Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
1998-12-31
2002-02-26
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C435S002000, C424S085100, C424S085400, C424S085600, C424S085700, C530S412000, C530S414000, C530S415000, C530S416000, C530S351000, C530S364000
Reexamination Certificate
active
06350589
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for isolating highly-purified mixtures of natural type I interferons from white blood cells, and particularly human white blood cells. The invention also relates to highly-purified mixtures of natural type I interferons which resemble natural type I interferon in that it includes 9 subtypes, i.e., alpha-1, alpha-2, alpha-5, alpha-7, alpha-8, alpha-10, alpha-14, alpha-21 and omega, giving rise to possibly 20 molecular species, including alpha-1a, alpha-1new, alpha-2a, alpha-2b, alpha-2c, alpha-5, alpha-5LG, alpha-7, alpha-8a, alpha-8c, alpha-10a, alpha-14a, alphal 14-b, 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 interferon, beta interferon, 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.
The type I interferons are believed to have three major functions. First, they inhibit viral replication by activating cellular genes that inhibit protein synthesis, thus contributing to the suppression of viral replication. Second, they downregulate the proliferation of specific cell types, a characteristic applied to the treatment of certain cancers. Finally, they have 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 interferons create a variety of potential pharmacological applications. While recognized for their antiviral capability, interferons are also effective in the treatment of some bacterial and eukaryotic pathogens. In addition, the immunomodulatory properties of the group 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 interferons 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 (5,256,410), breast cancer (U.S. Pat. No. 5,024,833), gastrointestinal malignancies (U.S. Pat. Nos. 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 (5,830,456), diabetes (WO09806431A2), 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 interferons 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 various 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 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 has traditionally involved ammonium chloride treatment of buffy coats to lyse the red blood cells and to isolate the leukocytes, followed by viral stimulation of 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 a low yield of a 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 functional activity and a full spectrum of natural interferon subtypes. Even immuno-based purification techniques have not produced a full spectrum of type I interferon subtypes because the various subtypes differ in their antigenicity.
One important reason for the low yield of type I interferon from the prior art technicques has been 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 believed that lysis of red blood cells with ammonium chloride can greatly reduce the yield of leukocytes, many of which may also be lysed. The remaining leukocytes are osmotically shocked and less effective in their protein synthesis, further reducing the yield of product. Further, the prior art usually employs serum in the culture of leukocytes, which significantly contributes to the resulting contamination of the secreted protein product. The use of viral preparations to induce interferon production also adds a significant source of contaminating material. The prior art has heretofore not addressed these problems.
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 stable pharmaceutical composition of interferon and thimerosal which is resistant to microorganism contamination and growth. U.S. Pat. No. 4,675,184 teaches a stabilized 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 can further contain an anionic surfactant and/or albumin as a stabilizer. U.S. Pat. No. 5,236,707 teaches the use of amine stabilizing agents such as primary aliphatic amines and anionic stabilizing agents such as lithium organo sulfates which protect human interferons from degradation and provide enhanced storage stability. Similarly, U.S. Pat. No.5,431,909 teaches the use of amine stabilizing agents such as primary aliphatic amines and anionic
Feldman Stephen B.
Hartman Hipolito
Kappelman James
McCabe Mead M.
Morris Joseph P.
Andres Janet L.
Eyler Yvonne
Gale James A.
Sherman Kenneth L.
Sherman & Sherman
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