AU2616502A - Methods for reducing hyperacute rejection of xenografts - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT a. a *aa.

a.

a Name of Applicants: Actual Inventors: Address for Service: Alexion Pharmaceuticals, Inc.

The Austin Research Institute Mauro S SANDRIN William L FODOR Russell P ROTHER Stephen P SQUINTO Ian F MCKENZIE CULLEN CO.

Patent Trade Mark Attorneys 240 Queen Street Brisbane, Qld. 4000 Australia.

Invention Title: Methods for Reducing Hyperacute Rejection of Xenografts The following statement is a full description of this invention, including the best method of performing it known to us -la- METHODS FOR REDUCING HYPERACUTE REJECTION OF XENOGRAFTS CROSS-REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of copending U.S. Patent Application Serial No. 08/260,201 filed June 15, 1994.

FIELD OF THE INVENTION This invention relates to xenotransplantation. More specifically, the invention relates to methods that will prevent or reduce hyperacute rejection of xenogeneic cells, tissues and organs following transplantation into human recipients. The invention provides methods for stably reducing the expression on the surface of a xenogeneic cell of the non-human antigen known as :galactose a(1,3) galactose. This prevents the phenomenon 20 of antibody-dependent rejection of xenogeneic cells that typically follows exposure to human blood, plasma, or serum following xenotransplantation into a human patient) as a result of the binding of preformed natural human antibodies to the surfaces of such cells.

25 BACKGROUND OF THE INVENTION *Soeo Xenotransplantation: Surgical problems related to the transplantation of allogeneic organs organs from donors of the same species as the transplant recipient), such as kidney, liver, heart, lung and pancreas, have been largely solved, and immunosuppression has been improved such that these procedures are now routinely performed with a high degree of success (Brent, 1991). However, a major problem in transplantation medicine today is the provision of sufficient allogeneic donor organs to satisfy the large numbers of patients awaiting a transplant. Given the increasing emphasis on the costs of dialysis and hospitalization incurred by patients awaiting transplantation, there is even greater emphasis on the transplantation of donor organs early in the course of disease. Additionally, it is clear that the supply of human donor allografts cannot satisfy this demand. Alternative sources for replacing diseased organs, tissues, or cells, are mechanical devices or animal organs. All clinical transplantations of animal organs have met with failure other than when closely related Old World primate species, such as, baboon, chimp or gorilla, were used as donors. Unfortunately, the supply of potential Old World primate donors is also limited, and ethical considerations further limit the use of organs from such species. Non-primate species, on the other hand, offer a vast potential source of donors.

15 The most likely donor species for xenotransplantation appears to be the pig (Cooper et al., 1991 and Niekrasz, et al., 1992). This animal is commonly used commercially, and therefore its use will engender fewer ethical problems than the use of primate 20 donors. Furthermore, the pig is considered a highly suitable donor for anatomical and physiological reasons (Cooper et al., 1991 and Niekrasz, et al., 1992).

Immunoloqical Relection of Xenoqrafts: The rejection of transplanted cells, tissues, or organs may S 25 involve both an extremely rapid hyperacute rejection S• (HAR) phase and a slower cellular rejection phase.

HAR

of non-human, non-Old World primate organs, tissues, or cells (referred to herein as "xenogeneic" organs, tissues, or cells, or "xenotransplants", or "xenografts") is initiated by preformed natural antibodies found in human blood, plasma, serum, lymph, and the like, that bind to donor cells, endothelial cells, and activate attack by the complement arm of the human immune system (Dalmasso, et al., 1992; and Tusso, et al., 1993).

While some xenograft tissues porcine pancreatic islets) do not appear to be rejected by this mechanism, HAR is the most significant impediment to the successful xenotransplantation of most cells and tissues, and of all vascularized organs. Methods for the control of the HAR are available. These include interference with the antibody antigen reactions responsible for initiating the HAR response, either by removing the preformed natural antibodies from the circulation or by interference with the binding of the natural antibodies to their specific epitopes (see copending

U.S.

application Serial No. 08/214,580, entitled "Xenotransplantation Therapies", filed by Mauro

S.

Sandrin and Ian F.C. McKenzie on March 15, 1994, and PCT publication No. 93/03735, entitled "Methods and Compositions for Attenuating Antibody-Mediated Xenograft Rejection") 15 A particularly desirable approach to the prevention of hyperacute rejection is to delete or inhibit the galactosyltransferase gene in xenogeneic cells, and to thus eliminate or significantly reduce expression of Gal a(1,3) Gal epitopes on the surface of such cells 20 (see copending U.S. patent application Serial No.

08/214,580, suEra). This approach eliminates or reduces the binding of preformed natural human antibodies to the xenogeneic cells and, therefore, prevents or reduces the activation of complement and subsequent hyperacute 25 rejection of xenogeneic cells, tissues and organs.

Inhibition of complement attack on the xenotransplant may be accomplished by several means, including the use of complement inhibitors such as the 18kDa C5b-9 inhibitory protein and monoclonal antibodies against human C5b-9 proteins as disclosed in U.S. Patent No. 5,135,916, issued August 4, 1992.

The foregoing methods are effective, but have certain drawbacks in practice, potentially requiring the continuous administration of pharmacologic agents, or, in some cases, requiring the technically difficult production of animals carrying a targeted disruption of a specific gene.

HAR and Complement: Activation of complement leads to the generation of fluid phase (C3a, C5a) and membrane bound (C3b and C5b-9) proteins with chemotactic, procoagulant, proinflammatory, adhesive, and cytolytic properties (Muler-Eberhard, 1988). Immunohistological analysis of hyperacutely rejected xenotransplants reveals antibody deposition, complement fixation, and vascular thrombosis as well as neutrophil infiltration (Zehr, et al., 1994; Auchincloss, 1988; Najarian, 1992; Somervile and d'Apice, 1993; and Mejia-Laguna, et al., 1972).

HAR and Xenoantigens: The targets of natural human antibodies have been the subject of investigations for a number of years, as the identification of these xenoantigens would enable the development of strategies 15 to circumvent hyperacute rejection of xenografts.

S

S

everal recent studies have convincingly demonstrated .i that the carbohydrate galactose a(1,3) galactose (Gal Gal) is the major xenoepitope recognized by natural human antibodies (see Sandrin, et al., 1993A; Sandrin, et al., 1993B; copending U.S. patent application Serial No. 08/214,580, supra; and PCT publication No. 93/03735, surra).

Galili and colleagues have shown that a large proportion of IgG in human serum is directed against 25 the Gal c(1,3) Gal epitope expressed as part of a variety S. of glycosylated molecules found on both cell surfaces and on secreted glycoproteins (Galili et al., 1984; and Thall and Galili, 1990). This disaccharide epitope is found in all mammals except humans and Old World primates, and naturally occurring preformed anti-Gal x(1,3) Gal antibodies are found only in humans and Old World primates, those species which do not themselves express the epitope (Galili et al., 1987 and Galili et al., 1988).

HAR and Preformed Natural Antibodies: The immunoglobulin class of an anti-Gal a(1,3) Gal antibody determines the biological role of that antibody in hyperacute rejection. On the basis of histological studies, Bach and Platt (Platt et al., 1990; Platt and Bach 1991; Platt et al., 1991; and Geller et al., 1993) consider that IgM is the most important class of immunoglobulin involved in hyperacute xenograft rejection.

However, natural human antibodies to Gal a(1,3) Gal are not exclusively of the IgM class, and several studies demonstrate the presence of IgG antibodies reactive with pig cells in human blood (Tusso et al., 1992; Fabian et al., 1992; Hammer et al., 1992; Cairns et al., 1993A; Cairns er al., 1993B; Fournier et al., 1993; Koren et al., 1993; and Zhao et al., 1993), in agreement with the original findings of Galili, et al., 1984 (see also 15 Galili, 1993). For example, by eluting antibodies from different xenogeneic organs after perfusion with normal .:human serum, Koren et al., 1992, have demonstrated the presence of IgM, IgG and IgA antibodies. Based on these various studies, there is a general consensus that both 20 IgM and IgG antibodies react with Gal a(1,3) Gal antigens.

The ability of different monosaccharides and oligosaccharides to inhibit the interaction of naturally occurring preformed human antibodies with pig cells and 25 to prevent the antibody-dependent and complement-mediated lysis of pig cells has been examined (Sandrin et al., 1993A; Sandrin et al., 1993B; PCT publication No.

93/03735, esra; and copending U.S. patent application Serial No. 08/214,580, Asura).

Inhibition of the binding of such antibodies to xenogeneic cells was obtained with galactose, or with moieties containing terminal galactose in an a linkage but not a f linkage. Various carbohydrates have also been shown to contain the target epitopes for several types of naturally occurring preformed human antibodies with other specificities ABO blood group antibodies). However, no monosaccharide tested, other -6than those containing the Gal a(1,3) Gal epitope, had any inhibitory effect on the binding of naturally occurring preformed human antibodies to xenogeneic cells.

Identical inhibition results were obtained when individual human serum samples from blood group A, B, AB or 0 individuals were used (Sandrin et al., 1993A and Sandrin et al., 1993B).

Similarly, Cooper and colleagues have demonstrated that, of a total of 132 carbohydrates screened for binding to preformed naturally occurring human IgG and IgM antibodies, each of the four carbohydrate molecules that they found could bind such antibodies contained a terminal a galactose (Good et al., 1992). The four carbohydrates were: 15 Gal a(1,3) Gal 8(1,4) GlcNAc, Gal a(1,3) Gal 8(1,4) Glc, 0 Gala Gal and Gal a(1,3) Gal.

Sugars such as melibiose (a disaccharide containing S. 20 a terminal galactose in an c linkage) coupled to a carrier such as SEPHAROSE can be used to purify anti-Gal a Gal antibodies (Galili et al, 1984 and Galili et al., 1985). In some antibody absorption experiments, human serum was passed over the carrier-sugar matrix in S 25 order to prepare serum from which the antibodies reactive with the sugar were removed. The results of testing the cytolytic activity of the sera prepared in these experiments indicate that the majority of the cytotoxic antibodies were removed from the serum by these means (Sandrin et al., 1993A; Sandrin et al., 1993B).

In sum, the results of the sugar inhibition studies, the studies of the binding of antibodies to terminal a galactose-containing molecules, and the studies of the 'absorption of antibodies by melibiose-SEPHAROSE, all lead to the conclusion that Gal a(1,3) Gal epitopes are the most important epitopes detected by naturally occurring human IgG and IgM antibodies.

Glycosyltransferases Mammalian cells display a complex variety of carbohydrate antigens on their surfaces. Carbohydrate epitopes are expressed on all mammalian cells by membrane glycoproteins and glycosphingolipids. Profound changes in the structures of these glycoconjugates frequently accompany important biological processes such as differentiation and development. The types and numbers of carbohydrate epitopes present on cells vary in different species and in different tissues within a given species (Yamakawa and Nagai, 1978).

The structures of these carbohydrate moieties are "is* 15 determined largely by the activities of the glycosyltransferases responsible for oligosaccharide synthesis. Therefore, the population of oligosaccharide molecules displayed on the surface of a given mammalian cell is largely determined by the repertoire of 20 glycosyltransferases active in the cell (Kornfeld and Kornfeld, 1985).

The glycosyltransferases comprise a family of enzymes that transfer sugars from nucleoside diphosphatesugar conjugates (donor molecules) to acceptor substrate molecules, forming covalent linkages. Acceptor substrates are often oligosaccharides or oligosaccharide moieties of larger molecules, but may also be specific proteins or lipids. Glycosyltransferases function in a sequential manner, such that the oligosaccharide product of a transferase activity often becomes the acceptor substrate for subsequent transferase activity. The final result generally contains a linear and/or branched polymer of component monosaccharides linked to one another.

