AU690100B2 - Use of porcine gal alpha (1,3) galactosyl transferase in xenograft therapies - Google Patents

Description

94/00 126 RECEIVED 1 9 APR 1994 -1- (32] Attorney Docket No.: ALX-137PCT Patent XENOTRANSPLANTATION THERAPIES This invention relates to xenotransp.antation (transplantation across species) and is particularly concerned with methods of alleviating xenotransplant rejection, maintenance of xenotransplanted tissue in an animal, nucleotide sequences useful in xenotransplant therapies, rejection resistant transgenic organs, and transgenic animals whose tissues are rejection-resistant on xenotransplantation.

The current shortage of tissues for human transplantation has led to recent close examination of xenografts as a possible source of organs. However, when tissues from non human-species are grafted to humans, hyperacute rejection occurs due to the existence of natural antibodies in human serum which react with antigeas present in these species, with rejection occurring within 10-15 minutes of transplantation. This phenomenon depends, in general, on the presence of some or all of antibody, complement, neutrophils, platelets and other mediators of inflammation. In transplantation of vascularized organs between "discordant" species (those in which natural antibodies occur) the first cells A to encounter natural antibodies are the endothelial cells SUBS7TTUTE SHEET (Rule 26) I II L I criAu 94/0 0 126 RECEIVED 1 9 APR 194 -2lining the blood vessels and it is likely that activation of these cells is induced by antibody binding to xenoantigens or other factors, leading to hyperacute rejection.

There is considerable uncertainty in the art ccncerning the nature of possible target xenoantigens on xenograft tissues. Platt et al (Transplantation 50:817-822,1990) and Yang et al (Transplant. Proc.

24:593-594, 1992) have identified a triad of glycoproteins of varying molecular weights as the major targets on the surface of pig endothelial cells. Other investigators (Holgersson et al, Transplant Proc 24:605-608, 1992) indicate glycolipids as key xenoantigens.

We have now found that xenograft rejection, particularly in the context of pig tissue, is associated with antibodies reactive with galactose in an a(1,3) linkage with galactose, (the Gal(l,3)Gal epitope) Modulating the interaction between antibodies reactive with the Gala(l,3)Gal epitope of xenotransplant tissue effects rejection.

In accordance with the first aspect of this invention, there is provided a method of inhibiting xenotransplant rejection in an animal patient, comprising administering to the patient an effective amount of an antagonist of antibody binding to xenotransplant antigers K having galactose in an a(L,3) linkage with galactose.

SUBSTITUTE SHEET (Rule 26) 1r/AU94/ 00 1 2 RECEIVED 1 9 APR 19S -3- Another aspect of this invention relates to the maintenance of xenograft tissue in an animal, which comprises administering to the animal a graft rejection effective amount of an antagonist to antibodies which bind to the xenograft antigen epitope Gal((l,3)Gal.

In another aspect of this invention there is provided a method of inhibiting the binding of antibodies to the Galc(1,3)Gal epitope which comprises modulating the interaction between the antibodies and the epitope with an antagonist which blocks the binding of the antibodies to the Gala(l,3)Gal epitope.

Preferably the xenograft recipient is a human. Age is not a determining factor for xenograft transplantation although transplants in the elderly over 75 years would normally not be carried out. The invention is directed particularly to human transplantation with xenograft tissue.

Xenografted tissue is preferably of pig origin.

Tissues from other mammals are also contemplated for use in this invention. Preferably the xenotransplanted tissue is in the form of an organ, for example, kidney, heart, lung or liver. Xenotransplant tissue may also be in the form of parts of organs, cell clusters, glands and the like. Examples include lenses, pancreatic islet cells, skin and corneal tissue. The nature of the xenotransplanted tissue is not of itself critical as any xenotransplanted tissue which expresses antigens having SUBSTITUTE SHEET (Rule 26) rCT7 V94 /00 0 1 RECEIVED 1 9 APR J8 Gala(l,3)Gal epitopes may be utilized in accordance with this inventio-i.

The binding of antibody to the Gala(1,3)Gal epitope expressed on xenotransplanted tissue provokes rejection of the tissue by humoral as well as cell-mediated immune effects leading to tissue rejection in a very short time scale, such as less than one hour. Antagonists which antagonize the binding of antibodies to the Gala(1,3)Gal epitope block antibody binding and therefore inhibit xenotransplant rejection. Because antibody binding is blocked, immune responses which give rise to tissue rejection are prevented.

In accordance with a further aspect of this invention, there is provided an antagonist which modulates the interaction of antibodies directed against Gale(1,3)Gal.

Any antagonist capable of modulating the interaction between antibodies directed to the Gala(l,3)Gal linkage iiay be utilized in this invention. By reference to modulation, is meant blockage of antibody binding or decrease in affinity reactivity of antibodies for the Gala(1,3)Gal epitope. Various mechanisms may be associated with the blockage of antibody binding or decreased affinity of antibodies for their respective epitope. These include binding or association with the antibody reactive site and change of conformation of the R AL 4antibody reactive site, such as by binding to residues r- mj associated with, adjacent to, or distanced from the SUBSTITUTE SHEET (Rule 26) 5 active site, which effect the conformation of the active site such that it is incapable of binding the Gala(l,3)Gal epitope or binds the epitope with reduced affinity. For example, in accordance with techniques well known in the art (see, for example, Coligan et al, eds, Current Protocols in Immunology, John Wiley Sons, New York, 1992; Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988; and Liddell and Cryer, A Practical Guide to Monoclonal Antibodies, John Wiley Sons, Chichester, West Sussex, England, 1991), such a change of the conformation of the antibody reactive site can be achieved through the use of an anti-idiotypic antibody raised against the natural antibody or fragments thereof. As is also well know in the art, these anti-idiotypic antibodies may be modified to enhance their 15 clinical usefulness, for example, by enzymatic techniques such as preparing chimeric, humanized, or single chain antibodies.

In accordance with a further aspect of the invention there is provided the method for blocking human anti-Gala(l,3)Gal antibodies comprising changing the conformation of the antibody reactive site so as to reduce the affinity of the antibody for the Gala(l,3)Gal epitope. The conformation of the antibody reactive site is preferably changed through the use of an antiidiotypic antibody.

This invention is not limited to any specific antagonist and any antagonist which is non-toxic and which modulates the interaction between antibodies specific for the Gala(1,3)Gal epitope may be used in this invention. Suitable examples of antagonists include D-galactose and melibiose, stachyose and methyl-a-D-galactopyranoside, D-galactosamine and derivatives thereof. The term derivatives encompasses, for example, any alkyl, alkoxy, alkylkoxy, aralkyl amine P:\WPDOCS\NEH\SPEC\6279294.SPE:9/2/98 -6hydroxyl, nitro, heterocycle, sulphate and/or cycloalkyl substituents whether taken alone or in combination, which derivatives have antagonist activities. This may be assessed according to methods as herein described.

Carbohydrate polymers containing one or more of the aforesaid carbohydrate moieties or derivatives may also be utilized in this invention.

The amount of antagonists which is effective to modulate interaction between antibodies reactive with Gala(l,3)Gal epitopes will vary depending upon a number of factors. These include the nature of the animal being treated, the nature of species of the transplanted tissue, the physical condition of the transplant recipient (age, weight, sex and health) and the like. In respect of human transplant recipients of tissue, for example from pigs, the amount of antagonists administered will generally depend upon the judgement of a consulting physician. As an example, a graft rejection effective amount of an antagonist in human subjects may be in the order of from O.Olmg to 1000gm per dose, more preferably to 500mg, more preferably 50mg to 300mg, and still more preferably 50mg to 200mg per dose.

