is given in FIG. 10.
It may also be desirable to insert a peptide linker between the HSA
part and G-CSF, for example in order to permit a better functional presentation of the transducing part. An MstII-HindIII restriction fragment is for example generated by substitution of the MstII-Apal fragment of the plasmid pYG1255 by the oligodeoxynucleotides Sq2742 (5'-TTAGGCTTA-GGTGGTGGCGGTACCCCCCTGGGCC-3', the codons encoding the glycine residues of this particular linker are underlined) and Sq2741 (5'-CAGGGGGGTACCGCCACCACCTAAGCC-3') which form, by pairing, an MstII-Apa1 fragment. The plasmid thus generated therefore contains an MstH-HindIII restriction fragment whose sequence is identical to that of FIG. 10 with the exception of the MstII-Apal fragment.
The ligation of the HindIIl-MstII fragment of the plasmid pYG404 to the MstII-HindIII fragment of the plasmid pYG1255 makes it possible to generate the Hindlll fragment of the plasmid pYG1259 which encodes a chimeric protein in which the B form of the mature G-CSF is positioned by genetic coupling in translational phase at the C-terminus of the HSA molecule (HSA-G.CSF).
An identical Hindlll restriction fragment, with the exception of the MstII-Apal fragment, may also be easily generated and which encodes a chimeric protein in which the B form of the mature G-CSF is positioned by genetic coupling in translational phase at the C-terminus of the HSA molecule and a specific peptide linker. For example, this linker consists of 4 glycine residues in the Hindlll fragment of the plasmid pYG1336 (chimera HSA-Gly4-G.CSF).
The Hindlll restriction fragment of the plasmid pYG1259 is cloned in the productive orientation and into the Hindlll restriction site of the expression plasmid pYG105, which generates the expression plasmid pYG1266 (HSA-G.CSF). In another exemplification, the cloning of the HindIII restriction fragment of the plasmid pYG1259 in the productive orientation and into the Hindlll site of the plasmid pYG 106 generates the plasmid pYG 1267. The plasmids pYG 1266 and pYG1267 are mutually isogenic with the exception of the SalI-HindIII
restriction fragment encoding the LAC4 promoter of K. lactis (plasmid pYG1266) or the PGK
promoter of S. cerevisiae (plasmid pYG1267).
In another exemplification, the cloning in the productive orientation of the Hindlll restriction fragment of the plasmid pYG1336 (chimera HSA-Gly4-G.CSF) into the Hindlll site of the plasmids pYG105 (LAC4) and pYG106 (PGK) generates the expression plasmids pYG1351 and pYG1352 respectively.
E.9.1.2. Coupling at the N-terminus of HSA
In a specific embodiment, the combined techniques of site-directed mutagenesis and PCR amplification make it possible to construct hybrid genes encoding a chimeric protein resulting from the translational coupling between a signal peptide (and for example the prepro region of HSA), a sequence including a gene having a G-CSF activity, and the mature form of HSA or one of its molecular variants (cf. chimera of panel B, FIG. 1). These hybrid genes are preferably bordered in 5' of the translational initiator ATG and in 3' of the translational stop codon by HindIII restriction sites. For example the oligodeoxynucleotide Sq2369 (5'-GTTCTACGCCACCTTG-CGCAGCCCGGTGGAGGCGGTGATGCACACAAGAGTGAGGTTGCTCAT-CGG-3' the residues underlined (optional) correspond in this particular chimera to a peptide linker composed of 4 glycine residues) makes it possible, by site-directed mutagenesis, to put in translational phase the mature form of the human G-CSF
of the plasmid BBG13 immediately upstream of the mature form of HSA, which generates the intermediate plasmid A. Likewise, the use of the oligodeoxynucleotide Sq2338 [5'-CAGGGAGCTGGCAGGGCCCAGGGGG-GTTCGACGAAACACACCCCTGGAATAAGCCGAGCT-3' (non-coding strand), the nucleotides complementary to the nucleotides encoding the first N-terminal residues of the mature form of the human G-CSF are underlined] makes it possible, by site-directed mutagenesis, to couple in translational reading phase the prepro region of HSA immediately upstream of the mature form of the human G-CSF, which generates the intermediate plasmid B. A HindIII fragment encoding a chimeric protein of the PEPTIDE-HSA type (cf. FIG. 1, panel B) is then generated by combining the HindIIl-Sstl fragment of the plasmid B(joining prepro region of HSA+N-terminal fragment of the mature G-CSF) with the Sstl-HindII1 fragment of the plasmid A[joining mature G-CSF-(glycine)ic4 - mature HSA]. The plasmid pYG1301 contains this specific HindIII restriction fragment encoding the chimera G.CSF-Gly4-HSA fused immediately downstream of the prepro region of HSA
(FIG. 11). The cloning of this HindIII restriction fragment in the productive orientation and into the HindIII site of the plasmids pYG105 (LAC4) and pYG106 (PGK) generates the expression plasmids pYG1302 and pYG 1303 respectively.
E.9.2. Secretion of the Hybrids.
After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the mature form of the chimeric proteins between HSA and G-CSF. A few clones corresponding to the strain K.
lactis CBS 293.91 transformed with the plasmids pYG1266 or pYG1267 (HSA-G.CSF), pYG1302 or pYG1303 (G.CSF-G1y4-HSA) or alternatively pYG1351 or pYG1352 (HSA-G1y4-G.CSF) are incubated in selective complete liquid medium at 28 C. The cellular supernatants are then tested after electrophoresis on an 8.5%
acrylamide gel, either directly by staining the gel with coomassie blue, or after immunoblotting using as primary antibodies rabbit polyclonal antibodies directed against the human G-CSF or a rabbit polyclonal serum directed against human albumin. The results of FIG. 12 demonstrate that the hybrid protein HSA-G.CSF
is recognized both by antibodies directed against human albumin (panel C) and human G-CSF (panel B). The results of FIG. 13 indicate that the chimera HSA-Gly4-G.CSF (lane 3) is particularly well secreted by the yeast Kluyveromyces, possibly because of the fact that the presence of the peptide linker between the HSA part and the G-CSF part is more favourable to an independent folding of these 2 parts during the transit of the chimera in the secretory pathway.
Furthermore, the N-terminal fusion (G.CSF-G1y4-HSA) is also secreted by the yeast Kluyveromyces (FIG. 13, lane 1).
E.9.3. Purification and Molecular Characterization of the Chimeras Between HSA and G-CSF.
After centrifugation of a culture of the CBS 293.91 strain transformed with the expression plasmids according to Example E.9.1., the culture supernatant is passed through a 0.22 mm filter (Millipore) and then concentrated by ultrafiltration (Amicon) using a membrane whose discrimination threshold is situated at 30 kDa.
The concentrate obtained is then adjusted to 50 mM Tris-HC1 from a 1M stock solution of Tris-HCI (pH 6), and then loaded in 20 ml fractions onto an ion-exchange column (5 ml) (Q Fast Flow, Pharmacia) equilibrated in the same buffer.
The chimeric protein is then eluted from the column by a gradient (0 to 1M) of NaCI. The fractions containing the chimeric protein are then pooled and dialysed against a 50 mM Tris-HCl solution (pH 6) and reloaded onto a Q Fast Flow column (1 ml) equilibrated in the same buffer. After elution of the column, the fractions containing the protein are pooled, dialysed against water and freeze-dried before characterization: for example, the sequencing (Applied Biosystem) of the protein HSA-G.CSF secreted by the yeast CBS 293.91 gives the N-terminal sequence expected for HSA (Asp-Ala-His ...), demonstrating a correct maturation of the chimera immediately at the C-terminus of the doublet of residues Arg-Arg of the "pro" region of HSA (FIG. 2).
EXAMPLE 10: CHIMERAS DERIVED FROM AN IMMUNOGLOBULIN
E.10.1. Constructs An Fv' fragment can be constructed by genetic engineering techniques, and which encodes the variable fragments of the heavy and light chains of an immunoglobulin (Ig), linked to each other by a linker peptide Bird et al., Science (1988) 242: 423; Huston et al., (1988) [Proc. 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 messenger RNA of the corresponding hybridoma, for example using the RT-PCR kit distributed by Pharmacia (Mouse ScFv module). In a second stage, the variable regions are genetically coupled by genetic engineering via a synthetic linkage peptide and for example the linker (GGGGS)x3. An MstII-HindIIl restriction fragment including the Fv' fragment of an immunoglobulin secreted by a murine hybridoma is given in FIG. 14. The ligation of the Hindlll-MstII fragment of the plasmid pYG404 to this MstII-HindIIl fragment makes it possible to generate the HindIII fragment of the plasmid pYG1382 which encodes a chimeric protein in which the HSA molecule is genetically coupled to the Fv' fragment of FIG. 14 (chimera HSA-Fv'). The cloning in the productive orientation of the HindIII restriction fragment of the plasmid pYG1382 into the HindIII site of the plasmids pYG105 (LAC4) and pYG106 (PGK) generates the expression plasmids pYG1383 and pYG1384 respectively.
E.10.2. Secretion of the Hybrids After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the mature form of the chimeric protein HSA-Fv'. A few clones corresponding to the strain K. lactis CBS 293.91 transformed with the plasmids pYG1383 or pYG1384 (HSA-Fv') are incubated in selective complete liquid medium at 28 C. The cellular supernatants are then tested after electrophoresis on an 8.5% acrylamide gel, either directly by staining of the gel with coomassie blue, or after immunoblotting using as primary antibodies a rabbit polyclonal serum directed against human albumin, or directly incubated with biotinylated antibodies directed against the immunoglobulins of murine origin.
The results of FIG. 15 demonstrate that the hybrid protein HSA-Fv' is recognized both by antibodies directed against human albumin (panel C) and reacts with biotinylated goat antibodies which are immunologically reactive towards mouse immunoglobulins (panel B).
EXAMPLE 11: BIOLOGICAL ACTIVITY OF THE CHIMERAS
E.11.1. Biological Activity In vitro.
E.11.1.1. Chimeras Between HSA and vWF.
The antagonistic activity of the products is determined by measuring the dose-dependent inhibition of the agglutination of human platelets fixed with paraformaldehyde according to the method described by Prior et al.
[Bio/Technology (1992) 10: 66]. The measurements are carried out in an aggregameter (PAP-4, Bio Data, Horsham, Pa., U.S.A.) which records the variations over time of the optical transmission, with stirring, at 37 C. in the presence of vWF, of botrocetin (8.2 mg/ml) and of the test product at various dilutions (concentrations). For each measurement, 400 ml (8 x 107 platelets) of a suspension of human platelets stabilized with paraformaldehyde (0.5%, and then resuspended in [NaCI (137 mM); MgC12 (1 mM); NaH2PO4 (0.36 mM); NaHCO3 (10 mM); KCl (2.7 mM); glucose (5.6 mM); HSA (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 and are then supplemented with 30 ml of the solution of the test product at various dilutions in apyrogenic formulation vehicle [mannitol (50 g/1); citric acid (192 mg/1); L-lysine monohydrochloride (182.6 mg/1); NaCI (88 mg/1); pH adjusted to 3.5 by addition of NaOH (1M)], or formulation vehicle alone (control assay).
The resulting suspension is then incubated for 1 min at 37 C. and 12.5 ml of human vWF [American Bioproducts, Parsippany, N.J., U.S.A.; 11% von Willebrand activity measured according to the recommendations for the use of PAP-4 (Platelet Aggregation ProfilerRTM) with the aid of platelets fixed with formaldehyde (2 x 105 platelets/ml), human plasma containing 0 to 100% vWF and ristocetin (10 mg/ml, cf. p. 36-45: vW ProgramTM] are added and incubated at 37 C. for 1 min before adding 12.5 ml of botrocetin solution purified from freeze-dried venom of Bothrops jararaca (Sigma) according to the procedure described by Sugimoto et al., [Biochemistry (1991) 266: 18172]. The recording of the reading of the transmission as a function of time is then carried out for 2 min with stirring by means of a magnetic bar (Wellcome Distriwell) placed in the tank and with a magnetic stirring of 1,100 rpm provided by the aggregameter. The mean variation of the optical transmission (n35 for each dilution) over time is therefore a measurement of the platelet agglutination due to the presence of vWF and botrocetin, in the absence or in the presence of variable concentrations of the test product. From such recordings, the % inhibition of the platelet agglutination due to each concentration of product is then determined and the straight line giving the % inhibition as a function of the reciprocal of the product dilution in log-log scale is plotted. The IC50 (or concentration of product causing 50% inhibition of the agglutination) is then determined on this straight line. The table of FIG. 6 compares the IC50 values of some of the HSA-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) 10: 66] and included in the assays as standard value. Identical tests for the inhibition of the agglutination of human platelets in the presence of vWF of pig plasma (Sigma) makes it possible, furthermore, to demonstrate that some of the hybrids of the present invention, and especially some type IIB variants, are very good antagonists of platelet agglutination in the absence of botrocetin-type cofactors. The botrocetin-independent antagonism of these specific chimeras can also be demonstrated according to the procedure initially described by Ware et al. [Proc. Natl.
Acad. Sci.
(1991) 88: 2946] by displacing the monoclonal antibody 125I-LJ-1B 1(10 mg/ml), a competitive inhibitor of the binding of vWF to the platelet GPIb Handa M. et al., (1986) [J. Biol. Chem. 261: 12579] after 30 min of incubation at 22 C. in the presence of fresh platelets (108 platelets/ml).
E.11.1.2. Chimeras between HSA and G-CSF
The purified chimeras are tested for their capacity to permit the in vitro proliferation of the IL3-dependant murine line NFS60, by measuring the incorporation of tritiated thymidine essentially according to the procedure described by Tsuchiya et al. [Proc. Natl. Acad. Sci. (1986) 83 7633]. For each chimera, the measurements are carried out between 3 and 6 times in a three-point test (three dilutions of the product) in a zone or the relation between the quantity of active product and incorporation of labelled thymidine (Amersham) is linear.
In each microtitre plate, the activity of a reference product consisting of recombinant human G-CSF expressed in mammalian cells is also systematically incorporated.
The results of FIG. 17 demonstrate that the chimera HSA-G.CSF (pYG 1266) secreted by the yeast Kluyveromyces and purified according to Example E.9.3.
is capable in vitro of transducing a signal for cellular proliferation for the line NFS60.
In this particular case, the specific activity (cpm/molarity) of the chimera is about 7 times lower than that of the reference G-CSF (non-coupled).
E.11.2. Biological Activity In vivo The activity of stimulation of the HSA-G-CSF chimeras on granulopoiesis in vivo is tested after subcutaneous injection in rats (Sprague-Dawley/CD, 250-300g, 8-9 weeks) and compared to that of the reference G-CSF expressed using mammalian cells. Each product, tested at the rate of 7 animals, is injected subcutaneously into the dorso-scapular region at the rate of 100 ml for 7 consecutive days, (D1-D7). 500 ml of blood are collected on days D-6, D2 (before the 2nd injection). D5 (before the 5th injection) and D8, and a blood count is performed. In this test, the specific activity (neutropoiesis units/mole injected) of the chimera HSA-G.CSF (pYG1266) is identical to that of the reference G-CSF
(FIG. 18). Since this specific chimera has in vitro a specific activity 7 times lower than that of the reference G-CSF (FIG. 17), it is therefore demonstrated that the genetic coupling of G-CSF onto HSA favourably modifies the pharmacokinetic properties thereof.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANTS:
(A) NAME: Rhone-Poulenc Rorer S.A.
(B) STREET: 20 Raymond ARON Avenue (C) CITY: Antony (E) COUNTRY: France (F) POSTAL CODE:92165 (ii) TITLE OF THE INVENTION: Novel Biologically Active Polypeptides, Preparation Thereof and Pharmaceutical Composition Containing Said Polypeptides (iii)NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS:
(A) NAME: MBM & Co.
(B) STREET: P.O. Box 809 (C) CITY: Ottawa (D) PROVINCE: ON
(E) COUNTRY: Canada (F) POSTAL CODE: K1P 5P9 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy Disk (B) COMPUTER: IBM-PC Compatible (C) OPERATING SYSTEM: Windows (D) SOFTWARE: Word (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,126,091 (B) FILING DATE: January 28, 1993 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SWAIN, Margaret (B) REGISTRATION NUMBER: 10926 (C) REFERENCE/DOCKET NUMBER:
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 613-567-0762 (B) TELEFAX: 613-563-7671 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1859 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no (iii) ANTISENSE: no (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 26..1855 (D) OTHER INFORMATION: chimera of type HSA-peptide (ix) FEATURE:
(A) NAME/KEY: miscfeature (B) LOCATION: 1842-1848 (D) OTHER INFORMATION: /standard name = "MstII Site"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser AAA CTG AAG GAA TGC TGT GAA AAA CCT CTG TTG GAA AAA TCC CAC TGC
Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu leu Glu Lys Ser His Cys 964 Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr GAA ACC ACT CTA GAG AAG TGC TGT GCC GCT GCA GAT CCT CAT GAA TGC 1209:
Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asn Ile Cys Thr Leu Ser Glu Lys Glu Arg Gin Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala GCA AGT CAA GGT GGC TTA GGC TTA (NNN)p TAAGCTT 18513 Ala Ser Gln Ala Ala Leu Gly Leu peptide (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 750 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no (iii) ANTISENSE: no (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..746 (D) OTHER INFORMATION:/product= "C-ter[ninal fragment of the HSA-vWF470 chimera"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Leu Gly Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser Glu Pro Pro Leu His Asp Phe Tyr Cys Ser Arg Leu Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe Glu Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu Arg Ile Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys Ile Asp Arg Pro Glu Ala Ser Arg Ile Ala Leu Leu Leu Met Ala Ser Gln Glu Pro Gln Arg Met Ser Arg Asn Phe Val Arg Tyr Val Gln Gly Leu Lys Lys Lys Lys Val Ile Val Ile Pro Val Gly Ile Gly Pro His Ala Asn Leu Lys Gln Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu Asn Lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln Arg Asp Glu Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala Pro Pro Pro Thr Leu Pro Pro Asp Met Ala Gln Val (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 423 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no (iii) ANTISENSE: no (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..419 (D) OTHER INFORMATION:/product = "C-terminal fragment of the HSA-UK1-135 chimera"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Leu Gly Leu Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn Cys Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile Asp Lys Ser Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val Leu Gin Gln Thr Tyr His Ala His Arg Ser Asp Ala Leu Gln Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met Val His Asp Cys Ala Asp Gly Lys (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 541 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no (iii) ANTISENSE: no (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..536 (D) OTHER INFORMATION: /product = "C-terminal fragment of the HSA-G.CSF chimera"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Leu Gly Leu Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gin Ala Leu Giu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Giy Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2455 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no (iii) ANTISENSE: no (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 26..2389 (D) OTHER INFORMATION:/product ="G.CSF-Gly4-HSA chimera downstream of`
HSA prepro region"
(ix) FEATURE:
(A) NAME/KEY: miscrecomb (B) LOCATION: 620-631 (D) OTHER INFORMATION: /standard name = "linker PolyGly"
(ix) FEATURE:
(A) NAME/KEY: miscfeature (B) LOCATION: 106-111 (D) OTHER INFORMATION: /standard name = "ApaI site"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala Tyr Ser Arg Gly Val Phe Arg Arg Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Gly Gly Gly Gly Asp Ala His Lys Ser, Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys AGG TAT AAA GCT GCT TTT ACA GAA TGT TGC CAA GCT GCT GAT AAA GCT 115Ei Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly GAA AGA GCT TTC AAA GCA TGG GCA GTA GCT CGC CTG AGC CAG AGA TTT 130C) Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 756 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no (iii) ANTISENSE: no (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..752 (D) OTHER INFORMATION:/product ="C-terminal fragment of the HSAFv chimera"
(D) OTHER INFORMATION: /standard_name = "Synthetic linker"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Leu Gly Leu Gln Val Gln Leu Glu Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Arg Ser Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Lys Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Val Gly Ser Ala Val Tyr Phe Cys Ala Lys Glu Asn Asn Arg Phe Asp Glu Arg Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ile Gln Leu Thr Gln Ser Pro Asn Ser Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asp Thr Ser Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Ser Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
More particularly, in the molecules of the invention, the polypeptide having a therapeutic activity is a polypeptide of human origin or a molecular variant. For example, this may be all or part of an enzyme, an enzyme inhibitor, an antigen, an antibody, a hormone, a factor involved in the control of coagulation, an interferon, a cytokine [the interleukins, but also their variants which are natural antagonists of their binding to the receptor(s), the SIS (small induced secreted) type cytokines and for example the macrophage inflammatory proteins (MIPs), and the like], of a growth factor and/or of differentiation [and for example the transformant growth factors (TGFs), the blood cell differentiation factors (erythropoietin, M-CSF, G-CSF, GM-CSF and the like), insulin and the growth factors resembling it (IGFs), or alternatively cell permeability factors (VPF/VEGF), and the like], of a factor involved in the genesis/resorption of bone tissues (OIF and osteospontin for example), of a factor involved in cellular motility or migration [and for example autocrine motility factor (AMF), migration stimulating factor (MSF), or alternatively the scatter factor (scatter factor/hepatocyte growth factor)], of a bactericidal or antifungal factor, of a chemotactic factor and for example platelet factor 4 (PF4), or alternatively the monocyte chemoattracting peptides (MCP/MCAF) or neutrophil chemoattracting peptides (NCAF), and the like, of a cytostatic factor (and for example the proteins which bind to galactosides), of a plasma (and for example von Willebrand factor, fibrinogen and the like) or interstitial (laminin, tenascin, vitronectin and the like) adhesive molecule or extracellular matrices, or alternatively any peptide sequence which is an antagonist or agonist of molecular and/or intercellular interactions involved in the pathologies of the circulatory and interstitial compartments and for example the formation of arterial and venous thrombi, cancerous metastases, tumour angiogenesis, inflammatory shock, autoimmune diseases, bone and osteoarticular pathologies and the like.
The active part of the polypeptides of the invention may consist for example of the polypeptide having a whole therapeutic activity, or of a structure derived therefrom, or alternatively of a non-natural polypeptide isolated from a peptide library. For the purposes of the present invention, a derived structure is understood to mean any polypeptide obtained by modification and preserving a therapeutic activity. Modification should be understood to mean any mutation, substitution, deletion, addition or modification of genetic and/or chemical nature.
Such derivatives may be generated for various reasons, such as especially that of increasing the affinity of the molecule for its binding sites, that of improving its levels of production, that of increasing its resistance to proteases, that of increasing its therapeutic efficacy or alternatively of reducing its side effects, or that of conferring on it new biological properties. As an example, the chimeric polypeptides of the invention possess pharmacokinetic properties and a biological activity which can be used for the prevention or treatment of diseases.
Particularly advantageous polypeptides of the invention are those in which the active part has:
(a) the whole peptide structure or, (b) a structure derived from (a) by structural modification (mutation, substitution addition and/or deletion of one or more residues) and possessing a therapeutic activity.
Among the structures of the (b) type, there may be mentioned more particularly the molecules in which certain N- or 0-glycosylation sites have been modified or suppressed, the molecules in which one or more residues have been substituted, or the molecules in which all the cystein residues have been substituted. There may also be mentioned molecules obtained from (a) by deletion of regions not involved or not highly involved in the interaction with the binding sites considered, or expressing an undesirable activity, and molecules containing, compared to (a), additional residues such as for example an N-terminal methionine and/or a signal for secretion and/or a joining peptide.
The active part of the molecules of the invention can be coupled either directly or via an artificial peptide to albumin. Furthermore, it may constitute the N-terminal end 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 repetitive within the chimera. A schematic representation of the molecules of the invention is given in FIG. 1.
Another subject of the invention relates to a process for preparing the chimeric molecules described above. More specifically, this process consists in causing a eukaryotic or prokaryotic cellular host to express a nucleotide sequence encoding the desired polypeptide, and then in harvesting the polypeptide produced.
Among the eukaryotic hosts which can be used within the framework of the present invention, there may be mentioned animal cells, yeasts or fungi. In particular, as regards yeasts, there may be mentioned yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula. As regards animal cells, there may be mentioned COS, CHO and C127 cells and the like. Among the fungi capable of being used in the present invention, there may be mentioned more particularly Aspergillus spp, or Trichoderma spp. As prokaryotic hosts, the use of bacteria such as Escherichia coli, or belonging to the genera Corynebacterium, Bacillus, or Streptomyces is preferred.
The nucleotide sequences which can be used within the framework of the present invention can be prepared in various ways. Generally, they are obtained by assembling, in reading phase, the sequences encoding each of the functional parts of the polypeptide. The latter may be isolated by the techniques of persons skilled in the art, and for example directly from cellular messenger RNAs (mRNAs), or by recloning from a complementary DNA (cDNA) library, or alternatively they may be completely synthetic nucleotide sequences. It is understood, furthermore, that the nucleotide sequences may also be subsequently modified, for example by the techniques of genetic engineering, in order to obtain derivatives or variants of the said sequences.
More preferably, in the process of the invention, the nucleotide sequence is part of an expression cassette comprising a region for initiation of transcription (promoter region) permitting, in the host cells, the expression of the nucleotide sequence placed under its control and encoding the polypeptides of the invention. This region may come from promoter regions of genes which are highly expressed in the host cell used, the expression being constitutive or regulatable. As regards yeasts, it may be the promoter of the gene for phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GPD), lactase (LAC4), enolases (ENO), alcohol dehydrogenases (ADH), and the like. As regards bacteria, it may be the promoter of the right-hand or left-hand genes from the lambda bacteriophage (PL, PR), or alternatively the promoters of the genes for the tryptophan (Pt,r) or lactose (Plaj operons. In addition, this control region can be modified, for example by in vitro mutagenesis, by the introduction of additional control elements or of synthetic sequences, or by deletions or substitutions of the original control elements. The expression cassette may also comprise a region for termination of transcription which is functional in the host envisaged, positioned immediately downstream of the nucleotide sequence encoding a polypeptide of the invention.
In a preferred mode, 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 product of expression of the said sequence into the culture medium. It is indeed particularly advantageous to be able to obtain, by the recombinant route, nlolecules directly in the culture medium. In this case, the nucleotide sequence encoding a polypeptide of the invention is preceded by a "leader" sequence (or signal sequence) directing the nascent polypeptide in the secretory pathways of the host used. This "leader" sequence may be the natural signal sequence of the biologically active polypeptide in the case where the latter is a naturally secreted protein, or that of the stabilizing structure, but it may also be any other functional "leader" sequence, or an artificial "leader" sequence.
The choice of one or the other of these sequences is especially guided by the host used.
Examples of functional signal sequences include those of the genes for the sexual pheromones or the "killer" toxins of yeasts.
In addition to the expression cassette, one or several markers which make it possible to select the recombinant host may be added, such as for example the URA3 gene from the yeast S. cerevisiae, or genes conferring the resistance to antibiotics such as geneticin (G418) or to any other toxic compound such as certain metal ions.
The unit formed by the expression cassette and by the selectable marker can be introduced directly into the considered host cells, or previously inserted in a functional self-replicating vector. In the first case, sequences homologous to regions present in the genome of the host cells are preferably added to this unit; the said sequences then being positioned on each side of the expression cassette and of the selectable gene so as to increase the frequency of integration of the unit into the genome of the host by targeting the integration of the sequences by homologous recombination. In the case where the expression cassette is inserted in a replicative system, a preferred replication system for yeasts of the genus Kluyveromyces is derived from the plasmid pKDI originally isolated from K.
drosophilarum; a preferred replication system for yeasts of the genus Saccharomyces is derived from the 2 plasmid from S. cerevisiae. Furthermore, this expression plasmid may contain all or part of the said replication systems, or may combine elements derived both from the plasmid pKDI and the 2 plasmid.
In addition, the expression plasmids may be shuttle vectors between a bacterial host such as Escherichia coli and the chosen host cell. In this case, a replication origin and a selectable 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 suppress these sequences by cutting and religation in vitro 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 the said hosts. For example, such restriction sites may correspond to sequences such as 5'-GGCCNNNNNGGCC-3' (Sfil) or 5'-GCGGCCGC-3' (Notl) in so far as these sites are extremely rare and generally absent from an expression vector.
After construction of such vectors or expression cassette, the latter are introduced into the host cells selected according to the conventional techniques described in the literature. In this respect, any method permitting the introduction of a foreign DNA into a cell can be used. This may be especially transformation, electroporation, conjugation, or any other technique known to persons skilled in the art. As an example of yeast-type hosts, the various strains of Kluyveromyces used were transformed by treating the 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.
Genet. 18 (1990) 7] using ethylene glycol and dimethyl sulphoxide was also used.
It is also possible to transform the yeasts by electroporation, according to the method described by Karube et al. [FEBS Letters 182 (1985) 90]. An alternative procedure is also described in detail in the examples below.
After selection of the transformed cells, the cells expressing the said polypeptides are inoculated and the recovery of the said polypeptides can be carried out, either during the cell growth for the "continuous" processes, or at the end of growth for the "batch" cultures. The polypeptides which are the subject of the present invention are then purified from the culture supernatant for their molecular, pharmacokinetic and biological characterization.