Glycosyltransferases differ from each other with respect to the nature of the nucleoside diphosphatecarbohydrate donor, the nature of the acceptor substrate, and the glycosidic linkage joining the donor sugar to the acceptor substrate (reviewed by Beyer and Hill, 1982).

Examples of glycosyltransferases include the following: galactosyltransferases, fucosyltransferases, sialyltransferases, N-acetylglucosaminyltransferases,

N-

acetylgalactosaminyltransferases, glucosyltransferases, sulfotransferases, acetylases, and mannosyltransferases.

Galactosvltransferases The galactosyltransferases are examples of glycosyltransferases that transfer galactose from a UDPgalactose donor molecule to an acceptor substrate. One such galactosyltransferase, UDP-Gal:Gal 8(1,4) Gal NAcGlc a galactosyltransferase (also referred to as a(1,3) Gal transferase), is a Golgi membrane-bound enzyme that 15 catalyzes the following reaction: UDP-Gal Gal or Glc NAc-R Gal a(1,3) Gal or Glc NAc-R UDP in which R may be a glycoprotein or a glycolipid (Blanken and Van den Eijnden, 1985). The resulting a(1,3) linked S 20 galactose occupies the terminal non-reducing position in N-acetyllactosamine-type carbohydrate chains and, as such, is a non-charged alternative to chain termination by sialic acid. As discussed above, such o(1,3) Gal structures are the most important epitopes of xenogeneic cells recognized by naturally occurring preformed human antibodies.

a(1,3) Gal transferase and the Gal a(1,3) Gal fl-R (herein referred to as Gal a(1,3) Gal) product of the activity of this enzyme show both species and tissuespecific expression (Galili et al., 1988). The c(1,3) Gal transferase is widely expressed in a variety of mammalian species, with the notable exception of Old World primates and humans. These mammals do not express the enzyme due to frameshift and nonsense mutations in their genomic sequences encoding this enzyme (Larsen et al., 1990a).

It is believed that humans and Old World primates have high levels of circulating natural preformed antibodies that bind specifically to the Gal a(1,3) Gal epitope as a consequence of these mutations and the resultant lack of the epitope in humans and Old World primates. The source of antigen exposure responsible for the natural preformed antibodies in these species has not been definitively established, but is believed to be certain bacteria bearing Gal a(1,3) Gal epitopes that are normally found in the intestines of humans and Old World primates.

The cDNA encoding the pig a(i,3) galactosyltransferase has been cloned using cross species hybridization (see copending U.S. patent application Serial No. 08/214,580, supra; and Dabkowski et al., 1993). Sequence comparison shows that at the amino acid level there is approximately 75% identity with the murine and approximately 82% identity with the bovine t(1,3) Gal transferase sequences, with the catalytic domains of the S 20 transferases having the highest identity.

Fucosvl transferases The carbohydrate antigens and glycosyltransferases of the human H blood group, as well as the specific details of the biosynthesis and distribution of the H 25 antigen, have been extensively reviewed (see, for S* example, Lowe, 1991). Several carbohydrates, including those associated with the H antigen, contain the terminal structure Pucose a(1,2) Galactose. The synthesis of the Fucose a(1,2) linkage is catalyzed by specific c(1,2) fucosyltransferase enzymes. The enzymatic activities of these transferases result in the covalent attachment of L-fucose by an a(1,2) linkage to a variety of acceptor molecules. The H transferase, for example, is a fucosyltransferase that catalyzes a transglycosylation reaction covalently linking a fucose to a specific oligosaccharide acceptor substrate. In this reaction the fucose is derived from the nucleotide sugar donor molecule GDP-fucose and connected by an a(1,2) linkage to the Galactose residue of Gal 8(1,3) GlcNAc-R or Gal 8(1,4) GlcNAc-R acceptor substrates galactose linked to N-acetylglucosamine in a 0(1,3) or a 0(1,4) linkage, where R represents a glycoprotein, protein, glycolipid, or lipid).

These acceptor substrates are also the acceptor substrates for the a(1,3) Gal transferase discussed above, although each transferase utilizes a different nucleotide sugar donor molecule (UDP galactose for a(1,3) Gal transferase vs. GDP fucose for H transferase). The a(1,3) Gal transferase and the H transferase have now been cloned (see copending U.S. patent application Serial No. 08/214,580, suPra; Stanley, 1992; and Lowe, 1991).

The recombinant expression of various glycosyltransferases, including the H transferase, in cells that would be expected to be expressing the a(1,3) Gal transferase has been reported (see, for example, Lowe, 1991). However, prior to the present invention, S 20 the effects of such recombinant expression on the expression of the Gal a Gal epitope have been unknown.

MSUMMARY OF THE INVENTION In view of the foregoing, it is an object of this S 25 invention to provide genetically modified xenogeneic organs, tissues, and cells that are less prone to hyperacute rejection when exposed to human blood, plasma, serum, lymph, or the like following xenotransplantation into human patients) than their unmodified precursors, and to provide methods for the preparation of such xenogeneic organs, tissues, and cells. In accordance with these methods, xenogeneic cells are genetically modified so that they express the glycosyltransferase activity of an exogenous glycosyltransferase a glycosyltransferase encoded by a recombinant nucleic acid molecule introduced into -11the xenogeneic cells or a parent cell of the xenogeneic cells). In particular, the genetically-modified xenogeneic cells of the invention exhibit reduced levels of the xenoantigen Gal a(1,3) Gal on their cell surfaces.

In another aspect of the invention, the geneticallymodified xenogeneic cells of the invention are inhibited from binding to preformed naturally occurring human antibodies and are therefore significantly less sensitive to HAR as demonstrated by reduced sensitivity to activation and/or lysis by human complement. In this way, when transplanted into human patients, the rejection of such cells by complement-mediated hyperacute rejection mechanisms is reduced or prevented.

In certain preferred embodiments, the invention 15 provides a method for reducing rejection of a xenogeneic cell following transplantation into a human or an Old SWorld primate comprising: producing a genetically altered cell by introducing an expression vector comprising a nucleic :20 acid sequence encoding a protein having fucosyltransferase activity into a recipient cell, the introduction of said expression vector causing a substantial reduction in the binding of naturally occurring preformed human antibodies or naturally 25 occurring preformed Old World primate antibodies to the genetically altered cell when compared to the binding of said antibodies to the recipient cell; and transplanting said genetically altered cell or a cell derived from said cell into a human or an Old World primate.

In other preferred embodiments the invention provides an ungulate cell which has been genetically altered by the introduction of an expression vector comprising a nucleic acid sequence encoding a protein having fucosyltransferase activity into a recipient ungulate cell, the introduction of said expression vector causing a substantial reduction in the binding of -12naturally occurring preformed human antibodies or naturally occurring preformed Old World primate antibodies to said genetically altered ungulate cell when compared to the binding of said antibodies to the recipient ungulate cell.

In further preferred embodiments, the invention provides a retroviral packaging or producer cell which has been genetically altered by the introduction of an expression vector comprising a nucleic acid sequence encoding a protein having fucosyltransferase activity into a recipient cell from which the genetically altered retroviral packaging or producer cell is derived, the introduction of said expression vector causing a substantial reduction in the binding of naturally 15 occurring preformed human antibodies or naturally occurring preformed Old World primate antibodies to said genetically altered retroviral packaging or producer cell when compared to the binding of said antibodies to the recipient cell from which the genetically altered 20 retroviral packaging or producer cell is derived.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1-4 are photomicrographs of African Green Monkey COS cells which have been fluorescently stained with anti-H antigen mAbs (FIGS. 2 and 3) or with lectins specific for the Gal a(1,3) Gal epitope (FIGS. 1 and 4).

In each figure, the bottom panel shows all cells, as seen by phase contrast illumination, and the top panel shows only those cells specifically binding to the mAb or lectin as seen by ultraviolet illumination. The cells in FIG. 1 have been transfected with a vector expressing the Gal a(1,3) Gal transferase; the cells in FIG. 2 have been transfected with a vector expressing H transferase; and the cells in FIGS. 3 and 4 have been transfected with equal amounts of both vectors. African Green Monkeys are Old World primates and thus their cells, including

COS

cells, do not express the Gal a(1,3) Gal epitope. In addition, COS cells do not express the H epitope.

-13- FIG. 5 illustrates the expression of the H epitope and reduced expression of the Gal a(1,3) Gal epitope in stably transfected porcine kidney cells as analyzed by lectin staining and fluorescence-based flow cytometric analysis of the cells. FIG. 5A is for clone PK1:H- Transferase #A3. FIG. 5B is for clone PK1:NEO #B6.

FIG. 5C is for clone PKl:H-Transferase #A3. FIG. 5D is for clone PK1:NEO #B6.

FIG. 6 demonstrates the loss of human serum IgG and IgM binding to porcine kidney cells associated with the expression of the H epitope and reduced expression of the Gal a(1,3) Gal epitope as analyzed by fluorescence staining and flow cytometry. FIG. 6A is for clone PKl:H- Transferase #A3. FIG. 6B is for clone PKl:H-Transferase 15 #A3. FIG. 6C is for clone PK1:NEO #B6. FIG. 6D is for clone PK1:NEO #B6.

FIG. 7 shows the results of a human serum dye release assay for H-Transferase and NEO control

PKI

cells. The figure illustrates the enhanced resistance to 20 human serum lysis associated with the expression of the H epitope and consequent reduced expression of the Gal a(1,3) Gal epitope in stably transfected porcine kidney Scells.

The foregoing drawings, which are incorporated in 25 and constitute part of the specification, illustrate the preferred embodiments of the invention, and together with the description, serve to explain the principles of the invention. It is to be understood, of course, that both the drawings and the description are explanatory only and are not restrictive of the invention.

-13/1- DESCRIPTION OF THE PREFERRED EMBODIMENT Glycosyltransferases: A variety of nucleic acid molecules encoding glycosyltransferases can be used in the practice of the present invention provided that the glycosyltransferase encoded by the nucleic acid molecule is able to reduce the levels of Gal a(1,3) Gal epitopes on the surface of a xenogeneic cell in which the exogenous transferase is expressed. In accordance with the present invention, this property can be determined by introducing an appropriate expression vector directing the expression of the candidate glycosyltransferase into xenogeneic cells and then testing the cells for cell surface levels of the Gal a(1,3) Gal epitope using, for example, human serum, as described below in Example 4.

oS *o

S

-14- Transferases suitable for use in the methods and cells of the present invention will cause a substantial reduction in the binding of naturally occurring preformed human antibodies to the xenogeneic cells after introduction of the expression vector compared to binding before introduction of the vector. An at least reduction in binding will, in general, comprise a "substantial reduction". Smaller reductions in binding are also considered "substantial" if they represent a statistically significant reduction, a reduction that, when analyzed by a standard statistical test, such as the students T test, will give a probability value, p, less than or equal to 0.05 and, preferably, less than or equal to 0.015. Examples of the construction of such vectors, production of such cells, and the testing of such cells for reduction of preformed natural antibody binding are given below in Examples In particular, reduction in the binding of naturally occur=rng preformed antibodies can be determined by staining and counting stained cells as described below in Example 2, or by FACS analysis as described in Example 4 below in which case a quantitative readout can be obtained by measuring the areas under the various

FACS

curves and the shifts in the positions of those curves, 25 or by measurement of changes in complement resistance as described in Example 5 below.

While not wishing to be bound by any particular theory of operation, it is believed that glycosyltransferases, which are able to reduce the levels of Gal a(1,3) Gal epitopes on the surface of a xenogeneic cell in which the transferase is expressed, effect this reduction by competition for a shared acceptor substrate.

Specifically, it is believed that transferases suitable for use in the methods and cells of the invention transfer donor sugars to 8(1,3) Glc NAc-R or 8(1,4) Glc NAc-R acceptor substrates. Thus, preferred tzansferases include those that transfer donor sugars to 8(1,3) Glc NAc-R or 8(1,4) Glc NAc-R acceptor substrates and create covalent linkages other than the Gal a(1,3) Gal linkage upon such transfer.

