| Non-glycosylated human alpha-fetoprotein, methods of production, and uses thereof -> Monitor Keywords |
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Non-glycosylated human alpha-fetoprotein, methods of production, and uses thereofRelated Patent Categories: Multicellular Living Organisms And Unmodified Parts Thereof And Related Processes, Nonhuman Animal, Transgenic Nonhuman Animal (e.g., Mollusks, Etc.), MammalNon-glycosylated human alpha-fetoprotein, methods of production, and uses thereof description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070169211, Non-glycosylated human alpha-fetoprotein, methods of production, and uses thereof. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional of, and claims priority from, U.S. Ser. No. 10/624,380, filed Jul. 22, 2003, which is incorporated by reference herein in its entirety. BACKGROUND OF THE INVENTION [0002] This invention relates to non-glycosylated human alpha-fetoprotein, its production in transgenic animals and plants, and uses thereof. [0003] Alpha-fetoprotein (AFP) is a 70 kDa glycoprotein produced by the yolk sac and fetal liver. AFP is present in fetal serum at milligram levels, and, at birth, declines to the nanogram levels normally found in adult serum: increased levels of AFP in adult serum are indicative of a yolk sac tumor, a hepatoma, or of liver regeneration. The role of AFP during fetal development is not known, although it has been suggested that AFP protects a gestating fetus from a maternal immune attack or from the effects of maternal estrogen. [0004] In vitro and in vivo experiments have shown that AFP has both cell growth-stimulatory and -inhibitory activities, depending upon the target cell, the relative concentration of AFP, and the presence of other cytokines and growth factors. For example, AFP can inhibit the growth of many types of tumor cells, and, in particular, inhibits estrogen-stimulated cell growth. Conversely, AFP stimulates the growth of normal embryonal fibroblasts. AFP has also been shown to have both immunosuppressive and immunoproliferative effects. [0005] In order to exploit the various biological properties of AFP, it will be necessary to obtain sufficient quantities of this molecule in an efficient and cost-effective manner. Expression of AFP in recombinant systems has proven difficult because expression of wild-type AFP in eukaryotic cells generally results in the production of several isoforms due to differential glycosylation of AFP at a single asparagine residue (amino acid 233). Expression of AFP in prokaryotic systems typically produces misfolded and inactive protein that is aggregated and does not have the correct internal disulfide bonds. This misfolded AFP must be purified and refolded under conditions that allow for the formation of 16 disulfide bonds, a difficult and time-consuming process, which results in a very low overall yield of active, useful protein. Because the non-glycosylated form of AFP exhibits the same biological properties as the glycosylated form and allows for a more standardized, consistent product due to the lack of glycosylation variability, non-glycosylated AFP is preferred for commercial production. Therefore, there exists a need for an efficient method to produce non-glycosylated human AFP for commercial and therapeutic applications. SUMMARY OF THE INVENTION [0006] The invention features a substantially pure nucleic acid molecule encoding non-glycosylated human alpha-fetoprotein (ng.HuAFP) or a non-glycosylated fragment thereof. In an embodiment, the nucleic acid molecule encoding ng.HuAFP includes nucleotides 45-1874 of the nucleic acid sequence set forth in SEQ ID NO: 5. [0007] The invention also features a polypeptide comprising non-glycosylated human alpha-fetoprotein. In an embodiment of this feature of the invention, the polypeptide is substantially pure and has the amino acid sequence set forth in SEQ ID NO: 6. In another embodiment, the polypeptide is substantially pure and has the amino acid sequence set forth in SEQ ID NO: 8. [0008] The invention further includes biologically active fragments and analogs of non-glycosylated recombinant HuAFP. In an embodiment, the biologically active fragments of non-glycosylated recombinant HuAFP include the amino acid sequence set forth in SEQ ID NO: 15 (Domain II), SEQ ID NO: 16 (Domain I+II), or SEQ ID NO: 17 (Domain II+III), or two or more of the amino acid sequences. [0009] The invention also features a substantially pure nucleic acid molecule that includes (i) a nucleic acid molecule encoding ng.HuAFP including nucleotides 45 through 1874 of the nucleic acid sequence set forth in SEQ ID NO: 5, (ii) a promoter that is operably linked to the ng.HuAFP-encoding sequence and that enables expression of ng.HuAFP, and (iii) a leader sequence encoding a protein secretory signal that enables secretion of ng.HuAFP by a cell. In an embodiment, the cell is a prokaryotic cell (e.g., E. coli) or a eukaryotic cell (e.g., a yeast cell (e.g., Pichia pastoris) or an