CA1322157C - Bifunctional proteins - Google Patents
Bifunctional proteinsInfo
- Publication number
- CA1322157C CA1322157C CA000564322A CA564322A CA1322157C CA 1322157 C CA1322157 C CA 1322157C CA 000564322 A CA000564322 A CA 000564322A CA 564322 A CA564322 A CA 564322A CA 1322157 C CA1322157 C CA 1322157C
- Authority
- CA
- Canada
- Prior art keywords
- protein
- constituent
- medicament
- asp
- thr
- 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 - Fee Related
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
Abstract
Abstract of the Disclosure Bifunctional proteins, obtainable by genetic manipulation, composed of an interleukin-2 and a granulocyte macro-phage colony stimulating factor constituent have the biological activity of both components but are distin-guished by increased stability. These proteins are thus medicaments which are suitable for the treatment of malignant neoplasms.
Description
~32~ ~7 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 T-1Q cells and LAK (lymphokine-activated killer) ceLls.
~y contrast~ granulocyte macrophage colony stimula~ing factor~ called GM-CSF hereinafter~ stimulates the forma-~ion of granulocytes and macrophages from hemopoietic precursor cells. Combination of the two bioLogical activities is of interest for human treatment with and without administra~ion of cytostatics~ However, the ~tabilities of IL-2 and GM-CSF differ, which may result in problems on direct administration of the two com-0 ponents and t-hus i-n 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 - ~ - Y - X
(Ia) (Ib) "
have aLready been proposed for the preparation, by genetic manipulation, of optionally modified GM SSF in ~hich X essentially denotes the amino acid sequence of approximately the first 100 amino acids of, preferably human,lL-2, Y denotes a direct bond if the amino acid or amino acid sequence adjacent to the desired protein allows the desired pro~ein to be cLeaved off, or . . :
., . :
: . . .
13~2~ ~7 -- 2 ~
otherwise denotes a bridging member which is composed of one or more genetically encodable amino acids and which allo~s the cLeavage off, and Z is a sequence wh;ch is composed of genetically encodable amino acids and which represents the desired GM~CSF protein. It is also pos-sible dur;ng this to make use - more or less - up to the end of the DNA sequence coding for IL-2~ and thus generate b;ologically active IL-2 - modified where appro-pr;ate - as a "by-product" (not prior~published European Patent Application with the publication number (EP-A) 0,228,018 and South African Patent ~6/~557).
In contras~ to the earlier proposal, the invention re-lates not to the use of the proteins as intermediate but to the use in ~ethods for the therapeutic treatment of the human body and to medicaments ~hich contain fusion proteins of this type or ~hich 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 mal;g-nant neoplasms.
The fus;on protein used according to the invention is thus composed of two b;ologically active components, namely of an IL-2 constituent~ ~hich can be modified in a manner known per s~, on the one hand, and of a GM-CSF constituPnt, which can likewise be mod;fied, on the other hand and, where appropria~e, of a bridging member corresponding to the defini~ion Y in the formulae g;ven above. The arrangement of the t~o 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 pB30 which codes for a bifunctional protein according to the invention.
' ' ~' ' :
~322~7 Mod;f;cat;ons of the IL-2 ~olecule have been disclosed, reference being made here only to EP-A 0,091~53~, 0,109,748, 0,118,617, 0,136,489 and 0~163,249 by way of example.
s Furthermore, the not prior-published EP-A 0,Z19,839 pro-poses an IL-2 derivati~e in which the first seven N-terminal amino acids are deleted.
Modi~ications of ~he GM-CSF molecule have been proposed in EP-A 0,228,018.
Further alterations to ~he two active constituents of the molecule can be carried out in a manner kno~n per se, ~ention being made here only of specific mutagenesis by ~ay of example.
The bridging ~ember Y advantageously has the formula II
- Asp - (aa)X - Pro - (II) d in which x denotes an integer up to about Z0, and aa denotes any desired genetically encodable amino acid with the exception of cyste;ne.
