Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1998-11-13
2003-06-24
Saunders, David (Department: 1644)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S193100, C424S194100, C436S518000, C436S543000, C530S403000, C536S123000
Reexamination Certificate
active
06582700
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the production of novel synthetic peptide constructs and the use of such constructs as antigens in the production of antibodies, vaccines, antiviral agents, and the like. The antibodies produced in accordance with the invention may be used diagnostically or therapeutically. The constructs may also be used as vaccines.
Antibodies are important products of the immune system. An antibody is an immunoglobulin, which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of another molecule. The antibody can be monoclonal or polyclonal and can be prepared by techniques that are well known in the art. Antibodies may include a complete immunoglobulin or fragment thereof, which immunoglobulins include the various classes and isotypes, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b, and IgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab′)
2
Gab′, and the like. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where appropriate so long as binding affinity for a particular molecule is maintained.
Antiserum containing antibodies, usually referred to as polyclonal antibodies, is obtained by well-established techniques involving immunization of an animal, such as a rabbit, guinea pig, or goat, with an appropriate immunogen and obtaining antisera from the blood of the immunized animal after an appropriate waiting period. State-of-the-art reviews are provided by Parker, “Radioimmunoassay of Biologically Active Compounds,” Prentice-Hall (Englewood Cliffs, N.J., U.S., 1976); Butler,
J. Immunol. Meth
. (1975) 7: 1-24; Broughton and Strong,
Clin. Chem
. 22: (1976) 726-732; and Playfair, et al.,
Br. Med. Bull
. (1974) 30: 24-31.
Antibodies can also be obtained by somatic cell hybridization techniques, such antibodies being commonly referred to as monoclonal antibodies. Monoclonal antibodies may be produced according to the standard techniques of Köhler and Milstein,
Nature
(1975) 265:495-497. Reviews of monoclonal antibody techniques are found in “Lymphocyte Hybridomas,” ed. Melchers, et al. Springer-Verlag (New York 1978),
Nature
(1977) 266:495
; Science
(1980) 208: 692, and
Methods of Enzymology
(1981) 73 (Part B):3-46. Samples of an appropriate immunogen preparation are injected into an animal such as a mouse and, after a sufficient time, the animal is sacrificed and spleen cells obtained. Alternatively, the spleen cells of a non-immunized animal can be sensitized to the immunogen in vitro. The spleen cell chromosomes encoding the base sequences for the desired immunoglobulins can be compressed by fusing the spleen cells, generally in the presence of a non-ionic detergent, for example, polyethylene glycol, with a myeloma cell line. The resulting cells, which include fused hybridomas, are allowed to grow in a selective medium, such as HAT-medium, and the surviving immortalized cells are grown in such medium using limiting dilution conditions. The cells are grown in a suitable container, e.g., microtiter wells, and the supernatant is screened for monoclonal antibodies having the desired specificity.
Various techniques exist for enhancing yields of monoclonal antibodies, such as injection of the hybridoma cells into the peritoneal cavity of a mammalian host, which accepts the cells, and harvesting the ascites fluid. Where an insufficient amount of the monoclonal antibody collects in the ascites fluid, the antibody is harvested from the blood of the host. Alternatively, the cell producing the desired antibody can be grown in a hollow fiber cell culture device or a spinner flask device, both of which are well known in the art. Various conventional ways exist for isolation and purification of the monoclonal antibodies from other proteins and other contaminants (see Köhler and Milstein, supra).
In another approach for the preparation of antibodies the sequence coding for antibody binding sites can be excised from the chromosome DNA and inserted into a cloning vector which can be expressed in bacteria to produce recombinant proteins having the corresponding antibody binding sites.
Vaccines often comprise an antigen on a natural carrier such as a protein, a carbohydrate, a lipid or a liposome. Such vaccines are useful and have been employed for many years. There are, however, a number of art recognized problems with them. Several of these problems are related to the carrier. Since the carriers are isolated from natural sources, they are often not of uniform quality. Additionally, despite expensive and arduous purification efforts, it is difficult, and often impossible, to provide products completely free of natural contaminants. Such contaminants may themselves be antigenic. They cause the undesirable side reactions often associated with the use of vaccines, particularly fevers and tissue swelling. Additionally, the concentration of antigen may vary from one batch to another because the amounts of antigen, which react with the carrier or are absorbed on its surface are not uniform.
It is known that synthetic peptides can induce antibodies reactive with their cognate sequences in the native proteins. Specific antipeptide antibodies are useful laboratory reagents for confirming proteins from recombinant DNA, exploring biosynthetic pathways and precursors, and probing structural functions of proteins. Synthetic peptide antigens, conveniently available through chemical synthesis, can also be used for producing immunogens and for passive immunoprophylaxis.
One approach to preparing antipeptide antibodies is conjugation of a peptide to a known immunogenic carrier such as a protein. Examples of such carriers include albumins, serum proteins, e.g., globulins, ocular lens proteins and lipoproteins, bovine serum albumin, keyhole limpet hemocyanin (“KLH”), egg ovalbumin and bovine gamma-globulin. The peptide may also be linked to a synthetic polymer carrier or a liposome to give a macromolecular structure to the antigen carrier. Methods designed to avoid the use of carrier by polymerizing synthetic peptide antigens to give peptide polymers are also known. Although such materials are effective in producing animal antibodies, these materials are ambiguous in composition and structure. This shortcoming is particularly troublesome, for example, for antipeptide antibodies used for a human vaccine.
In an effort to address this problem, multiple antigen carrying structures were developed. Such structures are known and available commercially under the name Multiple Antigen Peptide System (MAPS). A small peptidyl core matrix is utilized bearing radially branching synthetic peptides as dendride arms. These molecules are produced by solid phase peptide synthesis beginning with three or four lysine residues which have only one kind of side chain-protecting group. Upon deprotection both amino groups are freed and a new similarly protected lysine derivative is condensed to the two free amino groups. This produces a branch chain with up to eight free amino groups which can then be used to synthesize directly onto the scaffolding the desired antigen which is usually a relatively short peptide. These structures are typically subject to steric crowding, presenting disadvantages for synthesis, which suppress yields and lead to solutions of dendritic polymers with variable numbers of side chains. The practical limit for the MAPS approach is to include about 4 to 8 side chains, typically presenting the identical antigen. Moreover, the relatively tight steric crowding of side chains can interfere with antigen presentation.
2. Previous Disclosures
A synthetic peptide vaccine design and the synthesis and properties of a high-density multiple antigenic peptide system is described by Tam in
Proc. Natl. Acad. Sci. USA
(1988) 85:5409-5413.
A multiple antigen peptide system is disclosed in U.S. Pat. No. 5,229,490 issued Jul. 20, 1993 (Tam I).
A multiple antigen peptide system having adjuvant properties, vaccines prepared theref
Leitereg Theodore J.
Medical University of South Carolina
Saunders David
Verny Hana
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