Preferred transferases to be used in the practice of the invention include fucosyltransferases. With regard to these transferases, it is believed that the addition of a terminal fucose residue to the 8(1,3) Glc NAc-R or 8(1,4) Glc NAc-R acceptor substrate of the a(1,3) galactosyltransferase prevents the addition of a Gal U(1,3) Gal epitope to the acceptor substrate. Specific examples of fucosyltransferases that can be tested for use in the process of the present invention include the fucosyltransferase (H transferase; Larsen et al., 1990A) and the a(1,3/1,4) fucosyltransferase (Weston et 15 al., 1992). Of these, the H transferase is preferred and the human H transferase is particularly preferred. This transferase is responsible for synthesis of the H antigen which is the universal donor O-blood group antigen and utilizes the same acceptor substrates as the a(1,3) Gal transferase. Alternatively, although less preferred, sialyltransferases, the o(2,6) sialyltransferase (see Lowe, 1991), may be used in the practice of the invention.

S. Although the foregoing discussion and those that 25 follow are phrased in terms of nucleic acid molecules encoding glycosyltransferases, the cells and methods of the invention more generally comprise nucleic acid molecules encoding any and all proteins that have glycosyltransferase activity, including, in particular, fucosyltransferase activity. Such proteins may be in the form of intact glycosyltranferases, but may also be in the form of proteins comprising active mutant glycosyltranferases such as those comprising active fragments of glycosyltranferases. See, for example, Kukowska, et al., 1991.

-16- Vectors for expression of recombinant qlycosvltransferases: In addition to the foregoing, the present invention provides vectors for the expression of recombinant glycosyltransferases in xenogeneic cells at levels effective to reduce the expression of Gal a(1,3) Gal epitopes by the xenogeneic cells into which the vectors have been introduced. Recombinant polynucleotides encoding glycosyltransferases that are appropriate for use in such vectors include those encoding the transferases discussed above.

A

particularly preferred polynucleotide is that encoding human H transferase, SEQ ID NO: 3.

The nucleic acid encoding the desired exogenous .0 glycosyltransferase may be inserted into an appropriate 15 parent expression vector, an expression vector that contains a site for inserting protein-encoding nucleic -0 0 acid molecules, and also contains (in the appropriate orientation for expression) the necessary elements for i the transcription and translation of an inserted proteinencoding sequence. Particularly preferred transcriptional and translational signals allow for expression of the desired glycosyltransferase in a wide variety of xenogeneic cell types.

A candidate parent expression vector can be tested S 25 for suitability for use in the practice of the present invention by the insertion of a nucleic acid fragment encoding the human H transferase into a site appropriate for expression in the parent expression vector, as described below in Example 1 for the APEX-I vector, and testing cells containing the resulting expression vector for susceptibility to human complement-mediated damage as described below in Example The transcriptional and translational control sequences in mammalian expression vector systems to be used in genetically altering vertebrate cells may be provided by various sources, including viral sources.

For example, commonly used promoters and enhancers known -19- 5,124,263; as well as PCT Patent Publications Nos.

WO

85/05629, WO 89/07150, WO 90/02797, WO 90/02806,

WO

90/13641, WO 92/05266, WO 92/07943, WO 92/14829, and WO 93/14188.

In particular, retroviral vectors for use in the practice of the invention can be prepared and used as follows. First, a retroviral vector comprising a nucleic acid sequence encoding a glycosyltransferase is constructed from a parent retroviral vector. Examples of such parent retroviral vectors are found in, for example, Korman, et al., 1987; Morgenstern, et al., 1990;

U.S.

Patents Nos. 4,405,712, 4,980,289, and 5,112,767; and PCT Patent Publications Nos. W O 85/05629, WO 90/02797, and WO S 92/07943. A preferred parent retroviral vector is the 15 Moloney murine leukemia virus-derived expression vector pLXSN (Miller, et al., 1989).

St The parent retroviral vector used in the practice of the present invention will be modified to include a glycosyltransferase encoding sequence and will be packaged into non-infectious (replication incompetent) transducing retroviral particles (virions) using an amphotropic packaging system, preferably one suitable for use in gene therapy applications.

Examples of useful packaging systems are found in, 25 for example, Miller, et al., 1986; Markowitz, et al., 1988; Cosset, et al., 1990; U.S. Patents Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767, and 5,124,263, and PCT Patent Publications Nos. WO 85/05629, WO 89/07150,

WO

90/02797, WO 90/02806, WO 90/13641, WO 92/05266,

WO

92/07943, WO 92/14829, and WO 93/14188. A preferred packaging cell is the PA317 packaging cell line (ATCC

CRL

9078).

The generation of "producer cells" is accomplished by introducing retroviral vectors into the packaging cells. The producer cells generated by the foregoing procedures are used to produce the retroviral vector particles. This is accomplished by culturing of the cells in a suitable growth medium.

Preferably, the virions are harvested from the culture and administered to the target cells which are to be transduced. Examples of such target cells include isolated xenogeneic cells, cells of a xenogeneic organ or tissue, and other cells to be protected from antibody binding and complement attack, as well as xenogeneic progenitor cells, including stem cells such as embryonic or hematopoietic stem cells, which can be used to generate transgenic cells, tissues, or organs.

Alternatively, when practicable, virions are added to the target xenogeneic cells to be transduced by co-culture of the target cells with the producer cells.

15 Suitable buffers and conditions for stable storage and subsequent use of the virions can be found in, for example, Ausubel, et al., 1992.

Cells, tissues, and orans: In general, any :xenogeneic cell, tissue or organ may be utilized in the 20 practice of the present invention. Preferred cells are of ungulate origin, and particularly preferred cells are o f pig origin. The glycosyltransferase nucleic acid constructs of the invention can be used to engineer cultured cells of various types for subsequent use in S 25 transplantation. Examples of useful cell types include endothelial cells, fibroblastic and other skin cells, hepatic cells, neuronal and glial cells, pancreatic islet cells, hematopoietic cells, blood cells, lens cells, corneal cells, and stem cells.

Further, the glycosyltransferase nucleic acid constructs of the invention can be used to alter retroviral packaging cells or retroviral producer cells so that such cells exhibit a substantial reduction in the binding of naturally occurring preformed human antibodies or naturally occurring preformed Old World primate antibodies when compared to the binding of said antibodies to packaging or producer cells which have not -21been so altered. In the discussion that follows, the expression "altered retroviral packaging/producer cells" is used to describe either or both of said altered packaging or producer cells. Such altered retroviral packaging/producer cells may be from any species that expresses Gal a(1,3) Gal epitopes, including cells of rodent or canine origin.

Among other applications, such altered retroviral packaging/producer cells may be used to provide gene therapy treatment in a patient in need of such treatment, for therapeutic control of neoplastic tumors. In this embodiment of the invention, altered retroviral producer cells producing a retroviral vector particle providing a therapeutic benefit are implanted into the 15 patient. In the case of cancer therapy the implantation is preferably made into or adjacent to the tumor. In accordance with the invention, such altered producer Scells are protected from HAR upon transplantation (implantation) into a human or Old World primate patient.

20 In addition, as disclosed in copending U.S. patent application Serial No. 08/278,639, entitled "Retroviral Transduction of Cells in the Presence of Complement", which is being filed concurrently herewith in the names of Russell P. Rother, Scott A. Rollins, William L. Fodor, 25 and Stephen P. Squinto, the retroviral particles produced by the altered producer cells are protected from inactivation by complement in the body fluids of the patient. Other methods to protect retroviral vector particles from inactivation by complement in the body fluids of humans or Old World primates include those discussed in copending U.S. patent application Serial No.

08/278,550, entitled "Retroviral Transduction of Cells Using Soluble Complement Inhibitors", which is being filed concurrently herewith in the names of Russell

P.

Rother, Scott A. Rollins, James M. Mason, and Stephen

P.

Squinto, and in copending U.S. patent application Serial No. 08/278,630, entitled "Retroviral Vector Particles -22- Expressing Complement Inhibitor Activity", which is also being filed concurrently herewith in the names of James M. Mason and Stephen P. Squinto.

General discussions of packaging cells, retroviral vector particles and gene transfer using such particles can be found in various publications including PCT Patent Publication No. WO 92/07943, EPO Patent Publication No.

178,220, U.S. Patent No. 4,405,712, Gilboa, 1986; Mann, et al., 1983; Cone and Mulligan, 1984; Eglitis, et al., 1988; Miller, et al., 1989; Morgenstern and Land, 1990; Eglitis, 1991; Miller, 1992; Mulligan, 1993, and Ausubel, et al., 1992. The manipulation of retroviral nucleic acids to construct packaging vectors and packaging cells is discussed in, for example, Ausubel, et al., Volume 1, Section III (units 9.10.1 9.14.3), 1992; Sambrook, et al., 1989; Miller, et al., 1989; Eglitis, et al., 1988; U.S. Patents Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767, and 5,124,263; as well as PCT Patent Publications Nos. WO 85/05629, WO 89/07150, WO 90/02797, 20 WO 90/02806, WO 90/13641, WO 92/05266, WO 92/07943,

WO

92/14829, and WO 93/14188. To form packaging cells, packaging vectors are introduced into suitable host cells such as those found in, for example, Miller and Buttimore, Mol. Cell Biol.,, 6:2895-2902, 1986; Markowitz, 25 et al., J. Virol., 62:1120-1124, 1988; Cosset, et al., L Virol., 64:1070-1078, 1990; U.S. Patents Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767, and 5,124,263, and PCT Patent Publications Nos. WO 85/05629, WO 89/07150,

WO

90/02797, WO 90/02806, WO 90/13641, WO 92/05266,

WO

92/07943, WO 92/14829, and WO 93/14188. Once a packaging cell line has been established, producer cells are generated by introducing retroviral vectors into the packaging cells. Examples of such retroviral vectors are found in, for example, Korman, et al., 1987, Proc.

Natl. Acad. Sci. USA, 84:2150-2154; Miller and Rosman, Biotechniues, 7:980-990, 1989; Morgenstern and Land, 1990; U.S. Patents Nos. 4,405,712, 4,980,289, and -23- 5,112,767; and PCT Patent Publications Nos. WO 85/05629, WO 90/02797, and WO 92/07943. The retroviral vector includes a psi site and one or more exogenous nucleic acid sequences selected to perform a desired function, an experimental, diagnostic, or therapeutic function. These exogenous nucleic acid sequences are flanked by LTR sequences which function to direct high efficiency integration of the sequences into the genome of the ultimate target cell. (See also the discussion of transduction set forth above.) Many applications of gene therapy using retroviral vector particles (RVVPs) are known and have been p extensively reviewed (see, for example, Boggs, 1990; Kohn, et al., 1989; Lehn, 1990, Verma, 1990; Weatherall, *s 15 1991; and Felgner and Rhodes, 1991).

A variety of genes and DNA fragments can be incorporated into RVVPs for use in gene therapy. These DNA fragments and genes may encode RNA and/or protein molecules which render them useful as therapeutic agents.

S. 20 Protein encoding genes of use in gene therapy include those encoding various hormones, growth factors, enzymes, lymphokines, cytokines, receptors, and the like.

Among the genes which can be transferred are those encoding polypeptides that are absent, are produced in 25 diminished quantities, or are produced in mutant form in individuals suffering from a genetic disease. Other genes of interest are those that encode proteins that, when expressed by a cell, can adapt the cell to grow under conditions where the unmodified cell would be unable to survive, or would become infected by a pathogen. Genes encoding proteins that have been engineered to circumvent a metabolic defect are also suitable for transfer into the cells of a patient. Such genes include the transmembrane form of CD59 discussed in copending U.S. patent application No. 08/205,720, filed March 3, 1994, entitled "Terminal Complement Inhibitor Fusion Genes and Proteins" and copending U.S. patent -24application No. 08/206,189, filed March 3, 1994, entitled "Method for the Treatment of Paroxysmal Nocturnal Hemoglobinuria".

