AU613022B2 - Bifunctional proteins - Google Patents

Bifunctional proteins Download PDF

Info

Publication number
AU613022B2
AU613022B2 AU14661/88A AU1466188A AU613022B2 AU 613022 B2 AU613022 B2 AU 613022B2 AU 14661/88 A AU14661/88 A AU 14661/88A AU 1466188 A AU1466188 A AU 1466188A AU 613022 B2 AU613022 B2 AU 613022B2
Authority
AU
Australia
Prior art keywords
protein
asp
thr
pro
constituent
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.)
Ceased
Application number
AU14661/88A
Other versions
AU1466188A (en
Inventor
Paul Habermann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hoechst AG
Original Assignee
Hoechst AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hoechst AG filed Critical Hoechst AG
Publication of AU1466188A publication Critical patent/AU1466188A/en
Application granted granted Critical
Publication of AU613022B2 publication Critical patent/AU613022B2/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Description

1
V.
Form COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION
(ORIGINAL)
6130a22 Class Application Number: Lodged: .Qg plete Specification Lodged: Accepted: Published: Int. Class riqjity: o e.osted Art: Name of Applicant: HOECHST AKTIENGESELLSCHAFT o S Address of Applicant: Actual Inventor: Address for Service: 45 Bruningstrasse, D-6230 Frankfurt/Main 80, Federal Republic of Germany PAUL HABERMANN EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.
Complete Specification for the invention entitled: BIFUNCTIONAL PROTEINS The following statement is a full description of this invention, including the best method of performing it known to us 1.
i i i 1 \c HOECHST AKTIENGESELLSCHAFT HOE 87/F 113 Dr. KL/AW Specification Bifunctional proteins Interleukin-2, called IL-2 hereinafter, acts as T-cell growth factor. IL-2 potentiates the activity of killer cells such as NK (natural killer) cells, cytotoxic Tcells and LAK (lymphokine-activated killer) cells.
By contrast, granulocyte macrophage colony stimulating factor, called GM-CSF hereinafter, stimulates the formation of granulocytes and macrophages from hemopoietic 15 precursor cells. Combination of the two biological activities is of interest for human treatment with and without administration of cytostatics. However, the stabilities of IL-2 and GM-CSF differ, which may result in problems on direct administration of the two com- 20 ponents and thus in a decrease in the therapeutic success.
The problem of the difference in stability can be solved according to the invention by linking these two proteins to a bifunctional protein.
Fusion proteins of the general formula Met X Y Z or Met Z Y X (Ia) (Ib) have already been proposed for the preparation, by genetic manipulation, of optionally modified GM-CSF in which X essentially denotes the amino acid sequence of approximately the first 100 amino acids of, preferably human,IL-2, Y denotes a direct bond if the amino acid or amino acid sequence adjacent to the desired protein allows the desired protein to be cleaved off, or 1 i- i-MEW 2 otherwise denotes a bridging member which is composed of one or more genetically encodable amino acids and which allows the cleavage off, and Z is a sequence which is composed of genetically encodable amino acids and which represents the desired GM-CSF protein. It is also possible during this to make use more or less up to the end of the DNA sequence coding for IL-2, and thus generate biologically active IL-2 modified where appropriate as a "by-product" (not prior-published European Patent Application with the publication number (EP-A) 0,228,018 and South African Patent 86/9557).
In contrast to the earlier proposal, the invention re- Lates not to the use of the proteins as intermediate but 15 to the use in methods for the therapeutic treatment of the human body and to medicaments which contain fusion proteins of this type or which are composed of fusion proteins of this type. A further aspect of the invention relates to the use of these fusion proteins for the preparation of a medicament for the treatment of malignant neoplasms.
The fusion protein used according to the invention is thus composed of two biologically active components, namely of an IL-2 constituent, which can be modified in a manner known per se, on the one hand, and of a GM- S CSF constituent, which can likewise be modified, on the I other hand and, where appropriate, of a bridging member corresponding to the definition Y in the formulae given J 30 above. The arrangement of the two components preferably corresponds to the formula Ia. The principle according to the invention can also be used for the preparation of other novel bifunctional proteins.
The figure shows the construction of the plasmid which codes for a bifunctional protein according to the invention.
1~ 3 Modifications of the IL-2 molecule have been disclosed, reference being made here only to EP-A 0,091,539, 0,109,748, 0,118,617, 0,136,489 and 0,163,249 by way of example.