It is advantageous in the formula II for the IL-2 con-stituent to be arranged at the left-hand end, and con sequently the GM-CSF constituent to be arranged at the right-hand end.
Particularly preferred embodiments of Y have the amino acid se~uence -Asp-Pro-Met-Ile-Thr-Thr-Tyr-Ala-Asp-Asp-Pro or -Asp-Pro-Met-Ile-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr-Ile-Asp-Asp-Pro-it 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 snd~
~ . ~
~ 3 2 ~ 7 ~ 4 The bifunctional proteins accord;ng to the invent;on can be expressed ;n a manner kno~n per se. It is possible in bac~erial express;on systems for the route of direct expression to be followed. Suitable for this purpose are 3ll known host-vector systems with hosts such as bacter;a of the spec;es Streptomyces, B. subtilis, Salmonella typh;murium or Serrat;a marcescens, espec;ally E. coli.
The DNA sequence which codes for the desired protein is incorporated in a known manner into a vec~or which en-sures satisfactory expression in the chosen express;on system.
It is exped;ent to choose for this purpose the promoter and operator from the group trp, lac, tac, PL or PR of phage ~, hsp, omp or a synthetic promoter, as described in~ for example, German OffenlegungssGhrift 3D430,683 and in EP-A 0,173,149. The tac promoter-operator seq-uence is advantageous and is now commercially available (for example pKK~23-3 expression vector, Pharmacia, "Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1g84, page 63).
On expression of the protein according to the invent;on, it may prove expedient to moclify 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 modif;cations, such as deletions or additions of in-dividual a~ino acids~ are familiar to the exper~, and the invention also relates to them.
For expression in yeasts - preferably S. cerevisiae - it is expedient ~o use a secretion systemr for examPle heter-ologous expression via the ~-factor system, which has been described several times.
It is advantageous for the expression of the bifunc~ional molecule in yeast if dibasic peptide sequences and ,.: .. ..
: . ~
.:
1~22~
-glycosylation sites in the bifunctional prote;n have been destroyed by appropriate exchange of individual amino acidsO This results in many possible combinations which may also influence the biological actio~O
The expression of IL-2 in yeast is disclosed in EP-A
0,142,268, and that of GM-CSF in EP-A 0~188~350n The administration of the bifunctioRal proteins according to the invention corresponds to that of the two components.
Howeverr because of the greater stability a lower ~osage 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 indica~ed otherwise, percentage data and ratios relate to weight.
FxamPle 1 The plasmid p159i6 (EP-A2 0,163~49, Figure 5; (1) in the present figure) contains a synthet;c gene coding for IL-2 between an EcoRI and a SalI cleavage site. The DNA
sequence for this gene is represented in the sa;d EP-A2 as "DNA sequence I". A TaqI cleavage site is located in ~he region of triplets 127 and 1280 The IL-2 part-sequence (2) ;s cut out of this plasmid by cutting with EcoRI and TaqI~ and is isolated.
,~
The plasmid pHG23 (3) which codes for GM-CSF is disclosed in EP-A2 0,183,350~ The GM~CSF cDNA is representPd in Figure Z in this EP-A2. The plasmid pHG23 ;s 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 in~roduced at the 3' end by GC
tail;ng. The DNA sequence (4) ~hich contains most of the GM-CSF gene is isolated from this plasmid by cutting with SfaNI and PstI~
~.
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 T-1Q cells and LAK (lymphokine-activated killer) ceLls.