A preferred expression system for the polypeptides of the invention consists in using yeasts of the genus Kluyveromyces as host cell, transformed by certain vectors derived from the extrachromosomal replicon pKD 1 originally isolated from K. marxianus var. drosophilarum. These yeasts, and in particular K.
lactis and K. fragilis are generally capable of stably replicating the said vectors and possess, in addition, the advantage of being included in the list of G.R.A.S.
("Generally Recognized As Safe") organisms. Favoured yeasts are preferably industrial yeasts of the genus Kluyveromyces which are capable of stably replicating the said plasmids derived from the plasmid pKDI and in which has been inserted a selectable marker as well as an expression cassette permitting the secretion, at high levels, of the polypeptides of the invention.
The present invention also relates to the nucleotide sequences encoding the chimeric polypeptides described above, as well as the eukaryotic or prokaryotic recombinant cells comprising such sequences.
The present invention also relates to the application, as medicinal products, of the polypeptides according to the present invention. More particularly, the subject of the invention is any pharmaceutical composition comprising one or more polypeptides or nucleotide sequences as described above. The nucleotide sequences can indeed be used in gene therapy.
The present invention will be more fully described with the aid of the following examples, which should be considered as illustrative and non-limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
The representations of the plasmids indicated in the following figures are not plotted to scale and only the restriction sites important for the understanding of the clonings carried out have been indicated.
Figure 1: Schematic representation of the chimera of the HSA-PEPTIDE type (A), a chimera of the PEPTIDE-HSA type (B) and a chimera of the PEPTIDE-HSA-PEPTIDE type (C). Abbreviations used: M/LP, translational initiator methionine residue, optionally followed by a signal sequence for secretion;
HSA, mature albumin or one of its molecular variants; PEP, peptide of natural or artificial origin possessing a given therapeutic property. The PEP sequence may be present several times in the molecules of type A, B and C. The black arrow indicates the N-terminal end of the mature protein.
Figure 2: Examples of the nucleotide sequence of a HindIll restriction fragment encoding a chimeric protein of the prepro-HSA-PEPTIDE type. The black arrows indicate the end of the "pre" and "pro" regions of HSA. The MstII
restriction site is underlined and the codon specifying the termination of translation is in bold characters.
Figure 3: Restriction map for the plasmid pYG105 and generic strategy for construction of the plasmids for expression of the chimeric proteins of the present invention. Abbreviations used: P, transcriptional promoter; T, transcriptional terminator; IR, inverted repeat sequences of the plasmid pKDI;
LP, signal sequence for secretion; Apr and Kmr designate the genes for resistance to ampicillin (E. coli) and to G418 (yeasts), respectively.
Figure 4: Examples of nucleotide sequences of MstII-HindIII
restriction fragments derived from the von Willebrand factor. Representation of the structure of the Mstll-Hindlll fragment of the plasmid pYG1248 (panel A).
Representation of the structure of the Mstll-Hindlll fragment of the plasmid pYG1214 (panel B). Representation of the Mstll-Hindlll fragment of the plasmid pYG1206 (panel C); in this particular chimera, the Leu694 residue of the vWF
is also the last residue (Leu585) of the HSA. Representation of the Mstll-Hindlll fragment of the plasmid pYG1223 (panel D). The numbering of the amino acids corresponds to the numbering of the mature vWF according to Titani et al.
[Biochemistry 25 (1986) 3171-3184]. The Mstll and HindIII restriction sites are underlined and the translation termination codon is in bold characters. FIG.
4E is a nucleotide sequence (SEQ ID NO:3) of the Mstll-Hindlll restriction fragment of the plasmid pYG1248. The numbering of the amino acids (right-hand column) corresponds to the mature chimeric protein HSA-vWF470-*713 (829 residues).
The Thr470, Leu494, Asp498, Pro502, Tyr508, Leu694, Pro704 and Pro708 residues of the mature vWF are underlined.
Figure 5: The characterization of the material secreted after 4 days of culture (Erlenmeyers) of the strain CBS 293.91 transformed with the plasmids pYG1248 (plasmid for expression of a chimera of the HSA-vWF Thr470->Va1713) and pKan707 (control plasmid). In this experiment, the polypeptides for panels A, B and C were run on the same gel (8.5% SDS-PAGE) and then treated separately.
A: the results of Coomassie blue staining of a molecular weight standard (lane 2); of a supernatant equivalent to 50 l of the culture transformed with the plasmid pKan707 in YPL medium (lane 1); the plasmid pYG 1248 in YPD medium (lane 3) and the plasmid pYG 1248 in YPL medium (lane 4).
B: the results of immunological characterization of the secreted material after using mouse antibodies directed against human vWF. The lanes are the same as described for FIG. 5A except that biotinylated molecular weight standards were used (lane 2).
C: the results of immunological characterization of the secreted material after using rabbit antibodies directed against human albumin: supernatant equivalent to 50 l of the culture transformed with the plasmid pKan707 in YPL
medium (lane 1), the plasmid pYG1248 in YPD medium (lane 2) the plasmid pYG1248 in YPL medium (lane 3).
Figure 6: The kinetic analysis of secretion of a chimera of the invention by the strain CBS 293.91 transformed with the plasmid pYG1206 (HSA-vWF Leu694-Pro708).
A: Coomassie blue staining was employed. Lane 1 is the molecular weight standard, lane 2 is the supernatant equivalent to 2.5 l of a "Fed Batch"
culture in YPD medium after 24 hours of growth; lane 3 is the supernatant of the same culture after 40 hours; and lane 4 is the supernatant of the same culture after 46 hours of growth.
B: immunological characterization of the secreted material after using mouse antibodies directed against the human vWF. The lanes are the same as in A
except that biotinylated molecular weight standards were used.
Figure 7: Characterization of the material secreted by K. lactis transformed with the plasmids pKan707 (control plasmid, lane 2), pYG 1206 (lane 3), pYG1214 (lane 4) and pYG1223 (lane 5); molecular weight standard (lane 1).
The deposits correspond to 50 l of supernatant from a stationary culture after growing in YPD medium, running on an 8.5% acrylamide gel and staining with Coomassie blue.
Figure 8: Nucleotide sequence of the MstI1-Hindlll restriction fragment of the plasmid pYG1341 (HSA-UK1--),135). The limit of the EGF-like domain (UK1-46) present in the MstII-Hindlll restriction fragment of the plasmid pYG1340 is indicated. The numbering of the amino acids corresponds to the mature chimeric protein SAU-UK1- 135 (720 residues).
Figure 9: Secretion of the HSA-UK1-46 and HSA-UK1-135 chimeras by the strain CBS 293.91 respectively transformed with the plasmids pYG1343 (HSA-UKI-46) and pYG1345 (HSA-UK1-135), after 4 days of growth (YPL+G418 medium). The deposits (equivalent to 50 l of culture) are run on an 8.5% PAGE-SDS gel and stained with Coomassie blue: supernatant from a clone transformed with the plasmids pKan707 (lane 1), pYG1343 (lane 3) or pYG1345 (lane 4); molecular weight standard (lane 2).
Figure 10: Nucleotide sequence of the MstII-Hindlll restriction fragment of the plasmid pYG1259 (HSA-G.CSF). The limit of the G-CSF part (174 residues) is indicated. The Apal and Sstl (Sstl) restriction sites are underlined. The numbering of the amino acids corresponds to the mature chimeric protein HSA-G.CSF (759 residues).
Figure 11: The nucleotide sequence of the HindIII restriction fragment of the plasmid pYG1301 (chimera G.CSF-Gly4 -HSA). The black arrows indicate the end of the "pre" and "pro" regions of HSA. The Apal, Sstl (SacI) and MstIl restriction sites are underlined. The G.CSF (174 residues) and HSA (585 residues) domains are separated by the synthetic linker GGGG. The numbering of the amino acids corresponds to the mature chimeric protein G.CSF-Gly4-SAH (763 residues).
The nucleotide sequence between the translation termination codon and the HindIIl site comes from the HSA complementary DNA (cDNA) as described in Patent Application EP 361 991.
Figure 12: The characterization of the material secreted after 4 days of culture (erlenmeyers) of the strain CBS 293.91 transformed with the plasmids pYG1266 (plasmid for expression of a chimera of the HSA-G.CSF type) and pKan707 (control plasmid). In this experiment, the polypeptides for panels A, B
and C were run on the same gel (8.5% SDS-PAGE) and then treated separately.
A: coomassie blue staining of a molecular weight standard (lane 2);
supernatant equivalent to 100 l of culture transformed with the plasmid pKan707 in YPL medium (lane 1); the plasmid pYG1266 in YPD medium (lane 3) and the plasmid pYG 1266 in YPL medium (lane 4).
B: immunological characterization of the material secreted after using primary antibodies directed against human G-CSF. The lanes are as described above for A.
C: immunological characterization of the material secreted after using primary antibodies directed against human albumin. The lanes are as described above for A.
Figure 13: Characterization of the material secreted after 4 days of culture (erlenmeyers in YPD medium) of the strain CBS 293.91 transformed with the plasmids pYG1267 (chimera HSA-G.CSF), pYG1303 (chimera G.CSF-Gly4-HSA) and pYG1352 (chimera HSA-G1y4-G.CSF) after running on an 8.5% SDS-PAGE gel.
A: coomassie blue staining of a supernatant equivalent to 100 l of the culture transformed with the plasmid pYG1303 (lane 1), the plasmid pYG1267 (lane 2), and the plasmid pYG1352 (lane 3). Lane 4 is the molecular weight standard.
B: immunological characterization of the material secreted after using primary antibodies directed against the human G-CSF: same legend as in A.
Figure 14: Nucleotide sequence of the MstII-HindIIl restriction fragment of the plasmid pYG1382 (HSA-Fv'). The VH (124 residues) and VL (107 residues) domains of the Fv' fragment are separated by the synthetic linker (GGGGS)x3. The numbering of the amino acids corresponds to the mature chimeric protein HSA-Fv' (831 residues).
Figure 15: Characterization of the secretion of the chimera HSA-Fv' by the strain CBS 293.91 transformed with the plasmid pYG1383 (LAC4) after 4 days of growth in erlenmeyers at 28 C. in YPD medium (lane 2), and in YPL medium (lane 3). Lane 1 shows the molecular weight standard. The deposits, equivalent to 200 1 of culture (precipitation with ethanol), are run on a PAGE-SDS gel (8.5%).
A: coomassie blue staining of the gel.
B: immunological characterization of the material secreted after using primary antibodies directed against HSA.
Figure 16: Assay of the in vitro antagonistic activity of the agglutination of human platelets fixed with formaldehyde: IC50 of the hybrids HSA-vWF694-708, [HSA-vWF470-713 C471G, C474G] and [HSA-vWF470-704 C471G, C474G] compared with the standard RG12986. The determination of the dose-dependent inhibition of the platelet agglutination is carried out according to the method described by C. Prior et al. [Bio/Technology (1992) 10 66] using an aggregameter recording the variations in optical transmission, with stirring, at 37 C. in the presence of human vWF, botrocetin (8.2 mg/ml) of the test product at various dilutions. The concentration of the product which makes it possible to inhibit the control agglutination (in the absence of product) by half is then determined (IC50).
Figure 17: Activity on the in vitro cellular proliferation of the murine line NFS60. The radioactivity (3H-thymidine) incorporated into the cellular nuclei after 6 hours of incubation is represented on the y-axis (cpm); the quantity of product indicated on the x-axis is expressed in molarity (arbitrary units).
Figure 18: Activity on granulopoiesis in vivo in rats. The number of neutrophils (average for 7 animals) is indicated on the y-axis as a function of time.
The products tested are the chimera HSA-G.CSF (pYG1266), 4 or 40 mg/rat/day), the reference G-CSF (10 mg/rat/day), the recombinant HSA purified from Kluyveromyces lactis supernatant (HSA, 30 mg/rat/day, cf. EP 361 991), or physiological saline.
EXAMPLES
GENERAL CLONING TECHNIQUES
The methods conventionally used in molecular biology, such as the preparative extractions of plasmid DNA, the centrifugation of plasmid DNA in caesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, extractions of proteins with phenol or phenol-chloroform, DNA precipitation in saline medium with ethanol or isopropanol, transformation in Escherichia coli, and the like are well known to persons skilled in the art and are widely described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F. M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987].
The restriction enzymes were provided by New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Amersham and are used according to the recommendations of the suppliers.
The pBR322 and pUC type plasmids and the phages of the M13 series are of commercial origin (Bethesda Research Laboratories).
For the ligations, the DNA fragments are separated according to their size by electrophoresis on agarose or acrylamide gels, extracted with phenol or with a phenol/chloroform mixture, precipitated with ethanol and then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the recommendations of the manufacturer.
The filling of the protruding 5' ends is carried out by the Klenow fragment of DNA polymerase I of E. coli (Biolabs) according to the specifications of the supplier. The destruction of the protruding 3' ends is carried out in the presence of phage T4 DNA polymerase (Biolabs) used according to the recommendations of the manufacturer. The destruction of the protruding 5' ends is carried out by a controlled treatment with S 1 nuclease.
Site-directed mutagenesis in vitro with 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.
The enzymatic amplification of DNA fragments by the so-called PCR technique Polymerase-catalyzed Chain Reaction, [Saiki R. K. et al., Science 230 (1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth. Enzym. 155 (1987) 335-350] is carried out using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the specifications of the manufacturer.
The verification of the nucleotide sequences is carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 5467] using the kit distributed by Amersham.
The transformations of K. lactis with DNA from the plasmids for expression of the proteins of the present invention are carried out by any technique known to persons skilled in the art, and of which an example is given in the text.
Except where otherwise stated, the bacterial strains used are E. coli MC1060 (lacIPOZYA, X74, galU, galK, strA`), or E. coli TG1 (lac, proA,B, supE, thi, hsdD5/FtraD36, proA+ B+, IacI9, lacZ, M15).
The yeast strains used belong to the budding yeasts and more particularly to yeasts of the genus Kluyveromyces. The K. lactis MW98-8C (a, uraA, arg, lys, K, pKD1 ) and K. lactis CBS 293.91 strain were particularly used;
a sample of the MW98-8C strain was deposited on 16 Sep. 1988 at Centraalbureau voor Schimmelkulturen (CBS) at Baarn (the Netherlands) where it was registered under the number CBS 579.88.
A bacterial strain (E. coli) transformed with the plasmid pET-8c52K
was deposited on 17 Apr. 1990 with the American Type Culture Collection under the number ATCC 68306.
The yeast strains transformed with the expression plasmids encoding the proteins of the present invention are cultured in erlenmeyers or in 21 pilot fermenters (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.
EXAMPLE 1: COUPLING AT THE C-TERMINUS OF HSA
The plasmid pYG404 is described in Patent Application EP 361 991.
This plasmid contains a HindIII restriction fragment encoding the prepro-HSA
gene preceded by the 21 nucleotides naturally present immediately upstream of the initiator ATG for translation of the PGK gene of S. cerevisiae. The nucleotide sequence of this restriction fragment is included in that of FIG. 2. The Mstll site localized in the coding sequence, three residues from the codon specifying the end of translation is particularly useful as site for cloning a biologically active peptide which it is desired to couple in translational phase at the C-terminus of HSA.
In a specific embodiment, it is useful to use peptides whose sequence is encoded by an MstIl-Hindlll restriction fragment of the type: 5'-CCTTAGGCTTA [3xN]P
TAAGCTT-3', the sequence encoding the biologically active peptide (p residues) is [3xN]P). The ligation of this fragment to the HindIll-MstII restriction fragment corresponding to the entire gene encoding HSA, with the exception of the three C-terminal-most amino acids (leucine-glycine-leucine residues) generates a HindIII
restriction fragment containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro"
export region of HSA. In another embodiment, the biologically active peptide may be present more than once in the chimera.
EXAMPLE 2: COUPLING AT THE N-TERMINUS OF HSA
In a specific embodiment, the combined techniques of site-directed mutagenesis and PCR amplification make it possible to construct hybrid genes encoding a chimeric protein resulting from the translational coupling between a signal peptide (and for example the prepro region of HSA), a sequence including the biologically active peptide and the mature form of HSA or one of its molecular variants. These hybrid genes are preferably bordered in 5' of the translational initiator ATG and in 3' of the translational stop codon by HindIII restriction sites and encode chimeric proteins of the PEPTIDE-HSA type (FIG. 1, panel B). In a still more specific embodiment, the biologically active peptide may be present more than once in the chimera.
EXAMPLE 3: COUPLING AT THE N- AND C-TERMINUS OF HSA
The combined techniques of site-directed mutagenesis and PCR
amplification described in Examples 1 and 2 make it possible to construct hybrid genes encoding a chimeric protein resulting from the translational coupling between the mature form of HSA, or one of its molecular variants, and a biologically active peptide coupled to the N- and C-terminal ends of HSA.
These hybrid genes are preferably bordered in 5' of the translational initiator ATG
and in 3' of the translational stop codon by Hindlll restriction sites and encode chimeric proteins of the PEPTIDE-HSA-PEPTIDE type (FIG. 1, panel C), immediately preceded by the "prepro" export region of HSA. In a still more specific embodiment, the biologically active peptide may be present more than once in the chimera.
EXAMPLE 4: EXPRESSION PLASMIDS
The chimeric proteins of the preceding examples can be expressed in yeasts using functional, regulatable or constitutive promoters such as, for example, those present in the plasmids pYG105 (LAC4 promoter of Kluyveromyces lactis), pYG106 (PGK promoter of Saccharomyces cerevisiae), pYG536 (PHO5 promoter of S. cerevisiae), or hybrid promoters such as those described in Patent Application EP 361 991. The plasmids pYG105 and pYG106 are particularly useful here because they permit the expression of the genes encoded by the HindIII
restriction fragments as described in the preceding examples and cloned into the HindIII site and in the productive orientation (defined as the orientation which places the "prepro" region of albumin proximally relative to the promoter for transcription), using promoters which are functional in K. lactis, regulatable (pYG105) or constitutive (pYG106). The plasmid pYG105 corresponds to the plasmid pKan707 described in Patent Application EP 361 991 in which the HindIII
restriction site which is unique and localized in the gene for resistance to geneticin (G418) has been destroyed by site-directed mutagenesis while preserving an unchanged protein (oligodeoxynucleotide 5'-GAAA-TGCATAAGCTCTTGCCATTCTCACCG-3'). The Sall-Sacl fragment encoding the URA3 gene of the mutated plasmid was then replaced with a Sall-SacI
restriction fragment containing an expression cassette consisting of the LAC4 promoter of K. lactis (in the form of a SalI-HindIII fragment) and the terminator of the PGK gene of S. cerevisiae (in the form of a HindIII-Sac1 fragment). The plasmid pYG105 is mitotically very stable in the Kluyveromyces yeasts and a restriction map thereof is given in FIG. 3. The plasmids pYG105 and pYG106 differ from each other only in the nature of the promoter for transcription encoded by the SalI-HindI1I fragment.
EXAMPLE 5: TRANSFORMATION OF THE YEASTS
The transformation of the yeasts belonging to the genus Kluyveromyces, and in particular the strains MW98-8C and CBS 293.91 of K.
lactis is carried out for example by the technique for treating whole cells with lithium acetate Ito H. et al., [J. Bacteriol. 153 (1983) 163-168], adapted as follows.
The growth of the cells is carried out at 28 C. in 50 ml of YPD medium, with stirring and up to an optical density of 600 nm (OD600) of between 0.6 and 0.8; the cells are harvested by centrifugation at low speed, washed in a sterile solution of TE
(10 mM Tris HCl pH 7.4; 1 mM EDTA), resuspended in 3-4 ml of lithium acetate (0.1M in TE) in order to obtain a cellular density of about 2 x 108 cells/ml, and then incubated at 30 C. for 1 hour with moderate stirring. Aliquots of 0.1 ml 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 heat shock of 5 minutes at 42 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 in order to permit the phenotypic expression of the gene for resistance to G418 expressed under the control of the Pkl promoter (cf. EP 361 991); 200 l of the cellular suspension are then plated 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.
EXAMPLE 6:SECRETION OF THE CHIMERAS
After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the mature form of the chimeric proteins. Few clones, corresponding to the strain CBS 293.91 or MW98-8C transformed by the plasmids for expression of the chimeras between HSA and the biologically active part, are incubated in YPD or YPL medium at 28 C. The cellular supernatants are recovered by centrifugation when the cells reach the stationary growth phase, optionally concentrated 10 times by precipitation for minutes at -20 C. in a final concentration of 60% ethanol, and then tested after electrophoresis on an 8.5% SDS-PAGE gel, either directly by staining the gel with coomassie blue, or after immunoblotting using primary antibodies directed against the biologically active part or a rabbit polyclonal serum directed against HSA.
During the experiments for immunological detection, the nitrocellulose filter is first incubated in the presence of specific primary antibodies, washed several times, incubated in the presence of goat antibodies directed against the primary antibodies, and then incubated in the presence of an avidin-peroxidase complex using the "ABC kit" distributed by Vectastain (Biosys S. A., Compiegne, France). The immunological reaction is then revealed by the addition of 3,3'-diamino benzidine tetrahydrochloride (Prolabo) in the presence of hydrogen peroxide, according to the recommendations of the manufacturer.
EXAMPLE 7: CHIMERAS DERIVED FROM THE VON WILLEBRAND
FACTOR
E.7.1. Fragments Antagonizing the Binding of vWF to the Platelets E.7.1.1. Thr470-Va1713 Residues of vWF
The plasmid pET-8c52K contains a fragment of the vWF cDNA encoding residues 445 to 733 of human vWF and therefore includes several crucial determinants of the interaction between vWF and the platelets on the one hand, and certain elements of the basal membrane and the sub-endothelial tissue on the other, and especially the peptides G10 and D5 which antagonize the interaction between vWF and GPIb 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]. The amplification of these genetic determinants can be carried out using the plasmid pET-8c52K, for example by the PCR amplification technique, using as primer oligodeoxynucleotides encoding contiguous residues localized on either side of the sequence to be amplified.
The amplified fragments are then cloned into vectors of the M 13 type for their verification by sequencing using either the universal primers situated on either side of the multiple cloning site, or oligodeoxynucleotides specific for the amplified region of the vWF gene of which the sequence of several isomorphs is known Sadler J. E. et al., [Proc. Natl. Acad. Sci. 82 (1985) 6394-6398]; Verweij C.
L. et al., [EMBO J. 5 (1986) 1839-1847]; Shelton-Inloes B. B. et al., [Biochemistry (1986) 3164-3171]; Bonthron D. et al., [Nucleic Acids Res. 17 (1986) 7125-7127].
Thus, the PCR amplification of the plasmid pET-8c52K with the oligodeoxynucleotides 5'-CCCGGGATCCCTTAGGCTTAACCTGTGAAGCCTG
C-3' (Sq1969, the MstII site is underlined) and 5'-CCCGGGATCCAAGCTTA-GACTTGTGCCATGTCG-3' (Sq2029, the Hindlll site is underlined) generates an MstII-HindI1l restriction fragment including the Thr470 to Va1713 residues of vWF
(FIG. 4, panel E). The ligation of this fragment to the HindI1I-MstII
restriction fragment corresponding to the entire gene encoding HSA, with the exception of the three C-terminal most amino acids (cf. FIG. 2) generates a Hindlll restriction fragment containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro" export region of HSA. This restriction fragment is cloned in the productive orientation and into the Hindlll site of the plasmid pYG105, which generates the expression plasmid pYG 1248 (HSA-vWF470-713).
E.7.1.2. Molecular Variants:
In another embodiment, the binding site of vWF is a peptide including the Thr470 to Asp498 residues of the mature vWF. This sequence including 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 the GPIb of the human platelets. The sequence corresponding to the peptide G10 is first included in an MstII-Hindlll restriction fragment (FIG. 4, panel B), for example by PCR amplification of the plasmid pET-8c52K with the oligodeoxynucleotides Sq1969 and 5'-CCCGGGATCCAAGCTTAGTCCTCCACATACAG-3' (Sq1970, the HindIII site is underlined), which generates an MstII-HindI11 restriction fragment including the peptide G 10, and whose sequence is: 5'-CCTTAGGCTTAACCTGTGAAGCCTGCCAGGAGCCGGGAGGCCTGGT-GGTGCCTCCCACAGATGCCCCGGTGAGCCCCACCACTCTGTA-TGTGGAGGACTAAGCTT-3' (the sequence encoding the peptide G10 is in bold characters). The ligation of this fragment to the Hindlll-Mstll restriction fragment corresponding to the entire gene encoding HSA, with the exception of the three C-terminal most amino acids (cf. FIG. 2) generates a HindIII restriction fragment containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro" export region of HSA.
This restriction fragment is cloned in the productive orientation into the HindIIl site of the plasmid pYG105, which generates the expression plasmid pYG1214.
In another embodiment, the site for binding of vWF to GPlb is directly designed with the aid of synthetic oligodeoxynucleotides, and for example the oligodeoxynucleotides 5'-TTAGGCCTCTGTGACCTTGCCCCTGA-AG-CCCCTCCTCCTACTCTGCCCCCCTAAGCTTA-3' (SEQ ID NO:26) and 5'-GATCTAAG-CTTAGGGGGGCAGAGTAGGAGGAGGGGCTTCAGGG-GCAAGGTCACAGAGGCC-3' (SEQ ID NO:27). These oligodeoxynucleotides form, by pairing, a Mstll-Bg1Il restriction fragment including the MstII-Hindlll fragment (FIG. 4, panel C) corresponding to the peptide D5 defined by the Leu694 to Pro708 residues of vWF. The ligation of the MstII-HindIII fragment to the HindI1l-MstII restriction fragment corresponding to the entire gene encoding HSA
with the exception of the three C-terminal most amino acids (cf. FIG. 2) generates a Hindlll restriction fragment containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro" export region of HSA. This restriction fragment is cloned in the productive orientation into the Hindlll site of the plasmid pYG 105, which generates the expression plasmid pYG1206.
Useful variants of the plasmid pET-8c52K are deleted by site-directed mutagenesis between the peptides GIO and G5, for example sites for binding to collagen, and/or to heparin, and/or to botrocetin, and/or to sulphatides and/or to ristocetin. One example is the plasmid pMMB9 deleted by site-directed mutagenesis between the residues Cys509 and I1e662. The PCR amplification of this plasmid with the oligodeoxynucleotides Sq1969 and Sq2029 generates an Mstll-HindIIl restriction fragment (FIG. 4, panel D) including the Thr470 to Tyr508 and Arg663 to Va1713 residues and in particular the peptides G10 and D5 of vWF and deleted in particular of its site for binding to collagen localized between the residues G1u542 and Met622 Roth G. J. et al., [Biochemistry 25 (1986) 8357-8361]. The ligation of this fragment to the HindIII-Mst11 restriction fragment corresponding to the entire gene encoding HSA, with the exception of the three C-terminal most amino acids (cf. FIG. 2) generates a Hindlll restriction fragment containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro" export region of HSA.
This restriction fragment is cloned in the productive orientation into the Hindlll site of the plasmid pYG105, which generates the expression plasmid pYG1223.
In other embodiments, the use of combined techniques of site-directed mutagenesis and PCR amplification makes it possible to generate at will variants of the MstII-HindIIl restriction fragment of panel A of FIG. 4 but deleted of one or more sites for binding to sulphatides and/or to botrocetin and/or to heparin and/or to collagen, and/or substituted by any residue involved in the vWF-associated emergence of IIB type pathologies.
In other useful variants of the plasmid pET-8c52K, mutations are introduced, for example by site-directed mutagenesis, in order to replace or suppress all or part of the set of cysteines present at positions 471, 474, 509 and 695 of the human vWF. Specific examples are the plasmids p5E and p7E in which the cysteins present at positions 471 and 474, on the one hand, and at positions 471, 474, 509 and 695, on the other hand, have been respectively replaced by glycine residues. The PCR amplification of these plasmids with the oligodeoxynucleotides Sq2149 (5'-CCCGGGATCCCTTAGGCTTAACCGGTGAAGCCGGC-3' (SEQ ID
NO:28), the MstII site is underlined) and Sq2029 makes it possible to generate MstII-HindIlI restriction fragments including the Thr470 to Va1713 residues of the natural vWF with the exception that at least the cystein residues at positions and 474 were mutated to glycine residues. The ligation of these fragments to the HindIII-MstII restriction fragment corresponding to the entire gene encoding HSA
with the exception of the three C-terminal most amino acids (cf. FIG. 2) generates a HindIIl restriction fragment containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro" export region of HSA. These restriction fragments are cloned in the productive orientation into the HindIII site of the plasmid pYG105, which generates the expression plasmids pYG1283 (chimera HSA-vWF470-713, C471G, C474G) and pYG1279 (chimera HSA-vWF470-713, C471G, C474G, C509G, C695G).
Other particularly useful mutations affect at least one residue involved in vWF-associated type IIB pathologies (increase in the intrinsic affinity of vWF
for GP1b), such as the residues Arg543, Arg545, Trp550, Va1551, Va1553, Pro574 or Arg578 for example. The genetic recombination techniques in vitro also make it possible to introduce at will one or more additional residues into the sequence of vWF and for example a supernumerary methionine between positions Asp539 and G1u542.
