CA2064696A1 - Il-2 deletion mutants - Google Patents
Il-2 deletion mutantsInfo
- Publication number
- CA2064696A1 CA2064696A1 CA002064696A CA2064696A CA2064696A1 CA 2064696 A1 CA2064696 A1 CA 2064696A1 CA 002064696 A CA002064696 A CA 002064696A CA 2064696 A CA2064696 A CA 2064696A CA 2064696 A1 CA2064696 A1 CA 2064696A1
- Authority
- CA
- Canada
- Prior art keywords
- molecule
- mutant
- amino acid
- deleted
- toxin
- 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.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/55—IL-2
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
Abstract
A mutant IL-2 molecule capable of binding an IL-2 receptor-bearing cell, having a deletion of one to five amino acid residues of IL-2, the deletion resulting in active IL-2 molecules that have increased resistance to proteolysis.
Description
WO~t/02000 2 ~ PCT/US90/04258 Backqround of the Invention This invention relates to the use of recombinant DNA techniques to make mutant interleukin-2 (IL-2) molecules and chimeric IL-2/toxin molecules.
Il-2 is a protein secreted by human T-lymphocytes which is capable of binding to IL-2 receptors on activated T-lymphocytes and effecting T-lymphocyte proliferation. IL-~ has been shown to be a therapeutic immunostimulant in humans (Rosenberg, 1988, Immunology Today 9 2: 5a-62), and IL-2 or a specific binding portion thereof can be coupled to the enzymatically active portion of diphtheria toxin to form a hybrid molecule with a number of therapeutic applications ~Murphy U.S. Patent No. 4,675,382, hereby incorporated by reference). IL-2/diphtheria toxin hybrid proteins of Murphy '382, which were made using recombinant DNA techniques, have been shown to inhibit rejection of transplanted organs (Pankewycz et al., ~ransplantation 47:318-322 (1989)), and are also potential therapeutic agents in the treatment of certain cancers and autoimmune diseases in which the IL-2 receptor plays a role.
IL-2 encoding DNA sequences are reported in a 2~ number of publications, and in addition, a modified IL-2-encoding gene, in which a cysteine codon is changed to enhance stability, is described in U.S. Pat. No.
4,518,584, hereby incorporated by reference. U.S.S.N.
834,900, filed Feb. 28, 1986, hereby incorporated by reference, describes a synthetic IL-2-encoding DNA
. : -~ ' . :' ' - :'.
- . . - , ~ .
-W O 91/02000 - 2 - PC~r/US90~04258 sequence that dlffers ~rom the natural IL-2 encoding DNA
in that it contains more prokaryotic preferred translation codons than the naturally occurring sequence.
Amino acid deletions or substitutions have been made in the IL-2 amino acid sequence (European Pat.
Appln. Nos. 86114468.1 and 87101839.6, U.S. Pat. No.
4,604,377). Although the DNA and amino acid sequences of IL-2 and its crystal structure are known (Brandhuber lo et al., 1987, Science 238, 1707), there is little data available that allows accurate prediction of the regions of IL-2 that are responsible for biological activity or are sensitive to proteolytic breakdown; e.g., a single substitution of the cysteine residue at position 125 of the IL-2 amino acid sequence with a serine results in increased stability of the molecule (U.S. Patent No.
4,604,377); a substitution of the tryptophan residue at position 121 inactivates the molecule; deletion of amino acid residues 100-104 decreases the biological activity by two oders of magnitude; and deletion of amino acid residues 124-126 renders the molecule inactive (Collins et al., 198~, Proc. Nat. Aca. Sci. 85: 7709; Cohen et al., 1986, Science 234:349).
