WO1991002000A1 - Mutants de il-2 a action deletive - Google Patents

Mutants de il-2 a action deletive Download PDF

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Publication number
WO1991002000A1
WO1991002000A1 PCT/US1990/004258 US9004258W WO9102000A1 WO 1991002000 A1 WO1991002000 A1 WO 1991002000A1 US 9004258 W US9004258 W US 9004258W WO 9102000 A1 WO9102000 A1 WO 9102000A1
Authority
WO
WIPO (PCT)
Prior art keywords
molecule
mutant
amino acid
deleted
toxin
Prior art date
Application number
PCT/US1990/004258
Other languages
English (en)
Inventor
Francis S. Genbauffe, Jr.
Donna E. Akiyoshi
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 WO1991002000A1 publication Critical patent/WO1991002000A1/fr

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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.
PCT/US1990/004258 1989-08-02 1990-07-30 Mutants de il-2 a action deletive WO1991002000A1 (fr)

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
WO1991002000A1 true WO1991002000A1 (fr) 1991-02-21

Family

ID=23534610

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1990/004258 WO1991002000A1 (fr) 1989-08-02 1990-07-30 Mutants de il-2 a action deletive

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)

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0584251A1 (fr) * 1991-05-17 1994-03-02 Seragen, Inc. Molecules ciblees sur un recepteur de cytokine par le traitement de la croissance de cellules neoplasiques
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
WO2000004048A1 (fr) * 1998-07-16 2000-01-27 Institut Pasteur Peptides d'yl-2 et leurs derives et leur utilisation comme agents therapeutiques
US6146850A (en) * 1991-11-04 2000-11-14 Xoma Corporation Proteins encoding gelonin sequences
EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection
US20160229901A1 (en) * 2013-09-24 2016-08-11 Medicenna Therapeutics Pte Ltd Interleukin-2 fusion proteins and uses thereof
US10174092B1 (en) 2017-12-06 2019-01-08 Pandion Therapeutics, Inc. IL-2 muteins
WO2020057645A1 (fr) * 2018-09-21 2020-03-26 信达生物制药(苏州)有限公司 Nouvelle interleukine 2 et son utilisation
US10676516B2 (en) 2017-05-24 2020-06-09 Pandion Therapeutics, Inc. Targeted immunotolerance
US10946068B2 (en) 2017-12-06 2021-03-16 Pandion Operations, Inc. IL-2 muteins and uses thereof
US10961310B2 (en) 2017-03-15 2021-03-30 Pandion Operations, Inc. Targeted immunotolerance
WO2021185362A1 (fr) * 2020-03-19 2021-09-23 信达生物制药(苏州)有限公司 Mutant de l'interleukine-2 et son utilisation
WO2021185361A1 (fr) * 2020-03-19 2021-09-23 信达生物制药(苏州)有限公司 Mutant de l'interleukine-2 et son utilisation
US11384131B2 (en) 2014-04-24 2022-07-12 The Board Of Trustees Of The Leland Stanford Junior University Superagonists, partial agonists and antagonists of interleukin-2
US11542312B2 (en) 2017-06-19 2023-01-03 Medicenna Therapeutics, Inc. IL-2 superagonists in combination with anti-PD-1 antibodies
US11739146B2 (en) 2019-05-20 2023-08-29 Pandion Operations, Inc. MAdCAM targeted immunotolerance
US11981715B2 (en) 2020-02-21 2024-05-14 Pandion Operations, Inc. Tissue targeted immunotolerance with a CD39 effector
US12006347B2 (en) 2021-09-09 2024-06-11 The Board Of Trustees Of The Leland Stanford Junior University Superagonists and antagonists of interleukin-2

Citations (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

Patent Citations (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 (6)

* Cited by examiner, † Cited by third party
Title
Gene, Vol. 34, issued 1985, WELLS et al "Cassette Mutagenesis: An efficient Method for Generation of Multiple Mutations at Defined Sites", pages 315-23, see entire document. *
Nucleic Acids Research, Vol. 10, No. 20, issued 1982, ZOLLER et al, "Oligonucleotide-directed Mutagenesis Using M13-derived Vectors: an efficient and General Procedure for the Production of Point Mutations in any Fragment of DNA", pages 6487-6500, see entire document. *
Science, Vol. 234, issued 17 October 1986, COHEN et al "Structure-Activity Studies of Interleukin-2", pages 349-51, see page 351. *
Science, Vol. 238, issued 18 December 1987, BRANDHUBER et al, "Three Dimensional Structure of Interleukin-2", pages 1707-09, see entire document. *
See also references of EP0485497A4 *
The Journal of Biological Chemistry, Vol. 262, No. 12, issued 25 April 1987, JU et al, "Structure-Function Analysis of Human Interleukin-2", pages 5723-31, see entire document. *

