EP0485497A1 - Il-2 deletion mutants - Google Patents

Il-2 deletion mutants

Info

Publication number
EP0485497A1
EP0485497A1 EP19900912554 EP90912554A EP0485497A1 EP 0485497 A1 EP0485497 A1 EP 0485497A1 EP 19900912554 EP19900912554 EP 19900912554 EP 90912554 A EP90912554 A EP 90912554A EP 0485497 A1 EP0485497 A1 EP 0485497A1
Authority
EP
European Patent Office
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.)
Withdrawn
Application number
EP19900912554
Other languages
German (de)
French (fr)
Other versions
EP0485497A4 (en
Inventor
Francis S. Genbauffe, Jr.
Donna E. Akiyoshi
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.)
Seragen Inc
Original Assignee
Seragen Inc
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 Seragen Inc filed Critical Seragen Inc
Publication of EP0485497A1 publication Critical patent/EP0485497A1/en
Publication of EP0485497A4 publication Critical patent/EP0485497A4/en
Withdrawn legal-status Critical Current

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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

Definitions

  • This invention relates to the use of
  • 11-2 is a protein secreted by human
  • T-lymphocytes which is capable of binding to IL-2 receptors on activated T-lymphocytes and effecting
  • IL-2 has been shown to be a therapeutic immunostimulant in humans (Rosenberg, 1988, Immunology Today 9:2: 58-62), and IL-2 or a specific binding portion thereof can be coupled to the
  • IL-2 encoding DNA sequences are reported in a 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.
  • 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
  • lysine 76 is a proteolytic site in the IL-2 molecule (Cohen et al., 1986, Science 234:349). These mutants either delete this proteolytic site completely, or alter the structure 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, can be used to treat immune and other disorders
  • the invention thus generally features eight new mutant IL-2 polypeptides capable 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; 75, 78; and 79 (according to the numbering convention of the Figure, taken from Williams et al., Nucleic Acids Res., vol. 16, no. 22 (1988).
  • 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
  • a toxin portion e.g., derived from diphtheria toxin
  • diphtheria toxin portion is large enough to exhibit cytotoxic activity and small enough to fail to exhibit generalized eukaryotic cell binding.
  • the DNA sequence encoding the IL-2 polypeptide contains nucleotide substitutions designed to maximize gene expression in the cells used for expression; i.e., where prokaryotic cells such as E.
  • 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, and systemic lupus erythmatosis (SLE), or to prevent an immune response by IL-2 receptor bearing T cells that occurs in graft rejection.
  • 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
  • fragment A catalyzes the ADP-ribosylation of elongation factor 2, resulting in inhibition of protein synthesis and subsequent death of the IL-2-receptor bearing cell.
  • the Figure is a DNA sequence, encoding IL-2, in which preferred prokaryotic translation codons are employed; the numbers correspond to the numbering referred to in this specification.
  • Amino acids 74 through 79 are contained within the Xbal/Notl fragment of the synthetic IL-2 gene (see Figure). For each of the eight deletion mutants, an
  • Xbal/Notl fragment with a deletion 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 1/2 Xbal site at the 5' end and a 1/2 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 into the expression plasmid, pDWl5 (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
  • the genes encoding the IL-2/diphtheria toxin fusion proteins are constructed by standard recombinant DNA techniques, as follows.
  • the IL-2 portion of the fusion gene is contained within the Sphl/Hindlll
  • 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 into a suitable E. coli host and plated onto Luria broth plates plus an appropriate antibiotic for selection, according to conventional techniques. Transformants are screened by Ddel restriction digest analysis of
  • Proteins are electroblotted onto nylon membrane and iiranunoblot 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 by comparison of the size of 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.
  • C91/P1 cells (a high-affinity IL2 receptor-bearing cell line) were seeded in 96-well V-bottom plates (Nunc, Roskilde, Denmark) at a concentration of 10 5 per well in 100 ⁇ l complete medium.
  • Il-2-toxin was added at varying concentrations (10 -12 M to 10 -6 M) in complete
  • IC 50 refers to the concentration of IL2 required to inhibit protein synthesis to 50% of the untreated control.
  • deletion mutant IL-2 molecules can be used alone, in addition to their use in toxic hybrids, the deletions can advantagously provide
  • toxins other than diphtheria toxin can be coupled to the mutants, e.g., the enzymatically active portion of Pseudomonas exotoxin can be used.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Molécule mutante de IL-2 susceptible de lier une cellule porteuse d'un récepteur de IL-2, possédant une capacité délétive d'un à cinq résidus aminoacides de IL-2, cette délétion produisant des molécules actives de IL-2 qui possèdent une résistance accrue à la protéolyse.Mutant IL-2 molecule capable of binding a cell carrying an IL-2 receptor, having a deletion capacity of one to five amino acid residues of IL-2, this deletion producing active IL-2 molecules which have a increased resistance to proteolysis.

