Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-08-28
2004-08-03
Nguyen, Dave Trong (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S091400, C435S320100, C435S455000
Reexamination Certificate
active
06770631
ABSTRACT:
This invention relates to novel genetic constructs designed to permit expression or synthesis multidomain proteins containing extended repetitive sequences, particularly those useful for the creation of molecular adjuvants and immunogens.
The complement system consists of a set of serum proteins that are important in the response of the immune system to foreign antigens. The complement system becomes activated when its primary components are cleaved and the products, alone or with other proteins, activate additional complement proteins resulting in a proteolytic cascade. Activation of the complement system leads to a variety of responses including increased vascular permeability, chemotaxis of phagocytic cells, activation of inflammatory cells, opsonisation of foreign particles, direct killing of cells and tissue damage. Activation of the complement system may be triggered by antigen-antibody complexes (the classical pathway) or a normal slow activation may be amplified in the presence of cell walls of invading organisms such as bacteria and viruses (the alternative pathway). The complement system interacts with the cellular immune system through a specific pathway involving C3, a protein central to both classical and alternative pathways. The proteolytic activation of C3 gives rise to a large fragment (C3b and exposes a chemically reactive internal thiolester linkage which can react covalently with external nucleophiles such at the cell surface proteins of invading organisms or foreign cells. As a result, the potential antigen is ‘tagged’ with C3b and remains attached to that protein as it undergoes further proteolysis to iC3b and C3d,g. The latter fragments are, respectively, ligands for the complement receptors CR3 and CR2. Thus the labelling of antigen by C3b can result in a targeting mechanism for cells of the immune system bearing these receptors.
That such targeting is important for augmentation of the immune response is first shown by experiments in which mice were depleted of circulating C3 and then challenged with an antigen (sheep erythrocytes). Removal of C3 reduced the antibody response to this antigen. (M. B. Pepys, J.Exp.Med, 140, 126-145, 1974). The role of C3 was confirmed by studies in animals genetically deficient in either C3 or the upstream components of the complement cascade which generate C3b, i.e. C2 and C4, (J. M. Ahearn & D. T. Fearon, Adv.Immunol. 46, 183-219, 1989). More recently, it has been shown that linear conjugation of a model antigen with more than two copies of the murine C3d fragment sequence resulted in a very large (1000-10000-fold) increase in antibody response in mice compared with unmodified antigen controls (P. W. Deropsey et al, Science, 271: 348-350, 1996; WO96/17625, PCT/GB95/02851). The increase could be produced without the use of a conventional adjuvants such as Freund's complete adjuvant. The mechanism of this remarkable effect was demonstrated to be high-affinity binding of the multivalent C3d construct to CR2 on B-cells, followed by co-ligation of CR2 with another B-cell membrane protein, CD19 and with membrane-bound immunoglobulin to generate a signal to the B-cell nucleus.
In these experiments, the unmodified antigen control and linear fusions with one or two C3d domains were prepared by transfection of the appropriate coding plasmids into L cells followed by the selection of high-expressing clones. The most immunogenic construct that with three C3d units, had to be expressed transiently in COS cells and this procedure gave a very poor yield of the fusion protein. In part, the low yield could be attributed to the generation of species containing the antigen but with lower molecular weights, corresponding to fewer than three C3d units. It was unclear from the published work of Dempsey et al whether the latter molecules originated by proteolysis of the three-C3d construct or whether they were due to a recombination event in vivo.
Using another expression system but the same C3d constructs as Dempsey et al. we have now obtained evidence that the generation of molecules with <3 C3d units from DNA encoding 3× C3d repeats is due to loss of one or more C3d units by homologous recombination and not due to post-translational processing (see below). This observation has also identified an efficient system for the expression of the C3d monomer.
It is known generally that the production of high molecular weight polypeptides containing multiple repeating sequences is difficult because of the tendency of repeated DNA sequences to undergo rearrangement during replication. Some of the limitations on internal repetitiveness in plasmids have been discussed by Gupta (Bio/Technology 1. 602-609, 1983). Ferrari et al (U.S. Pat. No. 5,641,648) have described methods for expression of repetitive sequences using synthetic genes constructed from monomeric units which are concatenated by ligation. DNA sequences encoding the same repeated amino acid sequence but differing in nucleotide sequence either within or between monomers were constructed by exploiting the redundancy of the genetic code. The resulting lack of precise repetitiveness at the nucleotide level reduced homologous recombination to the point where the repeated oligopeptide sequence could be expressed. The work of Ferrari et al was restricted to relatively short repeating units of 4 to 30 codons (amino-acids) repeated a large number of times (typically ~30-fold).
The present invention describes a general method for introducing variability into entire genes or fragments of genes, particularly those encoding autonomously folding protein domains or motifs of>30 amino acids, in such a way that different DNA units encoding identical or near-identical amino acid sequences can be concatenated and expressed to give domain oligomers.
The invention comprises the following elements:
1. The construction of novel synthetic DNA sequences encoding an autonomously folding polypeptide domain and using in these DNA sequences the maximum third-base redundancy in each codon permitted by the genetic code which is consistent with a continuous reading frame and retention of the amino acid sequence. These mixtures of DNA molecules are termed ‘Fuzzy Gazes’.
2. Using these libraries to isolate or design concatamers in which the DNA repeats differ from each other in the third base positions. These concatamers may be made with or without in-frame coding regions for other proteins.
3. Placing these sequences either as mixed populations or single characterised concatamers into a suitable expression vector and expressing the population in a recombinant host cell.
4. The use of assays able to detect the presence of repeated-domain expressed protein products. Host cell clones are screened for those capable of producing useful levels of functionally active polypeptide concatamers/fusion proteins.
5. Where necessary, characterising one or more unique DNA sequences derived from these clones and encoding the expressed product.
6. Using the unique chemical reactivity of single cysteine residues in expressed proteins to assemble protein derivatives with multiple copies of a domain by post-translational chemical modification combined with concatamerisation at the DNA level.
In specific embodiments of the invention, the autonomously folding repeated protein domain is a ligand for one or mare cell surface receptors involved in the regulation of the immune system. One such example is human or murine C3d or C3d,g polypeptide sequence or another peptide ligand of CR2 (CD21) or CD 19.
In a second embodiment the additional domain may be an immunogen, particularly an antigenic protein or region of a protein. Examples of polypeptide immunogens include but are not restricted to: the Hepatitis B surface antigen, meningococcal surface proteins, proteins expressed at various stages of the life cycle of the malaria parasite, the glucan-binding region of streptococcal glucosyltransferases, the haemagglutinin (H) and neuraminidase (N) proteins of influenza virus strains and the D-repeat regions of the fibronectin binding pr
Cox Vivienne Frances
Rowling Pamela Jane Elizabeth
Smith Richard Anthony Godwin
Adprotech Limited
Heller Ehrman White & McAuliffe
Nguyen Dave Trong
LandOfFree
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