WO1999058157A1 - Method for inhibiting antibody-mediated rejection of xenogeneic tissues - Google Patents

Abstract

A method for inhibiting the rejection of tissue transplanted from one species to another, comprising modifying, eliminating or blocking the expression of a specific gene or fragment thereof, which when expressed encodes antibody or fragment thereof associated with transplanted tissue rejection.

Description

METHOD FOR INHIBITING ANTIBODY-MEDIATED REJECTION OF XENOGENEIC TISSUES

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to the transplantation of tissues from a first species to a second species for the purpose of preventing or curing a specific disease in the tissue recipient.

Description of the Background The use in humans of tissues for transplantation from one individual to another is severely limited by a lack of sufficient numbers of human donors. The use of organs and/or specialized tissue from non-human species (xenotransplantation) avoids the limitation on the availability of the tissues. Genetic and physiological characteristics point to the swine as the most appropriate species to act as tissue donor for xenotransplantation in humans. Specifically-defined pig donors may be produced in sufficient numbers to satisfy the need for clinical transplantation. In addition, the use of these animal donors permits their genetic manipulation to avoid host rejection of the donor tissues and/or provide for specific gene replacement therapy. The use of pigs as xenograft donors, however, is limited by the aggressive nature of the immune response of humans to tissues from the donor animals.

Clinical and experimental information on the nature and the severity of the human response to pig tissues suggests it to be an aggressive immunological reaction that causes a rapid loss of the _ transplanted tissue. The rapid loss of the transplanted tissue may be due to the presence of preformed antibodies to pig tissues present in the serum of normal humans. This immediate reaction is followed by the appearance of newly induced humoral and cellular immune responses that may cause acute failure of the graft within several days to a few weeks, independent of the graft damage due to pre-formed antibody. Traditional immunosuppressive treatment, e.g. cyclosporin or tacrolimus, fail to prevent either of these early host responses, primarily due to the failure of the drugs to prevent the production of human antibodies to the transplanted tissues. A more aggressive immunosuppressive regimen, particularly those that include drugs that inhibit antibody production by B cells permit a relatively long-term survival of xenografts in experimental animals. This improved survival usually comes with a heavy price, in the form of significant graft recipient.

The initial rejection of pig tissues by humans is a rapid (hyperacute) rejection of vascularized grafts due to the presence of pre-formed antibodies to donor tissues present in the sera of normal individuals. Normal individuals have variable levels of antibodies that bind to antigens expressed by the vascular endothelial cells in xenogeneic organs. Binding of these antibodies to the xenograft precipitates a direct injury to the endothelial cells, and a secondary widespread injury to the graft along with intravascular thrombosis. Pre-formed antibodies appear soon after birth, presumably due to exposure to bacterial and other microbial agents normally present in the environment. The exposure to common gut microbes stimulates the appearance of antibodies that react with xenogeneic tissues, presumably due to similarities in the structure of complex carbohydrate antigens and the protective structures of bacteria and viruses. Although a large number of potential protein/carbohydrate antigens may exist for any donor/recipient pair of species, the available information suggests that the primary targets for pre-formed xenoantibodies are cell surface- associated carbohydrate complexes, similar to the blood group antigens. The ability of pre-formed antibody to cause hyperacute rejection of organ xenografts may be effectively prevented by eliminating the antibody prior to transplantation and, in fact, when newborn animals exhibit low levels of anti-donor antibodies, they also display a delayed graft rejection (Minanov et al., 1997. Transplantation 63:182, 1997). Absorption of the antibody, either by perfusion of recipient blood through vascularized organs from the donor species or solid matrix columns containing the target antigens, removes the pre-formed antibody and, when combined with treatment strategies aimed at disrupting the activation of complement/inflammatory cascades, effectively prevents graft rejection for several days to a few weeks. Prevention of hyperacute rejection is a therapeutic approach that is limited by a rapid reappearance of new anti-donor antibody and/or alterations in the sensitivity of the graft endothelium to immune damage following xenotransplantation. Once a xenograft has been placed in the recipient, new anti-donor antibody is a rapid response of the host to the foreign graft. In experimental animals, the production of new xenoantibodies is rapid and graft rejection is closely associated with an increase in antibody levels. An effective suppression of antibody production, using either treatment or combinations of multiple immunosuppressive drugs, is critical to the development of techniques that allow for extended graft survival.