E.7.2. Fragments Antagonizing the Binding of vWF to the Sub-Endothelium In a specific embodiment, the sites for binding of vWF to the components of the sub-endothelial tissue, and for example collagen, are generated by PCR
amplification of the plasmid pET-8c52K, for example with the oligodeoxynucleotides Sq2258 (5'-GGATCCTTAGGGCT-GTGCAGCAGGCTACTGGACCTGGTC-3', the Mstll site is underlined) and Sq2259 (5'-GAATTCAAGCTTAACAGAGGTAGCTAA-CGATCTCGTCCC-3', the Hindlll site is underlined), which generates an MstII-HindIIl restriction fragment encoding the Cys509 to Cys695 residues of the natural vWF. Deletion molecular variants or modified variants are also generated which contain any desired combination between the sites for binding of vWF to the sulphatides and/or to botrocetin and/or to heparin and/or to collagen and/or any residue responsible for a modification of the affinity of vWF for GPIb (vWF-associated type II
pathologies). In another embodiment, the domain capable of binding to collagen may also come from the vWF fragment which is between the residues 911 and 1114 and described by Pareti et al. [J. Biol. Chem. (1987) 262: 13835-13841].
The ligation of these fragments to the HindIIl-MstII restriction fragment corresponding to the entire gene encoding HSA with the exception of the three C-terminal most amino acids (cf. FIG. 2) generates Hindlll restriction fragments containing a hybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the "prepro" export region of HSA. These restriction fragments are cloned in the productive orientation into the Hindlll site of the plasmid pYG105, which generates the corresponding expression plasmids, and for example the plasmid pYG1277 (HSA-vWF509-695).
E.7.3. Purification and Molecular Characterization of the Chimeras Between HSA and vWF
The chimeras present in the culture supernatants corresponding to the CBS
293.91 strain transformed, for example with the expression plasmids according to Examples E.7.1. and E.7.2., are characterized in a first instance by means of antibodies specific for the HSA part and for the vWF part. The results of FIGS. 5 to 7 demonstrate that the yeast K. lactis is capable of secreting chimeric proteins between HSA 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) and then concentrated by ultrafiltration (Amicon) using a membrane whose discrimination threshold is situated at 30 kDa. The concentrate obtained is then dialysed against a Tris-HCl solution (50 mM pH 8) and then purified on a column. For example, 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). A purification by ion-exchange chromatography can also be used. For example, in the case of the chimera HSA-vWF470-713, the concentrate obtained after ultrafiltration is dialysed against a Tris-HC1 solution (50 mM pH 8), and then loaded in 20 ml fractions onto a cation-exchange column (5 ml) (S Fast Flow, Pharmacia) equilibrated in the same buffer. The column is then washed several times with the Tris-HC1 solution (50 mM pH 8) and the chimeric protein is then eluted from the column by an NaC1 gradient (0 to 1M). The fractions containing the chimeric protein are then pooled and dialysed against a 50 mM Tris-HCI solution (pH 8) and then reloaded onto the S Fast Flow column. After elution of the column, the fractions containing the protein are pooled, dialysed against water and freeze-dried before characterization:
for example, sequencing (Applied Biosystem) of the protein [HSA-vWF470-704 C471G, C474G] secreted by the yeast CBS 293.91 gives the N-terminal sequence expected for HSA (Asp-Ala-His ...), demonstrating a correct maturation of the chimera immediately at the C-terminus of the doublet of residues Arg-Arg of the "pro" region of HSA (FIG. 2). The essentially monomeric character of the chimeric proteins between HSA and vWF is also confirmed by their elution profile on a TSK
3000 column [Toyo Soda Company, equilibrated with a cacodylate solution (pH 7) containing 0.2M Na2SO4]: for example the chimera [HSA-vWF 470-704 C471G, C474G] behaves under the conditions like a protein with an apparent molecular weight of 95 kDa, demonstrating its monomeric character.
EXAMPLE 8: CHIMERAS DERIVED FROM UROKINASE
E.8.1. Constructs A fragment corresponding to the amino-terminal fragment of urokinase (ATF: EGF-like domain + kringle domain) can be obtained from the corresponding messenger RNA of cells of certain human carcinoma, for example using the RT-PCR kit distributed by Pharmacia. An MstI1-HindIII restriction fragment including the ATF of human urokinase is given in FIG. 8. The ligation of the HindI11-MstII
fragment of the plasmid pYG404 to this MstII-HindIII fragment makes it possible to generate the Hindlll fragment of the plasmid pYG1341 which encodes a chimeric protein in which the HSA molecule is genetically coupled to the ATF
(HSA-UK1-*135). Likewise, the plasmid pYG1340 contains a Hindlll fragment encoding a chimera composed of HSA immediately followed by the first 46 residues of human urokinase (HSA-UK1-46, cf. FIG. 8). The cloning in the productive orientation, of the Hindlll restriction fragment of the plasmid pYG
(HSA-UK1-46) into the Hindlll site of the plasmids pYG105 (LAC4) and pYG106 (PGK) generates the expression plasmids pYG1343 and pYG1342 respectively. Likewise, the cloning, in the productive orientation, of the Hindlll restriction fragment of the plasmid pYG1341 (HSA-UK1--->135) into the Hindlll site of the plasmids pYG105 (LAC4) and pYG106 (PGK) generates the expression plasmids pYG1345 and pYG1344 respectively.
E.8.2. Secretion of the Hybrids After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the mature form of the chimeric proteins HSA-UK. A few clones corresponding to the strain K. lactis CBS
293.91, which is transformed with the expression plasmids according to Example E.9.1., are incubated in selective complete liquid medium at 28 C. The cellular supernatants are then tested after electrophoresis on an 8.5% acrylamide gel, either directly by staining of the gel with coomassie blue, or after immunoblotting using as primary antibodies a rabbit polyclonal serum directed against human albumin or against human urokinase. The results of FIG. 9 demonstrate that the hybrid proteins HSA-UK1--46 and HSA-UK1-*135 are particularly well secreted by the yeast Kluyveromyces.
E.8.3 Purification of the Chimeras Between HSA and Urokinase After centrifugation of a culture of the CBS 293.91 strain transformed with the expression plasmids according to Example E.8.1., the culture supernatant is passed through a 0.22 mm filter (Millipore) and then concentrated by ultrafiltration (Amicon) using a membrane whose discrimination threshold is situated at 30 kDa.
The concentrate obtained is then adjusted to 50 mM Tris-HCl starting with a stock solution of 1M Tris-HCi (pH 7), and then loaded in 20 ml fractions onto an anion-exchange column (3 ml) (D-Zephyr, Sepracor) equilibrated in the same buffer.
The chimeric protein (HSA-UK1--+46 or HSA-UK1-135) is then eluted from the column by a gradient (0 to 1M) of NaCI. The fractions containing the chimeric protein are then pooled and dialysed against a 50 mM Tris-HC1 solution (pH 6) and reloaded onto a D-Zephyr column equilibrated in the same buffer. After elution of the column, the fractions containing the protein are pooled, dialysed against water and freeze-dried before characterization of their biological activity and especially with respect to their ability to displace urokinase from its cellular receptor.
EXAMPLE 9: CHIMERAS DERIVED FROM G-CSF
E.9.1. Constructs E.9.1.1. Coupling at the C-terminus of HSA.
An MstII-HindIII restriction fragment including the mature form of human G-CSF is generated, for example according to the following strategy: a Kpnl-HindIII restriction fragment is first obtained by the enzymatic PCR
amplification technique using the oligodeoxynucleotides Sq2291 (5'-CAAGGATCCAAGCTTCAGGGCTGCGCAAGGTGGCGTAG-3', the HindIII
site is underlined) and Sq2292 (5'-CGGGGTACCTTAGGCTTAACCCCCCTG-GGCCCTGCCAGC-3', the KpnI site is underlined) as primer on the plasmid BBG13 serving as template. The plasmid BBG13 contains the gene encoding the B
form (174 amino acids) of mature human G-CSF, which is obtained from British Bio-technology Limited, Oxford, England. The enzymatic amplification product of about 550 nucleotides is then digested with the restriction enzymes KpnI and HindIII and cloned into the vector pUC19 cut with the same enzymes, which generates the recombinant plasmid pYG1255. This plasmid is the source of an MstII-HindlII restriction fragment which makes it possible to fuse G-CSF
immediately downstream of HSA (chimera HSA-G.CSF) and whose nucleotide sequence is given in FIG. 10.
It may also be desirable to insert a peptide linker between the HSA
part and G-CSF, for example in order to permit a better functional presentation of the transducing part. An MstII-HindIII restriction fragment is for example generated by substitution of the MstII-Apal fragment of the plasmid pYG1255 by the oligodeoxynucleotides Sq2742 (5'-TTAGGCTTA-GGTGGTGGCGGTACCCCCCTGGGCC-3', the codons encoding the glycine residues of this particular linker are underlined) and Sq2741 (5'-CAGGGGGGTACCGCCACCACCTAAGCC-3') which form, by pairing, an MstII-Apa1 fragment. The plasmid thus generated therefore contains an MstH-HindIII restriction fragment whose sequence is identical to that of FIG. 10 with the exception of the MstII-Apal fragment.
The ligation of the HindIIl-MstII fragment of the plasmid pYG404 to the MstII-HindIII fragment of the plasmid pYG1255 makes it possible to generate the Hindlll fragment of the plasmid pYG1259 which encodes a chimeric protein in which the B form of the mature G-CSF is positioned by genetic coupling in translational phase at the C-terminus of the HSA molecule (HSA-G.CSF).
An identical Hindlll restriction fragment, with the exception of the MstII-Apal fragment, may also be easily generated and which encodes a chimeric protein in which the B form of the mature G-CSF is positioned by genetic coupling in translational phase at the C-terminus of the HSA molecule and a specific peptide linker. For example, this linker consists of 4 glycine residues in the Hindlll fragment of the plasmid pYG1336 (chimera HSA-Gly4-G.CSF).
The Hindlll restriction fragment of the plasmid pYG1259 is cloned in the productive orientation and into the Hindlll restriction site of the expression plasmid pYG105, which generates the expression plasmid pYG1266 (HSA-G.CSF). In another exemplification, the cloning of the HindIII restriction fragment of the plasmid pYG1259 in the productive orientation and into the Hindlll site of the plasmid pYG 106 generates the plasmid pYG 1267. The plasmids pYG 1266 and pYG1267 are mutually isogenic with the exception of the SalI-HindIII
restriction fragment encoding the LAC4 promoter of K. lactis (plasmid pYG1266) or the PGK
promoter of S. cerevisiae (plasmid pYG1267).
In another exemplification, the cloning in the productive orientation of the Hindlll restriction fragment of the plasmid pYG1336 (chimera HSA-Gly4-G.CSF) into the Hindlll site of the plasmids pYG105 (LAC4) and pYG106 (PGK) generates the expression plasmids pYG1351 and pYG1352 respectively.
E.9.1.2. Coupling at the N-terminus of HSA
In a specific embodiment, the combined techniques of site-directed mutagenesis and PCR amplification make it possible to construct hybrid genes encoding a chimeric protein resulting from the translational coupling between a signal peptide (and for example the prepro region of HSA), a sequence including a gene having a G-CSF activity, and the mature form of HSA or one of its molecular variants (cf. chimera of panel B, FIG. 1). These hybrid genes are preferably bordered in 5' of the translational initiator ATG and in 3' of the translational stop codon by HindIII restriction sites. For example the oligodeoxynucleotide Sq2369 (5'-GTTCTACGCCACCTTG-CGCAGCCCGGTGGAGGCGGTGATGCACACAAGAGTGAGGTTGCTCAT-CGG-3' the residues underlined (optional) correspond in this particular chimera to a peptide linker composed of 4 glycine residues) makes it possible, by site-directed mutagenesis, to put in translational phase the mature form of the human G-CSF
of the plasmid BBG13 immediately upstream of the mature form of HSA, which generates the intermediate plasmid A. Likewise, the use of the oligodeoxynucleotide Sq2338 [5'-CAGGGAGCTGGCAGGGCCCAGGGGG-GTTCGACGAAACACACCCCTGGAATAAGCCGAGCT-3' (non-coding strand), the nucleotides complementary to the nucleotides encoding the first N-terminal residues of the mature form of the human G-CSF are underlined] makes it possible, by site-directed mutagenesis, to couple in translational reading phase the prepro region of HSA immediately upstream of the mature form of the human G-CSF, which generates the intermediate plasmid B. A HindIII fragment encoding a chimeric protein of the PEPTIDE-HSA type (cf. FIG. 1, panel B) is then generated by combining the HindIIl-Sstl fragment of the plasmid B(joining prepro region of HSA+N-terminal fragment of the mature G-CSF) with the Sstl-HindII1 fragment of the plasmid A[joining mature G-CSF-(glycine)ic4 - mature HSA]. The plasmid pYG1301 contains this specific HindIII restriction fragment encoding the chimera G.CSF-Gly4-HSA fused immediately downstream of the prepro region of HSA
(FIG. 11). The cloning of this HindIII restriction fragment in the productive orientation and into the HindIII site of the plasmids pYG105 (LAC4) and pYG106 (PGK) generates the expression plasmids pYG1302 and pYG 1303 respectively.
E.9.2. Secretion of the Hybrids.
After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the mature form of the chimeric proteins between HSA and G-CSF. A few clones corresponding to the strain K.
lactis CBS 293.91 transformed with the plasmids pYG1266 or pYG1267 (HSA-G.CSF), pYG1302 or pYG1303 (G.CSF-G1y4-HSA) or alternatively pYG1351 or pYG1352 (HSA-G1y4-G.CSF) are incubated in selective complete liquid medium at 28 C. The cellular supernatants are then tested after electrophoresis on an 8.5%
acrylamide gel, either directly by staining the gel with coomassie blue, or after immunoblotting using as primary antibodies rabbit polyclonal antibodies directed against the human G-CSF or a rabbit polyclonal serum directed against human albumin. The results of FIG. 12 demonstrate that the hybrid protein HSA-G.CSF
is recognized both by antibodies directed against human albumin (panel C) and human G-CSF (panel B). The results of FIG. 13 indicate that the chimera HSA-Gly4-G.CSF (lane 3) is particularly well secreted by the yeast Kluyveromyces, possibly because of the fact that the presence of the peptide linker between the HSA part and the G-CSF part is more favourable to an independent folding of these 2 parts during the transit of the chimera in the secretory pathway.
Furthermore, the N-terminal fusion (G.CSF-G1y4-HSA) is also secreted by the yeast Kluyveromyces (FIG. 13, lane 1).
E.9.3. Purification and Molecular Characterization of the Chimeras Between HSA and G-CSF.
After centrifugation of a culture of the CBS 293.91 strain transformed with the expression plasmids according to Example E.9.1., the culture supernatant is passed through a 0.22 mm filter (Millipore) and then concentrated by ultrafiltration (Amicon) using a membrane whose discrimination threshold is situated at 30 kDa.
The concentrate obtained is then adjusted to 50 mM Tris-HC1 from a 1M stock solution of Tris-HCI (pH 6), and then loaded in 20 ml fractions onto an ion-exchange column (5 ml) (Q Fast Flow, Pharmacia) equilibrated in the same buffer.
The chimeric protein is then eluted from the column by a gradient (0 to 1M) of NaCI. The fractions containing the chimeric protein are then pooled and dialysed against a 50 mM Tris-HCl solution (pH 6) and reloaded onto a Q Fast Flow column (1 ml) equilibrated in the same buffer. After elution of the column, the fractions containing the protein are pooled, dialysed against water and freeze-dried before characterization: for example, the sequencing (Applied Biosystem) of the protein HSA-G.CSF secreted by the yeast CBS 293.91 gives the N-terminal sequence expected for HSA (Asp-Ala-His ...), demonstrating a correct maturation of the chimera immediately at the C-terminus of the doublet of residues Arg-Arg of the "pro" region of HSA (FIG. 2).
EXAMPLE 10: CHIMERAS DERIVED FROM AN IMMUNOGLOBULIN
E.10.1. Constructs An Fv' fragment can be constructed by genetic engineering techniques, and which encodes the variable fragments of the heavy and light chains of an immunoglobulin (Ig), linked to each other by a linker peptide Bird et al., Science (1988) 242: 423; Huston et al., (1988) [Proc. 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 messenger RNA of the corresponding hybridoma, for example using the RT-PCR kit distributed by Pharmacia (Mouse ScFv module). In a second stage, the variable regions are genetically coupled by genetic engineering via a synthetic linkage peptide and for example the linker (GGGGS)x3. An MstII-HindIIl restriction fragment including the Fv' fragment of an immunoglobulin secreted by a murine hybridoma is given in FIG. 14. The ligation of the Hindlll-MstII fragment of the plasmid pYG404 to this MstII-HindIIl fragment makes it possible to generate the HindIII fragment of the plasmid pYG1382 which encodes a chimeric protein in which the HSA molecule is genetically coupled to the Fv' fragment of FIG. 14 (chimera HSA-Fv'). The cloning in the productive orientation of the HindIII restriction fragment of the plasmid pYG1382 into the HindIII site of the plasmids pYG105 (LAC4) and pYG106 (PGK) generates the expression plasmids pYG1383 and pYG1384 respectively.
E.10.2. Secretion of the Hybrids After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the mature form of the chimeric protein HSA-Fv'. A few clones corresponding to the strain K. lactis CBS 293.91 transformed with the plasmids pYG1383 or pYG1384 (HSA-Fv') are incubated in selective complete liquid medium at 28 C. The cellular supernatants are then tested after electrophoresis on an 8.5% acrylamide gel, either directly by staining of the gel with coomassie blue, or after immunoblotting using as primary antibodies a rabbit polyclonal serum directed against human albumin, or directly incubated with biotinylated antibodies directed against the immunoglobulins of murine origin.
The results of FIG. 15 demonstrate that the hybrid protein HSA-Fv' is recognized both by antibodies directed against human albumin (panel C) and reacts with biotinylated goat antibodies which are immunologically reactive towards mouse immunoglobulins (panel B).
EXAMPLE 11: BIOLOGICAL ACTIVITY OF THE CHIMERAS
E.11.1. Biological Activity In vitro.
E.11.1.1. Chimeras Between HSA and vWF.
The antagonistic activity of the products is determined by measuring the dose-dependent inhibition of the agglutination of human platelets fixed with paraformaldehyde according to the method described by Prior et al.
[Bio/Technology (1992) 10: 66]. The measurements are carried out in an aggregameter (PAP-4, Bio Data, Horsham, Pa., U.S.A.) which records the variations over time of the optical transmission, with stirring, at 37 C. in the presence of vWF, of botrocetin (8.2 mg/ml) and of the test product at various dilutions (concentrations). For each measurement, 400 ml (8 x 107 platelets) of a suspension of human platelets stabilized with paraformaldehyde (0.5%, and then resuspended in [NaCI (137 mM); MgC12 (1 mM); NaH2PO4 (0.36 mM); NaHCO3 (10 mM); KCl (2.7 mM); glucose (5.6 mM); HSA (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 and are then supplemented with 30 ml of the solution of the test product at various dilutions in apyrogenic formulation vehicle [mannitol (50 g/1); citric acid (192 mg/1); L-lysine monohydrochloride (182.6 mg/1); NaCI (88 mg/1); pH adjusted to 3.5 by addition of NaOH (1M)], or formulation vehicle alone (control assay).
The resulting suspension is then incubated for 1 min at 37 C. and 12.5 ml of human vWF [American Bioproducts, Parsippany, N.J., U.S.A.; 11% von Willebrand activity measured according to the recommendations for the use of PAP-4 (Platelet Aggregation ProfilerRTM) with the aid of platelets fixed with formaldehyde (2 x 105 platelets/ml), human plasma containing 0 to 100% vWF and ristocetin (10 mg/ml, cf. p. 36-45: vW ProgramTM] are added and incubated at 37 C. for 1 min before adding 12.5 ml of botrocetin solution purified from freeze-dried venom of Bothrops jararaca (Sigma) according to the procedure described by Sugimoto et al., [Biochemistry (1991) 266: 18172]. The recording of the reading of the transmission as a function of time is then carried out for 2 min with stirring by means of a magnetic bar (Wellcome Distriwell) placed in the tank and with a magnetic stirring of 1,100 rpm provided by the aggregameter. The mean variation of the optical transmission (n35 for each dilution) over time is therefore a measurement of the platelet agglutination due to the presence of vWF and botrocetin, in the absence or in the presence of variable concentrations of the test product. From such recordings, the % inhibition of the platelet agglutination due to each concentration of product is then determined and the straight line giving the % inhibition as a function of the reciprocal of the product dilution in log-log scale is plotted. The IC50 (or concentration of product causing 50% inhibition of the agglutination) is then determined on this straight line. The table of FIG. 6 compares the IC50 values of some of the HSA-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) 10: 66] and included in the assays as standard value. Identical tests for the inhibition of the agglutination of human platelets in the presence of vWF of pig plasma (Sigma) makes it possible, furthermore, to demonstrate that some of the hybrids of the present invention, and especially some type IIB variants, are very good antagonists of platelet agglutination in the absence of botrocetin-type cofactors. The botrocetin-independent antagonism of these specific chimeras can also be demonstrated according to the procedure initially described by Ware et al. [Proc. Natl.
Acad. Sci.
(1991) 88: 2946] by displacing the monoclonal antibody 125I-LJ-1B 1(10 mg/ml), a competitive inhibitor of the binding of vWF to the platelet GPIb Handa M. et al., (1986) [J. Biol. Chem. 261: 12579] after 30 min of incubation at 22 C. in the presence of fresh platelets (108 platelets/ml).
E.11.1.2. Chimeras between HSA and G-CSF
The purified chimeras are tested for their capacity to permit the in vitro proliferation of the IL3-dependant murine line NFS60, by measuring the incorporation of tritiated thymidine essentially according to the procedure described by Tsuchiya et al. [Proc. Natl. Acad. Sci. (1986) 83 7633]. For each chimera, the measurements are carried out between 3 and 6 times in a three-point test (three dilutions of the product) in a zone or the relation between the quantity of active product and incorporation of labelled thymidine (Amersham) is linear.
In each microtitre plate, the activity of a reference product consisting of recombinant human G-CSF expressed in mammalian cells is also systematically incorporated.
The results of FIG. 17 demonstrate that the chimera HSA-G.CSF (pYG 1266) secreted by the yeast Kluyveromyces and purified according to Example E.9.3.
is capable in vitro of transducing a signal for cellular proliferation for the line NFS60.
In this particular case, the specific activity (cpm/molarity) of the chimera is about 7 times lower than that of the reference G-CSF (non-coupled).
E.11.2. Biological Activity In vivo The activity of stimulation of the HSA-G-CSF chimeras on granulopoiesis in vivo is tested after subcutaneous injection in rats (Sprague-Dawley/CD, 250-300g, 8-9 weeks) and compared to that of the reference G-CSF expressed using mammalian cells. Each product, tested at the rate of 7 animals, is injected subcutaneously into the dorso-scapular region at the rate of 100 ml for 7 consecutive days, (D1-D7). 500 ml of blood are collected on days D-6, D2 (before the 2nd injection). D5 (before the 5th injection) and D8, and a blood count is performed. In this test, the specific activity (neutropoiesis units/mole injected) of the chimera HSA-G.CSF (pYG1266) is identical to that of the reference G-CSF
(FIG. 18). Since this specific chimera has in vitro a specific activity 7 times lower than that of the reference G-CSF (FIG. 17), it is therefore demonstrated that the genetic coupling of G-CSF onto HSA favourably modifies the pharmacokinetic properties thereof.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANTS:
(A) NAME: Rhone-Poulenc Rorer S.A.
(B) STREET: 20 Raymond ARON Avenue (C) CITY: Antony (E) COUNTRY: France (F) POSTAL CODE:92165 (ii) TITLE OF THE INVENTION: Novel Biologically Active Polypeptides, Preparation Thereof and Pharmaceutical Composition Containing Said Polypeptides (iii)NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS:
(A) NAME: MBM & Co.
(B) STREET: P.O. Box 809 (C) CITY: Ottawa (D) PROVINCE: ON
(E) COUNTRY: Canada (F) POSTAL CODE: K1P 5P9 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy Disk (B) COMPUTER: IBM-PC Compatible (C) OPERATING SYSTEM: Windows (D) SOFTWARE: Word (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,126,091 (B) FILING DATE: January 28, 1993 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SWAIN, Margaret (B) REGISTRATION NUMBER: 10926 (C) REFERENCE/DOCKET NUMBER:
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 613-567-0762 (B) TELEFAX: 613-563-7671 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1859 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no (iii) ANTISENSE: no (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 26..1855 (D) OTHER INFORMATION: chimera of type HSA-peptide (ix) FEATURE:
(A) NAME/KEY: miscfeature (B) LOCATION: 1842-1848 (D) OTHER INFORMATION: /standard name = "MstII Site"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser AAA CTG AAG GAA TGC TGT GAA AAA CCT CTG TTG GAA AAA TCC CAC TGC
Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu leu Glu Lys Ser His Cys 964 Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr GAA ACC ACT CTA GAG AAG TGC TGT GCC GCT GCA GAT CCT CAT GAA TGC 1209:
Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asn Ile Cys Thr Leu Ser Glu Lys Glu Arg Gin Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala GCA AGT CAA GGT GGC TTA GGC TTA (NNN)p TAAGCTT 18513 Ala Ser Gln Ala Ala Leu Gly Leu peptide (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 750 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no (iii) ANTISENSE: no (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..746 (D) OTHER INFORMATION:/product= "C-ter[ninal fragment of the HSA-vWF470 chimera"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Leu Gly Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser Glu Pro Pro Leu His Asp Phe Tyr Cys Ser Arg Leu Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe Glu Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu Arg Ile Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys Ile Asp Arg Pro Glu Ala Ser Arg Ile Ala Leu Leu Leu Met Ala Ser Gln Glu Pro Gln Arg Met Ser Arg Asn Phe Val Arg Tyr Val Gln Gly Leu Lys Lys Lys Lys Val Ile Val Ile Pro Val Gly Ile Gly Pro His Ala Asn Leu Lys Gln Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu Asn Lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln Arg Asp Glu Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala Pro Pro Pro Thr Leu Pro Pro Asp Met Ala Gln Val (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 423 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no (iii) ANTISENSE: no (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..419 (D) OTHER INFORMATION:/product = "C-terminal fragment of the HSA-UK1-135 chimera"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Leu Gly Leu Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn Cys Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile Asp Lys Ser Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val Leu Gin Gln Thr Tyr His Ala His Arg Ser Asp Ala Leu Gln Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met Val His Asp Cys Ala Asp Gly Lys (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 541 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no (iii) ANTISENSE: no (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..536 (D) OTHER INFORMATION: /product = "C-terminal fragment of the HSA-G.CSF chimera"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Leu Gly Leu Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gin Ala Leu Giu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Giy Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2455 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no (iii) ANTISENSE: no (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 26..2389 (D) OTHER INFORMATION:/product ="G.CSF-Gly4-HSA chimera downstream of`
HSA prepro region"
(ix) FEATURE:
(A) NAME/KEY: miscrecomb (B) LOCATION: 620-631 (D) OTHER INFORMATION: /standard name = "linker PolyGly"
(ix) FEATURE:
(A) NAME/KEY: miscfeature (B) LOCATION: 106-111 (D) OTHER INFORMATION: /standard name = "ApaI site"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala Tyr Ser Arg Gly Val Phe Arg Arg Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Gly Gly Gly Gly Asp Ala His Lys Ser, Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys AGG TAT AAA GCT GCT TTT ACA GAA TGT TGC CAA GCT GCT GAT AAA GCT 115Ei Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly GAA AGA GCT TTC AAA GCA TGG GCA GTA GCT CGC CTG AGC CAG AGA TTT 130C) Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 756 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no (iii) ANTISENSE: no (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..752 (D) OTHER INFORMATION:/product ="C-terminal fragment of the HSAFv chimera"
(D) OTHER INFORMATION: /standard_name = "Synthetic linker"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Leu Gly Leu Gln Val Gln Leu Glu Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Arg Ser Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Lys Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Val Gly Ser Ala Val Tyr Phe Cys Ala Lys Glu Asn Asn Arg Phe Asp Glu Arg Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ile Gln Leu Thr Gln Ser Pro Asn Ser Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asp Thr Ser Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Ser Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Claims (45)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A recombinant albumin fusion polypeptide comprising one or more therapeutically active polypeptides fused to an albumin or a variant thereof, wherein one or more of said therapeutically active polypeptides is a hormone or a therapeutically active fragment thereof and wherein said albumin fusion polypeptide has a higher plasma half-life than said hormone when not fused.
2. A recombinant albumin fusion polypeptide comprising one or more therapeutically active polypeptides fused to an albumin or a variant thereof, wherein one or more of said therapeutically active polypeptides is an interferon or a therapeutically active fragment thereof and wherein said albumin fusion polypeptide has a higher plasma half-life than said interferon when not fused.
3. A recombinant albumin fusion polypeptide comprising one or more therapeutically active polypeptides fused to an albumin or a variant thereof, wherein one or more of said therapeutically active polypeptides is an interleukin or a therapeutically active fragment thereof and wherein said albumin fusion polypeptide has a higher plasma half-life than said interleukin when not fused.
4. A recombinant albumin fusion polypeptide comprising one or more therapeutically active polypeptides fused to an albumin or a variant thereof, wherein one or more of said therapeutically active polypeptides is insulin or a therapeutically active fragment thereof and wherein said albumin fusion polypeptide has a higher plasma half-life than insulin when not fused.
