MXPA96003893A - Humanized milk - Google Patents

Abstract

The invention relates to humanized milk. the milk is produced by a non-human transgenic mammal, wherein the genome of this transgenic non-human mammal contains at least one heterologous gene encoding a human catalytic entity and wherein the catalytic entity produces oligosaccharides and glucoconjugates that are present in the milk of the non-human transgenic mammal. A particularly useful catalytic entity is that of human glycosyltransferases that produce oligosaccharides and glucocogons. The method comprises the steps of: (a) inserting into the genome of a non-human mammal, a heterologous gene encoding the production of a human catalytic entity, wherein the catalytic entity produces a secondary gene product in the milk of the mammal not human, and (b) milk the non-human mammal. Humanized milk can be used in the preparation of a nutritionally useful product in the nutritional maintenance of an animal

Description

HUMANIZED MILK Technical Field 5 The present invention relates to the in vivo production of secondary gene products of heterologous glycosyltransferases. These glycosyltransferases are expressed in the non-human breast tissue that leads to the production of heterologous oligosaccharides, as well as in different glycoconjugates that carry these oligosaccharides in the milk of the transgenic animal.

Background Art Carbohydrates are an important class of 5 biological compounds. The term "saccharides" encompasses a wide variety of carbohydrate-containing compounds.

These include polysaccharides, oligosaccharides, glycoproteins, • ~ and glycosides with aglucons that are not carbohydrate. Biological macromolecules composed of protein or lipids containing 0 fractions of oligosaccharide are collectively known as glucoconjugates. The carbohydrate moiety provides many biological functions. In cells, carbohydrates function as structural components where they regulate viscosity, stored energy, or are key components of the cell surface. Complex oligosaccharide chains of different glycoconjugates (especially glycoproteins and glycolipids) mediate or modulate a variety of biological processes. For a general review of the bioactivity of carbohydrates, see: (a) Biology of Carbohydrates, Volume 2, Ginsburg et al., Wiley, N.Y. (1984); and (b) P.W. Acher et al., Annual Review of Biochemistry, Volume 57, page 785, (1988). Among other things, it is known that: (a) carbohydrate structures are important for the stability, activity, localization, and degradation of glycoproteins; (b) certain oligosaccharide structures activate the secretion by the plants of antimicrobial substances; (c) glucoconjugates are often found on the surfaces of different cells, and are important, inter alia, for cellular interactions with the surroundings, since they function as receptors or regulators when they bind to cell surfaces of, for example , peptides, hormones, toxins, viruses, bacteria, and during cell-cell interaction; (d) the carbohydrate structures are antigenic determinants (e.g., blood group antigens); (e) carbohydrates function as cellular differentiation antigens during normal tissue development; (f) carbohydrates are important in oncogenesis, since it has been discovered that specific oligosaccharides are antigenic determinants associated with cancer; and 5 (g) oligosaccharides are important for sperm / egg interaction and for fertilization. Isolated oligosaccharides are known which inhibit the agglutination of the uropathogenic coliform bacteria with erythrocytes. It has been shown that other oligosaccharides possess a potent antitrobic activity, increasing the levels of plasminogen activator. This same biological activity has been used, by covalently linking these oligosaccharides with the surface of medical instruments, to produce surfaces that have anti-coagulation effects. These surfaces are useful in the collection, processing, storage, and use of blood. Still - • Other oligosaccharides have found utility as gram-positive antibiotics and disinfectants. In addition, certain free oligosaccharides have been used in the diagnosis and in the identification of specific bacteria. A considerable future market is foreseen for fine chemicals based on biologically active carbohydrates. Universities and Industry are currently working intensively on the development of additional uses of biologically active oligosaccharides.

These efforts include, but are not limited to: (a) the development of novel diagnostic and blood typing reagents; (b) the development of a novel type of therapy 5 as an alternative to antibiotics, based on the prevention of adhesion of bacteria and viruses to cell surfaces by means of specific oligosaccharides, and c) the use of oligosaccharides to stimulate the growth of plants and provide protection against certain plant pathogens. have identified and characterized a large number of oligosaccharide structures.The smallest block or unit of construction of an oligosaccharide is a monosaccharide.The major monosaccharides found in mammalian glycoconjugates are: D-glucose (Glc), D- - • galactose (Gal), D-mannose (Man), L-fucose (Fue), N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D- acid Neuraminic (NeuAc) The abbreviations between 0 parentheses are the standard terminology for monosaccharides according to the recommendations of the International Union of Physics, Chemistry, and Biology Council; Journal Biological Chemistry, volume 257, pages 3347-3354, (1982). These abbreviations will be used later in the present. Despite the relatively small number of fundamental building blocks, the number of possible combinations is very large because both the anore configuration (alpha- or β-glycosidic bond) and the position of the O-glycosidic bond can be varied. . Accordingly, a large variety of oligosaccharide structures can exist. It is known that the bioactivity of the oligosaccharides is specific in terms of both the conformation of the sugar and its composition. The individual monosaccharides provide an element of bioactivity, but also contribute to the overall conformation of the oligosaccharide, thus providing another level of specificity and bioactivity. It is the diversity of glucoconjugates and oligosaccharides that produces the biological specificity of certain oligosaccharide structures. However, this diversity also causes a particular problem for the practical utility of these compounds. Glucoconjugates are typically potent immunogens, and biospecificity, as noted above, is determined not only by the particular monosaccharide sequence, but also by the nature of the glycosidic linkage. Consequently, it is often not possible to use the oligosaccharide structures found in one species of animal, in another species. Similar restrictions on use may also apply, on an individual basis. For example, since it is known that certain blood group antigens are formed from specific oligosaccharides, it is necessary to be especially careful when conjugating a blood group oligosaccharide with a protein, and then that glycoprotein is therapeutically used. Careful consideration should be given to concerns about the potential immunogenicide. Despite these potential difficulties, it is well accepted that there is a need to produce large amounts of human oligosaccharides and / or glycoconjugates that carry those oligosaccharides. Numerous methods have been contemplated as adequate elements to achieve this goal. These methods include the synthesis of the oligosaccharides by conventional organic chemistry, or by the use of in vitro enzymes. Immobilized enzymes are currently in preferred mode for large-scale production of oligosaccharides in vitro. This is due to a high regio- and stereo-selectivity of the enzyme, as well as a high catalytic efficiency under light reaction conditions. The literature describes a synthesis number of oligosaccharides catalyzed above. For example, see the scientific review articles by Y. Ichikawa et al. "Enzyme-catalyzed Oligosaccharide Synthesis" in Analytical Biochemistry, volume 202, pages 215-238, (1992), and K.G. I. Nillson, "Enzymatic Synthesis of Oligosaccharides", Trends in Biotechnology, volume 6, pages 256-264 (1988). Both hydrolases and transferases have been used to facilitate the production of oligosaccharides. The glucosidase enzymes, a subclass of the hydrolases, are especially useful in the synthesis of oligosaccharides by an inversion process of the degradation cycle. However, in general, the enzymatic synthesis of oligosaccharides is based on the biosynthetic trajectory. Although the biosynthetic pathway of oligosaccharide synthesis is regulated primarily by the gene encoding the production of each glycosyltransferase, the actual oligosaccharide structures are determined by the specificity of the substrate and the acceptor of the individual glucosyltransferases. Oligosaccharides are synthesized by transferring the monosaccharides from sugar nucleotide donors to acceptor molecules. These acceptor molecules can be other free oligosaccharides, monosaccharides, or oligosaccharides linked with proteins or lipids. The enzymatic synthesis of oligosaccharides has been generally conducted only on a small scale, because enzymes, particularly glycosyltransferases from natural sources, have been difficult to isolate. Also, sugar nucleotide donors are very difficult to obtain from natural sources, and are very expensive when they are derived from organic chemistry synthesis.

/ * - • However, more recently, a recycling and reuse strategy has been developed to synthesize large quantities of oligosaccharides. U.S. Patent No. 5,180,674, incorporated herein by reference, discloses a novel affinity chromatography method, wherein the reaction products are recycled in a repetitive manner onto the glycosyltransferases bound to the matrix or resin. In addition, recent progress in gene cloning techniques has made available several glycosyltransferases in sufficient quality and quantity to make the enzymatic synthesis of oligosaccharides more practical. The literature is replete with descriptions of the recombinant or transgenic expression of a heterologous glycosyltransferase. However, before continuing with a discussion of the literature, it is necessary to clarify the meaning of different terms as used herein and in the claims: (a) Host, host cell, or host animal: These terms are used to refer to to the cell or mammal that is responsible for the biosynthesis of the biological material. (b) Homologous: This word means that the entity so characterized is normally present or is produced by the host. (c) Heterologous: This word means that the entity so characterized is not normally present or produced by the host. In other words, the entity so characterized is foreign to the host. (d) Catalytic activity: This term is used to refer to the inherent property of certain biological compounds to facilitate chemical change in other substances. (e) Catalytic entity: This term is used to refer to biological compounds that inherently possess a catalytic activity, which results in the production of new, different, or altered compounds. The examples are enzymes and antibodies. An enzyme is a biochemical catalyst of a specific biochemical reaction. An enzymatic product is formed as a result of the catalytic activity of the enzyme on a substrate material. (f) Genome: This word is used to refer to the complete genetic material found in the host. This material is configured in chromosomes. (g) Gene: This word refers to a functional portion of the genome, which is responsible for the biosynthesis of a specific biological entity. (h) Insertion: This word is used to refer to the process by which a portion of the heterologous DNA or a heterologous gene is introduced into the genome of a host. The DNA that is inserted is referred to as "insert". (i) Transgen: This refers to the heterologous genetic material that is transferred by inserting it from the genome of one species of animal to the genome of another species of animal. In a simpler way, a transgene is a gene that is heterologous to the host. The transgene codes for a specific biological material. (j) Transgenic mammal or transgenic host: These terms are used to refer to a mammal or cell that has had a transgene inserted into its genome. As a result of this insertion, the transgenic host produces a heterologous biological material that it would not normally synthesize. Heterologous entities are present or produced by a transgenic host as a result of the insertion of foreign genetic material into the genome of the host cell. (k) Primary gene product: This refers to a biological entity that is formed directly as a result of the transcription and translation of a homologous or heterologous gene. Examples include proteins, antibodies, enzymes, and the like. (1) Secondary gene product: This refers to a product that is formed as a result of the biological activity of a primary gene product. An example is an oligosaccharide that is formed as a result of the catalytic activity of an enzyme. (m) Biological products: This term is used to refer to products produced or synthesized by a transgenic host as a result of the insertion of a transgene into the mammalian genome. In a more specific way, the term means biological products that are secondary gene products. An example, as described below, is that of human oligosaccharides produced by transgenic cows. Human oligosaccharides are produced as a result of the catalytic activity of human glucosyltransferases. As discovered herein, when the gene encoding human glycosyltransferases is inserted into the murine genome, the resulting transgenic mouse produces a heterologous human glycosyltransferase as the primary gene product. Human glycosyltransferase, using homologous substrate materials, produces oligosaccharides and glycosylated proteins. The oligosaccharide, formed as a result of the enzymatic activity of the primary gene product, is also called a secondary gene product. Glucoconjugates are another example of the class of compounds referred to herein and in the claims as "biological products". (n) Product: This word is used to refer to the secondary gene products of the present invention, and is used as an alternative for "biological product". (o) Humanized milk: This refers to milk obtained from a non-human mammal that, through altering the host's genome, is caused to produce milk that closely resembles human milk. An example of humanized milk is cow's milk that contains products found in human milk, but not normally found in cow's milk. Human oligosaccharides are produced in cow's milk as a result of the insertion of the gene encoding human glycosyltransferases in the bovine genome. Humanized milk also contains glycosylated proteins with human oligosaccharides. As noted above, there is a considerable body of literature describing the recombinant or transgenic expression of heterologous glycosyltransferases. However, the literature does not disclose or suggest in any other way the production of secondary gene products in the milk of non-human transgenic mammals, as claimed in the present invention. Examples of the literature are: 1) US Pat. No. 5,032,519 to Paulson, discloses a method for genetically engineering cells, in such a way as to produce soluble and secretable Golgi processing enzymes in place of the enzymes linked with membrane that occur naturally. 2) United States Patent Number 5,047,335, by Paulson, describes the genetic engineering alteration of the genome of Chinese Hamster Ovary Cell (CHO) in such a way that the cells of Chinese Hamster ovary produce a sialyltransferase. 3) International Patent Application Number PCT / US91 / 08216 describes a transgene capable of producing heterologous recombinant proteins in the milk of transgenic bovine species. This published patent application describes a method for obtaining the primary gene product only. This published patent application also describes methods for producing and using the altered milk obtained from these transgenic animals. 4) International Patent Application Number PCT / US91 / 05917 describes methods for producing intracellular segments of DNA by homologous recombination of smaller overlapping DNA fragments. This published patent application describes a method for obtaining the primary gene product only. 5) International Patent Application Number PCT / GB87 / 00458 describes methods for producing a peptide, involving this method incorporating a DNA sequence encoding the peptide in the gene of a mammal coding for a whey protein, such that the DNA sequence is expressed in the mammary gland of the adult female mammal. This published patent application describes a method for obtaining only the primary gene product, the peptide, in the milk of the transgenic mammal, and also describes methods for producing and using the altered milk obtained from these transgenic animals. 6) International Patent Application Number PCT / GB89 / 01343 describes methods for producing proteinaceous materials in transgenic animals that have genetic constructs integrated into their genomes. The construct comprises a 5 'flanking sequence from a mammalian milk protein gene, and DNA encoding a heterologous protein other than a milk protein. This published patent application describes a method for obtaining only the product of the primary gene, the heterologous protein, in the milk of the transgenic mammal. 7) European Patent Application Number 88301112.4 describes methods for targeting specific genes to the mammary gland, which results in the efficient synthesis and secretion of biologically important molecules in the milk of these transgenic animals. This published patent application also describes methods for producing and using the altered milk obtained from y- "these transgenic animals, and a method for obtaining only the primary gene product in the milk of the transgenic mammal." 8) The Patent Application International Number PCT / DK93 / 00024 describes methods for producing human kappa-casein in the milk of transgenic animals The genetic construct comprises a 5 'flanking sequence from a mammalian milk protein gene, such as casein or serum acid, and DNA encoding human kappa-casein The DNA sequence contains at least one intron This published patent application describes a method to obtain only the product of the primary gene, the heterologous human kappa-casein, in the milk of the transgenic mammal 9) The International Patent Application Number PCT / US87 / 02069 describes a method for producing mammals capable of expressing recombinant proteins in their milk. Each of these publications teaches, in one way or another, a means to obtain the transgene primary gene product, this gene product being the active protein or the enzyme that is encoded by the transgene. This literature describes transgenic elements to obtain glucosyltransferases in non-human milk. However, none of the aforementioned publications describes or suggests the use of transgenic animals as a means to obtain a desired secondary gene product that is the product of the active enzyme. However, in a more particular way, none of the aforementioned publications describes or suggests, or otherwise reveals, the use of transgenic human glycosyltransferases in non-human milk to produce human or glucoconjugate oligosaccharides bearing those oligosaccharides. These oligosaccharides, which are the product of the active glucosyltransferases, are hereinafter referred to as the "secondary gene product". Accordingly, the different oligosaccharides found in human milk are formed as a direct result of the genetically regulated expression of certain specific glycosyltransferases. In this regard, oligosaccharides can appropriately be considered as "secondary gene products" since they are synthesized as a result of the biochemical activity of the primary gene product, the heterologous glycosyltransferase enzymes. Human milk contains a variety of oligosaccharides and proteins. Free, soluble oligosaccharides are not normally produced by animal cells and tissues, with the exception of highly differentiated lactation mammary glands. Oligosaccharides make up the bulk of the total carbohydrate content of human and bovine milk. The main constituent of carbohydrates in milk from mammals is the disaccharide lactose. Lactose is typically found at a concentration greater than 10 milligrams / milliliter, and is synthesized by the binding of galactose with glucose. This reaction is catalyzed by the enzyme, β-1,4-galactosyltransferase. The milk of most mammals, including cows, contains only very small amounts of a few additional oligosaccharides, in contrast, human milk contains substantial quantities of a number of additional soluble oligosaccharides that are larger than lactose. All human oligosaccharides are synthesized by sequential addition of monosaccharides to lactose. Representative oligosaccharides found in human milk are shown in Table 1.