In addition to protein-encoding genes, RVVPs can be used to introduce nucleic acid sequences encoding medically useful RNA molecules into cells. Examples of such RNA molecules include anti-sense molecules and catalytic molecules, such as ribozymes.

In order to expedite rapid transduction by eliminating the need to wait for target cells to divide and to allow transduction of cells that divide slowly or not at all, the use of RVVPs that can transduce non-dividing cells may be preferred. Such RVVPs are disclosed in copending U.S. patent applications Serial 15 Nos. 08/181,335 and 08/182,612, both entitled "Retroviral Vector Particles for Transducing Non-Proliferating Cells" and both filed January 14, 1994. These patent Sapplications also discuss specific procedures suitable for producing packaging vectors and retroviral vectors as 20 well as the use of such vectors to produce packaging cells and producer cells, respectively.

Transqenic animals: Transgenic xenogeneic animals provide a preferred source of the cells, tissues, and organs of the invention. In accordance with certain 25 aspects of the invention, the nucleic acid molecules of the invention are used to generate engineered transgenic animals, preferably ungulates hooved animals such as pigs, cows, goats, sheep, and the like), that express the carbohydrate products of glycosyltransferases on the surfaces of their cells endothelial cells) using techniques known in the art.

These techniques include, but are not limited to, microinjection of pronuclei), electroporation of ova or zygotes, electric field mediated transfer Baekonization, ME; see also Zhao and Wong, 1991), nuclear transplantation, and/or the stable transfection or transduction of embryonic stem cells derived from the animal of choice. Electric field mediated transfer, Baekonization, is a preferred method of producing the transgenic animals of the invention.

A common element of these techniques involves the preparation of a transgene transcription unit. Such a unit comprises a DNA molecule which generally includes: 1) a promoter, 2) the nucleic acid sequence of interest, the sequence encoding a glycosyltransferase, and 3) a polyadenylation signal sequence. Other sequences, such as enhancer and intron sequences, can be included if desired. The unit can be conveniently prepared by isolating a restriction fragment of a plasmid vector which expresses the glycosyltransferase protein in, for example, mammalian cells. Preferably, the restriction fragment is free of sequences which direct replication in bacterial host cells since such sequences are known to have deleterious effects on embryo viability.

The most well known method for making transgenic animals is that used to produce transgenic mice by superovulation of a donor female, surgical removal of the egg, injection of the transgene transcription unit into the pro-nuclei of the embryo, and introduction of the transgenic embryo into the reproductive tract of a pseudopregnant host mother, usually of the same species.

25 See Wagner, U.S. Patent No. 4,873,191, Brinster, et al., 1985, Hogan, et al., 1986, Robertson 1987, Pedersen, et al., 1990.

The use of this method to make transgenic ungulates is also widely practiced by those of skill in the art.

As an example, transgenic swine are routinely produced by the microinjection of a transgene transcription unit into pig embryos. See, for example, PCT Publication No.

W092/11757. In brief, this procedure may, for example, be performed as follows.

First, the transgene transcription unit is gel isolated and extensively purified through, for example, an ELUTIP column (Schleicher Schuell, Keene,

NH),

-26dialyzed against pyrogen free injection buffer Tris, pH7.4 0.1mM EDTA in pyrogen free water) and used for embryo injection.

Embryos are recovered from the oviduct of a hormonally synchronized, ovulation induced sow, preferably at the pronuclear stage. They are placed into a 1.5 ml microfuge tube containing approximately 0.5 ml of embryo transfer media (phosphate buffered saline with fetal calf serum). These are centrifuged for 12 minutes at 16,000 x g in a microcentrifuge. Embryos are removed from the microfuge tube with a drawn and polished Pasteur pipette and placed into a 35 mm petri dish for examination. If the cytoplasm is still opaque with lipid such that the pronuclei are not clearly visible, the embryos are centrifuged again for an additional minutes.

Embryos to be microinjected are placed into a drop of media (approximately 100 Al) in the center of the lid of a 100 mm petri dish. Silicone oil is used to cover 20 this drop and to fill the lid to prevent the medium from evaporating. The petri dish lid containing the embryos is set onto an inverted microscope equipped with both a heated stage (37.5-38oC) and Hoffman modulation contrast "optics (200X final magnification).

25 A finely drawn and polished micropipette is used to stabilize the embryos while about 1-2 picoliters of injection buffer containing approximately 200-500 copies of the purified transgene transcription unit is delivered into the nucleus, preferably the male pronucleus, with another finely drawn and polished micropipette. Embryos surviving the microinjection process as judged by morphological observation are loaded into a polypropylene tube (2 mm ID) for transfer into the recipient pseudopregnant sow.

Offspring are tested for the presence of the transgene by isolating genomic DNA from tissue removed from the tail of each piglet and subjecting this genomic -27- DNA to nucleic acid hybridization analysis with transgene specific probes or PCR analysis with transgene specific primers.

Another commonly used technique for generating transgenic animals involves the genetic manipulation of embryonic stem cells (ES cells) as described in PCT Patent Publication No. WO 93/02188 and Robertson, 1987.

In accordance with this technique, ES cells are grown as described in, for example, Robertson, 1987, and in U.S.

Patent No. 5,166,065 to Williams et al., 1988. Genetic material is introduced into the embryonic stem cells by, f o r example, electroporation according, for example, to the method of McMahon, et al., 1990, or by transduction with a retroviral vector according, for example, to the 15 method of Robertson, et al., 1986, or by any of the various techniques described by Lovell-Badge, 1987.

Chimeric animals are generated as described, for example, in Bradley, 1987. Briefly, genetically modified ES cells are introduced into blastocysts and the modified blastocysts are then implanted in pseudo-pregnant female animals. Chimeras are selected from the offspring, for example by the observation of mosaic coat coloration resulting from differences in the strain used to prepare the ES cells and the strain used to prepare the blastocysts, and are bred to produce non-chimeric transgenic animals.

Other methods for the production of transgenic animals are disclosed in U.S. Patent No. 5,032,407 to Wagner et al., and PCT Publication No. W090/08832.

Among other applications, transgenic animals prepared in accordance with the invention are useful as model systems for testing the xenotransplantation of their engineered cells, tissues, and organs and as sources of engineered cells, tissues, and organs for xenotransplantation. The expression of functional glycosyltransferases by endothelial cells and/or other cell types in the tissues and organs of the transgenic -28animals of the present invention will provide reduced susceptibility to hyperacute complement-mediated rejection following exposure of those cells, tissues, and organs to complement in human blood, plasma, serum, lymph, or the like, following xenotransplantation into humans or Old World primates. In accordance with the invention, reduced susceptibility to HAR is provided because naturally occurring preformed human or Old World primate antibodies have fewer binding sites on the transgenic cells of the invention.

Without intending to limit it in any manner, the .:"present invention will be more fully described by the following examples.

Examle 15 H Transferase The human H transferase gene was cloned from cDNA prepared from Human Epidermoid Carcinoma cells (HEC cells, ATCC CRL 1555 #A-431) utilizing the Polymerase Chain Reaction (PCR). Cytoplasmic RNA was prepared from approximately 5X10 6 cells, and first strand cDNA was synthesized from 5pg of RNA in a final volume of 100i using the following reaction conditions: 10mM Tris-HCI pH8.3; 50mM KC1; 1.5mM MgCl 2 500ng oligo(dT) 15 (Promega Corporation, Madison, Wisconsin); 10mM DTT; 0.25mM dNTPs (dG, dC, dA, dT); and 20U Avian Myeloblastosis Virus reverse transcriptase (Seikagaku of America, Inc., Rockville, Maryland) at 42 0 C for one hour.

PCR was performed following cDNA synthesis using 4 1l of first strand cDNA reaction mixture as template and the following primers: a 34 base 5' primer homologous to the untranslated region of the H transferase cDNA (SEQ ID NO:1; 5'-GGCCACGAAA AGCGGACTGT GGAT CCA CCTG-3'), where the underlined sequence represents a unique gamHI site; and a 38 base 3' primer homologous to the 3' UTR cf the H transferase cDNA (SEQ ID NO:2; ACCAAGCTTC TCAAGATGC CAGGCC-3'), in which the underlined sequence represents a unique XhaI site. PCR -29reactions consisted of 35 cycles of 95 0 C 1 minute, 52 0

C

minute, and 72°C 1.5 minutes. These 35 cycles were followed by a single ten minute extension at 720 C.

An approximately 1300 bp band representing the PCR product was seen following agarose gel electrophoresis of an aliquot of the PCR reaction. This PCR product was cloned into a plasmid vector using the T/A cloning kit (Invitrogen, San Diego, CA). The pCRII plasmid vector included in this kit served as the recipient, and the resulting plasmid construct was amplified in E coli and purified. Positive clones were identified by restriction endonuclease digestion and the insert was subsequently sequenced to confirm that the plasmid construct contained the human H transferase cDNA sequence shown in SEQ ID 15 NO:3. An approximately 1200 bp SamHI-XhoI DNA fragment, encoding the full length H transferase enzyme, was gel isolated from the pCRII plasmid construct, electroeluted and subcloned into a BmHI-XhaI cut pAPEX-l expression vector (see the following paragraph for a detailed description of this vector). Positive clones were identified by restriction mapping with AmHI-Xhol and S=I. Plasmid pAPEX1-HT, referred to hereinafter as pHT, was the result of these cloning and subcloning steps.

pAPEX-1 (SEQ. ID No:4) is a derivative of the vector S* 25 pcDNAI/Amp (Invitrogen, San Diego CA) which was modified as follows to increase protein expression in mammalian cells. First, since the intron derived from the gene encoding the SV40 small-t antigen has been shown to decrease expression of upstream coding regions (Evans and Scarpulla, 1989), this intron was removed from pcDNAI/Amp by digestion with XbaI-HElI, followed by treatment with the Klenow fragment of DNA polymerase and all four dNTPs.

The resulting blunt ended 4.2 kb fragment was gel purified and self ligated to yield a closed circular plasmid. A 5'-untranslated region adenovirus/immunoglobulin hybrid intron was introduced into the plasmid by replacing a 0.5 kb NdeI-NoLI fragment with the corresponding 0.7 kb NdeI-NotI fragment from the vector pRc/CMV7SB (obtained from Dr. Joseph Goldstein, University of Texas Southwest Medical Center, Dallas, TX). Finally, the resulting CMV promoter expression cassette was shuttled as an NdeI-Sfil fragment into the vector pGEM-4Z (Promega, Madison WI) by ligation to an deI-SfiI fragment (containing pGEM-4Z) obtained from a pGEM based expression vector containing a CMV-promoter and an SV40 origin of replication (Davis et al., 1991).

Example 2 Transient Transfection of COS Cells with Porcine Galactose c(1.3) Galactosyltransferase and man Transferase COS cells (ATCC CRL 1650) were transiently transfected with CMV-based expression vectors. These vectors were pGT, containing an insert comprising a S. sequence (SEQ ID NO:5) encoding the pig a(1,3) Gal transferase oriented for CMV promoter-driven expression in the parent vector pCDNAI (Invitrogen, San Diego,

CA),

and pHT (described above), encoding the human

H

transferase. Clones containing pig Gal transferase cDNAs have been deposited with the Australian Government Analytical Laboratories, 1 Suakin Street, 25 Pymble, N.S.W. 2073, Australia, and have been assigned 25 the designations N94/9030 and N94/9029, respectively (see copending U.S. application Serial No. 08/214,580, supra).

In a series of transfection experiments, the amount of pGT was kept constant at 3 Ag/well and the amount of pHT was varied between 0 pg and 3 pg (Table Transfection was carried out using the DEAE-dextran method (Seed and Aruffo, 1987).

