Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
2001-05-24
2003-11-25
Nashed, Nashaat T. (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C435S069100, C435S201000, C435S252300, C435S252330, C435S325000, C435S254110, C435S320100, C536S023200, C536S023500, C536S023100, C530S350000
Reexamination Certificate
active
06652851
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to nucleic acid molecules encoding Polistinae venom allergens, in particular enzymes such as phospholipase and hyaluronidase, or fragments thereof, recombinant vectors comprising such nucleic acid molecules, and host cells containing the recombinant vectors. The invention is further directed to expression of such nucleic acid molecules to produce a recombinant Polistinae venom enzyme, such as phospholipase or hyaluronidase, or recombinant fragments thereof. Such an allergen and fragments thereof are useful for diagnosis of allergy, for therapeutic treatment of allergy, for the treatment of immune system related diseases or disorders, or symptoms related thereto, and for the modulation of immune response towards an immunogen.
BACKGROUND OF THE INVENTION
Insect sting allergy to bees and vespids is of common occurrence. The vespids include hornets, yellow jackets and wasps (Golden, et al., 1989, Am. Med. Assoc. 262:240). Susceptible people can be sensitized on exposure to minute amounts of venom proteins; as little as 2-10 &mgr;g of protein is injected into the skin on a single sting by a vespid (Hoffman and Jacobson, 1984, Ann. Allergy. 52:276).
There are many species of hornets (genus Dolichovespula), yellow jackets (genus Vespula) and wasp (genus Polistes) in North America (Akre, et al., 1980, “Yellowjackets of America North of Mexico,” Agriculture Handbook No. 552, US Department of Agriculture). The vespids have similar venom compositions (King, et al., 1978, Biochemistry 17:5165; King, et al., 1983, Mol. Immunol. 20:297; King, et al., 1984, Arch. Biochem. Biophys. 230:1; King, et al., 1985, J. Allergy and Clin. Immunol. 75:621; King, 1987, J. Allergy Clin. Immunol. 79:113; Hoffman, 1985, J. Allergy and Clin. Immunol. 75:611). Their venom each contains three major venom allergens, phospholipase (37 kD), hyaluronidase (43 kD) and antigen 5 (23 kD) of as yet unknown biologic function. U.S. Pat. No. 5,593,877 describes cloning and expression of the vespid venom allergens phospholipase and hyaluronidase. As described in this patent, the recombinant allergens permit expression of a protein or fragments thereof for use in immunotherapy, dignostics, and to investigate T and B cell allergens, it sets forth in greater detail the rationale for cloning vespid venom enzymes. However, unique vespid venom cDNAs were not described.
In addition to the insect venom allergens described above, the complete amino acid sequence of several major allergens from different grass (Perez, et al., 1990, J. Biol. Chem. 265:16210; Ansari, et al., 1989, Biochemistry 26:8665; Silvanovich, et al., 1991, J. Biol. Chem. 266:1204), tree pollen (Breiteneder, 1989, EMBO J. 8:1935; Valenta, et al., 1991, Science, 253:557), weed pollen (Rafnar, et al., 1991, J. Biol. Chem. 266:1229; Griffith, et al., 1991, Int. Arch. Allergy Appl. Immunol. 96:296), mites (Chua, et al., 1988, J. Exp. Med. 167:175), cat dander (Griffith, et al., 1992, Gene. 113:263), and mold (Aruda, et al., 1990, J. Exp. Med. 172:1529; Han, et al., 1991, J. Allergy Clin. Immunol. 87:327) have been reported in the past few years. These major allergens are proteins of 10-40 kD and they have widely different biological functions. Nearly all allergens of known sequences have a varying extent of sequence similarity with other proteins in our environment.
Although U.S. Pat. No. 5,593,877 provides for cloning and expression of vespid venom enzymes, particularly hyaluronidase and phospholipase, there remains a need to identify unusual and unexpected sequences for such enzymes, and to design effective expression systems for them. There is a particular need to delineate the B and helper T cell epitopes of the paper wasp (e.g.,
Polistes annularis
). In particular, the major Polistinae venom allergens phospholipase and hyaluronidase are appropriate targets for determining the important B and T cell epitopes. In order to fully address the basis for allergic response to vespid allergens, and to develop allergen-based immunotherapies, the cDNA and protein sequences of several homologous allergens need to be investigated. Moreover, vectors suitable for high level expression in bacteria and eukaryotic cells of vespid allergens or their fragments should be developed. Recombinant vespid allergens and their fragments may then be used to map their B and T cell epitopes in the murine and, more importantly, human systems by antibody binding and T cell proliferation tests, respectively.
There is also a need in the art to use peptides having T or B cell epitopes of vespid venom allergens to study induction of tolerance in mice and induction of tolerance in humans.
There is a further need to test whether a modified peptide inhibits allergen T cell epitope binding to MHC class II molecule, or induces T cell anergy, or both.
Thus, there is a need in the art for unique sequence information about vespid venom allergens, and a plentiful source of such allergens for immunological investigations and for immunological therapy of the allergy.
Furthermore, due to the overuse of antibiotics throughout the world, and to the spread of numerous viruses, such as HIV, Ebolla, etc., efforts have been made to produce new “super” antibiotic medication, and compounds which have activity against viruses. For example, AZT has been developed, along with protease inhibitors to treat subjects suffering from HIV. However, the costs of developing new “super” antibiotics and anti-viral medications are enormous.
Hence, what is needed are agents and pharmaceutical compositions for treating immune system related diseases or disorders whose activity is not dependent necessarily on combating the particular virus or pathogen, but rather modulate or potentiate the immune system ability to combat the disease or disorder, thereby ameliorating the disease or disorder, or a symptom related thereto. Hymenoptera venoms, particularly vespid venoms, provide one possible source for such agents and pharmaceutical compositions, as described in U.S. Pat. Nos. 4,822,608 and 5,827,829.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.
SUMMARY OF THE INVENTION
The present invention provides a nucleic acid molecule encoding Polistinae venom enzymes, immunomodulatory fragments thereof, or derivatives or analogs thereof. In particular, the invention is directed to such nucleic acid molecules encoding a Polistinae venom phospholipase, and a Polistinae venom hyaluronidase. In specific embodiments, a nucleic acid molecule of the invention encodes an immunomodulatory portion of a T cell epitope of a Polistinae venom enzyme. In another embodiment, a nucleic acid molecule of the invention encodes an antigenic portion of a B cell epitope of a Polistinae venom enzyme.
The nucleic acids of the invention, which are not genomic, surprisingly are found, in one embodiment, to contain a non-coding, e.g., intronic sequences. In a specific embodiment, cDNA molecules for Polistinae venom enzyme contain what appears to be an intron. Thus, it has unexpectedly proved necessary to delete these “intronic” sequences in order to obtain a nucleic acid coding for a mature Polistinae venom enzyme, e.g., phosholipase or hyaluronidase.
Hence broadly, the present invention extends to an isolated nucleic acid molecule encoding a venom enzyme, conserved variant thereof, immunomodulatory fragment thereof, or derivative, or analog thereof. As noted above, the nucleic acid molecule contains internal non-coding sequences, i.e., in addition to 5
−
and 3
−
untranslated (UTR) sequences., but is not a genomic sequence. Examples of Polistinae venom enzymes which can be encoded by an isolated nucleic acid molecule of the invention include, but are not limited to phospholipase and hyaluronidase. Moreover, enzymes, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, from the venom of numerous Polistinae venoms can be encoded by an isolated nucleic acid molecule of the invention. A
Darby & Darby
Nashed Nashaat T.
The Rockefeller University
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