TABLE 1, OLIGOSACARIDOS PRESENT IN HUMAN MILK Structure Common Name Concentration (mg / 1. Gal-ß-l, 4-Glc Lactose 50,000 2. Fuc-al, 2-Gal-ß-l, 4-Glc 2-fucosyl-lactose 200 3. Gal-ß-l, 3-GlcNac-ß -l, 3-Gal-ß-l, 4-Glc Lacto-N-tetraose 400 4. Gal-ß-1, 4-GlcNAc- ß-1, 3 -Gal-ß-1, 4-Glc Lacto-N -neotetraose 60 5. Fuc-al, 2-Gal- / -1, 3-GlcNAc-ß-l, 3-Gal-10 ß-l, 4-Glc Lacto-N-fucopentaose I 200 6. Gal-ß- 1,3 [Fuc-al, 4] GlcNAc-ß-l, 3-Gal-ß-l, 4-Glc Lacto-N-fucopentase II 20 7. Gal-ß-l, 4 [Fuc-al, 3] GlcNAc-ß-l, 3-Gal-ß-l, 4-Glc Lacto-N-fucopentase III 50 15 8. Fuc-a-1,2-Gal-ß-l, 3 [Fuc-a-1,4 ] -GlcNAc- ß-l, 3-Gal-ß-l, 4-Glc Lacto-N-difucopentaose I 25 9. NeuAc-a-2, 6-Gal-al, 4-Glc 6-sialyl-lactose 25 . NeuAc-a-2,3-Gal-ß-l, 4-Glc 3-sialyl-lactose 10 11. NeuAc-a-2,3-Gal-ß-l, 3-R Sialyltetrasaccharide at 10 12. Gal-ß-1, 3- [NeuAc-a-2,6] GlcNAc-ß-l, 3-R Sialyltetrasaccharide b 35 13. NeuAc-a-2,6-Gal-ß-l, 4-GlcNAc-ß-l, 3-R Sialyltetrasaccharide c 50 14. NeuAc-a-2,3-Gal-ß-l, 3 [NeuAc-a-2, 6] - GlcNAc-ß-l, 3-Gal-ß-l, 4-Glc Disialyltetrasaccharide 60 . NeuAc-a-2,3-Gal-ß-l, 3 [Fuc-a-1,4] -GlcNAc-ß-l, 3-Gal-ß-l, 4-Glc Sialyl-Lacto-N-fucopentaose 50 -. 10 -a-: denotes an alpha-glucosidic bond. R: Gal-ß-l, 4-Glc. fifteen The oligosaccharides in human milk are present as a result of the activity of certain specific glycosyltransferases that are found in human breast tissue. For example, the alpha-1, 2-linked fucose residues in structures 2, 5, and 8, are produced by a single human fucosyltransferase, and characterize a phenotype known in the field of immunohematology, as "secretors". These individuals are therefore characterized, because they synthesize the substances of the human blood group in their salivary secretions and other mucous secretions wherein the oligosaccharides are covalently linked with different proteins. The alpha-1, 4-linked fucose residues in structures 6, 8, and 15 are formed as a result of the enzymatic action of a different fucosyltransferase. These oligosaccharides represent a phenotype present in individuals characterized by having a "Le is-positive" blood type. These individuals use this fucosyltransferase to synthesize an oligosaccharide structure corresponding to an antigen of the human blood group. This oligosaccharide is also found in saliva, and in other mucous secretions, and is covalently bound to the lipids that are found on the red blood cell membrane of "Lewis-positive" individuals. The structure 5 is related to the H antigen of the ABO blood group; structure 6 is the blood group antigen "Lewis a"; structure 8 is the blood group antigen "Lewis b". At least 15 human milk proteins have been identified. Some of these proteins are generally recognized as glycosylated, that is, they are covalently linked to certain specific oligosaccharides. Particular oligosaccharides that are covalently linked to the protein are the same as, or similar to, the oligosaccharides described above, and their formation is the result of the genetically regulated normal expression of certain specific glycosyltransferase genes. The presence of a heterologous glycosyltransferase would also affect the modification of proteins after translation. The heterologous glycosyltransferase proteins are also appropriately known as "secondary gene products". Both homologous and heterologous proteins would be modified by glycosyltransferases in a manner different from that resulting from the activity of homologous glycosyltransferases. It has long been known that these oligosaccharides and glycosylated proteins promote the growth of desirable bacteria in the human intestinal tract. It is also believed that the oligosaccharides in human milk inhibit the binding of harmful microorganisms to the mouth and throat. These human oligosaccharides and specifically glycosylated proteins are absent from, or present in markedly different amounts in, bovine milk. In addition, as noted above, bovine milk contains predominantly lactose only. Human milk contains not only lactose, but also numerous other oligosaccharides. Also, the amino acid composition of the proteins of human milk is significantly different from the amino acid composition of the corresponding cow's milk proteins. As a consequence, infants fed infant formula comprising cow's milk may be more susceptible to intestinal disorders such as diarrhea or its proportions, and amino acid levels in the blood plasma may differ from breast-fed infants. For the same reasons, the elderly, the immunocompromised, and the critically ill patients, also have an urgent need for the availability of a nutritional product that biochemically resembles the composition of human milk. The complicated chemistry of proteins and oligosaccharides in human milk has made it extremely difficult to synthesize on a large scale. Before they can be incorporated into a commercial nutritional product, a practical method must be devised to obtain large amounts of proteins and oligosaccharides from human glycated milk. A potential solution for this problem is the use of transgenic animals, more particularly transgenic cows that express genes or cDNAs that encode enzymes that catalyze the formation of oligosaccharides and / or glycosylated proteins with the same human oligosaccharides. Transgenic domestic animals that have milk, such as rabbits, pigs, sheep, goats, and cows, are proposed herein as a means to produce milk containing human oligosaccharides and proteins glycosylated with human oligosaccharides. More specifically, transgenic cows are highly suitable for the production of oligosaccharides and recombinant proteins because a single cow can produce more than 10,000 liters of milk containing as much as 300 kilograms of protein (mainly casein) per year at a minimal cost. Therefore, the transgenic cow appears to be a less expensive production route than other recombinant protein production methods, since the invention would not be required in fermentation facilities. Also, the mammary glands of the cow are more effective for the cost than the cultivated cells, they have possibilities to produce continuously, and since the milk is collected several times a day, the time between the real synthesis and the harvest can be so short like a few hours. The genetic stability of the cow is greater than that of microbial or cell-based production systems. Also, cows are relatively easy to reproduce using artificial insemination, embryo transfer, and embryo cloning techniques. In addition, the downstream processing of cow's milk containing human transgenic proteins may require little or no purification. The publications that teach these methods are referred to below. However, none of these publications teaches, describes, or suggests in any other way the production of secondary gene products in the milk of non-human transgenic mammals, as claimed in the present invention. "Molecular Farming: Transgenic Animáis as Bioreactors" by J. Van Brunt, Biotechnology, volume 6, pages 1149-1154, 1988, describes the alteration of the genome of different large domestic animals that have milk, which produce transgenic animals capable of producing different entities. heterologous This publication suggests methods to obtain the primary gene product only. International Patent Application Number PCT / US91 / 08216 describes a transgene capable of producing heterologous recombinant proteins in the milk of transgenic bovine species. This published patent application describes a method for obtaining the primary gene product only. This application also describes methods for producing and using the altered milk obtained from these transgenic animals. International Patent Application Number PCT / GB87 / 00458 describes methods for producing a peptide, this method involving incorporating a DNA sequence encoding the peptide in the gene of a mammal coding for a whey protein, in such a way that the DNA sequence is expressed in the mammary gland of the adult female mammal. This published patent application describes a method for obtaining only the primary gene product, the peptide, in the milk of the transgenic mammal. This application also describes methods for producing and using the altered milk obtained from these transgenic animals. International Patent Application Number PCT / GB89 / 01343, describes methods for producing proteinaceous materials in transgenic animals that have genetic constructs integrated in their genome. The construct comprises a 5 'flanking sequence from a mammalian milk protein gene, and DNA encoding a heterologous protein other than a milk protein. This published patent application describes a method for obtaining only the product of the primary gene, the heterologous protein, in the milk of the transgenic mammal. European Patent Application Number 88301112.4 describes methods for targeting specific genes to the mammary glands, which results in the efficient synthesis and secretion of biologically important molecules in the milk of these transgenic animals. This published application also describes methods for producing and using the altered milk obtained from these transgenic animals, and teaches a method for obtaining only the primary gene product in the milk of the transgenic mammal. International Patent Application Number PCT / US87 / 02069 describes a method for producing mammals capable of expressing recombinant proteins in the milk of lactating animals. This patent application does not describe or suggest in any other way the production of secondary gene products in the milk of non-human transgenic mammals, as claimed in the present invention. Although transgenic animáis can be used for the production of large amounts of human proteins, they have not been used for the production of secondary gene products, such as human oligosaccharides, or glycosylated proteins and lipids with certain specific oligosaccharides, or human milk proteins and glycosylated lipids with certain specific oligosaccharides. None of the aforementioned publications describes or suggests a method for producing human and glucoconjugate oligosaccharides in milk from non-human mammals. The aforementioned publications also do not describe or suggest a method for obtaining glucoconjugates in milk from non-human mammals, where the glycosylation is with the desired oligosaccharides. Achieving this result requires that the genome of non-human mammals having milk be altered to ensure that the breast tissue selectively expresses a desired human glycosyltransferase, which would then glycosylate certain proteins with the desired oligosaccharide. This approach requires incorporating the DNA encoding the desired human glycosyltransferases into the genome. The literature also does not describe or suggest a method for obtaining human glycosylated proteins in milk from non-human mammals, where thirst glycosylation with the desired oligosaccharides. The literature also does not disclose or suggest a method for obtaining glycosylated human milk proteins in the milk of a non-human mammal, where the glycosylation is with the desired oligosaccharide moieties. Achieving this result would require that the genome of non-human mammals having milk be altered to ensure that their breast tissue selectively expresses both the human glycosyltransferase and the desired human proteins, which are then appropriately glycosylated with the desired oligosaccharides by the glycosyltransferase active human This approach requires not only that the DNA encoding the desired glycosyltransferase be inserted into the genome, but also that the DNA encoding the desired human proteins be incorporated into that genome. In accordance with the foregoing, it is an aspect of the present invention to provide methods for detecting successful transgenesis of fertilized oocytes prior to implantation, in such a way that the transplanted oocytes contain the genetic constructions required to achieve the desired glycosylation and the production of the oligosaccharide. It is also an aspect of the present invention to provide non-human transgenic mammalian species that have milk, which are capable of producing human glycosyltransferases that are secreted extracellularly through the mammary tissue of these mammalian species. In addition, it is also an aspect of the present invention to provide non-transgenic mammalian species humans who have milk, who are capable of producing human glucosyltransferases that are secreted extracellularly - - through breast tissue to the milk produced by these mammalian species. In addition, it is an aspect of the present invention to provide non-human transgenic mammalian species having milk, which are capable of producing human glycosylated proteins and oligosaccharides that are secreted extracellularly through the mammary tissue into the milk produced by these mammalian species. The present invention also relates to transgenic non-human mammals having milk, which are capable of producing proteins and glycosylated human milk lipids, in the milk of those transgenic animals. It is also an aspect of the present invention to provide non-human transgenic mammalian species that have milk, which are capable of producing human oligosaccharides in the milk of those transgenic animals. The present invention also relates to food formulations containing human glycosylated proteins, lipids, and oligosaccharides from that transgenic milk. The present invention also relates to pharmaceutical, medical, diagnostic, and agricultural formulations containing glycosylated proteins, lipids, and oligosaccharides obtained from the milk of transgenic animals. It is also an aspect of the present invention to provide transgenic bovine species that are capable of producing glycosylated proteins, such as glycosylated human milk proteins and lipids, in their mammary glands. It is a further aspect of the present invention to provide transgenic bovine species that are capable of producing human oligosaccharides in the milk of those transgenic cows.

