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Interleukin-2 mutants with reduced toxicity

a technology of interleukin-2 and mutants, applied in the field of interleukin-2 (il -), can solve the problems of reduced toxicity of il-2, reduced vasopermeability activity, nausea and neuropathy, etc., and achieves similar binding affinity, reduced toxicity, and reduced vasopermeability activity

Inactive Publication Date: 2005-09-15
UNIV OF SOUTHERN CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007] In accordance with the present invention, novel IL-2 mutants with reduced toxicity as compared to native IL-2 are presented. Such mutants are characterized by substantially reduced vasopermeability activity and substantially similar binding affinity for an IL-2 receptor compared to a wildtype form of the IL-2 mutant. By reducing the vasopermeability activity of the IL-2, the present invention meets the need in the art for a low toxicity variant of IL-2 that avoids toxic side effects such as vascular leak syndrome. Thus, in one aspect of the present invention, the IL-2 mutant can be used to stimulate the immune system of an animal to achieve maximal therapeutic benefit with reduced side effects.

Problems solved by technology

However the efficacy of IL-2 has been restricted by the relatively severe toxicities associated with therapeutic dosages.
The native, form of IL-2 exhibits toxic side effects that may include myocardial infarction, renal failure requiring dialysis, fluid retention, nausea and neuropathy.
In addition, IL-2 administration is associated with generalized inflammatory changes which include the development of dose limiting capillary leak syndrome.
However, such approaches cannot be used in all anatomic locations and are not applicable to disseminated disease.

Method used

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  • Interleukin-2 mutants with reduced toxicity

Examples

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example 1

Reagents

[0067] This example provides the preferred reagents for practice of the embodied invention. One skilled in the art can readily appreciate comparable materials that can be substituted in place of these reagents.

[0068] The Glutamine Synthase Gene Amplification System, including the expression plasmids pEE6 / hCMV-B and pEE 12 as well as the NSO murine myeloma expression cell line, were purchased from Lonza Biologics (Slough, UK). Restriction endonucleases, T4 DNA ligase, Vent polymerase, and other molecular biology reagents were purchased from either New England Biolabs (Beverly, Mass.) or Boehringer Mannheim (Indianapolis, Ind.). Dialysed fetal bovine serum, crude DNA from salmon testes, single-stranded DNA from calf thymus, chloramine T, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) were purchased from Sigma Chemical Co. (St. Louis, Mo.). Recombinant human interleukin-2 was purchased from Chiron (Emeryville, Calif.). The Griess Reagent Syst...

example 2

Development and Characterization of IL-2 Mutant Polypeptides

[0070] This example provides methods of creating IL-2 mutant polypeptides and chimeric antibody / IL-2 fusion proteins (mutant or native). In addition, this example provides methods for determining the cytokine function and binding properties of resultant IL-2 molecules in vitro.

[0071] A. Construction and Expression of IL-2 and Antibody / IL-2 Fusion Proteins.

[0072] The construction of the chimeric monoclonal antibody TNT-3 (chTNT-3, IgG1, κ) and the fusion protein of this antibody with IL-2 have been previously described (Hornick et al., Cancer Biotherapy &Radiopharmaceuticals 13:255, 1998; Hornick et al., J. Nucl. Med. 41:355, 2000).

[0073] IL-2 mutant cDNA was prepared by site-directed mutagenesis to mutate amino acid 20 from aspartic acid to lysine (D20K), amino acid 38 from arginine to glycine (R38G) or tryptophan (R38W), amino acid 39 from methionine to valine (M39V) or leucine (M39L), amino acid 42 from phenylalanine ...

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Abstract

Interleukin-2 (IL-2) mutants having reduced toxicity, which include full-length IL-2, truncated forms of IL-2 and forms of IL-2 that are linked to another molecule are disclosed herein. Particular substitutions within IL-2, particularly within the permeability enhancing peptide region of IL-2 achieve substantial reduction of vasopermeability activity as compared to a wildtype form of the mutant IL-2 while retaining many of the immune activating properties of IL-2. Invention IL-2 mutants can be used to stimulate the immune system of an animal and may be used in the treatment of various disorders and conditions.

Description

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS [0001] This application is a Divisional of U.S. application Ser. No. 10 / 218,197, filed Aug. 12, 2002, which is a non-Provisional of U.S. Application 60 / 312,326, filed Aug. 13, 2001, from each of which priority is claimed, and each of which is fully incorporated by reference in their entirety, including any figures, tables, and drawings.FIELD OF THE INVENTION [0002] The present invention relates to the use of interleukin-2 (IL-2) as an immunotherapeutic agent and to IL-2 mutants that exhibit reduced vasopermeability and reduced toxicity compared to native IL-2. BACKGROUND OF THE INVENTION [0003] Cytokines play a. role in the growth and differentiation of all cells in the body but are especially important to cells of the immune system. A category of cytokines are called interleukins, of which 18 have been identified thus far. Interleukin-2 (IL-2) is an important cytokine for the regulation of T-cell function in the immune system. Because ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00C12N15/09A61K38/20A61K39/39A61K45/00A61P31/18A61P35/00A61P37/00A61P37/04C07K14/55C07K16/18C07K19/00C12N15/12C12P21/02G01N33/50
CPCC07K14/55A61K38/00A61P31/18A61P35/00A61P37/00A61P37/04
Inventor EPSTEIN, ALANHU, PEISHENG
Owner UNIV OF SOUTHERN CALIFORNIA
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