5. A recombinant albumin fusion polypeptide comprising one or more therapeutically active polypeptides fused to an albumin or a variant thereof, wherein one or more of said therapeutically active polypeptides is an erythropoietin or a therapeutically active fragment thereof and wherein said albumin fusion polypeptide has a higher plasma half-life than said erythropoietin when not fused.
6. A recombinant albumin fusion polypeptide comprising one or more therapeutically active polypeptides fused to an albumin or a variant thereof, wherein one or more of said therapeutically active polypeptides is a granulocyte colony-stimulating factor (G-CSF) or a therapeutically active fragment thereof and wherein said wherein said albumin fusion polypeptide has a higher plasma half-life than said G-CSF when not fused.
7. The recombinant albumin fusion polypeptide according to any one of Claims 1 to 6, wherein said one or more therapeutically active polypeptide is of human origin.
8. The recombinant albumin fusion polypeptide according to any one of Claims 1 to 7, wherein said one or more therapeutically active polypeptides is selected from the group of:
a) a full-length polypeptide;
b) a therapeutically active fragment of (a); and c) a therapeutically active variant of (a) or (b) obtained by one or more structural modification selected from the group of: a mutation, a substitution, an addition and a deletion of one or more residues.
a) a full-length polypeptide;
b) a therapeutically active fragment of (a); and c) a therapeutically active variant of (a) or (b) obtained by one or more structural modification selected from the group of: a mutation, a substitution, an addition and a deletion of one or more residues.
9. The recombinant albumin fusion polypeptide according to any one of Claims 1 to 8, wherein the albumin or the variant thereof is selected from the group of:
a) a mature albumin;
b) an albumin;
c) a fragment from (a) or (b); and d) a variant of (a) or (b) obtained by one or more structural modification selected from the group of: a mutation, a substitution, an addition and a deletion of one or more residues, wherein said fragment or variant has a high plasma half-life.
a) a mature albumin;
b) an albumin;
c) a fragment from (a) or (b); and d) a variant of (a) or (b) obtained by one or more structural modification selected from the group of: a mutation, a substitution, an addition and a deletion of one or more residues, wherein said fragment or variant has a high plasma half-life.
10. The recombinant albumin fusion polypeptide according to any one of Claims 1 to 9, wherein said recombinant albumin fusion polypeptide comprises a N-terminal methionine.
11. The recombinant albumin fusion polypeptide according to any one of Claims 1 to 10, wherein said recombinant albumin fusion polypeptide comprises a linker peptide.
12. The recombinant albumin fusion polypeptide according to any one of Claims 1 to 11, wherein said recombinant albumin fusion polypeptide comprises a secretion signal sequence.
13. The recombinant albumin fusion polypeptide according to Claim 12, wherein said secretion signal sequence is a natural secretion signal sequence of said therapeutically active polypeptide.
14. The recombinant albumin fusion polypeptide according to any one of Claims 1 to 13, wherein one or more of said therapeutically active polypeptides is coupled to the N-terminus of the albumin or variant thereof.
15. The recombinant albumin fusion polypeptide according to any one of Claims 1 to 13, wherein one or more of said therapeutically active polypeptides is coupled to the C-terminus of the albumin or variant thereof.
16. The recombinant albumin fusion polypeptide according to any one of Claims 1 to 15, wherein said recombinant albumin fusion polypeptide comprises two or more therapeutically active polypeptides.
17. The recombinant albumin fusion polypeptide according to Claim 16, wherein said two or more therapeutically active polypeptides are different.
18. A nucleotide sequence encoding the recombinant albumin fusion polypeptide according to any one of Claims 1 to 16.
19. An expression cassette comprising the nucleotide sequence according to Claim 18 under the control of a transcription initiation region.
20. The expression cassette according to Claim 19 further comprising a transcription termination element.
21. A self-replicating vector comprising the expression cassette according to Claim 19 or 20.
22. A recombinant cell comprising the nucleotide sequence according to Claim 18.
23. A recombinant cell comprising the expression cassette according to Claim 19 or 20.
24. A recombinant cell comprising the vector according to Claim 21.
25. The recombinant cell according to any one of Claims 22 to 24, wherein said recombinant cell is a yeast, animal, fungal or bacterial cell.
26. The recombinant cell according to Claim 25, wherein said recombinant cell is a yeast cell.
27. The recombinant cell according to Claim 26, wherein said yeast cell is from the genus Saccharomyces or Kluyveromyces.
28. The recombinant cell according to Claim 25, wherein said animal cell is a CHO or COS
cell.
cell.
29. A process for producing the recombinant albumin fusion polypeptide according to any one of Claims 1 to 16 comprising:
(a) culturing the recombinant cell according to any one of Claims 24 to 28 under conditions permitting expression of said recombinant albumin fusion polypeptide; and (b) recovering the recombinant albumin fusion polypeptide.
(a) culturing the recombinant cell according to any one of Claims 24 to 28 under conditions permitting expression of said recombinant albumin fusion polypeptide; and (b) recovering the recombinant albumin fusion polypeptide.
30. A pharmaceutical composition comprising one or more recombinant albumin fusion polypeptides according to any one of Claims 1 to 16 and a pharmaceutically acceptable carrier.
31. A pharmaceutical composition comprising one or more nucleotide sequences according to Claim 18 and a pharmaceutically acceptable carrier.
32. A pharmaceutical composition comprising one or more expression cassettes according to Claim 19 or 20 and a pharmaceutically acceptable carrier.
33. A pharmaceutical composition comprising one or more vectors according to Claim 21 and a pharmaceutically acceptable carrier.
34. Use of the recombinant albumin fusion protein according to Claim 1 in the treatment of a subject in need of hormone therapy.
35. Use of the recombinant albumin fusion protein according to Claim 2 in the treatment of a subject in need of interferon therapy.
36. Use of the recombinant albumin fusion protein according to Claim 3 in the treatment of a subject in need of interleukin therapy.
37. Use of the recombinant albumin fusion protein according to Claim 4 in the treatment of a subject in need of insulin therapy.
38. Use of the recombinant albumin fusion protein according to Claim 5 in the treatment of a subject in need of erythropoietin therapy.
39. Use of the recombinant albumin fusion protein according to Claim 6 in the treatment of a subject in need of granulocyte colony-stimulating factor therapy.
40. Use of the recombinant albumin fusion protein according to Claim 1 in the manufacture of a medicament for the treatment of a subject in need of hormone therapy.
41. Use of the recombinant albumin fusion protein according to Claim 2 in the manufacture of a medicament for the treatment of a subject in need of interferon therapy.
42. Use of the recombinant albumin fusion protein according to Claim 3 in the manufacture of a medicament for the treatment of a subject in need of interleukin therapy.
43. Use of the recombinant albumin fusion protein according to Claim 4 in the manufacture of a medicament for the treatment of a subject in need of insulin therapy.
44. Use of the recombinant albumin fusion protein according to Claim 5 in the manufacture of a medicament for the treatment of a subject in need of erythropoietin therapy.
45. Use of the recombinant albumin fusion protein according to Claim 6 in the manufacture of a medicament for the treatment of a subject in need of granulocyte colony-stimulating factor therapy.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR9201064A FR2686899B1 (en) | 1992-01-31 | 1992-01-31 | NOVEL BIOLOGICALLY ACTIVE POLYPEPTIDES, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM. |
FR9201064 | 1992-01-31 | ||
PCT/FR1993/000085 WO1993015199A1 (en) | 1992-01-31 | 1993-01-28 | Novel biologically active polypeptides, preparation thereof and pharmaceutical composition containing said polypeptides |
Publications (2)
Publication Number | Publication Date |
---|---|
CA2126091A1 CA2126091A1 (en) | 1993-08-05 |
CA2126091C true CA2126091C (en) | 2008-03-11 |
Family
ID=9426190
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002126091A Expired - Lifetime CA2126091C (en) | 1992-01-31 | 1993-01-28 | Novel biologically active polypeptides, preparation thereof and pharmaceutical composition containing said polypeptides |
Country Status (13)
Country | Link |
---|---|
US (12) | US5876969A (en) |
EP (2) | EP0624195B1 (en) |
JP (3) | JPH07503368A (en) |
AT (1) | ATE276361T1 (en) |
CA (1) | CA2126091C (en) |
DE (1) | DE69333622T2 (en) |
DK (1) | DK0624195T3 (en) |
ES (1) | ES2230541T3 (en) |
FI (1) | FI120355B (en) |
FR (1) | FR2686899B1 (en) |
NO (2) | NO325486B1 (en) |
PT (1) | PT624195E (en) |
WO (1) | WO1993015199A1 (en) |
Families Citing this family (517)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5824307A (en) | 1991-12-23 | 1998-10-20 | Medimmune, Inc. | Human-murine chimeric antibodies against respiratory syncytial virus |
FR2686899B1 (en) * | 1992-01-31 | 1995-09-01 | Rhone Poulenc Rorer Sa | NOVEL BIOLOGICALLY ACTIVE POLYPEPTIDES, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM. |
US6479055B1 (en) * | 1993-06-07 | 2002-11-12 | Trimeris, Inc. | Methods for inhibition of membrane fusion-associated events, including respiratory syncytial virus transmission |
US20070202127A1 (en) * | 1993-06-07 | 2007-08-30 | Duke University | Nucleic acids encoding DP-178 and other viral fusion inhibitor peptides useful for treating aids |
FR2719593B1 (en) * | 1994-05-06 | 1996-05-31 | Rhone Poulenc Rorer Sa | New biologically active polypeptides, their preparation and pharmaceutical composition containing them. |
US7597886B2 (en) * | 1994-11-07 | 2009-10-06 | Human Genome Sciences, Inc. | Tumor necrosis factor-gamma |
US7820798B2 (en) * | 1994-11-07 | 2010-10-26 | Human Genome Sciences, Inc. | Tumor necrosis factor-gamma |
US20030198640A1 (en) * | 1994-11-07 | 2003-10-23 | Human Genome Sciences, Inc. | Methods and compositions for treating inflammatory bowel diseases relating to human tumor necrosis factor-gamma-beta |
WO1996018412A1 (en) * | 1994-12-12 | 1996-06-20 | Beth Israel Hospital Association | Chimeric cytokines and uses thereof |
US6410008B1 (en) | 1994-12-12 | 2002-06-25 | Beth Israel Hospital Association | Chimeric IL-10 proteins and uses thereof |
GB9526733D0 (en) * | 1995-12-30 | 1996-02-28 | Delta Biotechnology Ltd | Fusion proteins |
US7888466B2 (en) | 1996-01-11 | 2011-02-15 | Human Genome Sciences, Inc. | Human G-protein chemokine receptor HSATU68 |
US6136311A (en) | 1996-05-06 | 2000-10-24 | Cornell Research Foundation, Inc. | Treatment and diagnosis of cancer |
US6423512B1 (en) | 1996-07-26 | 2002-07-23 | Novartis Ag | Fusion polypeptides |
US7285267B2 (en) * | 1997-01-14 | 2007-10-23 | Human Genome Sciences, Inc. | Tumor necrosis factor receptors 6α & 6β |
US20040013664A1 (en) * | 1997-01-14 | 2004-01-22 | Gentz Reiner L. | Tumor necrosis factor receptors 6 alpha & 6 beta |
AT406373B (en) * | 1997-02-27 | 2000-04-25 | Immuno Ag | METHOD FOR CLEANING FACTOR VIII / VWF COMPLEX BY CATION EXCHANGER CHROMATOGRAPHY |
AT405403B (en) | 1997-02-27 | 1999-08-25 | Immuno Ag | CLEANING OF WILLEBRAND FACTOR BY CATION EXCHANGER CHROMATOGRAPHY |
CA2288306A1 (en) | 1997-04-28 | 1998-11-05 | Rhone-Poulenc Rorer S.A. | Adenovirus-mediated intratumoral delivery of an angiogenesis antagonist for the treatment of tumors |
AT408443B (en) * | 1998-02-27 | 2001-11-26 | Immuno Ag | METHOD FOR OBTAINING PURIFIED FACTOR VIII: C / VWF COMPLEX |
US6656906B1 (en) | 1998-05-20 | 2003-12-02 | Trimeris, Inc. | Hybrid polypeptides with enhanced pharmacokinetic properties |
US6605222B1 (en) * | 1998-05-20 | 2003-08-12 | Baxter Aktiengesellschaft | Method for producing a factor VIII/von Willebrand factor complex |
US20050181482A1 (en) * | 2004-02-12 | 2005-08-18 | Meade Harry M. | Method for the production of an erythropoietin analog-human IgG fusion proteins in transgenic mammal milk |
CA2330527A1 (en) | 1998-06-15 | 1999-12-23 | Genzyme Transgenics Corporation | Erythropoietin analog-human serum albumin fusion |
US6475743B1 (en) * | 1998-10-02 | 2002-11-05 | Ischemia Technologies, Inc. | Marker useful for detection and measurement of free radical damage and method |
US7449338B2 (en) | 1998-10-02 | 2008-11-11 | Ischemia Technologies, Inc. | Tests for the rapid evaluation of ischemic states and kits |
US20050142613A1 (en) * | 1998-10-02 | 2005-06-30 | David Bar-Or | Test for the rapid evaluation of ischemic states and kits |
US20030215359A1 (en) * | 1998-10-02 | 2003-11-20 | Ischemia Technologies, Inc. | Tests for the rapid evaluation of ischemic states and kits |
US7070937B1 (en) | 1998-10-02 | 2006-07-04 | Ischemia Technologies, Inc. | Marker useful for detection and measurement of free radical damage and method |
US7297544B2 (en) * | 1998-10-02 | 2007-11-20 | Ischemia Technologies, Inc. | Tests for the rapid evaluation of ischemic states and kits |
US20030190740A1 (en) * | 1998-10-13 | 2003-10-09 | The University Of Georgia Research Foundation, Inc | Stabilized bioactive peptides and methods of identification, synthesis, and use |
GB9824632D0 (en) * | 1998-11-10 | 1999-01-06 | Celltech Therapeutics Ltd | Biological compounds |
DE60000665T3 (en) * | 1999-05-17 | 2009-10-29 | ConjuChem Biotechnologies Inc., Montreal | LONG-ACTING PEPTIDINHIBITORS OF VIRUS FUSION WITH BODY CELLS IN VIRAL INFECTIONS |
US20040266673A1 (en) * | 2002-07-31 | 2004-12-30 | Peter Bakis | Long lasting natriuretic peptide derivatives |
US7601691B2 (en) * | 1999-05-17 | 2009-10-13 | Conjuchem Biotechnologies Inc. | Anti-obesity agents |
US7087411B2 (en) | 1999-06-08 | 2006-08-08 | Regeneron Pharmaceuticals, Inc. | Fusion protein capable of binding VEGF |
US6951738B2 (en) * | 1999-07-16 | 2005-10-04 | Human Genome Sciences, Inc. | Human tumor necrosis factor receptors TR13 and TR14 |
US20020048571A1 (en) * | 1999-07-19 | 2002-04-25 | Jeno Gyuris | Chimeric polypeptides of serum albumin and uses related thereto |
US6797695B1 (en) | 1999-10-22 | 2004-09-28 | Kyoto University | Human FGF-20 gene and gene expression products |
EP2163626A1 (en) | 1999-11-18 | 2010-03-17 | Novartis Vaccines and Diagnostics, Inc. | Human FGF-21 gene and gene expression products |
US6716626B1 (en) | 1999-11-18 | 2004-04-06 | Chiron Corporation | Human FGF-21 nucleic acids |
DE60138535D1 (en) * | 2000-02-04 | 2009-06-10 | Children S Hospital Res Founda | USE OF LYSOSOMAL ACID LIPASE FOR THE TREATMENT OF ATHEROSCLEROSIS AND SIMILAR DISEASES |
CA2747325A1 (en) * | 2000-04-12 | 2001-10-25 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US6946134B1 (en) | 2000-04-12 | 2005-09-20 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US20050100991A1 (en) * | 2001-04-12 | 2005-05-12 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US20030031675A1 (en) | 2000-06-06 | 2003-02-13 | Mikesell Glen E. | B7-related nucleic acids and polypeptides useful for immunomodulation |
CA2413160A1 (en) | 2000-06-15 | 2001-12-20 | Human Genome Sciences, Inc. | Human tumor necrosis factor delta and epsilon |
CA2407910C (en) | 2000-06-16 | 2013-03-12 | Steven M. Ruben | Antibodies that immunospecifically bind to blys |
AU2001280705A1 (en) * | 2000-07-24 | 2002-02-05 | Human Genome Sciences, Inc. | Human tumor necrosis factor receptors tr21 and tr22 |
AU2001286688A1 (en) * | 2000-08-25 | 2002-03-13 | Human Genome Sciences, Inc. | Tumor necrosis factor receptors 6alpha and 6beta |
BR0116024A (en) * | 2000-12-07 | 2005-12-13 | Lilly Co Eli | Heterologous Fusion Protein and Use thereof |
CN1564826A (en) | 2001-02-09 | 2005-01-12 | 人类基因组科学公司 | Human G-protein chemokine receptor (CCR5) HDGNR10 |
WO2002074806A2 (en) | 2001-02-27 | 2002-09-26 | Maxygen Aps | New interferon beta-like molecules |
FR2822834B1 (en) * | 2001-04-02 | 2005-02-25 | Flamel Tech Sa | COLLOIDAL SUSPENSION OF NANOPARTICLES BASED ON AMPHIPHILIC COPOLYMERS FOR VECTORIZATION OF ACTIVE INGREDIENTS AND THEIR METHOD OF PREPARATION |
CN1558773A (en) * | 2001-04-06 | 2004-12-29 | 巴斯德研究院 | Conjugate vaccine composed of the polysaccharide moiety of the lipopolysaccharide of vibrio cholerae O139 bound to tetanus toxoid |
US6887462B2 (en) | 2001-04-09 | 2005-05-03 | Chiron Corporation | HSA-free formulations of interferon-beta |
US20050054051A1 (en) * | 2001-04-12 | 2005-03-10 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US20050244931A1 (en) * | 2001-04-12 | 2005-11-03 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US7507413B2 (en) | 2001-04-12 | 2009-03-24 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US20060084794A1 (en) * | 2001-04-12 | 2006-04-20 | Human Genome Sciences, Inc. | Albumin fusion proteins |
JP2004536579A (en) | 2001-04-13 | 2004-12-09 | ヒューマン ジノーム サイエンシーズ, インコーポレイテッド | Vascular endothelial growth factor 2 |
US7064189B2 (en) | 2001-05-25 | 2006-06-20 | Human Genome Sciences, Inc. | Antibodies that immunospecifically bind to trail receptors |
US20030143191A1 (en) * | 2001-05-25 | 2003-07-31 | Adam Bell | Chemokine beta-1 fusion proteins |
AU2002302277A1 (en) * | 2001-05-31 | 2002-12-09 | Conjuchem, Inc. | Long lasting fusion peptide inhibitors for hiv infection |
CA2457903C (en) | 2001-08-24 | 2018-04-17 | University Of Victoria Innovation And Development Corporation | Proaerolysin containing protease activation sequences and methods of use for treatment of prostate cancer |
US8129504B2 (en) | 2001-08-30 | 2012-03-06 | Biorexis Technology, Inc. | Oral delivery of modified transferrin fusion proteins |
US20030226155A1 (en) * | 2001-08-30 | 2003-12-04 | Biorexis Pharmaceutical Corporation | Modified transferrin-antibody fusion proteins |
US20040023334A1 (en) * | 2001-08-30 | 2004-02-05 | Biorexis Pharmaceutical Corporation | Modified transferrin fusion proteins |
US7176278B2 (en) * | 2001-08-30 | 2007-02-13 | Biorexis Technology, Inc. | Modified transferrin fusion proteins |
US20070031440A1 (en) * | 2001-08-30 | 2007-02-08 | Prior Christopher P | Modified transferin-antibody fusion proteins |
US20030228298A1 (en) * | 2001-09-04 | 2003-12-11 | Mark Nesbit | Abrogen polypeptides, nucleic acids encoding them and methods for using them to inhibit angiogenesis |
US6797493B2 (en) * | 2001-10-01 | 2004-09-28 | Lee-Hwei K. Sun | Fc fusion proteins of human granulocyte colony-stimulating factor with increased biological activities |
AU2002332041A1 (en) * | 2001-10-05 | 2003-04-22 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US20080194481A1 (en) * | 2001-12-21 | 2008-08-14 | Human Genome Sciences, Inc. | Albumin Fusion Proteins |
EP1463751B1 (en) * | 2001-12-21 | 2013-05-22 | Human Genome Sciences, Inc. | Albumin fusion proteins |
KR101271635B1 (en) * | 2001-12-21 | 2013-06-12 | 휴먼 게놈 사이언시즈, 인코포레이티드 | Albumin fusion proteins |
EP1463752A4 (en) * | 2001-12-21 | 2005-07-13 | Human Genome Sciences Inc | Albumin fusion proteins |
US20080167238A1 (en) * | 2001-12-21 | 2008-07-10 | Human Genome Sciences, Inc. | Albumin Fusion Proteins |
KR100441384B1 (en) * | 2002-01-08 | 2004-07-21 | 주식회사 안지오랩 | Expression system of human serum albumin-timp-2 fusion protein and recombinant human serum albumin- timp-2 protein |
WO2003062395A2 (en) * | 2002-01-18 | 2003-07-31 | Bristol-Myers Squibb Company | Predictor sets for tyrosine kinase pathways |
US20060241027A1 (en) * | 2002-02-07 | 2006-10-26 | Hans-Peter Hauser | Hiv inhibiting proteins |
US20050222023A1 (en) * | 2002-02-07 | 2005-10-06 | Hans-Peter Hauser | Albumin-fused kunitz domain peptides |
ZA200406124B (en) * | 2002-02-07 | 2008-09-25 | Delta Biotechnology Ltd | Albumin-fused kunitz domain peptides |
EP1497445A2 (en) * | 2002-04-01 | 2005-01-19 | Human Genome Sciences, Inc. | Antibodies that specifically bind to gmad |
EP1499352A4 (en) | 2002-04-12 | 2006-10-11 | Medimmune Inc | Recombinant anti-interleukin-9 antibodies |
MXPA04010522A (en) | 2002-04-23 | 2004-12-13 | Cargill Inc | Polypeptides and biosynthetic pathways. |
EP1837031B1 (en) * | 2002-06-07 | 2009-10-14 | Waratah Pharmaceuticals, Inc. | Compositions and methods for treating diabetes |
FR2840614B1 (en) | 2002-06-07 | 2004-08-27 | Flamel Tech Sa | POLYAMINOACIDS FUNCTIONALIZED BY ALPHA-TOCOPHEROL AND THEIR PARTICULARLY THERAPEUTIC APPLICATIONS |
US7563882B2 (en) * | 2002-06-10 | 2009-07-21 | University Of Rochester | Polynucleotides encoding antibodies that bind to the C35 polypeptide |
US7425618B2 (en) | 2002-06-14 | 2008-09-16 | Medimmune, Inc. | Stabilized anti-respiratory syncytial virus (RSV) antibody formulations |
US7132100B2 (en) | 2002-06-14 | 2006-11-07 | Medimmune, Inc. | Stabilized liquid anti-RSV antibody formulations |
AU2003256566A1 (en) * | 2002-07-16 | 2004-02-02 | Stuart Bussell | Methods to construct multimeric dna and polymeric protein sequences as direct fusions or with linkers |
FR2843117B1 (en) * | 2002-07-30 | 2004-10-15 | Flamel Tech Sa | POLYAMINOACIDS FUNCTIONALIZED BY AT LEAST ONE HYDROPHOBIC GROUP AND THEIR PARTICULARLY THERAPEUTIC APPLICATIONS |
CA2497338A1 (en) | 2002-08-30 | 2004-03-18 | Japan Science And Technology Corporation | Method of targeted gene disruption, genome of hyperthermostable bacterium and genome chip using the same |
CN1713920A (en) * | 2002-08-30 | 2005-12-28 | 比奥雷克西斯药物公司 | Modified transferrin fusion proteins |
US20060105387A1 (en) * | 2002-08-30 | 2006-05-18 | Prior Christopher P | Transferrin fusion proteins libraries |
US20040052777A1 (en) * | 2002-09-04 | 2004-03-18 | Mark Nesbit | Kringle polypeptides and methods for using them to inhibit angiogenesis |
US20040229810A1 (en) * | 2002-10-22 | 2004-11-18 | Antonio Cruz | Gastrin compositions and formulations, and methods of use and preparation |
US20060014248A1 (en) * | 2003-01-06 | 2006-01-19 | Xencor, Inc. | TNF super family members with altered immunogenicity |
US20050221443A1 (en) * | 2003-01-06 | 2005-10-06 | Xencor, Inc. | Tumor necrosis factor super family agonists |
US7553930B2 (en) * | 2003-01-06 | 2009-06-30 | Xencor, Inc. | BAFF variants and methods thereof |
ES2398460T3 (en) | 2003-02-06 | 2013-03-19 | Queen's University Of Belfast | Bradykinin B2 receptor antagonist peptide from the skin of an amphibian |
US20070060512A1 (en) * | 2003-03-04 | 2007-03-15 | Homayoun Sadeghi | Dipeptidyl-peptidase protected protein |
KR20110094361A (en) | 2003-04-11 | 2011-08-23 | 메디뮨 엘엘씨 | Recombinant il-9 antibodies and uses thereof |
US20050079546A1 (en) * | 2003-05-01 | 2005-04-14 | Dasa Lipovsek | Serum albumin scaffold-based proteins and uses thereof |
TWI353991B (en) | 2003-05-06 | 2011-12-11 | Syntonix Pharmaceuticals Inc | Immunoglobulin chimeric monomer-dimer hybrids |
US20080039379A1 (en) * | 2003-05-27 | 2008-02-14 | Waratah Pharmaceuticals, Inc. | Compositions Comprising Gastrin Compounds and Their Use in Diabetes |
FR2855521B1 (en) * | 2003-05-28 | 2005-08-05 | Flamel Tech Sa | POLYAMINOACIDES FUNCTIONALIZED BY AT LEAST ONE YDROPHOBIC GROUP AND THEIR PARTICULARLY THERAPEUTIC APPLICATIONS. |
CA2532250A1 (en) | 2003-07-15 | 2005-02-03 | Barros Research Institute | Compositions and methods for immunotherapy of cancer and infectious diseases |
WO2005012346A1 (en) * | 2003-07-25 | 2005-02-10 | Conjuchem, Inc. | Long lasting insulin derivatives and methods thereof |
US20060205037A1 (en) * | 2003-08-28 | 2006-09-14 | Homayoun Sadeghi | Modified transferrin fusion proteins |
EP1663278A4 (en) * | 2003-08-28 | 2009-07-29 | Biorexis Pharmaceutical Corp | Epo mimetic peptides and fusion proteins |
FR2860516B1 (en) * | 2003-10-03 | 2006-01-13 | Flamel Tech Sa | TELECHELIC HOMOPOLYAMINOACIDES FUNCTIONALIZED BY HYDROPHOBIC GROUPS AND THEIR PARTICULARLY THERAPEUTIC APPLICATIONS |