In general, two distinct pathways of antibody production are utilized to generate antibody responses. The most common is the traditional response of B cells to nominal antigen. The nature of this response depends upon the type and method of presentation of the target antigen to the host. The presentation of multiple, complex carbohydrate antigens directly to B cells may directly stimulate antibody production without the participation of T lymphocytes. This T cell-independent pathway of antibody production is thought to be a primitive response that provides a rapid, relatively nonspecific, response to infectious agents. The antibodies involved in this response are characterized by the use of their Ig genes in their original germline configuration. A second pathway for antibody production involves an indirect presentation by T cells of antigenic fragments to B cells, stimulation of B cell proliferation by T helper cells, and the production of antibodies with increased specificity and binding affinity for the target antigen. This T cell-dependent pathway initially includes the use of the same germline genes. The proliferative stimulus for B-2 cells provided by T helper cells, however, then results in the generation of more specific antibody due to the accumulation and preferential selection of somatic mutations in the variable regions of the antibody combining sites. Recently published data from rodent studies showed that the genetic control of antibody response to xenografts is controlled by a very restricted group of Ig genes, especially V_H and J_H immunoglobulin genes, involved in encoding the ability of antibody to bind to its target antigen. Rat monoclonal and polycional antibodies which arc produced in vivo by animals actively rejecting hamster grafts were shown to be encoded by the same group of closely-related V_H genes.

SUMMARY OF THE INVENTION

The invention relates to methods of preventing, reducing or inhibiting the production of antibody by a recipient and the rejection of tissues or organs transplanted from a different species donor. The method involves modifying, eliminating or blocking of the function of genes encoding antibody(ies) or fragments thereof produced by the recipient that are associated with the rejection of the transplanted tissue or organ. The transplantation of tissues or organs stimulates a rapid production of antibody that, by virtue of its structure, reacts with target antigens expressed by the graft and causes the activation of serum complement components and inflammatory/coagulation cascades, with a subsequent widespread damage to the graft. According to the invention, the modification of elimination or blocking of expression of a gene(s) that encode(s) these antibod(y)ies prevents this reaction. This invention may also be applied to (1) the treatment of diseases or conditions in which antibodies identical in V_H gene structure to xenoreactive antibodies play a dominant role and (2) to the use of antibodies and fragments of antibodies encoded by these genes diagnosis and treatment of the transplantation of porcine tissues to humans. Since modification of antibody gene expression may prevent any antibody-mediated tissue damage, the method of the invention may be applied to treating diseases or conditions in which antibody(ies) play(s) a predominant role, including the prevention of rejection of allogeneic tissues across ABO barriers, prevention of rejection of allografts by subjects presensitized to donor grafts, and treatment of autoimmune diseases mediated by antibody (ies).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the DP35 (IgHV3-l 1) germline gene expression in Patient No. 1 at days 0 and 10 following exposure to pig cells as determined by colony filter hybridization. A single DP35 (IgHV3-l 1) gene identified by an oligonucleotide probe specific for the CDR3 region, contributes to 63% of the increase in V_H genes related to the DP35 (IgHV3-l l) germline gene.

Figure 2 shows a similarly increased frequency of use of the IgHV3-74 genes in their original germline configuration expressed prior to and following pig xenoantigen exposure in Patient No. 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention arose from a desire by the inventors to improve on prior art technology in the field of prophylaxis and therapy of host antibody-mediated reaction to endogenous antigens as well as to xenogeneic tissue or graft transplantation. In the past, a host's immune system had to be suppressed in order to prevent an antibody reaction to a transplanted tissue or organ. The inventors reasoned that, based on their prior laboratory group's work in animals, they would undertake the search for immunoglobulin genes that control the reaction of patients to pig xenogeneic tissues and to subsequently propose methods to specifically prevent this reaction by interfering in the functional expression of these genes. Thus, once the gene sequences are known, they provide oligonucleotide segments that are targeted to general nucleic acid sequences involved in regulatory activities and specific nucleic acids involved in the expression of immunoglobulin genes or their fragments, and natural and synthetic aromatic amino acid oligomers comprising antibody (ies) fragments which are targeted to diagonal and minor grooves of the polynucleotide (s) encoding them. In this manner, they reasoned, they would prevent or lower the expression of antibody

(ies) specific to antigens located in the xenogeneic tissue which is transplanted into a host.

The humoral response of patients to pig tissues is encoded by two immunoglobulin heavy chain (V_H) variable region genes, IgHV_H3-l 1 and IgHV_H3-74. Exposure patients to pig tissues results in the rapid production of antibody to the galactose-α(l,3)galactose epitope and the concurrent expansion in the frequency of the specific use of these two genes. The figures accompanying this patent document this increased frequency of use.

The use of a restricted group of closely-related genes expressed in their original germline configuration indicates that the target antigens for the reaction must display limited variability and that it is possible to prevent the reaction by specifically targeting individual immunoglobulin genes to prevent their expression.

