EP0485497A1 - Mutants de il-2 a action deletive - Google Patents

Mutants de il-2 a action deletive

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Withdrawn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)
English (en)
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/fr
Publication of EP0485497A4 publication Critical patent/EP0485497A4/en
Withdrawn legal-status Critical Current

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

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.

Landscapes

  • 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.
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

Publications (2)

Publication Number Publication Date
EP0485497A1 true EP0485497A1 (fr) 1992-05-20
EP0485497A4 EP0485497A4 (en) 1992-07-08

Family

ID=23534610

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19900912554 Withdrawn EP0485497A4 (en) 1989-08-02 1990-07-30 Il-2 deletion mutants

Country Status (6)

Country Link
EP (1) EP0485497A4 (fr)
JP (1) JPH04507250A (fr)
AU (1) AU6179990A (fr)
CA (1) CA2064696A1 (fr)
NZ (1) NZ234674A (fr)
WO (1) WO1991002000A1 (fr)

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06507900A (ja) * 1991-05-17 1994-09-08 セラゲン・インコーポレイテッド 腫瘍性細胞増殖の治療用のサイトカイン受容体を標的とした分子
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 (fr) 2010-12-22 2015-07-01 Univ Leland Stanford Junior Super-agonistes et antagonistes de l'interleukine-2
EP3049445A4 (fr) 2013-09-24 2017-10-25 Medicenna Therapeutics, Inc. Protéines hybrides de l'interleukine-2 et leurs utilisations
JP6592505B2 (ja) 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 インターロイキン−２のスーパーアンタゴニスト、パーシャルアゴニスト及びアンタゴニスト
EP3596108A4 (fr) 2017-03-15 2020-12-23 Pandion Operations, Inc. Immunotolérance ciblée
KR20200010354A (ko) 2017-05-24 2020-01-30 팬디온 테라퓨틱스, 인코포레이티드 표적화된 면역관용
CA3067909A1 (fr) 2017-06-19 2018-12-27 Medicenna Therapeutics Inc. Utilisations et procedes pour des superagonistes et agonistes d'il-2 et des fusions de ceux-ci
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 (ko) 2019-05-20 2022-03-22 팬디온 오퍼레이션스, 인코포레이티드 Madcam 표적 면역관용
WO2021168079A1 (fr) 2020-02-21 2021-08-26 Pandion Operations, Inc. Immunotolérance ciblée sur un tissu avec un effecteur cd39
EP4122951A4 (fr) * 2020-03-19 2024-05-29 Innovent Biologics (Singapore) Pte. Ltd. Mutant de l'interleukine-2 et son utilisation
EP4122952A4 (fr) * 2020-03-19 2024-05-29 Innovent Biologics (Singapore) Pte. Ltd. Mutant de l'interleukine-2 et son utilisation

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1985000817A1 (fr) * 1983-08-10 1985-02-28 Amgen Expression microbienne de l'interleukine 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' *

Also Published As

Publication number Publication date
AU6179990A (en) 1991-03-11
EP0485497A4 (en) 1992-07-08
WO1991002000A1 (fr) 1991-02-21
NZ234674A (en) 1992-02-25
CA2064696A1 (fr) 1991-02-03
JPH04507250A (ja) 1992-12-17

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