COS cells maintained in DMEM with 10% FBS were seeded into 6-well tissue culture plates and were subsequently transfected with pHT and/or pGT.

Transfected cells were examined for the expression of the Gal a(1,3) Gal epitope or the H epitope 48 hours after transfection. The cell surface expression of these two -31epitopes was assessed using a fluoresceinated IB4 lectin, which binds specifically to the Gal a(1,3) Gal sugar structure, or by indirect immunofluorescence using a monoclonal antibody specific for the human H epitope (ASH-1952, obtained from the Austin Research Institute, Heidelberg, Victoria, Australia; see Devine, et al., 1990) and an immunopurified, fluorescein-conjugated Sheep anti-mouse IgG (Selenus Laboratories, Melbourne, Australia) as the secondary antibody. FIGS. 1-4 show the results of phase contrast and fluorescence

(F)

microscopy of the cells obtained in these experiments.

In particular, FIGS. 1-4 are photomicrographs of African Green Monkey COS cells which have been fluorescently stained with ASH-1952 (FIGS. 2 and 3) or 15 with IB4 (FIGS. 1 and In each figure, the bottom panel shows all cells, as seen by phase contrast illumination, and the top panel shows only those cells specifically binding to the mAb or lectin as seen by ultraviolet illumination. The cells in FIG. 1 have been transfected with pGT; the cells in FIG. 2 have been transfected with pHT; and the cells in FIGS. 3 and 4 have been transfected with equal amounts of both vectors.

To assess the percentage of cells staining for the carbohydrate epitopes, 600-800 cells from each well were 25 counted after staining. As shown in Table 1 and in FIGS.

1-2, most COS cells transiently transfected with the porcine Gal Gal transferase alone (3 ig) were positive for IB4 staining, and most COS cells transfected with the human H transferase alone (3 pg) were positive for anti-H staining. When equal amounts of the two expression plasmids were used (3 Ag each), COS cells stained predominately for the H epitope (68% of cells), with only weak staining observed for the Gal a(1,3) Gal epitope of cells). See Table 1 and FIGS. 3-4. In fact, even at a DNA ratio of 10:1 (3 ig pGT to 0.3 jg pHT), COS cells still stained predominately for the H -32epitope (50.2 relative to the Gal a(1,3) Gal epitope As a control, cotransfections were also done using expression vectors derived from a parent CMV-based expression vector (pCDM8; Seed and Aruffo, 1987) encoding either Ly-9 (Sandrin et al., 1992) or CD48 (Vaughan et al., 1991). Staining for the Ly-9 epitope was carried out using monoclonal antibody anti-Ly-9.2 (Sandrin et al., 1992). Staining for the CD48 epitope was carried out using an anti-CD48 monoclonal antibody (HuLy-m3; Vaughan et al., 1991). When equal amounts of the Ly-9 or CD48 expression vectors were cotransfected with either pGT or pHT, COS cells demonstrated intense staining for the appropriate carbohydrate epitope and for either CD48 15 or Ly-9, respectively. These results indicate that two different CMV-based expression vectors can function equally well when cotransfected into COS cells.

Example 3 Sbession of H transferase in xeno eneic cells 20 results in down-reulation of Gal a(1.3) Gal exDression A porcine kidney cell line (LLC-PK,: ATCC# CRL 1392) was transfected with plasmid pHT (directing the expression of H transferase) and plasmid pSV2neo (directing the expression of the neomycin resistance gene 25 encoding neomycin phosphotransferase) at a molar ratio of 20:1. Transfection was carried out by the calcium phosphate co-precipitation method and transfected cells were cultured in DMEM 10% fetal bovine serum G418 (500mg/ml, active). Stable neomycin resistant colonies were selected and expanded.

The cell surface expression of the H epitope was analyzed on G418 resistant colonies by indirect immunofluorescence performed with ASH-1952 (identified as "anti-H mAB" in FIG. 5) or with the H epitope specific lectin UEAI (EY Laboratories, Inc., San Mateo,

CA.)

directly conjugated to FITC. The Gal a(1,3) Gal cell surface epitope was visualized by staining control and -33transfected cells with the FITC-conjugated lectin, IB4 (EY Laboratories, Inc., San Mateo, CA). As a control, transfected

LLC-PK

1 cells were also stained with the anti-SLA class I (anti-pig major histocompatibility antigen class I) mAb, PT85A (VMRD, Inc., Pullman WA), as a positive control. Goat anti-mouse IgG antisera (monoclonal sera, Zymed Laboratories, South San Francisco, CA) directly conjugated to FITC was used to detect specific antibody binding to the cell surface by flow cytometry as shown in FIG. These data demonstrate that G418 resistant control LLC-PKI cells (clone PKl:neo #B6) normally express low levels of both the Gal a Gal epitope and the H epitope (FIG. 5B) compared to staining with secondary antibody alone (FIG. 5D; 20 curve). However, cells transfected with the human H transferase vector (clone #A3) express high levels of the H epitope (FIG. 5A) and reduced levels of the Gal a(1,3) Gal epitope (FIG. Transfection of these cells with H transferase, however, did not alter the cell surface expression of the SLA class I gene product (FIG. 5C) relative to G418 resistant control cells (FIG. Examole 4 Stable expression of H transferase in 25 xenogeneic ells results in sinificantly reduced binding of human IqG and IqM antibodies Cell surface reactivity of human serum on the LLC- PKI transfectants was measure by incubation with 0% or human whole serum followed by incubation with

FITC

conjugated goat anti-human antibodies specific for either human IgG or human IgM (Zymed Laboratories, South San Francisco, CA). Cell surface antibody binding was then measured by flow cytometry on a FACSort instrument (Becton-Dickinson Immunocytometry Systems, San Jose,

CA).

As shown in FIG. 6, LLC-PK 1 cells stably transfected with pHT demonstrate little to no reactivity to either human IgG (FIG. 6A) or IgM (FIG. 6B) relative to G418 resistant -34control cells which demonstrate significant binding to human IgG (FIG. 6C) and IgM (FIG. 6D) present in human serum. The binding of human IgG and IgM present in 20% human serum to H transferase-expressing

LLC-PK

1 cells is similar to the binding observed with 0% whole human serum. These data together with the data presented in Example 3 indicate that expression of the H epitope on the surface of the LLC-PK, cells results in downregulation of the expression of the Gal Gal epitope to such low levels that preformed naturally occurring human antibodies no longer bind to the cells.

Example Stable eression of H transe rase in xenoeneic cells results in sinifican reduced sensitivity to human complement The functional significance of recombinant

H

transferase expression by LLC-PK, cells was assessed by measuring the efflux of the trapped cytoplasmic indicator dye, Calcein AM (Molecular Probes, Inc.), from cells 20 subjected to human complement-mediated damage by human serum. Transfected cells expressing the human

H

transferase and the neomycin resistance gene (clone #A3; see Examples 3 and 4 above) or the neomycin resistance gene alone (clone #C6; prepared in the same manner as 25 clone B6 described above in Examples 3 and 4) were grown to confluence in 96-well plates. Cells were washed 2X with 200 A1 of HBSS containing 1% BSA (HBSS/BSA).

Calcein AM was added (10M final) and the plates were incubated at 37oC for 30 minutes. Subsequently, the cells were incubated at 37 0 C for 30 minutes in the presence of increasing concentrations of human whole serum.

Dye released from the cells was determined by the fluorescence in the supernatant. Total cell associated dye was determined from a 1% SDS cell lysate. The dye release was calculated as a percent of total, correcting for non-specific dye release and background fluorescence measured for identically matched controls without the addition of serum. Fluorescence was measured using a Millipore Cytofluor 2350 fluorescence plate reader (490nm excitation, 530nm emission).

As shown in FIG. 7, LLC-PK 1 cells stably transfected with pHT (clone #A3; open triangles) were significantly less sensitive to the lytic activity of human complement relative to control LLC-PK, cells (clone #C6; closed circles) at all concentrations of human serum tested between 1% and Throughout this application various publications, patents, and patent applications are referred to. The teachings and disclosures of these publications, patents, and patent applications in their entireties are hereby 15 incorporated by reference into this application to more fully describe the state of the art to which the present invention pertains.

Although preferred and other embodiments of the invention have been described herein, further embodiments may be perceived by those skilled in the art without departing from the scope of the invention as defined by the following claims.

-36vGT (US) 0.0 Positive k Positive 3.0 3.0 1.5 1.0 0.3 0.15 0.03 .01 1.5 4.5 4.6 17.5 43.4 61.5 69 .0 68.0 70.3 65.8 50.2 34.0 68.4 PGT =porcine galactose galactosyl tranf erase 5 cDNA subcloned into CMV-based expression plasmid pCDNAI (Invitrogen, Sand Diego,

CA).

pHT =human H transferase cDNA subcloned into CMV-based expression plasmid pAPEX-1.

1B4 binds to the Gal a(1,3) Gal epitope.

ASH-1952 binds to the H epitope.

-37-

REFERENCES

Auchincloss, 1988. Transpiantation 46, pp. 1.

Ausubel et al., 1992. Current Protocols in Mol Bio, Volume 1, Section III (Units 9.10.1 John Wiley Sons, New York.

Beyer and Hill, 1982, in Horowitz, The Glvcocon-iugrates Volume 3. Academic Press, NY, pp. 25-45.

Blanken and Van den Eijnden, 1985. J Biol _Chem 260, pp.

12927-12934.

Boggs SS, Int. LT. Cell Cloning, 8.80-96; 1990.

Bradley, 1987, in Robertson Teratocarcino)ms and *.*Embryonic Stem- Cells a PracticalAroach. IRL.Press, EynsamOxford, England.

Brent, 1991. Curr Opin Immunol 3, pp. 707.

Brinteret l.,1985. Proc Nati Acad Si. USA 82, pp.

4438-4442.

Cairns et al., 1993A. Secon Ine!toa oncrress on too* Xenotransplantation, pp. 56 (astr).

Cairns et al., 1993B. SeodItentoal Congress on Xenotransplantation, pp. 156 (abstr).

Cone and Mulligan, Proc:. tl. Acad. Sci, USA, 81:6349- 6353 1984.

Cooper et al., 1991, in Cooper et al., (eds). Xenotransplantation Heidelberg, Springet-Verlag, pp.

*Vo* 481-500.

Cosset et al., 1990. J Virol 64, pp. 1070-1078.

Dabkowski et al., 1993. Transplant Pros 25, pp. 2921.

Dalinasso et al., 1992. Am J Path 140, pp. 1157.

Davis et al., 1991. Science 253, pp. 59.

Devine, et al., 1990. J Tumor Marker Oncol pp.

321-339.

Eglitis et al., 1988. Bitchnae 6, pp. 608-614.

Eglitis, 1991. Human Gene Terapy, 2:195-201.

Evans and Scarpulla, 1989. Gene 84, pp. 135.

-38- Fabian et al., 1992. TransplatProc 24p. 604.

Feigner P'L, Rhodes G, Nature, 349:351-352; 1991.

Fournier et al., 1993. Second International Concress on Xenotransplpntation, p 142 (abstr).

Galili et al., 1984. J Exp) Med 160, pp. 1519.

Galili et al., 1985. J ExD Med 162, pp. 573.

Galili et al., 1987. Proc-Nati Acad Sci. USA 84, pp.

1369.

Galili et al., 1988. J Biol Chem 263, pp. 17755-17762.

Galili, 1993. In-ununolocry Today 14, pp. 480.

:Geller et al., 1993. Transplit-ation 55, pp. 168.

Gilboa, Biotechniqrues, 4:504-512, 1986.

Good et al., 1992. Transpat procs 24, pp. 558.

Hammer et al., 1992. Transplant Procs 24, pp. 590.