The schedule of administration of antagonists to inhibit rejection and maintain xenografts will depend upon varying factors as mentioned above. Varying dosage regimes may be contemplated, such as daily, weekly, monthly or the like.

:SUBSTITUTE SHEET (Rule 26) 7 The mode of administration of antagonists and dosage forms thereof are not critical to this invention. Antagonists may be administered parenterally (intravenous, intramuscular or intraorgan injection), orally, transdermally, or by vaginal or anal routes, or by other routes of administration, as are well known in the art. Antagonists may be in solid or liquid form and would generally include pharmaceutically acceptable excipients and/or carriers. Examples of dosage forms which may be used in this invention are those well known in the art as mentioned previously such as described in Remington's Pharmaceutical Sciences (Mack Publishing Company, 10th Edition, which is incorporated herein by reference).

In accordance with another aspect of this invention, there 15 is provided an isolated nucleic acid molecule comprising: the nucleic acid sequence of SEQ ID NO:1; or an antisense sequence complementary to or both and In another aspect of the invention there is provided an isolated nucleic acid molecule comprising:

C.

the nucleic acid sequence of SEQ ID NO:2; or

C.

an antisense sequence complementary to or both and Nucleotide sequences may be in the form of DNA, RNA or mixtures thereof. Nucleotide sequences or isolated nucleic acids may be inserted into replicating DNA, RNA or DNA/RNA vectors as are well known in the art, such as plasmid, viral vectors, and the like (Sambrook et al, Molecular Clongin: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, Second Edition 1989).

Nucleotide sequences encoding Gala(l,3) galactosyl transferase may include promoters, enhancers and other "N j P:\WPDOCSWBNEI\SPEC\6279294.SPF9/2/98 0 r'c O a PCT/AU9 4/ 0 1 2 RECEIVED 1 9 APR 199: -8regulatory sequences necessary for expression, transcription and translation. Vectors encoding such sequences may include restriction enzyme sites for the insertion of additional genes and/or selection markers, as well as elements necessary for propagation and maintenance of vectors within cells.

Mutants of nucleotide sequences encoding a(1,3)galactosyl transferase are particularly preferred as they may be used in homologous recombination techniques as are well known in the art (Capecchi M R, Altering the Genome by Homologous Recombination, Science 244:1288-1292, 1989; Merlino G T, Transgenic Animals in Biomedical research, FASEB J 5:2996-3001, 1991; Cosgrove et al, Mice Lacking MHC Class II Molecules, Cell 66:1051- 1066, 1991; Zijlstra et al, Germ-line Transmission of a disrupted B2-microglobulin gene produced by homologous recombination in embryonic stem cells, Nature 342:435, 1989) for the inactivation of wild type a(1,3) galactosyl transferase genes.

Mutant a(1,3) galactosyl transferase nucleotide sequences include nucleotide deletions, insertions, substitutions and additions to wild type a(1,3) galactosyl transferase such that the resultant mutant does not encode a functional galactosyl transferase.

These nucleotide sequences may be utilized in homologous recombination techniques. In such techniques, mutant sequences are recombined with wild type genomic sequences in stem cells, ova or newly fertilized cells comprising SUBSTITUTE SHEET (Rule 26) llr 9 r r e o oc from 1 to about 500 cells. Nucleotide sequences utilized in homologous recombination may be in the form of isolated nucleic acid sequences, or in the context of vectors. Recombination is a random event and on recombination, destruction of the functional gene takes place.

Transgenic animals produced by homologous recombination and other such techniques to destroy wild type gene function are included within this invention, as are organs derived therefrom. By way of example, transgenic pigs may be produced utilizing homologous recombination techniques to produce a transgenic animal having non-functional Galc(l,3) galactosyl transferase genomic sequences. Tissues derived from such transgenic animals may then be utilized in xenotransplantation 15 into human patients with the avoidance of immune reaction between circulating human antibodies reactive with Gala(l,3)Gal epitopes. Such transplants are contemplated to be well tolerated by transplant recipients. Whilst transplanted tissue may comprise other antigens which provoke immune reaction beyond those associated with Gala(l,3)Gal epitopes, removing the major source of the immune reaction with such transplanted tissues should lead to xenotransplants being relatively well tolerated in conjunction with standard rejection therapy (treatment with immune suppressants such as cyclosporin).

In accordance with another aspect of this invention there is provided a porcine cell comprising an inactivated porcine a(1,3) galactosyl transferase gene, said inactivated porcine a(1,3) galactosyl transferase gene comprising a wild type porcine o(1,3) galactosyl transferase sequence disrupted by a cloned mutant porcine a(1,3) galactosyl transferase sequence, wherein the cloned mutant porcine a(1,3) galactosyl transferase sequence comprises a mutation of SEQ ID NO:1, wherein the mutation is selected from the group consisting of a deletion, an insertion, a substitution, and an addition such that the cloned mutant porcine u(1,3) galactosyl transferase sequence does not encode a functional galactosyl transferase so that P:\WPDOCSNE\SPEC\6279294.SPE:9/2/98 Uj

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'AIT 0C 9a immune reaction of the cell with human antibodies reactive with Galo(1,3)Gal epitopes is avoided.

In accordance with another aspect of this invention there is provided a porcine cell comprising an inactivated porcine u(1,3) galactosyl transferase gene, said inactivated porcine u(1,3) galactosyl transferase gene comprising a wild type porcine a(1,3) galactosyl transferase sequence disrupted by a cloned mutant porcine u(1,3) galactosyl transferase sequence, wherein the cloned mutant porcine a(1,3) galactosyl transferase sequence comprises a mutation SEQ ID NO:2, wherein the mutation is selected from the group consisting of a deletion, an insertion, a substitution, and an addition such that the cloned "i mutant porcine u(1,3) galactosyl transferase sequence does not 15 encode a functional galactosyl transferase so that immune reaction of the cell with human antibodies reactive with Gala(1,3)Gal epitopes is avoided.

This invention will now be described with reference to the following non-limiting Figures and Examples.

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r P:\WPDOCS\NEH\SPEC\6279294.SPE:9/2/98 cr/AU 9 4 0 0 1 26 RECEIVED 1 9 APR 1994 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Figure 1A shows titer of pooled human serum before and after absorption. Titer obtained by hemagglutination on RBC (hatched bars) and rosetting assay on PBL (open bars) and spleen cells (solid bars).

Absorption studies demonstrated that the same xeno antigens were present on all of these tissues (Figure 1 and Figure as absorption with RBC, spleen cells or PBL, removed reactivity for the other cells (Figure 1A and Figure Absorption of the serum pool with EC, while removing all of the EC reactive antibodies (Figure 2A), completely removed all PBL reactive antibodies and almost all the RBC hemagglutinating antibodies (titer fell from 1/128 to 1/2) (Figure 1A). AbsorptliL with RBC removed 75% (Figure 2B3 and spleen cells all (Figure 2C) of the EC reacrive -n-._bodies shown by flow cytometry.

Thus, common epiitpes -re present on pig red cells, PBL, spleen and endothel al cells. Serum absorbed with EC was not tested on ?L zr spleen cells. Figure 1B see Figure 3.

Figure 2: Testing of pig EC with pooled human serum before and after absorption. In each panel EC tested with absorbed serum (dotted line) or non absorbed serum (solid line). Serum absorbed with EC (panel RBC (panel B) or spleen cells (panel Binding of human antibody was detected using sheep anti-human IgM and analysis by flow cytometry.