This invention is predicatd on studies by the inventors on the nature of the immunological response of a host to xenogeneic tissues including patients exposed to pig tissues. The results obtained demonstrate that the humoral response mounted by humans to pig tissues consists of a rapid production of both IgM and IgG antibodies, and that these antibodies primarily react with carbohydrate antigenic file, e.g. the galactose-α(l,3)galctose carbohydrate antigen described by Baquerizo et al. See Baquerizo et al., Transplantation 67:5, 1999. The inventors are reporting on their genetically cloned, sequenced and identified immunoglobulin genes responsible for encoding the ability of these antibodies to react with pig tissues. They isolated one of the genes and shown that single chain antibody fragments encoded by this gene display specific binding affinity for the galactose-α(l,3)galactose epitope and blocks the ability of preformed antibodies to bind to the target antigen. Their report on the identification of two genes that encode for the humoral response to xenografts and the isolation and characterization of the expression products permit the specific targeting of the function of these genes and, therefore, to preventing the rejection of pig xenografts in humans.

The present invention, thus provides a method of preventing, reducing or inhibiting expression of anti-xenogeneic antigen antibody, which comprises selecting a gene (s) or messenger ribonucleic acid (s) (mRNA) or protein product of this gene encoding antibody (ies) to xenogeneic tissue, organ or antigen (s) thereof; obtaining oligonucleotide (s) which are anti-sense to a target selected from the group consisting of gene (s), mRNA (s) and fragment (s) thereof which are associated with expression or regulation of expression of the antibodies or pyrrole-imidazole polyamides targeted to minor grooves of the DNA sequence encoding them; administering the thus obtained oligonucleotide (s) to a subject in need of such treatment in an amount and under conditions effective to bind to its target and prevent, reduce or inhibit expression of anti-xenogeneic antigen antibody.

In one preferred embodiment, the method of the invention employs oligonucleotides which are about 7 to 400 base pairs (bp) long, preferably about 10 to 200 bp long, more preferably about 13 to 100 bp long, and still more preferably up to about 40 bp long.

In the present method, the oligonucleotide (s)' targets may be selected from the group consisting of transcription initiation, enhance function, antigen contact sites that mediate antibody/xenoantigen interaction and intra-genic sequences. One preferred embodiment includes target sequences found within the coding region(s) of a gene or its mRNA. In another preferred embodiment, the oligonucleotide targets may be nucleotide segments located within the CDR1 , CDR2, CDR3 and FR3 regions of the immunoglobulin V_H gene (s) shown in Table 5 and the amino acid sequence (s) shown in Table 6 below. Amongst these, still more preferred are oligonucleotide targets such as nucleotide segments located within the HI loop in the CDR1, CDR2, FR3 and CDR3 regions of the the immunoglobulin VH gene (s) shown Tables 4 and 5, and the amino acid sequences shown in Table 6 below, each selected segment within any one group either alone or in combination with one another. A still more preferred embodiment is one where the transcription initiation target is made of gene sequences in the promoter region between positions -154 and +57. Another preferred group of oligonucleotide targets is that comprising nucleotide segments located within the HI loop, CDR2, FR3 and CDR3 regions of the the immunoglobulin VH gene (s) of the IgHV3-l l and IgHV3-74 nucleic acids, more preferably within the HI loop and the CDR2 region. Another of the more preferred oligonucleotides are those directed to sequences within the FR3 and CDR3 regions of the immunoglobulin V_H gene (s). Another preferred group is composed of target regions such as the nucleotide sequences shown in Table 5 below and fragments thereof, as well as the ones encoding amino acid sequences shown in Table 6 below, and more preferably amino acids 24-35 and 50-68 shown in Table 6 below. Preferred aromatic amino acid oligomers are those having about 5 to 50 amino acids, more preferred are aromatic amino acid oligomers comprising up to about 40 amino acids, still more preferred aromatic amino acid oligomerss comprise up to about 15 amino acids, and even more preferred are aromatic amino acid oligomers comprise up to about 10 aromatic amino acids. A most preferred group of aromatic amino acid oligomers comprise about 5 to 13 amino acids. The aromatic amino acid oligomer may be a natural aromatic amino acid or a natural amino acid substituted by halogen, alkyl, alkenyl, alkynyl, azido, amino, primary and secondary amines, alkoxy, thiol, thioalkyl, azo and alkylazo, among other ring or chain substituted amino acids.