~y contrast~ granulocyte macrophage colony stimula~ing factor~ called GM-CSF hereinafter~ stimulates the forma-~ion of granulocytes and macrophages from hemopoietic precursor cells. Combination of the two bioLogical activities is of interest for human treatment with and without administra~ion of cytostatics~ However, the ~tabilities of IL-2 and GM-CSF differ, which may result in problems on direct administration of the two com-0 ponents and t-hus i-n 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 - ~ - Y - X
(Ia) (Ib) "
have aLready been proposed for the preparation, by genetic manipulation, of optionally modified GM SSF in ~hich X essentially denotes the amino acid sequence of approximately the first 100 amino acids of, preferably human,lL-2, Y denotes a direct bond if the amino acid or amino acid sequence adjacent to the desired protein allows the desired pro~ein to be cLeaved off, or . . :
., . :
: . . .
13~2~ ~7 -- 2 ~
otherwise denotes a bridging member which is composed of one or more genetically encodable amino acids and which allo~s the cLeavage off, and Z is a sequence wh;ch is composed of genetically encodable amino acids and which represents the desired GM~CSF protein. It is also pos-sible dur;ng this to make use - more or less - up to the end of the DNA sequence coding for IL-2~ and thus generate b;ologically active IL-2 - modified where appro-pr;ate - as a "by-product" (not prior~published European Patent Application with the publication number (EP-A) 0,228,018 and South African Patent ~6/~557).
In contras~ to the earlier proposal, the invention re-lates not to the use of the proteins as intermediate but to the use in ~ethods for the therapeutic treatment of the human body and to medicaments ~hich contain fusion proteins of this type or ~hich 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 mal;g-nant neoplasms.
The fus;on protein used according to the invention is thus composed of two b;ologically active components, namely of an IL-2 constituent~ ~hich can be modified in a manner known per s~, on the one hand, and of a GM-CSF constituPnt, which can likewise be mod;fied, on the other hand and, where appropria~e, of a bridging member corresponding to the defini~ion Y in the formulae g;ven above. The arrangement of the t~o 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 pB30 which codes for a bifunctional protein according to the invention.
' ' ~' ' :
~322~7 Mod;f;cat;ons of the IL-2 ~olecule have been disclosed, reference being made here only to EP-A 0,091~53~, 0,109,748, 0,118,617, 0,136,489 and 0~163,249 by way of example.
s Furthermore, the not prior-published EP-A 0,Z19,839 pro-poses an IL-2 derivati~e in which the first seven N-terminal amino acids are deleted.
Modi~ications of ~he GM-CSF molecule have been proposed in EP-A 0,228,018.
Further alterations to ~he two active constituents of the molecule can be carried out in a manner kno~n per se, ~ention being made here only of specific mutagenesis by ~ay of example.
The bridging ~ember Y advantageously has the formula II
- Asp - (aa)X - Pro - (II) d in which x denotes an integer up to about Z0, and aa denotes any desired genetically encodable amino acid with the exception of cyste;ne.
It is advantageous in the formula II for the IL-2 con-stituent to be arranged at the left-hand end, and con sequently the GM-CSF constituent to be arranged at the right-hand end.
Particularly preferred embodiments of Y have the amino acid se~uence -Asp-Pro-Met-Ile-Thr-Thr-Tyr-Ala-Asp-Asp-Pro or -Asp-Pro-Met-Ile-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr-Ile-Asp-Asp-Pro-it 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 snd~
~ . ~
~ 3 2 ~ 7 ~ 4 The bifunctional proteins accord;ng to the invent;on can be expressed ;n a manner kno~n per se. It is possible in bac~erial express;on systems for the route of direct expression to be followed. Suitable for this purpose are 3ll known host-vector systems with hosts such as bacter;a of the spec;es Streptomyces, B. subtilis, Salmonella typh;murium or Serrat;a marcescens, espec;ally E. coli.
The DNA sequence which codes for the desired protein is incorporated in a known manner into a vec~or which en-sures satisfactory expression in the chosen express;on system.
It is exped;ent to choose for this purpose the promoter and operator from the group trp, lac, tac, PL or PR of phage ~, hsp, omp or a synthetic promoter, as described in~ for example, German OffenlegungssGhrift 3D430,683 and in EP-A 0,173,149. The tac promoter-operator seq-uence is advantageous and is now commercially available (for example pKK~23-3 expression vector, Pharmacia, "Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1g84, page 63).