SUBSTITUTE SHEET (Rule 26) L I S/A 9 4/0 0 12 6 RECEIVED 9 APR 199 -11- Figure 3: Hemagglutination titer of treated and untreated human serum. Untreated human serum protein-A non binding immunoglobulin protein-A eluted immunoglobulin serum treated with 2-mercaptoethanol Figure 1B shows the same data with the addition of data obtained using a high molecular weight immunoglobulin fraction. Figure 1B: Untreated human serum protein-A non binding immunoglobulin high molecular weight fraction protein-A eluted immunoglobulin serum treated with 2-mercaptoethanol Figure 4: Carbohydrate inhibition of hemagglutination of normal human serum. Human serum was titered in the presence of 300mM solutions of carbohydrates.

Figure 5: Concentration of carbohydrate giving inhibition of hemagglutination titer of normal human serum. Only carbohydrates inhibiting hemagglutination in Figure 4 were used in this experiment, with glucose and methyl-f-galactopyranoside as negative controls.

Figure 6: Hemagglutination titer of human serum on pig RBC pre and post absorption on a melibiose column.

Human serum was absorbed with equal volumes of melibiose-sepharose (solid bars) or sepharose (open bars), a number of times as indicated in the figure axis.

Figure 7: Southern blot of pig genomic DNA probed with the cDNA insert of clone pPGT-4.

SUBSTITUTE SHEET (Rule 26) rCT/A 94/00 126 RECEIVED 1 9 APR 1994 -12- BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS SEQ ID NO:1 Partial nucleotide and predicted amino acid sequence of the pig Gala(1,3) transferase.

SEQ ID NO:2 Complete nucleotide and predicted amino acid sequence of the pig Gala(1,3) transferase.

SEQ ID NO:3 Nucleotide sequence for PCR primer aGT-1.

SEQ ID NO:4 Nucleotide sequence for PCR primer aGT-2.

With regard to SEQ ID NOS:1-2, it should be noted that the present invention is not limited to the specific sequences shown, but, in addition to the mutations discussed above, also includes changes that are found as naturally occurring allelic variants of the porcine Gal c(l,3) galactosyl transferase gene, as well as nucleic acid mutations which do not change the amino acid sequences set forth in these sequences, Lhird nucleotide changes in degenerate codons.

EXAMPLE 1 Materials and Methods Cells. Pig cells and tissues were obtained from an abattoir from freshly slaughtered animals. Whole blood was centrifuged at 800g, and erythrocytes (RBC) obtained and were washed three times in phosphate buffered saline (PBS); pig peripheral blood lymphocytes (PBL) were isolated by density gradient centrifugation using ISOPAQUE FICOLL (Vaughan et al, (1983) Transplantation 36:446-450). Pig splenocytes were obtained from whole RA4/ N'rCf SUBSTITUTE SHEET (Rule 26) II rcr/AU 9 4/ u Z b RECEIVED 1 9 APR t994 -13spleen by teasing tissue through a sieve to give i single cell suspension. Endothelial cell (EC) cultures were established after treatment of sterile pig aorta with Collagenase Type 4 (Worthington Biochemical Corporation, New Jersey) and the isolated cells were grown in Dulbeccc's modified Eagles medium (DMEM) (ICN Biomedicals Australasia Pty Ltd, Seven Hills, NSW) on gelatin coated plates at 37 0 C. The endothelial origin of EC cultures was verified using rabbit anti human von Willebrand factor antibody (Dako A/S, Copenhagen) and indirect immunofluorescence. COS cells used were maintained in fully supplemented DMEM medium.

Antibodies. Human serum was obtained from a panel of normal volunteers, heat inactivated and pooled before use. The mAb HuLy-m3 (CD48), was used as a negative control (Vaughan Supra). Equal volumes of human serum and 5 to 200mM 2-mercaptoethanol were incubated at 37 0

C

for one hour to destroy IgM.

Absorptions. Pooled serum was absorbed with equal volumes of washed, packed cells for 15 minutes at 37 0

C,

for 15 minutes at 4 0 C, serum obtained and the procedure repeated three times. For the absorption with melibiose-agarose (Sigma, St Louis, MO) and sepharose (Pharmacia LKB Biotechnology, Uppsala, Sweden), equal volumes packed beads and serum were incubated at 37 0 C for 16 hours, the beads removed by centrifugation, and the absorption repeated several times.

SUBSTITUTE SHEET (Rule 26) cr 9 U94/00 1 26 RECEIVED 1 9 APR 1994 -14- Serological Assays. a) Hemagglutination: 50p1 of 0.1% pig RBC were added to 50Al of human serum in 96 well plates, incubated at 37 0 C for 30 minutes, room temperature for 30 minutes and on ice for 60 minutes prior to both macroscopic and microscopic evaluation of hemagglutination; b) Rosetting: Sheep anti human IgG was coupled to sheep RBC with chromic chloride and used in a rosetting assay (Parish et al (1978) J Immunol. Methods 20:173-183); c) Cytofluorographic analysis was performed on FACScan (Becton Dickinson, San Jose, CA) (Vaughan et al (1991) Immunogenetics 33:113-117); d) Indirect immunofluorescence was performed on cell monolayers in 6 well tissue culture plates using fluoresceinated sheep anti human IgM or IgG (Silenus Laboratories Pty Ltd, Hawthorn, Victoria, Australia) (Vaughan Supra).

Sugar Inhibitions. Two types of sugar inhibition assays were performed: a) 50p1 of sugars (300mM in PBS) were added to 50Al of doubling dilutions of human serum in 96 well plates, incubated overnight at 46 0 C and then 501 of 0.1% pig RBC added and the hemagglutination assay performed; b) Human serum, diluted in PBS at one dilution less than that of the 50% hemagglutination titer, was added to 50A1 of doubling dilutions of sugars (starting at 300mM) and incubated overnight at 4 0 C, after which 50j1 of 0.1% pig RBC was added and the hemagglutination assay performed.

Murine Gal o(l-3) Transferase cDNA construct. A cDNA clone, encoding the mouse a(1,3)galactosyl SUBSTITUTE SHEET (Rule 26) i I I II I rcr/A94/00 1 2 6 RECEIVED 1 9 APR 1994 transferase was produced using the published sequence of this transferase (Larsen et al (1989) J Biol. Chem 264:14290-14297) and the polymerase chain reaction (PCR) technique. Briefly two oligonucleotides were synthesized; GT-1 (5'-GAATTCAAGC TTATGATCAC TATGCTTCAA which is the sense oligonucleotide encoding the first six amino acids of the mature aGT and contains a HindIII restriction site, and aGT-2 AGTCAGACAT TATTCTAAC-3') which is the anti-sense oligonucleotide encoding the last 5 amino acids of the mature aGT and the in phase termination codon and contains a PstI restriction site. This oligonucleotide pair was used to amplify a 1185 bp fragment from a C57BL/6 spleen cell NA library (Sandrin et al (1992) J Immunol. 194:1636-1 41). The 1185 bp fragment was purified from a L-w -lling point agarose gel, digested with HindIII id PstI (Pharmacia) restriction endonucleases, and 1i:rectionally cloned into HindIII/PstI digested CDM8 vector Seed B (1987) Nature 329:840 842) using T4 ligase (Pharmacia). The product of the ligation was used to transform MC1061/p3, and DNA prepared from resultant colonies for further examination. One plasmid (paGT-3) having the 1185 bp fragment was selected for further studies. Plasmid DNA was prepared, sequenced to confirm the correct DNA sequence, and used for COS cells transfection experiments using DEAE/Dextran (Vaughan et al (1991) Immunogenetics 33: 113-117; Sandrin et al -tU

C.,

AI 0 'v SUBSTITUTE SHEET (Rule 26) I rcr/A 94 /O 0 126 RECEIVED 1 9 APR 1994 -16- (1992) J Immunol. 194:1636-1641, Seed B (1987) Nature 329:840-842).