Although the method of this invention is applicable to any and all animal species which produce antibodies to interspecies foreign antigens, tissues and/or organs, it is preferably intended for application to human subjects, either for transplantation of tissues and organs of other species, amongst which preferred are swine tissues and organs.

The method of this invention is particularly suitable for application to preventing, reducing or inhibiting xenograft rejection which normally occurs during transplantation into the subject, such as a human subject, of a tissue or organ comprising the xenogeneic antigen. The present method is generally suitable for preventing rejection of transplanted organs and tissues where the xenograft is, for example, heart, kidney and liver, among other organs, and islet of Langerhans and skin tissues, among others.

Another application of the method of the invention is to treat autoimmune diseases or conditions, where the xenogeneic antigen comprises an endogenous antigen which is not recognized as a self antigen by the subject. Examples of autoimmune diseases are systemic lupus erythematosus, rheumatoid arthritis, anti-β amyloid antibodies (Alzheimer's disease) and anti-insulin auto-antibodies (diabetes), among others. When the method of the invention is applied to facilitating transplantation, the oligonucleotide (s) may be contacted with the tissue or organ comprising a xenogeneic antigen prior to, during or subsequent to transplantation, or a combination thereof. In one preferred mode, the oligonucleotide (s) are administered to the subject prior and subsequent to transplantation, to maintain the effect for a prolonged period of time. In a particularly preferred form of the method of the invention, the oligonucleotide is operatively linked the oligonucleotide to a vector or a ribozyme. Suitable vectors are viral vectors and plasmids, such as murine leukemia viruses and adeno-associated viral vectors, among others. Of particular interest are target sequences which contain at least one unmethylated CpG, and preferably multiple un ethylated CpG motifs. The number of these motifs may be increased by demethylation after the oligonucleotide is prepared or synthesized. The design of oligonucleotide and/or gene constructs which are specific for the regulation of xenoantibody gene expression, in accordance with this invention, includes constructs varying in size from about 7, preferably 13 bp in length to 400, preferably 200, more preferably 100, and still more preferably 40 bp in length. The constructs are preferably targeted at coding and regulatory regions, including targets of transcription initiation, enhancer function identified in genomic DNA and antigen contact sites that mediate antibody/xenoantigen interaction and intragenic sequences identified in cDNA clones, among others. The nucleotide sequences located in the CDRl, CDR2, CDR3, and framework 3 (FR3) regions in Tables 5 and 6 below are preferred for the synthesis and targeting of the oligonucleotide of the invention. In addition, also part of this invention are aromatic amino acid polymers designed to down-regulate immunoglobulin gene expression related to the production of xenoantibodies.

Transcriptional tissue specificity may be influenced by gene sequences in the promoter region , e.g. between positions -154 and +57. Particularly suitable are anti-sense constructs directed at additional unique sequences within the immunoglobulin V_H genes that respond to xenografts, e.g. regions within the HI loop of the CDRl, CDR2, framework 3 and CDR3 as shown in Tables 4, 5 and 6 below. These sequences may be part of an anti-sense molecule by themselves or in combination, and may be designed with specificity for the immunoglobulin VH genes associated with, for example, the DP35 (IgHV3-l 1) and IgHV3-74 nucleic acids. These and other target regions may be identified in the nucleotide sequence presented in Table 5 and the amino acid sequence presented in Table 6, e.g. amino acids 24-35 and 50-68, which include short stretches of amino acid sequences that are unique features of immunoglobulin genes encoding antibody responses to pig cells in patients. The expression of immunoglobulin genes associated with T cell-independent responses is generally associated with certain regulatory interactions (motifs) that are potentially unique to this population of VH genes. Krieg, A. M., A. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, G. A. Koretzky, and D. M. Klinman, "CpG motifs in bacterial DNA trigger direct B-cell activation, Nature 574:546 (1995). This application includes the manipulation of gene sequences responding to unmethylated CpG motifs to inhibit the rapid reponse of human xenoantibodies to pig antigens.