Description

IL-2 DELETION MUTANTS
Background 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.
11-2 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-2 has been shown to be a therapeutic immunostimulant in humans (Rosenberg, 1988, Immunology Today 9:2: 58-62), 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., Transplantation 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 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 sequence that differs from 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 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., 1988, 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 site in the IL-2 molecule (Cohen et al., 1986, Science 234:349). These mutants either delete this proteolytic site completely, or alter the structure 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, can 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 capable 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; 75, 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 enough 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 expression; 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, and systemic lupus erythmatosis (SLE), or to prevent an immune response by IL-2 receptor bearing T cells that occurs in graft rejection. 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 the endosome causes a conformational change in the toxin, allowing its membrane-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 subsequent 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.
Description of the Preferred Embodiments The drawing is first described.
Drawing
The Figure is a DNA sequence, encoding IL-2, in which preferred prokaryotic translation codons are employed; the numbers correspond to the numbering referred to in this specification.
Construction of the Genes Encoding IL-2 Deletion
Mutants/Toxin
Amino acids 74 through 79 are contained within the Xbal/Notl fragment of the synthetic IL-2 gene (see Figure). For each of the eight deletion mutants, an
Xbal/Notl fragment with a deletion 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 1/2 Xbal site at the 5' end and a 1/2 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 into the expression plasmid, pDWl5 (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 of the IL-2 deletion mutants are confirmed by the dideoxy method of Sanger et al. (1977, Proc. Nat. Acad. Sci., 74:5463).
The genes encoding the IL-2/diphtheria toxin fusion proteins are constructed by standard recombinant DNA techniques, as follows. The IL-2 portion of the fusion gene is contained within the Sphl/Hindlll
fragment of the IL-2 deletion mutant derived from pDW15. This DNA fragment is ligated to Sphl/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 into a suitable E. coli host and plated onto Luria broth plates plus an appropriate antibiotic for selection, according to conventional techniques. Transformants are screened by Ddel restriction digest analysis of
minilysate DNA and by Western blot analy is, as follows. Western Blot Analysis
Total bacterial cell lysates are analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970, Nature 227:680) for the production of IL-2/toxin
protein. Proteins are electroblotted onto nylon membrane and iiranunoblot 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 by comparison of the size of 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.
Cytotoxicity assay
Referring to Table II, C91/P1 cells (a high-affinity IL2 receptor-bearing cell line) were seeded in 96-well V-bottom plates (Nunc, Roskilde, Denmark) at a concentration of 105 per well in 100 μl complete medium. Il-2-toxin was added at varying concentrations (10 -12M to 10-6M) in complete
medium. Cells cultured with medium alone were included as the control. Following 18 hours incubation at 37°C in a 5% Co2 atmosphere, the plates were centrifuged for 5 minutes at 170 × g, the medium was removed and replaced with 100 μl leucine-free medium (DMEM
Selectamine, Gibco) containing 2.5 μCi/ml
[14C]-leucinec (New England Nuclear, Boston, MA).
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. T
Table I I
Plasmid amino acid(s)
deleted C91/PL IC50 psI133 Փ74 6×10-1M psI134 Փ75 1×10-10M
PsI136 Փ78 5×10-11M psI137 Փ79 2×10-10M psI143 Փ75-77 2X10-10M psI141 Փ74-78 1×10-10M psI145 Փ76-78 2×10-10M psI150 Փ76-79 7×1011M
(psI129 no deletion typically control 5×10-11M)
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 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