Furthermore, the not prior-published EP-A 0,219,839 proposes an IL-2 derivative in which the first seven Nterminal amino acids are deleted.
Modifications of the GM-CSF molecule have been proposed in EP-A 0,228,018.
:Further alterations to the two active constituents of the Smolecule can be carried out in a manner known per se, 15 mention being made here only of specific mutagenesis by way of example.
The bridging member Y advantageously has the formula II Asp (aa)x Pro (II) in which x denotes an integer up to about 20, and aa denotes any desired genetically encodable amino acid with the exception of cysteine.
It is advantageous in the formula II for the IL-2 constituent to be arranged at the left-hand end, and consequently the GM-CSF constituent to be arranged at the right-hand end.
Particularly preferred embodiments of Y have the amino acid sequence -Asp-Pro-Met-Ilehr-Thr-Thr-Tyr-ALa-Asp-Asp-Pro or -Asp-Pro-Met-ILe-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr- ILe-Asp-Asp-Proit again being preferable for the IL-2 constituent to be arranged at the left-hand end and the GM-CSF constituent to be arranged at the right-hand end.
I---1 4 The bifunctional proteins according to the invention can be expressed in a manner known per se. It is possible in bacterial expression systems for the route of direct expression to be followed. Suitable for this purpose are all known host-vector systems with hosts such as bacteria of the species Streptomyces, B. subtilis, Salmonella typhimurium or Serratia marcescens, especially E. coli.
The DNA sequence which codes for the desired protein is incorporated in a known manner into a vector which ensures satisfactory expression in the chosen expression system.
OSSS It is expedient to choose for this purpose the promoter 15 and operator from the group trp, lac, tac, PL or PR of phage X, hsp, omp or a synthetic promoter, as described in, for example, German Offenlegungsschrift 3,430,683 and in EP-A 0,173,149. The tac promoter-operator sequence is advantageous and is now commercially available (for.example pKK223-3 expression vector, Pharmacia, "Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1984, page 63).
On expression of the protein according to the invention, it may prove expedient to modify individual triplets for the first few amino acids after the ATG start codon in order to prevent any base-pairing at the level of the mRNA.
Such modifications, such as deletions or additions of individual amino acids, are familiar to the expert, and the invention also relates to them.
For expression in yeasts preferably S. cerevisiae it is expedient to use a secretion system, for example heterologous expression via the a-factor system, which has been described several times.
1 It is advantageous for the expression of the bifunctional molecule in yeast if dibasic peptide sequences and i 5 glycosylation sites in the bifunctional protein have been destroyed by appropriate exchange of individual amino acids. This results in many possible combinations which may also influence the biological action.
The expression of IL-2 in yeast is disclosed in EP-A 0,142,268, and that of GM-CSF in EP-A 0,188,350.
The administration of the bifunctional proteins according to the invention corresponds to that of the two components.
However, because of the greater stability a lower dosage is possible in many cases, the dosage being in the lower part of the range of those hitherto proposed.
The invention is illustrated in detail in the examples which follow. Unless indicated otherwise, percentage data and ratios relate to weight.
Example 1 The-plasmid p159/6 (EP-A2 0,163,249, Figure 5; in the present figure) contains a synthetic gene coding for IL-2 between an EcoRI and a Sail cleavage site. The DNA sequence for this gene is represented in the said EP-A2 as "DNA sequence A TaqI cleavage site is located in the region of triplets 127 and 128. The IL-2 partsequence is cut out of this plasmid by cutting with EcoRI and TaqI, and is isolated.
9*99** The plasmid pHG23 which codes for GM-CSF is disclosed in EP-A2 0,183,350. The GM-CSF cDNA is represented in Figure 2 in this EP-A2. The plasmid pHG23 is obtained when the cDNA sequence is incorporated in the PstI cleavage site of pBR322, use being made of, on the one hand, the PstI cleavage site at the 5' end and, on the other hand, a PstI site introduced at the 3' end by GC tailing. The DNA sequence which contains most of the GM-CSF gene is isolated from this plasmid by cutting with SfaNI and PstI.