The present invention also relates to food formulations containing glycosylated proteins, lipids, and oligosaccharides from that transgenic bovine milk. The present invention also relates to pharmaceutical, medical, diagnostic, and agricultural formulations containing glycosylated proteins, lipids, and oligosaccharides obtained from the milk of transgenic cows.

Description of the Invention The present invention utilizes transgenes encoding a heterologous catalytic entity to produce secondary gene products in the milk of non-human transgenic mammals. More particularly, the present invention uses transgenes encoding heterologous glycosyltransferases to produce heterologous oligosaccharides and glycosylated glucoconjugates in the milk of transgenic non-human mammals. A milk is described from a non-human transgenic mammal, this milk being characterized in that it contains heterologous components produced as the secondary gene products of at least one heterologous gene contained in the genome of the non-human transgenic mammal. Also described is a product produced in the milk of transgenic non-human mammals, wherein the product results from the action of a catalytic entity selected from the group consisting of heterologous enzymes and heterologous antibodies, and wherein the non-human transgenic mammal contains in its genome at least one heterologous gene that codes for that catalytic entity. Examples of the aforementioned product are oligosaccharides and glucoconjugates. The production of transgenic milk containing human oligosaccharides and / or glycosylated proteins with certain oligosaccharides, is desirable, since it provides a milk matrix where little or no additional purification is needed for human consumption, and where the transgenic milk resembles human milk biochemically. Humanized milk is described wherein the milk is produced by a non-human transgenic mammal, wherein the genome of the non-human transgenic mammal contains at least one heterologous gene encoding a human catalytic entity. The catalytic entity produces oligosaccharides and glucoconjugates that are present in the milk of the non-human transgenic mammal. A method for obtaining a humanized milk is also described, this method comprising the steps of: (a) inserting into the genome of a non-human mammal, a heterologous gene that encodes the production of a human catalytic entity, where this catalytic entity produces a secondary gene product in non-human mammalian milk; and (b) milking the non-human mammal. Also disclosed is a method for obtaining a biological product from humanized milk, this method comprising the steps of: (a) inserting into the genome of a non-human mammal, a heterologous gene encoding the production of a heterologous catalytic entity, in where the catalytic entity produces a secondary gene product in the milk of the non-human mammal; and (b) milk the non-human mammal, and (c) isolate the biological product from the milk. Also disclosed is a non-human transgenic mammal characterized in that the mammalian genome contains at least one heterologous gene which codes for the production of the heterologous catalytic entity selected from the group consisting of enzymes and antibodies, and wherein the catalytic entity produces a second heterologous product in the milk of the mammal. Also described is a transgenic cow characterized in that the cow genome contains at least one heterologous gene encoding the production of a heterologous glycosyltransferase selected from the group consisting of fucosyltransferase, galactosyltransferase, glucosyltransferase, xylosyltransferase, acetylases, glucuronyltransferases, glucuronylepimerases, sialyltransferases, mannosyltransferases, sulfotransferases, β-acetylgalactosaminyltransferases, and N -acetylglucosaminyltransferases, and wherein the milk of the cow contains heterologous oligosaccharides and glucoconjugates produced by said glucosyltransferase. Representative non-human mammals useful in the present invention are mice, rats, rabbits, pigs, goats, sheep, horses, and cows. Representative heterologous genes useful in the present invention are the genes encoding human enzymes and human antibodies.

(Human enzymes and human antibodies are also referred to herein and in the claims as a catalytic entity). Examples of human enzymes useful in the present invention are enzymes selected from the group consisting of glycosyltransferases, phosphorylases, hydroxylases, peptidases, and sulfotransferases. Glucosyltransferases are especially useful in the practice of the present invention. Exemplary glycosyltransferases especially useful in the practice of the present invention are enzymes selected from the group consisting of fucosyltransferase, ga 1-actsi-1-triaphenes, glucosyltransferase, xylosyltransferase, acetylases, glucuronyltransferase, glucuronylepimerases, sialyltransferases, mannosyltransferases, sulfotransferases, β-acetylgalactosaminyltransferases, and N-acetylglucosaminyltransierases. Examples of the desired heterologous gene products of the present invention are oligosaccharides and glucoconjugates. (Heterologous secondary gene products are also referred to herein and in the claims as "a biological product," or more simply as a "product"). Representative heterologous oligosaccharides produced as secondary gene products are lactose, 2-fucosyl lactose, lacto-N-tetraose, lactp-N-neotetraose, lacto-N-fucopentase I, lacto-N-fucopentase II, lacto-N-fucopentaose III, lacto-N-difucopentase I, sialyl lactose, 3-sialyl lactose, sialyltetrasaccharide a, sialyltetrasaccharide b, sialyltetrasaccharide c, disialyltetrasaccharide, and sialyl-lacto-N-fucopentase. Exemplary heterologous glycoconjugates produced as secondary gene products described herein, are glycosylated homologous proteins, glycosylated heterologous proteins, and glycosylated lipids. Representative or desirable glycosylated heterologous proteins according to the practice of the present invention are proteins selected from the group of proteins consisting of human serum proteins and human milk proteins. Examples of human milk proteins are proteins selected from secretory immunoglobulins, lysozyme, lactoferrin, kappa-casein, alpha-lactalbumin, beta-lactalbumin, lactoperoxidase and lipase stimulated by the bile salt. Also described is an enteral nutritional product containing humanized milk useful in. the nutritional maintenance of an animal. Also disclosed is a pharmaceutical product containing the product of the present invention, useful in the treatment of an animal. In addition, a medical diagnosis containing the product of the invention, useful in the diagnosis of an animal, is described. Also described are agricultural products that contain the product of the invention, useful in the maintenance of crops. Also disclosed is a method for producing a non-human transgenic mammal species, capable of producing heterologous secondary gene products in the milk of said species, the method comprising the steps of: (a) preparing a transgene, this transgene consisting at least an expression regulation DNA sequence, functional in the mammary secretory cells of the transgenic species, a secretory DNA sequence, functional in the mammary secretory cells of the transgenic species, and a recombinant DNA sequence encoding a recombinant heterologous catalytic entity , the secretory DNA sequence being operably linked to the recombinant DNA sequence to form a secretory-recombinant DNA sequence, and the at least one expression regulation sequence being operably linked to a sequence of secretory-recombinant DNA, wherein the transgene is able to direct the expression of the secretory-recombinant DNA sequence in the mammary secretory cells of the transgenic species containing the transgene, to produce - * a recombinant heterologous catalytic entity which, when expressed by mammary secretory cells, catalyzes the production of secondary gene products in the milk of the transgenic species; (b) introducing the transgene into the objective embryonic cell; transplant the transgenic embryonic cell objective formed by the same, or the embryo formed from the same, in a receiving female mother; and (c) identifying at least one female progeny that is capable of producing the secondary gene products in the milk of the progeny. Also disclosed is a useful method for producing large non-human transgenic mammals such as pigs, goats, sheep, horses, and cows, capable of producing heterologous secondary gene products in their milk. The method described comprises the steps of: (a) preparing a transgene capable of conferring said phenotype when incorporated into the cells of the transgenic non-human mammal, - (b) methylating this transgene; (c) introducing the methylated transgene into fertilized oocytes of the non-human mammal, to allow integration of the transgene into the genomic DNA of the fertilized oocytes. (d) cultivate the individual oocytes formed by the same, for the preimplantation of embryos, thus replicating the genome of each of the fertilized oocytes, - (e) removing at least one cell from each of the embryos of the preimplantation, and lysing the at least one cell to release the DNA contained therein; (f) contacting the released DNA with a restriction endonuclease capable of dissociating the methylated transgene, but unable to dissociate the non-methylated form of this transgene, formed after integration into, and replication of, the genomic DNA; and (g) detecting which of the cells from the embryos of the preimplantation contain a transgene that is resistant to dissociation, by means of the restriction endonuclease, as an indication of which embryos of the preimplantation have integrated the transgene. In accordance with the above method, the removal of the first semi-embryos that are Used and analyzed according to steps (d) to (f) are also described, this method also comprising: (g) cloning at least one of the Semi-embryos seconds; and (h) forming a multiplicity of transgenic embryos. Transplantation of more than one of the transgenic embryos in female host mothers is also described, to produce a population that contains at least two non-human transgenic mammals that have the same genotype, and to transplant the rest of the pre-implantation embryos containing a genomically integrated transgene, in a recipient female mother, and identify at least one progeny having the desirable phenotype, this phenotype having the ability to produce a heterologous secondary gene product in the milk of that species, with heterologous secondary gene products being selected for from the group consisting of oligosaccharides and glucoconjugates. The DNA sequence forming the transgene useful in the present invention comprises at least three functional parts: (a) A portion encoding human glycosyltransferase. This portion of the transgene is hereinafter referred to as the "recombinant portion" or the "recombinant sequence", - (b) A signal portion; and (c) A portion of expression regulation. The recombinant portion of the transgene comprises a DNA sequence encoding the desired glycosyltransferase enzyme. The signal portion may be naturally present, or it may be genetically engineered into the DNA sequence. This signal encodes a secretory sequence that ensures that the glycosyltransferase is transported to the Golgi apparatus of the cell. In the present invention, the signal DNA sequence is functional in mammary secretory cells. These sequences are operably linked to form a recombinant signal-expression DNA sequence. The expression sequence ensures that the transgene is expressed in certain types of tissue only. In the present invention, the expression is regulated towards the mammary secretory tissue. At least one expression regulation sequence, functional in the mammary secretory cells of the transgenic species, is operably linked to the recombinant signal DNA sequences. The transgene thus constructed is capable of directing the expression of the recombinant signal DNA sequence in the mammary secretory cells containing the transgene. This expression results in the production of the glycosyltransferase that is secreted from the mammary secretory cells into the milk of the transgenic species. In addition to the functional parts described above, the transgene can also comprise additional elements. For example, the recombinant portion can code for more than one protein. Accordingly, in addition to encoding the glycosyltransferase, it can also code for one or more human proteins. Also, multiple transgenes encoding other glycosyltransferases and other heterologous proteins can be transfected simultaneously. All additional transgenes are also operably linked to the secretory and expression regulation sequences of the glycosyltransferase transgene. The expression of multiple transgenes results not only in the production of the glycosyltransferase, but also in the other proteins, all of which are secreted from the mammary secretory cells into the milk of the transgenic species. In the presence of suitable substrate materials, the glycosyltransferase will convert the individual monosaccharide units into the desired oligosaccharides. The desired oligosaccharides will be present in the milk of the transgenic species. The same glycosyltransferase enzyme will also covalently link the monosaccharides to the proteins via the available glycosylation sites. These proteins glycosylated with the desired oligosaccharides will also be present in the milk of the transgenic species. The advantages of the present invention will come to be better understood by reference to the following detailed description, when taken in conjunction with the accompanying Figures.