SummarY of the Invention The present invention provides IL-2 mutant polypeptides that bear a deletion of one to five amino acids, yet retain the ability to bind to IL-2 receptor-bearing cells. It is known that lysine 76 is a proteolytic sîte in the IL-2 molecule (Cohen et al., 1986, Science 234:349). These mutants either delete this proteolytic site completely, or alter the s.ructure of that area in an effort to reduce proteolysis. The IL-2 mutants can be used as immunostimulants or, when coupled to a toxin to form a hybrid IL2-toxin molecule, 3 2 ~ ~ 4 ~ ~ ~ ` PcT/us9o/o4258 an be used to treat immune and other disorders characterized by the presence of the IL-2 receptor.
The invention,thus generally features eight new mutant IL-2 polypeptides capabl~ of binding to the IL-2 receptor; the IL-2 polypeptides have deletions of one or more amino acid residues, as follows; 74; 74-78; 75-77;
76-78; 76-79; 15, 78; and 79 (according to the numbering convention of the Figure, taken from Williams et al., Nucleic Acids Res., vol. 16, no. 22 (1988).
In some preferred embodiments, the mutant IL-2 polypeptide may be part of a fusion protein consisting of a toxin portion (e.g., derived from diphtheria toxin) covalently linked, preferably through a peptide bond at its carboxy terminal end, to the mutant IL-2 polypeptide. The diphtheria toxin portion is large enough to exhibit cytotoxic activity and small enouah to fail to exhibit generalized eukaryotic cell binding Preferably, the DNA sequence encoding the IL-2 polypeptide contains nucleotide substitutions designed to maximize gene expression in the cells used for expressior i.e., where prokaryotic cells such as E.
coli are used, preferred prokaryotic codons are substituted for some of the natural codons (this has been done in the sequence shown in the Figure).
The hybrid molecules of the invention are useful for treating diseases in which the IL-2 receptor plays a role, e.g., IL-2 receptor positive malignancies, allergic reactions, ~nd systemic lupus erythmatosis (SLE), or to prevent an immune response by IL-2 receptor bearing T cells that occurs in graft rejec~ion. This targeted toxin functions by the following mechanism: the IL-2/toxin, by virtue of the IL-2 domain, binds to high affinity IL-2 receptor-bearing cells. The IL-2-toxin is internalized into endocytic vesicles by IL-2 receptor-mediated endocytosis. Acidification of t~e .. - ' ........... . ~ ,. .
.. , , . . ~ .
- -: : ' : - . . ~ -2 ~ g ~ fi 9 ~ 4 PCT/US90/~4258 endosome causes a conformational change in the toxin, allowing its me~orane-associating domains to interact with the endocytic vesicle's membrane and facilitate translocation of the enzymatically active fragment A
into the cytosol. Once delivered to the cytosol, fragment A catalyzes the ADP-ribosylation of elongation factor 2, resulting in inhibition of protein synthesis and subseguent death of the IL-2-receptor bearing cell.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Descri~ticn _ the Preferred Embodiments The drawing is first described.
Drawinq The Figure is a DNA sequence, encoding IL-2, in which preferred prokaryotic translation codons are employed; the numbers correspond to the nu~oering referred to in this specification.
Construction of the Genes Encodinq IL-2 Deletion Mutants/Toxin Amino acids 74 through 79 are contained within the Xbal/Notl fragment of ~he synthetic IL-2 gene (see Figure). For each of the eight deletion mutants, an Xbal/Notl fragment with a dele~ion of DNA encoding between one and five amino acids is synthesized using an automated DNA synthesizer according to conventional techniques. The DNA sequences of the oligonucleotides are shown in Table I., Each Xbal/Notl fragment is synthesized as two complementary strands with a l/2 Xb 1 site at the 5' end and a l/2 Notl site at the 3' end. The synthetic DNA's are gel purified on a denaturing polyacrylamide-urea gel and complementary strands are annealed according to conventional methods. The annealed DNA's are ligated WOgl/02000 - 5 - 2 ~ 9 ~ PCT/US90/04258 into the expression plasmid, pDW15 (Williams et al., 1987, Prot. Engineering 1: 493 ), which contains the synthetic IL-2 gene shown in the Figure. Ligation reactions are transformed into a suitable E. coli host according to conventional techniques.