EXAMPLE 2 Human Anti-pig Antibodies Detect Epitopes Present on Different Cells To establish that human serum contains antibodies to pig cells which are predominantly of the IgM class, a pool of human serum was made (from 10 donors) and found to contain antibodies which reacted with pig red cells (by hemagglutination); pig lymphocytes (rosetting assay and flow cytometry); pig spleen cells (rosetting); and pig endothelial cells (flow cytometry) (Figures 1 and 2).

Absorption studies demonstrated that the same xeno antigens were present on all of these tissues (Figure 1 and Figure as absorption with RBC, spleen cells or PBL, removed reactivity for the other cells (Figure 1A and Figure Absorption of the serum pool with EC, while removing all of the EC reactive antibodies (Figure 2a), completely removed all PBL reactive antibodies and almost all the RBC hemagglutinating antibodies (titer fell from 1/128 to 1/2) (Figure 1A). Absorption with RBC removed 75% (Figure 2B) and spleen cells all (Figure 2C) of the EC reactive antibodies shown by flow cytometry.

Thus, common epitopes are present on pig red cells, PBL, spleen and endothelial cells.

Most of the activity in the serum pool was due to IgM rather than IgG antibodies as indicated by the inability of a protein A-sepharose column, which does not SUBSTITUTE SHEET (Rule 26) I i 1 rCr/At 94/ 0 0 126 RECEIVED 1 9 APR 1994 -17bind IgM, to alter the titer of the serum after passage through the column (Figure and IgG antibodies eluted from the protein A-sepharose column reacted only weakly with RBC (Figure Furthermore, treatment of the serum with 2-mercaptoethanol, which destroys IgM but leaves IgG intact, led to a complete loss of antibody activity (Figure When the serum was fractionated by SEPHACRYL gel chromatography, the high molecular weight fractions (IgM) were reactive with RBC, whereas the low molecular weight fractions (IgG) were not (data not shown). Thus the different pig cells carry similar epitopes, all reacted with IgM antibodies and in our assays there was little IgG activity found in the human serum for pig cells.

EXAMPLE 3 Human Anti-pig Antibodies React Predominantly With Terminal Galactose Residues The ability of different carbohydrates to inhibit the hemagglutination reaction (Figure 4) was examined.

Of the sugars tested, inhibition as measured by decrease in titer, was observed with 300mM galactose, methyl-a-D-'Talactopyranoside, melibiose and stachyose, all of which decreased the titer of the serum pool by (Figure and with 300mM D-galactosamine, for which a 50% decrease in titer was observed (Figure None of the other monosaccharides tested (listed in the Aigure Slegend) had any effect on hemagglutination titer (Figure w- These studies demonstrated that galactose is the SUBSTITUTE SHEET (Rule 26) l ooU.9 4/00 126 RECEIVED 1 9 APR 1994 -18part of the epitope, as both melibiose and stachyose have terminal galactose residues. It is of interest to note the difference in the ability of galactose in the a(methyl-a-D-galactopyranoside, melibiose and stachyose) but not 3(methyl-O-D-galactopyranoside) configuration to inhibit the serum.

The relative avidity of the antibodies for the sugars which inhibited agglutination was estimated from the concentration of sugar giving 50% inhibition of the agglutination titer (Figure Both D-galactose and melibiose achieved this inhibition at <1.5mM, stachyose and methyl-a-D-galactopyranoside at 4.7mM and D-galactosamine at 18.7mM (Figure By contrast, D-glucose and methyl--D-galactopyranoside had no effect even at 300mM concentration. Thus D-galactose is an important part of the epitope, as it is a potent inhibitor of the xenoantibodies at low concentration 5mM); the ability of methyl-a-D-galactopyranoside to inhibit agglutination at low concentrations (<1.15mM), compared with the failure of methyl-o-D-galactopyranoside (300mM) to inhibit, demonstrates that the galactose residue (which is likely to be a terminal sugar) is in an a-linkage rather than a 0-linkage with the subterminal residue. The results obtained with melibiose (Gala(l,6)Glc) and stachyose (Gala(l,6)Gal(1l,6)Glco(l,2)Fru), which have a-linked terminal galactose residues, are in accord with this IJHA4/\

I,

I

SUBSTITUTE SHEET (Rule 26) M4 u4 U l RECIVED 1 9 APR 1994 -19conclusion. The inhibition of hemagglutination observed with galactosamine, which has an additional amine side chain on galactose, (50% inhibition of titer at 18.7mM) could be due to a second carbohydrate involved in the epitope, or a lower affinity of the xenoantibodies for this sugar.

To further examine the reaction with galactose, the serum pool was absorbed four times with equal volumes of packed melibiose sepharose or with sepharose as the control (Figure one absorption with melibiosesepharose decreased the titer of the antibody from 1/32 to 1/4, and two sequential absorptions decreased the titer further to 1/2 (Figure This absorption was specific for melibiose, as using sepharose beads had no effect (Figure Thus the majority of the antibody reactive with xenoantigens reacts with galactose in an a-linkage.

EXAMPLE 4 Human Anti-Piq Antibodies React with COS Cells After Transfection with a(1,3) Galactosyl Transferase The cDNA coding for the a(l,3)galactosyl transferase which transfers a terminal galactose residue with e.n a(1,3) linkage to a subterminal galactose has been cloned for both mouse (Larsen et al (1989) J Biol Chem 264:14290-14297) and ox (Joziasse et al (1989) J Biol Chem 264:14290-14297). Using this data we used transfection experiments to determine the role of the Gal(1,3)Gal epitope in isolation of others. The mouse SSUBSTITUTE SHEET (Rule 26) I I ErCA'94 0 0 12 6 RECEIV 1 9 APR 194 transferase was isolated from a cDNA library using the PCR technique, and the PCR product was directionally cloned into the _3M8 vector for expression studies in COS cells. The cDNA insert was sequenced in both directions and shown to be identical to the published nucleotide sequence (Larsen et al (1989) J Biol Chem 264:14290-14297). COS cells, derived from Old World Monkeys, were chosen as they do not react with human serum nor with the IB-4 lectin (which is specific for the Gal(l1,3)Gal epitope) (Table After transfection of COS cells with the a(1,3)galactosyl transferase, the Gala(l,3)Gal epitope was detected on the cell surface by binding of the IB-4 lectin (Table these cells were also strongly reac:ive with the serum pool. Absorbing the human sera wi:. pig RBC removed the reactivity for Gala(l,3)Gal COS -:ils, (Table Passage of the serum over a protein-A rnarose column had no effect on the reactivity of the ezrn for Gala(1,3)Gal+COS cells, when using an FITC cnr-.; u:aed sheep anti-human IgM as the second antibody (this was reflected in the same number of reactive cells, the intensity of staining and the titer of the serum (Table In contrast to this, eluted antibodies reacted only weakly with the Gala(l,3)Gal'COS cells, and this reaction was only observed when using FITC conjugated sheep anti-human IgG or FITC conjugated sheep anti-human Ig, but not FITC conjugated sheep anti human IgM (Table Thus human serum has IgM antibodies I. to the Gala(l,3)Gal epitope which was expressed on P~ALti PIT 0 SUBSTITUTE SHEET (Rule 26)

I

rCT/AU 4/00 12 6 RECEfIVED I APR199 -21- Gala(1,3)Gal*COS cells. The reaction of the serum with Gala (1,3)Gal+COS cells is specific and not due to the transfection procedure as CD48 COS cells were not reactive with either the serum-nor the IB-4 lectin (Table Furthermore, the reactivity for both pig RBC (as detected by hemagglutination) and EC (as detected by FACS analysis) could be removed by absorption with Gala(l,3)Gal COS cells but not untransfected COS cells.