Hogan et al., 1986. ManpulQAtijng the Mouse mbryo:

A

**Lbrtr anual. Cold Spring Harbor Laboratory, Cold ***Spring Harbor,

KY.

Kohn DE, Anderson WF, Blaese RM, Cancer InvestigAtion, V ~7:179-192;1989.

Koren et al., 1992. TranspiAnt_Proc 24, pp. 598.

0. Koren et al., 1993. SeodItra ial ConcweCgsson XenotransR3 antaton, pp. 178 (abstr).

Kormuan et al., 1987. Proc Nati Aad Sci. USA 84, pp.

2150-2154.

Kornfeld and Kornfeld, 1985. Ann Rev Biochem 54, pp.

631-664.

Kukowska et al., 1990. Genes Dev 4, pp. 11288-13039.

Larsen et al., 1990a. J Biol Chem 265, pp. 7055-7061.

Larsen et al., 1990b. Proc Nati Acad SpiUA 87, pp.

6674-6678.

Lehn PM, Bone Marrow Translantation, 5:287-293;1990.

-39- Lovell-Badge, 1987, in Robertson Teratocarcinomas and EmbrKYonic Stem Cells a Practical Aproach.

IRL

Press, Eynshan, Oxford, England.

Lowe, 1991. Sem Cell Biol 2, pp. 289-307.

Mann, et al., Cell, 33:153-159, 1983.

Markowitz et al., 1988. J Virol 62, pp. 1120-1124.

McMahon et al., 1990. Cell 62, pp. 1073-1085.

Mejia-Laguna et al., 1972. Am J Path 69, pp. 71.

Miller et al., 1986. Mol Cell Bio 6, pp. 2895-2§02.

Miller et al., 1989. Biotechniques 7, pp. 981-990.

Miller, 1992, Naue 357:455-460.

9Morgenstern et al., 1990. Nucleic Acids Res 18, pp. 3587-3596.

Muler-Eberhard, 1988. Ann Rev Biochem 57, pp. 321.

Mulligan, 1993, S-cience, 260:926-932.

9* Najarian, 1992. Transplant Proc 24, pp. 733.

9999 Niekrasz et al., 1992. Transplant Procs 24, pp. 625-626.

Pedersen et al., 1990. Transcrenic Techniaues in Mice A Video GUe Cold Spring Harbor Laboratory, Cold Spring Harbor,

KY.

Platt and Bach, 1991. Curr Opin lInmunol pp. 735.

Platt et al., 1990. Immunologry Today 11, pp. 450.

Platt et al., 1991. Transplantation 52, pp. 214.

Robertson et al., 1986. Nature 323, pp. 445-448.

Robertson, 1987, in Robertson (ed) Teratocarcinomas and Embryonic-Stem, Cells a Practcal Aproach. IRL Press, Eynsham, Oxford, England.

Samnbrook et al., 1989. Molecular C1 ingr: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,

NY.

Sandrin et al., 1992. J Inmunoi 149, pp. 1636-1641.

Sandrin et al., 1993A. Proc Natl1 Acad Sci. USA 90, pp.

11391-11395.

Sandrin et al., 1993B. Transplant Proc 25, pp. 2917.

Seed and Aruffo, 1987. Proc Nati Acad Sci. USA 84, pp.

3365.

Somervile and d'Apice, 1993. Kidney Intl 44, pp. 112.

Stanley, 1992. Glycobiol pp. 99-107.

Talib et al., 1991. Gene 98, pp. 289-293.

Thall and Galili, 1990. Biochemistry 29, pp. 3959.

Tusso et al., 1992. Transplant Procs 24, pp. 596.

Tusso et al., 1993. Transplantation 55, pp. 1375.

Vaughan et al., 1991. Irmunocenet-ics 33, pp. 113-117.

Verina IM, Scientific American, PP. 68-84;1990.

Weatherall DJ, Nature, 349:275-276; 1991.

S..Weston et al., 1992. J ilCe 267, pp. 4152-4160.

a 0Williams et al., 1988. Inmiunorenetics 27, pp. 265-272.

:Yamaicawa and Nagai, 1978. Trends- Biol. Sci 3, pp. 128- 131.

Zehr et al., 1994. Transplantation 57, pp. 900.* *Zhao and Wong, 1991. J Cell Biol 115, pp. 83a.

Zhao et al., 1993. Second Inter-national Concrress on Xenotransplantation, pp. 138 (abstr).

Page(s) _55 5 are claims pages They appear after the sequence listing(s) -41- SEQUENCE

LISTING

GENERAL

INFORMATION:

APPLICANT: Sandrin, Mauro S.

Fodor, William

L.

Rother, Russell

P.

Squinto, Stephen

P.

McKenzie, Ian F. C.

(ii) TITLE OF INVENTION: Methods for Reducing Hyperacute Rejection of Xenografts (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Maurice M. Klee STREET: 1951 Burr Street CITY: Fairfield STATE: Connecticut COUNTRY:

USA

ZIP: 06430 COMPUTER READABLE

FORM:

MEDIUM TYPE: 3.5 inch, 750 Kb storage COMPUTER: Dell 486/50 OPERATING SYSTEM: DOS 6.2 SOFTWARE: WordPerfect (vi) CURRENT APPLICATION

DATA:

APPLICATION

NUMBER:

FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: 08/260,201 FILING DATE: June 15, 1994

CLASSIFICATION:

-42- APPLICATION NUMBER: 08/278,282 FILING DATE: July 21, 1994

CLASSIFICATION:

(Viii) ATTORNEY/AGN

INFORMATION:

NAME: Klee, Maurice

M.

REGISTRATION NUMBER: 30,399 REFERENCE/DOCKET NUMBER: ALX-144.lPCT (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (203) 255-1400 TELEFAX: (203) 254-1101 -43- INFORMATION FOR SEQ ID NO:l: Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 34 bases TYPE: Nucleic Acid STRAliDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: Oligonucleotide primer (iii) HYPOTHETICALj: No ~.(iv)ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION. SEQ ID NO:l: GGCCACGAAA, AGCGGACTGT GGATCCGCCA CCTG 34 -44- INFORMATION FOR SEQ ID NO:2: Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 38 bases TYPE: Nucleic Acid STRAN'DEDNESS: Single TOPOLOGY: Linear MOLECULE TYPE: Other nudE DESCRIPTION: Oligonuc (iii) HYPOTHETICAL~: No (iv) ANTI-.SENSE: Yes ic acid :leotide primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CAGGAACACC ACCAAGCTTC TCGAGAAGAT

GCCAGGCC

INFORMATION FOR SEQ ID NO:3: SEQUENCE

CHARACTERISTICS:

LENGTH: 1174 base pairs TYPE: Nucleic Acid STRANDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA to mRNA DESCRIPTION: Human H-transferase (iii) HYPOTHETICAL: No ANTI-SENSE: No PUBLICATION

INFORMATION:

AUTHORS: Larsen, R.D.

Ernst, L.K.

Nair, R.P.

Lowe, J.B.

TITLE: Molecular cloning, sequence, and expression of a human GDP-L-fucose: -D-galactoside 2-alpha-Lfucosyltransferase cDNA that can form the H blood group antigen.

JOURNAL: Proceedings of the National Academy of Sciences, USA VOLUME: 87 PAGES: 6674-6678 DATE: SEP-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CAAGCAGCTC GGCC 14 ATG TGG CTC CGG AGC CAT CGT CAG CTC TGC CTG GCC TTC CTG 56 Met Trp Leu Arg Ser His Arg Gln Leu Cys Leu Ala Phe Leu 1 5 -46- CTA GTC Leu Val TGT GTC CTC TCT GTA ATC TI'C TTC CTC CAT Cys Val Leu Ser Val Ile Phe Phe Leu His 20 25 ATC CAT le His

CAA

Gin

CCA

Pro

CTG

Leu ccc Pro

CCC

Pro 85

CTG

Leu

CTG

Leu

GAC

Asp

GAC

Asp

CCG

Pro

CAG

Gin

AAT

Asn

CTG

Leu 100

CCT

Pro

AGC

Ser

CGC

Arg

GGT

Giy

CAC

His

GGC

Gly

GCT

Ala

GCC

~TT

Phe

CGC

Arg

ACT

Thr

CCT

Pro

CGG

Arg

CTG

Leu,

CCA

Pro

CTG

Leu

GCG

Ala

GCT

Ala 75 ~Tr Phe

GCC

Ala

CAT

IHis

GTG

Val

ATG

Met

TCC

Ser

GGT

Gly 90

CAG

Gin

GGC

Gly 35

ACA

Thr

GGC

Gly

CTC

Leu

AAT

Asn

CTC

Leu 105

CTA

Leu

CCC

Pro 50

CCC

Pro

TCC

Ser

CAG

Gin

AAC

Asn

GGC

Gly

CCA

Pro

AAC

Asn 65

GGC

Gly

ATG

Met

GGC

Giy CTG TCG ATC CTG Leu Ser Ile Leu a.

a a a.

a GTG GCC ATO Vai Ala Ile GCC TCC TCT Ala Ser Ser ACC TGG ACT Thr Trp Thr GGA CAG TAT Gly Gin Tyr CGC CGG GCC Arg Arg Ala 110 CCG GTA TTC

TTC

Phe

TCC

Ser

GTC

Vai

GCC

Ala TTr Phe

TGT

Cys

TGC

Cyrs

TGT

Cys

TAC

Tyr

ACG

Thr

ATC

le CTG CC Ala Met His Ala Ala Leu Ala 140 182 224 266 308 350 392 434 476 518 560 602 644 Pro Val 115 120 CG ATCIl

ACC

Thr

TGG

Trp

GCG

Ala 155

TGC

Cys

AGA

Arg

AGT

Ser

CTG

Leu

CGG

Arg

GAC

Asp

TCT

Ser 170

GAG

Giu

GTG

Val

CCC

Pro

GAG

Giu

TTG

Leu

TGG

Trp 7TC Phe 185

CTG

Leu

GTG

Vai 130

CTG

Leu

AGA

Arg

ACT

Thr

ACC

Thr

GGT

Gly 200

CTG

Leu

CAG

Gin 145

GAT

Asp

TTC

Phe

CTG

Leu

CAG

Gin

GCC

Ala err Leu

CCT

Pro 160

TTC

Phe

CAC

His

CTC

Leu

CCA

Pro

CAC

His

TTC

Phe

CAC

His 175

GAC

Asp

CGC

Arg

GAA

Giu

GAC

Asp

CTG

Leu

CAT

His

CAC

His 190

CTG

Leu

GTG

Vai 135

TGG

Tip

AAG

Lys

CTC

Leu err Leu

GGC

Gly 205 GAC AGC Asp Ser ATG TCG Met Ser 150 CTC TCT Leu Ser 165 CGG GAA Arg Glu CGG GAA Arg Glu CGC ACA Arg Thr CGC ACG CCG Arg Thr Pro 140 GAG GAG

TAC

GGC 'rrC CCC Gly Phe Pro CAG ATC CGC Gin Ile Arg 180 GAG GCG CAG Giu Ala Gin 195 GGG GAC CC Gly Asp Arg 210 -47-

CCG

Pro CGC ACC TI'T GTC GGC GTC CAC GTG Arg Thr Phe Val Gly Val His Val

CGC

Arc 22C

CTG

Leu 225

AGC

Ser

CAC

His

TGG

Trp Phe

GCC

Ala 295

ACC

Thr

GTC'