On expression of the protein according to the invent;on, it may prove expedient to moclify 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 modif;cations, such as deletions or additions of in-dividual a~ino acids~ are familiar to the exper~, and the invention also relates to them.
For expression in yeasts - preferably S. cerevisiae - it is expedient ~o use a secretion systemr for examPle heter-ologous expression via the ~-factor system, which has been described several times.
It is advantageous for the expression of the bifunc~ional molecule in yeast if dibasic peptide sequences and ,.: .. ..
: . ~
.:
1~22~
-glycosylation sites in the bifunctional prote;n have been destroyed by appropriate exchange of individual amino acidsO This results in many possible combinations which may also influence the biological actio~O
The expression of IL-2 in yeast is disclosed in EP-A
0,142,268, and that of GM-CSF in EP-A 0~188~350n The administration of the bifunctioRal proteins according to the invention corresponds to that of the two components.
Howeverr because of the greater stability a lower ~osage 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 indica~ed otherwise, percentage data and ratios relate to weight.
FxamPle 1 The plasmid p159i6 (EP-A2 0,163~49, Figure 5; (1) in the present figure) contains a synthet;c gene coding for IL-2 between an EcoRI and a SalI cleavage site. The DNA
sequence for this gene is represented in the sa;d EP-A2 as "DNA sequence I". A TaqI cleavage site is located in ~he region of triplets 127 and 1280 The IL-2 part-sequence (2) ;s cut out of this plasmid by cutting with EcoRI and TaqI~ and is isolated.
,~
The plasmid pHG23 (3) which codes for GM-CSF is disclosed in EP-A2 0,183,350~ The GM~CSF cDNA is representPd in Figure Z in this EP-A2. The plasmid pHG23 ;s 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 in~roduced at the 3' end by GC
tail;ng. The DNA sequence (4) ~hich contains most of the GM-CSF gene is isolated from this plasmid by cutting with SfaNI and PstI~
~.
2 2 ~ 5 ~
The folLo~;ng oligonucleotide (5) 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) 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) The oligonucLeotide (5? extends at:the 5' end the DNA
sequence of IL-2, there being, however, Asp in place of Thr in position 133. At the 3' end of this o~igonucleo-tide are located the nucleotides which have been deleted from the cDNA by cutting w;th SfaNI.
The preparation of the expression plasmid pE~1000 (6) is proposed in the (not prior-pubLished) EP-A 0,227,938 (Figure 1). This plasmid is a derivative of the plasmid ptac 11 tAmann et al., Gene 25 t1983) 167 - 178), in ~hich a synthetic sequence which contains a SalI cleavage site has been incorporated in the recognition site for coRI~ The eKpression plasm;d pKK 177.3 is obta;ned in this way. Insertion of the lac repressor ~Farabaughv Nature 274 (1978) 765 - 769) results in the plasmid pJF118. The latter is opened at the unique restriction cleavage site for AvaI, and is shortened by about 1000 bp in a known manner by exonuclease treatment and ;s Ligated. The ~lasmid pE~100D ~6) is obtained. Opening of this plasmid in the polylinker using the enzymes EcoRI
and PstI results in the linear;zed expression plasmid (7).
Th;s linearized plasm;d DNA (7~ is now ligated with the DNA fragment (2) which codes ~or th~ IL-2 sequence, with _ 7 _ ~ 3 22~ ~t~
the synthetic oligonucleotide (5) and ~ith the cDNA
fragment (4). The result is the plasm;d pB30 (8) whish is transformed ;nto the E. coli strain Mc10~1. The plasmid DNA from individual clones is isolated and character;zed by restriction analysis.