animal cell (e.g., a mammalian cell, such as a Chinese hamster ovary (CHO) cell). In a desired embodiment, the cell secretes ng.HuAFP into cell culture medium (i.e., a non-biological fluid). In another embodiment, the eukaryotic cell is in a transgenic animal (e.g., a mammal, such as a goat, sheep, camel, cow, pig, rabbit, horse, or llama). In yet another embodiment, the cell is a biological fluid-producing cell in a transgenic animal, the promoter enables expression of ng.HuAFP in the biological fluid-producing cell, and the leader sequence enables secretion of ng.HuAFP into a biological fluid (e.g., milk, urine, blood, or lymph) of the transgenic animal. [0010] In an embodiment, the cell expressing ng.HuAFP is in a transgenic animal, the promoter driving expression of ng.HuAFP is a milk-producing cell-specific promoter that enables expression of ng.HuAFP in a milk-producing cell of the animal, and the leader sequence enables secretion of ng.HuAFP into the milk of the animal. In another embodiment, the cell expressing ng.HuAFP is in a transgenic animal, the promoter driving expression of ng.HuAFP is a urine-producing cell-specific promoter that enables expression of ng.HuAFP in a urine-producing cell of the animal, and the leader sequence enables secretion of ng.HuAFP into the urine of the animal. In yet another an embodiment, the cell expressing ng.HuAFP is in a transgenic animal, the promoter driving expression of ng.HuAFP is a blood-producing cell-specific promoter that enables expression of ng.HuAFP in a blood-producing cell of the animal, and the leader sequence enables secretion of ng.HuAFP into the blood of the animal. In still another embodiment, the cell expressing ng.HuAFP is in a transgenic animal, the promoter driving expression of ng.HuAFP is a lymph-producing cell-specific promoter that enables expression of ng.HuAFP in a lymph-producing cell of the animal, and the leader sequence enables secretion of ng.HuAFP into the lymph of the animal. [0011] Another feature of the invention is a non-human transgenic organism that expresses and secretes ng.HuAFP into a biological fluid (e.g., milk, urine, saliva, seminal or vaginal fluid, syiovial fluid, lymph fluid, amniotic fluid, the fluid within the yolk sac, the chorion, or the allantois of an egg, blood, sweat, and tears; or an aqueous solution produced by a plant, including, for example, exudates or guttation fluid, xylem, phloem, resin, and nectar). In an embodiment, the transgenic organism is a mammal (e.g., a goat, sheep, camel, cow, pig, rabbit, horse, or llama), a bird, a reptile, an amphibian, or a plant. In another embodiment, the ng.HuAFP is expressed from a transgene that includes: (i) a nucleic acid molecule encoding ng.HuAFP including nucleotides 45 through 1874 of the nucleic acid sequence set forth in SEQ ID NO: 5, (ii) a promoter that is operably linked to the ng.HuAFP-encoding sequence such that the promoter enables expression of ng.HuAFP by cells of the organism that secrete a protein into a biological fluid, and (iii) a leader sequence encoding a protein secretory signal that enables secretion of ng.HuAFP into the biological fluid by the cells of the organism. In yet another embodiment, the promoter is a milk-, urine-, blood-, or lymph-specific promoter and the leader sequence enables secretion of ng.HuAFP into the milk, urine, blood, or lymph, respectively, of the organism. In still another embodiment, the organism is a mouse or a goat. [0012] The invention also features a non-human mammal's milk, urine, blood, or lymph that includes ng.HuAFP. In an embodiment, the ng.HuAFP is soluble and is produced by a non-human transgenic mammal whose milk-, urine-, blood-, or lymph-producing cells express a transgene that comprises: (i) a nucleic acid molecule encoding ng.HuAFP including nucleotides 45 through 1874 of the nucleic acid sequence set forth in SEQ ID NO: 5, (ii) a milk-, urine-, blood-, or lymph-specific promoter such that the promoter is operably linked to the ng.HuAFP-encoding sequence and enables expression of ng.HuAFP by the milk-, urine-, blood-, or lymph-producing cells of the mammal, and (iii) a leader sequence encoding a protein secretory signal that enables secretion of ng.HuAFP by the milk-, urine-, blood-, or lymph-producing cells into milk, urine, blood, or lymph, respectively, of the mammal. [0013] The invention also features a method of producing ng.HuAFP by the following steps: (a) providing a cell transduced with a transgene that comprises: (i) a nucleic acid molecule encoding ng.HuAFP including nucleotides 45 through 1874 of the nucleic acid sequence set forth in SEQ ID NO: 5, (ii) a promoter that is operably linked to the ng.HuAFP-encoding sequence such that the promoter enables expression of ng.HuAFP by the cell, and (iii) a leader sequence encoding a protein secretory signal that enables secretion of ng.HuAFP by the cell; and (b) growing the transduced cell such that the cell expresses and secretes ng.HuAFP. [0014] In an embodiment, the cell is a