Transformants are screened by restriction digest analysis of minilysate DNA using the restriction enzyme Ddel. The Ddel restriction digest profile of the IL-2 mutants differs from that of non-deleted IL-2 due to elimination of a Ddel site within the Xbal/Notl fragment of the deletion mutants. The DNA sequence o~
the rL-2 deletion mutants are confirmed by the dideoxy method of Sanger et al. (1977, Proc. Nat. Acad. Sci., 74:S463).
The genes encoding the IL-2/diphtheria toxin fusion proteins are constructed by standard recombinant D~A techniques, as follows. The IL-2 portion of the fusion gene is contained within the S~hl/Hindlll fragment of the IL-2 deletion mutant derived from pDW15. This DNA fragment is ligated to S~hl/Hindlll digested plasmid pABM6508 (Bishai et al., 1987, J.
Bacteriol, 169:5140), which contains the diphtheria toxin-related portion of the fusion up to and including the amino acid residue Ala 486. The DNA is transformed 2S into a suitable E. coli host an~ plated onto Luria broth plates plus an appropriate antibiotic for selection, according to conventional techniques. Transformants are screened by _del restriction digest analysis of minilysate DNA and by Western blot analysis, as follows.
Western Blot Anal~sis -Total bacterial cell lysates are analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970, Nature 227:680) for the production of IL-2ttoxin protein. Proteins are electro~lotted onto nylon . . . ; .: . .
. .: ~ : . : . :
.
. .
WO91/02000 2 ~ 9 6 - 6 - PCT/US90/04258 membrane and immunoblot analysis is performed according to conventional techniques. Confirmation of the expected construct is made by positive cross-reactivity to both anti-diphtheria toxin (Connaught Laboratories, Toronto, Ontario, Canada) and to a monoclonal anti-IL-2 antibody, as well as ~y comparison of the size cf the expressed protein to known IL-2/toxin standard. Final confirmation of the construct is made by DNA sequence analysis of the IL-2//toxin gene.
CvtotoxicitY assaY
Referring to Table II, C9l/Pl cells (a high-affinity IL2 receptor-bearing cell line) were seeded in 96-well V-bottom plates (Nunc, Roskilde, Denmark) at a concentration of l05 per well in l00 ~1 complete medium. Il-2-toxin was added at varying concentrations (l0-l2M to l0-6M) in complete medium. Cells cultured with medium alone were included as the control. Following 18 hours incubation at 37C
in a 5~ Co2 atmosphere, the plates were centrifuged for 5 minutes at 170 x g, the medium was removed and replaced with l00 ~l leucine-free medium (DMEM
Selectamine, Gibco) containing 2.5 ~Ci/ml ~14Cl-leucine ~New England Nuclear, Boston, ~A).
Cells were then incubated at 37 for 90 minutes and collected on glass fiber filters using a cell harvester (Skatron, Sterling, VA). Filters were washed, dried, and counted according to standard methods. All determinations were performed in pentuplicate. IC50 refers to the concentration of IL2 required to inhibit protein synthesis to 50~ of the untreated control.
WO 91/02000 2 ~ P~/US90/04258 ~8 8 ~ 8 ~ :~ L~ L~ b 2 ~ 8 ~ ~
3 ~ ~ ~
Ç} k ~ } k ~, ~ <~ a ~ ~ o S ~ ~ ~ ,.