The present invention leads to the design and/or selection of constructs that are functionally effective in the regulation of antibody gene expression based on the functional in vitro examination of several gene constructs designed to target various regions of the xenoantibody molecules. Suitable for different applications or the present technology, in combination with the described oligonucleotide are a plurality of vectors and/or modifications to the oligonucleotide (s) to impart increased efficiency in the down-regulation of xenoantibody expression. The present technology includes modifications such as selecting for demethylated CpG sites or regions, or demethylating CpG sites or regions to increase efficacy. In addition, also contemplated is the design of small molecules to modify gene expression associated with xenoantibody production, which includes the use of oligonucleotides in a backbone-modified form to enhance therapeutic efficiency, delivery using plasmids or viral vectors, e.g. murine leukemia viral vectors or adeno-associated viral vectors, or the use of ribozymes. Rossi, J. J., "Therapeutic applications of catalytic antisense RNAs (ribozymes)", Ciba Found. Symp. 209:195 (1997); Dachs, G. U., D. J. Chaplin, I. J. Stratford, and G. J. Dougherty, "Targeting gene therapy to cancer: a review", Oncol. Res. 9:313 (1997); Suzuki, Y. and T. Funato, "Specific inhibition of anti-DNA antibody production by an anti-DH ribozyme", Nippon Rinsho 55:1557 (1997). The relative efficiency of downregulation of gene expression of each oligonucleotide, with or without a vector or ribozyme, may be tested by ELISA assay and different functions may be tested in cell lines expressing these immunoglobulin genes. Any constructs that are functionally effective in vitro may then be tested in small or large animal models of xenograft rejection in vivo known in the art. The gene sequences in Table 5 and 6 may be applied to obtain primers to monitor the course of xenograft rejection by assessing relative xenoantibody levels in the peripheral blood of graft recipients. The location of the primer includes any regions identified and selected based on the above teachings. These primers have been used in the identification of the frequency of these xenoantibodies in subjects in Tables 1, 2, 5 and 6 of this patent, and others targeting somewhat different regions may be similarly designed. I. Human Ig V_H and J_π Genes Encoding the Antibody Response to Pig Tissues

Wang et al. suggested that antibodies to the galα(l,3)gal epitope expressed by pig tissues may be encoded by genes within the V_H3 family. Wang et al., J. Immunol. 155:1276 (1995). The inventors generated three DNA libraries specific for genes in the immunoglobulin V_H3 family. These libraries include:

(1) a Cμ, V_H3 library that includes all genes in the V_H3 family (constant region of the heavy chain),

(2) a Cμ library that selectively amplifies 20 germline genes in the V_H3 family, and (3) a Cμ library that amplifies genes related to the IgHV3-74 germline progenitor.

These libraries were designed to include all V_H3 germline genes thought to encode antibody binding to galα(l,3)gal epitopes. The present technology uses the colony filter hybridization technique to identify the specific genes within the V_H3 family that demonstrate an increase in expression following the exposure of individual patients to pig cells. By applying their technology, the inventors have demonstrated that at least one specific V_H3 family gene (DP35 (IgHV3-l 1)) evidences an increase in the frequency of its expression following B AL treatment. They have shown that exposure to porcine hepatocytes stimulates an increase in the expression of DP35 (IgHV3-l 1) genes from 12.7% of the V_H3 family in the peripheral blood of Patient No. 1 at day 0 to 32.4% at day 10 (39% post BAL in Patient No. 3) following exposure to pig cells as shown in Table 1 below.

Table 1 : Rearranged μ Chain V„3 Gene Segments from 2 BAL-treated Humans

Library V„3 Frequency of V_H3 Germline Gene

Origin Germline Gene Expression (% Identity)

(Day) (%)

0 DP35 (IgHV3-l l) 12.7 93.5 - 96.9

10 DP35 (IgHV3-l l) 32.4 98.11- 99.32

In addition to the increased frequency of expression, the similarity of the response in two different patients provides support for the use of this gene for mediating the antibody response to pig cells presented in this fashion.

The inventors conducted nucleic acid sequencing of individual DP35 (IgHV3-l l) clones and this analysis has provided evidence for the clonal expansion of a specific VDJ gene configuration due to the identification of a large number of clones expressing identical CDR3 regions at day 10. To extend this observation, they designed a unique, CDR3 specific, oligonucleotide probe to examine the frequency of expression of this gene following exposure to pig cells using the colony filter hybridization technique. The CDR3 specific probe is unique for these genes and displays no similarity with any of the Ig genes present in the Vbase or Genebank databases. This probe was used to screen cDNA V_H3 libraries from Patient No. 1 and more than 63%) of the DP35 (IgHV3-l 1) clones present at day 10 are represented by this single gene as shown in Figure 1. Similar results were obtained with Patient No. 3 following exposure to pig cells. Colony filter hybridization experiments were also used to demonstrate that genes related to the IgHV3-74 germline progenitor display a significant increase in frequency following BAL treatment. This is shown in Table 6 below. Two oligonucleotide probes that demonstrate specificity for specific V_H3 genes (DP58 and DP29) do not increase in frequency of expression following BAL treatment. An anchored PCR-ELISA assay was then applied to independently confirm the expansion of the V_H3 family genes in cDNA libraries representing each of six human V_H gene families in patient samples at various time points following BAL treatment. This technique demonstrates that only genes within the V_H3 family display an increase in frequency of expression in patients exposed to pig cells (data not included).