WO2005035564A2 (en) | 2003-10-10 | 2005-04-21 | Xencor, Inc. | Protein based tnf-alpha variants for the treatment of tnf-alpha related disorders |
EP2256134B1 (en) | 2003-11-13 | 2014-01-08 | Hanmi Science Co., Ltd. | IgG Fc fragment for a drug carrier and method for the preparation thereof |
FR2862536B1 (en) * | 2003-11-21 | 2007-11-23 | Flamel Tech Sa | PHARMACEUTICAL FORMULATIONS FOR THE PROLONGED DELIVERY OF ACTIVE (S) PRINCIPLE (S) AND THEIR PARTICULARLY THERAPEUTIC APPLICATIONS |
FR2862535B1 (en) * | 2003-11-21 | 2007-11-23 | Flamel Tech Sa | PHARMACEUTICAL FORMULATIONS FOR PROLONGED RELEASE OF INTERLEUKINS AND THEIR THERAPEUTIC APPLICATIONS |
EP1708751B1 (en) * | 2003-12-04 | 2011-09-28 | Vaccinex, Inc. | Methods of killing tumor cells by targeting internal antigens exposed on apoptotic tumor cells |
US7371381B2 (en) | 2003-12-12 | 2008-05-13 | Amgen Inc. | Anti-galanin antibodies and uses thereof |
JP2007518423A (en) * | 2004-01-23 | 2007-07-12 | イントロン、インコーポレイテッド | Expression of ApoA-1 and its variants using spliceosome-mediated RNA trans-splicing |
PL1729795T3 (en) * | 2004-02-09 | 2016-08-31 | Human Genome Sciences Inc | Albumin fusion proteins |
US7973139B2 (en) * | 2004-03-26 | 2011-07-05 | Human Genome Sciences, Inc. | Antibodies against nogo receptor |
EP1750754A4 (en) * | 2004-03-31 | 2010-09-22 | Centocor Ortho Biotech Inc | Human glp-1 mimetibodies, compositions, methods and uses |
JP2008505059A (en) * | 2004-05-06 | 2008-02-21 | コンジュシェム バイオテクノロジーズ インコーポレイティド | Specific virus targeting compounds |
EP2287310B1 (en) | 2004-07-22 | 2015-05-06 | Five Prime Therapeutics, Inc. | Compositions and methods using MGD-CSF in disease treatment |
EP1786831A4 (en) | 2004-07-30 | 2008-01-23 | Cargill Inc | Alanine 2, 3 aminomutases |
JP2008512097A (en) * | 2004-09-07 | 2008-04-24 | アーケミックス コーポレイション | Aptamer medicinal chemistry |
US7566701B2 (en) * | 2004-09-07 | 2009-07-28 | Archemix Corp. | Aptamers to von Willebrand Factor and their use as thrombotic disease therapeutics |
EP1789096A4 (en) | 2004-09-07 | 2009-07-08 | Archemix Corp | Aptamers to von willebrand factor and their use as thrombotic disease therapeutics |
WO2006034292A2 (en) | 2004-09-21 | 2006-03-30 | Medimmune, Inc. | Antibodies against and methods for producing vaccines for respiratory syncytial virus |
AU2005286662B2 (en) | 2004-09-23 | 2011-10-06 | Vasgene Therapeutics, Inc. | Polypeptide compounds for inhibiting angiogenesis and tumor growth |
AU2005326784B2 (en) * | 2004-10-08 | 2012-03-15 | Virxsys Corporation | Use of RNA trans-splicing for antibody gene transfer and antibody polypeptide production |
CA2583254A1 (en) * | 2004-10-08 | 2006-04-20 | Intronn, Inc. | Targeted trans-splicing of highly abundant transcripts for in vivo production of recombinant proteins |
JP5058822B2 (en) | 2005-01-25 | 2012-10-24 | ファイブ プライム セラピューティクス, インコーポレイテッド | Compositions and methods for treating cardiac conditions |
AU2006214121B9 (en) | 2005-02-15 | 2013-02-14 | Duke University | Anti-CD19 antibodies and uses in oncology |
US7723472B2 (en) * | 2005-02-28 | 2010-05-25 | The Regents Of The University Of California | Extracellular matrix binding chimeric proteins and methods of use thereof |
KR100754667B1 (en) | 2005-04-08 | 2007-09-03 | 한미약품 주식회사 | Immunoglobulin Fc fragment modified by non-peptide polymer and pharmaceutical composition comprising the same |
US7833979B2 (en) * | 2005-04-22 | 2010-11-16 | Amgen Inc. | Toxin peptide therapeutic agents |
UA95446C2 (en) * | 2005-05-04 | 2011-08-10 | Іллюміджен Байосайєнсіз, Інк. | Mutations in oas1 genes |
CA2607281C (en) | 2005-05-05 | 2023-10-03 | Duke University | Anti-cd19 antibody therapy for autoimmune disease |
EP1891111A1 (en) | 2005-05-06 | 2008-02-27 | ZymoGenetics, Inc. | Il-31 monoclonal antibodies and methods of use |
US20060292609A1 (en) * | 2005-05-19 | 2006-12-28 | Schering Aktiengesellschaft | Interferon-beta gene therapy using an improved, regulated expression system |
US20080076729A1 (en) * | 2005-05-19 | 2008-03-27 | Schering Aktiengesellachaft | Interferon-beta gene therapy using an improved, regulated expression system |
CA2608764A1 (en) * | 2005-05-19 | 2006-11-23 | Schering Ag | Treatment of disease using an improved regulated expression system |
US20070179113A1 (en) * | 2005-05-19 | 2007-08-02 | Schering Aktiengesellachaft | GM-CSF gene therapy for Crohn's disease using an improved regulated expression system |
US20090275481A1 (en) * | 2005-06-17 | 2009-11-05 | Pfizer, Inc. | Anchored Transferrin Fusion Protein Libraries |
ES2336832T3 (en) | 2005-07-22 | 2010-04-16 | Five Prime Therapeutics, Inc. | COMPOSITIONS AND PROCEDURES TO TREAT DISEASES WITH FGFR FUSION PROTEINS. |
CA2618681C (en) | 2005-08-10 | 2015-10-27 | Macrogenics, Inc. | Identification and engineering of antibodies with variant fc regions and methods of using same |
EP3037544A1 (en) | 2005-10-13 | 2016-06-29 | Human Genome Sciences, Inc. | Methods and compositions for use in treatment of systemic lupus erythematosus (sle) patients with autoantibody positive diseases |
CA2562249A1 (en) * | 2005-10-20 | 2007-04-20 | University Of Ottawa Heart Institute | Anf analogue |
US8168592B2 (en) * | 2005-10-21 | 2012-05-01 | Amgen Inc. | CGRP peptide antagonists and conjugates |
EP3058972A1 (en) | 2005-11-17 | 2016-08-24 | Zogenix, Inc. | Delivery of viscous formulations by needle-free injection |
US20080280328A1 (en) * | 2005-11-18 | 2008-11-13 | Novozymes A/S | Glucoamylase Variants |
CA2634034A1 (en) | 2005-12-20 | 2007-06-28 | Duke University | Methods and compositions for delivering active agents with enhanced pharmacological properties |
CN101384623B (en) * | 2005-12-22 | 2013-07-24 | 常山凯捷健生物药物研发(河北)有限公司 | Process for the production of preformed conjugates of albumin and a therapeutic agent |
US7625564B2 (en) * | 2006-01-27 | 2009-12-01 | Novagen Holding Corporation | Recombinant human EPO-Fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo |
EP1816201A1 (en) | 2006-02-06 | 2007-08-08 | CSL Behring GmbH | Modified coagulation factor VIIa with extended half-life |
US8389688B2 (en) | 2006-03-06 | 2013-03-05 | Aeres Biomedical, Ltd. | Humanized anti-CD22 antibodies and their use in treatment of oncology, transplantation and autoimmune disease |
CA2646329C (en) | 2006-03-20 | 2018-07-03 | The Regents Of The University Of California | Engineered anti-prostate stem cell antigen (psca) antibodies for cancer targeting |
AU2007258609B2 (en) * | 2006-06-07 | 2013-01-24 | Human Genome Sciences, Inc. | Albumin fusion proteins |
GB0611405D0 (en) * | 2006-06-09 | 2006-07-19 | Univ Belfast | FKBP-L: A novel inhibitor of angiogenesis |
EP2046826B1 (en) | 2006-07-24 | 2011-09-14 | Biorexis Pharmaceutical Corporation | Exendin fusion proteins |
TW200817438A (en) * | 2006-08-17 | 2008-04-16 | Hoffmann La Roche | A conjugate of an antibody against CCR5 and an antifusogenic peptide |
BRPI0715660B8 (en) | 2006-09-01 | 2021-05-25 | Zymogenetics Inc | isolated antibody that binds to human IL-31; and, use of a monoclonal antibody |
EP2354254A1 (en) | 2006-09-06 | 2011-08-10 | Ortho-McNeil Pharmaceutical, Inc. | Biomarkers for assessing response to C-met treatment |
JP5840345B2 (en) | 2006-09-08 | 2016-01-06 | アンブルックス, インコーポレイテッドAmbrx, Inc. | Modified human plasma polypeptides or modified human Fc scaffold proteins and uses thereof |
EP2114437A2 (en) * | 2006-10-16 | 2009-11-11 | ConjuChem Biotechnologies Inc. | Modified corticotropin releasing factor peptides and uses thereof |
US8268347B1 (en) | 2006-10-24 | 2012-09-18 | Aradigm Corporation | Dual action, inhaled formulations providing both an immediate and sustained release profile |
WO2008052043A2 (en) * | 2006-10-24 | 2008-05-02 | Cogenesys, Inc. | Opioid receptor agonist fusion proteins |
WO2008088422A2 (en) | 2006-10-25 | 2008-07-24 | Amgen Inc. | Toxin peptide therapeutic agents |
WO2008140472A2 (en) * | 2006-10-27 | 2008-11-20 | The Regents Of The University Of California | Functionalized molecules comprising an autosilification moiety and methods of making and using same |
AU2007351813B2 (en) | 2006-10-31 | 2013-10-10 | East Carolina University | Fusion proteins comprising an anti-inflammatory cytokine and an antigen for treatment of immune disorders |
WO2008056961A1 (en) * | 2006-11-10 | 2008-05-15 | Boryung Pharmaceutical Co., Ltd | A novel fusion protein, cell lines expressing the same and preparation method thereof |
EP2120998B1 (en) | 2006-11-28 | 2013-08-07 | HanAll Biopharma Co., Ltd. | Modified erythropoietin polypeptides and uses thereof for treatment |
EP3231440A1 (en) * | 2006-12-22 | 2017-10-18 | CSL Behring GmbH | Modified coagulation factors with prolonged in vivo half-life |
US20090099074A1 (en) * | 2007-01-10 | 2009-04-16 | Conjuchem Biotechnologies Inc. | Modulating food intake |
US20090088378A1 (en) * | 2007-01-12 | 2009-04-02 | Omar Quraishi | Long lasting inhibitors of viral infection |
EP2450371B1 (en) | 2007-01-30 | 2015-04-29 | Epivax, Inc. | Regulatory t cell epitopes, compositions and uses thereof |
HUP0700203A2 (en) * | 2007-03-08 | 2008-12-29 | Univ Szegedi | Sustained release, core-shell structure precipitated citokine bionanocomposit, a process for producing the same and use thereof |
EP2136832B1 (en) | 2007-03-26 | 2015-09-02 | General Regeneratives Limited | Methods for promoting protection and regeneration of bone marrow using cxcl9 and anti-cxcl9 antibodies |
KR101476472B1 (en) | 2007-03-30 | 2015-01-05 | 암브룩스, 인코포레이티드 | Modified fgf-21 polypeptides and their uses |
WO2008136790A1 (en) | 2007-05-02 | 2008-11-13 | Merial Limited | Dna plasmids having improved expression and stability |
JP2010525821A (en) | 2007-05-02 | 2010-07-29 | アンブルックス,インコーポレイテッド | Modified IFN beta polypeptides and their use |
EP2068925A4 (en) | 2007-05-07 | 2011-08-31 | Medimmune Llc | Anti-icos antibodies and their use in treatment of oncology, transplantation and autoimmune disease |
ES2558689T3 (en) | 2007-05-14 | 2016-02-08 | Medimmune, Llc | Methods to reduce eosinophil levels |
AU2008254767A1 (en) * | 2007-05-16 | 2008-11-27 | Conjuchem Biotechnologies Inc. | Cysteic acid derivatives of anti-viral peptides |
US8420779B2 (en) * | 2007-05-22 | 2013-04-16 | Amgen Inc. | Compositions and methods for producing bioactive fusion proteins |
WO2008150495A2 (en) * | 2007-06-01 | 2008-12-11 | Archemix Corp. | Vwf aptamer formulations and methods for use |
EP1997830A1 (en) | 2007-06-01 | 2008-12-03 | AIMM Therapeutics B.V. | RSV specific binding molecules and means for producing them |
WO2008157824A2 (en) * | 2007-06-21 | 2008-12-24 | Conjuchem Biotechnologies Inc. | Thrombopoietin peptide conjugates |
CL2008002054A1 (en) * | 2007-07-17 | 2009-05-29 | Hoffmann La Roche | Method for the regeneration of a cation exchange chromatography column after elusion of monopeglated erythropoietin and method to obtain a monopeglated erythropoietin, incorporating the regeneration method of the cation exchange column. |
CL2008002053A1 (en) * | 2007-07-17 | 2009-05-22 | Hoffmann La Roche | Method for the purification of a monopeglated erythropoietin (epompeg) which consists of providing a solution containing mono, poly and non-peglated erythropoietin and passing it through two steps of cation exchange chromatography and a method to produce epo mpeg that includes a purification method. |
CL2008002092A1 (en) * | 2007-07-20 | 2009-05-29 | Hoffmann La Roche | Conjugate containing two or more antifusogenic peptides and an anti-cd-4 antibody; Method of production; pharmaceutical composition comprising it; antifusogenic polypeptides and use of the conjugate to treat viral infections. |
WO2009012600A1 (en) | 2007-07-26 | 2009-01-29 | Novagen Holding Corporation | Fusion proteins |
WO2009023549A2 (en) * | 2007-08-09 | 2009-02-19 | University Of Rochester | Vaccine against botulism |
AU2008287340A1 (en) * | 2007-08-15 | 2009-02-19 | Amunix, Inc. | Compositions and methods for modifying properties of biologically active polypeptides |
NZ583605A (en) | 2007-08-29 | 2012-10-26 | Sanofi Aventis | Humanized anti-cxcr5 antibodies, derivatives thereof and their uses |
CA2698343C (en) | 2007-09-04 | 2018-06-12 | The Regents Of The University Of California | High affinity anti-prostate stem cell antigen (psca) antibodies for cancer targeting and detection |
US8273561B2 (en) * | 2007-10-05 | 2012-09-25 | Nuron Biotech, Inc. | High pressure treatment of aggregated interferons |
EP2050764A1 (en) | 2007-10-15 | 2009-04-22 | sanofi-aventis | Novel polyvalent bispecific antibody format and uses thereof |
DK2217265T3 (en) | 2007-11-20 | 2017-07-03 | Ambrx Inc | Modified insulin polypeptides and their use |
JP5490714B2 (en) | 2007-11-28 | 2014-05-14 | メディミューン,エルエルシー | Protein preparation |
LT2796466T (en) | 2007-12-07 | 2018-02-26 | Zymogenetics, Inc. | Humanized antibody molecules specific for IL-31 |
US8092804B2 (en) | 2007-12-21 | 2012-01-10 | Medimmune Limited | Binding members for interleukin-4 receptor alpha (IL-4Rα)-173 |
EP2245064B1 (en) | 2007-12-21 | 2014-07-23 | Medimmune Limited | BINDING MEMBERS FOR INTERLEUKIN-4 RECEPTOR ALPHA (IL-4Ralpha) |
AU2008341050B2 (en) | 2007-12-26 | 2013-10-24 | Vaccinex, Inc. | Anti-C35 antibody combination therapies and methods |
US20110020368A1 (en) | 2008-03-25 | 2011-01-27 | Nancy Hynes | Treating cancer by down-regulating frizzled-4 and/or frizzled-1 |
AU2009241847B2 (en) | 2008-04-28 | 2014-07-10 | Zogenix, Inc. | Novel formulations for treatment of migraine |
EP2383292A1 (en) | 2008-05-02 | 2011-11-02 | Novartis AG | Improved fibronectin-based binding molecules and uses thereof |
US8575104B2 (en) | 2008-06-24 | 2013-11-05 | Csl Behring Gmbh | Factor VIII, von willebrand factor or complexes thereof with prolonged in vivo half-life |
US20100048488A1 (en) * | 2008-08-01 | 2010-02-25 | Syntonix Pharmaceuticals, Inc. | Immunomodulatory peptides |
DK2328616T3 (en) | 2008-08-05 | 2015-07-20 | Novartis Ag | Compositions and Methods for Antibodies to Complement Protein C5 |
US8163497B2 (en) | 2008-09-07 | 2012-04-24 | Glyconex Inc. | Anti-extended type I glycosphingolipid antibody, derivatives thereof and use |
KR20110093775A (en) * | 2008-11-03 | 2011-08-18 | 바이엘 헬스케어 엘엘씨 | Method for the treatment of hemophilia |
JP5933975B2 (en) | 2008-11-12 | 2016-06-15 | メディミューン,エルエルシー | Antibody preparation |
CN102282168A (en) * | 2008-11-18 | 2011-12-14 | 梅里麦克制药股份有限公司 | Human serum albumin linkers and conjugates thereof |
WO2010078325A2 (en) | 2008-12-29 | 2010-07-08 | Mayo Foundation For Medical Education And Research | Natriuretic polypeptides for reducing or preventing restenosis |
WO2010083439A2 (en) | 2009-01-16 | 2010-07-22 | Teva Biopharmaceuticals Usa, Inc. | Recombinant human albumin-human granulocyte colony stimulating factor for the prevention of neutropenia |
WO2010087927A2 (en) | 2009-02-02 | 2010-08-05 | Medimmune, Llc | Antibodies against and methods for producing vaccines for respiratory syncytial virus |
PT2393828T (en) | 2009-02-03 | 2017-01-18 | Amunix Operating Inc | Extended recombinant polypeptides and compositions comprising same |
SG2014012918A (en) | 2009-02-11 | 2014-04-28 | Novozymes Biopharma Dk As | Albumin variants and conjugates |
ES2712732T3 (en) | 2009-02-17 | 2019-05-14 | Cornell Res Foundation Inc | Methods and kits for the diagnosis of cancer and the prediction of therapeutic value |
WO2010096394A2 (en) | 2009-02-17 | 2010-08-26 | Redwood Biosciences, Inc. | Aldehyde-tagged protein-based drug carriers and methods of use |
EP2405920A1 (en) | 2009-03-06 | 2012-01-18 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Novel therapy for anxiety |
EP2413958A4 (en) | 2009-03-31 | 2014-04-02 | Univ East Carolina | Cytokines and neuroantigens for treatment of immune disorders |
EP2241323A1 (en) | 2009-04-14 | 2010-10-20 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Tenascin-W and brain cancers |
EP2423233B1 (en) | 2009-04-22 | 2015-03-11 | Alteogen, Inc | In vivo half life increased fusion protein or peptide maintained by sustained in vivo release, and method for increasing in vivo half-life using same |
EP2427486B1 (en) | 2009-05-07 | 2015-02-25 | Novozymes Biopharma DK A/S | Method for purifying albumin |
US11512326B2 (en) | 2009-05-26 | 2022-11-29 | University Of Florida Research Foundation, Incorporated | Small angiotensin peptide expression system in mammalian cells |
WO2011005939A2 (en) | 2009-07-09 | 2011-01-13 | Mayo Foundation For Medical Education And Research | Long acting atrial natriuretic peptide (la-anp) and methods for use thereof |
US8945895B2 (en) * | 2009-07-31 | 2015-02-03 | Baxter International Inc. | Methods of purifying recombinant ADAMTS13 and other proteins and compositions thereof |
EP2464661B1 (en) | 2009-08-13 | 2018-01-17 | The Johns Hopkins University | Methods of modulating immune function with anti-b7-h7cr antibodies |
EP2292266A1 (en) | 2009-08-27 | 2011-03-09 | Novartis Forschungsstiftung, Zweigniederlassung | Treating cancer by modulating copine III |
US8451450B2 (en) * | 2009-09-14 | 2013-05-28 | Bio-Rad Laboratories, Inc. | Near real time optical phase conjugation |
WO2011036118A1 (en) | 2009-09-22 | 2011-03-31 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Treating cancer by modulating mex-3 |
US8568726B2 (en) | 2009-10-06 | 2013-10-29 | Medimmune Limited | RSV specific binding molecule |
US9096877B2 (en) | 2009-10-07 | 2015-08-04 | Macrogenics, Inc. | Fc region-containing polypeptides that exhibit improved effector function due to alterations of the extent of fucosylation, and methods for their use |
WO2011045352A2 (en) | 2009-10-15 | 2011-04-21 | Novartis Forschungsstiftung | Spleen tyrosine kinase and brain cancers |
NZ598465A (en) | 2009-10-30 | 2013-10-25 | Boehringer Ingelheim Int | Dosage regimens for hcv combination therapy comprising bi201335, interferon alpha and ribavirin |
GB2488077A (en) | 2009-10-30 | 2012-08-15 | Novozymes Biopharma Dk As | Albumin variants |
US20120213801A1 (en) | 2009-10-30 | 2012-08-23 | Ekaterina Gresko | Phosphorylated Twist1 and cancer |
WO2011051466A1 (en) | 2009-11-02 | 2011-05-05 | Novartis Ag | Anti-idiotypic fibronectin-based binding molecules and uses thereof |
CN102164949B (en) | 2009-11-19 | 2013-10-23 | 浙江大学 | Nonnatural protein |
BR112012012526A2 (en) * | 2009-11-24 | 2016-05-03 | Syngenta Participations Ag | stable mixtures and related methods |
MX354143B (en) | 2009-12-02 | 2018-02-14 | Imaginab Inc | J591 minibodies and cys-diabodies for targeting human prostate specific membrane antigen (psma) and methods for their use. |
PL3326643T3 (en) | 2009-12-06 | 2021-10-25 | Bioverativ Therapeutics Inc. | Factor viii-fc chimeric and hybrid polypeptides, and methods of use thereof |
TWI409082B (en) | 2010-01-19 | 2013-09-21 | Hanmi Science Co Ltd | Liquid formulation for long-acting g-csf conjugate |
WO2011092233A1 (en) | 2010-01-29 | 2011-08-04 | Novartis Ag | Yeast mating to produce high-affinity combinations of fibronectin-based binders |
EP2542578A1 (en) | 2010-03-05 | 2013-01-09 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Smoc1, tenascin-c and brain cancers |
KR20130070576A (en) | 2010-04-09 | 2013-06-27 | 노보자임스 바이오파마 디케이 에이/에스 | Albumin derivatives and variants |
GB201105584D0 (en) | 2011-04-01 | 2011-05-18 | Imp Innovations Ltd | Cancer methods |
US20130034543A1 (en) | 2010-04-19 | 2013-02-07 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Resear | Modulating xrn1 |
KR20130066631A (en) | 2010-05-06 | 2013-06-20 | 노파르티스 아게 | Compositions and methods of use for therapeutic low density lipoprotein - related protein 6 (lrp6) multivalent antibodies |
EP4234698A3 (en) | 2010-05-06 | 2023-11-08 | Novartis AG | Compositions and methods of use for therapeutic low density lipoprotein-related protein 6 (lrp6) antibodies |
EP2580239A1 (en) | 2010-06-10 | 2013-04-17 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Treating cancer by modulating mammalian sterile 20-like kinase 3 |
JP2013529627A (en) | 2010-06-24 | 2013-07-22 | パンメド リミテッド | Treatment of hepatitis C virus-related disease using hydroxychloroquine or a combination of hydroxychloroquine and an antiviral agent |
EP2585098B1 (en) | 2010-06-28 | 2014-08-27 | Five Prime Therapeutics, Inc. | Fzd8 extracellular domains and fzd8 extracellular domain fusion molecules for use in treating obesity and obesity-related disorders |
NZ605348A (en) | 2010-07-09 | 2015-01-30 | Biogen Idec Hemophilia Inc | Factor ix polypeptides and methods of use thereof |
NZ605400A (en) | 2010-07-09 | 2015-05-29 | Biogen Idec Hemophilia Inc | Chimeric clotting factors |
WO2012019061A2 (en) | 2010-08-05 | 2012-02-09 | Stem Centrx, Inc. | Novel effectors and methods of use |
DK2605789T3 (en) | 2010-08-17 | 2019-09-16 | Ambrx Inc | MODIFIED RELAXIN POLYPEPTIDES AND APPLICATIONS THEREOF |
BR112013004012B1 (en) | 2010-08-20 | 2021-03-23 | Novartis Ag | ISOLATED MONOCLONAL ANTIBODY OR ANTIGEN BINDING FRAGMENT OF THE SAME TO THE HER3 RECEPTOR, ITS USE AND PHARMACEUTICAL COMPOSITION |
EP2608807A1 (en) | 2010-08-27 | 2013-07-03 | Stem Centrx, Inc. | Notum protein modulators and methods of use |
EP2611464B1 (en) | 2010-09-03 | 2018-04-25 | AbbVie Stemcentrx LLC | Novel modulators and methods of use |
EP2614080A1 (en) | 2010-09-10 | 2013-07-17 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Phosphorylated twist1 and metastasis |
WO2012035037A1 (en) | 2010-09-14 | 2012-03-22 | F. Hoffmann-La Roche Ag | Method for purifying pegylated erythropoietin |
DK2635607T3 (en) | 2010-11-05 | 2019-11-18 | Zymeworks Inc | STABLE HETERODIMED ANTIBODY DESIGN WITH MUTATIONS IN THE FC DOMAIN |
EP2640738A1 (en) | 2010-11-15 | 2013-09-25 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Anti-fungal agents |
AU2011329127B2 (en) | 2010-11-15 | 2015-09-03 | Five Prime Therapeutics, Inc. | FGFR1 extracellular domain combination therapies |
WO2012069466A1 (en) | 2010-11-24 | 2012-05-31 | Novartis Ag | Multispecific molecules |
KR20190112175A (en) | 2010-12-01 | 2019-10-02 | 앨더바이오 홀딩스 엘엘씨 | Anti-ngf compositions and use thereof |
SG190990A1 (en) | 2010-12-08 | 2013-07-31 | Stem Centrx Inc | Novel modulators and methods of use |
US20120171195A1 (en) | 2011-01-03 | 2012-07-05 | Ravindranath Mepur H | Anti-hla-e antibodies, therapeutic immunomodulatory antibodies to human hla-e heavy chain, useful as ivig mimetics and methods of their use |
AU2012205301B2 (en) | 2011-01-14 | 2017-01-05 | Redwood Bioscience, Inc. | Aldehyde-tagged immunoglobulin polypeptides and method of use thereof |
WO2012097238A2 (en) | 2011-01-14 | 2012-07-19 | Five Prime Therapeutics, Inc. | Il-27 antagonists for treating inflammatory diseases |
SA112330278B1 (en) | 2011-02-18 | 2015-10-09 | ستيم سينتركس، انك. | Novel modulators and methods of use |
AU2012222833B2 (en) | 2011-03-03 | 2017-03-16 | Zymeworks Inc. | Multivalent heteromultimer scaffold design and constructs |
AR085911A1 (en) | 2011-03-16 | 2013-11-06 | Sanofi Sa | SAFE THERAPEUTIC DOSE OF A SIMILAR PROTEIN TO AN ANTIBODY WITH VUAL REGION |
CA2836800A1 (en) | 2011-05-20 | 2012-11-29 | Alderbio Holdings Llc | Use of anti-cgrp antibodies and antibody fragments to prevent or inhibit photophobia or light aversion in subjects in need thereof, especially migraine sufferers |
US9855332B2 (en) | 2011-05-20 | 2018-01-02 | Alderbio Holdings Llc | Use of anti-CGRP antibodies and antibody fragments to treat diarrhea in subjects with diseases or treatments that result in elevated CGRP levels |
CN107602700B (en) | 2011-05-20 | 2021-12-17 | H.伦德贝克公司 | anti-CGRP compositions and uses thereof |
WO2012168259A1 (en) | 2011-06-06 | 2012-12-13 | Novartis Forschungsstiftung, Zweigniederlassung | Protein tyrosine phosphatase, non-receptor type 11 (ptpn11) and triple-negative breast cancer |
US9244074B2 (en) | 2011-06-07 | 2016-01-26 | University Of Hawaii | Biomarker of asbestos exposure and mesothelioma |
US9561274B2 (en) | 2011-06-07 | 2017-02-07 | University Of Hawaii | Treatment and prevention of cancer with HMGB1 antagonists |
SI2717898T1 (en) | 2011-06-10 | 2019-07-31 | Bioverativ Therapeutics Inc. | Pro-coagulant compounds and methods of use thereof |
WO2012172495A1 (en) | 2011-06-14 | 2012-12-20 | Novartis Ag | Compositions and methods for antibodies targeting tem8 |
AR087020A1 (en) | 2011-07-01 | 2014-02-05 | Bayer Ip Gmbh | RELAXIN FUSION POLIPEPTIDES AND USES OF THE SAME |
CN103649111B (en) | 2011-07-05 | 2018-03-13 | 阿尔布梅迪克斯医疗公司 | Albumin preparation and purposes |