~3 ~ ~ 3 ~ 3 ~ ~ ~ ~ ~ 3 3 ~ 3 ~ ~
3 ~ 3 ~ 3 ~ ~ ~ 3 ~ 3 L~ O n P O ~ ~ O O ~ ~ :
~ .S !~ ~ ~ ~
'~ a ~ ~ 5 ~ ~ ~ 3 ~ ~ ~ e ~ ~ 5 ~ i 8 ~ 3 ~ ~
s~ ~ ~
~3~ ~ 3~ 3B ~1~ a~
s 3 ~ 3 ~
~8 3 1~ 3 e 5 ~ 3 e ~ ~3 3 ~ ~ ~ 3 ~ ~ ~ 3 ~ 3 e 5 ~ e 3 3~ ~a a~ 3a 3~ 0 ~, a ~ ". o ~, ~ o 5 ~o3~ ~ 3~ ~a ~ 3~ ~ a~
~a3 ~3 ~3 ~ ~5 ~5 ~ a~
t ~ ~ ~ ~ ~ ~ h ~ t ~ ~ o~ -- ~
c - E '4 '~ ~ r1 , ~ ., ` :, WO910~000 _ ~/_ PCT/US90/04258 Plasmid amino acid(s) deleted C9l/PL IC50 ~;
psI133 ~74 6xlO lM
psI134 ~75 lxlO lOM
PsI136 ~78 5xlO llM
psIl37 ~79 2xlO lOM
psI143 ~75-77 2xlO lOM
psI141 ~74-78 lxlO 1OM
psIl45 ~76-78 2xlO 1OM
psI150 ~76-79 7xlO1lM
(psI129 no deletion typically control 5 1o~ll . . . :
WO91/02000 - 8 ~ 2 ~ q ~ PCT/US90/04258 Other Embodiments Other embodiments are within the following claims. For example, the deletion mutant IL-2 molecules can be used alone, in addition to their use in toxic S hybrids, the deletions can advantagously provide resistance to proteolysis in both contexts. In addition, toxins other than diphtheria toxin can be coupled to the mutants, e.g., the enzymatically active portion of Pseudomonas exotoxin can be used.
. ~'. ' -' .
Il-2 is a protein secreted by human T-lymphocytes which is capable of binding to IL-2 receptors on activated T-lymphocytes and effecting T-lymphocyte proliferation. IL-~ has been shown to be a therapeutic immunostimulant in humans (Rosenberg, 1988, Immunology Today 9 2: 5a-62), and IL-2 or a specific binding portion thereof can be coupled to the enzymatically active portion of diphtheria toxin to form a hybrid molecule with a number of therapeutic applications ~Murphy U.S. Patent No. 4,675,382, hereby incorporated by reference). IL-2/diphtheria toxin hybrid proteins of Murphy '382, which were made using recombinant DNA techniques, have been shown to inhibit rejection of transplanted organs (Pankewycz et al., ~ransplantation 47:318-322 (1989)), and are also potential therapeutic agents in the treatment of certain cancers and autoimmune diseases in which the IL-2 receptor plays a role.
IL-2 encoding DNA sequences are reported in a 2~ number of publications, and in addition, a modified IL-2-encoding gene, in which a cysteine codon is changed to enhance stability, is described in U.S. Pat. No.
4,518,584, hereby incorporated by reference. U.S.S.N.
834,900, filed Feb. 28, 1986, hereby incorporated by reference, describes a synthetic IL-2-encoding DNA
. : -~ ' . :' ' - :'.
- . . - , ~ .
-W O 91/02000 - 2 - PC~r/US90~04258 sequence that dlffers ~rom the natural IL-2 encoding DNA
in that it contains more prokaryotic preferred translation codons than the naturally occurring sequence.
Amino acid deletions or substitutions have been made in the IL-2 amino acid sequence (European Pat.
Appln. Nos. 86114468.1 and 87101839.6, U.S. Pat. No.
4,604,377). Although the DNA and amino acid sequences of IL-2 and its crystal structure are known (Brandhuber lo et al., 1987, Science 238, 1707), there is little data available that allows accurate prediction of the regions of IL-2 that are responsible for biological activity or are sensitive to proteolytic breakdown; e.g., a single substitution of the cysteine residue at position 125 of the IL-2 amino acid sequence with a serine results in increased stability of the molecule (U.S. Patent No.
4,604,377); a substitution of the tryptophan residue at position 121 inactivates the molecule; deletion of amino acid residues 100-104 decreases the biological activity by two oders of magnitude; and deletion of amino acid residues 124-126 renders the molecule inactive (Collins et al., 198~, Proc. Nat. Aca. Sci. 85: 7709; Cohen et al., 1986, Science 234:349).