The inventors then screened immunoglobulin V_H3 gene libraries obtained at days 0 and 10 with an oligonucleotide probe designed to hybridize with the majority of genes within the V_H3 library that were excluded from the previous colony filter hybridization experiments. Oligonucleotide primer RVH20 was designed to hybridize with the majority of germline genes within the V_H3 family, and was shown to have at least 95% identity with at least 320 additional V_H genes identified in the human database. The RVH20 primer, in conjunction with the 29IC primer that identifies a germline gene that differs in canonical structure, was used in colony filter hybridization experiments to identify immunoglobulin genes associated with other germline progenitors that may be amplified in patients exposed to pig cells.

The increases in frequency of V_H gene expression were related to antibodies encoded by the DP35 (IgHV3-l l) and Cos6 (IgHV3-74) genes detected following exposure to pig cells, indicating that the response is specific and restricted as shown in Table 2 below. Table 2: Frequency of Immunoglobulin Gene Expression in BAL Patients

Oligo Specificity Frequency

(Day)

0 10

RVH1 1 DP35 (IgHV3-l l ) 12.7 32.4

583IC DP58 1.96 0.0

29IC DP29 22.0 0.9

543IC DP54 2.4 4.2

RVH20 43 germline genes 34.3 12.7

193WS Cos6 (IgHV3-74) 3.1 49.4

Several cDNA clones were isolated and sequenced following colony filter hybridization to identify whether the immunoglobulin genes related to DP35 (IgHV3-l 1) and IgHV3-74 demonstrated evidence of somatic mutation in the patients' immune response to porcine xenoantigens. Comparison of the nucleic acid sequences of these genes with their genomic progenitors indicates that these genes are expressed in a germline configuration as shown in Table 3 below.

Table 3: Nucleotide Substitutions in V„ Genes Expressed in Porcine Hepatocyte Administered Patients v„ v„ Nucleotide Nucleotide Differences

Clone Germline (amino acid) CDR Framework CDR Framework Gene Identify (%) Region Region Region Region

R^a/S^b R / S

793 IgHV3-l l 99.32 (98.0) 1/0 1/0 0.0 0.0

802 IgHV3-l l 98.98 (96.9) 1/0 2/0 0.0 0.0

D105 IgHV3-l l 98.54 (96.9) 1/1 2/0 1.0 2.0

D103 IgHV3- l l 98.16 (98.0) 1/1 1/2 1.0 0.5

D104 IgHV3-l l 98.1 1 (95.9) 1/0 3/1 0.0 3.0

31939 IgHV3-l l 98.64 (98.0) 1/1 1/1 1.0 1.0

1385 IGHV3-74 99.66 (100) 0/0 0/0 0.0 0.0

1362 IGHV3-74 97.62 (98.0) 0/0 2/3 0.0 0.7

1401 IGHV3-74 100 (100) 0/0 0/0 0.0 0.0 a. "R" refers to replacement mutation. b. "S" refers to silent mutation.

An analysis of the CDR3 regions of the xenoantibodies that were isolated from two patients following exposure to pig cells indicates that 63% of the cDNA clones related to

DP35 (IgHV3-l 1) had identical amino acid sequences in the CDR3 regions represented by clone 793 in Table 4 below. Nucleic acid sequencing of nine cDNA clones related to IgHV3- 74 were compared in the CDR3 region. Two groups of cDNA clones with identical CDR3 regions (1935 and 1399; 1358 and 1932) were identified within this group as shown in Table 4 below. The majority of the cDNA clones derived from the IgHV_H3-74 germline progenitor were expanded as a population of independent antibodies, including many clones that displayed differences in the CDR3 region.

Table 4: CDR3 Regions of V„ Genes Isolated from

Subjects Exposed to Porcine Hepatocytes

V_h Clone N-D-N J Region FR4

_____

1362 TCHYYF J_H5

1385 EGAGIAA J_H5 WGQ 1 1993355 G GAASSGGGGGGWWDDSSGGYYDD J J_H„33 WGQ

1399 GASGGGWDSGYD J_H3 WGQ

1358 SARYNWF J_H5 WGQ

1932 SARYNWF J_H5 WGQ

1933 EGDYYGSGSYST J_H3 WGQ 1 1993344 D DRRSSAAPPGGWWLLRRNNYY J J_HH66 WGQ

1945 YYDILTGP J_H6 WGQ

793 EYLSSL J„3 WGQ

The characteristic features associated with the CDR3 regions of xenoantibodies identified in these patients include an enhanced representation of amino acids tyrosine, glycine and serine, and a relatively short length. The data obtained clearly indicates that a closely-related group of genes are expanded following BAL exposure, and these genes lack evidence for T cell-induced somatic mutation.