Thus human serum pool contains IgM antibodies reactive with the Gala(1,3)Gal epitope.

The level of antibodies in human serum reactive with the Gale(l,3)Gal epitope can be used to determine the propensity of a patient to hyperacutely reject a porcine xenotransplant. In addition, the level of such antibodies can be used to determine the amount of antibody antagonist that should be administered to a patient prior to such xenotransplantation.

The level of these antibodies can be effectively determined using the transfected and untransfected COS cells described above as matched Gala(l,3)Gal' and Gala(1,3)Gal' absorbants, followed by a measurement of the reactivity of the absorbed serum for pig RBC and/or EC.

Higher levels of serum antibody will result in a larger difference in reactivity of the serum absorbed against the Gala(1,3)Gal absorbant versus that absorbed against the Gala(1,3}Gal" absorbant. Cells from other species, human cells, can be used in such an assay. Also, rather than using a DNA sequence encoding the murine SUBSTITUTE SHEET (Rule 26) ZcX7AU 4/0 U uI b RECEIVED 9 APR 1994 -22transferase, a DNA sequence encoding the porcine transferase (see Example 5) can be used. Such a porcine transferase is preferred since there may be differences in the action of the murine and porcine transferases, altered sensitivity to the macromolecular environment of the galactose substrate of the enzyme, and for a porcine xenotransplantation, it is 'the level of antibodies against the Gala(l,3)Gal epitope in the porcine macromolecular environment that is of interest.

In addition to the foregoing, the transfected Gale(l,3)Gal cells described above can also be used as absorbants to remove anti-Gala(l,3)Gal antibodies from human serum, by binding such cells to a solid support and passing the serum over the immobilized cells.

EXAMPLE Cloning of Porcine a(1,3) Galactosyl Transferase Utilizing the murine cDNA clone for the a(1,3) galactosyl transferase as a hybridization probe we have cloned the pig a(1,3) galactosyl transferase from a XGT11 pig spleen cDNA library (Clontech Laboratories, Palo Alto, CA) according to standard methods as described in Sambrook et al (supra). This clone, pPGT-4, has been deposited with the AGAL and assigned accession number N94/9030. SEQ ID NO:1 shows a partial nucleotide sequence and predicted amino acid sequence of pig Gala(l,3) transferase as determined by sequencing of clone pPGT-4. The sequence shown is incomplete at the end.

(RAQ

I 4-'.F cU 6." lu SUBSTITUTE SHEET (Rule 26) /Au 9 4/00 1 2 6 RECEIVED 19 APR 1994 -23- Utilizing the cDNA insert of the pPGT-4 clone as a hybridization probe we have also cloned the 5' end of the pig u(1,3) galactosyl transferase from a 5' STRECH pig liver cDNA library in Xgtl0, according to standard methods as described in Sambrook et al (suora). The insert was obtained by the PCR technique using a X oligonucleotide, and an oligonucleotide made to the pig sequence. This PCR product was subcloned into SmaI cut pBLUESCRIPT KS This clone, pPGT-2, has been deposited with the AGAL and assigned accession number N94/9029.

SEQ ID NO:2 shows a complete nucleotide sequence and predicted amino acid sequence of pig Gal(1,3) trareferase as determined by sequencing of clones pPGT-4 and pPGT-2. The pig transferase has high sequence homology with both the murine and bovine a(1,3) galactosyl transferase genes.

Both the partial and complete cDNA sequences of SEQ ID NOS:1-2 can be used in the xenotransplant therapies discussed above. For example, using techniques well known in the art, all or a part of any of the nucleotide sequences of SEQ ID NOS:1-2, when inserted into replicatin DNA, RNA or DNA/RNA vectors, can be used to reduce the expression of the Gala(l,3) transferase in porcine cells by directing the expression of anti-sense RNAs in transgenic cells or animals. See, for example, Biotechniques, 6(10):958-976, 1988.

R 1 j SUBSTITUTE SHEET (Rule 26) "IT O Scr/ A94/00 126 RECEIVED 19 APR 1994 -24- In addition, as illustrated in the fol.owing example, the sequences of SEQ ID NOS:1-2 can be used as hybridization probes for the characterization and isolation of genomic clones encoding the porcine Gal(1,3) transferase. Mutants of the genomic nucleotide sequence, in turn, can be used in homologous recombination techniques of the types described above so that destruction of the functional gene takes place in porcine cells.

EXAMPLE 6 Characterization and Isolation of the Porcine Gene Encoding a(1,3) Galactosyl Transferase Genomic DNA prepared from pig spleen tissue was digested with EcoRl, BamHl, Pstl, HindIII, Kpnl and BstEII, electrophoresed on a 0.8% agarose gel and transferred to a nylon filter, the final wash was at 65 0

C

in 0.1x SSC, 0.1% SDS. As shown in Figure 7, the genomic Southern blot demonstrated a simple pattern suggesting that the gene exists as a single copy with a genomic size of Utilizing the cDNA insert of the pPGT-4 clone as a hybridization probe, we have cloned the porcine a(1,3) galactosyl transferase gene from a pig genomic DNA EMBL library (Clontech Laboratories, Inc., Palo Alto, CA) according to standard methods as described in Sambrook et al (suDra). This cloning has resulted in the isolation of two lambda phage clones, XPGT-gl and XPGT-g5 that RA4,

O

4/T O SUBSTITUTE SHEET (Rule 26) I I r/A 9 4 0 0 1 2 0 RECEIVED 9 APR 1994 contain different regions of the porcine transferase gene.

As discussed above, the gene for the U(1,3) galactosyl transferase can be used to effect targeted destruction o. the native gene for this enzyme using homologous recombination technology. In accordance with the conventional techniques used in this art, such gene knockout is performed using fragments obtained from genomic clones of the type provided by this example. The gene destruction can be performed in somatic or stem cells (Capecchi, 1989, supra). Because such genetically engineered cells do not produce the Gala(1,3)Gal epitope, they and their progeny are less likely to induce hyperacute rejection in humans and are thus suitable for xenotransplantaticn :n:o human patients.

EXAMPLE 7 Producti.n Anti-idiotypic Antibodies Against Hu'ran Arni-Gala(l,3)Gal Antibodies Polyclonal anti-i.iotypic antibodies against human anti-Gala(1,3)Gal antibodies are prepared following the procedures of Coligan, et al., 1992, supra; Harlow and Lane, 1988, supra; and Liddell and Cryer, 1991, supra.

