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Stabilized virus-like particles and epitope display systems

A virus and epitope technology, applied in the interdisciplinary field of immunology and peptide engineering, can solve problems such as immune interference

Inactive Publication Date: 2008-11-19
CELLDEX THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first potential problem is inadvertent transfer of nucleic acid from the chimeric vaccine to the vaccinated host
The second potential problem is the interference of pre-existing immunity to HB
A third potential issue concerns the repeated preparation requirements of intact chimeric particles that can also withstand long-term storage
Although this region of the M2 protein was found to be useful as an antigen in various recombinant influenza vaccines when bound to VLPs or self-associated peptides [see e.g. Neirynck et al., (1999) Nat. Med., 5(10): 1157-1163 and WO 99 / 07839, and WO 02 / 74795, De Filette et al. and their citations], but have not previously been reported to be beneficial for stabilizing VLPs, and rather surprisingly, this stabilization does not require the corresponding position between positions 17 and 19 disulfide bonds

Method used

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  • Stabilized virus-like particles and epitope display systems
  • Stabilized virus-like particles and epitope display systems
  • Stabilized virus-like particles and epitope display systems

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0289] Example 1: Construction of stabilized HBc polypeptide VLP with M2 ectodomain

[0290] To illustrate the ability of the N-terminal region of the influenza virus M2 protein to non-covalently stabilize hepatitis B virus core-like particles, the extracellular domain of M2e was fused to the C-terminus of a C-terminally truncated HBc polypeptide. To ensure that no intrinsic cysteine ​​residues contributed to the stabilization, a version of M2 (positions 2-24) in which cysteines were mutated to serines was used (CV-1895, V7.M2e (2C > 2S). The expression vector V7.M2e (2C>2S) was constructed, and a pair of oligonucleotides were annealed and inserted into the expression vector V7 (see below), and the vector V7 accepted the insertion after the amino acid V149 of the HB capsid polypeptide gene. This is shown in Fig. 2 is shown schematically.

[0291] Therefore, a new vector was constructed to enable the fusion of the self-binding peptide to the C-terminus of the HBc chimera. Uni...

Embodiment 2

[0305] Example 2: Analysis of Stabilized HB Capsid Polypeptide VLP

[0306] After expression and purification of the particles, their ability to maintain particle-like morphology was analyzed by analytical size-exclusion chromatography. When analyzed in this manner, non-C-terminally stabilized particles (eg, CV-1048) were present as a mixture of particulate and non-particulate material.

[0307] Similar analysis of the CV-1895 particles revealed that they elute as a uniform particle structure ( Figure 4 ). These data suggest that the M2e(2-24, C17S, C19S) sequence, when present, fuses to the C-terminus of HBc, forming an intermolecular interaction that stabilizes the granular structure. These data further suggest that intermolecular stabilization occurs in the absence of cysteine ​​residues at positions 17 and 19 of M2.

Embodiment 3

[0308] Example 3: Construction of stabilized HBc polypeptide VLP with GCN4-p1 leucine zipper

[0309] In order to study the ability of the leucine zipper domain of the yeast GCN4 transcriptional regulator to stabilize hepatitis B capsid polypeptide particles in a non-covalent manner, a protein derived from GCN4 that can simultaneously form dimers and trimers in solution The modified leucine zipper GNC4-VL of the body was fused to the C-terminus of the C-terminal truncated HBc particle by genetic engineering. According to the method for constructing the expression vector in Example 1, the synthetic oligonucleotide encoding GCN4-VL was annealed and inserted into the expression vector V7, and the vector V7 was inserted after amino acid V149 of the HB capsid polypeptide gene. The peptide linked to the HB capsid in this way has the following sequence:

[0310] RVKQLEDKVEELLSKVYHLENEVARLKKLVGER

[0311] SEQ ID NO: 282

[0312] The obtained particles are more s...

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Abstract

A chimeric polypeptide is disclosed that comprises: i) a first portion that self-assembles into an organized, repetitive, supramolecular structure that contains at least about 9 subunits, covalently linked to ii) a second polypeptide portion that comprises a peptide having a length of about 15 to about 80 amino acid residues. The second portion peptide self-assembles to form parallel multimers. A contemplated chimeric polypeptide forms a particle that is more stable than is a particle formed from a first polypeptide that is otherwise identical in sequence, but lacks the covalently linked self-binding peptide sequence of the second polypeptide portion.

Description

technical field [0001] The present invention relates to the cross field of immunology and polypeptide engineering, in particular to genetically engineered chimeric polypeptide and its supramolecular assembly, which are used to reduce nucleic acid binding, enhance stability and display immunogenic epitopes. Background of the invention [0002] Although virus particles are usually composed of one or more different polypeptides, they can trigger a stronger immune response than their isolated components. For B cell responses, it is known that an important factor in the immunogenicity of viral particles may be the repetition and order of surface epitopes. The surface of most virus particles contains polypeptides in a neat, symmetrical quasi-crystalline form, displaying regularly arranged epitopes, thereby allowing sufficient cross-linking of epitope-specific immunoglobulins of B cells [Bachmann et al., (1996) Immunol. Today 17:553-558]. This surface crosslinking of B cell immun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00C07K14/00
CPCC12N7/00A61K2039/5256A61K2039/6075C07K2319/40C07K2319/73C12N2730/10123C12N2760/16123
Inventor A·J·伯克特
Owner CELLDEX THERAPEUTICS INC