II. Modification of Gene Function

The present invention relies on the use of oligonucleotides or aromatic amino acid polymers for down-regulating the expression of immunoglobulin genes associated with the production of xenoantibodies. Pyrrole-imidazole polyamides are small, cell permeable molecules that may be designed to bind to any predetermined DNA sequence. They are generally 5 to 13 base pairs (bp) in length and have been demonstrated to function as selective and efficient regulators of gene expression by binding to the minor groove of DNA. See, Gottesfeld et al., Nature 387:202 (1995); White et al., Nature 391 : 468 (1998). This invention also involves the use of oligonucleotides designed for specific regulation of gene expression that may function by one of three major mechanisms:

1) antisense oligonucleotides designed to be in complementary orientation to their target RNA sequence that react in a strictly base pair specific manner (Watson Crick base pairing ) and function to block translation,

2) oligonucleotides that bind double stranded DNA in the nucleus and block transcription by the formation of Hoogsten-type base triplets function by an antigene mechanism, and

3) oligonucleotides that demonstrate reactivity with a target protein (aptamer binding) as described by Oberbauer, Wien Klin. Wochenschr

109:40 (1997).

This invention provides a technology for modifying, inhibiting or blocking the expression of specific immunoglobulin genes we have identified that encode xenoreactive pantibodies, by means of oligonucleotide, aromatic amino acid polymers and/or a combination of these tools to facilitate the acceptance of xenografted organs in transplant recipients. Hairpin polyamides containing imidazole, pyrrole and 3-hydroxypyrrole rings inhibit the transcription of specific genes when targeted to sequences in the minor groove of the DNA double helix as described by Helene, C, Nature, 391 :436 (1997). The ability of hairpin polyamides to inhibit transcription of immunoglobulin genes is determined by the accession to regulatory regions and gene-specific intergenic sequences by these synthetic ligands.

The design and implementation of oligonucleotides that bind to the major groove of a complementary homopyrimidine-homopurine stretch by triple-helix formation represents an alternative method for controlling genes at the transcriptional level. Oligonucleotides engaged in a triple helix targeted to a transcription factor binding site or located down-stream of the transcription initiation site have been demonstrated to function in preventing transcriptional activation and/or by blocking movement of RNA polymerase along the DNA by Duval-Valentin et al., Proc. Natl. Acad. Sci. (USA) 89: 504 (1992). Oligodeoxynucleotides have been successfully applied to the regulation of gene expression in several small animal models of human disease. See Ding et al., J. Immunol. 160:2560 (1998); Smith et al., Cancer Gene Ther., 2:207 (1995). Clinical trials have been initiated to examine the potential of these novel molecular drugs for therapeutic use in inflammatory bowel disease, HIV, cancer and rheumatoid arthritis. See, Robertson, D., Nat. Biotechnol. 15:209 (1997); de Clercq. E., Clin. Microbiol. Rev. 8:200 (1995); Mercola & Cohen, Cancer Metastasis Rev. 15:287 (1996); Gibson, I., Cancer Metastasis Rev. 15:287 (1996); Wagner & Flanagan, Mol. Med. Today 3:31 (1997); Putnam, D.A., Health System Pharm. 53:151 (1996).

Anti-sense oligonucleotides are short (15-25 bp), single stranded DNA fragments that may be targeted to any specific region of a desired gene. The present method targets anti- sense constructs to several regions of the immunoglobulin genes that mediate xenoantibody responses. The targeted regions include

1) regulatory regions of xenoreactive antibodies,

2) nudeotides that encode the first hypervariable loop and/or antigen contact sites that mediate antibody/xenoantigen interaction, and 3) the CDR3 region of the antibody.

This invention also provides the means for producing antibodies and their fragments that bind to pig xenograft target antigens. This invention, thus, provides specific antibody proteins suitable for diagnosis and therapy of transplant rejection, e.g. of humoral responses of humans to pig xenografts.