SummarY of the Invention The present invention provides IL-2 mutant polypeptides that bear a deletion of one to five amino acids, yet retain the ability to bind to IL-2 receptor-bearing cells. It is known that lysine 76 is a proteolytic sîte in the IL-2 molecule (Cohen et al., 1986, Science 234:349). These mutants either delete this proteolytic site completely, or alter the s.ructure of that area in an effort to reduce proteolysis. The IL-2 mutants can be used as immunostimulants or, when coupled to a toxin to form a hybrid IL2-toxin molecule, 3 2 ~ ~ 4 ~ ~ ~ ` PcT/us9o/o4258 an be used to treat immune and other disorders characterized by the presence of the IL-2 receptor.
The invention,thus generally features eight new mutant IL-2 polypeptides capabl~ of binding to the IL-2 receptor; the IL-2 polypeptides have deletions of one or more amino acid residues, as follows; 74; 74-78; 75-77;
76-78; 76-79; 15, 78; and 79 (according to the numbering convention of the Figure, taken from Williams et al., Nucleic Acids Res., vol. 16, no. 22 (1988).
In some preferred embodiments, the mutant IL-2 polypeptide may be part of a fusion protein consisting of a toxin portion (e.g., derived from diphtheria toxin) covalently linked, preferably through a peptide bond at its carboxy terminal end, to the mutant IL-2 polypeptide. The diphtheria toxin portion is large enough to exhibit cytotoxic activity and small enouah to fail to exhibit generalized eukaryotic cell binding Preferably, the DNA sequence encoding the IL-2 polypeptide contains nucleotide substitutions designed to maximize gene expression in the cells used for expressior i.e., where prokaryotic cells such as E.
coli are used, preferred prokaryotic codons are substituted for some of the natural codons (this has been done in the sequence shown in the Figure).
The hybrid molecules of the invention are useful for treating diseases in which the IL-2 receptor plays a role, e.g., IL-2 receptor positive malignancies, allergic reactions, ~nd systemic lupus erythmatosis (SLE), or to prevent an immune response by IL-2 receptor bearing T cells that occurs in graft rejec~ion. This targeted toxin functions by the following mechanism: the IL-2/toxin, by virtue of the IL-2 domain, binds to high affinity IL-2 receptor-bearing cells. The IL-2-toxin is internalized into endocytic vesicles by IL-2 receptor-mediated endocytosis. Acidification of t~e .. - ' ........... . ~ ,. .
.. , , . . ~ .
- -: : ' : - . . ~ -2 ~ g ~ fi 9 ~ 4 PCT/US90/~4258 endosome causes a conformational change in the toxin, allowing its me~orane-associating domains to interact with the endocytic vesicle's membrane and facilitate translocation of the enzymatically active fragment A
into the cytosol. Once delivered to the cytosol, fragment A catalyzes the ADP-ribosylation of elongation factor 2, resulting in inhibition of protein synthesis and subseguent death of the IL-2-receptor bearing cell.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Descri~ticn _ the Preferred Embodiments The drawing is first described.
Drawinq The Figure is a DNA sequence, encoding IL-2, in which preferred prokaryotic translation codons are employed; the numbers correspond to the nu~oering referred to in this specification.
Construction of the Genes Encodinq IL-2 Deletion Mutants/Toxin Amino acids 74 through 79 are contained within the Xbal/Notl fragment of ~he synthetic IL-2 gene (see Figure). For each of the eight deletion mutants, an Xbal/Notl fragment with a dele~ion of DNA encoding between one and five amino acids is synthesized using an automated DNA synthesizer according to conventional techniques. The DNA sequences of the oligonucleotides are shown in Table I., Each Xbal/Notl fragment is synthesized as two complementary strands with a l/2 Xb 1 site at the 5' end and a l/2 Notl site at the 3' end. The synthetic DNA's are gel purified on a denaturing polyacrylamide-urea gel and complementary strands are annealed according to conventional methods. The annealed DNA's are ligated WOgl/02000 - 5 - 2 ~ 9 ~ PCT/US90/04258 into the expression plasmid, pDW15 (Williams et al., 1987, Prot. Engineering 1: 493 ), which contains the synthetic IL-2 gene shown in the Figure. Ligation reactions are transformed into a suitable E. coli host according to conventional techniques.