Example 2 If the following synthetic oligonucleotide 128 (133) Ile Ile Ser Thr Leu Asp Pro Met Ile Thr Thr Tyr CG ATC ATC TCT ACC CTG GAC CCG A~6 ATC ACC ACC TAT
TAG TAG AGA TGG ~AC CTG GGC TAC TAG TGG TGG ATA
(TaqI) 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
(SfaNI) is used in place cf oligonucleotide (5) in the example 1, the result is the plasmid pB310 ExampLe 3 ~ompetent cells of the E. coli strain W3110 are trans-formed with the plasmid pB30 or p~31. An overnight culture of the strain is diluted in the ratio of about 1:100 with L~ medium (J. H. M;ller~ Experim~nts in Molec.
Gen., Cold Spring Harbor Lab~, 1972), which contains 5a ~glml ampicillin, and the growth ;s followed by measure-~ent of the OD. At OD = 0~5 the culture is adjusted to a concentration of 2 mM in isopropyl-~-D-thiogalactopyra-noside (IPTG) and, after 150 - 180 m;nutes, the bacteria are spun down. These bacteria are treated in a buffer m;~ture ~7M urea, 0.1% SDS, 0.1M sodium phosphate, pH
7.0) for about 5 minutes, and samples are applied to an SDS polyacrylamide gel electrophoresis pLate. This confirms 4he expression of the b;fun~tional protein.
- 8 - ~322~57 The stated cond;tions apply to shake cultures; for larger fermentations it is expedient to modify the OD values and nutrient media and vary the IPTG concentrations appro-priately.
s Example 4 E~ coli ~3110 cells which contain the plasmid pB30 or p931 are, after inductionr spun down, resuspended in sodium phosphate buffer (pH 7) and again spun down. Th8 bacteria are taken up in the same buffer and then dis-rupted ~French Press, (R)Dynomi71~. The disrupted cells are spun do~n. The supernatant and sediment are analyzed by SDS polyacrylam;de gel electrophorese as described in Example 3. Staining of the protein bands reveals that the bifunctional protein ;s located in the sediment from the disruption. The sediment is washed several times with chaotropic buffers and finally ~ith 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 concen-tration of 5 M in guanidinium hydrochloride and 2 mM in dithiothreitol (DTT jD The mixture is stirred under nitrogen for about 30 m;nutes and then diluted with 50 mM
tris buffer (pH 8.5) so that the protein concentration is 100 ~g/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 fil-tered and its biological activity is checked~ It s~ full bio-logical action both in the interleukin-2-dependent CTLL 2 cell proliferation assay and in thehuman bone marrow assay. Mixed colonies of granulocytes and ~acrophages are observed in these.
The bifunctional protein can be further purified by interleukin-2-specific affinity chromatography~ The protein is st;ll active in both assays. In contrast, an . coli extract of the untransformed strain ~3110 whish has been treated as described shows no act;vity.
- - ::
: ~ : : : . ~;, ~ ~2~
Other conditions are expedient for the industrial pre-paration of the product, for exa~ple for the folding of the protein and its purification~ Suitable purification processes - wh;ch are known per se - are ion e~change, adsorption, gel fil~ration and preparative HPLC chroma-tography.
.
The folLo~;ng oligonucleotide (5) 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) 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) The oligonucLeotide (5? extends at:the 5' end the DNA
sequence of IL-2, there being, however, Asp in place of Thr in position 133. At the 3' end of this o~igonucleo-tide are located the nucleotides which have been deleted from the cDNA by cutting w;th SfaNI.
The preparation of the expression plasmid pE~1000 (6) is proposed in the (not prior-pubLished) EP-A 0,227,938 (Figure 1). This plasmid is a derivative of the plasmid ptac 11 tAmann et al., Gene 25 t1983) 167 - 178), in ~hich a synthetic sequence which contains a SalI cleavage site has been incorporated in the recognition site for coRI~ The eKpression plasm;d pKK 177.3 is obta;ned in this way. Insertion of the lac repressor ~Farabaughv Nature 274 (1978) 765 - 769) results in the plasmid pJF118. The latter is opened at the unique restriction cleavage site for AvaI, and is shortened by about 1000 bp in a known manner by exonuclease treatment and ;s Ligated. The ~lasmid pE~100D ~6) is obtained. Opening of this plasmid in the polylinker using the enzymes EcoRI
and PstI results in the linear;zed expression plasmid (7).