WO2013007563A1 (en) | 2011-07-08 | 2013-01-17 | Bayer Intellectual Property Gmbh | Fusion proteins releasing relaxin and uses thereof |
DK3513804T3 (en) | 2011-07-08 | 2022-06-20 | Bioverativ Therapeutics Inc | CHIMARY AND HYBRID FACTOR VIII POLYPEPTIDES AND METHODS OF USING IT |
EP2737311B1 (en) | 2011-07-25 | 2020-12-02 | Bioverativ Therapeutics Inc. | Assays to monitor bleeding disorders |
EP2751136B1 (en) | 2011-08-30 | 2017-10-18 | Mayo Foundation For Medical Education And Research | Natriuretic polypeptides |
US20130058947A1 (en) | 2011-09-02 | 2013-03-07 | Stem Centrx, Inc | Novel Modulators and Methods of Use |
KR102434073B1 (en) | 2011-10-11 | 2022-08-18 | 비엘라 바이오, 인크. | Cd40l-specific tn3-derived scaffolds and methods of use thereof |
CA2853951A1 (en) | 2011-11-01 | 2013-05-10 | Bionomics, Inc. | Antibodies and methods of treating cancer |
US10598653B2 (en) | 2011-11-01 | 2020-03-24 | Bionomics Inc. | Methods of blocking cancer stem cell growth |
EP2773667A1 (en) | 2011-11-01 | 2014-09-10 | Bionomics, Inc. | Anti-gpr49 antibodies |
US9220774B2 (en) | 2011-11-01 | 2015-12-29 | Bionomics Inc. | Methods of treating cancer by administering anti-GPR49 antibodies |
WO2013067355A1 (en) | 2011-11-04 | 2013-05-10 | Novartis Ag | Low density lipoprotein-related protein 6 (lrp6) - half life extender constructs |
CN109897103A (en) | 2011-11-04 | 2019-06-18 | 酵活有限公司 | There is the antibody design of the stabilization heterodimeric of mutation in Fc structural domain |
US20140314787A1 (en) | 2011-11-08 | 2014-10-23 | Novartis Forschungsstiftung, Zweigniederlassung, Friedrich Miescher Institute | Treatment for neurodegenerative diseases |
EP2776838A1 (en) | 2011-11-08 | 2014-09-17 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Early diagnostic of neurodegenerative diseases |
WO2013075066A2 (en) | 2011-11-18 | 2013-05-23 | Eleven Biotherapeutics, Inc. | Proteins with improved half-life and other properties |
US9493515B2 (en) | 2011-11-29 | 2016-11-15 | Proclara Biosciences, Inc. | Bacteriophage gene 3 protein compositions and use as amyloid binding agents |
JP2015500829A (en) | 2011-12-05 | 2015-01-08 | ノバルティス アーゲー | HER3 antibody against domain II of epidermal growth factor receptor 3 (HER3) |
WO2013084147A2 (en) | 2011-12-05 | 2013-06-13 | Novartis Ag | Antibodies for epidermal growth factor receptor 3 (her3) |
ES2728278T3 (en) | 2011-12-21 | 2019-10-23 | Novartis Ag | Compositions comprising antibodies directed to factor P and C5 |
US20140363448A1 (en) | 2012-01-02 | 2014-12-11 | Novartis Ag | Cdcp1 and breast cancer |
US9611305B2 (en) | 2012-01-06 | 2017-04-04 | Mayo Foundation For Medical Education And Research | Treating cardiovascular or renal diseases |
US20130177574A1 (en) | 2012-01-11 | 2013-07-11 | Paul I. Terasaki Foundation Laboratory | ANTI-HLA CLASS-Ib ANTIBODIES MIMIC IMMUNOREACTIVITY AND IMMUNOMODULATORY FUNCTIONS OF INTRAVENOUS IMMUNOGLOBULIN (IVIg) USEFUL AS THERAPEUTIC IVIg MIMETICS AND METHODS OF THEIR USE |
US10800847B2 (en) | 2012-01-11 | 2020-10-13 | Dr. Mepur Ravindranath | Anti-HLA class-IB antibodies mimic immunoreactivity and immunomodulatory functions of intravenous immunoglobulin (IVIG) useful as therapeutic IVIG mimetics and methods of their use |
ES2812849T3 (en) | 2012-02-24 | 2021-03-18 | Abbvie Stemcentrx Llc | Anti-DLL3 antibodies and procedures for using them |
WO2013137869A1 (en) | 2012-03-14 | 2013-09-19 | Boehringer Ingelheim International Gmbh | Combination therapy for treating hcv infection in an hcv-hiv coinfected patient population |
US9944691B2 (en) | 2012-03-16 | 2018-04-17 | Albumedix A/S | Albumin variants |
US9592289B2 (en) | 2012-03-26 | 2017-03-14 | Sanofi | Stable IgG4 based binding agent formulations |
EP2830646B1 (en) | 2012-03-27 | 2018-03-07 | NGM Biopharmaceuticals, Inc. | Compositions and methods of use for treating metabolic disorders |
JP2015512900A (en) | 2012-03-28 | 2015-04-30 | ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング | Combination therapy to treat HCV infection in a special patient genotype subpopulation |
US20150266961A1 (en) | 2012-03-29 | 2015-09-24 | Novartis Forschungsstiftung, Zweigniederlassung, Fridrich Miescher Institute | Inhibition of interleukin-8 and/or its receptor cxcr1 in the treatment of her2/her3-overexpressing breast cancer |
US10064951B2 (en) | 2012-03-30 | 2018-09-04 | Hanmi Science Co., Ltd. | Liquid formulation of highly concentrated long-acting human growth hormone conjugate |
WO2013166290A1 (en) | 2012-05-04 | 2013-11-07 | Abbvie Biotherapeutics Inc. | P21 biomarker assay |
CA2872540A1 (en) | 2012-05-10 | 2013-11-14 | Zymeworks Inc. | Heteromultimer constructs of immunoglobulin heavy chains with mutations in the fc domain |
KR20220051197A (en) | 2012-05-17 | 2022-04-26 | 익스텐드 바이오사이언시즈, 인크. | Carriers for improved drug delivery |
WO2013177386A1 (en) | 2012-05-24 | 2013-11-28 | Abbvie Biotherapeutics Inc. | Biomarkers for predicting response to tweak receptor (tweakr) agonist therapy |
EP2854834A4 (en) | 2012-05-30 | 2016-05-18 | Biostrategies LC | Plant lectins as carriers of associated drug substances into animal and human cells |
EP2857417B1 (en) | 2012-06-05 | 2018-02-14 | CJ Healthcare Corporation | Highly glycosylated long-acting human growth hormone protein and production method for same |
EP2866831A1 (en) | 2012-06-29 | 2015-05-06 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Treating diseases by modulating a specific isoform of mkl1 |
US10656156B2 (en) | 2012-07-05 | 2020-05-19 | Mepur Ravindranath | Diagnostic and therapeutic potential of HLA-E monospecific monoclonal IgG antibodies directed against tumor cell surface and soluble HLA-E |
EP2870242A1 (en) | 2012-07-05 | 2015-05-13 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | New treatment for neurodegenerative diseases |
WO2014008480A2 (en) | 2012-07-06 | 2014-01-09 | Biogen Idec Ma Inc. | Cell line expressing single chain factor viii polypeptides and uses thereof |
WO2014012082A2 (en) | 2012-07-13 | 2014-01-16 | Zymeworks Inc. | Multivalent heteromultimer scaffold design an constructs |
AR091902A1 (en) | 2012-07-25 | 2015-03-11 | Hanmi Pharm Ind Co Ltd | LIQUID FORMULATION OF A PROLONGED INSULIN CONJUGATE |
US20150202287A1 (en) | 2012-08-30 | 2015-07-23 | Merrimack Pharmaceuticals, Inc. | Combination therapies comprising anti-erbb3 agents |
EP3738605A1 (en) | 2012-09-10 | 2020-11-18 | Xencor, Inc. | Methods of treating neurological diseases |
EP2900328A4 (en) | 2012-09-25 | 2016-05-11 | Biogen Ma Inc | Methods of using fix polypeptides |
DK2906235T3 (en) | 2012-10-02 | 2017-09-25 | Proclara Biosciences Inc | USE OF P3 OF BACTERIOPHAGIC FUSION PROTEINS AS AMYLOID BINDING AGENTS |
EP3446700A1 (en) | 2012-10-30 | 2019-02-27 | Bioverativ Therapeutics Inc. | Methods of using fviii polypeptide |
BR112015010318A2 (en) | 2012-11-08 | 2017-08-22 | Albumedix As | ALBUMIN VARIANTS |
WO2014084859A1 (en) | 2012-11-30 | 2014-06-05 | Novartis Ag | Molecules and methods for modulating tmem16a activities |
EP3851454A1 (en) | 2012-12-05 | 2021-07-21 | Novartis AG | Compositions and methods for antibodies targeting epo |
US9161966B2 (en) | 2013-01-30 | 2015-10-20 | Ngm Biopharmaceuticals, Inc. | GDF15 mutein polypeptides |
JP6272907B2 (en) | 2013-01-30 | 2018-01-31 | エヌジーエム バイオファーマシューティカルズ インコーポレイテッド | Compositions and methods of use in the treatment of metabolic disorders |
EP2954048A1 (en) | 2013-02-08 | 2015-12-16 | Friedrich Miescher Institute for Biomedical Research | Novel methods for the targeted introduction of viruses into cells |
WO2014127120A1 (en) | 2013-02-15 | 2014-08-21 | Mayo Foundation For Medical Education And Research | Insulin secreting polypeptides |
RS60026B1 (en) | 2013-02-18 | 2020-04-30 | Vegenics Pty Ltd | Ligand binding molecules and uses thereof |
USRE48805E1 (en) * | 2013-03-06 | 2021-11-02 | Vision Global Holdings Ltd. | Method for cancer targeting treatment and detection of arginine using albumin-binding arginine deiminase fusion protein |
US9255262B2 (en) * | 2013-03-06 | 2016-02-09 | Vision Global Holdings Ltd. | Albumin-binding arginine deminase and the use thereof |
CA2940513C (en) | 2013-03-11 | 2023-08-15 | University Of Florida Research Foundation, Inc. | Delivery of card protein as therapy for ocular inflammation |
EP3611189A1 (en) | 2013-03-14 | 2020-02-19 | Novartis AG | Antibodies against notch 3 |
WO2014144795A1 (en) | 2013-03-15 | 2014-09-18 | Biogen Idec Ma Inc. | Factor viii polypeptide formulations |
SG10201913874TA (en) | 2013-03-15 | 2020-03-30 | Biogen Ma Inc | Factor ix polypeptide formulations |
WO2014147489A2 (en) | 2013-03-15 | 2014-09-25 | Teva Pharmaceutical Industries Ltd. | Recombinant human albumin-human granulocyte colony stimulating factor for the prevention of neutropenia in pediatric patients |
EP3473272A1 (en) | 2013-03-29 | 2019-04-24 | Alexion Pharmaceuticals, Inc. | Compositions and methods for increasing the serum half-life of a therapeutic agent targeting complement c5 |
JP2016519108A (en) | 2013-04-18 | 2016-06-30 | アルモ・バイオサイエンシーズ・インコーポレイテッド | Method for using interleukin-10 for the treatment of diseases and disorders |
ES2657291T3 (en) | 2013-04-22 | 2018-03-02 | Csl Ltd. | A covalent complex of von Willebrand factor and factor VIII associated by a disulfide bridge |
SG11201508878WA (en) | 2013-05-01 | 2015-11-27 | Five Prime Therapeutics Inc | Methods of treating cancer |
EP2999799A4 (en) | 2013-05-23 | 2016-12-28 | Five Prime Therapeutics Inc | Methods of treating cancer |
CN105473153A (en) | 2013-05-28 | 2016-04-06 | 神经噬菌体制药股份有限公司 | Polypeptides comprising a modified bacteriophage G3P amino acid sequence with reduced immunogenicity |
JP2016528879A (en) | 2013-06-17 | 2016-09-23 | アルモ・バイオサイエンシーズ・インコーポレイテッド | Methods for assessing protein identity and stability |
UY35620A (en) | 2013-06-21 | 2015-01-30 | Novartis Ag | ANTIBODIES OF LEXINED OXIDATED LDL RECEIVER 1 AND METHODS OF USE |
AR096601A1 (en) | 2013-06-21 | 2016-01-20 | Novartis Ag | ANTIBODIES OF LEXINED OXIDATED LDL RECEIVER 1 AND METHODS OF USE |
TWI712419B (en) | 2013-07-03 | 2020-12-11 | 美商艾爾德生物製藥股份有限公司 | Regulation of glucose metabolism using anti-cgrp antibodies |
ES2761587T3 (en) | 2013-08-07 | 2020-05-20 | Friedrich Miescher Institute For Biomedical Res | New screening method for the treatment of Friedreich's ataxia |
CN105792836A (en) | 2013-08-28 | 2016-07-20 | 施特姆森特克斯股份有限公司 | Novel SEZ6 modulators and methods of use |
AU2014312215B2 (en) | 2013-08-28 | 2020-02-27 | Abbvie Stemcentrx Llc | Site-specific antibody conjugation methods and compositions |
JP6509867B2 (en) | 2013-08-30 | 2019-05-08 | アルモ・バイオサイエンシーズ・インコーポレイテッド | Methods of using interleukin-10 to treat diseases and disorders |
WO2015066550A1 (en) * | 2013-10-31 | 2015-05-07 | Resolve Therapeutics, Llc | Therapeutic nuclease-albumin fusions and methods |
RU2016122957A (en) | 2013-11-11 | 2017-12-19 | Армо Байосайенсиз, Инк. | Methods of using interleukin-10 for the treatment of diseases and disorders |
EP4332839A2 (en) | 2013-12-06 | 2024-03-06 | Bioverativ Therapeutics Inc. | Population pharmacokinetics tools and uses thereof |
US8980273B1 (en) | 2014-07-15 | 2015-03-17 | Kymab Limited | Method of treating atopic dermatitis or asthma using antibody to IL4RA |
US8986691B1 (en) | 2014-07-15 | 2015-03-24 | Kymab Limited | Method of treating atopic dermatitis or asthma using antibody to IL4RA |
US9682123B2 (en) | 2013-12-20 | 2017-06-20 | The Trustees Of Columbia University In The City Of New York | Methods of treating metabolic disease |
EA038573B1 (en) | 2014-03-24 | 2021-09-16 | Биовератив Терапьютикс Инк. | Lyophilized factor ix formulation for preventing bleeding episodes |
CN106536556B (en) | 2014-04-04 | 2020-02-07 | 生态学有限公司 | Humanized antibodies that bind LGR5 |
WO2015168207A1 (en) | 2014-04-29 | 2015-11-05 | Mayo Foundation For Medical Education And Research | Butyrylcholinesterases having an enhanced ability to hydrolyze acyl ghrelin |
MX2016014306A (en) | 2014-05-02 | 2017-06-12 | Cerenis Therapeutics Holding Sa | Hdl therapy markers. |
US10293043B2 (en) | 2014-06-02 | 2019-05-21 | Armo Biosciences, Inc. | Methods of lowering serum cholesterol |
WO2015189816A1 (en) | 2014-06-13 | 2015-12-17 | Friedrich Miescher Institute For Biomedical Research | New treatment against influenza virus |
DK3173420T3 (en) * | 2014-07-23 | 2021-10-18 | Nat Ct Nanoscience & Technology China | POLYPEPTID AND POLYPEPTID COMPLEX FOR SUPPRESSING TUMOR METASTASE AND TREATMENT OF LEUKEMIA AND PROCEDURE FOR MANUFACTURING ITS AND USING IT |
PE20170771A1 (en) | 2014-07-30 | 2017-07-04 | Ngm Biopharmaceuticals Inc | COMPOSITIONS AND METHODS OF USE TO TREAT METABOLIC DISORDERS |
EP3194437B1 (en) | 2014-08-07 | 2021-01-20 | Novartis AG | Angiopoietin-like 4 (angptl4) antibodies and methods of use |
DK3177642T3 (en) | 2014-08-07 | 2022-02-21 | Novartis Ag | ANGIOPOIETIN-LIKE 4 ANTIBODIES AND METHODS OF USING IT |
US20170224777A1 (en) | 2014-08-12 | 2017-08-10 | Massachusetts Institute Of Technology | Synergistic tumor treatment with il-2, a therapeutic antibody, and a cancer vaccine |
JP6800141B2 (en) | 2014-08-12 | 2020-12-16 | マサチューセッツ インスティテュート オブ テクノロジー | Synergistic tumor treatment with IL-2 and integrin-binding FC fusion protein |
EP3197557A1 (en) | 2014-09-24 | 2017-08-02 | Friedrich Miescher Institute for Biomedical Research | Lats and breast cancer |
MA41044A (en) | 2014-10-08 | 2017-08-15 | Novartis Ag | COMPOSITIONS AND METHODS OF USE FOR INCREASED IMMUNE RESPONSE AND CANCER TREATMENT |
CA2909044A1 (en) * | 2014-10-10 | 2016-04-10 | Queen's University At Kingston | Rhomboid proteins and uses thereof |
KR20170068553A (en) | 2014-10-14 | 2017-06-19 | 아르모 바이오사이언시스 인코포레이티드 | Interleukin-15 compositions and uses thereof |
WO2016065052A1 (en) | 2014-10-22 | 2016-04-28 | Extend Biosciences, Inc. | Insulin vitamin d conjugates |
US9789197B2 (en) | 2014-10-22 | 2017-10-17 | Extend Biosciences, Inc. | RNAi vitamin D conjugates |
JP6675394B2 (en) | 2014-10-22 | 2020-04-01 | アルモ・バイオサイエンシーズ・インコーポレイテッド | Use of interleukin-10 for the treatment of diseases and disorders |
US9585934B2 (en) | 2014-10-22 | 2017-03-07 | Extend Biosciences, Inc. | Therapeutic vitamin D conjugates |
EP3209681A4 (en) | 2014-10-23 | 2018-10-31 | NGM Biopharmaceuticals, Inc. | Pharmaceutical compositions comprising peptide variants and methods of use thereof |
UY36370A (en) | 2014-10-24 | 2016-04-29 | Bristol Myers Squibb Company Una Corporación Del Estado De Delaware | MODIFIED FGF-21 POLIPEPTIDES AND ITS USES |
EP3701969A1 (en) | 2014-10-31 | 2020-09-02 | NGM Biopharmaceuticals, Inc. | Compositions and methods of use for treating metabolic disorders |
MX2017006663A (en) | 2014-11-19 | 2017-08-21 | Axon Neuroscience Se | Humanized tau antibodies in alzheimer's disease. |
AU2015358504A1 (en) | 2014-12-03 | 2017-06-29 | Proclara Biosciences, Inc. | Polypeptides comprising a modified bacteriophage g3p amino acid sequence lacking a glycosylation signal |
EP3227319B1 (en) * | 2014-12-04 | 2019-11-27 | Novartis AG | Methods and compositions using klotho variant polypeptides |
EP3835319A1 (en) | 2014-12-19 | 2021-06-16 | Alder Biopharmaceuticals, Inc. | Humanized anti-acth antibodies and use thereof |
UY36449A (en) | 2014-12-19 | 2016-07-29 | Novartis Ag | COMPOSITIONS AND METHODS FOR ANTIBODIES DIRECTED TO BMP6 |
WO2016126615A1 (en) | 2015-02-03 | 2016-08-11 | Armo Biosciences, Inc. | Methods of using interleukin-10 for treating diseases and disorders |
EP3842451A1 (en) | 2015-03-12 | 2021-06-30 | MedImmune, LLC | Method of purifying albumin-fusion proteins |
CA2984947A1 (en) | 2015-05-19 | 2016-11-24 | Yale University | Compositions for treating pathological calcification conditions, and methods using same |
JP7121496B2 (en) | 2015-05-28 | 2022-08-18 | アルモ・バイオサイエンシーズ・インコーポレイテッド | Pegylated interleukin-10 for use in cancer therapy |
WO2016196211A1 (en) | 2015-05-29 | 2016-12-08 | Armo Biosciences, Inc. | Methods of using interleukin-10 for treating diseases and disorders |
PE20180041A1 (en) | 2015-06-05 | 2018-01-09 | Novartis Ag | ANTIBODIES TARGETING BONE MORPHOGENETIC PROTEIN (BMP9) AND METHODS FROM THESE |
CA2988588A1 (en) | 2015-06-12 | 2016-12-15 | Georgia State University Research Foundation, Inc. | Compositions and methods for treating opioid tolerance |
AU2016279804B2 (en) | 2015-06-15 | 2019-03-07 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for treating aging-associated conditions |
JOP20200312A1 (en) | 2015-06-26 | 2017-06-16 | Novartis Ag | Factor xi antibodies and methods of use |
EP3331914A1 (en) | 2015-08-03 | 2018-06-13 | Novartis AG | Methods of treating fgf21-associated disorders |
EP4137158A1 (en) | 2015-08-07 | 2023-02-22 | Imaginab, Inc. | Antigen binding constructs to target molecules |
CA2995838A1 (en) | 2015-08-19 | 2017-02-23 | Rutgers, The State University Of New Jersey | Novel methods of generating antibodies |
JP7007261B2 (en) | 2015-08-20 | 2022-01-24 | アルブミディクス リミティド | Albumin variants and conjugates |
CN108025040A (en) | 2015-08-25 | 2018-05-11 | 阿尔莫生物科技股份有限公司 | The method that disease and illness are treated using interleukin-10 |
JP6886920B2 (en) | 2015-09-08 | 2021-06-16 | Jcrファーマ株式会社 | New human serum albumin mutant |
TN2018000076A1 (en) | 2015-09-09 | 2019-07-08 | Novartis Ag | Thymic stromal lymphopoietin (tslp)-binding molecules and methods of using the molecules |
DK3347377T3 (en) | 2015-09-09 | 2021-05-10 | Novartis Ag | Thymic stromal lymphopoietin (TSLP) -binding antibodies and methods of using the antibodies |
WO2017058923A1 (en) | 2015-09-28 | 2017-04-06 | East Carolina University | Aluminum based adjuvants for tolerogenic vaccination |
WO2017072669A1 (en) | 2015-10-28 | 2017-05-04 | Friedrich Miescher Institute For Biomedical Research | Tenascin-w and biliary tract cancers |
EP3888672A1 (en) | 2015-11-09 | 2021-10-06 | NGM Biopharmaceuticals, Inc. | Methods for treatment of bile acid-related disorders |
UY37030A (en) | 2015-12-18 | 2017-07-31 | Novartis Ag | ANTIBODIES DIRECTED TO CD32B AND METHODS OF USE OF THE SAME |
CN116003593A (en) | 2016-01-11 | 2023-04-25 | 苏黎世大学 | Immunostimulatory humanized monoclonal antibodies directed against human interleukin-2 and fusion proteins thereof |
ES2847155T3 (en) | 2016-01-21 | 2021-08-02 | Novartis Ag | Multispecific molecules targeting CLL-1 |
JP2017165713A (en) | 2016-03-14 | 2017-09-21 | Jcrファーマ株式会社 | Serum albumin-20K growth hormone fusion protein |
CA3018081A1 (en) | 2016-03-22 | 2017-09-28 | Bionomics Limited | Administration of an anti-lgr5 monoclonal antibody |
KR102370762B1 (en) | 2016-03-31 | 2022-03-04 | 엔지엠 바이오파마슈티컬스, 아이엔씨. | Binding proteins and methods of use thereof |
CA3020990A1 (en) | 2016-04-15 | 2017-10-19 | Alder Biopharmaceuticals, Inc. | Humanized anti-pacap antibodies and uses thereof |
CN115975044A (en) | 2016-04-25 | 2023-04-18 | 戊瑞治疗有限公司 | NOPE for the treatment of pathological muscle loss and weakness |
WO2017189432A1 (en) | 2016-04-26 | 2017-11-02 | R.P. Scherer Technologies, Llc | Antibody conjugates and methods of making and using the same |
JP7138567B2 (en) | 2016-04-27 | 2022-09-16 | ノバルティス アーゲー | Antibodies against growth differentiation factor 15 and their uses |
AU2017267047B2 (en) * | 2016-05-20 | 2023-11-09 | Octapharma Ag | Glycosylated VWF fusion proteins with improved pharmacokinetics |
WO2017216724A1 (en) | 2016-06-15 | 2017-12-21 | Novartis Ag | Methods for treating disease using inhibitors of bone morphogenetic protein 6 (bmp6) |
CA3027143A1 (en) | 2016-07-15 | 2018-01-18 | F. Hoffmann-La Roche Ag | Method for purifying pegylated erythropoietin |
WO2018053216A1 (en) | 2016-09-15 | 2018-03-22 | Mayo Foundation For Medical Education And Research | Methods and materials for using butyrylcholinesterases to treat cancer |
EP3538131A4 (en) | 2016-11-09 | 2020-07-01 | Mayo Foundation for Medical Education and Research | Manp analogues |
EP3558370A2 (en) | 2016-12-23 | 2019-10-30 | Novartis AG | Methods of treatment with anti-factor xi/xia antibodies |
IL267538B1 (en) | 2016-12-23 | 2024-01-01 | Novartis Ag | Anti-factor xi/xia antibodies for use in preventing, treating, managing or reducing the risk of a thromboembolic disorder or stroke in a subject |
US10350266B2 (en) | 2017-01-10 | 2019-07-16 | Nodus Therapeutics, Inc. | Method of treating cancer with a multiple integrin binding Fc fusion protein |
AU2018207303A1 (en) | 2017-01-10 | 2019-07-25 | xCella Biosciences, Inc. | Combination tumor treatment with an integrin-binding-Fc fusion protein and immune modulator |
MX2019009498A (en) | 2017-02-08 | 2019-10-02 | Novartis Ag | Fgf21 mimetic antibodies and uses thereof. |
US11266745B2 (en) | 2017-02-08 | 2022-03-08 | Imaginab, Inc. | Extension sequences for diabodies |
CN110869386A (en) * | 2017-02-10 | 2020-03-06 | 维维巴巴公司 | Compositions and methods for recombinant nerve growth factor |
US20200131225A1 (en) * | 2017-04-20 | 2020-04-30 | Novo Nordisk A/S | Methods of purification of albumin fusion proteins |
JP2020517271A (en) | 2017-04-22 | 2020-06-18 | イミュノミック セラピューティックス, インコーポレイテッドImmunomic Therapeutics, Inc. | Improved LAMP construct |
JP7222915B2 (en) | 2017-05-02 | 2023-02-15 | イミュノミック セラピューティックス, インコーポレイテッド | Improved LAMP constructs containing cancer antigens |
WO2018229715A1 (en) | 2017-06-16 | 2018-12-20 | Novartis Ag | Compositions comprising anti-cd32b antibodies and methods of use thereof |
EP3684811A2 (en) | 2017-08-17 | 2020-07-29 | Massachusetts Institute of Technology | Multiple specificity binders of cxc chemokines and uses thereof |
US20200172933A1 (en) | 2017-08-18 | 2020-06-04 | Friedrich Miescher Institute For Biomedical Research | Novel methods for the targeted introduction of viruses into cells and tissues |
CA3073537A1 (en) | 2017-08-22 | 2019-02-28 | Sanabio, Llc | Soluble interferon receptors and uses thereof |
US20210040205A1 (en) | 2017-10-25 | 2021-02-11 | Novartis Ag | Antibodies targeting cd32b and methods of use thereof |
US20200353050A1 (en) | 2017-11-10 | 2020-11-12 | Armo Biosciences, Inc. | Compositions and methods of use of interleukin-10 in combination with immune check-point pathway inhibitors |
JP7165193B2 (en) | 2017-11-27 | 2022-11-02 | パーデュー、ファーマ、リミテッド、パートナーシップ | Humanized antibody targeting human tissue factor |
WO2019154985A1 (en) | 2018-02-12 | 2019-08-15 | Biontech Rna Pharmaceuticals Gmbh | Treatment using cytokine encoding rna |
EP3774877A1 (en) * | 2018-03-28 | 2021-02-17 | Bioxodes | Anticoagulant fusion proteins and uses thereof |
WO2019186344A1 (en) | 2018-03-28 | 2019-10-03 | Institut Pasteur De Tunis | Anti-cancer and disintegrin scorpion venoms |
TW202015726A (en) | 2018-05-30 | 2020-05-01 | 瑞士商諾華公司 | Entpd2 antibodies, combination therapies, and methods of using the antibodies and combination therapies |
EP3802611A2 (en) | 2018-06-01 | 2021-04-14 | Novartis AG | Binding molecules against bcma and uses thereof |
US20210347842A1 (en) | 2018-06-19 | 2021-11-11 | Eli Lilly And Company | Compositions and methods of use of il-10 agents in conjunction with chimeric antigen receptor cell therapy |
AU2019311233A1 (en) | 2018-07-24 | 2021-01-21 | BioNTech SE | IL2 agonists |
CN110818793A (en) * | 2018-08-14 | 2020-02-21 | 中山康方生物医药有限公司 | Antibodies against IL-1 β, pharmaceutical compositions thereof and uses thereof |
EP3856763A1 (en) | 2018-09-28 | 2021-08-04 | Massachusetts Institute of Technology | Collagen-localized immunomodulatory molecules and methods thereof |
UY38407A (en) | 2018-10-15 | 2020-05-29 | Novartis Ag | TREM2 STABILIZING ANTIBODIES |
WO2020109978A1 (en) | 2018-11-26 | 2020-06-04 | Novartis Ag | Lpl-gpihbp1 fusion polypeptides |
KR20210113261A (en) | 2019-01-04 | 2021-09-15 | 리졸브 테라퓨틱스, 엘엘씨 | Treatment of Sjogren's Disease Using Nuclease Fusion Proteins |
EP3914289A1 (en) | 2019-01-23 | 2021-12-01 | Massachusetts Institute of Technology | Combination immunotherapy dosing regimen for immune checkpoint blockade |
AU2020218615A1 (en) | 2019-02-08 | 2021-09-30 | Biontech Cell & Gene Therapies Gmbh | Treatment involving car-engineered T cells and cytokines |
AU2020242254A1 (en) | 2019-03-18 | 2020-09-24 | Biontech Cell & Gene Therapies Gmbh | Lnterleukin-2 receptor (IL2R) and interleukin-2 (IL2) variants for specific activation of immune effector cells |
CN113874392A (en) | 2019-03-28 | 2021-12-31 | 丹尼斯科美国公司 | Engineered antibodies |
