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Novel immunogenic mimetics of multimer proteins

An immunogenic and polyprotein technology, applied in the direction of antibody mimics/scaffolds, animal/human proteins, immunoglobulins, etc., can solve the problems of protein sequence stability, poor storage period and in vivo stability

Inactive Publication Date: 2005-05-11
PHARMEXA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In addition, the stability of genetically modified protein sequences may be less than optimal (in terms of both shelf life and in vivo stability)

Method used

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  • Novel immunogenic mimetics of multimer proteins
  • Novel immunogenic mimetics of multimer proteins
  • Novel immunogenic mimetics of multimer proteins

Examples

Experimental program
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Effect test

Embodiment 1

[0241] Design of Four New Diepitope (P2+P30) Monomeric IL5 Constructs

[0242] IL5 is an antiparallel homodimer in which the C-terminal and N-terminal ends of the monomer are closely located in the molecule. This makes it possible to link the two monomers into a single monomer that closely resembles the quaternary structure of the wild-type dimer.

[0243] We have approached this using the p2 / P30 epitope as a linker or by inserting a diglycine linker as previously described in Li et al. 1997, PNAS USA 94(13):6694-9.

[0244] Native hIL5 encoding DNA molecules used for all research work were purchased from R&D systems (BBG16). This DNA sequence did not include the hIL5 leader sequence; therefore a synthetic DNA sequence encoding the native hIL5 leader peptide was added. Sequences encoding P2 and P30 T cell epitopes were derived from tetanus toxoid. These sequences were inserted into the native sequence of the gene, thereby providing immunogenic variants of IL5. The reading ...

Embodiment 2

[0248] hIL5.34 and hIL5.35

[0249] To make the T-cell epitope internal to the molecule, the P2 and P30 head-to-tail were inserted as a linker between the two IL5 monomers, thus generating two constructs hIL5-P30-P2-hIL5 (hIL5.34, in SEQ ID NO : mature peptide in 5 and 6) and hIL5-P2-P30-hIL5 (hIL5.35, mature peptide in SEQ ID NO: 7 and 8) - both DNA constructs encode the native IL5 leader sequence, resulting in 266 The mature expression product of amino acids. The translation products resulting from these designs are intended to fold into "monomeric IL5 dimers", ie monomeric molecules with a tertiary structure that closely resembles the full 3-dimensional structure of dimeric IL5.

Embodiment 3

[0251] hIL5.36 & hIL5.37

[0252] Similar but immunogenic constructs incorporating T-cell epitopes were designed based on the previous successful production of biologically active monomeric "IL5 dimer mimics" by J. Li et al. by insertion of a diglycine linker. Variant hIL5.36 thus has the structure of the mature peptide in SEQ ID NO:9 and 10 and variant hIL5.37 has the structure of the mature peptide in SEQ ID NO:11 and 12. Both constructs encode the native IL5 leader sequence, followed by the first 4 amino acids in IL5, which are followed by the first inserted epitope - the other epitope is at the C-terminus.

[0253] There are 2 main reasons why the N-terminal epitope is not located N-terminal to the full IL5 sequence in these two constructs rather than to preserve the hIL5 sequence. By using the native hIL5 leader peptide together with the N-terminus of hIL5, we ensured that the leader peptide was cleaved correctly. Also, since the N-terminus of IL5 constitutes a flexible...

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Abstract

The present invention relates to novel immunogenic variants of multi-subunit proteins such as interleukin 5 (IL5) and tumor necrosis factor alpha (TNF, TNF alpha). In addition to being immunogenic in the autologous host, the variants also highly resemble the native 3D structure of the protein from which they were derived. Certain variants are monomeric mimics of multimers in which a peptide linker (containing an inert or T helper epitope) ensures spatial organization of the monomeric unit that facilitates correct folding. One subtype of variant is the monomeric TNFα variant, which displays a superior ability to assemble into multimers with a high structural similarity to the native protein. Methods of processing and producing variants as well as DNA fragments, vectors and host cells are also disclosed.

Description

field of invention [0001] The present invention relates to the field of therapeutic immunotherapy, in particular active immunotherapy targeting the downregulation of autologous ("self") proteins and other weakly immunogenic antigens. The present invention thus provides new and improved immunogenic variants of multi-subunit proteins as well as the necessary tools for making such variants. The invention additionally relates to methods of immunotherapy and compositions effective in such methods. Background of the invention [0002] Active immunotherapy ("vaccination") has received increasing attention in the last 20+ years as a means of treating or ameliorating disease. Remarkably, active immunotherapy has been known since the late 1970s when the first experiments using anti-fertilization vaccines were reported as a way to destroy resistance to self-proteins that are somehow associated with pathological (or otherwise undesirable) physiological disease. The application of rece...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/74A61K35/76A61K39/00A61K39/39A61K47/36C12N15/09A61K48/00C07K14/525C07K14/54C07K14/74C07K16/24
CPCA61K39/00C07K14/525C07K14/54C07K14/5409C07K16/244C07K2319/00Y02A50/30C12N15/11
Inventor 斯腾·克雷斯内尔芬恩·斯陶舒姆·尼尔森托马斯·布拉特比耶恩·沃尔博格索尔恩·穆里特森
Owner PHARMEXA
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