Transformants are screened by restriction digest analysis of minilysate DNA using the restriction enzyme Ddel. The Ddel restriction digest profile of the IL-2 mutants differs from that of non-deleted IL-2 due to elimination of a Ddel site within the Xbal/Notl fragment of the deletion mutants. The DNA sequence o~
the rL-2 deletion mutants are confirmed by the dideoxy method of Sanger et al. (1977, Proc. Nat. Acad. Sci., 74:S463).
The genes encoding the IL-2/diphtheria toxin fusion proteins are constructed by standard recombinant D~A techniques, as follows. The IL-2 portion of the fusion gene is contained within the S~hl/Hindlll fragment of the IL-2 deletion mutant derived from pDW15. This DNA fragment is ligated to S~hl/Hindlll digested plasmid pABM6508 (Bishai et al., 1987, J.
Bacteriol, 169:5140), which contains the diphtheria toxin-related portion of the fusion up to and including the amino acid residue Ala 486. The DNA is transformed 2S into a suitable E. coli host an~ plated onto Luria broth plates plus an appropriate antibiotic for selection, according to conventional techniques. Transformants are screened by _del restriction digest analysis of minilysate DNA and by Western blot analysis, as follows.
Western Blot Anal~sis -Total bacterial cell lysates are analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970, Nature 227:680) for the production of IL-2ttoxin protein. Proteins are electro~lotted onto nylon . . . ; .: . .
. .: ~ : . : . :
.
. .
WO91/02000 2 ~ 9 6 - 6 - PCT/US90/04258 membrane and immunoblot analysis is performed according to conventional techniques. Confirmation of the expected construct is made by positive cross-reactivity to both anti-diphtheria toxin (Connaught Laboratories, Toronto, Ontario, Canada) and to a monoclonal anti-IL-2 antibody, as well as ~y comparison of the size cf the expressed protein to known IL-2/toxin standard. Final confirmation of the construct is made by DNA sequence analysis of the IL-2//toxin gene.
CvtotoxicitY assaY
Referring to Table II, C9l/Pl cells (a high-affinity IL2 receptor-bearing cell line) were seeded in 96-well V-bottom plates (Nunc, Roskilde, Denmark) at a concentration of l05 per well in l00 ~1 complete medium. Il-2-toxin was added at varying concentrations (l0-l2M to l0-6M) in complete medium. Cells cultured with medium alone were included as the control. Following 18 hours incubation at 37C
in a 5~ Co2 atmosphere, the plates were centrifuged for 5 minutes at 170 x g, the medium was removed and replaced with l00 ~l leucine-free medium (DMEM
Selectamine, Gibco) containing 2.5 ~Ci/ml ~14Cl-leucine ~New England Nuclear, Boston, ~A).
Cells were then incubated at 37 for 90 minutes and collected on glass fiber filters using a cell harvester (Skatron, Sterling, VA). Filters were washed, dried, and counted according to standard methods. All determinations were performed in pentuplicate. IC50 refers to the concentration of IL2 required to inhibit protein synthesis to 50~ of the untreated control.
WO 91/02000 2 ~ P~/US90/04258 ~8 8 ~ 8 ~ :~ L~ L~ b 2 ~ 8 ~ ~
3 ~ ~ ~
Ç} k ~ } k ~, ~ <~ a ~ ~ o S ~ ~ ~ ,.