Val

AAG~

Lys

GGC

Gly

CAG

Gin

GCC

Ala 240

GAA

Glu

TGT

Cys

GCT

Ala

CTG

Leu TTvC Phe 310 rAC T'yr kTC Ile kT'T Ele

GTT

Val

TAC

Tyr

GCC

Ala 255

AAA

Lys

GGC

Gly cTC Leu

GGC

Gly CTG4 Leu 325

TTT~

Phe

AAT

ATG

Met

CTC

Leu

CCC

Pro

GAA

Glu 270

GAT

Asp

ACA

Thr TTrC Phe

GCC

Ala RiAG Lays 340 .7CA 215 Prc

CGG

Arg

GTT

Val

AAC

Asn

GGA

Giy 285

CAG

Gin

TGG

Trp

AAC

Asn

CCG

Pro

CAG

Gin 230

CAG

Gin TrC Phe

ATC

Ile

CAG

Gin

TGC

Cys 300

GCT

Ala 7TC Phe

GAG

Giu

*CGC

*Arg

GCC

Ala 245

GTG

Val

GAC

Asp

GAG

Giu

AAC

Asn

GCC

Ala 315

ACC

Thr

GCG(

Ala TGG AAG GGT Trp Lys Gly ATG GAC TGG Met Asp Trp GTC ACC AGC Vai Thr Ser 260 ACC TCC C-AG Thr Ser Gin 275 GCT ACA CCG Ala Thr Pro 290 CAC ACC ATT His Thr le TAC CTG GCT Tyr Leu Ala 'TG CCA GAC' L~eu Pro Asp 330 ;CC TrC CTGC kla Phe Leu 345 ~CA CTC TGG ?ro Leu Trp 360

CGT

Arg

GTG

Val 235 T'rC Phe

AAC

Asn

GGC

Gly

TGG

Trp AiTG Met 305 3GC Giy

['CT

Ser :CcC Pro

LCA

['hr

GGG

Gly

GTG

Val

CGG

Arg 250

GGC

Gly

GAT

Asp

AAA

Lys

ACC

Thr 3GA Gly 320

AG

G1u

:AG

flu

GAC

Asp

GGC

Gly

GCA

Ala

ATG

Met 265

GTG

Val

GAC

Asp

ATT

Ile

GAC

Asp TrC Phe 335

TGG

Trp

TAT

Tyr

GAC

Asp

CGG

Arg

GAG

Giu

ACG

Thr 280 Trr Phe

GGC

Gly

ACT

Thr

CTG

Leu

GTG

Val 686 728 770 812 854 896 938 980 1022 1064 1106 Asn Ala Asp 355 Leu Ser 7MT GCT AAG Lieu Ala Lys CCT TGAGAGCCAG GGAGACTTTC TGAAGTAGCC TGATCTITrCT Pro 365 AGAGCCAGCA GTACGTGGCT

TCAGA

1149 1174 -48- INFORMATION FOR SEQ ID NO:4: Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 4059 base pairs TYPE: Nucleic Acid STRAliDEDNESS: Double TOPOLOGY: Circular (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: Apex-i Eukaryotic (CMV) Expression Vector (iv) ANTI-SENSE: No 9* 9 9* 9 *99*

S

9 9 9*9* (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 9. 9 .9 9 9* 99 9 9.

99 9 *99999 9

ACGCG'ITGAC

GTCATTAGTr

TAAATGGCCC

AAT.AATGACG

GTCAATGGGT

GTGTATCATA

GCCCGCCTGG

GGCAGTACAT

TGGCAGTACA

AAGTCTCCAC

AACGGGACTr

GGCGGT.AGGC

GAACCGTCAG

TCTTTCCAGT

CGCCACCGAG

ATTGATTATT

CATAGCCCAT

CGCCTGGCTG

TATGTI'CCCA

GGACTA ITTA

TGCCAAGTAC

CATTATGCCC

CTACGTA'rTA

TCAATGGGCG

CCCATTGACG

TCCAAAATGT

GTGTACGGTG

AATTCTGTTG

ACTCTrGGAT

GGACCTGAGC

GACTAGTrAT

ATATGGAGTT

ACCGCCCAAC

TAGTAACGCC

CGGTAAACTG

GCCCCCTA'rr

AGTACATGAC

GTCATCGCTA

TGGATAGCGG

TCAATGGGAG

CGTAACAACT

GGAGGTCTAT

GGCTCGCGGT

CGGAAACCCG

GAGTCCGCAT

T.AATAGTAAT

CCGCGTTACA

GACCCCCGCC

AATAGGGACT

CCCACTTGGC

GACGTCAATG

CTTATGGGAC

TTACCATGGT

TTTGACTC.AC

TITGTTGG

CCGCCCCArr

ATAAGCAGAG

TGATTACAAA~

TCGGCCTCCG

CGACCGGATC

CAATTACGGG

TAACTTACGG

CATTGACGTC

TTCCATTGAC

AGTACATcAA,

ACGGTAAATG

TTI'CC!TACTI'

GATGCGGTTT

GGGGATTTCC

CACCAAAATC

GACGCAAATG

CTCGTITTAGT

CTCT1'CGCGG AACGGTACcC GGAAAACCTc s0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 -49-

S.

S

S

TCGACTGTfl CTAAGATrGr,

CGCGGTGAT(

TCT1TrTGr

ACTTGAGTGI

TCCCAGGTCC

TAGTAACGGC

CGGCCGCTcc

TACAAATAAA

ACTGCATrc7

TCTGGATCGA

GGACCTC'rrC GCACC'rrGAA

AAACACAGGC

TAGTTGCTAG

GTAATTrCGC

TGCCGAGAGT

CTACTAGAAT

CAGCTTCAGA

CTC7TAAAAT

ACGCAGCTGG

CTCCAGAGGG

GGCCTAGAAT

TAGTArTTAAG

AAGAAGAGAG

CTGCTTCTAT

TCCATACCCC

GCCCATCCCG

"T GGGTGAGTAC r' CAGTTTCCAA 4 CTrTAGGG r' GTCAAGCerrG kCAATGACATC 4AACTGCAGGT

-CGCCAGTGTG

AGCATGCATC

LGCAATAGCAT

'AGTTGTGGTT'

TCCCGCCATG

GTTGTGTAGG

CTGTCTGCAT

ACAGTACTGA

GGCTGTCTCC

CATCAAGGGC

CCCGTAAGGG

AGTCAGTGCG

AGATGGCGGAC

AGAAAATGTC1 CCGTGCGACA

I~

CGTGTGGTT']

GTTTCCACCC

CAGAGGCCGGG

GCA'rrGTAGAG

TTCTGTCACAC

CTITAATAAGC

CCCCTAACTCC

TCCCTCTCAA

AAACGAGGAG

TGGCCGCGTC

AGGTGTGGCA

CACTTTGCCT

CGACCGGCTT

CTGGAATTCT

TAGAACTTGT

CACAAATTTC

TGTCCAAACT

GTATCAACGC

TACCGCTGTA'

CAGCCATATA

CAAACCCATA

"AACTCATTA

kGCGAGGGCT' rAGACACTrc 1 7CTccCATrT

;GGCCTCCAAC

LAGTCAGTTA

'CCTCTITI'A

'GCAAGAGGA

LATCATTACTA

IGACCCCTGG G

~GCTTCCAGAG

TGTCTGGCCC

AGTT'TGGGAA

'GCCCAGTrc 0

AAGCGGGCAT

GATTrGATAT

CATCTGGTCA

GGCTTGAGAT

2VrCTCTCCAC

GGTACCGAGC

GCAGATATCC

TTATTGCAGC

ACAAATAAAG

CATCAATGTA

CATATTTCTA

ITCCTAGGGA

"CCCCCGC'rG

:ACCTCCTCT

2ACCCTCCAA r'CTCCAGATA kGCTAATCCC nGAAAATTCA

ACAGTAATT

LGCAGGAAGT

LTTAGTTGCT

LGCAAAAGCC

TI

frGACAACAGC CCCGC7TAC T

GCAACTTGTC

TGTCACAAG

G

CGGGTGCGG G ~CCCA7TCT

C

GACTrCTGCG

TCACCTGGCC

GAAAAGACAA

CTGGCCATAC

AGGTGTCCAC

TCGGATCCAC

ATCACACTGG

TTATAATGGT

CATIWTTC

TCTTATCATG

TrTACAGTAG

AATAGTAGAG

ITCGACTrAC "AAATA CCA kGTCAGAGCT kAATAGCTTc rCGATGAGGT '"TrACTTGAT T7CCTCCCGA

;GACTAACTG

LGGCAACGCC

'CTCCACCCA

WG~Iur

'CTGGAGAAA

AAAACAGGA

TCCAGCACC

TCTTACTCC

CGCCCCATG

800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150 GCTGACTAAT 71I'r1-rTTATr

TATGCAGAG

a a a a.

a. a a a a a a a.a a..

a

GAGCTATTC

CAAAAAGGA

CTGGCGT

ACGCTCAAG,

CGTTrCcCCC

CTI'ACCGGA!

TCAATGCTO~

AGCTGGGCT

TCCGGTAAcr,,

ACTGGCAGC)

GTGCTACAGI

ACAGTA77r'C.

AGTTGGTAGC

TI-llwGTTG GATCC'rrTGA ACG7TAAGGG TCCTT7TAAA

TAAACTTGGT

AGCGATCTGT

AGATAACTAC

ATACCGCGAG

GCCAGCCGGA

CCATCCAGTC

G7TAATAGTr

ACGCTCGTCG

GGCGAGTTAC

C AGAAGTAGN G CTCCCAGCAJ T TCCALTAGGC~ T CAGAGGTGG( C TGGAAGcTc( r ACCTGTCCG( k CGCTGTAGGI 47 TGTGCACGAZ r' ATCGTCTrGI k GCCACTGGT,

LGTCTTGAAG

GTATCTGCGC

TCTTGATCCG

CAAGCAGCAG

TCTTTrCTAC

ATTTTGGTCA

TTAAAAATGA

CTGACAGTTA

CTATTrCGTr

GATACGGGAG

ACCCACGCTC

AGGGCCGAGC

TATTAATrGT

TGCGCAACGT

TI'TGGTATGG

ATGATCCCCC

"AGGAGGC

AL AAGGCCAGG r CCGcccccc

SGAAACCCGAI

7 CTCGTGCGC SC7-r-rCTcC rATCTCAGTr( kCCCCCCGTT( LGTCCAACCcC LACAGGATrAC.

TGGTGGCCT;

TCTGCTGAAG.

GCAAACAAAC

ATTrACGCGCA

GGGGTCTGAC

TGAGATATC

AGTTTTAAAT

CCAATGC7TA

CATCCATAGT

GGC7TACCAT

ACCGGCTCCA

GCAGAAGTGG

TGCCGGGAAG

TGTrGCCA7wr C7TCATTCAG

ATGTTGTGCA

OCCGAGGCCG

T TTrGGAGGC A ACCGTAAAA T GACGAGCATi C AGGACTATA& r CTCCTGTTC( r TCGGGAAGCC

GGTGTAGGT(

SAGCCCGACCC.

GTAAGACACG.

CAGAGCGAGG.

LACTACGGCTA

CCAGT'ACCTI

CACCGCTGGT

6GAAAAAAAGG

IGCTCAGTGGA

AAAAAGGATC

CAATCTAAAG

ATCAGTGAGG

TGCCTGACTC

CTGGCCCCAG

GATrATCAG

TCCTGCAALCT

CTAGAGTAAG

GCTACAGGCA

CTCCGGrrrcc C CTCGGCCTCT C TAGGCTTrTG P, GGCCGCGTrG

ACAAAAATCG

-k AGATACCAGcG

SGACCCTGCCG

TGGCGC7Ti'rC

GTTCGCTCCA

CTGCGCCTrA AC'rrATCGCC

TATGTAGGCG

CACTAGAAGG

'AGCGGTGGrr

ATCTCAAGAA

ACGAAAACTC

7rCACCTAGA

TATATATGAG

CACCTATCTC

CCCGTCGTGT

TGCTGCAATG

CAATAAACCA

TI'ATCCGCCT

TAGTrCGCCA

TCGTGGTGTC

CAACGATCAA

2200 2250 2300 2350 2400 2450 2500 2550 2600 2650 2700 2750 2800 2850 2900 2950 3000 3050 3100 3150 3200 3250 3300 3350 3400 3450 GGTCCTCCGA TCGTTGTCAG AAGTAAGTrG GCCGCAGTGT

TATCACTCAT

3550 -51- GGTTATGGCA GCACTGCATA

ATCTCTTAC

GCTITrCTGT

GACTGGTGAG

ATGCGGCGAC

GCCACATAGC

GGCGAAAACT

CCCACTCGTG

TTCTGGGTGA

GGGCGAcAcG

TGAAGCATTT

TATTTAGAAA.