Human anti-Gala(l,3)Gal antibodies are absorbed from pooled human serum onto immobilized melibiose (melibiosesepharose or melibiose-agarose) as described above in Example 3. The antibodies are eluted using standard methods, such as, high or low pH, high salt, and/or N chaotropic agents. Fab' fragments are prepared following SUBSTITUTE SHEET (Rule 26) rcr/AU 9 4/0 0 1 2 ,CIVED 1 9 APR 1994 -26dialysis into an appropriate buffer. The Fab' fragments are used to immunize rabbits, goats, or other suitable animals, along with conventional adjuvants.

The resulting polyclonal antisera are tested for their ability to change the conformation of the human antibody reactive site so as to reduce its affinity for the Galc(l,3)Gal epitope. Those sera that produce such reduced affinity constitute the desired anti-idiotypic antibodies.

Monoclonal antibodies are produced using the same Fab' fragments as antigens to immunize appropriate strains of mice. Hybridomas are prepared by fusing spleen cells from such immunized mice with murine myeloma cells. Supernatants are tested for antibodies having the ability to change the conformation of the human antibody reactive site so as to reduce its affinity for the Gal(l,3)Gal epitope. Those antibodies that produce such reduced affinity constitute the desired monoclonal antiidiotypic antibodies.

The finding that the majority of xenoreactive IgM is directed to the enzymatic product of the single transferase raises the possibility of producing transgenic pigs lacking the epitope, by targeted destruction of the a(1,3) galactosyl transferase genes using homologous recombination technology. Such genetically modified pigs could be used for transplantation. The destruction of the gene is likely ,-A72j to have no deleterious effect on the pig humans live SSUBSTITUTE SHEET (Rule 26) N,-rO PCLAU9 4/ U 1 z0 RECEIVED 19 APR 1991 -27a, normally in its absence.

This invention has been described by way of example only and is in no way limited by the specific examples herewith.

DEPOSITS

Clones pPGT-4, pPGT-2, XPGT-gl, and discussed above, have been deposited with the Australian Government Analytical Laboratories, (AGAL), 1 Suakin Street, Pymble, N.S.W. 2073, Australia, and have been assigned the designations N94/9030, N94/9029, N94/9027, and N94/9028, respectively. These deposits were made under the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure (1977). These deposits were made on March 11, 1994.

,I)

SUBSTITUTE SHEET (Rule 26) ldl I xCx1IALU 9

RECEIVED

4 1Q 12 6 1 9 APR 1994 -28-

I

TABLE 1 Rernloav n TrAns f~cr Pc COS rel 1,z Serum

NH{S

NHS abs RBC NIHS Tx 2-ME NHS abs Protein A NHS Eluted Protein A CD4 8 Target

GT+COS

GT+COS

GT

4

COS

GTVCOS

GTVCOS

GT+COS

CD4 8+COS CD4 8+COS

COS

COS

Reaction 1 4.+4+2 CD4 8

NHS

CD4 8 IB4 4

GTVCOS..

IB4 CD4 8+COS IB4 COS 1Reactivity detected by indirect immunofluorescence using FITC conjugated sheep anti-human Ig or FITC conjugated sheep anti-mouse Ig unless otherwise stated.

2 No difference in titer was observed when tested with FITC conjugated sheep anti-human IgM.

3 Reaction detected on protein A purified immunoglobulin using FITC conjugated sheep anti-human Ig or FITC conjugated sheep anti-human IgG, but not with FITC conjugated sheep anti-human 1gM.

4 Reactivity detected using FITC conjugated IB4 lectin.

SLTBSTITUT SHEET (Rule 26) Pcarl'A 94 0 0 126 RECEIVED 19 APR 1994 -29- SEOUENCE LISTING CENERAL INFORMATION: APPLICANT: The Austin Research Institute (ii) TITLE OF INVENTION: XENOTRANSPLANTATION

THERAPIES

(iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Peter A. Stearne STREET: Level 10, 10 Barrack Street CITY: Sydney STATE: New South Wales COUNTRY: Australia Postal Code 2001 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Peter A. Stearne REFERENCE/DOCKET NUMBER: 462552/pas (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 612 262 2611 TELEFAX: 612 262 1080 SUBSTITUTE SHEET (Rule 26) i rciA 9 4 9 !0 U ih RECEIVED 1 9 APR 1994 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1353 base pairs TYPE: Nucleic Acid STRANDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA to mRNA DESCRIPTION: galactosyl transferase, 3' clone (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: ORGANISM: Sus scrofa SUBSTITUTE SHEET (Rule 26) i i aiWl494/00 126 RECEIVED 19 APR 199i -31- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GTA CCG AGC TCG AAT TCC GCA AGC CAG TCA CCA CAA GCC ATG 42 Val Pro Ser Ser Asn Ser Ala Ser Gin Ser Pro Gin Ala Met 55 ACT GAC CCA TGT TCC CCC AGA CTG TCG TAC CTT AGC AAA GCC 84 Thr Asp Pro Cys Ser Pro Arg Leu Ser Tyr Leu Ser Lys Ala ATC CTG ACT CTA TGT TTT GTC ACC AGG AAA CCC CCA GAG GTC 126 Ile Leu Thr Leu Cys Phe Val Thr Arg Lys Pro Pro Glu Val 80 GTG ACC ATA ACC AGA TGG AAG GCT CCA GTG GTA TGG GAA GGC 168 Val Thr Ile Thr Arg Trp Lys Ala Pro Val Val Trp Glu Gly 95 i.00 ACT TAC AAC AGAJGCC GTC TTA GAT AAT TAT TAT GCC AAA CAG 210 Thr Tyr Asn Arg Ala Val Leu Asp Asn Tyr Tyr Ala Lys Gin 105 110 115 AAA ATT ACC GTG GGC TTG ACG GTT TTT GCT GTC GGA AGA TAC 252 Lys Ile Thr Val Gly Leu Thr Val Phe Ala Val Gly Arg Tyr 120 125 130- ATT GAG CAT TAC fTG GAG GAG TTC TTA ATA TCT GCA AAT ACA 294 Ile Glu His Tyr Leu Glu Glu Phe Leu Ile Ser Ala Asn Thr 135 140 TAC TTC ATG GTT GGC CAC AAA GTC ATC TTT TAC ATC ATG GTG 336 Tyr Phe Met Val Gly His Lys Val Ile Phe Tyr Ile Met Val 145 150 155 GAC GAT ATC TCC AGG ATG CCT TTG ATA GAG CTG GGT CCT CTG 378 Asp Asp Ile Ser Arg Met Pro Leu Ile Glu Leu Gly Pro Leu 160 165 170 CGT TCC TTT AAA GTG TTT GAG ATC AAG TCC GAG AAG AGG TGG 420 Arg Ser Phe Lys Val Phe Glu Ile Lys Ser Glu Lys Arg Trp 175 180 185 CAA GAC ATC AGC ATG ATG CGC ATG AAG ACC ATC GGG GAG CAC 462 Gin Asp Ile Ser Met Met Arg Met Lys Thr Ile Gly Glu His 190 195 200 ATC CTG GCC CAC ATC CAG CAC GAG GTG GAC TTC CTC TTC TGC 504 Ile Leu Ala His Ile Gin His Glu Val Asp Phe Leu Phe Cys 205 210 SSS SHEET (ule 26)

C)