Cited By (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0584251A1 (fr) * 1991-05-17 1994-03-02 Seragen, Inc. Molecules ciblees sur un recepteur de cytokine par le traitement de la croissance de cellules neoplasiques
EP0584251A4 (fr) * 1991-05-17 1995-08-02 Seragen Inc Molecules ciblees sur un recepteur de cytokine par le traitement de la croissance de cellules neoplasiques.
US6649742B1 (en) 1991-11-04 2003-11-18 Xoma Technology Ltd. Immunotoxins comprising ribosome-inactivating proteins
US5744580A (en) * 1991-11-04 1998-04-28 Xoma Corporation Immunotoxins comprising ribosome-inactivating proteins
US5756699A (en) * 1991-11-04 1998-05-26 Xoma Corporation Immunotoxins comprising ribosome-inactivating proteins
US5837491A (en) * 1991-11-04 1998-11-17 Xoma Corporation Polynucleotides encoding gelonin sequences
US5621083A (en) * 1991-11-04 1997-04-15 Xoma Corporation Immunotoxins comprising ribosome-inactivating proteins
US6146631A (en) * 1991-11-04 2000-11-14 Xoma Corporation Immunotoxins comprising ribosome-inactivating proteins
US6146850A (en) * 1991-11-04 2000-11-14 Xoma Corporation Proteins encoding gelonin sequences
US6376217B1 (en) 1991-11-04 2002-04-23 Xoma Technology Ltd. Fusion proteins and polynucleotides encoding gelonin sequences
US6825334B1 (en) 1998-07-16 2004-11-30 Institut Pasteur Peptides of IL-2 and derivatives thereof and their use as therapeutic agents
WO2000004048A1 (fr) * 1998-07-16 2000-01-27 Institut Pasteur Peptides d'yl-2 et leurs derives et leur utilisation comme agents therapeutiques
EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection
US20160229901A1 (en) * 2013-09-24 2016-08-11 Medicenna Therapeutics Pte Ltd Interleukin-2 fusion proteins and uses thereof
US11680090B2 (en) 2013-09-24 2023-06-20 Medicenna Therapeutics, Inc. Interleukin-2 fusion proteins and uses thereof
US10781242B2 (en) * 2013-09-24 2020-09-22 Medicenna Therapeutics Inc. Interleukin-2 fusion proteins and uses thereof
US11384131B2 (en) 2014-04-24 2022-07-12 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
US11466068B2 (en) 2017-05-24 2022-10-11 Pandion Operations, Inc. Targeted immunotolerance
US10676516B2 (en) 2017-05-24 2020-06-09 Pandion Therapeutics, Inc. Targeted immunotolerance
US11542312B2 (en) 2017-06-19 2023-01-03 Medicenna Therapeutics, Inc. IL-2 superagonists in combination with anti-PD-1 antibodies
US10946068B2 (en) 2017-12-06 2021-03-16 Pandion Operations, Inc. IL-2 muteins and uses thereof
US10174091B1 (en) 2017-12-06 2019-01-08 Pandion Therapeutics, Inc. IL-2 muteins
US11091526B2 (en) 2017-12-06 2021-08-17 Pandion Operations, Inc. IL-2 muteins and uses thereof
US11965008B2 (en) 2017-12-06 2024-04-23 Pandion Operations, Inc. IL-2 muteins and uses thereof
US11945852B2 (en) 2017-12-06 2024-04-02 Pandion Operations, Inc. IL-2 muteins and uses thereof
US11091527B2 (en) 2017-12-06 2021-08-17 Pandion Operations, Inc. IL-2 muteins and uses thereof
US11779632B2 (en) 2017-12-06 2023-10-10 Pandion Operation, Inc. IL-2 muteins and uses thereof
US10174092B1 (en) 2017-12-06 2019-01-08 Pandion Therapeutics, Inc. IL-2 muteins
AU2019344875B2 (en) * 2018-09-21 2021-12-23 Innovent Biologics (Suzhou) Co., Ltd. Novel interleukin 2 and use thereof
TWI801664B (zh) * 2018-09-21 2023-05-11 大陸商信達生物製藥（蘇州）有限公司 新型白介素２及其用途
WO2020057645A1 (fr) * 2018-09-21 2020-03-26 信达生物制药(苏州)有限公司 Nouvelle interleukine 2 et son utilisation
CN112105634A (zh) * 2018-09-21 2020-12-18 信达生物制药(苏州)有限公司 新型白介素2及其用途
CN112105634B (zh) * 2018-09-21 2024-03-12 信达生物制药(苏州)有限公司 新型白介素2及其用途
US11739146B2 (en) 2019-05-20 2023-08-29 Pandion Operations, Inc. MAdCAM targeted immunotolerance
US11981715B2 (en) 2020-02-21 2024-05-14 Pandion Operations, Inc. Tissue targeted immunotolerance with a CD39 effector
TWI790573B (zh) * 2020-03-19 2023-01-21 大陸商信達生物製藥（蘇州）有限公司 白介素２突變體及其用途
WO2021185361A1 (fr) * 2020-03-19 2021-09-23 信达生物制药(苏州)有限公司 Mutant de l'interleukine-2 et son utilisation
WO2021185362A1 (fr) * 2020-03-19 2021-09-23 信达生物制药(苏州)有限公司 Mutant de l'interleukine-2 et son utilisation
US12006347B2 (en) 2021-09-09 2024-06-11 The Board Of Trustees Of The Leland Stanford Junior University Superagonists and antagonists of interleukin-2

Also Published As

Publication number Publication date
JPH04507250A (ja) 1992-12-17
EP0485497A4 (en) 1992-07-08
AU6179990A (en) 1991-03-11
CA2064696A1 (fr) 1991-02-03
EP0485497A1 (fr) 1992-05-20
NZ234674A (en) 1992-02-25

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