Brief Description of the Drawings Figure 1. Nucleotide and amino acid sequence of human alpha-1, 2-fucosyltransferase. Figure 2. Illustration of the protocol to achieve the amplification and expression of the fucosyltransferase cDNA. Figure 3. Illustration of the construction of the plasmid pWAP-polyA, using the regulatory sequence (promoter) of the whey acid protein (WAP). Figure 4. Illustration of the pWAP-fucosyltransferase plasmid used for microinjection in mouse embryos. Figure 5. Photograph of a Western blot illustrating the presence of human alpha-1, 2-fucosyltransferase in the milk of transgenic mice. Figure 6A to 6F. High-pressure liquid chromatography profiles of milk samples obtained from normal or non-transgenic mice (Frames A and B) and transgenic, expressing human alpha-1, 2-fucosyltransferase (Frames C, D, E, and F) . Figure 7. Photograph of a fluorophore-assisted carbohydrate electrophoresis gel of an oligosaccharide material pooled after separation by high pressure liquid chromatography. Figure 8. Photograph of a fluorophore-assisted carbohydrate electrophoresis gel following the digestion of the oligosaccharide samples with a specific fucosidase for alpha-1,2-fucose bonds. Figure 9. Photograph of a fluorophore-assisted carbohydrate gel electrophoresis, showing the monosaccharide composition of the oligosaccharide samples isolated from milk, followed by exhaustive digestion with a mixture of fucosidase and β-galactosidase. Released monosaccharide units were labeled with 8-aminonaphthalen-2,3,6-trisulfonic acid (ANTS) to facilitate detection. Figure 10. Photograph of a Western blot of milk protein isolated from normal (non-transgenic) and transgenic mice expressing human alpha-1,2-fucosyltransferase. The glycosylation of the stained proteins was detected by immunofluorescence using a specific lectin for the alpha-1,2-fucose bond. The figure tests the presence of glycosylated milk proteins with the H antigen product of the transgenic enzyme.

Figures 1 through 10 are provided in accordance with title 37 of the Code of Federal Regulations, section 1.81.

Detailed Description of the Invention The present invention relates to the expression in vivo in mammary tissue of non-human mammals, of catalytically active heterologous glycosyltransferases that control the production of secondary gene product or resulting from the activity of the specific glucosyltransferase enzyme. . These glycosyltransferase enzymes control the synthesis of the free oligosaccharides, or the covalent attachment of the oligosaccharides with proteins or lipids. This expression is achieved in a cell by the use of genetic engineering to instruct the cell to produce specific heterologous glycosyltransferases (gene product). , - primary), and then employs the specific catalytic activity associated with each glycosyltransferase, to produce a specific product, the secondary gene product. In the case of glycosyltransferases, the secondary gene product includes not only the synthesized oligosaccharides, but also the glycosylated proteins and lipids. The oligosaccharides and the glycosylated proteins / lipids are secreted and found in the free form in the milk of the transgenic mammalian species. As used herein, and in the claims, the term "glycosylation" is understood to mean the modification after translation of a protein or lipid by an enzymatic process facilitated by expressed glycosyltransferase, which results in covalent attachment to the protein or the lipid of one or more monosaccharide units. This glycosylation is done instructing the cell to produce both the glucosyltransferases and the protein or lipid of interest. The protein or lipid of interest can be a homologous or heterologous entity. As used herein and in the claims, the term "homologous" is understood to refer to a composition or molecular form normally produced by the host cell or animal. As used herein and in the claims, the term "heterologous" is understood to refer to a composition or molecular form not normally produced by the host cell or animal. Genetic engineering techniques are used to incorporate foreign genetic material into the genome of the host animal, that is, genetic material derived from another species. As used herein and in the claims, the terms "transgenic cell" or "transgenic animal" are intended to refer to a host cell line or an animal that contains those transformed genomes. As used herein and in the claims, "transgenic products" are intended to refer to products derived from those transgenic entities; for example, milk derived from a transgenic cow is referred to as transgenic milk. The present invention is based, in part, on the production of a non-human transgenic mammal, wherein the cells comprising the mammary gland contain a transgene that expresses a desired glycosyltransferase. (The genome of the transgenic mammary cell can also be transfected with a gene that encodes a human protein). The resulting glycosyltransferase, when expressed in the transgenic host mammary cells, is useful to produce free soluble oligosaccharides in the milk produced by this transgenic animal. The expressed glycosyltransferase is also useful in the glycosylation of milk proteins homologues, or heterologous human proteins, when the transgenic mammary cell also expresses those proteins. The same concept can be applied to the modification of lipids. The present invention has wide application in the synthesis of oligosaccharides by different glycosyltransferases, such as fucosyltransferase, galactosyltransferase, glucosyltransferase, sialyltransferases, mannosyltransferases, xylosyltransferases, sulfotransferases, glucuronyltransferases, β-acetylgalactosaminyltransferases and N-acetylglucosaminyltransferases. The products of other classes of Golgi apparatus enzymes, such as acetylases, glucuronylepimerases, glucosidases, acetyltransferases, mannosidases, and phosphotransferases, can also be synthesized by the method described.

BEST MODE FOR CARRYING OUT THE INVENTION The following describes the incorporation of DNA encoding the production of a fucosyltransferase, particularly human alpha-1, 2-fucosyltransferase (hereinafter also referred to as Fuc-T), into the genome of forming cells. the non-human mammary glands. An example of a Fuc-T product is 2'-fucosyl lactose. This is one of the oligosaccharides in human milk, and has the chemical formula of fucose-alpha-1,2-Gal-β-1,4-Glc. Other Fuc-T products will include glycoproteins containing β-linked terminal galactose residues, which can be fucosylated by Fuc-T. The resulting carbohydrate structures of fucose-alpha-1,2-galactose-β-R, wherein R is selected from the group consisting of β-1,3-GlcNAc, β-1,4-GlcNAc, and the like , they are known in the field of blood group serology as the "H Antigen". It is well recognized by those skilled in the art that other glycosyltransferases and Golgi processing enzymes may also be used in accordance with the present invention. In the non-limiting examples described below, transgenic mice were employed. Mouse genomes do not contain or express the DNA encoding Fuc-T. Accordingly, if the transgenic mice produce either Fuc-T, 2 '-fucosyl-lactose, or H antigen, then a successful incorporation of the gene encoding Fuc-T must have occurred in the murine genome. It is well known in the art that it is possible to insert the DNA encoding the glycosyltransferases into the genome of the transgenic host cells. Some of the cell lines that could be used for transgenic expression of glycosyltransferases, are Chinese Hamster Ovary (CHO) cells, mouse L cells, A9 mouse cells, Baby Hamster kidney cells, C-127 cells, cells PC8, insect cells, yeast, and other eukaryotic cell lines. In a preferred embodiment of the present invention, the host cells are mammary cells, these cells comprising the mammary gland tissue of non-human transgenic mammals. Preferred embodiments of the present invention utilize mice, rats, rabbits, pigs, sheep, goats, horses, or transgenic cows. Particularly preferred embodiments use sheep, goats, or transgenic cows. A particularly preferred embodiment of the present invention is the use of bovine mammary tissue in lactating transgenic cows. The precise procedure used to introduce the altered genetic material into the host cell is not critical. Any of the well-known procedures for introducing the foreign nucleotide sequences into the host cells can be employed. These include the use of plasmid vectors, viral vectors, and any other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into the host cell. It is only necessary that the particular genetic engineering method used be able to successfully introduce at least one transgene in the host cell, which is then capable of expressing the desired glycosyltransferase. A preferred technique in the practice of the present invention is the transfection of an objective embryonic cell, transplanting the objective transgenic embryonic cell formed by the same to a recipient surrogate mother, and identifying at least one female progeny that is capable of producing the human oligosaccharides. free or recombinant human glycosylated protein in their milk. A more preferred embodiment of the present invention comprises the steps of transfecting an objective embryonic cell of a bobbin species, transplanting the objective transgenic embryonic cell, formed by the same to a recipient bovine mother, and identifying at least one female bovine offspring that is capable of of producing the free human oligosaccharides or the homologous or heterologous glycosylated recombinant protein in their milk. The following examples demonstrate the alteration of the genome of non-human mammalian host cells, by inserting therein heterologous DNA encoding specific glycosyltransferases. The transgenic host then expresses catalytically active specific glycosyltransferases that facilitate the production of a desirable secondary gene product, more specifically, a specific oligosaccharide. The glycosylation of milk proteins is also demonstrated. If in addition to DNA encoding the oligosaccharides, heterologous DNA encoding human milk proteins is also inserted into the host genome, then that host will also express human milk proteins. Since the same host will also express the glycosyltransferase, the glycosylation of human milk proteins will be presented with certain specific oligosaccharides. Human milk proteins of interest include secretory immunoglobulins, lysozyme, lactoferrin, kappa-casein, lactoperoxidase, alpha-lactalbumin, β-lactalbumin, and lipase stimulated by the bile salt. This approach to oligosaccharide synthesis and protein / lipid glycosylation has several advantages over other currently available methods. This approach is based on the novel combination of: (a) the use of transgenic mammary cells for the synthesis of sugar nucleotides from sources of natural carbon, such as glucose; (b) the expression of heterologous recombinant glycosyltransferase genes in transgenic mammalian cells; (c) the production of heterologous oligosaccharides of the desired structure by the lactating or natural mammary glands of transgenic animals, this production being the result of the enzymatic activity of the expressed heterologous glycosyltransferase enzymes; and (d) the use of the heterologous glycosyltransferase enzyme to glycosylate homologous or heterologous proteins or lipids. The experiments described below illustrate the following points: (a) A human alpha-1, 2- fucosyltransferase gene was isolated and cloned from a cell line of 0 human epidermal carcinoma. This enzyme is responsible for the synthesis of the oligosaccharide 2 '-fucosyl lactose, and the glycosylation of proteins with the blood group-specific antigen H; (b) The functional nature of the gene was demonstrated by its ability to express catalytically active alpha-1, 2-fucosyltransferase in cultured non-human cell lines. The presence of 1,2-fucosyltransferase was demonstrated by assays of enzymatic activity specific for this enzyme. The presence of the catalytically active alpha-1,2-fucosyltransferase was also demonstrated by the use of the immunofluorescence technique, to show the presence of the H antigen on the surface of the cells expressing the enzyme; (c) The utility of this gene in the formation of non-human transgenic animals capable of expressing the alpha-1,2-fucosyltransferase gene product was demonstrated by the successful development of transgenic mice carrying the alpha-1 gene, Human 2-fucosyltransferase which is capable of expressing the catalytically active alpha-1, 2-fucosyltransferase; (d) The expression in the breast tissue of. a non-human transgenic animal of catalytically active human alpha-1, 2-fucosyltransferase. The presence of the enzyme was established through tests of direct enzymatic activity and immunofluorescence using antibodies that exhibit binding specificity for the enzyme. (e) The formation of secondary gene products resulting from the catalytic activity of human alpha-1, 2-fucosyltransferase expressed in non-human milk. These products include the release of human oligosaccharide, 2'-fucosyl lactose, in milk, and the glycosylation of milk proteins with the H antigen product of the enzyme. The presence of the secondary gene products was established through the biochemical analysis of the compounds 5 and immunofluorescence using lectins exhibiting binding specificity for the H antigen. The following examples are provided as representative of the scope of the invention, and should not be considered to limit the invention claimed in the present one. Examples 1 and 2 use tissue culture systems. These in vitro experiments were undertaken to prove that the expression of enzymatically active heterologous glycosyltransferases was possible. Examples 1 and 2 are not critical to enable the present invention, and are provided exclusively for the purpose of securing a - .. understanding and appreciation of the invention. Examples 3, 4, 5, and 6 prove that the production in vivo of heterologous secondary gene products in the milk of 0 transgenic non-human mammals is possible. Examples 3 to 6 are provided for the purpose of making the teachings, scope, and claims of the invention possible. In light of the above, the Requesters believe that a deposit of biological material is not required in accordance with Title 37 of the Code of Federal Regulations, section 1.802.

