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IL-2 selective agonists and antagonists

An IL-2, IL-2R technology, applied in the field of pharmacology and immunology, can solve the problem of not describing the potential of low toxicity mutant protein, not including

Inactive Publication Date: 2008-12-10
AICURIS GMBH & CO KG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these references refer to "selective agonist" IL-2 muteins, they do not describe their potential as hypotoxic muteins, but rather as potential antagonists of endogenous IL-2
[0019] EP 0267795A2 (Zurawski et al.) discloses various mouse IL-2 muteins throughout the sequence, including some mutations containing deletions and / or substitutions in the first 30 N-terminal amino acid residues that are biologically active protein, but did not include, discuss or suggest the amino acid substitutions disclosed herein at the same mouse IL-2 residue

Method used

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  • IL-2 selective agonists and antagonists
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  • IL-2 selective agonists and antagonists

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1. Production of muteins in E. coli

[0102] Essentially as described in Kunkel TA, Roberts JD and Zakour RA, "Rapid and efficient site-specific mutagenesis without phenotypic selection", (1987), Methods Enzymol 154:367-382, using Primers for the desired mutated codons were subjected to site-directed mutagenesis to generate muteins. Briefly, human IL-2 cDNA containing restriction enzyme sites Bam HI and Xba I was subcloned into the M13 phage vector M13 mp19 (New England Biolabs, Beverly, MA) using the same sites. Wild-type IL-2 cDNA was obtained using polymerase chain reaction ("PCR") from a cDNA library generated from human peripheral mRNA isolated from blood lymphocytes. The PCR primer used is the 5' end of the IL-2 open reading frame,

[0103] 5'-CCT CAA CTC CTG AAT TCA TGT ACA GGA TGC-3' (SEQ ID NO: 3);

[0104] and the 3' end of the IL-2 open reading frame,

[0105] 5'-GGA AGC GGA TCC TTA TCA AGT CAG TGT TGA G-3' (SEQ ID NO: 4).

[0106] Restriction e...

Embodiment 2

[0112] Example 2. Extraction and purification of IL-2 mutein from Escherichia coli

[0113] After 3 hours of induction, cells were collected by centrifugation at 10,000 xg. Recombinant IL-2 muteins were first refolded and purified by dispersing cells in 10 volumes (volume / wet weight) of sucrose / Tris / EDTA buffer (0.375M sucrose, 10 mM Tris / HCl pH 8.0, 1 mM EDTA). The dispersed cells were sonicated 3 times at 300W with 30 second rest intervals on an ice bath using a Missonix XL2020 model sonicator equipped with a 1 inch standard probe. The sonicated material was then centrifuged at 17,000 xg, 4°C for 20 minutes. The pellet, which should be white at this point, is washed by resuspending and centrifuging once in sucrose / Tris / EDTA buffer, resuspending and centrifuging twice in Tris / EDTA buffer (50 mM Tris / HCL pH 8.0, 1 mM EDTA) , finally resuspended in 10 volumes of 0.1M Tris / HCL, pH 8.0 buffer (at which time a sample was taken for gel analysis) and centrifuged at 17,000 xg for 2...

Embodiment 3

[0115] Example 3. T cell proliferation analysis

[0116] Peripheral blood mononuclear cells (PBMC) were isolated from approximately 100 mL of normal human blood (Irwin Memorial Blood Bank, San Francisco, CA) in cold Dulbecco's phosphate-buffered saline (Ca 2+ and Mg 2+ free; DPBS) at a 1:2 dilution. Ficoll-Paque (Pharmacia) was placed in the lower layer, and the samples were centrifuged to separate PBMCs, followed by extensive washing in cold DPBS. PHA blasts (activated T cells) were generated by the following method: 1×10 6 Cells / ml density Cells were resuspended in RPMI 1640 containing 10% fetal bovine serum (Hyclone) supplemented with 1% (w / v) of the following: L-glutamine, non-essential amino acids, sodium pyruvate and Antibiotic-antimycotic (RPMI medium). Phytohemagglutinin (PHA-P; Sigma) was added at a final concentration of 10 μg / mL, and the cells were incubated at 37°C, 5% CO 2 Cultured for 3 days. Cells were harvested and washed twice in DPBS, resuspended in RPM...

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Abstract

The invention is directed to a polypeptide comprising a human IL-2 mutein numbered in accordance with wild-type IL-2 wherein said human IL-2 is substituted at at least one of positions 20, 88 or 126, whereby said mutein preferentially activates T cells over NK cells. D20H and I, N88G, I, and R, in particular have a relative T cell-differential activity much greater than native IL-2, with predicted associated reduced in vivo toxicity. The invention also includes polynucleotides coding for the muteins of the invention, vectors containing the polynucleotides, transformed host cells, pharmaceutical compositions comprising the muteins, and therapeutic methods of treatment.

Description

[0001] This application is a Chinese patent application (national application number is No.99808650.9, international application number is PCT / US99 / 10643) divisional application. [0002] Cross References to Related Applications [0003] This patent is a continuation-in-part of US Serial No. 09 / 080,080, filed May 15,1998. 1. Technical field [0004] The present invention relates generally to the fields of pharmacology and immunology. More specifically, the present invention relates to novel compositions that selectively activate T cells (PHA-blasts) while reducing the activation of natural killer cells ("NK"). The novel compositions include variants of a family of cytokines, particularly human interleukin-2 ("IL-2"). 2. Background technology [0005] Interleukin 2 (IL-2) is a potent immunostimulant that activates different cells of the immune system, including T cells, B cells and monocytes. IL-2 is also a potent and critical T cell growth factor. With these activities...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N33/50A61K38/00A61K38/20A61P35/00A61P37/00C07K14/55C12N15/09C12N15/26G01N33/15
CPCA61K38/00C07K14/55A61P31/00A61P31/18A61P35/00A61P37/00A61P37/04
Inventor A·B·沙纳菲尔特J·M·格雷维G·杰斯莫克K·J·伦巴赫G·D·韦策尔
Owner AICURIS GMBH & CO KG
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