TGCCACCTG

CGAGTTGCTC

AGAACTTTA

CTCAAGGATC

CACCCAACTG

GCAAAAAcAG GAAATGTrGA ATCAGGGrrTA

AATAAACAAA

TACTCAACCA

TTGCCCGGCG

AAGTGCTCAT

TTACCGCTGT

ATCTTCAGCA

GAAGGCAAAA

ATACTCATAC

TTGTCTCATG

TAGGGGTTCC

TGTCATGCCA

AGTCATTCTG

TCAATACGGG

CATTGGAAAA

TGAGATCCAG

TCTTITACTT

TGCCGCAAAA

TCTrCCT'T AGCGGATAcA GCGCACAT'rr

TCCGTAAGAT

AGAATAGTGT

ATAATACCGC

CGTrCTrCGG

TTCGATGTAA

TCACCAGCGT

AAGGGAATAA

TCAATATTAT

TATTTGAAG

CCCCGAAAAG

3600 3650 3700 3750 3800 3850 3900 3950 4000 4050 4059 4@

S

S

*SSI**

P

0* S S S. S 54 4* *5 S

S

S.

*5*

S

*5 V 0

S

SO

0e S -52- INFORMATION FOR SEQ ID Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 1423 base pairs TYPE: Nucleic Acid STRA1NDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA to znRNA DESCRIPTION: galactosyl transferase, full coding sequence (iii) HYPOTHETICAL: No ANTI-SENSE: No (vi) ORIGINAL

SOURCE:

ORGANISM: Sus scrofa (xi) SEQUENCE DESCRIPTION: SEQ ID CGGGGGCCAT CCCCGAGCGC ACCCAGCTI'C TGCCGATCAG GAGAAAATA 49 ATG AAT GTC AAA GGA AGA GTG GTT CTG TCA ATG CTG CT!' GTC 91 *Met Asn Val Lys Gly Arg Val Val Leu Ser Met Leu Leu Val *TCA ACT GTA ATG GT1' GTG T'IT TGG GAA TAC ATC AAC AGA AAC 133 Ser Thr Val Met Val Val Phe Trp Glu Tyr le Asn Arg Asn 20 CCA GAA GTT GGC AGC AGT GCT CAG, AGG GGC TGG TGG TIT CCG 175 Pro Glu Val Gly Ser Ser Ala Gin Arg Gly Trp, Trp Phe Pro 35 AGC TGG rrr AAC AAT GOG ACT CAC AGT TAC CAC GAA GAA GAA 217 Ser Trp, Phe Asn Asn Gly Thr His Ser Tyr His Glu Glu Glu 50 GAC GCT ATA GGC AAC GAA AAG GAA CAA AGA AAA GAA GAC AAC 259 Asp Ala Ile Gly Asn Glu Lys Glu Gin Arg Lys Glu Asp Asn 65 -53- AGA GGA GAG Arg Gly Giu CTT CCG CTA GTG GAC TGG TI'r AAT CCT GAC Leu Pro Leu Val Asp Trp Phe Asn Pro Glt CGC CCA GAG

S.

S.

*5 S *5 S

*SS*

S

*SSSS.

Arg

GTA

Val

TAT

Tyr

GTC

Val

TCT

Ser

TAC

Tyr 155

CTG

Leu

GAG.

Giu,

ATC

Phe Phe 225

GCC

Ala Prc

TGC

Trr 100

GCC

Ala GG1A Gly

GCA

Ala

ATC

le

GGT

Gly 170

AAG

Lys

~GG

Gly

CTC

Lieu

;GG

rrp Giu

GAA

Glu

~AAA

Lys 115

LAGA

*Arg

*AAT

Asn

ATG

Met

CCT

Pro

AGG

Arg 185

GAG

Giu

TTC

Phe GTG4 Val

TGG'

Trp'

GTC

Val

GGC

Gly

CAG

Gin

TAC

Tyr 130

ACA

Thr

GTG

Val

CTG

Leu

TGG

Trp

CAC

His 200

TGC

Cys

GAG

Giu rAC I'yr GTG ACC *Val Thr 90 ACT TAC *Thr Tyr AAA AT *Lys Ile A'IT GAG Ile Giu TAC 'rrc Tyr Phe 145 GAT GAT Asp Asp 160 CGT TCC Arg Ser CAA GAC Gin Asp ATC CTG Ile Leu ATT GAC Ile Asp 215 ACC CTG Thr Leu 230 AAG GCA Lys Ala

AT;~

le

AAC

Asn 105

ACC

Thr

CAT

His

ATG

Met

ATC

Ile Trr Phe 175

ATC

le

GCC

Ala

GTG

Val

GGC

Gly

CAT

His 245

L

AGA

Arg

*GTG

Val 120

TAC

Tyr

GTT~

Val

TCC

Ser

AAA

Lys

AGC.

Ser 190

CAC

His GAT4 Asp

CAG

Gin

CT

Pro

TACJ

Tyr 260

GCC

Ala

GGC

Gly

TTG

Leu 135

GGC

Gly

AGG

Arg

GTG

Vai

ATG

Met

ATC

Ile 205

CAG

3er xAC

GTC

Val

TTG

Leu

GAG

Giu

CAG

His 150

ATG

Met Trr Phe

ATG

Met

CAG

Gin

GTC

Val 220

GTG

Val

!AG

i7"u ACC AGA.TGC Thr Arg Trp

AAG

4Lys

TTA

Leu

ACG

Thr

GAG

Glu

AAA

Lys

CCT

Pro 165

GAG

Glu

CGC,

Arg

CAC

His

TI'C

Phe

GCT

Ala 235

TTC

Phe

GM'

Ala

GA]

Asr

GTI

Val

TTC

Phe

GTC

Val Leu

ATC

le 180

ATG

Met

GAG

Glu

CAA

Glm

CAG

fin kLCC m'r so0 E'CC-4 Prc

AA'I

Phe 125

TA

Leu

ATC

le

ATA

le

AAG

Lys

AAG

Lys 195

GTG

Val

AAC

CTA

Leu

TAC

Tyr

AAA

Lye

LGTG

Val

'TAT

Tyr

GCT

Ala

ATA

Ile 140 Phe

GAG

Giu

TCC

Ser

ACC

Thr

GAC

Asp 210

AAC

Asn

GAG

Gin

GAG

Glu 301 343 385 427 469 511 553 595 637 679 721 763 805 847 AGG CGG Arg Arg AAG GAG TCC GCA GCC Lys 255 Glu Ser Ala Ala kLTT CCG T1'T GGC Ile Pro Phe Giy GAG GGG Gin Giy 265 -54- GAT TTT TAT TAC CAC GCA Asp Phe Tyr Tyr His Ala 270 GCC ATT TTT GGG GGA Ala Ile Phe Gly Gly 275 ACA CCC ACT Thr Pro Thr 280 GGA ATC CTC Gly Ile Leu CAG GTT CTA AAC Gin Val Leu Asn

ATC

Ile 285

CAG

Gin 295 GAC AAG GAA AAT Asp Lys Giu Asn ACT CAG GAG Thr Gin Giu GAC ATA GAA Asp leGiu 300 TAT TTC CTT Tyr Phe Leu

GCC

Ala 290

GAG

Giu TGC 7TC AAG Cys Phe Lys

TGG

Trp 305

AAA

Lys AGC CAT Ser His 310 CTA AAC AAG Leu Asn Lys CTC AAC Leu Asn C7A-. GAT GAA His Asp Giu C C C ACT

AAA,

320 ATA GGC ATG Ile Glv Met 889 931 973 1015 1057 1099 ATC TTA TCC le Leu Ser 325 TCT GTG GAT Ser Val Asp CCA GAA TAC Pro Giu Tyr 315

TGC

Cys

TGG

Trp 330

AAG

Lys GAT TAT CAT Asp Tyr His

ATT

le 340 AGG ATT GTC Arg Ile Val

ATA

le 345 GCT TGG CAG Ala Trp Gin 335 AAA AAA Lye Lye 350 0..

GAG TAT AAT TMG GWI AGA AAT AAC ATC TGAC'rrTAAA Glu Tyr Asn Leu Val Arg Asn Asn le 355 'FrGTGCCAGC AGTTCTGA ATrTGAAAGA GTATTACTCT

GG(

TCAGAGAAGT AGCACTrAAT TTTAACTTr

AAAAAAATACTA

CCAACACAGT AAGTACATAT TA7TC'ITCCT

TGCAAC

T

rrrG AGC AATGGGAGAA TGACTCTGTA GTAATCAGAT GTAAA'rrCCC

A

TATCTGCGGA A7TCCAGCTG AGCGCCGGTC GCTACCATTA

CC

TGGTGTCGAC GACTCCTGGA GCCCGTCAcGT ATCGr" 1136

"'TACTTCC

I-CAAAATA

7CTTGTCA

L'GATTTCT

LGTTGGTC

1186 1236 1286 1336 1386 1423

Claims (7)

1. A method for reducing rejection of a xenogeneic cell following transplantation into a human or an Old World primate comprising: producing a genetically altered cell by introducing an expression vector comprising a nucleic acid sequence encoding a protein having fucosyltransferase activity into a recipient cell, the introduction of said expression vector causing a substantial reduction in the binding of naturally occurring preformed human antibodies or naturally occurring preformed Old World primate antibodies to the genetically altered cell when compared to the binding of said antibodies to the recipient cell; and transplanting said genetically altered cell or a cell derived from said cell into a human or an Old World primate.

2. The method of Claim 1 wherein the genetically altered cell is an ungulate cell.

3. The method of Claim 1 wherein the genetically altered cell is a retroviral producer cell.

4. An ungulate cell which has been genetically altered by the introduction of an expression vector 0:0. comprising a nucleic acid sequence encoding a protein having fucosyltransferase activity into a recipient ungulate cell, the introduction of said expression vector causing a substantial reduction in the binding of naturally occurring preformed human antibodies or naturally occurring preformed Old World primate antibodies to said genetically altered ungulate cell when compared to the binding of said antibodies to the recipient ungulate cell.

An ungulate cell, tissue, or organ derived from the genetically altered ungulate cell of Claim 4.

6. A retroviral packaging cell which* has been genetically altered by the introduction of an expression vector comprising a nucleic acid sequence encoding a -56- protein having fucosyltransferase activity into a recipient cell from which the genetically altered retroviral packaging cell is derived, the introduction of said expression vector causing a substantial reduction in the binding of naturally occurring preformed human antibodies or naturally occurring preformed Old World primate antibodies to said genetically altered retroviral packaging cell when compared to the binding of said antibodies to the recipient cell from which the genetically altered retroviral packaging cell is derived.

7. A retroviral producer cell which has been genetically altered by the introduction of an expression vector comprising a nucleic acid sequence encoding a protein having fucosyltransferase activity into a recipient cell from which the genetically altered retroviral producer cell is derived, the introduction of said expression vector causing a substantial reduction in the binding of naturally occurring preformed human antibodies or naturally occurring preformed Old World primate antibodies to said genetically altered retroviral producer cell when compared to the binding of said antibodies to the recipient cell from which the genetically altered retroviral producer cell is derived. Dated this eighteenth day of March 2002 Alexion Pharmaceuticals, Inc. The Austin Research Institute By their Patent Attorneys Cullen Co.