'IT0 I I I~CI,~J4/0O0 12 6 RE jVED 1 9 APR 1994 -32- ATT GAC GTG GAT CAG GTC TTC CAA AAC AAC ITGGG GTG GAG 546 Ile Asp Val Asp Gin Vai Phe Gin Asn Asn Phe Gly Vai Giu 215 20225 ACC CTG GGC CAG TCG GTC GCT CAG CTA CAG GCC TGG TGG TAC 588 T r Leu Giy Gin Ser Vai Ala Gin Leu Gin Ala Trp, Trp Tyr 230 325 240 AAG GCA CAT CCT GAC GAG TTC ACC TAC GAG CGG CCG AAG GAG 630 Lys Ala His Pro Asp Giu Phe Thr Tyr Giu Arg Pro Lys Giu 245 250 255 TCC GCA GCC TAC ATT CCG TTT CGC CAG GGG, GAT TTT TAT TAC 672 Ser Ala Ala Tyr Ile Pro Phe Arg Gin Gly Asp Phe Tyr Tyr 260 FR 25 F: X270 CAC GCA GCC ATT TTG GGG GGA ACA CCC ACT CAG GrE' CTA AAC 714 His Ala Ala Ie Leu Giy Gly Thr Pro Thr Gin Val Leu Asn 275 280 ATC ACT CAG GAG TGC TTC AAG GGA ATC CTC CAG GAC AAG GAA 756 Ile Thr Gin Giu Cys Phe Lys Gly Ile Leu Gin Asp Lys Giu 285 290 295 AAT GAC ATA GAA GCC GAG TGG CAT GAT GAA AGC GGG CTA AAC 798 Asn Asp Ie Giu Ala Giu Trp, His Asp Giu Ser Giy Leu Asn 300 305 310 AAG TAT TTC CTT CTC AAC AAA CCC ACT AAA ATC TTA TCC CCA 840 Lys Tyr Phe Leu Leu Asn Lys Pro Thr Lys Ile Leu Ser Pro 315 320 325 GAA TAC TGC TGG GAT TAT CAT ATA GGC ATG TCT GTG GAT AT P 882 Giu Tyr Cys Trp Asp Tyr His Ile Giy Met Ser Vai Asp Ile 330 335 340 AGG ATT GTC AAG GGG GCT TGG CAG AAA AAA GAG TAT AAT TTG 924 Arg Ile Vai Lys Giy Ala Trp Gin Lys Lys Giu Tyr Asn Leu 345 350 G'N' AGA AAT AAC ATC TGACTTTAAA TTGTGCCAGC AGTI= CTGA 969 Val Arg Asn Asn Ile 355 ATTTGAAAGA GTATTACTCT GGCTACT-TCC TCAGAGAAGT AGCACTTAAT 1019 TTAACTTTT CAAAAA.;TAC TAACAAAATA CCAACACAGT AAGTACATAT 1069 TATTCTTCCT TGCAACTrTTG, AGCCTTGTCA. AATGGGAGAA TGACTCTGTA 1119 GTAATCAGAT GTAAA'FI'CCC AATGA'TI'CT TATCTGCGGA. ATTCCAGCTG 1169 AGCGCCGGTC CTACCATTAC CAGTTGGTCT GGTGTCGACG, ACTCCTGGAG 1219 jl; RAZ%"'.

0' \,-SUBSTITUTrE SKE-ET (Rule 26) m 1/0 042 6 -33- ATTCCGCCCT ATAGTGAGTC GTAI'ACAAT TCACTGGCCG TGFTT=ACAA CCTCGTGACT GGGAAAACCC TGGCCTTACC CAAC 1319 1353

RA

4

AVT

SUBSTITUTE SHEET (Rule 2u) /A 9 4/ 0 0 1 2 6 RECEIVED 1 9 APR 1994 -34- INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 1423 base pairs TYPE: Nucleic Acid STRANDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA to mRNA DESCRIPTION: galactosyl transferase, full coding sequence (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: ORGANISM: Sus scrofa SUBSTITUTE SHEET (Rule 26) p =JcV 9 4/ 0 e RECEIVED 19 APR 1994' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: COOGGGCCAT CCCCGAGCGC ACCCAGCTTC TGCCGATCAG GAOAAAATA 49 ATG AAT GTC AAA OGA AGA GTG GTT CTO TCA ATO CTG CTT GTC 91 Met Asn Val. Lys Gly Arg Val Vai Leu Ser Met Leu Leu Val TCA ACT GTA ATG GTT GTG TTT TOG GAA TAC ATC AAC AGA AAC 133 Ser Thr Val Met Val Vai Phe Tr-p GTh Tyr Ile Asn Arg Asn 20 CCA OAA GTT OGC AGC AGT GCT CAG AGG GOC TOG TOO TTT CCG 175 Pro Giu Val Gly Ser Ser Ala Gin Arg Giy Trp Trp Phe Pro 35 AGC TOG TTT AAC AAT G ACT CAC AGT TAC CAC GAA GAA GAA 217 Ser Trp Phe Asn Asn Gly Thr His Ser Tyr His Olu Oiu Glu 50 GAC OCT ATA OGC AAC OAA AAG GAA CAA AGA AAA GAA GAC AAC 259 A9p Ala Ile Oly Asn Oiu Lys Giu Gin Arg Lys Oiu Asp Asri 65 AGA OGA GAO CTT CCO CTA OTO GAC TOG TTT AAT CCT GAO AAA 301 Arg Oiy Oiu Leu Pro Leu Va'. Asp Trp Phe Asn Pro Gl.u Lys COC CCA GAO GTC OTO AC:: A ACC AGA TOG AAO OCT CCA GTO 343 Arg Pro Oiu Val Val Tnr Thr Arg Trp Lys Ala Pro Val OTA TOG OAA GOC ACT TA AGA 0CC OTC TTA OAT AAT TAT 385 Val Trp Olu Oly Thr 'Ty s'Ar9' Ala Val Leu Asp Asn Tyr 100 110 TAT 0CC AAA CAG AAA A:Z STG OGC TOG ACO OTT TTT OCT 427 Tyr Ala Lys Gin Lys 1ile Th' al Oly Leu Thr Vai Phe Ala 115 120 125 OTC GGA AGA TAC ATT GAG CAT TAC TTO GAG GAG TTC TTA ATA 469 Val Gly Arg Tyr Ile Oiu His Tyr Leu 0Th Oiu Phe Leu Ile 130 135 1.40 TCT OCA AAT ACA TAC TTC ATO OTT OGC CAC AAA OTC ATC TTT 511 Ser Ala Asn Thr Tyr Phe Met Val Oly His Lys Val Ile Phe 145 150 TAC ATC ATO OTO OAT OAT ATC TCC AGO ATO CCT TTG ATA GAO 553 Tyr Ile Met Val Asp Asp Ile Ser Arg Met Pro Leu Ile Oiu q 160 165 SUBSTITUTE SHEET (Rule 26) mg RECEIVED J9 APR ins' CTG GGT Leu Gly 170 CCT CTG CGT TCC Pro Leu Arg Ser

TTT

Phe 175 AAA GTG TTT GAG Lys Val Phe Glu ATC AAG TCC Ile Lys Ser 180 595 GAG AAG AGG Glu Lys Arg 185 ATC GGG GAG Ile Gly Glu TGG CAA GAC ATC Trp Gin Asp Ile

AGC

Ser 190 ATG ATG CGC ATG Met Met Arg Met AAG ACC Lys Thr 195

CAC

His 200 ATC CTG GCC CAC Ile Leu Ala His

ATC

Ile 205 CAG CAC GAG GTG Gin His Giu Val

GAC

Asp 210 TTC CTC TTC TGC Phe Leu Phe Cys

ATT

Ile 215 GAC GTG GAT CAG Asp Val Asp Gin

GTC

Vai 220 TTC CAA*AAC AAC Phe Gin Asn Asn 637 679 721 763 805

TTT

Phe 225 GGG GTG GAG ACC Gly Val Giu Thr

CTG

Leu 230 GGC CAG TCG GTG Gly Gin Ser Vai

GCT

Ala 235 CAG CTA CAG Gin Leu Gin ACC TAC GAG Thr Tyr Glu 250 GCC TGG Ala Trp 240 TGG TAC AAG GCA Trp Tyr Lys Ala