~3 ~ ~ 3 ~ 3 ~ ~ ~ ~ ~ 3 3 ~ 3 ~ ~
3 ~ 3 ~ 3 ~ ~ ~ 3 ~ 3 L~ O n P O ~ ~ O O ~ ~ :
~ .S !~ ~ ~ ~
'~ a ~ ~ 5 ~ ~ ~ 3 ~ ~ ~ e ~ ~ 5 ~ i 8 ~ 3 ~ ~
s~ ~ ~
~3~ ~ 3~ 3B ~1~ a~
s 3 ~ 3 ~
~8 3 1~ 3 e 5 ~ 3 e ~ ~3 3 ~ ~ ~ 3 ~ ~ ~ 3 ~ 3 e 5 ~ e 3 3~ ~a a~ 3a 3~ 0 ~, a ~ ". o ~, ~ o 5 ~o3~ ~ 3~ ~a ~ 3~ ~ a~
~a3 ~3 ~3 ~ ~5 ~5 ~ a~
t ~ ~ ~ ~ ~ ~ h ~ t ~ ~ o~ -- ~
c - E '4 '~ ~ r1 , ~ ., ` :, WO910~000 _ ~/_ PCT/US90/04258 Plasmid amino acid(s) deleted C9l/PL IC50 ~;
psI133 ~74 6xlO lM
psI134 ~75 lxlO lOM
PsI136 ~78 5xlO llM
psIl37 ~79 2xlO lOM
psI143 ~75-77 2xlO lOM
psI141 ~74-78 lxlO 1OM
psIl45 ~76-78 2xlO 1OM
psI150 ~76-79 7xlO1lM
(psI129 no deletion typically control 5 1o~ll . . . :
WO91/02000 - 8 ~ 2 ~ q ~ PCT/US90/04258 Other Embodiments Other embodiments are within the following claims. For example, the deletion mutant IL-2 molecules can be used alone, in addition to their use in toxic S hybrids, the deletions can advantagously provide resistance to proteolysis in both contexts. In addition, toxins other than diphtheria toxin can be coupled to the mutants, e.g., the enzymatically active portion of Pseudomonas exotoxin can be used.
. ~'. ' -' .
Claims (15)
1. A mutant IL-2 molecule in which only amino acid residue 74 has been deleted.
2. A mutant IL-2 molecule in which only amino acid residues 74-78 have been deleted.
3. A mutant IL-2 molecule in which only amino acid residues 76-78 have been deleted.
4. A mutant IL-2 molecule in which only amino acid residues 76-79 have been deleted.
5. A mutant IL-2 molecule in which only amino acid residue 75 has been deleted.
6. A mutant IL-2 molecule in which only amino acid residue 78 has been deleted.
7. A mutant IL-2 molecule in which only amino acid residues 75-77 have been deleted.
8. A mutant IL-2 molecule in which only amino acid residue 79 has been deleted.
9. A DNA sequence encoding the mutant IL-2 molecule of any of claims 1-8.
10. The DNA sequence of claim 9, contained in an expression vector.
11. A cell containing the expression vector of claim 10.
12. The DNA sequence of claim 9 wherein said DNA sequence is a synthetic sequence containing more prokaryotic preferred translation codons than naturally occurring IL-2 encoding DNA,
13. A method of producing mutant IL-2 comprising culturing the cell of claim 12 and recovering mutant IL-2 therefrom.
14. The mutant IL-2 molecule of any of claims 1-8, covalently linked to a portion of a toxin molecule which is large enough to exhibit cytotoxic activity and small enough to fail to exhibit generalized eukaryotic cell binding.