WO2020200481A1 (en) | 2019-04-05 | 2020-10-08 | Biontech Rna Pharmaceuticals Gmbh | Treatment involving interleukin-2 (il2) and interferon (ifn) |
EP3972998A1 (en) | 2019-05-21 | 2022-03-30 | Novartis AG | Cd19 binding molecules and uses thereof |
EP3972993A1 (en) | 2019-05-21 | 2022-03-30 | Novartis AG | Variant cd58 domains and uses thereof |
JP2022532928A (en) | 2019-05-24 | 2022-07-20 | サノフイ | Methods for treating systemic scleroderma |
TW202115105A (en) | 2019-06-24 | 2021-04-16 | 德商拜恩迪克Rna製藥有限公司 | Il2 agonists |
JP2022538974A (en) | 2019-06-26 | 2022-09-07 | マサチューセッツ インスチテュート オブ テクノロジー | Immunomodulatory fusion protein-metal hydroxide complexes and methods thereof |
TW202124446A (en) | 2019-09-18 | 2021-07-01 | 瑞士商諾華公司 | Combination therapies with entpd2 antibodies |
US20220348651A1 (en) | 2019-09-18 | 2022-11-03 | Novartis Ag | Entpd2 antibodies, combination therapies, and methods of using the antibodies and combination therapies |
WO2021058091A1 (en) | 2019-09-24 | 2021-04-01 | Biontech Rna Pharmaceuticals Gmbh | Treatment involving therapeutic antibody and interleukin-2 (il2) |
CN114641306A (en) | 2019-10-18 | 2022-06-17 | 免疫治疗有限公司 | Improved LAMP constructs comprising cancer antigens |
EP4069200A1 (en) | 2019-12-04 | 2022-10-12 | Albumedix Ltd | Methods and compositions produced thereby |
WO2021129927A1 (en) | 2019-12-23 | 2021-07-01 | Biontech Cell & Gene Therapies Gmbh | Treatment with immune effector cells engineered to express an antigen receptor |
EP4081241A1 (en) * | 2019-12-24 | 2022-11-02 | Juvena Therapeutics, Inc. | Regenerative polypeptides and uses thereof |
WO2021129945A1 (en) | 2019-12-27 | 2021-07-01 | Biontech Cell & Gene Therapies Gmbh | In vitro and in vivo gene delivery to immune effector cells using nanoparticles functionalized with designed ankyrin repeat proteins (darpins) |
US20230076768A1 (en) | 2020-01-14 | 2023-03-09 | Synthekine, Inc. | IL2 Orthologs and Methods of Use |
US20230405046A1 (en) | 2020-03-16 | 2023-12-21 | Biontech Cell & Gene Therapies Gmbh | Antigen-specific t cell receptors and t cell epitopes |
WO2021202473A2 (en) | 2020-03-30 | 2021-10-07 | Danisco Us Inc | Engineered antibodies |
WO2021197589A1 (en) | 2020-03-31 | 2021-10-07 | BioNTech SE | Treatment involving non-immunogenic rna for antigen vaccination |
SG10202003296VA (en) | 2020-04-06 | 2021-11-29 | H Lundbeck As | Treatment of most bothersome symptom (mbs) associated with migraine using anti-cgrp antibodies |
CA3165342A1 (en) | 2020-06-29 | 2022-01-06 | James Arthur Posada | Treatment of sjogren's syndrome with nuclease fusion proteins |
AU2021320233A1 (en) | 2020-08-05 | 2023-03-23 | Synthekine, Inc. | IL10Ra binding molecules and methods of use |
EP4192877A1 (en) | 2020-08-05 | 2023-06-14 | Synthekine, Inc. | Il2rb/il2rg synthetic cytokines |
EP4192495A1 (en) | 2020-08-07 | 2023-06-14 | Bristol-Myers Squibb Company | Fgf21 combined with ccr2/5 antagonists for the treatment of fibrosis |
EP4208197A1 (en) | 2020-09-04 | 2023-07-12 | Rutgers, The State University of New Jersey | Sars-cov-2 vaccines and antibodies |
US20230382978A1 (en) | 2020-10-15 | 2023-11-30 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Antibody specific for sars-cov-2 receptor binding domain and therapeutic methods |
WO2022087274A1 (en) | 2020-10-21 | 2022-04-28 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Antibodies that neutralize type-i interferon (ifn) activity |
EP4240491A1 (en) | 2020-11-06 | 2023-09-13 | Novartis AG | Cd19 binding molecules and uses thereof |
US20240123031A1 (en) | 2020-11-25 | 2024-04-18 | Bristol-Myers Squibb Company | Methods of treating liver diseases |
TW202245808A (en) | 2020-12-21 | 2022-12-01 | 德商拜恩迪克公司 | Therapeutic rna for treating cancer |
WO2022135667A1 (en) | 2020-12-21 | 2022-06-30 | BioNTech SE | Therapeutic rna for treating cancer |
WO2022135666A1 (en) | 2020-12-21 | 2022-06-30 | BioNTech SE | Treatment schedule for cytokine proteins |
EP4294834A2 (en) | 2021-02-19 | 2023-12-27 | The United States of America, as represented by The Secretary, Department of Health and Human Services | Single domain antibodies that neutralize sars-cov-2 |
JP2024514364A (en) | 2021-04-12 | 2024-04-01 | ビオンテック・ソシエタス・エウロパエア | RNA compositions containing buffer substances and methods for their production, storage and use |
WO2022223617A1 (en) | 2021-04-20 | 2022-10-27 | BioNTech SE | Virus vaccine |
AU2022333323A1 (en) | 2021-08-27 | 2024-02-29 | H. Lundbeck A/S | Treatment of cluster headache using anti-cgrp antibodies |
WO2023051926A1 (en) | 2021-09-30 | 2023-04-06 | BioNTech SE | Treatment involving non-immunogenic rna for antigen vaccination and pd-1 axis binding antagonists |
WO2023066496A1 (en) | 2021-10-21 | 2023-04-27 | BioNTech SE | Coronavirus vaccine |
EP4238577A3 (en) | 2021-10-22 | 2023-12-06 | BioNTech SE | Compositions for administration of different doses of rna |
WO2023083434A1 (en) | 2021-11-09 | 2023-05-19 | BioNTech SE | Rna encoding peptidoglycan hydrolase and use thereof for treating bacterial infection |
WO2023126053A1 (en) | 2021-12-28 | 2023-07-06 | BioNTech SE | Lipid-based formulations for administration of rna |
WO2023165681A1 (en) | 2022-03-01 | 2023-09-07 | BioNTech SE | Rna lipid nanoparticles (lnps) comprising a polyoxazoline and/or polyoxazine polymer |
WO2023193892A1 (en) | 2022-04-05 | 2023-10-12 | BioNTech SE | Nucleic acid compositions comprising an inorganic polyphosphate and methods for preparing, storing and using the same |
TW202400658A (en) | 2022-04-26 | 2024-01-01 | 瑞士商諾華公司 | Multispecific antibodies targeting il-13 and il-18 |
WO2024002985A1 (en) | 2022-06-26 | 2024-01-04 | BioNTech SE | Coronavirus vaccine |
WO2024015953A1 (en) | 2022-07-15 | 2024-01-18 | Danisco Us Inc. | Methods for producing monoclonal antibodies |
WO2024017479A1 (en) | 2022-07-21 | 2024-01-25 | BioNTech SE | Multifunctional cells transiently expressing an immune receptor and one or more cytokines, their use and methods for their production |
WO2024028325A1 (en) | 2022-08-01 | 2024-02-08 | BioNTech SE | Nucleic acid compositions comprising amphiphilic oligo ethylene glycol (oeg)-conjugated compounds and methods of using such compounds and compositions |
WO2024028445A1 (en) | 2022-08-03 | 2024-02-08 | BioNTech SE | Rna for preventing or treating tuberculosis |
WO2024027910A1 (en) | 2022-08-03 | 2024-02-08 | BioNTech SE | Rna for preventing or treating tuberculosis |
US11773160B1 (en) | 2022-08-05 | 2023-10-03 | Anaveon AG | Immune-stimulating IL-2 fusion proteins |
Family Cites Families (291)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US507367A (en) * | 1893-10-24 | Electro-thermal bathing apparatus | ||
US5625041A (en) | 1990-09-12 | 1997-04-29 | Delta Biotechnology Limited | Purification of proteins |
DE2449885C3 (en) | 1974-10-21 | 1980-04-30 | Biotest-Serum-Institut Gmbh, 6000 Frankfurt | Process for the production of chemically modified, long-life hemoglobin preparations as well as the modified hemoglobin preparation produced by this process |
US4264731A (en) | 1977-05-27 | 1981-04-28 | The Regents Of The University Of California | DNA Joining method |
US4440859A (en) | 1977-05-27 | 1984-04-03 | The Regents Of The University Of California | Method for producing recombinant bacterial plasmids containing the coding sequences of higher organisms |
US4363877B1 (en) | 1977-09-23 | 1998-05-26 | Univ California | Recombinant dna transfer vectors |
US4283489A (en) | 1977-09-23 | 1981-08-11 | The Regents Of The University Of California | Purification of nucleotide sequences suitable for expression in bacteria |
US4407948A (en) | 1977-09-23 | 1983-10-04 | The Regents Of The University Of California | Purification of nucleotide sequences suitable for expression in bacteria |
US4366246A (en) | 1977-11-08 | 1982-12-28 | Genentech, Inc. | Method for microbial polypeptide expression |
US4652525A (en) | 1978-04-19 | 1987-03-24 | The Regents Of The University Of California | Recombinant bacterial plasmids containing the coding sequences of insulin genes |
US4447538A (en) | 1978-04-19 | 1984-05-08 | Regents Of The University Of California | Microorganism containing gene for human chorionic somatomammotropin |
US5514567A (en) | 1979-01-30 | 1996-05-07 | Juridical Foundation, Japanese Foundation For Cancer Research | DNA and recombinant plasmid |
US5326859A (en) | 1979-10-30 | 1994-07-05 | Juridical Foundation, Japanese Foundation For Cancer Research | DNA and recombinant plasmid |
US4898830A (en) | 1979-07-05 | 1990-02-06 | Genentech, Inc. | Human growth hormone DNA |
US4342832A (en) | 1979-07-05 | 1982-08-03 | Genentech, Inc. | Method of constructing a replicable cloning vehicle having quasi-synthetic genes |
US4431740A (en) | 1979-09-12 | 1984-02-14 | The Regents Of The University Of California | DNA Transfer vector and transformed microorganism containing human proinsulin and pre-proinsulin genes |
AU538665B2 (en) | 1979-10-30 | 1984-08-23 | Juridical Foundation, Japanese Foundation For Cancer Research | Human interferon dna |
US4530901A (en) | 1980-01-08 | 1985-07-23 | Biogen N.V. | Recombinant DNA molecules and their use in producing human interferon-like polypeptides |
US6610830B1 (en) | 1980-07-01 | 2003-08-26 | Hoffman-La Roche Inc. | Microbial production of mature human leukocyte interferons |
FR2490675B1 (en) | 1980-09-25 | 1985-11-15 | Genentech Inc | MICROBIAL PRODUCTION OF HUMAN FIBROPLASTER INTERFERON |
US4456748A (en) | 1981-02-23 | 1984-06-26 | Genentech, Inc. | Hybrid human leukocyte interferons |
NZ199722A (en) | 1981-02-25 | 1985-12-13 | Genentech Inc | Dna transfer vector for expression of exogenous polypeptide in yeast;transformed yeast strain |
JPS57149228A (en) | 1981-03-11 | 1982-09-14 | Ajinomoto Co Inc | Novel erythropoietin and its preparation |
US4792602A (en) | 1981-06-19 | 1988-12-20 | Cornell Research Foundation, Inc. | Adaptors, and synthesis and cloning of proinsulin genes |
CA1204682A (en) | 1981-06-19 | 1986-05-20 | Saran A. Narang | Adaptors, and synthesis and cloning of proinsulin genes |
IL66614A (en) | 1981-08-28 | 1985-09-29 | Genentech Inc | Method of constructing a dna sequence encoding a polypeptide,microbial production of human serum albumin,and pharmaceutical compositions comprising it |
EP0079739A3 (en) | 1981-11-12 | 1984-08-08 | The Upjohn Company | Albumin-based nucleotides, their replication and use, and plasmids for use therein |
EP0091527A3 (en) | 1981-12-14 | 1984-07-25 | The President And Fellows Of Harvard College | Dna sequences, recombinant dna molecules and processes for producing human serum albumin-like polypeptides |
WO1983002461A1 (en) | 1982-01-19 | 1983-07-21 | Cetus Corp | Multiclass hybrid interferons |
US4450103A (en) | 1982-03-01 | 1984-05-22 | Cetus Corporation | Process for recovering human IFN-β from a transformed microorganism |
US4775622A (en) * | 1982-03-08 | 1988-10-04 | Genentech, Inc. | Expression, processing and secretion of heterologous protein by yeast |
US4670393A (en) | 1982-03-22 | 1987-06-02 | Genentech, Inc. | DNA vectors encoding a novel human growth hormone-variant protein |
EP0091539B2 (en) | 1982-03-31 | 1996-11-27 | Ajinomoto Co., Inc. | Gene coding for interleukin-2 polypeptide, recombinant DNA carrying said gene, cell lines possessing the recombinant DNA,and method for producing interleukin-2 using said cells |
US4925919A (en) | 1984-04-25 | 1990-05-15 | Roland Mertelsmann | Purified interleukin 2 |
US4778879A (en) | 1982-04-20 | 1988-10-18 | Sloan-Kettering Institute For Cancer Research | Highly purified human interleukin 2 and method |
US5824330A (en) | 1982-04-20 | 1998-10-20 | Sloan-Kettering Institute For Cancer Research | Highly purified interleukin-2 and method |
US4499188A (en) | 1982-05-05 | 1985-02-12 | Cetus Corporation | Bacterial production of heterologous polypeptides under the control of a repressible promoter-operator |
US4490289A (en) | 1982-09-16 | 1984-12-25 | Hoffmann-La Roche Inc. | Homogeneous human interleukin 2 |
US4992271A (en) | 1982-09-23 | 1991-02-12 | Cetus Corporation | Formulation for lipophilic IL-2 proteins |
US4462940A (en) | 1982-09-23 | 1984-07-31 | Cetus Corporation | Process for the recovery of human β-interferon-like polypeptides |
US4853332A (en) | 1982-10-19 | 1989-08-01 | Cetus Corporation | Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of biologically active proteins |
US4966843A (en) | 1982-11-01 | 1990-10-30 | Cetus Corporation | Expression of interferon genes in Chinese hamster ovary cells |
US5618697A (en) | 1982-12-10 | 1997-04-08 | Novo Nordisk A/S | Process for preparing a desired protein |
US5700913A (en) | 1982-12-15 | 1997-12-23 | Ajinomoto Co., Inc. | Unglycosylated human interleukin-2 polypeptides |
EP0116201B1 (en) | 1983-01-12 | 1992-04-22 | Chiron Corporation | Secretory expression in eukaryotes |
US4840934A (en) | 1983-01-25 | 1989-06-20 | Eleanor Roosevelt Institute For Cancer Research, Inc. | Therapeutic method using T cell growth factor |
US5015575A (en) | 1983-04-07 | 1991-05-14 | Chiron Corporation | Hybrid DNA synthesis of insulin |
US4914026A (en) | 1983-04-07 | 1990-04-03 | Chiron Corporation | Alpha factor leader sequence directed secretion of insulin |
US4518584A (en) | 1983-04-15 | 1985-05-21 | Cetus Corporation | Human recombinant interleukin-2 muteins |
JPS60501140A (en) | 1983-04-22 | 1985-07-25 | アムジエン | Secretion of exogenous polypeptides by yeast |
NZ207926A (en) | 1983-04-25 | 1988-04-29 | Genentech Inc | Use of yeast #a#-factor to assist in expression of proteins heterologus to yeast |
US5010003A (en) | 1983-04-25 | 1991-04-23 | Genentech, Inc. | Use of yeast homologous signals to secrete heterologous proteins |
CA1196863A (en) | 1983-06-08 | 1985-11-19 | Mattheus F.A. Goosen | Slow release injectable insulin composition |
JPS6087792A (en) | 1983-09-23 | 1985-05-17 | ジェネックス・コーポレイション | Variant control region |
KR850004274A (en) | 1983-12-13 | 1985-07-11 | 원본미기재 | Method for preparing erythropoietin |
NZ210501A (en) | 1983-12-13 | 1991-08-27 | Kirin Amgen Inc | Erythropoietin produced by procaryotic or eucaryotic expression of an exogenous dna sequence |
US4703008A (en) | 1983-12-13 | 1987-10-27 | Kiren-Amgen, Inc. | DNA sequences encoding erythropoietin |
GB8334261D0 (en) | 1983-12-22 | 1984-02-01 | Bass Plc | Fermentation processes |
IT1185503B (en) * | 1984-01-11 | 1987-11-12 | Univ New York | HUMAN Erythropietine ODNA CLONES |
US4569790A (en) | 1984-03-28 | 1986-02-11 | Cetus Corporation | Process for recovering microbially produced interleukin-2 and purified recombinant interleukin-2 compositions |
US4908433A (en) | 1984-04-25 | 1990-03-13 | Sloan-Kettering Institute For Cancer Research | Uses of interleukin-2 |
US4908434A (en) | 1984-04-25 | 1990-03-13 | Sloan-Kettering Institute For Cancer Research | Process for preparing purified interleukin-2 |
CA1213537A (en) | 1984-05-01 | 1986-11-04 | Canadian Patents And Development Limited - Societe Canadienne Des Brevets Et D'exploitation Limitee | Polypeptide expression method |
DK58285D0 (en) | 1984-05-30 | 1985-02-08 | Novo Industri As | PEPTIDES AND MANUFACTURING AND USING THEREOF |
JPS61227526A (en) * | 1984-07-25 | 1986-10-09 | Chugai Pharmaceut Co Ltd | Novel csf and method of collecting same |
US4716217A (en) | 1984-08-31 | 1987-12-29 | University Patents, Inc. | Hybrid lymphoblastoid-leukocyte human interferons |
US4734491A (en) | 1984-08-31 | 1988-03-29 | University Patents, Inc. | DNA sequences encoding hybrid lymphoblastoid-leukocyte human interferons |
JPS6191131A (en) | 1984-10-09 | 1986-05-09 | Chugai Pharmaceut Co Ltd | Method and composition for preventing adsorption of pharmaceutical |
JPS6197229A (en) | 1984-10-18 | 1986-05-15 | Chugai Pharmaceut Co Ltd | Stable erythropoietin preparation |
US4959314A (en) | 1984-11-09 | 1990-09-25 | Cetus Corporation | Cysteine-depleted muteins of biologically active proteins |
IL77081A (en) | 1984-12-04 | 1999-10-28 | Genetics Inst | Dna sequence encoding human erythropoietin process for the preparation thereof and a pharmaceutical composition of human erythropoietin |
US4970300A (en) | 1985-02-01 | 1990-11-13 | New York University | Modified factor VIII |
US4732889A (en) | 1985-02-06 | 1988-03-22 | Chugai Seiyaku Kabushiki Kaisha | Pharmaceutical composition for the treatment of the anemia of rheumatoid arthritis |
US4745099A (en) | 1985-02-06 | 1988-05-17 | Chugai Seiyaku Kabushiki Kaisha | Pharmaceutical composition for the treatment of the anemia of malignant tumors |
GB8504099D0 (en) | 1985-02-18 | 1985-03-20 | Wellcome Found | Physiologically active substances |
FR2579224B1 (en) | 1985-03-25 | 1987-05-22 | Genetica | PROCESS FOR THE MICROBIOLOGICAL PREPARATION OF HUMAN SERUM-ALBUMIN |
US4751180A (en) | 1985-03-28 | 1988-06-14 | Chiron Corporation | Expression using fused genes providing for protein product |
US5532341A (en) * | 1985-03-28 | 1996-07-02 | Sloan-Kettering Institute For Cancer Research | Human pluripotent hematopoietic colony stimulating factor |
DE3679343D1 (en) | 1985-03-28 | 1991-06-27 | Chiron Corp | EXPRESSION BY USING FUSION GENES FOR PROTEIN PRODUCTION. |
GB8510219D0 (en) | 1985-04-22 | 1985-05-30 | Bass Plc | Isolation of fermentation products |
AU5890286A (en) | 1985-06-17 | 1986-12-24 | Genex Corp. | Cloned human serum albumin gene |
JPS63500636A (en) * | 1985-08-23 | 1988-03-10 | 麒麟麦酒株式会社 | DNA encoding multipotent granulocyte colony stimulating factor |
US4810643A (en) * | 1985-08-23 | 1989-03-07 | Kirin- Amgen Inc. | Production of pluripotent granulocyte colony-stimulating factor |
US5102872A (en) | 1985-09-20 | 1992-04-07 | Cetus Corporation | Controlled-release formulations of interleukin-2 |
CA1295566C (en) | 1987-07-21 | 1992-02-11 | Robert T. Garvin | Characterization and structure of genes for protease a and protease b from streptomyces griseus |
US5641663A (en) | 1985-11-06 | 1997-06-24 | Cangene Corporation | Expression system for the secretion of bioactive human granulocyte macrophage colony stimulating factor (GM-CSF) and other heterologous proteins from steptomyces |
US5013824A (en) * | 1985-11-19 | 1991-05-07 | Schering Corporation | Human interleukin-4 peptides and conjugates thereof |
FR2594846B1 (en) | 1986-02-21 | 1989-10-20 | Genetica | PROCESS FOR THE PREPARATION OF MATURE HUMAN ALBUMIN SERUM |
IT1203758B (en) | 1986-03-27 | 1989-02-23 | Univ Roma | CLONING AND EXPRESSION VECTORS OF HETEROLOGICAL GENES IN YEASTS AND YEASES TRANSFORMED WITH SUCH CARRIERS |
DK179286D0 (en) | 1986-04-18 | 1986-04-18 | Nordisk Gentofte | INSULIN PREPARATION |
US4765980A (en) | 1986-04-28 | 1988-08-23 | International Minerals & Chemical Corp. | Stabilized porcine growth hormone |
US4859609A (en) | 1986-04-30 | 1989-08-22 | Genentech, Inc. | Novel receptors for efficient determination of ligands and their antagonists or agonists |
US5118666A (en) | 1986-05-05 | 1992-06-02 | The General Hospital Corporation | Insulinotropic hormone |
US5120712A (en) | 1986-05-05 | 1992-06-09 | The General Hospital Corporation | Insulinotropic hormone |
US5614492A (en) | 1986-05-05 | 1997-03-25 | The General Hospital Corporation | Insulinotropic hormone GLP-1 (7-36) and uses thereof |
US5028422A (en) | 1986-05-27 | 1991-07-02 | Schering Corporation | Treatment of basal cell carcinoma intralesionally with recombinant human alpha interferon |
IT1196484B (en) * | 1986-07-11 | 1988-11-16 | Sclavo Spa | YEAST EXPRESSION AND SECRETION VECTOR, USEFUL FOR THE PREPARATION OF HETEROLOGICAL PROTEINS |
GR871067B (en) | 1986-07-18 | 1987-11-19 | Chugai Pharmaceutical Co Ltd | Process for producing stable pharmaceutical preparation containing granulocyte colony stimulating factor |
US4801575A (en) | 1986-07-30 | 1989-01-31 | The Regents Of The University Of California | Chimeric peptides for neuropeptide delivery through the blood-brain barrier |
US5002764A (en) | 1986-08-12 | 1991-03-26 | Schering Corporation | Treatment of actinic keratoses with alpha2 interferon |
GB8620926D0 (en) * | 1986-08-29 | 1986-10-08 | Delta Biotechnology Ltd | Yeast promoter |
US4881175A (en) * | 1986-09-02 | 1989-11-14 | Genex Corporation | Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides |
US4954437A (en) | 1986-09-15 | 1990-09-04 | Integrated Genetics, Inc. | Cell encoding recombinant human erythropoietin |
US4929442A (en) | 1986-09-26 | 1990-05-29 | Exovir, Inc. | Compositions suitable for human topical application including a growth factor and/or related materials |
US5508031A (en) | 1986-11-21 | 1996-04-16 | Cetus Oncology Corporation | Method for treating biological damage using a free-radial scavenger and interleukin-2 |
US4835260A (en) | 1987-03-20 | 1989-05-30 | Genetics Institute, Inc. | Erythropoietin composition |
EP0286424B1 (en) * | 1987-04-09 | 1994-03-16 | Delta Biotechnology Limited | Yeast vector |
KR880012235A (en) | 1987-04-10 | 1988-11-26 | 벤자민 에프.람버트 | How to increase hematocrit in normal mammals |
FI107939B (en) | 1987-07-28 | 2001-10-31 | Dsm Nv | Kluyveromyces as a host strain |
SE459586B (en) | 1987-09-14 | 1989-07-17 | Mta Szegedi Biolog Koezponti | THE STRUCTURES CODING FOR AUTHENTIC HUMAN SERUM ALBUMIN AND PROCEDURES FOR ITS PREPARATION |
NZ226414A (en) | 1987-10-02 | 1992-07-28 | Genentech Inc | Cd4 peptide adhesion variants and their preparation and use |
US5336603A (en) | 1987-10-02 | 1994-08-09 | Genentech, Inc. | CD4 adheson variants |
GB8725529D0 (en) | 1987-10-30 | 1987-12-02 | Delta Biotechnology Ltd | Polypeptides |
JP2791418B2 (en) | 1987-12-02 | 1998-08-27 | 株式会社ミドリ十字 | Method for producing heterologous protein, recombinant DNA, transformant |
ZA89430B (en) | 1988-01-22 | 1989-10-25 | Gen Hospital Corp | Cloned genes encoding ig-cd4 fusion proteins and the use thereof |
JPH01240191A (en) * | 1988-02-16 | 1989-09-25 | Green Cross Corp:The | Novel signal peptide functioning by yeast and secretory manifestation of foreign protein using same |
JPH01215289A (en) | 1988-02-22 | 1989-08-29 | Toa Nenryo Kogyo Kk | Production of normal human serum albumin a through gene recombination |
US4999339A (en) | 1988-03-28 | 1991-03-12 | Cetus Corporation | Combination therapy of IL-2 and DTIC for the treatment of melanoma |
US5066489A (en) | 1988-03-28 | 1991-11-19 | Cetus Corporation | Combination therapy of IL-2 and DTIC for the treatment of melanoma |
US5096707A (en) | 1988-04-15 | 1992-03-17 | The United States Of America As Represented By The Department Of Health And Human Services | Flavone-8-acetic acid and interleukin-2 in a method of treating certain cancers |
US5763394A (en) * | 1988-04-15 | 1998-06-09 | Genentech, Inc. | Human growth hormone aqueous formulation |
US5096885A (en) | 1988-04-15 | 1992-03-17 | Genentech, Inc. | Human growth hormone formulation |
US5061488A (en) | 1988-04-15 | 1991-10-29 | The United States Of America As Represented Department Of Health & Human Services | Flavone-8-acetic acid and interleukin-2 for cancer therapy |
IL89992A0 (en) | 1988-04-25 | 1989-12-15 | Phillips Petroleum Co | Expression of human serum albumin in methylotrophic yeasts |
US5126129A (en) | 1988-05-23 | 1992-06-30 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health & Human Services | Cancer therapy using interleukin-2 and flavone compounds |
ATE135045T1 (en) | 1988-07-23 | 1996-03-15 | Delta Biotechnology Ltd | SECRETORY LEADER SEQUENCES |
FR2649991B2 (en) | 1988-08-05 | 1994-03-04 | Rhone Poulenc Sante | USE OF STABLE DERIVATIVES OF PLASMID PKD1 FOR THE EXPRESSION AND SECRETION OF HETEROLOGOUS PROTEINS IN YEASTS OF THE GENUS KLUYVEROMYCES |
FR2635115B1 (en) | 1988-08-05 | 1992-04-03 | Rhone Poulenc Sante | PROCESS FOR THE PREPARATION OF HUMAN ALBUMIN SERUM FROM YEAST |
US5260202A (en) * | 1988-09-07 | 1993-11-09 | Delta Biotechnology Limited | Fermentation method |
US5298243A (en) | 1988-10-20 | 1994-03-29 | Denki Kagaku Kogyo Kabushiki Kaisha | Colony stimulating factor-gelatin conjugate |
US5759802A (en) | 1988-10-26 | 1998-06-02 | Tonen Corporation | Production of human serum alubumin A |
US5256410A (en) | 1988-12-01 | 1993-10-26 | Schering Corporation | Treatment of squamous cell carcinoma intralesionally with recombinant human alpha interferon |
DE68925966T2 (en) | 1988-12-22 | 1996-08-29 | Kirin Amgen Inc | CHEMICALLY MODIFIED GRANULOCYTE COLONY EXCITING FACTOR |
DK105489D0 (en) | 1989-03-03 | 1989-03-03 | Novo Nordisk As | POLYPEPTIDE |
