Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
1999-01-12
2002-08-13
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
C530S409000, C530S412000, C530S415000, C530S417000, C530S419000, C424S085100, C424S085200, C424S085400, C424S085600, C424S085700, C514S002600, C514S008100, C514S012200, C514S021800
Reexamination Certificate
active
06433144
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for producing highly purified mixtures of natural Type I interferon from white blood cells, with a protein purity in excess of 95% as determined by standard analytical methods. The invention also relates to highly-purified mixtures of Type I interferon which resemble natural Type I interferon in that the highly purified mixtures of natural Type I interferons includes at least 9 subtypes, i.e., alpha-1, alpha-2, alpha-5, alpha-7, alpha-8, alpha-10, alpha-14, alpha-21 and omega. These subtypes may be present as varying proportions of the following subspecies: alpha-1a, alpha-1new, alpha-2a, alpha-2b, alpha-2c, alpha-5, alpha-5LG, alpha-7, alpha-8a, alpha-8c, alpha-10a, alpha-14a, alpha14b, alpha 14-c, alpha-14LG, alpha-21a, alpha-21b, alpha-21c, omega and omega LG.
BACKGROUND
The interferons are a family of proinflammatory cytokines important in mediating nonspecific host defense. While of critical importance in initiating anti-viral immunity, the family also acts as a potent initiator of cell growth and differentiation. Type I interferon is a designation for a family of related interferons that can include multiple subtypes of alpha, beta and omega interferons, and in some species the related trophoblast tau interferon. The proteins are structurally similar, share common receptors, have common biological activities and may share a common genetic locus.
Type I interferon is believed to have three major functions. First, it inhibits viral replication by activating cellular genes that inhibit protein synthesis, thus contributing to the suppression of viral replication. Second, Type I interferon downregulates the proliferation of specific cell types, a characteristic applied to the treatment of certain cancers. Finally, Type I interferon has an immunomodulatory effect, which can influence the nature of the immune response (i.e. cellular or humoral) while activating innate components such as NK cells or monocytes.
The plurality of effector functions of Type I interferon creates a variety of potential pharmacological applications. While recognized for its antiviral capability, Type I interferon is also effective in the treatment of some bacterial and eukaryotic pathogens. In addition, the immunomodulatory properties of the subtypes in Type I interferon have proven useful in the treatment of some cancers and autoimmune disorders. The literature describing the uses of interferon preparations is vast and includes the use of Type I interferon in the treatment of cancers, including leukemias (U.S. Pat. No. 5,830,455), basal cell carcinomas (U.S. Pat. No. 5,028,422), squamous cell carcinomas (U.S. Pat. No. 5,256,410), breast cancer (U.S. Pat. No. 5,024,833), gastrointestinal malignancies (U.S. Pat. No. 5,444,064; 5,814,640), actinic keratoses (U.S. Pat. No. 5,002,764), as well as macular degeneration (U.S. Pat. No. 5,632,984), autoimmune disorders (U.S. Pat. No. 5,830,456), diabetes (WO0980643 1A2), bacterial infections (U.S. Pat. No 5,817,307), and viral infections (U.S. Pat. No. 5,830,456), including genital warts (U.S. Pat. No. 4,959,210), hepatitis B (WO09823285A1), and herpes zoster and psoriasis (U.S. Pat. No. 4,957,734). While the pharmaceutical applications of this family of cytokines is only beginning to be understood, the problems related to obtaining an inexpensive and highly purified preparation containing a comprehensive spectrum of Type I interferon have limited the therapeutic potential of Type I interferon.
A number of different techniques have been utilized to produce quantities of interferons. The successful cloning and sequencing of genes encoding specific members of the family have allowed for the recombinant production of individual Type I interferon subtypes. While it is possible to produce individual recombinant Type I subtypes, these individual recombinant products are limited because (1) their structures may vary from the natural state, and (2) their individual activities may lack the therapeutic potential of all subtypes collectively. Further, individual interferon subtypes may cause negative host reactions, including fever, nausea, tissue necrosis and psychopharmacological effects. These side effects have in some cases limited the efficacy of interferon treatment.
Natural interferon production heretofore has traditionally involved ammonium chloride treatment of buffy coats to lyse red blood cells and to isolate leukocytes, followed by viral stimulation of the isolated leukocytes with subsequent large scale harvesting of culture medium. The interferons are then isolated by various precipitation, adsorption, or immuno-affinity techniques.
Despite the use of a variety of purification techniques, the quality, quantity and subtype diversity of the interferons obtained using these methods has remained unsatisfactory. These techniques have generally required tremendous quantities of culture media with processing resulting in low yields of product having limited subtype distribution. It is presently believed that the methods described heretofore are unable to achieve easily and economically a sufficiently high recovery rate with a high degree of purity, full spectrum of subtypes and full functional activity of natural interferon subtypes. Even immuno-based purification techniques have not produced a full spectrum of individual Type I interferon subtypes because the various subtypes differ in their antigenicity.
It is currently thought that one important reason for the low yield of Type I interferon from the prior art techniques is due to ineffective methods of leukocyte collection, transport, separation, culture and stimulation to secrete interferons. For example, transport mechanisms for whole blood or buffy coats are not optimal for retaining active leukocyte cells, being subject to a wide range of temperatures, high osmolarity, low oxygenation levels and variable transport times. Also, it is presently believed that lysis of red blood cells with ammonium chloride may greatly reduce the yield of leukocytes, many of which may also be lysed. The remaining leukocytes are then osmotically shocked which is thought to render the captured leukocytes less effective in their protein synthesis, further reducing the yield of product. Further, the prior art utilized hitherto usually employs serum in the culture of leukocytes, which is believed to significantly contribute to the resulting contamination of the secreted protein product. Moreover, it is thought that the use of viral preparations to induce interferon production also adds a significant source of contaminating material. To date, the prior art methods available up to now have not addressed these problems.
Turning now to representative interferon disclosures, U.S. Pat. No. 5,503,828 describes an alpha-interferon composition characterized by having at least 50% of alleles of &agr;2 and &agr;8, and one or more additional alpha interferon species selected from the group consisting of &agr;4, &agr;7, &agr;10, &agr;16, &agr;17, and &agr;21. While U.S. Pat. No. 4,503,035 teaches a preparation of certain interferon species, the preparation does not include, for example, alpha-1, alpha-5, alpha-14 and omega subtypes. Thus, a natural mixture of highly pure interferon having a full spectrum of subtypes is not taught by this U.S. Pat. No. 4,503,035.
U.S. Pat. No.5,762,923 teaches an aqueous interferon composition dissolved in water with a non-ionic detergent and benzyl alcohol in amounts sufficient to stabilize the interferon-alpha. The composition also contains an acidic buffer which provides a pH of 4.5 to 6.0, and may also contain an isotonizing agent. U.S. Pat. No. 4,847,079 teaches a pharmaceutical composition of interferon and thimerosal which is resistant to microorganism contamination and growth. U.S. Pat. No. 4,675,184 teaches an interferon with 15 to 60% by weight of a tri or higher polyhydric sugar alcohol and an organic acid buffer as stabilizers, and a conventional pharmaceutical carrier or diluent at pH about 3 to 6. Optionally, the composition may further contain
Feldman Stephen
Hartman Hipolito
Kappelman James
McCabe Mead M.
Morris Joseph P.
Kershishzadeh Milord
Low Christopher S. F.
Mohamed Abdel A.
Sherman Kenneth L.
Sherman & Sherman
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