Drug – bio-affecting and body treating compositions – Nonspecific immunoeffector – per se ; or nonspecific...
Reexamination Certificate
1999-06-30
2004-07-20
Saunders, David (Department: 1640)
Drug, bio-affecting and body treating compositions
Nonspecific immunoeffector, per se ; or nonspecific...
C424S192100, C424S193100, C530S324000, C530S350000, C530S403000
Reexamination Certificate
active
06764689
ABSTRACT:
CITED REFERENCES
A full bibliographic citation of the references cited in this application can be found in the section preceding the claims.
DESCRIPTION OF THE PRIOR ART
The present invention generally relates to markets where the detection or purification of molecules is involved. Representative markets include in vitro and in vivo diagnostics, research products, clinical products, clinical research products, pharmaceuticals and many industrial markets. These markets necessarily require a tight binding and specific affinity ligand that recognizes the biomolecule of interest. Given the importance of affinity recognition for proteins in a wide variety of markets, new methods for generating “immunoaffinity” or “macromolecular recognition” ligands using DNA technology are sought.
The production of proteins and peptides by recombinant DNA technology is now relatively common. Recombinant expression of peptides often is accomplished by inserting a DNA sequence encoding the desired peptide into an expression vector. The expression vector generally contains regulatory sequences which are recognized by the host cell, and which provide for transcription and translation of the inserted DNA to produce the peptide. The expression vector is inserted into a suitable whole cell, typically a procaryotic organism, in culture. The expression vector also usually includes a selectable marker, so that one may identify those cells which have been transformed successfully and carry the vector, and separate them from those which do not carry the vector.
Peptides may be expressed either directly or in the form of a fusion protein. Direct expression involves the production of the desired protein or peptide without modification. However, this form of expression often results in low yields and degradation of the product, particularly with small peptides.
One method commonly employed to increase the yield of the desired peptide is to express it as part of a fusion protein. In this approach, a leader protein, hereafter referred to as a carrier segment, is selected which is expressed easily in the host of choice. Often this protein is native to the host, as in the case of &bgr;-galactosidase. An expression vector encoding the carrier segment then is modified using standard molecular cloning techniques to express the desired peptide linked to the carrier segment. Most often, the peptide is linked to the carboxy terminus of the carrier segment but, in principle, it could be linked anywhere through a normal peptide linkage.
The expression of the peptide as a fusion protein with the carrier segment may imbue it with new, favorable properties. For instance, the fusion protein can be injected into a host animal to create antibodies to the peptide or to produce a vaccine. The technology of using fusion proteins as antigens is well-known to the art (
Current Protocols
, 1994, Chapter 16). Other favorable properties of the fusion protein may include ease of purification, the ability to be immobilized on surfaces, and the creation of bifunctional molecules (Santo, C. et al. 1992).
Although peptides with desired additional properties are commonly produced by the use of fusion proteins, it is also common to produce a peptide separately through chemical synthesis. The peptide then can be covalently coupled to purified carrier segments using chemical crosslinking agents. The resultant carrier protein conjugate can be used in many of the same applications as the fusion protein described above.
For example, peptides are sometimes covalently coupled to keyhole limpet hemocyanin for immunizations or to alkaline phosphatase for use as a detection reagent. In the case of the fusion protein, the only molecules which can be linked to the carrier segment are peptides, and the linkage must be through the normal polypeptide backbone.
When the second molecule is coupled chemically to the carrier segment after expression, the nature of both the second molecules, called ligands, and of the chemical linkages to the carrier segment is much broader. Any ligand and crosslinker which is chemically allowed can be contemplated.
In some cases, covalent association of the ligand (peptide, hapten, or other) with the carrier segment is not required to be effective. The two molecules associate with each other by any number of means. In this case, the mixture of the two is called a carrier protein complex.
The following paragraphs illustrate some uses of prior art fusion proteins. For example, U.S. Pat. No. 4,743,679 to Cohen et al. discloses the expression of epidermal growth factor (EGF) as a fusion protein with a leader sequence of up to 200 amino acids (preferably up to 75). The fusion protein is expressed in bacteria as an insoluble inclusion body.
U.S. Pat. No. 5,302,526 to Keck et al. is directed to recombinant DNA encoding amphophilic leader sequences (carrier segments) for the production and purification of fusion proteins. The polypeptide comprises an amphophilic helix designed to have hydrophilic charged amino acid residues on one side and nonpolar amino acid residues on the other side of the helix. When a gene encoding a protein of interest is attached to the helix, an inclusion body is formed. The inclusion bodies may be collected and purified.
U.S. Pat. No. 5,322,930 to Tarnowski et al. describes a method for expressing proteins as fusion proteins by using the portion of human pro-atrial natriuretic peptide (proATP) as the carrier for a heterologous peptide, wherein each of the Glu residues normally present in the proATP protein portion is altered to Gln.
U.S. Pat. No. 5,008,373 to Kingsman et al. describes a fusion protein system useful in vaccines or in diagnostic or purification applications. The fusion protein includes a first amino acid sequence derived from a retrotransposon or an RNA retrovirus encoded for by a yeast TYA gene sequence. The second amino acid sequence is a biologically active amino acid sequence, acting as the antigen.
U.S. Pat. No. 5,322,769 to Bolling, et al. is directed to a method for using CKS fusion proteins in assays.
Lin et al. (1987) disclose the use as an antigen of a fusion protein containing a carrier segment consisting of the gene 10 molecule of phage T7.
A major problem with prior art carrier segments is that the carrier segments also are known to be antigenic; that is, antibodies are produced in response to the carrier segment. Thus, there is a competition between the production of antibodies to the desired ligand and the production of antibodies to the carrier segment which may result in a lower production of antibodies with specificity for the target segment. Since the immune system usually reacts to surface exposed peptide segments which are often charged, it is likely that the existence of charged residues on the carrier segment exacerbates such problems with antigenicity.
Another problem with prior art carrier segments is that they often require the use of adjuvants. Adjuvants serve a variety of purposes (Klein, 1990). Adjuvants trap the antigen by causing the formation of an emulsion, precipitate or small vesicles at the injection site from which the antigen is released slowly over a prolonged period. The clearance of the antigen is thus delayed and the organism's exposure to the antigen is lengthened. Adjuvants also stimulate the nonspecific migration of cells to the site of antigen injection and increase the probability of interaction of the antigen with cells of the immune system. Further, adjuvants increase antigen dispersion in the recipient's body by continually delivering the antigen in small amounts from the injection site to the regional lymph nodes or spleen. Some adjuvants also have a mitogenic effect and so stimulate the proliferation of lymphocytes nonspecifically. Some adjuvants also help to stimulate lymphocytes by activating adenylate cyclase and other chemical messengers. Adjuvants may increase the probability of contact among T- and B-cells, macrophages, and antigens through the activation of lymphocyte-trapping mechanisms. The main problem with adjuvants is that
Haak-Frendscho Mary
Knuth Mark W.
Lesley Scott A.
Shultz John W.
Villars Catherine E.
Frenchick Grady J.
Michael & Best & Friedrich LLP
Promega Corporation
Saunders David
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