Claims 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.
EP19900912554 1989-08-02 1990-07-30 Il-2 deletion mutants Withdrawn EP0485497A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US38855789A 1989-08-02 1989-08-02
US388557 1995-02-14

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EP0485497A1 true EP0485497A1 (en) 1992-05-20
EP0485497A4 EP0485497A4 (en) 1992-07-08

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JP (1) JPH04507250A (en)
AU (1) AU6179990A (en)
CA (1) CA2064696A1 (en)
NZ (1) NZ234674A (en)
WO (1) WO1991002000A1 (en)

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06507900A (en) * 1991-05-17 1994-09-08 セラゲン・インコーポレイテッド Molecules targeting cytokine receptors for the treatment of neoplastic cell proliferation
US6146850A (en) * 1991-11-04 2000-11-14 Xoma Corporation Proteins encoding gelonin sequences
US5621083A (en) 1991-11-04 1997-04-15 Xoma Corporation Immunotoxins comprising ribosome-inactivating proteins
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
US20110104186A1 (en) 2004-06-24 2011-05-05 Nicholas Valiante Small molecule immunopotentiators and assays for their detection
EP2655409A4 (en) 2010-12-22 2015-07-01 Univ Leland Stanford Junior Superagonists and antagonists of interleukin-2
EP3049445A4 (en) 2013-09-24 2017-10-25 Medicenna Therapeutics, Inc. Interleukin-2 fusion proteins and uses thereof
JP6592505B2 (en) 2014-04-24 2019-10-16 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー Interleukin-2 super antagonist, partial agonist and antagonist
EP3596108A4 (en) 2017-03-15 2020-12-23 Pandion Operations, Inc. Targeted immunotolerance
KR20200010354A (en) 2017-05-24 2020-01-30 팬디온 테라퓨틱스, 인코포레이티드 Targeted Immune Tolerance
CA3067909A1 (en) 2017-06-19 2018-12-27 Medicenna Therapeutics Inc. Uses and methods for il-2 superagonists, agonists, and fusions thereof
US10174092B1 (en) 2017-12-06 2019-01-08 Pandion Therapeutics, Inc. IL-2 muteins
US10946068B2 (en) 2017-12-06 2021-03-16 Pandion Operations, Inc. IL-2 muteins and uses thereof
US20210213102A1 (en) * 2018-09-21 2021-07-15 Innovent Biologics (Suzhou) Co., Ltd. Novel interleukin-2 and use thereof
KR20220035333A (en) 2019-05-20 2022-03-22 팬디온 오퍼레이션스, 인코포레이티드 MADCAM Targeted Immune Tolerance
WO2021168079A1 (en) 2020-02-21 2021-08-26 Pandion Operations, Inc. Tissue targeted immunotolerance with a cd39 effector
EP4122951A4 (en) * 2020-03-19 2024-05-29 Innovent Biologics (Singapore) Pte. Ltd. Interleukin-2 mutant and use thereof
EP4122952A4 (en) * 2020-03-19 2024-05-29 Innovent Biologics (Singapore) Pte. Ltd. Interleukin-2 mutant and use thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1985000817A1 (en) * 1983-08-10 1985-02-28 Amgen Microbial expression of interleukin ii

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PROTEIN ENGINEERING. vol. 1, no. 6, 1987, EYNSHAM, OXFORD, ENGLAND GB pages 493 - 498; D. P. WILLIAMS ET AL: 'Diphtheria toxin receptor binding domain substitution with interleukin-2: genetic construction and properties of a diphtheria toxin-related interleukin-2 fusion protein' *
See also references of WO9102000A1 *
TRANSPLANTATION vol. 47, no. 2, February 1989, BALTIMORE pages 318 - 322; O. PANKEWYCZ ET AL: 'Interleukin-2-diphteria toxin fusion protein prolongs murine islet cell engraftment' *

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JPH04507250A (en) 1992-12-17

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