Th;s linearized plasm;d DNA (7~ is now ligated with the DNA fragment (2) which codes ~or th~ IL-2 sequence, with _ 7 _ ~ 3 22~ ~t~
the synthetic oligonucleotide (5) and ~ith the cDNA
fragment (4). The result is the plasm;d pB30 (8) whish is transformed ;nto the E. coli strain Mc10~1. The plasmid DNA from individual clones is isolated and character;zed by restriction analysis.
Example 2 If the following synthetic oligonucleotide 128 (133) Ile Ile Ser Thr Leu Asp Pro Met Ile Thr Thr Tyr CG ATC ATC TCT ACC CTG GAC CCG A~6 ATC ACC ACC TAT
TAG TAG AGA TGG ~AC CTG GGC TAC TAG TGG TGG ATA
(TaqI) 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
(SfaNI) is used in place cf oligonucleotide (5) in the example 1, the result is the plasmid pB310 ExampLe 3 ~ompetent cells of the E. coli strain W3110 are trans-formed with the plasmid pB30 or p~31. An overnight culture of the strain is diluted in the ratio of about 1:100 with L~ medium (J. H. M;ller~ Experim~nts in Molec.
Gen., Cold Spring Harbor Lab~, 1972), which contains 5a ~glml ampicillin, and the growth ;s followed by measure-~ent of the OD. At OD = 0~5 the culture is adjusted to a concentration of 2 mM in isopropyl-~-D-thiogalactopyra-noside (IPTG) and, after 150 - 180 m;nutes, the bacteria are spun down. These bacteria are treated in a buffer m;~ture ~7M urea, 0.1% SDS, 0.1M sodium phosphate, pH
7.0) for about 5 minutes, and samples are applied to an SDS polyacrylamide gel electrophoresis pLate. This confirms 4he expression of the b;fun~tional protein.
- 8 - ~322~57 The stated cond;tions apply to shake cultures; for larger fermentations it is expedient to modify the OD values and nutrient media and vary the IPTG concentrations appro-priately.
s Example 4 E~ coli ~3110 cells which contain the plasmid pB30 or p931 are, after inductionr spun down, resuspended in sodium phosphate buffer (pH 7) and again spun down. Th8 bacteria are taken up in the same buffer and then dis-rupted ~French Press, (R)Dynomi71~. The disrupted cells are spun do~n. The supernatant and sediment are analyzed by SDS polyacrylam;de gel electrophorese as described in Example 3. Staining of the protein bands reveals that the bifunctional protein ;s located in the sediment from the disruption. The sediment is washed several times with chaotropic buffers and finally ~ith 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 concen-tration of 5 M in guanidinium hydrochloride and 2 mM in dithiothreitol (DTT jD The mixture is stirred under nitrogen for about 30 m;nutes and then diluted with 50 mM
tris buffer (pH 8.5) so that the protein concentration is 100 ~g/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 fil-tered and its biological activity is checked~ It s~ full bio-logical action both in the interleukin-2-dependent CTLL 2 cell proliferation assay and in thehuman bone marrow assay. Mixed colonies of granulocytes and ~acrophages are observed in these.
The bifunctional protein can be further purified by interleukin-2-specific affinity chromatography~ The protein is st;ll active in both assays. In contrast, an . coli extract of the untransformed strain ~3110 whish has been treated as described shows no act;vity.
- - ::
: ~ : : : . ~;, ~ ~2~
Other conditions are expedient for the industrial pre-paration of the product, for exa~ple for the folding of the protein and its purification~ Suitable purification processes - wh;ch are known per se - are ion e~change, adsorption, gel fil~ration and preparative HPLC chroma-tography.