1 3 1 6 The following oligonucleotide is synthesized by the phosphite method: 128 (133) ILe ILe Ser Thr Leu Asp Pro Met ILe CG ATC ATC TCT ACC CTG GAC CCG ATG ATC TAG TAG AGA TGG GAC CTG GGC TAC TAG (TaqI) 1 2 Thr Thr Tyr Ala Asp Asp Pro (ALa) (Pro) ACC ACC TAT GCG GAC GAT CCG GC TGG TGG ATA CGC CTG CTA GGC CGT GGG (SfaNI) 9*S** The oligonucleotide extends at the 5' end the DNA sequence of IL-2, there being, however, Asp in place of So:" Thr in position 133. At the 3' end of this oligonucleotide are located the nucleotides which have been deleted from the cDNA by cutting with SfaNI.
The preparation of the expression plasmid pEW1000 is proposed in the (not prior-published) EP-A 0,227,938 (Figure This plasmid is a derivative of the plasmid 25 ptac 11 (Amann et al., Gene 25 (1983) 167 178), in which a synthetic sequence which contains a SalI cleavage site has been incorporated in the recognition site for EcoRI. The expression plasmid pKK 177.3 is obtained in this way. Insertion of the lac repressor (Farabaugh, Nature 274 (1978) 765 769) results in the plasmid pJF118. The latter is opened at the unique restriction cleavage site for Aval, and is shortened by about 1000 bp in a known manner by exonuclease treatment and is ligated. The plasmid pEW1000 is obtained. Opening of this plasmid in the polylinker using the enzymes EcoRI and PstI results in the linearized expression plasmid This linearized plasmid DNA is now ligated with the DNA fragment which codes for the IL-2 sequence, with -7the synthetic oligonucleotide and with the cDNA fragment The result is the plasmid pB30 which is transformed into the E. coli strain Mc1061. The plasmid DNA from individual clones is isolated and characterized by restriction analysis.
Example 2 If the following synthetic oligonucleotide 128 (133) ILe lie Ser Thr Leu Asp Pro Met lie Thr Thr Tyr CG ATC ATC TCT ACC CTG GAC CCG ATG ATC ACC ACC TAT TAG TAG AGA TGG GAC CTG GGC TAC TAG TGG TGG ATA (TaqI) 15 1 2 Leu Glu Glu Leu Thr ILe Asp Asp Pro (Ala) (Pro) CTA GAA GAG CTC ACG ATC GAC GAT CCG GC GAT CTT CTC GAG TGC TAG CTG CTA GGC CGT GGG 0 (SfaNI) is used in place of oligonucleotide in the example 1, the result is the plasmid pB31.
Example 3 Competent cells of the E. coli strain W3110 are transformed with the plasmid pB 3 0 or pB31. An overnight culture of the strain is diluted in the ratio of about 1:100 with LB medium H. Miller, Experiments in Molec.
Gen., Cold Spring Harbor Lab., 1972), which contains pg/ml ampicillin, and the growth is followed by measurement of the OD. At 00 0.5 the culture is adjusted to a concentration of 2 mM in isopropyl-B-D-thiogalactopyranoside (IPTG) and, after 150 180 minutes, the bacteria are spun down. These bacteria are treated in a buffer mixture (7M urea, 0.1% SDS, 0.1M sodium phosphate, pH for about 5 minutes, and samples are applied to an SDS polyacrylamide gel electrophoresis plate. This confirms the expression of the bifunctional protein.
I 1. i 8 The stated conditions apply to shake cultures; for larger fermentations it is expedient to modify the OD values and nutrient media and vary the IPTG concentrations appropriately.
Example 4 E. coli W3110 cells which contain the plasmid pB30 or pB31 are, after induction, spun down, resuspended in sodium phosphate buffer (pH 7) and again spun down. The bacteria are taken up in the same buffer and then dis-
(R)
rupted (French Press, Dynomill). The disrupted cells are spun down. The supernatant and sediment are analyzed by SDS polyacrylamide gel electrophorese as described in Example 3. Staining of the protein bands reveals that the 15 bifunctional protein is located in the sediment from the disruption. The sediment is washed several times with chaotropic buffers and finally with water, resulting in further enrichment of the desired protein. The protein concentration is then determined in the aqueous protein suspension. The suspension is .now -adjusted to a concentration of 5 M in guanidinium hydrochloride and 2 mM in dithiothreitol (DTT). The mixture is stirred under bS nitrogen for about 30 minutes and then diluted with 50 mM tris buffer (pH 8.5) so that the protein concentration is 100 pg/ml. It is now dialyzed against this tris buffer and, after two changes of the buffer, dialyzed against water. The protein treated in this way is sterile filtered and its biological activity is checked. It shows full bio-
S
logical action both in the interleukin-2dependent CTLL 2 cell proliferation assay and in the human bone marrow assay. Mixed colonies of granulocytes and macrophages are observed in these.
The bifunctional protein can be further purified by interleukin-2-specific affinity chromatography. The protein is still active in both assays. In contrast, an E. coli extract of the untransformed strain W3110 which has been treated as described shows no activity.
i 9 Other conditions are expedient for the industrial preparation of the product, for example for the folding of the protein and its purification. Suitable purification processes which are known per se are ion exchange, adsorption, gel filtration and preparative HPLC chromatography.
9 9 I i *9 S .t