15. The molecule of claim 14 wherein said toxin molecule is diptheria toxin, and said portion of diptheria toxin is linked to said mutant IL-2 molecule by a peptide bond.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US38855789A | 1989-08-02 | 1989-08-02 | |
| US388,557 | 1989-08-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2064696A1 true CA2064696A1 (en) | 1991-02-03 |
Family
ID=23534610
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002064696A Abandoned CA2064696A1 (en) | 1989-08-02 | 1990-07-30 | Il-2 deletion mutants |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0485497A4 (en) |
| JP (1) | JPH04507250A (en) |
| AU (1) | AU6179990A (en) |
| CA (1) | CA2064696A1 (en) |
| NZ (1) | NZ234674A (en) |
| WO (1) | WO1991002000A1 (en) |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2103258A1 (en) * | 1991-05-17 | 1992-11-18 | Cory A. Waters | Cytokine receptor targeted molecules for treatment of neoplastic cell growth |
| US5621083A (en) * | 1991-11-04 | 1997-04-15 | Xoma Corporation | Immunotoxins comprising ribosome-inactivating proteins |
| US6146850A (en) | 1991-11-04 | 2000-11-14 | Xoma Corporation | Proteins encoding gelonin sequences |
| US5837491A (en) * | 1991-11-04 | 1998-11-17 | Xoma Corporation | Polynucleotides encoding gelonin sequences |
| US6168785B1 (en) | 1998-07-16 | 2001-01-02 | Institut Pasteur | Biological applications of new peptides of IL-2 and derivatives and use as therapeutic agents |
| CA2571710A1 (en) | 2004-06-24 | 2006-11-02 | Nicholas Valiante | Small molecule immunopotentiators and assays for their detection |
| WO2015042707A1 (en) | 2013-09-24 | 2015-04-02 | Medicenna Therapeutics Pte Ltd | Interleukin-2 fusion proteins and uses thereof |
| EP3134102B1 (en) | 2014-04-24 | 2019-07-03 | The Board of Trustees of The Leland Stanford Junior University | Superagonists, partial agonists and antagonists of interleukin-2 |
| US10961310B2 (en) | 2017-03-15 | 2021-03-30 | Pandion Operations, Inc. | Targeted immunotolerance |
| BR112019024127A2 (en) | 2017-05-24 | 2020-06-23 | Pandion Therapeutics, Inc. | TARGETED IMMUNOTOLERANCE |
| WO2018234862A1 (en) | 2017-06-19 | 2018-12-27 | Medicenna Therapeutics Inc. | Uses and methods for il-2 superagonists, agonists, and fusions thereof |
| US10946068B2 (en) | 2017-12-06 | 2021-03-16 | Pandion Operations, Inc. | IL-2 muteins and uses thereof |
| US10174092B1 (en) | 2017-12-06 | 2019-01-08 | Pandion Therapeutics, Inc. | IL-2 muteins |
| EP3854805A4 (en) * | 2018-09-21 | 2022-08-24 | Innovent Biologics (Suzhou) Co., Ltd. | Novel interleukin 2 and use thereof |
| CN114679909A (en) | 2019-05-20 | 2022-06-28 | 潘迪恩运营公司 | MAdCAM-targeted immune tolerance |
| AU2021237518A1 (en) * | 2020-03-19 | 2022-10-13 | Innovent Biologics (Singapore) Pte. Ltd. | Interleukin-2 mutant and use thereof |
| WO2021185361A1 (en) * | 2020-03-19 | 2021-09-23 | 信达生物制药(苏州)有限公司 | Interleukin-2 mutant and use thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1985000817A1 (en) * | 1983-08-10 | 1985-02-28 | Amgen | Microbial expression of interleukin ii |
-
1990
- 1990-07-27 NZ NZ234674A patent/NZ234674A/en unknown
- 1990-07-30 CA CA002064696A patent/CA2064696A1/en not_active Abandoned
- 1990-07-30 WO PCT/US1990/004258 patent/WO1991002000A1/en not_active Application Discontinuation
- 1990-07-30 AU AU61799/90A patent/AU6179990A/en not_active Abandoned
- 1990-07-30 JP JP2511867A patent/JPH04507250A/en active Pending
- 1990-07-30 EP EP19900912554 patent/EP0485497A4/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| EP0485497A1 (en) | 1992-05-20 |
| JPH04507250A (en) | 1992-12-17 |
| EP0485497A4 (en) | 1992-07-08 |
| AU6179990A (en) | 1991-03-11 |
| WO1991002000A1 (en) | 1991-02-21 |
| NZ234674A (en) | 1992-02-25 |
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