US5116964A (en) | 1989-02-23 | 1992-05-26 | Genentech, Inc. | Hybrid immunoglobulins |
US5705363A (en) | 1989-03-02 | 1998-01-06 | The Women's Research Institute | Recombinant production of human interferon τ polypeptides and nucleic acids |
DE59000270D1 (en) | 1989-04-11 | 1992-10-01 | Boehringer Ingelheim Int | USE OF AT LEAST ONE CYTOKIN FOR THE PRODUCTION OF A MEDICINAL PRODUCT FOR THE SYSTEMIC TREATMENT OF PRAENEOPLASTIC LESIONS. |
EP0395918A3 (en) | 1989-04-13 | 1991-10-23 | Vascular Laboratory, Inc. | Plasminogen activator complex of pure pro-urokinase covalently bound by a disulfide bridge to human serum albumin |
ATE92107T1 (en) | 1989-04-29 | 1993-08-15 | Delta Biotechnology Ltd | N-TERMINAL FRAGMENTS OF HUMAN SERUM ALBUMIN-CONTAINING FUSION PROTEINS. |
US5766883A (en) | 1989-04-29 | 1998-06-16 | Delta Biotechnology Limited | Polypeptides |
GB8909916D0 (en) * | 1989-04-29 | 1989-06-14 | Delta Biotechnology Ltd | Polypeptides |
CA2033176C (en) | 1989-06-09 | 1999-12-14 | Julian Richard Este Wells | Growth hormone fusion proteins |
DE3923963A1 (en) | 1989-07-20 | 1991-01-31 | Behringwerke Ag | MUTEINE OF HUMAN ERYTHROPOETIN, THEIR PRODUCTION AND THEIR USE |
DE3924746A1 (en) | 1989-07-26 | 1991-01-31 | Behringwerke Ag | ERTHROPOIETIN (EPO) PEPTIDES AND ANTIBODIES THEREFOR |
CU22222A1 (en) | 1989-08-03 | 1995-01-31 | Cigb | PROCEDURE FOR THE EXPRESSION OF HETEROLOGICAL PROTEINS PRODUCED IN A FUSION FORM IN ESCHERICHIA COLI, ITS USE, EXPRESSION VECTORS AND RECOMBINANT STRAINS |
FR2650598B1 (en) | 1989-08-03 | 1994-06-03 | Rhone Poulenc Sante | DERIVATIVES OF ALBUMIN WITH THERAPEUTIC FUNCTION |
US5073627A (en) | 1989-08-22 | 1991-12-17 | Immunex Corporation | Fusion proteins comprising GM-CSF and IL-3 |
US6063373A (en) * | 1989-09-19 | 2000-05-16 | Maxim Pharmaceuticals, Inc. | Enhanced activation of NK cells using an NK cell activator and a hydrogen peroxide scavenger or inhibitor |
CA1340994C (en) * | 1989-09-21 | 2000-05-16 | Rudolf Edgar Dr. Falk | Treatment of conditions and disease |
US5856298A (en) | 1989-10-13 | 1999-01-05 | Amgen Inc. | Erythropoietin isoforms |
WO1991005867A1 (en) | 1989-10-13 | 1991-05-02 | Amgen Inc. | Erythropoietin isoforms |
FR2653020B1 (en) | 1989-10-17 | 1993-03-26 | Roussel Uclaf | USE OF A POLYPEPTIDE HAVING THE ACTIVITY OF HUMAN INTERLEUKIN 2 FOR THE PREPARATION OF PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF LEUKEMIA. |
US5667986A (en) * | 1989-10-18 | 1997-09-16 | Delta Biotechnology Limited | Yeast promoter for expressing heterologous polypeptides |
JPH03151399A (en) | 1989-11-07 | 1991-06-27 | Snow Brand Milk Prod Co Ltd | Mutant human erythropoietin |
US5116944A (en) | 1989-12-29 | 1992-05-26 | Neorx Corporation | Conjugates having improved characteristics for in vivo administration |
JPH03201987A (en) * | 1989-12-29 | 1991-09-03 | Tonen Corp | Human serum albumin fragment |
US5545618A (en) | 1990-01-24 | 1996-08-13 | Buckley; Douglas I. | GLP-1 analogs useful for diabetes treatment |
US5208018A (en) | 1990-03-19 | 1993-05-04 | Brigham And Women's Hospital | Treatment of cachexia with interleukin 2 |
FR2660863B1 (en) | 1990-04-17 | 1994-01-21 | Roussel Uclaf | USE OF A POLYPEPTIDE HAVING THE ACTIVITY OF HUMAN INTERLEUKIN 2 FOR THE PREPARATION OF A PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF PRIMITIVE CANCERS OF THE POWDER. |
US5374620A (en) * | 1990-06-07 | 1994-12-20 | Genentech, Inc. | Growth-promoting composition and its use |
US5202239A (en) | 1990-08-07 | 1993-04-13 | Scios Nova Inc. | Expression of recombinant polypeptides with improved purification |
US5071872A (en) | 1990-08-14 | 1991-12-10 | The Ohio State University Research Foundation | Method for improving interleukin-2 activity using aci-reductone compounds |
FR2668368B1 (en) | 1990-10-30 | 1995-03-10 | Roussel Uclaf | USE OF A POLYPEPTIDE HAVING THE ACTIVITY OF HUMAN INTERLEUKIN 2 FOR PREPARING A PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF MALIGNANT EPITHELIAL TUMORS. |
US5830452A (en) | 1990-11-20 | 1998-11-03 | Chiron Corporation | Method for enhancing the anti-tumor therapeutic index of interleukin-2 |
JP3366947B2 (en) | 1990-11-29 | 2003-01-14 | アンスティテュ ナシオナル ド ラ ルシェルシュ アグロノミクーイ.エン.エル.アー. | Novel mutants derived from type 1 interferon, their production method and their application |
US5272070A (en) | 1991-03-08 | 1993-12-21 | Board Of Regents, The University Of Texas System | Method for the preparation of cell lines producing Man3 GlcNac 2 asparagine-linked gylcans and cell lines produced thereby |
CA2062659A1 (en) | 1991-03-12 | 1992-09-13 | Yasutaka Igari | Composition for sustained-release of erythropoietin |
JPH0748378B2 (en) * | 1991-03-28 | 1995-05-24 | 日本碍子株式会社 | Air electrode for solid electrolyte fuel cell and solid electrolyte fuel cell having the same |
EP0509841A3 (en) | 1991-04-18 | 1993-08-18 | Tonen Corporation | Co-expression system of protein disulfide isomerase gene and useful polypeptide gene and process for producing the polypeptide using its system |
CA2058820C (en) | 1991-04-25 | 2003-07-15 | Kotikanyad Sreekrishna | Expression cassettes and vectors for the secretion of human serum albumin in pichia pastoris cells |
US5330901A (en) | 1991-04-26 | 1994-07-19 | Research Corporation Technologies, Inc. | Expression of human serum albumin in Pichia pastoris |
US5646012A (en) | 1991-04-30 | 1997-07-08 | Rhone-Poulenc Rorer S.A. | Yeast promoter and use thereof |
FR2676070B1 (en) | 1991-04-30 | 1994-09-30 | Rhone Poulenc Rorer Sa | YEAST PROMOTER AND ITS USE. |
US5304473A (en) | 1991-06-11 | 1994-04-19 | Eli Lilly And Company | A-C-B proinsulin, method of manufacturing and using same, and intermediates in insulin production |
FR2677996B1 (en) * | 1991-06-21 | 1993-08-27 | Rhone Poulenc Rorer Sa | CLONING AND / OR EXPRESSION VECTORS PREPARATION AND USE. |
US5851795A (en) | 1991-06-27 | 1998-12-22 | Bristol-Myers Squibb Company | Soluble CTLA4 molecules and uses thereof |
US5844095A (en) | 1991-06-27 | 1998-12-01 | Bristol-Myers Squibb Company | CTLA4 Ig fusion proteins |
US5223408A (en) | 1991-07-11 | 1993-06-29 | Genentech, Inc. | Method for making variant secreted proteins with altered properties |
PT101031B (en) * | 1991-11-05 | 2002-07-31 | Transkaryotic Therapies Inc | PROCESS FOR THE SUPPLY OF PROTEINS BY GENETIC THERAPY |
DE69226197T2 (en) | 1991-11-08 | 1999-02-11 | Somatogen Inc | HEMOGLOBINE AS A MEDICINE DELIVERY SYSTEM |
US5540923A (en) | 1991-12-06 | 1996-07-30 | Landsforeningen Til Kraeftens Bekaemplse | Interferon proteins |
US6348327B1 (en) | 1991-12-06 | 2002-02-19 | Genentech, Inc. | Non-endocrine animal host cells capable of expressing variant proinsulin and processing the same to form active, mature insulin and methods of culturing such cells |
GB9200417D0 (en) | 1992-01-09 | 1992-02-26 | Bagshawe Kenneth D | Cytotoxic drug therapy |
FR2686620B1 (en) | 1992-01-27 | 1995-06-23 | Rhone Poulenc Rorer Sa | HUMAN SERUM-ALBUMIN, PREPARATION AND USE. |
FR2686900B1 (en) | 1992-01-31 | 1995-07-21 | Rhone Poulenc Rorer Sa | NOVEL POLYPEPTIDES HAVING GRANULOCYTE COLONY STIMULATION ACTIVITY, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM. |
FR2686899B1 (en) | 1992-01-31 | 1995-09-01 | Rhone Poulenc Rorer Sa | NOVEL BIOLOGICALLY ACTIVE POLYPEPTIDES, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM. |
US5230886A (en) | 1992-03-18 | 1993-07-27 | Trustees Of Boston University | Tumor cell suppression |
DK36392D0 (en) * | 1992-03-19 | 1992-03-19 | Novo Nordisk As | USE OF CHEMICAL COMPOUND |
US5460954A (en) | 1992-04-01 | 1995-10-24 | Cheil Foods & Chemicals, Inc. | Production of human proinsulin using a novel vector system |
SE9201073D0 (en) | 1992-04-03 | 1992-04-03 | Kabi Pharmacia Ab | PROTEIN FORMULATION |
US5229109A (en) | 1992-04-14 | 1993-07-20 | Board Of Regents, The University Of Texas System | Low toxicity interleukin-2 analogues for use in immunotherapy |
US5686268A (en) | 1992-06-19 | 1997-11-11 | Pfizer Inc. | Fused proteins |
JP3269504B2 (en) | 1992-07-08 | 2002-03-25 | 三菱ウェルファーマ株式会社 | Method for producing human serum albumin |
FR2694294B1 (en) | 1992-07-30 | 1994-09-09 | Rhone Poulenc Rorer Sa | Yeast promoter and its user. |
DE4244915C2 (en) | 1992-08-14 | 1998-12-03 | Widmar Prof Dr Tanner | Fungal cells contg. mutated DPM2 mannosyl transferase gene |
US5728553A (en) | 1992-09-23 | 1998-03-17 | Delta Biotechnology Limited | High purity albumin and method of producing |
WO1994008599A1 (en) | 1992-10-14 | 1994-04-28 | The Regents Of The University Of Colorado | Ion-pairing of drugs for improved efficacy and delivery |
FR2697752B1 (en) * | 1992-11-10 | 1995-04-14 | Rhone Poulenc Rorer Sa | Antitumor compositions containing taxane derivatives. |
US6221958B1 (en) * | 1993-01-06 | 2001-04-24 | Societe De Conseils De Recherches Et D'applications Scientifiques, Sas | Ionic molecular conjugates of biodegradable polyesters and bioactive polypeptides |
US5441734A (en) | 1993-02-25 | 1995-08-15 | Schering Corporation | Metal-interferon-alpha crystals |
US5780021A (en) | 1993-03-05 | 1998-07-14 | Georgetown University | Method for treating type 1 diabetes using α-interferon and/or β-i |
DK82893D0 (en) * | 1993-07-08 | 1993-07-08 | Novo Nordisk As | PEPTIDE |
HUT74425A (en) | 1993-08-09 | 1996-12-30 | Baral | A method for sensitization of cancer for killer cell mediated lysis |
US5521086A (en) * | 1993-09-16 | 1996-05-28 | Cephalon, Inc. | Secretion sequence for the production of a heterologous protein in yeast |
US5459031A (en) | 1993-11-05 | 1995-10-17 | Amgen Inc. | Methods for controlling sialic acid derivatives in recombinant glycoproteins |
GB9404270D0 (en) | 1994-03-05 | 1994-04-20 | Delta Biotechnology Ltd | Yeast strains and modified albumins |
US5629286A (en) | 1994-03-31 | 1997-05-13 | Brewitt; Barbara | Homeopathic dilutions of growth factors |
US5646113A (en) | 1994-04-07 | 1997-07-08 | Genentech, Inc. | Treatment of partial growth hormone insensitivity syndrome |
FR2719593B1 (en) | 1994-05-06 | 1996-05-31 | Rhone Poulenc Rorer Sa | New biologically active polypeptides, their preparation and pharmaceutical composition containing them. |
GB9411356D0 (en) | 1994-06-07 | 1994-07-27 | Delta Biotechnology Ltd | Yeast strains |
US5639642A (en) | 1994-06-16 | 1997-06-17 | Novo Nordisk A/S | Synthetic leader peptide sequences |
US5574008A (en) | 1994-08-30 | 1996-11-12 | Eli Lilly And Company | Biologically active fragments of glucagon-like insulinotropic peptide |
US6071923A (en) * | 1994-09-16 | 2000-06-06 | Bar-Ilan University | Retinoyloxy aryl-substituted alkylene butyrates useful for the treatment of cancer and other proliferative diseases |
US5512549A (en) | 1994-10-18 | 1996-04-30 | Eli Lilly And Company | Glucagon-like insulinotropic peptide analogs, compositions, and methods of use |
FR2726471B1 (en) | 1994-11-07 | 1997-01-31 | Pf Medicament | PROCESS FOR IMPROVING THE IMMUNOGENICITY OF AN IMMUNOGENIC COMPOUND OR A HAPTENA AND APPLICATION TO THE PREPARATION OF VACCINES |
AT403167B (en) | 1994-11-14 | 1997-11-25 | Immuno Ag | SELECTION AND EXPRESSION OF FOREIGN PROTEINS BY MEANS OF A SELECTION-AMPLIFICATION SYSTEM |
US6387365B1 (en) * | 1995-05-19 | 2002-05-14 | Schering Corporation | Combination therapy for chronic hepatitis C infection |
US5741815A (en) | 1995-06-02 | 1998-04-21 | Lai; Ching-San | Methods for in vivo reduction of nitric oxide levels and compositions useful therefor |
US5728707A (en) * | 1995-07-21 | 1998-03-17 | Constantia Gruppe | Treatment and prevention of primary and metastatic neoplasms with salts of aminoimidazole carboxamide |
US5840542A (en) | 1995-07-28 | 1998-11-24 | Mogam Biotechnology Research Institute | Method for manufacture of proinsulin with high export yield |
US5766620A (en) * | 1995-10-23 | 1998-06-16 | Theratech, Inc. | Buccal delivery of glucagon-like insulinotropic peptides |
US5702717A (en) | 1995-10-25 | 1997-12-30 | Macromed, Inc. | Thermosensitive biodegradable polymers based on poly(ether-ester)block copolymers |
DE69632062T2 (en) | 1995-11-02 | 2004-11-18 | Schering Corp. | CONTINUOUS, LOW-DOSE CYTOKINE INFUSION THERAPY |
US5744095A (en) * | 1995-11-14 | 1998-04-28 | Smith; Henry J. | Medical assay cassette |
US6048964A (en) | 1995-12-12 | 2000-04-11 | Stryker Corporation | Compositions and therapeutic methods using morphogenic proteins and stimulatory factors |
GB9526733D0 (en) * | 1995-12-30 | 1996-02-28 | Delta Biotechnology Ltd | Fusion proteins |
US5767097A (en) * | 1996-01-23 | 1998-06-16 | Icn Pharmaceuticals, Inc. | Specific modulation of Th1/Th2 cytokine expression by ribavirin in activated T-lymphocytes |
US6150337A (en) | 1996-01-23 | 2000-11-21 | Icn Pharmaceuticals, Inc. | Specific modulation of Th1/Th2 cytokine expression by Ribavirin in activated T-lymphocytes |
US5616724A (en) | 1996-02-21 | 1997-04-01 | Cephalon, Inc. | Fused pyrrolo[2,3-c]carbazole-6-ones |
US6045788A (en) * | 1996-02-28 | 2000-04-04 | Cornell Research Foundation, Inc. | Method of stimulation of immune response with low doses of IL-2 |
US5912229A (en) * | 1996-03-01 | 1999-06-15 | Novo Nordisk Als | Use of a pharmaceutical composition comprising an appetite-suppressing peptide |
JP3794748B2 (en) * | 1996-03-04 | 2006-07-12 | 第一アスビオファーマ株式会社 | Method for culturing microorganisms with methanol metabolism |
ATE241695T1 (en) | 1996-03-13 | 2003-06-15 | Delta Biotechnology Ltd | FERMENTATION CONTROL SYSTEMS |
WO1997044026A1 (en) * | 1996-05-22 | 1997-11-27 | Neuromedica, Inc. | Compositions comprising conjugates of cis-docosahexaenoic acid and taxotere |
US5919815A (en) | 1996-05-22 | 1999-07-06 | Neuromedica, Inc. | Taxane compounds and compositions |
US6110891A (en) * | 1996-06-21 | 2000-08-29 | Alizyme Therapeutics Ltd. | Lectin compositions and uses thereof |
US5922761A (en) | 1996-09-06 | 1999-07-13 | Medinox, Inc. | Methods for in vivo reduction of iron levels and compositions useful therefor |
US20020052309A1 (en) | 1996-09-11 | 2002-05-02 | Athanasius A. Anagnostou | Method of treating endothelial injury |
US5876774A (en) * | 1996-10-11 | 1999-03-02 | Nestec S.A. | Method of making fat-based confection |
US5955508A (en) * | 1996-10-15 | 1999-09-21 | Loyola University Of Chicago | Method for the enhancement of lymphocyte activity against opportunistic microbial pathogens |
US5908830A (en) * | 1996-10-31 | 1999-06-01 | Merck & Co., Inc. | Combination therapy for the treatment of diabetes and obesity |
UA65549C2 (en) * | 1996-11-05 | 2004-04-15 | Елі Ліллі Енд Компані | Use of glucagon-like peptides such as glp-1, glp-1 analog, or glp-1 derivative in methods and compositions for reducing body weight |
WO1998032867A1 (en) * | 1997-01-24 | 1998-07-30 | Novo Nordisk A/S | Synthetic leader peptide sequences |
US5939455A (en) | 1997-03-11 | 1999-08-17 | Beacon Laboratories, Inc. | Therapeutic augmentation of oxyalkylene diesters and butyric acid derivatives |
US6110970A (en) * | 1997-03-11 | 2000-08-29 | Beacon Laboratories, Inc. | Nitrogen-containing oxyalkylene esters and uses thereof |
US6030961A (en) * | 1997-03-11 | 2000-02-29 | Bar-Ilan Research & Development Co., Ltd. | Oxyalkylene phosphate compounds and uses thereof |
US6124495A (en) * | 1997-03-11 | 2000-09-26 | Beacon Laboratories, Inc. | Unsaturated oxyalkylene esters and uses thereof |
MY118835A (en) * | 1997-04-18 | 2005-01-31 | Ipsen Pharma Biotech | Sustained release compositions and the process for their preparation |
US5921996A (en) * | 1997-05-02 | 1999-07-13 | Cardio Thoracic Systems, Inc. | Surgical clamp applier/remover and detachable clamp |
JP3201987B2 (en) | 1997-09-09 | 2001-08-27 | 株式会社オーレック | Mowing equipment |
US6172046B1 (en) | 1997-09-21 | 2001-01-09 | Schering Corporation | Combination therapy for eradicating detectable HCV-RNA in patients having chronic Hepatitis C infection |
US6472373B1 (en) | 1997-09-21 | 2002-10-29 | Schering Corporation | Combination therapy for eradicating detectable HCV-RNA in antiviral treatment naive patients having chronic hepatitis C infection |
US6117949A (en) * | 1998-10-01 | 2000-09-12 | Macromed, Inc. | Biodegradable low molecular weight triblock poly (lactide-co-glycolide) polyethylene glycol copolymers having reverse thermal gelation properties |
US6004573A (en) | 1997-10-03 | 1999-12-21 | Macromed, Inc. | Biodegradable low molecular weight triblock poly(lactide-co-glycolide) polyethylene glycol copolymers having reverse thermal gelation properties |
US6201072B1 (en) * | 1997-10-03 | 2001-03-13 | Macromed, Inc. | Biodegradable low molecular weight triblock poly(lactide-co- glycolide) polyethylene glycol copolymers having reverse thermal gelation properties |
BR9813391C1 (en) | 1997-12-03 | 2021-05-25 | Roche Diagnostics Gmbh | process to produce an epo composition and process to increase the specific activity of an epo composition |
EP1037927B1 (en) * | 1997-12-08 | 2004-05-19 | Lexigen Pharmaceuticals Corp. | Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation |
WO1999029314A1 (en) * | 1997-12-08 | 1999-06-17 | Bristol-Myers Squibb Company | Novel salts of metformin and method |
KR100253916B1 (en) | 1997-12-29 | 2000-05-01 | 김충환 | A process for preparing human proinsulin |
US5951996A (en) * | 1998-02-04 | 1999-09-14 | Czeizler Zaharia; Veronica L. | Treatment of chronic diffuse GI bleeding with erythropoietin |
US6017545A (en) * | 1998-02-10 | 2000-01-25 | Modi; Pankaj | Mixed micellar delivery system and method of preparation |
US6221378B1 (en) * | 1998-02-10 | 2001-04-24 | Generex Pharmaceuticals Incorporated | Mixed micellar delivery system and method of preparation |
CN1119352C (en) | 1998-05-15 | 2003-08-27 | 中国科学院上海生物化学研究所 | Express and purification of human serum albumin in pichia |
CN1105727C (en) | 1998-06-17 | 2003-04-16 | 上海海济医药生物工程有限公司 | Process for preparing recombined human serum albumin |
GB9817084D0 (en) | 1998-08-06 | 1998-10-07 | Wood Christopher B | A method for promoting extra-heptic production of proteins for the correction of hypoalbuminaemia,anaemia,thrombocytopenia and/or coagulation disorders |
US6346543B1 (en) * | 1998-08-17 | 2002-02-12 | Aventis Pharma S.A. | Use of a taxoid to treat abnormal cell proliferation in the brain |
US6193997B1 (en) * | 1998-09-27 | 2001-02-27 | Generex Pharmaceuticals Inc. | Proteinic drug delivery system using membrane mimetics |
ES2283139T5 (en) * | 1998-10-23 | 2018-03-05 | Amgen Inc. | Methods and compositions for the prevention and treatment of anemia |
CA2348822A1 (en) * | 1998-10-30 | 2000-05-11 | Novozymes A/S | Glycosylated proteins having reduced allergenicity |
US6245690B1 (en) * | 1998-11-04 | 2001-06-12 | Applied Materials, Inc. | Method of improving moisture resistance of low dielectric constant films |
JP2002531089A (en) * | 1998-11-30 | 2002-09-24 | イーライ・リリー・アンド・カンパニー | Erythropoietic compounds |
US6048891A (en) * | 1998-12-17 | 2000-04-11 | Loma Linda University Medical Center | Use of γ-tocopherol and its oxidative metabolite LLU-α in the treatment of natriuretic disease |
US6116964A (en) * | 1999-03-08 | 2000-09-12 | Lucent Technologies Inc. | High frequency communications connector assembly with crosstalk compensation |
US6348192B1 (en) * | 1999-05-11 | 2002-02-19 | Bayer Corporation | Interleukin-2 mutein expressed from mammalian cells |
US6514500B1 (en) * | 1999-10-15 | 2003-02-04 | Conjuchem, Inc. | Long lasting synthetic glucagon like peptide {GLP-!} |
US6319691B1 (en) * | 1999-06-15 | 2001-11-20 | Usa Universe Bioengineering, Inc. | Fusion proteins comprising IFN-alpha2b and TM-alpha1 |
JO2291B1 (en) * | 1999-07-02 | 2005-09-12 | اف . هوفمان لاروش ايه جي | Erythopintin derivatives |
US20020048571A1 (en) * | 1999-07-19 | 2002-04-25 | Jeno Gyuris | Chimeric polypeptides of serum albumin and uses related thereto |
AU1573601A (en) * | 1999-10-21 | 2001-04-30 | Monsanto Company | Post-translational modification of recombinant proteins produced in plants |
AU1226901A (en) * | 1999-10-22 | 2001-05-08 | Amgen, Inc. | Biodegradable microparticles with novel erythropoietin stimulating protein |
US6569832B1 (en) * | 1999-11-12 | 2003-05-27 | Novo Nordisk A/S | Inhibition of beta cell degeneration |
WO2001036489A2 (en) * | 1999-11-12 | 2001-05-25 | Merck Patent Gmbh | Erythropoietin forms with improved properties |
CA2747325A1 (en) * | 2000-04-12 | 2001-10-25 | Human Genome Sciences, Inc. | Albumin fusion proteins |
ES2306711T3 (en) * | 2000-04-21 | 2008-11-16 | Amgen Inc. | METHOD AND COMPOSITION INTENDED FOR THE PREVENTION AND TREATMENT OF THE ANEMIA. |
DK1311285T4 (en) * | 2000-05-15 | 2017-07-24 | Hoffmann La Roche | Liquid pharmaceutical composition containing an erythropoietin derivative |
US6580893B2 (en) * | 2000-09-06 | 2003-06-17 | Canon Kabushiki Kaisha | Fixing device and image forming apparatus including the device |
KR100399337B1 (en) * | 2001-02-07 | 2003-09-26 | 드림바이오젠 주식회사 | Method for Cell-free Protein Post-translational Modification |
US20020169128A1 (en) * | 2001-04-09 | 2002-11-14 | Geroge Sigounas | Erythropoietin ameliorates chemotherapy-induced toxicity in vivo |
AU2002332041A1 (en) * | 2001-10-05 | 2003-04-22 | Human Genome Sciences, Inc. | Albumin fusion proteins |
CN1241946C (en) * | 2002-07-01 | 2006-02-15 | 美国福源集团 | Human serum albumins recombined merge protein having hyperplasia stimulation function to multiple cells |
US6913890B2 (en) | 2002-12-18 | 2005-07-05 | Palo Alto Research Center Incorporated | Process for preparing albumin protein conjugated oligonucleotide probes |
-
1992
- 1992-01-31 FR FR9201064A patent/FR2686899B1/en not_active Expired - Lifetime
-
1993
- 1993-01-28 EP EP93904129A patent/EP0624195B1/en not_active Expired - Lifetime
- 1993-01-28 CA CA002126091A patent/CA2126091C/en not_active Expired - Lifetime
- 1993-01-28 AT AT93904129T patent/ATE276361T1/en active
- 1993-01-28 JP JP5512986A patent/JPH07503368A/en not_active Withdrawn
- 1993-01-28 DE DE69333622T patent/DE69333622T2/en not_active Expired - Lifetime
- 1993-01-28 PT PT93904129T patent/PT624195E/en unknown
- 1993-01-28 EP EP04075986A patent/EP1449921A3/en not_active Withdrawn
- 1993-01-28 ES ES93904129T patent/ES2230541T3/en not_active Expired - Lifetime
- 1993-01-28 WO PCT/FR1993/000085 patent/WO1993015199A1/en active IP Right Grant
- 1993-01-28 DK DK93904129T patent/DK0624195T3/en active
-
1994
- 1994-07-29 NO NO19942839A patent/NO325486B1/en not_active IP Right Cessation
- 1994-07-29 FI FI943563A patent/FI120355B/en not_active IP Right Cessation
-
1997
- 1997-01-31 US US08/797,689 patent/US5876969A/en not_active Expired - Lifetime
-
2001
- 2001-10-29 US US09/984,186 patent/US6686179B2/en not_active Expired - Fee Related
-
2002
- 2002-09-10 US US10/237,708 patent/US7081354B2/en not_active Expired - Fee Related
- 2002-09-10 US US10/237,866 patent/US6972322B2/en not_active Expired - Fee Related
- 2002-09-10 US US10/237,871 patent/US7094577B2/en not_active Expired - Fee Related
- 2002-09-10 US US10/237,667 patent/US7041478B2/en not_active Expired - Fee Related
- 2002-09-10 US US10/237,624 patent/US7056701B2/en not_active Expired - Fee Related
-
2003
- 2003-01-16 JP JP2003008385A patent/JP2003235589A/en not_active Withdrawn
- 2003-11-07 US US10/702,636 patent/US6987006B2/en not_active Expired - Fee Related
- 2003-11-07 US US10/702,536 patent/US6989365B2/en not_active Expired - Fee Related
-
2005
- 2005-06-07 US US11/146,077 patent/US7435410B2/en not_active Expired - Fee Related
-
2006
- 2006-01-12 US US11/330,353 patent/US7410779B2/en not_active Expired - Fee Related
- 2006-09-14 NO NO20064159A patent/NO20064159L/en not_active Application Discontinuation
-
2007
- 2007-08-20 JP JP2007214102A patent/JP2007306939A/en active Pending
- 2007-10-29 US US11/927,628 patent/US7833521B2/en not_active Expired - Fee Related
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2126091C (en) | Novel biologically active polypeptides, preparation thereof and pharmaceutical composition containing said polypeptides | |
US5665863A (en) | Polypeptides having granulocyte colony stimulating activity, their preparation and pharmaceutical compositions containing them | |
CA2240292C (en) | Recombinant fusion proteins to growth hormone and serum albumin | |
JPH07503369A (en) | Antithrombotic polypeptides that are antagonists of vWF binding to platelets and/or subendothelium |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
MKEX | Expiry |
Effective date: 20130128 |