Claims (3)

  1. 6. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: prot cons 1. A bifunctional protein composed of a biologically codi active interleukin-2 (IL-2) constituent and a granulocyte macrophage colony stimulating factor (GM-CSF) constituent.
  2. 7. any 2. A bifunctional protein having a biologically active IL-2 constituent and a GM-CSF constituent, wherein the two biologically active protein constituents are linked via a bridge composed of 1 to about 20 genetically encodable amino8 to acids. *of S i 3. A protein as claimed in Claim 2, wherein the bridge corresponds to the formula (II) 0** Asp (aa) x Pro (II) WATI x denoting an integer from 1 to 18, and aa being a THE genetically encodable amino acid with the exception of Cys. 290 i j 4. A protein as claimed in Claim 3, wherein the** AUS' bridging member (aa) x denotes the amino acid sequence* DBM I -Pro-Met-Ile-Thr-Thr-Tyr-Ala-Asp-Asp- (7/ Sor -Pro-Met-Ile-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr-Ile-Asp-Asp-. A protein as claimed in one or more of Claims 2 to 4, wherein the IL-2 constituent is N-terminal and the GM-CSF constituent is C-terminal. I rY~ 11 6. A process for the preparation of bifunctional proteins as claimed in any of Claims 1 to 5, which comprises construction, and expression in a host cell, of a gene coding for these proteins. 7. A medicament composed of a protein as claimed in any of Claims 1 to 5, where appropriate combined with a pharmacologically suitable vehicle.
  3. 8. The use of a protein as claimed in any of Claims 1 to 5 for the preparation of a medicament for the treatment of malignant neoplasms. s**e DATED this 28th day of March, 1991 HOECHST AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN, VICTORIA 3122 AUSTRALIA o.. DBM/JMW:JJC (7/10) o* I I TaqI AA ITC AIG EcoRi G TAC (EcoRI) Ser) AU C (TaqI) fr i.e C 4'. C S SC eCC* 4C S. 4 *5 5 4 S C SfaNI ic r pHG23 c- PstI ori 128 (133) IleIlie Ser Thr Leu Asp CU AIC AIC ICT ACC CTG UACI TAS TAG AGA TU G GAC CTUG (Taq I 2 Sfa NI (Pro) Psl ACCC (Sfa NI) (CSF4 -127)-IA- CTUCA U (PstI) 1 2 Pro Met Ile Thr Thr Tyr Ala Asp Asp Pro (Ala) (Pro) CCU ATU AIC ACC ACC TAT UCU GAC GAT CCU GC GUC TAC TAG TUG TUG ATA CUC CTU CIA UGC CUT GU (SfaNI) :cI, Hinc 11 U U EcoRI TTAA ACUIC]' Pstl L (EcoRI) (PstI) 1(7 6) tac Eco RI PvuI nR~l Taq I GM -CSF PVUII-
AU14661/88A 1987-04-16 1988-04-15 Bifunctional proteins Ceased AU613022B2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE3712985 1987-04-16
DE19873712985 DE3712985A1 (en) 1987-04-16 1987-04-16 BIFUNCTIONAL PROTEINS

Publications (2)

Publication Number Publication Date
AU1466188A AU1466188A (en) 1988-10-20
AU613022B2 true AU613022B2 (en) 1991-07-25

Family

ID=6325799

Family Applications (1)

Application Number Title Priority Date Filing Date
AU14661/88A Ceased AU613022B2 (en) 1987-04-16 1988-04-15 Bifunctional proteins

Country Status (20)

Country Link
EP (1) EP0288809B1 (en)
JP (1) JP2667193B2 (en)
KR (1) KR970000187B1 (en)
AR (1) AR242991A1 (en)
AT (1) ATE79135T1 (en)
AU (1) AU613022B2 (en)
CA (1) CA1322157C (en)
DE (2) DE3712985A1 (en)
DK (1) DK170741B1 (en)
ES (1) ES2033981T3 (en)
FI (1) FI98830C (en)
GR (1) GR3006141T3 (en)
HU (1) HU204303B (en)
IE (1) IE61574B1 (en)
IL (1) IL86086A (en)
NO (1) NO176922C (en)
NZ (1) NZ224247A (en)
PH (1) PH25327A (en)
PT (1) PT87237B (en)
ZA (1) ZA882659B (en)

Families Citing this family (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5662896A (en) * 1988-03-21 1997-09-02 Chiron Viagene, Inc. Compositions and methods for cancer immunotherapy
US5225538A (en) * 1989-02-23 1993-07-06 Genentech, Inc. Lymphocyte homing receptor/immunoglobulin fusion proteins
US6406697B1 (en) 1989-02-23 2002-06-18 Genentech, Inc. Hybrid immunoglobulins
US5116964A (en) * 1989-02-23 1992-05-26 Genentech, Inc. Hybrid immunoglobulins
WO1990012877A1 (en) * 1989-04-19 1990-11-01 Cetus Corporation Multifunctional m-csf proteins and genes encoding therefor
ES2055445T3 (en) * 1989-08-22 1994-08-16 Immunex Corp FUSION PROTEINS INCLUDING GM-CSF AND IL-3.
US5108910A (en) * 1989-08-22 1992-04-28 Immunex Corporation DNA sequences encoding fusion proteins comprising GM-CSF and IL-3
US5073627A (en) * 1989-08-22 1991-12-17 Immunex Corporation Fusion proteins comprising GM-CSF and IL-3
US5376367A (en) * 1991-11-22 1994-12-27 Immunex Corporation Fusion proteins comprising MGF and IL-3
US5723125A (en) * 1995-12-28 1998-03-03 Tanox Biosystems, Inc. Hybrid with interferon-alpha and an immunoglobulin Fc linked through a non-immunogenic peptide
DE69824039T2 (en) 1997-12-08 2005-08-18 Lexigen Pharmaceuticals Corp., Lexington HETERODIMARY FUSION PROTEINS FOR THE USE OF TARGETED IMMUNOTHERAPY AND GENERAL IMMUNE REGION
SK782002A3 (en) 1999-07-21 2003-08-05 Lexigen Pharm Corp FC fusion proteins for enhancing the immunogenicity of protein and peptide antigens
BR0013231A (en) 1999-08-09 2002-07-23 Lexigen Pharm Corp Multiple cytokine-antibody complexes
WO2001040311A1 (en) * 1999-11-30 2001-06-07 Shionogi & Co., Ltd. Chemokine slc-il2 fused protein and gene thereof
ES2269366T3 (en) 2000-02-11 2007-04-01 Merck Patent Gmbh IMPROVEMENT OF AVERAGE LIFE IN CIRCULATION OF FUSION PROTEINS BASED ON ANTIBODIES.
BR0207854A (en) 2001-03-07 2004-08-24 Merck Patent Gmbh Expression technology for proteins containing a hybrid isotype antibody moiety
US6992174B2 (en) 2001-03-30 2006-01-31 Emd Lexigen Research Center Corp. Reducing the immunogenicity of fusion proteins
DE60239454D1 (en) 2001-05-03 2011-04-28 Merck Patent Gmbh RECOMBINANT, TUMOR-SPECIFIC ANTIBODY AND ITS USE
NZ532027A (en) 2001-10-10 2008-09-26 Neose Technologies Inc Remodeling and glycoconjugation of peptides
ES2381025T3 (en) 2001-12-04 2012-05-22 Merck Patent Gmbh Immunocytokines with modulated selectivity
JP4494977B2 (en) 2002-12-17 2010-06-30 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング Humanized antibody (H14.18) of mouse 14.18 antibody that binds to GD2 and its IL-2 fusion protein
EP1648931B1 (en) * 2003-07-21 2011-02-09 Transgene S.A. Multifunctional cytokines
DE602004031341D1 (en) 2003-07-21 2011-03-24 Transgene Sa MULTIFUNCTIONAL CYTOKINE
WO2010001414A1 (en) * 2008-07-03 2010-01-07 Lupin Limited Expression of heterologous proteins in bacterial system using a gm-csf fusion tag

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0136489A1 (en) * 1983-08-10 1985-04-10 Amgen Inc. Analogs of human interleukin II and their preparation
EP0183350A2 (en) * 1984-10-29 1986-06-04 Immunex Corporation DNA Encoding human colony stimulating factor, peptide encoded thereby,vectors and transformed hosts containing such DNA, and the production of all thereof
AU6675686A (en) * 1985-12-21 1987-06-25 Hoechst Aktiengesellschaft GM-CSF protein, its derivatives, the preparation of proteins of this type, and their use

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8327880D0 (en) * 1983-10-18 1983-11-16 Ajinomoto Kk Saccharomyces cerevisiae
EP0158198A1 (en) * 1984-03-29 1985-10-16 Takeda Chemical Industries, Ltd. DNA and use thereof
WO1985004673A1 (en) * 1984-04-10 1985-10-24 Takeda Chemical Industries, Ltd. Novel dna and its use
DE3419995A1 (en) * 1984-05-29 1985-12-05 Hoechst Ag, 6230 Frankfurt GENE TECHNOLOGICAL METHOD FOR PRODUCING HUMAN INTERLEUKIN-2 AND MEANS FOR CARRYING OUT THIS METHOD
JPS61128889A (en) * 1984-11-27 1986-06-16 Green Cross Corp:The Recombinant dna and transformant by same
JPH0646957B2 (en) * 1985-03-11 1994-06-22 武田薬品工業株式会社 Method for producing interleukin-2
EP0238655A4 (en) * 1985-10-03 1989-09-11 Biogen Nv Human granulocyte-macrophage colony stimulating factor-like polypeptides and processes for producing them in high yields in microbial cells.
DE3541856A1 (en) * 1985-11-27 1987-06-04 Hoechst Ag EUKARYOTIC FUSION PROTEINS, THEIR PRODUCTION AND USE, AND MEANS FOR CARRYING OUT THE PROCESS

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0136489A1 (en) * 1983-08-10 1985-04-10 Amgen Inc. Analogs of human interleukin II and their preparation
EP0183350A2 (en) * 1984-10-29 1986-06-04 Immunex Corporation DNA Encoding human colony stimulating factor, peptide encoded thereby,vectors and transformed hosts containing such DNA, and the production of all thereof
AU6675686A (en) * 1985-12-21 1987-06-25 Hoechst Aktiengesellschaft GM-CSF protein, its derivatives, the preparation of proteins of this type, and their use