Example 1: Isolation of the Gene for Human Alpha-1,2-Fucosyltransferase from the Cellular Line of Human Epidermal Carcinoma. The cDNA encoding alpha-1, 2-fucosyltransferase was isolated from a cDNA library of an epidermal carcinoma cell line (A 431), since alpha-1, 2-fucosyltransferase was previously cloned from this source (V.P. Rajan et al., J. Biological Chemistry, volume 264, pages 11158-11167, 1989). This reference is incorporated herein by reference. After amplification mediated by polymerase chain reaction (PCR) of the protein encoding the sequence, the cDNA was cloned into a bacterial vector to determine the cDNA sequence of the amplified gene. The DNA sequence was determined from each of six independently isolated clones of human alpha-1,2-fucosyltransferase. This nucleotide sequence and the corresponding amino acid sequence are shown in Figure 1. In order to determine the sequence of cDNA previously noted, two alpha-1, 2-fucosyltransferase preparers were designed, each containing 31 nucleotides (31- mers), based on the published alpha-1, 2-fucosyltransferase cDNA sequence. The BigNH2 preparer contained the methionine initiator residue at position 27, where transcription of Fuc-T (start of open reading frame) begins. The second preparer, BigCOOH, contained a stop codon at position 10. The primers are indicated in Figure 2. The polymerase chain reaction included approximately 1 μM of each preparator, 1 microgram of template with chain reaction regulator. polymerase, and polymerase Taq. The polymerase chain reaction was performed in a thermal cycler (Perkin and Elmer, Model 840) using a temperature cycle of 94 ° C for 1 minute, 60 ° C for three minutes, 72 ° C for three minutes, for 30 cycles , followed by an extension of 5 minutes at 72 ° C. The product of the polymerase chain reaction was electrophoresed, about 0.8 weight percent / volume of low melting point agarose. A 1.1 kilobase fragment was detected. This fragment was separated and subcloned into the PCR II cloning vector. One of the transformants, hereinafter referred to as the selectant, was selected and characterized both by restriction analysis and by nucleotide sequence analysis.

DNA sequencing was performed using an Applied Biosystems Model 373A automatic DNA sequencer. The restriction pattern of the insert indicated similarity with the coding region of alpha-1,2-fucosyltransferase. The nucleotide sequence of this candidate clone was identical to the published sequence, except at position 640. In vitro site-directed mutagenesis was employed to correct this single defective base, thereby forming the wild-type sequence that was used in the transfection experiments described later.

Example 2; Expression in the Host Cell of Human Glucosyltransferases. This example describes the transfection of cultured mouse L cells and Chinese Hamster Ovary (CHO) cells with a gene capable of expressing the specific human glycosyltransferase, alpha-1, 2-fucosyltransferase or Fuc-T. These cell lines were selected for transflection, since their natural genomes do not carry the DNA encoding the Fuc-T. If after transfection, it is shown that the cell lines produce either Fuc-T or the enzymatic product thereof (2 '-fucosyl-lactose or the H antigen bound to the glycoproteins), then a transfection of success. This is demonstrated by the immunofluorescence technique, using specific antibodies and / or a specific lectin that binds selectively with the H antigen. The Fuc-T gene used for transfection was obtained as described in Example 1. The transfection and The materials used in it are described below.

Phenyl-β-D-galactoside was obtained from Sigma Chemical Co. The nucleotide sugar, GDP-L- (U-14C) fucose, with a specific activity of 278 mCi / millimole, was purchased from Amersham Corporation. The human epidermal carcinoma cDNA library A431 was obtained as a present from Dr. Nevis Frigien, The University of Miami, Oxford, Ohio. The PCR II vector was purchased from Invitrogen Corporation. The pQEll expression vector was purchased from Qiagen Inc. Plasmid pSV2-neo was obtained from Pharmacia Fine Chemicals Corporation. Plasmid pMet-FucT-bGH was obtained from Drs. Xhou Chen and Bruce Kelder at the University of Ohio, Athens, Ohio. This construction contains the cDNA that encodes Fuc-T. The primers were synthesized by Operon Technology or Fischer Scientific Corporation. The mouse monoclonal antibody for H antigen was purchased from Dako Corporation. Goat-to-mouse antibodies labeled with fluorescein isothiocyanate were purchased from Sigma Chemical Company. Rabbit polyclonal antibodies to alpha-1, 2-fucosyltransferase were cultured as a means to detect the expression of this enzyme. In order to cultivate enough enzyme to act as the antigen in the induction of the antibody, the insert of the selector was subcloned into an inducible expression vector in the frame with a 6XHis tag (pQEll). A protein labeled with 6XHis was easily purified with a nickel affinity chromatography column. To avoid possible cellular toxicity, the hydrophobic region of alpha-1,2-fucosyltransferase was deleted. To do this, two new trainers were built. The first, a BamHI-NH2, hybridizes to the template at position 60; the second preparer, Salí COO, extends into the stop codon. The BamHI site and the Sal I site were designed on the upstream and downstream trainers. The product of the polymerase chain reaction was subcloned into the frame at a BamHI / Sal I site of the pQEll expression vector, allowing fusion with the 6XHis tag. Three milligrams of the fusion protein (alpha-1, 2-fucosyltransferase-6XHis) was purified using a Ni-agarose affinity column. This material was used to culture polyclonal rabbit antibodies that exhibited specificity against Fuc-T.

Cell Line v Cultivation; Mouse L-cells and Chinese Hamster Ovary cells were obtained from the American Tissue Culture Collection (ATCC) in Washington, D.C. Cells were cultured in an alpha minimal essential medium (alpha-MEM, GIBCO, Gran Island, New York) supplemented with 10 percent fetal calf serum (GIBCO), penicillin, 80 micrograms / milliliter (Sigma), streptomycin 80 micrograms / milliliter (Sigma), and L-glutamine (Sigma), hereinafter referred to as alpha-MEM / 10 percent FCS. The transfected L cells were cultured on alpha-MEM containing G418 (GIBCO) at 400 micrograms / milliliter. The transfected Chinese Hamster Ovary cells were cultured on alpha-MEM containing G418 (GIBCO) at 1000 micrograms / milliliter.

Transient Transfection: L cells were cultured on 8-well chamber slides (Lab-Tek) to a confluence level of 75 percent. A transfection cocktail was added to each DNA chamber pMet-Fuc-bGH (2 micrograms), lipofection (2 microliters), and 200 microliters of Opti-MEM medium (GIBCO). After 6 hours of incubation at 37 ° C, 200 microliters of alpha-MEM / 10 percent FCS was added, and after 48 hours of another incubation at 37 ° C, the slides were processed for indirect immunofluorescence as described further ahead. The ability of the cloned cDNA fragment to encode the functional alpha-1, 2-fucosyltransferase was tested by demonstrating the presence of the catalytic product of this enzyme, i.e., the H antigen, on the cell surface of the cultured mouse L cells. (L cells do not normally have the H antigen on their membrane). The wild type insert of the selector noted in Example 1 was subcloned into the plasmid pMet-bGH at an EcoRI site. In this construct, the expression of alpha-1,2-fucosyltransferase activity is under the control of the metallothionein promoter. This promoter is zinc-inducible. Mouse L cells were transiently transfected with the pMet-Fuc-bGH construct, and the presence of the structure of the H antigen on the cell surface was confirmed using the immunofluorescence technique with mouse monoclonal antibodies against the primary H antigen as described later. Secondary antibodies labeled with fluorescein were goat against mouse antibodies. The presence of the H antigen was further confirmed using the fluorescein-labeled lectin, Ulex europaeus agglutinin 1, which binds specifically to the fucose-alpha-1,2-galactose structures.

Indirect immunofluorescence. Successful transfection was demonstrated by the presence on the cell surface of the H antigen. Indirect immunofluorescence assays were performed using 8-well tissue culture chamber slides. Cells were coated in each chamber to an appropriate density, incubated overnight at 37 ° C, and then assayed for H antigen. Chamber slides were washed with phosphate buffered saline (PBS), fixed with 100 microliters of a 2 percent solution of formalin in Hanks Balanced Salt Solution (HBSS), and permeabilized with saponin (2 milligrams / milliliter; Sigma) in 1 percent FCS, and incubated with a dilution of 1 1,000 of the anti-H antibody for 60 minutes in a humid chamber at room temperature. Subsequently, the slides were washed three times with PBS, and incubated for an additional 60 minutes with a 1: 1000 dilution of goat anti-mouse antibody labeled with FITC at room temperature in a humid chamber. Humidification prevented drying of the sample. The efficiency of transfection gives L cells, or the percentage of transformed L cells expressing H antigen, based on immunofluorescence, was about 30 percent. The previously noted results clearly demonstrate the successful transfection of non-human mammalian cell lines with the DNA encoding Fuc-T. The transfected cultured cell lines produce not only the primary gene product, Fuc-T, but also the modified glycoproteins. As a result of the activity of Fuc-T, the modified proteins carry the H antigen. These results prove that the cloned cDNA fragment encoding Fuc-T is capable of expressing the enzymatically active Fuc-T. Accordingly, this cDNA was used for the production of transgenic animals as described below.