.
Claims (16)
1. A medicament comprising a bifunctional protein consisting of a biologically active interleukin-2 (IL-2) constituent and granulocyte macrophage colony stimulating factor (GM-CSF) constituent.
2. A bifunctional protein having a biologically active IL-2 constituent and GM-CSF constituent, wherein the two biologically active protein constituents are linked via a bridge consisting of 1 to about 20 genetically encodable amino acids.
3, A protein as claimed in Claim 2, wherein the bridge corresponds to the formula (II) - Asp - (aa)x- Pro - (II) wherein x is an integer from 1 to 18, and aa is a genetically encodable amino acid with the exception of Cys.
4. A protein as claimed in Claim 3, wherein (aa)x is the amino acid sequence -Prb-Met-Ile-Thr-Thr-Tyr-Ala-Asp-Asp-or -Pro-Met-Ile-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr-Ile-Asp-Asp-.
5. A protein as claimed in claim 2, wherein the IL-2 constituent is arranged N-terminal and the GN-CSF constituent is arranged C-terminal.
6. A protein as claimed in claim 3, wherein the IL-2 constituent is arranged N-terminal and the GM-CSF constituent is arranged C-terminal.
7. A protein as claimed in claim 4, wherein the IL-2 constituent is arranged N-terminal and the GM-CSF constituent is arranged C-terminal.
8. A process for the preparation of a bifunctional protein as claimed in any one of claims 1 to 7, which comprises constructing a gene coding for this protein and expressing it in a host cell.
9. A medicament comprising a protein as claimed in claim 2 and a pharmacologically suitable vehicle.
10. A medicament comprising a protein as claimed in claim 3 and a pharmacologically suitable vehicle.
11. A medicament comprising a protein as claimed in claim 4 and a pharmacologically suitable vehicle.
12. A medicament comprising a protein as claimed in claim 5 and a pharmacologically suitable vehicle.
13. A medicament comprising a protein as claimed in claim 6 and a pharmacologically suitable vehicle.
14. A medicament comprising a protein as claimed in claim 7 and a pharmacologically suitable vehicle.
15. Use of a medicament as claimed in any one of claims 9 to 14 in the treatment of malignant neoplasms.
16. A medicament as claimed in any one of claims 9 to 14 for use in the treatment of malignant neoplasms.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3712985.6 | 1987-04-16 | ||
| DE19873712985 DE3712985A1 (en) | 1987-04-16 | 1987-04-16 | BIFUNCTIONAL PROTEINS |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1322157C true CA1322157C (en) | 1993-09-14 |
Family
ID=6325799
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000564322A Expired - Fee Related CA1322157C (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)
| 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 |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1985000817A1 (en) * | 1983-08-10 | 1985-02-28 | Amgen | Microbial expression of interleukin ii |
| 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 |
| AU588819B2 (en) * | 1984-10-29 | 1989-09-28 | Immunex Corporation | Cloning of human granulocyte-macrophage colony stimulating factor gene |
| 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 |
| DE3545568A1 (en) * | 1985-12-21 | 1987-07-16 | Hoechst Ag | GM-CSF-PROTEIN, ITS DERIVATIVES, PRODUCTION OF SUCH PROTEINS AND THEIR USE |
-
1987
- 1987-04-16 DE DE19873712985 patent/DE3712985A1/en not_active Withdrawn
-
1988
- 1988-04-09 AT AT88105693T patent/ATE79135T1/en not_active IP Right Cessation
- 1988-04-09 DE DE8888105693T patent/DE3873397D1/en not_active Expired - Fee Related