Also Published As

Publication number Publication date
ES2033981T3 (en) 1993-04-01
NO881658D0 (en) 1988-04-15
PT87237A (en) 1988-05-01
FI98830C (en) 1997-08-25
AR242991A1 (en) 1993-06-30
JPS63301898A (en) 1988-12-08
DE3873397D1 (en) 1992-09-10
NO881658L (en) 1988-10-17
IL86086A (en) 1995-01-24
NZ224247A (en) 1990-04-26
KR970000187B1 (en) 1997-01-06
DK170741B1 (en) 1996-01-08
FI881743A0 (en) 1988-04-14
IE881146L (en) 1988-10-16
DK209188D0 (en) 1988-04-15
HU204303B (en) 1991-12-30
NO176922C (en) 1995-06-21
AU1466188A (en) 1988-10-20
PH25327A (en) 1991-04-30
ZA882659B (en) 1988-10-14
DK209188A (en) 1988-10-17
EP0288809A1 (en) 1988-11-02
FI98830B (en) 1997-05-15
ATE79135T1 (en) 1992-08-15
CA1322157C (en) 1993-09-14
GR3006141T3 (en) 1993-06-21
KR880012760A (en) 1988-11-29
NO176922B (en) 1995-03-13
HUT47319A (en) 1989-02-28
DE3712985A1 (en) 1988-11-03
IE61574B1 (en) 1994-11-16
FI881743A (en) 1988-10-17
IL86086A0 (en) 1988-09-30
PT87237B (en) 1992-07-31
EP0288809B1 (en) 1992-08-05
JP2667193B2 (en) 1997-10-27

Similar Documents

Publication Publication Date Title
AU613022B2 (en) Bifunctional proteins
US5359035A (en) Bifunctional proteins including interleukin-2 (IL-2) and granuloctyte macrophage colony stimulating factor (GM-CSF)
CA1341240C (en) Dna encoding human tumor necrosis factor and human tumor necrosis factor polypeptide
US7361738B2 (en) Megakaryocyte stimulating factors
JP2659530B2 (en) Production of streptavidin-like polypeptide
CA1339892C (en) Preparation of polypeptides having human gamma enterferon activity
IE49255B1 (en) Interferons and process for their preparation
JPH0720431B2 (en) DNA encoding a polypeptide showing the activity of a Mullerian duct inhibitor, recombinant DNA and transformed host
US5098703A (en) Interferon-alpha 76
DE60021188T2 (en) MODIFIED HUMAN GRANULOCYTE COLONY STIMULATING FACTOR AND METHOD FOR THE PRODUCTION THEREOF
IE863335L (en) Gm-csf protein, derivatives and preparation
JPH09118697A (en) Mature human interleukin-1 protein
US4973479A (en) Interferon-α61
US4975276A (en) Interferon-alpha 54
HU196846B (en) Process for producing biologically active derivatives of human gamma-interferon and pharmaceutical compositions comprising the same
EP0306870A2 (en) Gamma-interferon derivatives, process for their manufacture, vectors and medicines obtained therefrom
US5246699A (en) Maturation of hemopoietic cells
AU621051B2 (en) Method for purifying granulocyte-macrophage colony stimulating factor
Steinkasserer et al. Human Interleukin‐1 receptor antagonist High yield expression in E. coli and examination of cysteine residues
EP0234592A1 (en) Plasmid containing DNA fragment coding for human immunoglobulin G Fc region protein and use thereof for production of said protein
DE3910084A1 (en) POLYPEPTIDES WITH GROWTH FACTOR ACTIVITY AND NUCLEAR SEA SEQUENCES
US5047504A (en) Method for purifying granulocyte-macrophage colony stimulating factor
JPH022390A (en) Novel stimulation factor for human granulocyte macrophage colony
JPH0159874B2 (en)
US5290917A (en) Modified polypeptides of IL-1α

Legal Events

Date Code Title Description
MK14 Patent ceased section 143(a) (annual fees not paid) or expired