CAT

His 245 CCT GAC GAG TTC Pro Asp Giu Phe AGG CGG AAG Arg Arg Lys 255 GAG TCC GCA GCC Glu Ser Ala Ala

TAC

Tyr 260 ATT CCG TT GGC Ile Pro Phe Gly CAG GGG Gin Gly 265 GAT TTT TAT TAC Asp Phe Tyr Tyr 270 CAG GTT CTA AAC Gin Val Leu Asn CAC GCA GCC ATT His Aia Aia Ile

TT

Phe 275 GGG GGA ACA CCC Gly Giy Thr Pro

ACT

Thr 280

ATC

Ile 285 ACT CAG GAG TGC Thr Gin Glu Cys

TTC

Phe 290 AAG GGA ATC CTC Lys Giy Ile Leu 847 889 931 973 1015

CAG

Gin 295 GAC AAG GAA AAT Asp Lys Giu Asn

GAC

Asp 300 ATA GAA GCC GAG Ile Giu Ala Glu

TGG

Trp 305 CAT GAT GAA His Asp Glu CCC ACT AAA Pro Thr Lys 320 AGC CAT Ser His 310 CTA AAC AAG TAT Leu Asn Lys Tyr

TTC

Phe 315 CTT CTC AAC AAA Leu Leu Asn Lys ATC TTA TCC Ile Leu Ser 325 TCT GTG GAT Ser Vai Asp CCA GAA TAC TGC Pro Glu Tyr Cys

TGG

Trp 330 GAT TAT CAT ATA Asp Tyr His Ile GGC ATG Gly Met 335 1057 1099 ATT Ile 340 AGO ATT GTC AAG Arg lie Val Lys

ATA

Ile 345 OCT TGG CAG AAA AAA Ala Trp Gin Lys Lys 350 TGACTTTAAA GAG TAT AAT IrC Giu Tyr Asn 'eu

GTT

Val 355 AGA AAT AAC ATC Arg Asn Asn Ile 1136 SUBSTITUTE SHEET (Rule 26) c-- ?c/Au 9 4 0OO 12 6 RECEIVED 19 A PR 19 9 -37- TFTGTGCCAGC AG'm-rCTGA ATPTTGAAAGA GTATTACTCT GGCTAC VFCC TCAGAGAAGT AGCACTTAAT TAACTTTT AAAAAAATAC TAACAAAATA CCAACACAGT AAGTACATAT TATTCTTCCT TGCAACTPTG AGCC'FFGTCA AATGGGAGAA TGACTCTGTA GTAATCAGAT GTAAATTCCC AATGA'TTCT TATCTGCGGA ATTCCAGCTG AGCGCCGGTC GCTACCATTA CCAGTTGGTC TGGTGTCGAC GACTCCTGGA GCCCGTCAGT ATCGGCG 1186 1236 1286 1336 1386 1423 1-. SuBSTITUT SIBET (Rule 26) 00 126 61rE E V\I E APR 199 -38- INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH 30 bases TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYDE: Other Nucleic Acid DESCRIPTION: PCR primer aGT-1 /t SUBSTITUTE SHEET (Rule 26) I i i i I RECEVED9 APR 1084s (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GAATTCAAGC TTATGATCAC TATGCTTCAA SUBSTITUTE SHEET (Rule 26) OaEAU i 4 0 26 RECEIED 9 APR 199 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 29 bases TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: Other Nucleic Acid DESCRIPTION: PCR primer cGT-2 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes SUBSTITUTE SHEET (Rule 26) I locr/Au 94 /O0O 12 6

RKECV

1 9 APR 1994 .41- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GAATTCCTGC AGTCAGACAT TATTCTAAC SUBSTITUTE, SHEET (Rule

Claims (9)

1. An isolated nucleic acid molecule comprising: the nucleic acid sequence of SEQ ID NO:1; or an antisense sequence complementary to or both and

2. An isolated nucleic acid molecule comprising: the nucleic acid sequence of SEQ ID NO:2; or an antisense sequence compl uentary to or both and

3. A method for blocking human anti-Gala(l,3)Gal antibodies comprising changing the conformation of the antibody reactive site so as to reduce the affinity of the antibody for the Gala(1,3)Gal epitope.

4. The method of claim 3 wherein the conformation of the antibody reactive site is changed through the use of an anti-idiotypic antibody. at C a a a a bea bG

5. Clone pPGT-4 having deposit N94/9030.

6. Clone pPGT-2 having deposit N94/9029.

7. Clone XPGT-gl having deposit N94/9027.

8. Clone XPGT-g5 having deposit N94/9028. designation designation designation designation number AGAL number AGAL number AGAL number AGAL N i ,c\

9. A porcine cell comprising an inactivated porcine a(1,3) galactosyl transferase gene, said inactivated porcine a(1,3) galactosyl transferase gene comprising a wild type porcine a(1,3) galactosyl transferase sequence disrupted P:\WPDOCS\NEH\SPEC\6279294.SPE:9/2/98 I -r I I 43 by a cloned mutant porcine a(1,3) galactosyl transferase sequence, wherein the cloned mutant porcine a(1,3) galactosyl transferase sequence comprises a mutation of SEQ ID NO:1, wherein the mutation is selected from the group consisting of a deletion, an insertion, a substitution, and an addition such that the cloned mutant porcine a(1,3) galactosyl transferase sequence does not encode a functional galactosyl transferase so that immune reaction of the cell with human antibodies reactive with Gala(1,3)Gal epitopes is avoided. A porcine cell comprising an inactivated porcine a(1,3) galactosyl transferase gene, said inactivated porcine u(1,3) galactosyl transferase gene comprising a wild type 15 porcine a(1,3) galactosyl transferase sequence disrupted S* by a cloned mutant porcine a(1,3) galactosyl transferase I' sequence, wherein the cloned mutant porcine u(1,3) galactosyl transferase sequence comprises a mutation SEQ NO:2, wherein the mutation is selected from the group consisting of a deletion, an insertion, a substitution, and an addition such that the cloned mutant porcine u(1,3) galactosyl transferase sequence does not encode a g functional galactosyl transferase so that immune reaction of the cell with human antibodies reactive with 25 Gala(1,3)Gal epitopes is avoided. *oo Dated this 9th day of February 1998. AUSTIN RESEARCH INSTITUTE By their Patent Attorneys DAVIES COLLISON CAVE P" Jj :\WPDOCS\NEH\SPEC\6279294.SPE:9n/S8 (7 L~gJ T9~1C llt I I REC1EIVED 19 A PR 1994 A13STP-ACT OF THE DISCLOSURE DNA sequtences encoding a porcine Galu(1,3) galactosyl transferase and clones containing such sequiences are provided. The porcine Galu(1,3) galactosyl transferase produces the Gala(1,3)Gal epitope on the surfaces of porcine cells. This epitope is tecognized by humnan anti-Galu(1,3)Gal antibodies which are responsible for hyperacute rejection of xenotransplanted pig cells, tissues and organs. Methods of reducing such hyperacute rejection are also provided. SUBSTITUT SHBET (Rule 26)