- 1988-04-09 ES ES198888105693T patent/ES2033981T3/en not_active Expired - Lifetime
- 1988-04-09 EP EP88105693A patent/EP0288809B1/en not_active Expired - Lifetime
- 1988-04-14 NZ NZ224247A patent/NZ224247A/en unknown
- 1988-04-14 PT PT87237A patent/PT87237B/en active IP Right Grant
- 1988-04-14 FI FI881743A patent/FI98830C/en not_active IP Right Cessation
- 1988-04-15 IE IE114688A patent/IE61574B1/en not_active IP Right Cessation
- 1988-04-15 NO NO881658A patent/NO176922C/en not_active IP Right Cessation
- 1988-04-15 AR AR88310585A patent/AR242991A1/en active
- 1988-04-15 AU AU14661/88A patent/AU613022B2/en not_active Ceased
- 1988-04-15 DK DK209188A patent/DK170741B1/en not_active IP Right Cessation
- 1988-04-15 CA CA000564322A patent/CA1322157C/en not_active Expired - Fee Related
- 1988-04-15 JP JP63093321A patent/JP2667193B2/en not_active Expired - Fee Related
- 1988-04-15 IL IL8608688A patent/IL86086A/en not_active IP Right Cessation
- 1988-04-15 HU HU881966A patent/HU204303B/en not_active IP Right Cessation
- 1988-04-15 ZA ZA882659A patent/ZA882659B/en unknown
- 1988-04-16 KR KR1019880004345A patent/KR970000187B1/en not_active IP Right Cessation
-
1989
- 1989-04-14 PH PH36799A patent/PH25327A/en unknown
-
1992
- 1992-11-04 GR GR920402185T patent/GR3006141T3/el unknown
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 |
| GR3006141T3 (en) | 1993-06-21 |
| KR880012760A (en) | 1988-11-29 |
| NO176922B (en) | 1995-03-13 |
| AU613022B2 (en) | 1991-07-25 |
| 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 |
|---|---|---|
| CA1322157C (en) | Bifunctional proteins | |
| US5359035A (en) | Bifunctional proteins including interleukin-2 (IL-2) and granuloctyte macrophage colony stimulating factor (GM-CSF) | |
| US5563046A (en) | Fusion polypeptides and proteins | |
| CA1339894C (en) | Fusion proteins with a eukaryotic ballast portion | |
| EP0188920A2 (en) | Interleukin 1 and its derivative | |
| KR940005585B1 (en) | Gm-csf protein its derivatives the preparation of proteins of this type and their use | |
| JP3217698B2 (en) | Mature human interleukin 1 protein | |
| EP0204527A1 (en) | Removal of the N-terminal methionine from methionylproteins | |
| CA2000309A1 (en) | Recombinant pdgf and methods for production | |
| AU621051B2 (en) | Method for purifying granulocyte-macrophage colony stimulating factor | |
| US5157004A (en) | Polypeptides and production thereof | |
| EP0134673A1 (en) | Improved plasmid vector and use thereof | |
| US5047504A (en) | Method for purifying granulocyte-macrophage colony stimulating factor | |
| EP0871470A1 (en) | Haemopoietic growth factor antagonists and uses therefor | |
| AU703052B2 (en) | Haemopoietic growth factor antagonists and uses therefor | |
| JPS60137292A (en) | Polydeoxyribonucleic acid having gene information of novel physiologically active polypeptide, replicable recombinant dna containing said polydeoxyribonucleic acid, microorganism or cell transformed by said replicable recombinant dna, novel physiologically active polypeptide, and their preparation | |
| JP2544255B2 (en) | Process for producing polypeptide having human interleukin 1 activity | |
| JPH02119793A (en) | Novel polypeptide | |
| JPH0673095A (en) | Polypeptidfe having human interleukin 1 activity | |
| JPS62135493A (en) | Carcinonecrotic factor | |
| AU3497499A (en) | Haemopoietic growth factor antagonists and uses therefor II | |
| EP0750667A1 (en) | Use of a recombinant inhibitor from erythrina caffra for purifying serine proteases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed |