WO1993014769A1 - Method for making universal donor cells - Google Patents

Abstract

Transplantation antigen-depleted cells and methods for making such cells from a target cell are provided. The transplantation antigen-depleted cell is made by exposing a target cell to an oligonucleotide capable of binding to a transplantation antigen nucleotide sequence. The oligonucleotide is capable of binding to the nucleotide sequence according to Watson-Crick or triplex binding rules. Preferred transplantation antigens are the MHC class I or II antigens. Oligonucleotides useful in practicing this method are also provided.

Description

METHOD FOR MAKING UNIVERSAL DONOR CELLS

Technical Field

This invention is related to therapeutics, transplantation and immunology. More specifically, it relates to a method for making cells that are more easily transplanted into a recipient host using oligonucleotides that interact with genes and gene products relating to transplantation antigens expressed on the cell surface of transplanted cells.

Background Art Anti-gene code molecules are short RNA or DNA transcripts that are "antisense" (i.e., complementary to a DNA or RNA strand in a Watson-Crick pairing manner) to a portion of the normal RNA and are not translated. Regulation of expression of genes by anti-gene code RNA, one of the natural modes of gene regulation, was first recognized in prokaryotes. Green, P.M. et al., Ann. Rev. Bioche (1986) 5_5:569. Natural anti-gene codes and artificial anti-gene codes have been used in prokaryotes to downregulate prokaryotic proteins. Simmons, R.w. et al., Cell (1983) 3.4:683; Mizuno, T. et al. , Proc. Natl. Acad. Sci. (1984) 8J.:1966; Okamoto, K. et al., Proc. Natl. Acad. Sci. (1986) 3:5000; Pestka, S. et al., Proc. Natl. Acad. Sci. (1984) 8JL:7525; Coleman, J. et al. , Cell (1984) 37:429; Farnham, P^'.-J. et al. , Proc. Nat1. Acad. Sci. (1985) 82.:3978; Kindy, M.S. et al. , Mol. Cell. Biol. (1987) 1:2857. Artificial anti-gene codes have also been synthesized and used to regulate eukaryotic gene expression. Microinjection or transfection of thymidine kinase (TK) anti-gene codes has been shown to inhibit expression of the TK protein. Izant, J.G. et al., Cell (1984) 3_6:1007; Kim, S.K. et al., Cell (1985) 42:129. Additionally, short anti-gene codes to the 5 ^r untranslated region of the thymidine kinase gene successfully downregulates protein expression. Izant, J.G. et al. , Science (1985) 229:345. Other examples of the regulation of εukaryotic gene expression by anti-gene codes are the pp66 c-src gene (by transfected full length anti-gene codes) , and the c-fos gene (by an anti-gene code spanning the 5' untranslated region of the first exon) . Amini, S. et al., Mol. Cell. Biol. (1986) j5:2305; Holt, J.T. et al., Proc. Natl. Acad. Sci. (1986) 83.:4794. These anti-gene codes have been introduced under constitutive or heterologous inducible promoters.

Synthetic oligomers have also been used to downregulate the expression of c-myc in promyelocytic leukemia cells, and T-lymphocytes. Wickstrom, E.L. et al., Proc. Natl. Acad. Sci. (1988) 8_5:1028; Heikkila, R. et al.. Nature (1987) 328:445. C-myc anti-gene code oligonucleotides have been shown to inhibit proliferation in normal hematopoietic cells. Gewirtz, A.M. et al. , Science (1988) 242:1303. An anti-gene code to a CD8 fragment downregulated the expression of CD8 molecules on the surface of human cytotoxic T-cells. Hambor, J.E. et al., Proc. Natl. Acad. Sci. (1988) 8_5:4010. Lotteau et al. , J. Exp. Med. (1989) 169:351 used episomal vectors to introduce DR A coding sequences in B-lymphoblastoid cell lines and downregulated the expression of DR A-DQ B mixed isotype heterodimers, but did not observe any changes in the levels of isotype matched DR A-DQ B heterodimers. Anti-gene code oligonucleotides may act to prevent transcription by inhibiting DNA or RNA poly erase, by binding to mRNA and preventing ribosomal translation, by decreasing the stability of mRNA through enhancement of mRNA degradation by RNase H, or by preventing or inhibiting the processing to mature mRNA. Maher, L.J. et al., Science (1989) 4_5:725; Moser, H.E. et al.. Science (1987) 238:645; Melton, D.A. et al. , Proc. Natl. Acad. Sci. (1985) 82.:144; Gewirtz, A.M. et al.. Science (1989) 245:180: Walder, R.Y. et al., Proc. Natl. Acad. Sci. (1988) 85:5011. Absolute homology between the target and the antisense sequences is preferred but not required for the inhibition. Holt, J.T. , supra.

Anti-gene code oligonucleotides may also form a triplex DNA structure with the intact duplex gene. Moffat, A.S., Science (1991) 252:1374-1375. This technique of making anti-gene code oligonucleotides involves the formation of a triplex structure according to certain binding rules. When this triplex structure is formed in the promoter region of a gene, it has been shown to disrupt transcription of that gene. Orson, F.M. et al., Nuc. Acids Res. (1991) 19:3435-3441.

One set of genes potentially subject to regulation by anti-gene codes is the Human Leukocyte Antigen (HLA) complex, located on the short arm of chromosome 6. The HLA antigens are divided into two classes depending on their structure. The genetic loci denoted HLA-A -B, and -C code for the HLA Class I antigens, and HLA-DP, -DQ and -DR code for the HLA Class

II antigens. HLA Class II molecules are composed of two non- covalently linked glycoproteins, the α chain and the highly polymorphic β chain. Each chain contains one extracellular domain, a transmembrane segment and a cytoplasmic tail. The structure of the and β chains and their genes have been elucidated. All known Class II genes are similar in structure and encoded by exons 1 - 4, with exon 5 coding for an untranslated region. The DP, DQ and DR loci all consist of multiple genes. A total of twelve class II genes have been identified. In some haplotypes, some class II genes do not code for a functional peptide and are classified as pseudogenes. Regulation of HLA class II antigen expression by binding anti-gene oligonucleotides to the structural region of the gene has not been reported in the literature.

Regulation of HLA class II antigen expression occurs in part through a series of promoter regions such as the J, W, X (including X- and X₂) , and Y boxes, and the gamma interferon response element. The X (including Xj and X₂) and Y boxes are known to be required in the transcriptional regulation of all class II promoters. Ono, S.J. et al., Proc. Natl. Acad. Sci. CUSA) (1991) 88: 4304-4308.

HLA antigens are implicated in the survival of cell grafts or transplants in host organisms. Although there is acceptable graft survival in the first year for nearly all types of transplants, by five and ten years after transplantation only 40-50% of all grafts are still functioning. This low rate is due to the relentless attack of the immune system on the graft. In addition, death rates of 1-5% are recorded even at the best transplant centers. Drugs are commonly used to control immune responses and prevent graft rejection, and death is often an indirect result of this drug administration. The drugs used to control immune responses usually cause a non-specific depression of the immune system. A patient with a depressed immune system is far more susceptible to develop life-threatening infections and a variety of neoplasia. The low rate of long term success, and serious risks of infection and cancer are the two main challenges now facing the entire field of tissue and organ transplantation.

It has been suggested that graft rejection can be prevented or reduced by reducing the levels of exposed HLA antigens on the surface of transplant cells. Faustman, D. et al.. Science (1991) 252:1700-1702. observed that xenograft survival was increased by masking HLA class I surface antigens with F(ab^,)₂ antibody fragments to HLA class I or tissue specific epitopes.

This invention contemplates the development of a "universal donor cell" reduced in one or more HLA antigens. The absence of certain HLA antigens on the surface of donor cells, tissues or organs comprising these cells will cause them not to be recognized as foreign and not to elicit a rejection response. By the selective introduction of anti-gene codes into a cell it is possible to block the expression of targeted HLA genes, thereby rendering a graft "invisible" to the immune system. Thus, the problem of rejection is eliminated without nonspecific suppression of the immune system, and the immune system remains active to defend against infection and neoplasia.

Summary of the Invention

A superior method of providing transplantation antigen-depleted/reduced cells has now been found. In accord with this invention, oligonucleotides that reduce the antigenicity of cells are designed to be able to bind in some fashion to a nucleotide sequence relating to a transplantation antigen, and prevent the expression of that antigen. Cells treated with these oligonucleotides will express significantly less of the targeted antigen, and when transplanted will be more easily tolerated by the recipient host. Although these oligonucleotides are designed to be capable of binding to a transplantation antigen nucleotide sequence, it is contemplated that their ultimate mode of action may be different.

The present invention gives physicians an improved source of transplantable cells. These transplantation antigen-depleted cells give rise to improved graft survival rates in the recipient or require lower levels of immunosuppressant drug administration in the recipient. These cells may also be useful in treating patients with autoimmune diseases.

In one aspect, this invention provides a method for making a transplantation antigen-depleted cell from a target cell comprising obtaining the target cell, and then exposing the target cell to an oligonucleotide capable of binding to a transplantation antigen nucleotide sequence, wherein the oligonucleotide is presented or produced locally in an amount sufficient to make the target cell a transplantation antigen-depleted cell. The oligonucleotide is capable of binding to the nucleotide sequence according to Watson-Crick or triplex binding rules (which includes Hoogsteen-like bonds) . In preferred embodiments the transplantation antigen is an MHC class I or II antigen.

In another aspect of this invention, a transplantation antigen-depleted cell is provided, prepared by obtaining a target cell, and exposing the target cell to an oligonucleotide capable of binding to a transplantation antigen nucleotide sequence, wherein the oligonucleotide is presented or produced locally in an amount sufficient to make the target cell a transplantation antigen-depleted cell.

In yet another aspect of this invention, an oligonucleotide capable of binding to a double-stranded transplantation antigen nucleotide sequence is provided. In still another aspect of this invention, a universal donor organ is provided, prepared by obtaining a target organ from an individual, and exposing the target organ to an oligonucleotide capable of binding to a transplantation antigen nucleotide sequence, wherein the oligonucleotide is presented or produced locally in an amount sufficient to make the target organ a universal donor organ.

In a further aspect of this invention, a method of treating an individual with an autoimmune disease characterized by dysfunctional expression of a transplantation antigen is provided, comprising administering to that individual an oligonucleotide capable of binding to a portion of the transplantation antigen nucleotide sequence, in an amount sufficient to inhibit expression of the transplantation antigen.

Brief Description of the Drawings

Figure 1 shows the DNA sequence for the X and X₂ boxes of the DR A promoter, and the structure and binding pattern of the triplex-forming oligonucleotides T, and T₂.

Figure 2 shows the fluorescence profile of HeLa cells incubated with gamma interferon and various amounts of T₂ and then labelled with anti-DR monoclonal antibody.

Figure 3 shows the fluorescence profile of HeLa cells incubated with gamma interferon and various amounts of ^~~ ₂ ^and then labelled with anti-DP monoclonal antibody.

Figure 4 is a Northern blot analysis using an anti-sense RNA probe that specifically binds to sense DR A mRNA. Cells were blotted at 3 and 7 days with the indicated treatments (CO. indicates control oligonucleotide) .

Figure 5 contains the nucleotide sequences for the X and X₂ promoter regions for various transplantation antigens.

Figure 6(a) shows the fluorescence profile of gamma interferon induced Colo 38 cells incubated with control antibody (mouse IgG₂) and anti-DR monoclonal antibody.

Figure 6(b) shows the fluorescence profile of gamma interferon induced Colo 38 cells treated with (a) nothing, (b) 50 μM oligo A, or (c) 100 μM oligo A, and followed by incubation with anti-DR monoclonal antibody. Figure 6(c) shows the fluorescence profile of gamma interferon induced Colo 38 cells treated with (a) nothing, or (b) 50 μM control oligo Al and followed by incubation with anti-DR monoclonal antibody.

Figure 7 shows the fluorescence profile from flow cytometry of HeLa cells incubated with gamma interferon and various amounts of TSl and then labeled with anti-DR monoclonal antibody fluorescein.

Figure 8 shows the Dose Response Percent suppression of cell surface DR antigen as a function of concentration of TSl as it affects HeLa cells.

Figure 9 shows the duration of TSl effect on HeLa cells.

Figure 10a shows the effect of TSl on constitutive DR Colo cells. Figure 10b is the accumulative integration along the fluorescence axis of the data shown in Figure lOa.

Figure 11 is a bar graph that shows the effect of anti-sense oligonucleotides ANTI-B, AB, ACAT, ATCT and T₂ on the induction of MHC Class I antigen expression by IFN- . Figure 12 is a graph showing the effect of T₂ and of A₃ on the IFN-7 mediated enhancement of tryptophan degradation.

Figure 13 is a graph showing the effect of oligonucleotides T₂ and A₃ on kynurenine production.

Figure 14 is a bar graph showing the effect of T₂ on HLA Class I induction by IFN-α, IFN-/3, and IFN-7.

Figure 15 is a bar graph showing the effect of T₂ on IFN-7 induced MHC-II in WEHI-3 cells. Figures 16A and 16B are graphs showing the effect of T2 on IFN-7 and TNF-α induced ICAM-1 cell surface expression, respectively.

Figure 17 is a graph showing the effect of T₂ on antigen-induced proliferation of human monocytes. Figure 18 is a graph showing the effect of T₂ on T cell activation using an IL-2 production assay.

Detailed Description of the Invention

The practice of the present invention encom- passes conventional techniques of chemistry, molecular biology, biochemistry, protein chemistry, and recombinant

DNA technology, which are within the skill of the art.

Such techniques are explained fully in the literature.

See, e.g.. Oligonucleotide Synthesis (M.J. Gait ed. 1984) ; Nucleic Acid Hybridization (B.D. Hames & S.J.

Higgins, eds., 1984); Sambrook, Fritsch & Maniatis,

Molecular Cloning: A Laboratory Manual. Second Edition

(1989); PCR Technology (H.A. Erlich ed., Stockton Press);

R.K. Scope, Protein Purification Principles and Practice (Springer-Verlag) ; and the series Methods in Enzvmology

(S. Colowick and N. Kaplan eds., Academic Press, Inc.).

All patents, patent applications and publications mentioned herein, whether supra or infra, are hereby incorporated by reference in their entirety. Definitions:

As used herein, the term "transplantation antigen" is used to refer to antigenic molecules that are expressed on the cell surface of transplanted cells, either at the time of transplantation, or at some point following transplantation. Generally these antigenic molecules are proteins and glycoproteins. The primary transplantation antigens are products of the major histocompatibility complex (MHC) , located on chromosome 6 in humans. The human MHC complex is also called the human leukocyte antigen (HLA) complex. MHC antigens are divided into MHC class I antigens (in humans, this class includes HLA-A, -B, and -C antigens) and MHC class II antigens (in humans, this class includes HLA-DP, -DQ, and -DR antigens) . Transplantation antigens also include cell surface molecules other than MHC class I and II antigens. These antigens include the following: (1) the ABO antigens involved in blood cell recognition; (2) cell adhesion molecules such as ICAM, which is involved in leukocyte cell-cell recognition; and (3) β₂- microglobulin, a polypeptide associated with the 44 kd heavy chain polypeptide that comprises the HLA-I antigens but is not encoded by the MHC complex.

As used herein, the term "transplantation antigen nucleotide sequence" refers to nucleotide sequences associated with genes encoding transplantation antigens. Nucleotide sequences associated with genes include the region of the gene encoding the structural product, including intron and exon regions, and regions upstream of the structural gene associated with transcription, transcription initiation, translation initiation, operator and promoter regions, ribosome binding regions, as well as regions downstream of the . . . . . structural gene, including termination sites. Nucleotide sequences associated with genes also include sequences found on any form of messenger RNA (mRNA) derived from the gene, including the pre-mRNA, spliced mRNA, and polyadenylated mRNA.

As used herein, the term "transplantation antigen-depleted cell" refers to cells that are in some way depleted in the expression of at least one transplantation antigen. This depletion may be manifested by a reduced amount of antigen present on the cell surface at all times. Preferably, at least 90% of the antigen is eliminated at the cell surface. Most preferably, this depletion results in essentially total absence of the antigen at the cell surface.

Certain transplantation antigens are not always constitutively expressed on the cell surface. These antigens have their expression increased at some point shortly after transplant. In these cases, the depletion is manifested by a reduced amount of antigen or complete lack of antigen at the cell surface at the post- transplant point of normal increased expression. A transplantation antigen-depleted cell will have at least one of two properties: (1) the cell will survive in the transplant recipient for time periods significantly longer than normal cells; or (2) the cell will survive in the transplant recipient for time periods commensurate to normal or untreated cells, but will require lower doses of immunosuppressive agents to the transplant recipient.

As used herein, "oligomers" or "oligo¬ nucleotides" include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. "Nucleic acids", as used herein, refers to RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form.

As used herein, the term "binding" refers to an interaction or complexation between an oligonucleotide and a target transplantation antigen nucleotide sequence, mediated through hydrogen bonding or other molecular forces. As used herein, the term "binding" more specifically refers to two types of internucleotide binding mediated through base-base hydrogen bonding. The first type of binding is "Watson-Crick-type" binding interactions in which adenine-thymine (or adenine-uracil) and guanine-cytosine base-pairs are formed through hydrogen bonding between the bases. An example of this type of binding is the binding traditionally associated with the DNA double helix.

The second type of binding is "triplex binding" which follows a set of still-developing binding rules. In general, triplex binding refers to any type of base- base hydrogen bonding of a third oligonucleotide strand with a duplex DNA (or DNA-RNA hybrid) that is already paired in a Watson-Crick manner. Triplex binding is more fully described in PCT Application No. WO 90/15884 (published 27 December 1990) . In one set of triplex binding rules, the third strand is designed to match each A or T in one of the duplex strands with T, and each C or G with C, and the third strand runs antiparallel to the matched strand. In another set of triplex rules, the third strand is designed to match each A or T in one of the duplex strands with T, and each C or G with G, also running antiparallel to the matched strand. Other types of triplex binding rules are described in PCT Application No. WO 90/15884. One or more types of triplex binding may occur for a given oligonucleotide. As used herein, Hoogsteen-like bonds refers to hydrogen bonding between bases.

The Oligonucleotide:

According to this invention, oligonucleotides are synthesized that are capable of binding to a transplantation antigen nucleotide sequence. The binding may occur between the oligonucleotide and a single- stranded sequence through Watson-Crick-type binding, or between the oligonucleotide and a duplex sequence through triplex binding. In either case, the binding capability results in a transplantation antigen-depleted cell which has reduced expression of at least one transplantation antigen at some point after transplant.

Because it is contemplated that there may be cross reactivity and homology between structural and control regions of various transplantation antigens, it is also contemplated that the transplantation antigens that are ultimately depleted in the treated cell may be different than the antigen whose nucleotide sequence was originally targeted.

One specific target sequence is the well- characterized DR A promoter region. The DR A promoter region contains a number of subregions known to be specific binding sites for DNA binding proteins, called the J, W, X (including X, and X₂) , and Y boxes, and the gamma interferon response element. Particularly significant are the X and X₂ boxes, as described herein. Other specific target sequences are within the structure gene. in general, a minimum of approximately 5 nucleotides, preferably at least 10 nucleotides, are necessary to effect the necessary binding to a specific target sequence within the intron region of the structural gene. By targeting the structural gene region, only two target DNA sequences per cell are required to be bound by this oligonucleotide. Furthermore, short strands of oligonucleotides

(approximately 26 nucleotides or less) are readily taken up by cells. The only apparent limitations on the required binding length of the target/oligonucleotide complexes of the invention concern making the oligonucleotide of sufficient binding length to be capable of binding to the target transplantation antigen sequence, and not to bind to other undesirable non-target sequences and disrupt other cellular mechanisms.

Oligonucleotides of sequences shorter than 15 nucleotides may be feasible if the appropriate interaction can be obtained.

As further explained below, the oligonucleotides need to contain the sequence-conferring specificity, but may be extended with flanking regions and otherwise derivatized or modified. The oligonucleotide may be prepared by any known method, including synthetic, recombinant, and purification methods, and may be used alone or in combination with other oligonucleotides specific for the same or different target transplantation antigens.

The oligonucleotide may also contain "interior flanking sequences", which are sequences within a binding sequence that are not capable of binding to the target through Watson-Crick or triplex binding rules. Thus, the oligonucleotide may comprise two or more binding regions separated by nonbinding interior flanking sequences. It is also contemplated that the binding sequences may contain one or more mismatches that do not conform to the binding rules. These substitutions are contemplated as part of the invention as long as the oligonucleotide retains its binding capability as described herein.

The oligonucleotide may also be amplified by PCR. The PCR method is well known in the art and described in, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202 and Saiki, R.K. , et al., Science (1988) 239:487-491. and European patent applications 86302298.4, 86302299.2 and 87300203.4, as well as Methods in

Enzvmology (1987) 155:335-350. The amplified DNA may then be recovered as DNA or RNA, in the original single- stranded or duplex form, using conventional techniques. The oligonucleotides of the invention usually comprise the naturally-occurring bases, sugars and phosphodiester linkages. However, any of the hydroxyl groups ordinarily present in the sugars may be replaced by phosphonate groups, phosphate groups, protected by a standard protecting group, or activated to prepare ad¬ ditional linkages to additional nucleotides, or may be conjugated to solid supports. The 5' and 3" terminal OH groups are conventionally free but may be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to embodiments wherein phosphate is replaced by P(0)S ("thioate"), P(S)S ("dithioate") , P(0)NR₂ ("amidate") , P(0)R, P(0)OR', CO or CH₂ ("formacetal") , wherein each R or R' is independently H or substituted or unsubstituted alkyl (1-20C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all linkages in an oligomer need to be identical.

Also included within this invention are synthetic procedures in which the resultant oligonucleotides incorporate analogous forms of purines and pyrimidines. "Analogous" forms of purines and pyrimidines are those generally known in the art, many of which are used as chemotherapeutic agents. An exemplary but not exhaustive list includes aziridinylcytosine,

4-acetylcytosine, 5-fluorouracil, 5-bromouracil,

5-carboxymethylaminomethyl-2-thιouracιl, 5-carboxymethyl- aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, l-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-meth ladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyl- uracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5-methoxyuracil, 2-methyl- thio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid, 5-pentynyluracil and 2, 6-diaminopurine. The use of uracil as a substitute base for thymine in deoxyribonucleic acid (hereinafter referred to as "dϋ") is considered to be an "analogous" form of pyrimidine in this invention. The oligonucleotides may contain analogous forms of ribose or deoxyribose sugars that are generally known in the art. An exemplary, but not exhaustive list includes 2 ' substituted sugars such as 2'-O-methyl-, 2'- O-allyl, 2'-fluoro- or 2 -azido-ribose, carbocyclic sugar analogs-, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.

Although the conventional sugars and bases will be used in applying the method of the invention, substi¬ tution of analogous forms of sugars, purines and pyrimidines can be advantageous in designing the final product, as can alternative backbone structures like a polyamide backbone. Oligonucleotides containing the designed binding sequences discerned through the method of the invention can also be derivatized in various ways.

- Primarily, the oligonucleotides will be derivatized by . . . . attaching a nuclear localization signal to it to improve targeted delivery to the nucleus. One well-characterized nuclear localization signal is the heptapeptide PKKKRKV (pro-lys-lys-lys-arg-lys-val) . Also, if the oligonucleotide is to be used for separation of the target substance, conventionally the oligonucleotide will be derivatized to a solid support to permit chromato- graphic separation. If the oligonucleotide is to be used to label the target or otherwise attach a detectable moiety to target, the oligonucleotide will be derivatized to include a radionuclide, a fluorescent molecule, a chromophore or the like. If the oligonucleotide is to be used in specific binding assays, coupling to solid support or detectable label is also desirable. If it is to be used therapeutically, the oligonucleotide may be derivatized to include ligands which provide targeting to specific cellular sites or permit easier transit of cellular barriers, toxic moieties which aid in the therapeutic effect, or enzymatic activities which perform desired functions at the targeted site.

One desired function that may be performed by the oligonucleotide at the targeted site is alteration of the targeted DNA. The oligonucleotide may be derivatized to attach to the targeted sequence, to crosslink the targeted sequence (e.g., through psoralen crosslinks), or to alter, modify or delete all or part of the targeted sequence. In this manner, the oligonucleotide may cause a permanent depletion of a transplantation antigen on a cell and its daughter cells.

The oligonucleotide sequence may also be included in a suitable expression system that would provide in situ generation of the desired oligonucleotide.

The Methods:

According to this invention, the oligonucleotides described above are used in a method of treatment to make a transplantation antigen-depleted cell from a normal target cell. The cells are created by incubation of the cell with one or more of the above- described oligonucleotides under standard conditions for uptake of nucleic acids, including electroporation or lipofection.

Alternatively, the oligonucleotides can be modified or co-administered for targeted delivery to the nucleus. The cell nucleus is the likely preferred site for action of the triplex-forming oligonucleotides of this invention, due to the location therein of the cellular transcription and replication machinery. Also, improved oligonucleotide stability is expected in the nucleus due to: (1) lower levels of DNases and RNases; (2) higher oligonucleotide concentrations due to lower total volume; (3) higher concentrations of key enzymes such as RNase H implicated in the mechanism of action of these oligonucleotides. The cytoplasm, however, is the likely preferred site for action of the traditional antisense oligonucleotides of this invention.

A primary path for nuclear transport is the nuclear pore. Targeted delivery can thus be accomplished by derivatizing the oligonucleotides by attaching a nuclear localization signal. One well-characterized nuclear localization signal is the heptapeptide PKKKRKV (pro-lys-lys-lys-arg-lys-val) .

Any transplantable cell type is a potential target cell for this invention. Preferably, the target cell is selected from corneal endothelial cells, thyroid cells, parathyroid cells, brain cells, adrenal gland cells, bone marrow cells, pancreatic islet cells, hepatic cells, lymphoid cells, fibroblasts, epithelial cells, chondrocytes, endocrine cells, renal cells, cardiac muscle cells, and hair follicle cells. Most preferably, the target cell is selected from corneal endothelial cells, thyroid cells, parathyroid cells, brain cells, adrenal gland cells, bone marrow cells, pancreatic islet cells and hepatic cells.

In another aspect of this invention, the above- described oligonucleotides may be incorporated into an expression vector through methods well known in the art, and then inserted into the target cell via standard techniques such as electroporation, lipofection, or calcium phosphate or calcium salt mediation. In this fashion, the desired oligonucleotides are produced in situ by the expression vector, and the target cell will continue to express the oligonucleotides for at least a period of time following transplant.

Furthermore, this invention is applicable to the field of solid organ transplants. Organs are normally perfused ex vivo prior to transplantation. By adding an amount of the above-described oligonucleotides to the perfusion medium, transplantation antigen-depleted cells can be created from perfusion-accessible cells in the organ to create a transplantation antigen-depleted organ useful in solid organ transplants.

Finally, local administration of the anti-gene oligonucleotides directly into the transplanted organ during the first day after the transplant is within the scope of this invention. Also, sustained releases of the of these drugs are also contemplated.

Methods of Treatment and Administration

The oligonucleotides of this invention are useful in creating the transplantation antigen-depleted cells of this invention. These cells are then directly transplanted to an individual. This technique can be used for any individual with an immune system, including humans. The oligonucleotides of this invention are also useful in treating autoimmune diseases characterized by dysfunctional or aberrant expression of a transplantation antigen. In such a case, the oligonucleotides described herein may be administered in an amount sufficient to inhibit expression of the transplantation antigen.

It may be commented that the mechanism by which the oligonucleotides of the invention interfere with or inhibit the production of one or more transplantation antigens is not always established, and is not a part of the invention. The oligonucleotides of the invention are characterized by their capability to bind to a specific target nucleotide sequence regardless of the mechanisms of binding or the mechanism of the effect thereof. Described below are examples of the present invention which are provided for illustrative purposes, and not to limit the scope of the present invention. In light of the disclosure, numerous embodiments within the scope of the claims will be apparent to those of ordinary skill in the art.

Examples

Materials and Methods Cell Strains and Culture Media

HeLa S3 cells (human cervical carcinoma cell line ATCC CCL 2.2), K562 cell lines (UCSF Cell Culture Facility) , and BJAB cells (human lymphoblastoid cell line, UCSF Cell Culture Facility) and Colo 38 (human cervical carcinoma cell line) are grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum heat inactivated at 65°C for 30 minutes. Fibroblast 143B cells (human osteosarcoma cell line ATCC/crl 8303) is grown in MEM Eagle's BSS medium (UCSF Cell Culture Facility) supplemented with 10% fetal calf serum heat inactivated at 65°C for 30 minutes.

Oligonucleotide design Phosphodiester oligonucleotide A consisting of

19 nucleotides was designed to base-pair in Watson-Crick fashion to the mRNA base sequence of the DR A structural gene beginning 13 nucleotides upstream (5') of the translational initiation codon (AUG) , A: 5' GCC ATT TTC TTC TTG GGC G 3' and ordered from the UCSF Biomolecular Resource Facility. Two control phosphodiester oligonucleotides (Al and A2) , consisting of random sequences of the same base composition as above but that would not bind to DR A mRNA were also ordered from the Resource Facility: Al: 5' TTG CCA GAC TAT TGT CCC A 3' A2: 5' TAT CGG CTT TGT TGC CCG T 3'

Triplex-forming oligonucleotides (TFOs) were designed to match the HLA DR A X and X₂ box promoter. T,, τ₂, T₂C and T₇ were ordered from American Synthesis Inc.. T,, T₂, T₂C were designed according to the formula shown in Figure l. T, was designed to have a C to match each GC base pair in the duplex and a T to match each AT pair. T₂ was designed to have a G to match each GC base pair in the duplex and a T to match each AT pair. T, and T₂ were modified with a 3' amino group to increase stability. T₂C is the same as T₂ except T₂C is unmodified. T₇ was designed as a control oligonucleotide with the same overall nucleotide composition as T₂ and T₂C but with its sequence altered to have less triplex-type pairing with the X and X, boxes:

T₇: 5' TGT TGG TGT GGG TTG TGG TTG GTT GC 3' Al and A2 are unmodified oligonucleotide sequences that would not form triplex structures with the promoters. TS1 is a 26 nucleotide oligonucleotide consisting of a phosphodiester backbone and a ine modified 3' terminus. TSl was designed to be anti-parallel to the coding strand with the maximum number of Hoogstein bonds that can form between TSl and the targeted sequence.

Under physiologic conditions the formation of GGC and TAT bare triplets are favored, giving rise to a triplex helix with one DNA strand of the DR A gene at residue positions 5'-85l to 3'-876 as numbered in Schamboeck, A., et al., Nuc. Acids Res. (1983) 11:8663-8675. TSl is unique in that it will bind either parallel or antiparallel because of the palindrome character of duplex DNA. The DR A gene was selected because it is monomorphic between individuals, thereby minimizing the variability of gene sequence which normally occurs in polymorphic genes. The method to construct the TSl sequence was to select a C for every C or G in the DNA target sequence and a T for every A or T. T_con and GT_con are control oligonucleotides that are also 26 nucleotides in length and amine modified at the 3' terminus. The sequences are compared to the segment of the DR A intron of the structural gene below: DR A: 5'-GGG GGT GGG GGT GGG GGT GGG GGA GG-3' TSl: 3'-GGG GGT GGG GGT GGG GGT GGG GGT GG-5' T_con: 3'-TTT GTG TTT TGT TTT TTT GTT TTT TT-5' GT_coα: 3'-GGT GTG TGT GTG TGT GTG TGT GTG TG-5' The oligonucleotides were ordered from Keystone Laboratories.

Oligonucleotide A₃ is a control oligonucleotide that showed relatively lower ability to inhibit the IFN-7 enhanced MHC-I expression in HeLa S3 cells. The X at the end of the oligonucleotide represents the 3'-amino linker modification discussed for T₂ oligonucleotide. The sequence of A₃ is the following.

A,: 5' TTG CCA GAC TAT TGT CCC A X 3' Other oligonucleotides used are the following. Oligonucleotide CL, is designed to be antisense to the ATG site in HLA-A2 mRNA. Oligonucleotide ANTI-B is designed to be identical to one strand in the KBF binding site in the enhancer A region of the MHC-I HLA-A2 promoter. Oligonucleotide AB is directed toward the Enhancer B in the 5'-region of MHC-I A2 gene. ACAT is an 18 mer directed towards the CAAT box of the MHC-I A2 gene. ATCT is directed towards the MHC-I A2 equivalent of the TATA box. These oligonucleotides and the A₃ oligonucleotide were purchased from Keystone Laboratories, Menlo Park, CA. The sequences of these nucleotides are the following.

CL,: 5' AGG GTT CGG GGC GCC ATG ACG GC X 3' ANTI-B: 5' CCC AGC CTT GGG GAT TCC CCA ACT CC X 3' AB: 5' CCG ACA CCC AAT GGG AGT X 3' ACAT: 5' CGA CAC TGA TTG GCT TCT 3' ATCT: 5' TGC GTG CGG ACT TTA GAA X 3'

Antibodies

Mouse Anti-human HLA-A,B,C, anti-β₂ microglobulin and control IgG₂b antibodies were purchased as fluorescein isothiocyanate conjugates from Olympus, Lake Success, N.Y.. Mouse anti ICAM-1 and IgG, antibodies were purchased from AMAC, Westbrook ME as fluorescein isothiocyanate conjugates.

Oligonucleotide Uptake and Gamma Interferon Addition

Oligonucleotides were added to the cell medium as described by Orson et al., Nuc. Acids Res. (1991)

11:3435-3441. Cells were initially concentrated to 2-6 x

10⁶ cells/ml and incubated with various oligonucleotides at various concentrations (5μM, lOμM, 20μM) with gentle shaking every 30 minutes for 2 hours. Cells were then diluted to 0.2 x 10⁶/ml in order to optimize cell growth. At Days 1 and/or 2 cells were again concentrated to 2-6 x 10⁶ cells/ml, and incubated with the oligonucleotide and diluted as above. Where appropriate, gamma interferon (IFN7, Collaborative Research, Inc.) at 200 units/ml was added at Days 0, 2, 4 and 6 to induce HLA-DR expression.

Cell surface HLA detection.

At days 3 and 7, cells were isolated and stained with fluorescein isothiocyanate conjugated (FITC) anti-HLA-DR monoclonal antibodies. HLA-DP was detected by indirect staining with mouse anti-DP monoclonal antibody followed by FITC goat anti-mouse (Becton- Dickinson) . About 0.1 x 10° cells were used per assay. 10 μl of mouse IgG₂-FITC served as background control and 10 μl of monoclonal anti-DR-FITC IgG₂ was added to detect cell surface HLA DR expression. Monoclonal antibody was incubated with cells on ice for 30 min. The mixture was then washed with phosphate buffered saline (PBS) with 0.1% sodium azide, the supernatant was removed after centrifugation, and the pellet was resuspended in 150 ml of PBS and 50 υl of 0.05% propidium iodide. Flow cyto etry analysis (FACScan - Becton-Dickinson) was used to detect cell surface antigen expression. A similar procedure using anti-HLA-DP specific monoclonal antibody was used to measure surface HLA-DP expression.

Northern Hybridization and RNA Isolation

Approximately 2 x 10⁶ cells treated with oligonucleotide were isolated at Day 3 and Day 7. The cells were washed with cold PBS and resuspended in 200 ml cold lysis buffer (0.5% Nonidet P-40, 150 mM NaCl, 10 mM

Tris pH 8.0, 2 mM MgCl) . Cell nuclei were removed by spinning at 15,000 RPM for 5 min. An equal volume of protein denaturing buffer (10 mM EDTA, 450 mM NaCl, 7 M urea, 10 mM Tris pH 7.4, 1% SDS) was added to the supernatant. The resulting solution was extracted with equal volumes of phenol/chloroform and the aqueous phase transferred to 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2 volumes of 100% ethanol. The solution was kept at -20°C overnight. Supernatant was removed after centrifugation and the RNA pellet was washed with 1 ml 70% ethanol. RNA was then dissolved in 50μl of TE buffer (10 mM Tris pH 7.4, 1 mM EDTA) . RNA concentration was ascertained by optical density at 260 nm (OD₂₆₀) .

Plasmid containing the DR A gene (DR A PBS M13) was obtained from Lars Karlsson at the Scripps Research Institute, and linearized by incubation with EcoRI. T3 RNA polymerase was added along with ATP, CTP, GTP, and digoxigen-coupled UTP to synthesize DR A RNA probes. The resulting antisense DR A RNA probe was used to detect sense DR A mRNA. A similar method was used to prepare the control antisense β actin RNA probe.

7 to 10 μg of the prepared RNA extract were separated by 1.2% agarose formaldehyde gel along with RNA size markers (Pharmacia or Gibco BRL) at 125 volts for 5- 6 hours. Separated RNA in the gel was blotted onto nylon paper overnight, and baked at 80°C for 2 hrs. The paper was then put into a prehybridizing solution (Genius protocol) for 6-8 hours and hybridized with antisense DR A RNA probe (lμg/ml) and antisense 0-actin probe (0.5 μg/ml) for about 48 hours. Anti-digoxigen alkaline phosphatase-conjugated monoclonal antibody was added to the blotted paper, followed by Lumi-Phos 530 (Boehringer Mannheim, Indianapolis, IN) . Lumi-Phos light emission was detected by autoradiography.

RNase Protection Assay

³²P-Riboprobes were generated using a MaxiScript in vitro transcription kit (Ambion, Austin TX) and alpha ³²P-UTP (NEN Dupont, Boston MA) . Analyses of RNA were performed using an RNAse protection assay kit (Ambion, Austin, TX) . A 0.16-1.77 kb RNA ladder (Gibco, Grand Island, NY) was dephosphorylated using calf intestinal alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN) , ³²P-labeled with gamma ³²P-ATP (NEN Dupont, Boston, MA) and used as molecular weight markers in RNAse protection assays. Isolated RNA from HeLa S3 was hybridized to a T3 polymerase generated ³²P-RNA probe antisense to a 1.2 kb fragment of HLA DR A gene (same fragment as used in Northern analysis) probe protected from RNAse digestion after hybridization to HLA-DR A mRNA was recovered and electrophoresed on a denaturing 7% polyacrylamide gel. Each RNA sample from HeLa S3 was simultaneously hybridized to a T3 polymerase generated ³²P-riboprobe antisense to a glyceraldehyde-3-phosphate dehydrogenase (GPD) transcript (0.14kb). Hybridization with the GPD probe was used to compare amounts of initial total RNA in the samples analyzed by RNAse protection.

Detection of ICAM-1

The presence of ICAM-1 sites on cells was determined as follows. Samples containing 0.1ml of cells and reactants were drawn from each tube at various times and stained for flow cyto etry using anti-ICAM-1 antibody. The cell suspension was washed once with 0.75 ml phosphate buffered saline (PBS) containing 2% fetal calf serum and 0.1% sodium azide. The antibody conjugate (lOμg) was added and the mixture was agitated. The cells were incubated for 30 minutes on ice in the dark, washed twice with 0.75 ml of PBS containing 0.1% sodium azide to remove unbound antibody, and resuspended in 0.1ml PBS containing sodium azide (0.1%). Propidium iodide was added to exclude dead cells from the analysis. The mean number of ICAM-1 sites was estimated by first determining the fluorescence to antibody (F/P) ratio for the ICAM-1 antibody and its cognate IgG_! on Simply Cellular beads

(Flow Cytometry Standards Corporation, Research Triangle, N.C.) containing a fixed number of goat anti-mouse sites. The flow cytometer was calibrated with QuickCal beads

(Flow Cytometry Standards Corporation, Research Triangle, NC) . The calibration curve was used in conjunction with the F/P ratio to estimate the mean number of ICAM-1 sites from the mean channel fluorescence according to the manufacturer's directions.

Indolea ine 2.3 dioxygenase assay

Indoleamine 2,3 dioxygenase (IDO) of cells was estimated using a spectrophotometric assay. IDO converts tryptophan to kineurenine. Tryptophan was measured at its absorption maximum of 280 nm, a wave length at which kynurenine does not absorb significantly. Kynurenine was measured at 360 nm, a wave length at which tryptophan does not absorb significantly.

Example 1 Two 26 base pair (bp) triplex-forming oligo¬ nucleotides (TFOs) were designed. Oligonucleotide T, was designed by using a T to match each A-T bp and a C for each G-C bp. Oligonucleotide T₂ was designed by using a T to match each A-T bp and a G for each G-C bp. Each oligonucleotide was modified with a 3' amino group to prolong its half-life. In each experiment HeLa cells or fibroblast 143B cells were pre-incubated with T,, T₂, or τ₇ for 2 hours before exposure to 200 units of recombinant gamma interferon, which was added on day 0 and day 2 of culture. Tj and T₂ were also added on day 1 and day 2. Induction of DR expression was then measured . . on day 3 by flow cytometry using anti-DR monoclonal antibody which binds to those HLA class II antigens on the cell surface. Although cells cultured with the control TFO T₇ showed normal induction of HLA-DR and DP antigens, T* inhibited expression of DR and DP by 50%, while T₂ showed about 100% inhibition of both DR and DP at 20 μM concentration (Figs. 2-3, T₂ data only). At lower doses of oligonucleotide (5 and 10 μM) , both T, and T₂ showed inhibitory effects in a dose responsive fashion.

The mechanism of this inhibitory effect was investigated further. Transcription of the DR A gene was measured by Northern blot analysis (Figure 4) using an anti-sense RNA probe that specifically binds to sense mRNA. At a concentration of 20 μM, T₂ completely suppressed DR A mRNA expression measured at day 3. This suppression was reversible with the continued addition of gamma interferon to the culture, as shown at day 7.

While T] and T₂ were able to block gamma interferon-induced HLA DR and DP expression, they had no effect on constitutive DR expression. This was proven by treating BJAB (B lymphoblastoid) cells and Colo 38

(malignant melanoma) cells, both of which express DR constitutively, with 5-20 μM of T_t and/or T₂ for up to 7 continuous days. No decrease in the constitutive expression of cell surface DR was observed. The effects of T₂ appear to be somewhat specific for promoter sequences that share homology with HLA-DR A (Figure 5) . ICAM-1, an adhesion molecule which is constitutively expressed on HeLa cells, can be increased by gamma interferon treatment. T₂ blocks the augmentation of ICAM expression but leaves the constitutive expression intact and has no effect on either constitutive or inducible expression of HLA Class I genes. Example 2 The above protocols were followed to test the antisense nucleotide A and controls Al and A2 with the following differences. Colo 38 cells were subcultured at 0.1 to 0.3 x 10⁶ cells/ml and incubated with various oligonucleotides at concentrations of 1 - 100 μM (added twice a day, approximately 9 a.m. and 5 p.m.), presuming complete depletion prior to addition. Gamma interferon (Collaborative Research, Inc.) was added at 200 units/ml on days 0 and 2 to induce DR A expression and the cells were harvested for flow cytometry analysis on day 3.

Results are shown in Figure 6. Figure 6(a) shows that gamma interferon induced cells are specifically bound by DR specific antibody. Figure 6(b) indicates that increased levels of added oligo A reduces the amount of specifically bound antibody, signifying decreased expression of DR A antigen. Figure 6(c) shows that the addition of control oligos Al or A2 does not reduce DR A antigen expression.

Example 3 The above protocols used for oligonucleotides T, and T₂ were followed to test the antisense oligonucleotide TSl and controls T_coo and GT_COO, except that the following differences were used. The oligonucleotides were added to the media for 3 days in the case of HeLa cells, and for 5 days in the case of other types of cells. Gamma interferon was added to the HeLa and keratinocytes cells on days 0, 2, 4, and 6. No gamma interferon was added to the Colo cells. TSl was added daily to all the cells at 20μM except for the dose response experiment; in the dose response experiment, the

TSl concentration varied from 0.1 to 40 μM.

The TSl dose responsiveness of HeLa cells as indicated by the binding of fluorescent anti-DR A monoclonal antibody is shown in Figure 7. Figure 8 compares the dose response of HeLa cells to TSl with that to the control oligonucleotide GT_C00. As indicated in both Figure 7 and Figure 8, TSl at levels of 5 μM and 10 μM give greater than fifty and ninety percent inhibition, respectively, of the expression of the DR A antigen.

The duration of TSl inhibition of DR A expression in HeLa cells is shown in Figure 9. The HeLa cells were treated with 200 units/ml gamma interferon on days 0, 2, 4, and 6. 20 μM TSl or GT_coα was added on days

0, 1, and 2. On days 3 and 7, the cells were treated with anti-DR monoclonal antibody-fluorescein and analyzed by flow cytometry. Figure 9 shows that TSl completely suppresses the surface expression of DR after 3 days and 7 days. The effect of TSl remains at least 4 days after it is removed from the media. Unlike T₂ where the cells begin to resynthesize DR after removal of the oligonucleotide, TSl has a longer lasting effect. The effect of TSl on the constitutive expression of DR expression on Colo 38 cells was also examined. The results on the binding of anti-DR monoclonal antibody-fluorescein to TSl treated and Tcon treated cells is shown in Figure 10. Figure 10 depicts the flow cytometry data when cells are treated with Tcon (Peak B) and TSl (Peak A) . Peak B has been mathematically reduced so that its peak height coincides with the right peak height of curve A. Peak C is the mathematical result of subtracting reduced Peak B from Peak A. Figure 10b (Percent vs. Fluorescence) is the accumulative integration along the fluorescence axis of Peak C. This figure indicates that TSl decreased the expression of cell surface DR antigen in part of the cells, as represented by the shaded area. This shaded area represents approximately 20% of the cells, based upon the integration curves in the lower figure. TSl has a partial effect in constitutively expressed DR antigen, but the treatments have not been optimized yet.

To further elucidate the mechanism of action of TSl, DR A RNA levels were determined in untreated, TSl treated, and Tcon treated Colo cells. RNA was extracted and incubated with a 32P-DR A probe. The probe was prepared from a plasmid containing the DR A gene (DR A PBS M13) obtained from Lars Karlsson at the Scripps Research Institute, and linearized by incubation with EcoRI. The RNase Protection Assay was previously described on page 23.

Table l

The G3PDH (glycerol aldehyde-3-phosphate dehydrogenase) probe was used as a control to determine the levels of RNA loaded onto each gel lane. When the RNA is loaded unequally into each lane, the labelled bands for each probe can be excised and counted. Normalizing to the G3PDH radioactivity allows rough comparison of the DR A RNA. Table 1 shows that when DR A results are normalized to G3PDH, 20 μM TSl decreased the DR A RNA level by approximately 50% when compared to untreated or Tcon treated cells. The cross reactivity of TSl to other gamma interferon genes (IFN-7) was determined in HeLa cells.

Table 2

Table 2 shows the cross reactivity of TSl to other gamma interferon genes. TSl completely suppresses gamma interferon induced DR and DP expression. TSl suppresses gamma interferon ICAM suppression to levels of constitutive expression. It is unknown whether TSl reduces constitutive as well as gamma interferon induced ICAM antigen. TSl has very little effect on HLA Class I expression, both constitutive and gamma interferon induced.

The effect of TSl on keratinocytes, a type of "normal" primary cell, not an immortal cell line, was also evaluated.

Table 3 TSl EFFECT ON KERATINOCYTES Mean Channel Shift (Experimental - Control) Untreated 6 IFN-7 31

20μM TSl 0

20μM T_coo 11

Table 3 shows that TSl completely suppresses the gamma interferon inducible DR antigen expression on keratinocytes. T_con also shows some suppression.

In summary, these results show that, in contrast to the earlier studies with oligonucleotide T₂, treatment with TSl inhibits the transcription and expression of constitutively synthesized HLA DR A antigens. In addition, TSl effects appear to have longer duration than those of T₂.

Example 4 The above protocols were followed to test the effect of anti-sense nucleotides on the induction of MHC Class I antigens by IFN-7. K562 cells were treated with 25 μM of the following oligonucleotides for 2 hours prior to the addition of 500 U/ml of IFN-7: ANTI-B, AB, ACAT, ATCT and T₂. Fresh oligonucleotides were added at 24,

48, 72, and 92 hours after the addition of IFN-7, and the cells were analyzed by antibody staining and flow cytometry at 100 hours for the presence of MHC-I antigens. The results presented in Figure 11 show that the anti-sense nucleotides tested are all capable of preventing the upregulation of MHC Class I antigens by IFN-7.

Example 5 Antiproliferative and antitumor effects of IFN-7 are thought to be mediated primarily by induction of an enzyme in the tryptophan catabolism pathway, indoleamine 2,3 dioxygenase (IDO). (Taylor and Feng, FASEB J. , 5_:2516 (1991)). IFN-7 and IDO induction have been observed in tumor allografts undergoing rejection suggesting that activation of the catabolism pathway may be one of the factors involved in graft rejection. (Takikawa et al, J. Immunol. 145:1246 (1990)). The protocols described above were used to examine the effect of oligonucleotide T₂ on the induction of IDO by IFN-7. At 0, 24, and 48 hours HeLa S3 cells in RPMI 1640 were incubated for 2 hours at 37°C with 25 μM of either oligonucleotide T₂ or A₃ (control) and then stimulated with 500 U/ml of IFN-7. Two aliquots, each containing 5 x 10⁴ cells were removed; one aliquot was transferred to Hank's balanced salt solution (HBSS) and the other to HBSS containing 50 μM L-tryptophan (Sigma, St. Louis, MO) . A_2g0 and A₃₆₀ measurements were taken at intervals on supernatants from both samples.

Figure 12 is a graph showing the effect of T₂ and of A₃ on the IFN-7 mediated enhancement of tryptophan degradation. As seen from the results, T₂ but not A₃ inhibited the increase in the rate of tryptophan degradation induced by IFN-7.

IDO converts tryptophan to kynurenine. The results in Figure 13 confirm that the decrease in tryptophan is accompanied by a corresponding increase in material that absorbs at 360 nm, the wave length at which kynurenine absorbs. Figure 13 is a graph showing the effect of oligonucleotides T₂ and A₃ on kynurenine production. As seen from the graph, T₂ but not A₃ inhibited the increase in the rate of kynurenine production induced by IFN-7. Example 6 The above-described protocols were used to examine the effect of T₂ on HLA Class I induction by IFN- α, IFN-/3, and IFN-7. The cells were incubated with the interferons indicated in Figure 14, and with 25 μM T₂ or without oligonucleotide (Control) . The amount of MHC Class I antigens was determined by cell sorting after staining with two antibodies, one directed to the heavy chain and the other directed to /3₂-microglobulin. As seen from the results, shown in the bar graph in Figure

14, T2 inhibits the induction by IFN-7. However, it does not inhibit induction by IFN-α or IFN-jS. In Figure 14 MCS is the mean channel shift and the change from cells not treated with interferon is plotted on the y-axis.

Example 7 The following illustrates that T2 prevents the induction of mouse MHC class II by gamma interferon.

It has been shown that the promoter region of the mouse MHC displays a large degree of homology with its human counterpart. In order to investigate the effect of T2* on the expression of mouse MHC class II molecule, we utilized the myelomonocytic cell line, WEHI- 3. This cell line derived from BALB/c mice (H-2^d) expresses MHC class II molecules (A^d,E^d) at very low levels (4%) . However, both Ad and Ed molecules can be induced on these cells following 72 hours treatment with gamma interferon. Consequently, gamma Interferon- mediated MHC class II expression on WEHI-3 restores the capacity of these cells to stimulate class Il-restricted

T cell proliferation in an antigen specific manner.

WEHI-3 were preincubated with the oligonucleotide T2

(final concentration 25, 50, 100 mM) for 2 hours, then cultured in the presence of gamma interferon (100 U/ml) for 48 hours. Cell surface expression of MHC class II Ad and Ed was then measured by cytofluorometry analysis (FACS) using direct staining with FITC-labelled anti-MHC class II monoclonal antibodies (I-Ed) . Figure 15 is a bar graph showing the effect of T₂ on IFN-7 induced MHC- II in WEHI-3 cells. The results are indicated as mean channel shift corresponding to: number of channel for anti-I-Ed mAB - number of channels for a control IgG2 mAb.. As seen from the results in Figure 15, T2, completely abolished the induction by gamma interferon of urine MHC class II on WEHI-3 cell lines (95% reduction) .

Example 8 The oligonucleotide (T2) designed to form a triplex helix with the promoter region of the human major histocompatibility complex (HLA) locus has been shown to prevent the induction by gamma Interferon of HLA class II (DR) cell surface molecules on different cells (Hela, fibroblasts, keratinocytes) (See above) . The effect of T2 on the surface expression of another gamma interferon- inducible immune receptor was examined.

Hela cells were preincubated for 2 hours with the oligonucleotide T2 (20uM) or with a control oligonucleotide (Al) or with medium alone. Then, the cells were cultured for different periods of time in the presence of gamma interferon (50 U/ml) (Figure 16A) or

Tumor Necrosis factor (TNF) alpha (150 U/ml) (Figure 16B) . Following this step, the cells were stained with a FITC- labelled anti-ICAM-1 and analyzed by cytofluorometry (FACS) . The results are indicated as mean channel shift corresponding to: number of channel for anti-I-Ed AB - number of channels for control IgG2 mAb.

As seen from the results in Figure 16A, there was a complete suppression of the gamma Interferon- induced expression of the adhesion molecule ICAM-1.

However, as seen from Figure 16B, T2 had no effect on and TNF-a mediated ICAM-1 cell surface expression on these human cells. In other studies, a lack of effect was seen both with IL-1 and IL-4 mediated ICAM-1 cell surface expression. Therefore, we conclude that the oligonucleotide T2 is specific for gamma Interferon- mediated functions.

Example 9 This example illustrates that T₂ inhibits T cell proliferation and IL-2 production by preventing antigen presentation by accessory cells.

a) T2 blocks anti-CD3 mediated human T cell proliferation by preventing the expression of Fc receptors on monocytes.

Whether other gamma interferon-induced immune receptors would be suppressed by T2 and whether this would impact human T cell responses was examined. More specifically, 1) the influence of T2 on gamma interferon- induced expression of Fc receptors on human monocytes, and 2) the capacity of T2-treated monocytes to fulfill their accessory functions such as supporting anti-CD3- mediated human T cell proliferation were examined.

It is well established that human T cells can be stimulated to proliferate when incubated with monoclonal antibodies (mAb) directed to the CD3 complex (0KT3) . To mediate this effect, anti-CD3 mAb need first to bind through their Fc portion to Fc receptors (FcR) on monocytes in order to stimulate IL-1 secretion by these cells and to aggregate TCR/CD3 complexes on T lymphocytes. Both signals (IL-l and CD3/TCR aggregation) are necessary to trigger T cell proliferation and IL-2 production. Gamma interferon regulates the expression of FcR on human monocytes. Gamma interferon-mediated induction of FcR on monocytes restores IgGl anti-CD3 (Leu-4, UCHT-1)-mediated T cell proliferation in non- responder individuals as well as enhances IgG2 anti-CD3 (0KT3)-mediated T cell mitogenesis (G. Benichou et al. , Eur. J. Immunol 1987, 17:1175-1181).

In the study, the results of which are shown in Figure 17, human monocytes were purified by adherence from human peripheral blood mononuclear cells (PBMC) . They were preincubated in the presence of the oligonucleotide T2 at different final concentrations ranging from 5 uM to 50 uM, or in the absence of oligonucleotide (dashed line) . Then, the cells were washed and treated for 48 hours with different concentrations of gamma interferon. Following this step, the mononuclear cells were cocultured with syngeneic peripheral T lymphocytes in the presence of anti-CD3 monoclonal antibodies (0KT3) in 96-well culture dishes for 4 days. Anti.gen-i.nduced proli.ferati.on was assessed by the incorporation of 1 Ci [³H]-thymidine during the last 18 hours of culture. Results are expressed as counts per minute (cpm) obtained with cells stimulated in vitro with anti-CD3 mAb.

The results of the study indicate that: 1) T2 completely abolished (100% inhibition) the induction by gamma interferon of FcR at the surface of monocytes; and 2) the lack of FcR rendered human monocytes incapable of supporting 0KT3-mediated in vitro T cell proliferation of human T lymphocytes (Figure 17) .

•b) T2 inhibits antigen-mediated IL-2 release by mouse T cell hvbridomas. The effect of inhibition of gamma interferon- induced MHC class II on mouse cells on antigen presentation for T cell activation was examined. T lymphocytes recognize the antigen in the form of peptide presented in association with self-MHC molecules at the surface of antigen presenting cells (APC) . Following immunization, CD4+ T helper cells initiate the immune response by interacting through their antigen receptor (TCR) with the bimolecular complex formed by the MHC class II and the peptide antigen. Antigen recognition by T cells triggers their proliferation and the secretion interleukin-2. Here we tested whether the oligonucleotide T2 would influence antigen presentation by a cloned APC, WEHI3. WEHI3 is a myelomonocytic cell line whose level of MHC expression was very low (4%) but could be increased up to 90% following exposure to gamma interferon. We have shown that the induction of MHC class II (A^d,E^d) on WEHI3 by gamma interferon can be blocked by preincubating the murine cell line with T2 (20 mM final concentration) . In order to measure the effect on T activation we used a T cell hybridoma, 1E1 specific for the lambda repressor peptide 12-24 presented in association with the murine MHC class II molecule, E. Following preincubation with gamma interferon, WEHI3 displays high levels of surface Ed molecule and presents efficiently the peptide to the T cell hybridoma, 1E1. The oligonucleotide T2 suppressed antigen presenting functions of WEHI-3 in that it prevented the in vitro interleukin 2 (IL-2) production of the CD4+, class Il- restricted T cell hybridoma (1E1) to its specific antigen, the lambda repressor peptide 12-24 (Figure 18) .

In the study for Figure 18, 1 x 10⁵ 1E1 T hybridoma cells specific for the lambda repressor peptide, 12-24, in association with I-Ed were used. They were cocultured for 24 h with the A20 (A^d,E^d) B cell lymphoma (10⁵ cells) as APC control, or with WEHI-3 myelomonocytic cell line treated with gamma interferon (100 u/ml) in the presence of the oligonucleotide T2 or with medium alone. Then the relevant peptide was added to the cell culture at different concentrations. The 96- well microplates were then centrifuged, and the culture supernatants (100 ml) were aspirated and transferred to a new microtiter tray which was frozen and thawed before assay for IL-2 production. IL-2 was assayed by [³H]- thymidine incorporation of the IL-2-dependent cell line, HT2. Briefly, 0.04 ml of culture supernatants were further incubated with 10⁴ HT-2 for 24 h in a total volume of 0.2 ml HL-1 medium. Incorporation of 1 mCi [³H]-thymidine was assayed during the last 4 h of culture.

The results from the studies indicate that the oligonucleotide T2 can block in both human and murine systems, the induction of different cell surface receptors by gamma interferon, but not other lymphokines. This results in the abolition of the capacity of a murine cell line to present the antigen and to stimulate in vitro antigen specific T cell proliferation.

Claims

ClaimsWe claim:

1. A method for making a transplantation antigen-depleted cell from a target cell, the method comprising:

(a) obtaining the target cell; and

(b) exposing the target cell to an oligo¬ nucleotide capable of binding to a transplantation antigen nucleotide sequence, said oligonucleotide being present in an amount sufficient to make the target cell a transplantation antigen-depleted cell.

2. The method of claim 1 wherein the oligonucleotide is capable of binding to the transplantation antigen nucleotide sequence through Watson-Crick binding or through triplex binding.

3. The method of claim 2 wherein the transplantation antigen nucleotide sequence is a ICAM gene sequence or an MHC class I gene sequence or an MHC class II gene sequence.

4. The method of claim 3 wherein the MHC class II gene sequence is located at the DR A, DP, or DQ locus.

5. The method of claim 4 wherein the MHC class II gene sequence is located at the DR A locus.

6. The method of claim 5 wherein the oligonucleotide comprises the sequence

5' GCC ATT TTC TTC TTG GGC G 3' or fragments thereof which retain binding capability.

7. The method of claim 2 wherein the transplantation antigen nucleotide sequence is a promoter sequence or a structural gene sequence.

8. The method of claim 7 wherein the transplantation antigen nucleotide sequence is a DR A structural gene sequence or a DR A promoter sequence.

9. The method of claim 8 wherein the DR A promoter sequence comprises the X-box or X₂-box.

10. The method of claim 9 or claim 1 wherein the oligonucleotide comprises the sequence

5' GGGGTTGGTTGTGTTGGGTGTTGTGT 3' or fragments thereof which retain binding capability.

11. The method of claim 7 wherein the oligonucleotide comprises the sequence

5' GGTGGGGGTGGGGGTGGGGGTGGGGG 3' or fragments thereof which retain binding capability.

12. The method of claim 1 wherein the target cell is selected from the group consisting of corneal endothelial cells, thyroid cells, parathyroid cells, brain cells, adrenal gland cells, bone marrow cells, pancreatic islet cells and hepatic cells.

13. The method of claim 1 wherein the transplantation antigen nucleotide sequence is an ICAM gene sequence.

14. A transplantation antigen-depleted cell prepared by a method comprising:

(a) obtaining a target cell; and (b) exposing the target cell to an oligonucleotide capable of binding to a transplantation antigen nucleotide sequence, said oligonucleotide being present in an amount sufficient to make the target cell a transplantation antigen-depleted cell.

15. The transplantation antigen-depleted cell of claim 14 wherein the oligonucleotide is capable of binding to the transplantation antigen nucleotide sequence through Watson-Crick binding or through triplex binding.

16. The transplantation antigen-depleted cell of claim 15 wherein the transplantation antigen nucleotide sequence is a ICAM gene sequence or a MHC class I gene sequence or a MHC class II gene sequence.

17. The transplantation antigen-depleted cell of claim 16 wherein the MHC class II gene sequence is located at the DR A, DQ or DP locus.

18. The transplantation antigen-depleted cell of claim 17 wherein the MHC class II gene sequence is located at the DR A locus.

19. The transplantation antigen-depleted cell of claim 15 wherein the transplantation antigen nucleotide sequence is a promoter sequence or a structural gene sequence.

20. The transplantation antigen-depleted cell of claim 19 wherein the structural gene sequence is a DR A structural gene sequence or the promoter sequence is a DR A promoter sequence.

21. The transplantation antigen-depleted cell of claim 20 wherein the DR A promoter sequence comprises the X-box or X₂-box.

22. The transplantation antigen-depleted cell of claim 21 or of claim 14 wherein the oligonucleotide comprises the sequence

5' GGGGTTGGTTGTGTTGGGTGTTGTGT 3' or the sequence

5' GGTGGGGGTGGGGGTGGGGGTGGGGG 3' or fragments thereof which retain binding capability.

23. The transplantation antigen-depleted cell of claim 14 wherein the target cell is selected from the group consisting of corneal endothelial cells, thyroid cells, parathyroid cells, brain cells, adrenal gland cells, bone marrow cells, pancreatic islet cells and hepatic cells.

24. The transplantation antigen-depleted cell of claim 14 wherein the transplantation antigen nucleotide sequence is a ICAM gene sequence.

25. An oligonucleotide capable of binding to a transplantation antigen nucleotide sequence through Watson-Crick binding or through triplex binding.

26. The oligonucleotide of claim 25 wherein the transplantation antigen nucleotide sequence is a MHC class II gene sequence.

27. The oligonucleotide of claim 26 wherein the MHC class II gene sequence is located at the DR A, DP or DQ locus.

28. The oligonucleotide of claim 27 wherein the MHC class II gene sequence is a DR A structural gene sequence or a DR A promoter sequence.

29. The oligonucleotide of claim 28 wherein the DR A promoter sequence comprises the X-box or X₂-box.

30. The oligonucleotide of claim 29 or of claim 28 wherein the oligonucleotide comprises the sequence 5' GGGGTTGGTTGTGTTGGGTGTTGTGT 3' or the sequence

5' GGTGGGGGTGGGGGTGGGGGTGGGGG 3' or fragments thereof which retain binding capability.

31. An oligonucleotide for use in the preparation of a composition for treating target cells to make them transplantation antigen-depleted, wherein the oligonucleotide is capable of binding to a portion of the transplantation antigen nucleotide sequence.

32. A universal donor organ prepared by the method comprising:

(a) obtaining a target organ from an individual; and

(b) exposing the target organ to an oligonucleotide capable of binding to a transplantation antigen nucleotide sequence, said oligonucleotide being preset in an amount sufficient to make the target organ a universal donor organ.

33. A method of treating an individual with an autoimmune disease characterized by dysfunctional expression of a transplantation antigen, the method comprising administering to that individual an oligonucleotide capable of binding to a portion of the transplantation antigen nucleotide sequence, in an amount sufficient to inhibit expression of the transplantation antigen.