Example 3: Non-Human Transgenic Mammal that Has the Gene that Encodes a Specific Human Glucosyltransferase. This experiment proves that transgenic non-human mammals are capable of producing a catalytically active heterologous glycosyltransferase. In a more specific way, the production by transgenic animals of human alpha-1, 2-fucosyltransferase is tested. Transgenic mice were produced by microinjection of human Fuc-T cDNA into the genome of mouse embryos. Fertilized mouse eggs were isolated at the single-cell stage, and the male pronuclei were injected with the transgenic construct containing the human alpha-1, 2-fucosyltransferase gene as shown in Example 1. These embryos were implanted then in pseudo-pregnant mice that had previously been coupled with sterile males. The founder pups of transgenic mice were identified after approximately 25 days after birth, using polymerase chain reaction amplification to analyze the chromosomal DNA obtained from a tail fragment with probes specific for the inserted human gene. Standard techniques were used in this field to achieve the desired transformation. These details have been described more fully in the following references, which are incorporated herein by reference, and which were also discussed hereinabove. (a) International Patent Application Number PCT / US90 / 06874; (b) International Patent Application Number PCT / DK93 / 00024; (c) International Patent Application Number PCT / GB87 / 00458; and (d) International Patent Application Number PCT / GB89 / 01343. One aspect of the present invention relates to the expression of a catalytically active human glycosyltransferase in the milk of a non-human mammal, and the use of that glycosyltransferase to effect the formation of the desired secondary gene product. In order to achieve the specific expression by the mammary gland of the human gene during lactation of the transgenic mice, the regulatory sequence (promoter) of the serum acid protein (WAP) of a mouse was used to generate a transgenic construct for the expression of human alpha-1, 2-fucosyltransferase. The urine whey protein acid promoter was received as a present from Dr. L. Henninghauser of the National Institutes of Health, Bethesda, Maryland. This material was used to construct the plasmid pWAP-polyA shown in Figure 3. This plasmid contains the polyadenylation signal sequence (polyA) of bovine growth hormone at the 3 'end of the fusion gene, which results in the expression effective, processing, and stability of messenger RNA. The human alpha-1, 2-fucosyltransferase (Fuc-T) gene was inserted into this plasmid to result in the formation of plasmid pWAP-polyA-Fuc-T illustrated in Figure 4. This plasmid was used for the microinjection of mouse embryos as described above. Using microinjections of DNA in concentrations of 2 and 4 micrograms / milliliter, a total of 85 pups were obtained from 16 injections. Only two injections did not result in pregnancy. The size of the bait of a single injection was normal, averaging three to ten pups per litter. Tail biopsies were performed on all 85 mouse pups. It was determined, by the tail biopsy assay, that nine of the founder population, hereinafter referred to as F0, possessed the gene encoding human alpha-1, 2-fucosyltransferase. This corresponds to a production efficiency of transgenic mice of approximately 11 percent. This falls within the scale of expected production efficiency of between 5 and 25 percent. The F0 progeny comprised eight males and one female. Six of the founders were then bred with normal mice, resulting in a total of one progeny out of a total of 98. Progeny 38, (hereinafter referred to as Fl) possessed the gene encoding alpha-1, 2-fucosyltransferase human, as determined from tail biopsies and polymerase chain reaction analysis. This corresponds to an efficiency Fl of approximately 36 percent. The Fl generation is comprised of 19 males and 19 females. Table 2 summarizes the results obtained.

TABLE 2 PRODUCTION EFFICIENCY OF THE GENERATION Fl FROM SIX FOUNDING MICE Founder # Total Number Progeny Number Efficiency of the Trans-Male Proenia of the Trans-Male Female agenesis (%) 6 16 4 2 37.5 28 18 2 2 22.2 29 18 4 6 55.6 34 13 3 6 69.2 54 15 2 1 20.0 72 18 4 2 33.3 Fifteen of the F (second generation) females were allowed to mature, and were reared with normal mice. Pregnant females were allowed to give birth. The milk of four of these Fl mothers was harvested ten days after birth. The collection of the milk was done using one of two techniques that are standard in this field: (a) breast suction using a vacuum line connected to a trap flask and a suction cup; or (b) anesthetize and sacrifice the animal, and then pierce the teats to release the fluid content of the mammary gland. The milk samples were kept frozen on dry ice until they were subjected to the analytical procedures described below. The collected milk samples were prepared to initially separate the oligosaccharides from the milk proteins and lipids. This was achieved using the methods described by A. Kobata (Methods in Enzymology, chapter 24, volume 28, pages 262-271, 1972), and a. Kobata et al. (Methods in Enzymology, chapter 21, pages 211-226, 1978). The milk samples were treated as follows. Samples typically of 90 to 100 microliters, obtained from control animals (non-transgenic), and transgenic, were centrifuged at 10,000 relative centrifugal force (RCF) for 20 minutes in conical polypropylene centrifuge tubes. The centrifugation resulted in the separation of the milk into two layers: an upper layer of cream consisting mostly of lipids, and a lower layer. The lower layer, which contained soluble material, was removed and transferred to a new centrifuge tube. Two equivalent volumes of ice-cold ethanol were added, mixed by vortexing, and centrifuged at 10,000 RCF. The supernatants soluble in ethanol were recovered and concentrated by evaporating the alcohol using a Speed-Vac concentrator. The granule of insoluble protein in ethanol was kept frozen at -70 ° C until another analysis. Following the IX) concentration, the extracts containing the oligosaccharide were resuspended in water to the exact volume of the original milk sample. These resuspended samples were kept at 4 ° C in an autosampler, refrigerated until another use. When appropriate, these samples were submitted to composition analysis, as described in Examples 4 to 7. One aspect of the present invention is the transgenic expression of heterologous glycosyltransferases in the mammary gland of non-human mammals having milk. The The expression of heterologous glycosyltransferases can be demonstrated in two ways: (a) directly, by determining the presence of the enzyme (primary gene product) itself, - and (b) indirectly, by determining the presence of the product enzyme (secondary gene product: oligosaccharide or glycosylated protein) in the milk of the transgenic animal. As noted above, the murine genome does not encode the specific alpha-1, 2-fucosyltransferase responsible for the synthesis of the H antigen. Therefore, if either Fuc-T or the Fuc-T products are present in the milk of the transgenic mice, then successful transgenesis has been presented, which provides a unique means to synthesize, and consequently, to obtain secondary gene products. An important aspect of the present invention is the production of heterologous secondary gene products in the milk of non-human animals. As noted above, the secondary gene products can comprise not only the immediate product of the enzyme, the oligosaccharide, but also the glycosylated homologous or heterologous protein or lipids, which are glycolized through the covalent attachment of the oligosaccharide with the protein or lipid. The milk harvested from Example 3 was analyzed for the presence of human alpha-1, 2-fucosyltransferase, and also for the presence of secondary gene products, specifically 2 '-fucosyl lactose and proteins glycosylated with the H antigen. Examples 4 , 5, 6, and 7 test the production of human Fuc-T and Fuc-T products in the milk of non-human animals.

Example 4: Analysis Testing the Production of a Specific Glucosyltransferase in the Milk of Non-Human Transgenic Mammals. This example demonstrates the feasibility of obtaining human alpha-1, 2-fucosyltransferase in the milk of transgenic mice. As noted above, the murine serum acidic protein promoter was employed to ensure expression by the site-specific mammary gland of human alpha-1, 2-glucosyltransferase. The milk protein precipitate insoluble in ethanol, obtained from the mice as described above in Example 3, was resuspended in a polyacrylic amide gel electrophoresis regulator (PAGE) containing sodium dodecyl sulfate (SDS). The SDS-PAGE regulator volume used to resuspend the protein granule was exactly equal to the original volume of the milk sample. The reconstituted samples were assayed for the presence of alpha-1,2-fucosyltransferase. This presence was determined using immunoblot technology as described below. More specifically, Western blots were employed. Five microliter samples of the resuspended protein granule were electrophoresed in SDS-PAGE on a 12.5 percent polyacrylic amide gel. Electrophoresis was performed at 150 volts. Following the electrophoresis, the resolved proteins were transferred to a nitrocellulose membrane. Transmigration was performed for one hour at 100 volts using a 12.5 mM Tris-HCL buffer, pH 7.5, containing 96 mM glycine, 20 percent methanol, and 0.01 percent SDS. Following the transfer, the remaining unbound reactive groups on the nitrocellulose membranes were blocked by incubation in a 50 mM Tris-HCL buffer, pH 7.5, containing 0.5 M NaCl and 2 percent gelatin, hereinafter referred to as TBS. Then, the membranes were washed three times in TBS containing 0.05 percent Tween-20. The membranes were incubated for 18 hours in 1 percent gelatin / TBS containing a 1: 500 dilution of polyclonal rabbit antibody with a specificity against alpha-1,2-fucosyltransferase. This polyclonal antibody was obtained as described in Example 2. Following the rinsing with TBS-Tween, the membrane was then incubated with a solution of 1% gelatin-TBS containing goat anti-rabbit IgG previously conjugated with horseradish peroxidase. . Then the membrane was washed with TBS-Tween. The presence and position of the proteins on the nitrocellulose membrane were visualized by incubating the membrane in a volume of 50 milliliters of TBS containing 0.018 percent hydrogen peroxide and 10 milliliters of methanol containing 30 milligrams 4-chloro-naphthol . Figure 5 shows the result of this experiment for milk samples obtained from a control animal (non-transgenic) and two transgenic animals. The transgenic animals are referred to in Figure 5 as 28-89 and 29-119. Non-transgenic animals are referred to in Figure 5 as the control. Figure 5 indicates that a very intense band is clearly present in the milk samples obtained from the two transgenic animals, but is absent from milk obtained from the non-transgenic control animal. Clearly there are intense bands present at a relative molecular weight of approximately 46 kilodaltons, corresponding to the predicted molecular weight of alpha-1,2-fucosyltransferase. There are also intense bands present in the positions corresponding to the lowest relative molecular weights on the scale of approximately 30 to 25 kilodaltons. These bands are absent in the sample of milk derived from the non-transgenic sample. Without being bound to the inventors, it is speculated that these lower molecular weight bands probably correspond to Fuc-T fragment. These results prove that the milk samples from the transgenic samples contain Fuc-T, while the milk samples from the non-transgenic animal do not contain Fuc-T.

Example 5: Analysis Testing the Production of Specific Heterologous Secondary Gene Products in the Milk of Non-Human Transgenic Mammals. This example proves the feasibility of obtaining heterologous secondary gene products in the milk of non-human transgenic mammals. More specifically, this example demonstrates the ability to obtain the secondary gene product of Fuc-T in the milk of a non-human animal. More specifically, the presence of the secondary gene product, 2 '-fucosyl lactose, was demonstrated in the transgenic milk. Control non-transgenic mouse milk does not contain 2 '-fucosyl lactose. Evaporated oligosaccharide extracts, obtained as described in Example 3, were analyzed and separated using a combination of high pressure liquid chromatography and ion exchange chromatography on a Dionex apparatus as previously described by Reddy and Bush (Analytical Biochemistry , volume 198, pages 278-284, 1991) and Townsend and Hardy (Glycobiology, volume 1, pages 139-147, 1991). These techniques are well known and standard in this field. The specifics of the experimental establishment, the elution profiles and the conditions for the separation and analysis of the oligosaccharide extracts were as follows: The Dionex apparatus was adapted with a degasser to remove the C02 from the elution regulators, a suppressor of ions to remove the ions from the eluents in the column, and an online conductivity meter to ensure the removal of the ions by the ion suppressor. The parameters of the chromatography were as follows: Test time: 45 minutes Peak width: 50 seconds Peak threshold: 0.500 Peak Area rejection: 500 Injection Volume: 20 liters Flow rate: 1.0 milliliters / minute.

The gradient elution program, presented in Table 3, comprised the following three eluents: Eluent 1: 600 mM sodium acetate in 100 mM sodium hydroxide. Eluent 2: Water Milli-Q NanoPure Eluent 3: 200 mM sodium hydroxide TABLE 3 ELUTION GRADIENT PROGRAM Time (minute) Fli: (ml / min)% # 1% # 2% # 3 0.0 1.0 0 50 50 12.0 1.0 0 50 50 12. 1 1.0 7 46 47 20.0 1.0 7 46 47 20.1 1.0 10 45 45 27.0 1.0 10 45 45 27.1 1.0 50 25 25 32.0 1.0 50 25 25 32.0 1.0 0 50 50 45.0 1.0 0 50 50 90.0 0.1 0 50 50 The eluted fractions were collected every 0.5 minutes. The chromatographic profiles of the milk samples obtained from two control mice and four transgenic animals expressing alpha-1,2-fucosyltransferase are shown in Figures 6A to 6F. It was determined that the 2 '-fucosyl lactose (which is the oligosaccharide product synthesized by the enzyme encoded by the transgene) is eluted after the lactose. A review of the profiles revealed that only the transgenic animals produce milk containing a carbohydrate that co-elutes with the standard 2 '-fucosyl lactose. Based on the chromatographic peak areas, it was possible to calculate the concentrations of 2 '-fucosyl lactose present in the milk samples from transgenic animals using standard techniques. The data is summarized in Table 4.

TABLE 4 Concentration of 2 '-fucosyl lactose in different non-human milk samples. Donor Concentration of 2 '-fucosyl lactose (mq / L) 1. Control (non-tranegenic) 0 2. Transgenic 28-29 711 29-119 468 34-34 686 72-66 338 These data prove, in accordance with the present invention, that a secondary gene product, ie, a 2 '-fucosyl lactose, can be produced in the milk of transgenic non-human mammals. To further characterize the oligosaccharide, a different method was used for carbohydrate analysis. Fluorophore-assisted carbohydrate (FACE) electrophoresis is a technology first described by P. Jackson, J. Chromatography, volume 270, page 705-713, 1990. The FACE technique was employed to unequivocally demonstrate that the carbohydrate that co-elutes with 2 '-fucosyl-lactose, has the same mobility as the authentic 2 '-fucosyl-lactose standards in an electrophoresis system. This provides further confirmation that the identity of the oligosaccharide contained in the transgenic milk sample is 2 '-fucosyl lactose. In order to conduct FACE experiments with the putative 2 '-fucosyl lactose, the separated fractions were pooled during Dionex-HPLC chromatography. The fractions between the arrows (indicated on the abscissa) in Figures 6A to 6F were grouped from each sample. Portions of each group (1/8) were obtained from the control and from two transgenic mice, and labeled for 3 hours at 45 ° C using 8-aminonaphthalen-2,3,6-trisulfonic acid (ANTS) from Glyko Inc. (Novato, California). The dried samples were resuspended in 5 microliters of the labeling reagent, and 5 microliters of reducing reagent solution (sodium cyanoborohydride) was incubated at 45 ° C for 3 hours. The resulting labeled samples were dried and resuspended in 6 microliters of deionized water. From this solution, a 2 microliter aliquot was transferred to a fresh microcentrifuge tube. Two microliters of charge buffer containing glycerol was added, and then the mixture was mixed vigorously. Then the total mixture (4 microliters) was subjected to electrophoresis in an "O-linked oligosaccharide gel" (Glyko). Electrophoresis was conducted at a constant current of 20 milliamps and at 15 ° C. The profile of the migrated gel pattern thus obtained was imaged using a Millipore imaging apparatus. Figure 7 shows the image of the gel obtained in this way. The sample from a control mouse (lane 2) shows a single band that migrates at the position of a standard lactose marker. Samples obtained from transgenic mice (lanes 3 and 4) contained an additional band of a higher molecular weight. This band indicated in the figure with an arrow, migrates in the position of a standard 2 '-fucosil-lactose (track 6). Another characterization of the oligosaccharide was performed by the incubation of aliquots equivalent to 1/8 of the groups illustrated in Figures 6A to 6F in the presence of the enzyme fucosidase, which is specifically dissociated in the alpha-1,2-fucose linkages. This enzyme used was derived from Corynebacterium sp. and was purchased at Panvera Corp. Madison, Wisconsin. The dried oligosaccharides were incubated overnight in the presence of 20 microliters of sodium phosphate buffer, pH 6.0, containing 20 milliunits of the enzyme at 37 ° C. Then the digestions were labeled with ANTS, and subjected to electrophoresis as described above. The gels in Figure 8 show the results of this experiment. It can be easily seen that the material that is co-eluted with 2 '-fucosyl lactose in Dionex-HPLC chromatography and that co-migrates with the same molecule after being labeled with ANTS and electrophoresis, is also susceptible to action of the specific hydrolytic enzyme, alpha-1,2-fucosidase. The 3 '-fucosyl-lactose (which is the most similar isomer of the 2' -fucosyl-lactose) is not affected by the enzyme. This experiment further confirms the identity of the oligosaccharide in the transgenic milk sample as 2'-fucosyl lactose. In contrast, milk samples obtained from non-transgenic control animals (tracks 6 and 14) following the hydrolysis, they produce only a single band (lanes 7 and 15) that migrate at the position of the galactose standard.

Example 6: Analysis to Test the Identity of Oligosaccharide Produced in the Milk of Non-Human Transgenic Mammals. This experiment evaluated the monosaccharide units comprising the oligosaccharide. For this purpose, the pooled milk samples obtained from the control and the transgenic mice were thoroughly treated with a mixture of glucosidases. The aliquots (1/8 of the total in 20 microliters of water) of the groups illustrated in Figures 6A to 6F were dried by evaporation in conical tubes. The dry content was resuspended in 20 microliters of a solution containing 20 milliunits of alpha 1, 2-fucosidase (Panvera, Madison, Wisconsin) and 20 microliters of a suspension containing 30 units of E. coli β-galactosidase ( Boehringer Mannheim, Indianapolis, Indiana). The resulting suspensions were incubated for 18 hours at 37 ° C under an atmosphere of toluene. In this manner, only oligosaccharides susceptible to sequential actions of fucosidase and β-galactosidase were hydrolysed in their corresponding monosaccharide units. After incubation, the mixtures were dried in a Speed Vac concentrator. The oligosaccharides resulting from this hydrolysis were labeled as described above in Example 5. The labeled monosaccharides were subjected to electrophoresis in a "monosaccharide gel" (Glyko). Electrophoresis was performed at a constant voltage of 30 milliamps for 1 hour and 10 minutes. Figure 9 shows the results of this experiment. Milk samples obtained from transgenic animals (lanes 2 and 4) contain three bands corresponding to fucose, galactose, and glucose. The monosaccharides released from a standard 2 '-fucosyl lactose (lane 1) are identical to the monosaccharides released from the oligosaccharide groups obtained from two transgenic animals (lanes 2 and 4). The 3 '-fucosyl lactose is not affected by the enzymatic action of the glucosidases (lane 3). This result unequivocally establishes the identity of the oligosaccharide in the transgenic milk as 2 '-fucosyl lactose. Collectively, these results discussed above prove that the invention, as described and claimed, makes possible the production of secondary gene products in the milk of transgenic animals. More specifically, the experimental data prove the feasibility of obtaining oligosaccharides in the milk of transgenic animals that contains a transgene comprised, in part, of DNA encoding glycosyltransferases. To further corroborate the invention, it was decided to demonstrate the presence of other glucoconjugates, such as glycoproteins in the milk of the transgenic animals. These glycoproteins are covalent adducts of protein and oligosaccharide, wherein the oligosaccharide is the product of the glucosyltransferase. The oligosaccharide is covalently bound to the protein by the glycosyltransferase.

Example 7: Analysis Testing the Production of Glucoconjugates in the Milk of Non-Human Transgenic Mammals. This example demonstrates the feasibility of obtaining glycoproteins in the milk of non-human transgenic animals. The oligosaccharide fraction is the same oligosaccharide produced as a result of the activity of the primary gene product, the glycosyltransferase. The resulting glycosylated protein is an example of a secondary gene product. Western blots were prepared from the milk proteins of transgenic and control animals in the manner described in Example 4. However, instead of incubating the transferred membrane with polyclonal rabbit antibodies, the membrane was incubated with the Ulex lectin europaeus agglutinin 1 (UEA 1). This lectin specifically binds to the alpha-1,2-fucose bond. For this purpose, the protein granules described in Example 3, were centrifuged at 13,000 xg for 10 minutes, the supernatant (excess of ethanol and water) was removed, and the resulting granules were resuspended in a sample regulator volume of SDS- PAGE equal to the original volume of milk. Five microliters of these extracts were electrophoresed on 12.5 percent polyacrylic amide SDS-PAGE, as described in detail in Example 3. Following electrophoresis, the proteins were transferred to nitrocellulose membranes for 1 hour at 100 minutes. volts in 12.5 mM Tris-HCl, 96 mM glycine, 20 percent methanol, 0.01 percent SDS, pH 7.5. The nitrocellulose membranes were blocked for 1 hour with 2 percent gelatin in TBS (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl), and washed 3 x 5 minutes in TBS containing 0.05 percent Tween-20. . The membranes were then incubated for 18 hours in 1 percent gelatin / TBS containing a 1: 500 dilution of UEA-1 labeled with peroxidase (Sigma, St. Louis Mo.). The resulting membrane was then washed, and the proteins visualized by incubation in a mixture of 50 milliliters of TBS containing 0.018 percent hydrogen peroxide, and 10 milliliters of methanol containing 30 milligrams of 4-chloro-naphthol (Bio Rad, Richmond, California). Figure 10 shows a photograph of the proteins visualized using this technique. It is clear that only the transgenic animals produced milk containing fucosylated proteins specifically recognized by the UEA-1 lectin. These proteins migrated with a relative molecular weight of 35-40 kilodaltons, and are believed to be casein. These results indicate that glycoproteins carrying the 2'-fucosyl lactose oligosaccharide (H antigen) have been produced in the milk of transgenic animals that carry a transgene encoding alpha-1,2-pucosyltransferase. The milk of the non-transgenic control animals did not contain glycoproteins carrying 2 '-fucosyl lactose. Examples 3 to 7 have proven that it is possible to produce non-human transgenic mammals capable of synthesizing secondary gene products in their milk. More specifically, it is possible to produce non-human transgenic mammals that express human glucosyltransferases in the mammary tissue, resulting in the presence of human oligosaccharides and glycosylated glucoconjugates in the milk of these animals.

Industrial Applicability The invention as described and claimed herein, solves a need felt for a long time in that it provides a means to obtain large quantities of desired oligosaccharides and glycoconjugates. The desired oligosaccharides and glycoconjugates can be isolated from the milk of the transgenic mammals, and can be used in the preparation of pharmaceutical products, diagnostic kits, nutritional products, and the like. Whole transgenic milk can also be used to formulate nutritional products that provide special advantages.

Transgenic milk can also be used in the production of specialized nutritional food products. The invention as described and claimed, eliminates laborious organic chemistry and the synthesis of immobilized enzymatic chemistry of these very important materials that have use in pharmaceutical, research, diagnostic, nutritional, and agricultural formulas. Having described the preferred embodiments of the present invention, it will be obvious to those of ordinary skill in the art that various modifications may be made to the embodiments described, and that those modifications are intended to be within the scope of the present invention.

SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANT: Abbott Laboratories, ROSS Products Division (ii) TITLE OF THE INVENTION: Humanized Milk (iii) SEQUENCE NUMBER: 1 (iv) CORRESPONDENCE ADDRESS: (A) RECIPIENT: Donald O. Nickey ROSS Products Division Abbott Laboratories (B) STREET: 625 Cleveland Avenue (C) CITY: Columbus (D) STATE: Ohio (E) COUNTRY: United States of America (F) POSTAL CODE: 43215 (v) LEGIBLE FORM COMPUTER: (A) - TYPE OF MEDIUM: 3.5-inch diskette, storage of 1.44 Mb. (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: MS-DOS Version 6.21 (D) SOFTWARE: WordPerfect Version 6.0a (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: Not applicable (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (614) 624-7080 (B) TELEFAX: (614) 624-3074 (C) TELEX: none (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1,155 base pairs. (B) TYPE: Nucleic acid (C) TYPE OF CHAIN: Simple. (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cloned cDNA representing the product of a segment of human genomic DNA. (A) DESCRIPTION: GDP-L-fucose-ß-D-galactosida2-alpha-fucosyltransferase. (iii) HYPOTHETICAL: (iv) ANTI-SENSE: (v) TYPE OF FRAGMENT: The entire amino acid sequence is provided. (vi) ORIGINAL SOURCE: Carcinoma Cell Line Epidermal Human. (A) ORGANISM: (B) CEPA: (C) INDIVIDUAL INSULATED: (D) DEVELOPMENT STAGE: (E) HAPLOTIPO: (F) TYPE OF TISSUE: (G) TYPE OF CELL: (H) CELLULAR LINE: (I) ) ORGANELO: (vii) IMMEDIATE SOURCE: Cell Line of Human Epidermal Carcinoma (A) LIBRARY: (B) CLON: (viii) POSITION IN THE GENOME: (A) CHROMOSOME / SEGMENT: 19 (B) POSITION OF THE MAP: (C) UNITS: (ix) CHARACTERISTICS: (A) NAME / KEY: (B) LOCATION: (C) IDENTIFICATION METHOD: DNA sequencing and restriction analysis. (D) OTHER INFORMATION: The encoded product of nucleotide SEQ ID NO: 1: is the enzyme, GDP-L-fucose-β-D-galactoside 2-alpha-fucosyltransferase, having the amino acid sequence described in SEQ. ID NO: 1 :. This enzyme is responsible for the synthesis of 2 '-fucosyl lactose. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAATTCGGCT TATCTGCCAC CTGCAAGCAG CTCGGCC ATG TGG CTC CGG AGC CAT 55 Met Trp Leu Arg Ser His 1 5 CGT CAG CTC TGC CTG GCC TTC CTG CTA GTC TGT GTC CTC TCT GTA ATC 103 Arg Gln Leu Cys Leu Ala Phe Leu Leu Val Cys Val Leu Ser Val lie 10 15 20 TTC TTC CTC CAT ATC CAT CAA GAC AGC TTT CCA CAT GGC CTA GGC CTG 151 Phe Phe Leu His lie His Gln Asp Ser Phe Pro His Gly Leu Gly Leu 25 30 35 TCG ATC CTG TGT CCA GAC CGC CGC CTG GTG ACÁ CCC CCA GTG GCC ATC 199 Ser lie Leu Cys Pro Asp Arg Arg Leu Val Thr Pro Pro Val Ala lie 40 45 50 TTC TGC CTG CCG GGT ACT GCG ATG GGC CCC AAC GCC TCC TCT TCC TGT 24 '/ Phe Cys Leu Pro Gly Thr Wing Mee Gly Pro Asn Wing Being Ser Cys 55 60 65 70 CCC CAG CAC CCT GCT TCC CTC TCC GGC ACC TGG ACT GTC TAC CCC AAT 295 Pro Gln His Pro Wing Ser Leu Ser Gly Thr Trp Thr Val Tyr Pro Asn 75 80 85 GGC CGG TTT GGT AAT CAG ATG GGA CAG TAT GCC ACG CTG CTG GCT CTG 343 Gly Arg Phe Gly Asn Gln Mee Gly Gln Tyr Wing Thr Leu Leu Wing Leu 90 95 100 GCC CAG CTC AAC GGC CGC CGG GCC TTT ATC CTG CCT GCC ATG CAT GCC 391 Ala Gln Leu Asn Gly Arg Arg Ala Phe lie Leu Pro Ala Met His Ala 105 110 115 GCC CTG GCC CCG GTA TTC CGC ATC ACC CTG CCC GTG CTG GCC CCA GAA 439 Ala Leu Ala Pro Val Phe Arg lie Thr Leu Pro Val Leu Ala Pro Glu 120 125 130 GTG GAC AGC CGC ACG CCG TGG CGG GAG CTG CAG CTT CAC GAC TGG ATG 487 Val Asp Ser Arg Thr Pro Trp Arg-Glu Leu Gln Leu His Asp Trp Met 135 140 145 150 TCG GAG GAG TAC GCG GAC TTG AGA GAT CCT TTC CTG AAG CTC TCT GGC 535 Ser Glu Glu Tyr Wing Asp Leu Arg Asp Pro Phe Leu Lys Leu Ser Gly 155 '160 165 TTC CCC TGC TCT TGG ACT TTC TTC CAC CAT CTC CGG GAA CAG ATC CGC 583 Phe Pro Cys Be Trp Thr Phe Phe His His Leu Arg Glu Gln He Arg 170 175 • 180 AGA GAG TTC ACC CTG CAC GAC CAC CTT CGG GAA GAG GCG CAG AGT GTG 631 Arg Glu Phe Thr Leu His Asp His Leu Arg Glu Glu Wing Gln Ser Val 185 190 195 CTG GGT CAG CTC CGC CTG GGC CGC ACA GGG GAC CGC CCG CGC ACC TTT 679 Leu Gly Gln Leu Arg Leu Gly Arg Thr Gly Asp Arg Pro Arg Thr Phe 200 205 210 GTC GGC GTC CAC GTG CGC CGT GGG GAC T AT CTG CAG GTT ATG CCT CAG 727 Val Gly Val His Val Arg Arg Gly Asp Tyr Leu G n Val Met Pro Gln 215 220 225 230 CGC TGG AAG GGT GTG GTG GGC GAC AGC GCC TAC CTC CGG CAG GCC ATG 775 Arg Trp Lys Gly Val Val Gly Asp Be Wing Tyr Leu Arg Gln Wing Met 235 240 245 GAC TGG TTC CGG GCA CGG CAC GAA GCC CCC GTT TTC GTG GTC ACC AGC 823 Asp Trp Phe Arg Wing Arg His Glu Wing Pro Val Phe Val Val Thr Ser 250 255 260 AAC GGC ATG GAG TGG TGT AAA GAA AAC ATC GAC ACC TCC CAG 'GGC GAT 871 Asn Gly Met Glu Trp Cys Lys Glu Asn He Asp Thr Ser Gln Gly Asp 265 270 275 GTG ACG TTT GCT GGC GAT GGA CAG GAG GCT ACÁ CCG TGG AAA GAC TTT 919 Val Thr Phe Wing Gly Asp Gly Gln Glu Wing Thr Pro Trp Lys Asp Phe 280 285 290 GCC CTG CTC ACA CAG TGC AAC CAC ACC ATT ATG ACC ATT GGC ACC TTC 967 Wing Leu Leu Thr Gln Cys Asn His Thr He Met Thr He Gly Thr Phe 295 300 305 310 GGC TTC TGG GCT GCC TAC CTG GCT GGC GAC ACT GTC TAC CTG GCC 1015 Gly Phe Trp Wing Wing Tyr Leu Wing Gly Gly Asp Thr Val Tyr Leu Wing 315 320 235 AAC TT C ACC CTG CCA GAC TCT GAG TTC CTG AAG ATC TTT AAG CCG GAG 1063 Asn Phe Thr Leu Pro Asp Ser Glu Phe Leu Lys He Phe Lys Pro Glu 330 335 340 GCG GCC TTC CTG CCC GAG TGG GTG GGC ATT AAT GCA GAC TTG TCT CCA 1111 Wing Wing Phe Leu Pro Glu Trp Val Gly He Aen Wing Asp Leu Ser Pro 345 350 355 CTC TGG ACÁ TTG GCT AAG CCT TGAGAGCCAG GGAAGCCGAA TTC 1155 Leu Trp Thr Leu Wing Lys Pro 360 365

Claims (33)

1. A humanised milk, where milk is produced by a transgenic nonhuman mammal in which the genome of the transgenic nonhuman mammal containing at least one heterologous gene encoding a human catalytic entity, and wherein the catalytic entity produces oligosaccharides and glycoconjugates they are present in the milk of this non-human transgenic mammal.

2. The humanized milk according to claim 1, wherein the non-human transgenic mammal is selected from the group consisting of mice, rats, rabbits, pigs, goats, sheep, horses, and cows.

3. The humanized milk according to claim 1, wherein the non-human transgenic mammals are cows.

4. The humanized milk according to claim 1, wherein the human catalytic entity is selected from the group consisting of enzymes and antibodies.

5. The humanized milk according to claim 4, wherein the enzymes are selected from the group consisting of glucosyltransferases, phosphorylases, hydroxylases, peptidases, and sulfotransferases.

6. The humanized milk according to claim 5, wherein the glucosyltransferases are selected from the group consisting of fucosyltransferase, galactosyltransferase, glucosyltransferase, xylosyltransferase, acetylases, glucuronyltransferases, glucuronylepimerases, sialyltransferase, mannosyltransferase, sulfotransferase, beta-acetylgalactosaminyltransferase. and N -acetylglucosaminyltransfrases.

7. The humanized milk according to claim 1, wherein the oligosaccharides are selected from the group consisting of lactose, 2-fucosyl-lactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-difucopentaosa I, sialyl lactose, 3-sialyl lactose, sialiltetrasacárido to, sialilte rasacárido b, c sialiltetrasacárido, disialiltetrasacárido, and sialyl-lacto N-f ucopentaosa.

8. The humanized milk according to claim 1, wherein the glucoconjugates are selected from the group consisting of glycosylated homologous proteins, glycosylated heterologous proteins, and glycosylated lipids.

9. The humanized milk according to claim 8, wherein the glycosylated heterologous proteins are selected from the group of proteins consisting of human serum proteins and human milk proteins.

10. The humanized milk according to claim 9, wherein the human milk proteins are selected from the group consisting of secretory immunoglobulins, lysozyme, lactoferrin, kappa casein, alpha-lactalbumin, beta-lactalbumin, lactoperoxidase and lipase stimulated by bile salt.

11. An enteral nutritional product useful in the nutritional maintenance of an animal, said enteral nutritional product containing the humanized milk according to claim 1.

12. A method for obtaining a humanized milk, this method comprising the steps of: (a) inserting into the genome of a non-human mammal a heterologous gene that encodes the production of a human catalytic entity, wherein the catalytic entity produces a product of secondary gene in the milk of the non-human mammal, and (b) milking the non-human mammal.

The method according to claim 12, wherein the non-human mammal is selected from the group consisting of mice, rats, rabbits, pigs, goats, sheep, horses, and cows.

14. The method according to claim 13, wherein the non-human mammals are cows.

15. The method according to claim 12, wherein the human catalytic entity is selected from the group consisting of enzymes and antibodies.

16. The method according to claim 15, wherein the enzymes are selected from the group consisting of glycosyltransferases, phosphorylases, hydroxylases, peptidases, and sulfotransferases.

17. The method according to claim 16, wherein the glucosyltransferases are selected from the group consisting of fucosyltransferase, galactosyltransferase, glucosyltransferase, xylosyltransferase, acetylases, glucuronyltransferases, glucuronylepimerases, sialyltransferases, mannosyltransferases, sulfotransferases, β-acetylgalactosaminyltransferases and N ~ acetylglucosaminyltransferases.

18. The method according to claim 12, wherein the secondary gene products are selected from the group consisting of oligosaccharides and glucoconjugates. The method according to claim 18, wherein the oligosaccharides are selected from the group consisting of lactose, 2-fucosyl lactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I , lacto-N-fucopentase II, lacto-N-fucopentase III, lacto-N-difucopentaose I, sialyl-lactose, 3-sialyl-lactose, sialyl-tetrasaccharide-a, sialyl-tetra-saccharide-b, sialyl-tetra-saccharide-c, disialyl-tetra-saccharide, and sialyl-lacto-N- fucopenta The method according to claim 18, wherein the glucoconjugates are selected from the group consisting of glycosylated homologous proteins, glycosylated heterologous proteins, and glycosylated lipids. The method according to claim 20, wherein the glycosylated heterologous proteins are selected from the group of proteins consisting of human serum proteins and human milk proteins. 22. The method according to claim 21, wherein the human milk proteins are selected from the group consisting of secretory immunoglobulins, lieozyme, lactoferrin, kappa-casein, alpha-lactalbumin, beta-lactalbumin, lactoperoxidase and stimulated lipase. by the bile salt. 23. A method for obtaining a biological product, this method comprising the steps of: (a) inserting into the genome of a non-human mammal, a heterologous gene that encodes the production of a heterologous catalytic entity, wherein the catalytic entity produces a secondary gene product in the milk of the non-human mammal, - (b) milking the non-human mammal; and (c) isolate the biological product from the milk. The method according to claim 23, wherein the non-human mammal is selected from the group consisting of mice, rats, rabbits, pigs, goats, sheep, horses, and cows. 25. The method according to claim 24, wherein the non-human mammals are cows. 26. The method according to claim 23, wherein the human catalytic entity is selected from the group consisting of enzymes and antibodies. The method according to claim 26, wherein the enzymes are selected from the group consisting of glycosyltransferases, phosphorylases, hydroxylases, peptidases, and sulfotransferases. 28. The method according to claim 27, wherein the glucosyltransferases are selected from the group consisting of fucos il t rans frasas, galactosyl transferase, glucosyl transferase, »Xylosyltransferase, acetylases, glucuronyltransferases, glucuronilepimerases, sialyltransferases, mannosyltransferases, sulfotransferases, β-acetylgalactosaminyltransferases and N-acetylglucosaminyltransferases. 29. The method according to claim 23, wherein the biological product is selected from the group consisting of oligosaccharides and glucoconjugates. The method according to claim 29, wherein the oligosaccharides are selected from the group consisting of lactose, 2-fucosyl lactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentase I , lacto-N-fucopentase II, lacto-N-fucopentase III, lacto-N-difucopentaose I, sialyl-lactose, 3-sialyl-lactose, sialyltetrasaccharide a, sialyltranstracharide b, sialyltetrasaccharide c, disialyltetrasaccharide, sialyl-lacto-N-fucopenta . 31. The method according to claim 29, wherein the glucoconjugates are selected from the group consisting of glycosylated homologous proteins, glycosylated heterologous proteins, and glycosylated lipids. 32. The method according to claim 31, wherein the glycosylated heterologous proteins are selected from the groups of proteins consisting of human serum proteins and human milk proteins. The method according to claim 32, wherein the human milk proteins are selected from the group consisting of secretory immunoglobulins, lysozyme, lactoferrin, kappa-casein, alpha-lactalbumin, beta-lactalbumin, lactoperoxidase, and lipase stimulated by bile salt.