prokaryotic cell (e.g., E. coli) or a eukaryotic cell (e.g., a yeast cell (e.g., Pichia pastoris) or a mammalian cell (e.g., a CHO cell, or a milk-, urine-, blood-, or lymph-producing cell)). [0015] The invention also features a method of producing ng.HuAFP by the following steps: (a) providing a transgenic organism (e.g., a mammal (e.g., a goat, sheep, camel, cow, pig, rabbit, horse, or llama), a bird, a reptile, an amphibian, or a plant) that includes a transgene having (i) a nucleic acid molecule encoding ng.HuAFP including nucleotides 45 through 1874 of the nucleic acid sequence set forth in SEQ ID NO: 5, (ii) a promoter that is operably linked to the ng.HuAFP-encoding sequence such that the promoter enables expression of ng.HuAFP in a biological fluid-producing cell of the transgenic organism, and (iii) a leader sequence encoding a protein secretory signal that enables secretion of ng.HuAFP by the biological fluid-producing cell into a biological fluid of the transgenic organism; and (b) collecting the biological fluid that includes ng.HuAFP from the transgenic organism. [0016] In an embodiment, the biological fluid is milk, urine, saliva, seminal or vaginal fluid, synovial fluid, lymph fluid, amniotic fluid, the fluid within the yolk sac, the chorion, or the allantois of an egg, blood, sweat, or tears; or an aqueous solution produced by a plant, including, for example, exudates or guttation fluid, xylem, phloem, resin, and nectar. In a desired embodiment, the biological fluid is milk, urine, blood, or lymph, and ng.HuAFP is purified from the milk, urine, blood, or lymph, respectively. In another embodiment, the promoter is a milk-, urine-, blood-, or lymph-specific promoter that enables expression of ng.HuAFP in milk-, urine-, blood-, or lymph-producing cells, respectively, of the transgenic organism. In yet another embodiment, the transgenic organism expresses and secretes ng.HuAFP in two or more biological fluids (e.g., milk and urine, milk and blood, urine and blood, or milk, urine, and blood). [0017] Another feature of the invention is a method of treating a patient in need of ng.HuAFP by administering to the patient a therapeutically-effective amount of ng.HuAFP that is purified from a cell culture medium. [0018] Another feature of the invention is a method of treating a patient in need of ng.HuAFP by administering to the patient a therapeutically-effective amount of a biological fluid (e.g., milk, urine, saliva, seminal or vaginal fluid, synovial fluid, lymph fluid, amniotic fluid, the fluid within the yolk sac, the chorion, or the allantois of an egg, blood, sweat, and tears; or an aqueous solution produced by a plant, including, for example, exudates or guttation fluid, xylem, phloem, resin, and nectar), or extract thereof, that includes ng.HuAFP that is obtained from a transgenic non-human organism (e.g., a mammal (e.g., a mouse, goat, sheep, camel, cow, pig, rabbit, horse, ox, or llama), a bird, a reptile, an amphibian, or a plant). In a desired embodiment, n.g.HuAFP has the sequence set forth in SEQ ID NO: 8. In another embodiment, the biological fluid is milk. In yet another embodiment, ng.HuAFP is purified from the transgenic non-human organism's biological fluid (e.g., ng.HuAFP purified from the milk, urine, blood, or lymph of a mammal). In various desired embodiments, the method may be used to inhibit or treat an immunologic disorder, e.g., infection with the human immunodeficiency virus (HIV), cancer cell growth, to induce bone marrow cell proliferation (for example, after a bone marrow transplant or after administration of a myelotoxic treatment such as chemotherapy or radiation treatment), or as an immunosuppressive agent (for example, to inhibit autoreactive immune cell proliferation, to inhibit rejection of a transplanted organ (e.g., graft-versus-host disease), or to inhibit or treat an autoimmune disorder, e.g., rheumatoid arthritis, muscular dystrophy, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, or psoriasis). [0019] The invention also features a therapeutic composition that includes ng.HuAFP having the amino acid sequence set forth in SEQ ID NO: 8. [0020] The invention also features the use of ng.HuAFP having the amino acid sequence set forth in SEQ ID NO: 8 in the manufacture of a medicament for treating an individual diagnosed with or suffering from a disease (e.g., cancer, rheumatoid arthritis, muscular dystrophy, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, or psoriasis). [0021] The invention also features the use of ng.HuAFP having the amino acid sequence set forth in SEQ ID NO: 8 in the manufacture of a medicament for enhancing cell proliferation (e.g., to induce bone marrow cell proliferation (for example, after a bone marrow transplant or after administration of a myelotoxic treatment such as chemotherapy or radiation treatment)). Continue reading about Non-glycosylated human alpha-fetoprotein, methods of production, and uses thereof... 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