EXAMPLES Immunoglobulin V_H gene promoters, enhancers and intragenic sequences control the regulation of gene expression as shown by Grosschedl & Baltimore, Cell 41:885 (1985). A wide range of sequences associated with regulatory functions may be targeted with anti-sense oligonucleotides with specificities ranging from broad (sequences associated with the binding of nuclear regulatory molecules such as NF-κB or BSAP) to narrow (promoter and/or intragenic sequences). See, Snapper et al., J. Immunol. 156:183 (1996); Khaled et al., Clin. Immunol. Immunopathol. 83:254 (1997); Max et al., Cur. Top. Microbiol. Immunol. 194:449 (1995). Transcriptional tissue specificity is determined by gene sequences in the promoter region (between positions -154 and +57), intragenic sequences,-and immunoglobulin gene enhancer sequences Grossschedl & Baltimore (1985), supra. Anti-sense oligos directed at additional unique sequences within the immunoglobulin V_H genes that respond to xenografts (regions within the HI loop, CDR2, framework 3 and CDR3 as demonstrated in Tables 4, 5 and 6 may be designed, either alone or in combination, with specificity for the immunoglobulin V_H genes associated with DP35 (IgHV3-l l) and IgHV3-74 progenitors. These potential target regions may be identified in the nucleotide sequence presented in Table 5 and the amino acid sequence presented in Table 6 (amino acids 24-35 and 50-68 include short stretches of amino acid sequences that are unique features of immunoglobulin genes responding to pig cells in patients).

The expression of immunoglobulin genes associated with T cell-independent responses appears to be associated with certain regulatory interactions (motifs) that are potentially unique to this population of V_H genes. Krieg et al., Nature 374:546 (1995). This application includes the manipulation of gene sequences responding to unmethylated CpG motifs to inhibit the rapid reponse of human xenoantibodies to pig antigens.

The proposed methodology for the design of small molecules to modify the expression of specific genes associated with xenoantibody production includes the use of oligonucleotides in a backbone-modified form to enhance therapeutic efficiency, delivery using viral vectors (murine leukemia virus or adeno-associated viral vectors) or the use of ribozymes. Rossi, J. J., Therapeutic applications of catalytic antisense RNAs (ribozymes), Ciba Found. Symp. 209:195 (1997); Dachs, G. U., D. J. Chaplin, I. J. Stratford, and G. J. Dougherty, Targeting gene therapy to cancer: a review, Oncol. Res. 9:313 (1997); Suzuki, Y. and T. Funato, Specific inhibition of anti-DNA antibody production by an anti-DH ribozyme, Nippon Rinsho 55:1557 (1997). The manipulation of the human antibody response to xenoantigens by regulating the expression of a select group of xenoantibodies at the nucleic acid level offers the advantage of selectivity for specific nucleic acid targets compared with traditional drugs. Recent modifications in oligonucleotide design for therapeutic strategies have led to the development of oligos that are safe and effective at pharmacologically relevant doses. This proposal extends the application of the use of antisense oligonucleotide to a therapeutic strategy that will enhance the survival of xenografted organs into human patients.

Table 5: V_H Gene Sequences Binding to Target Nucleic Acids

A. F 1

DP₃5 (IgHV3-ll) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTC 60

Day0(64₂) * Q* *************** *

DaylO(D10₃) _{***************}*_{************ *********************************}

DaylO(D105) _{*************** *******************************************}

Dayl0(793) _{* ******************************}*_{********************************************}

Dayl0(80₂) ************************* + *******"p**********************

CDRl FW 2

DP₃5 (IgHV3-ll) TCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCT 120

Day0(64₂) *****£***************** _{******* *************}

DaylO(D103) * *********£fj********** ******************<J>************* _**_**_**_**_**_**_**_**_**_**_**_**_****** ******

DaylO(D105) *****Q*****************'P************************************

Dayl0(793) _{*********************************************************}*_**

Dayl0(802) _****_*******_{*************************************************} _FW_2 ^CDR2

DP₃5 (IgHV3-ll) CCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTAC 180

Day0(642) *******************************************************£****

DaylO(D103) *************************** ****************************_Q****

DaylO(D105) _************_***********************************_********** ****

Dayl0(793) _************_*

Dayl0(802) _{****************} ft_{******************************} ******** ****

B. FW 1

Cos6(IgHV3-74) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTTCAGCCTGGGGGGTCCCTGAGACTC 60

DayO _{************************************************************}

DaylQ(1385)

Dayl0{1401) _{************************************************************}

Dayl0(1362)

CDRl FW_2

Cos6(IgHV3-74) TCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGCACTGGGTCCGCCAAGCT 120

DayO *****r-*************_*****P****************_****_****************

DayO (1385) _{************************************************************}

Dayl0(1401) _{************************************************************}

Dayl0(l₃6₂) _{**********************}*_*

FW 2 CDR2

Cos6(IgHV3-74) CCAGGGAAGGGGCTGGTGTGGGTCTCACGTATTAATAGTGATGGGAGTAGCACAAGCTAC 180

DayO ****************_************************_*************** ****

Dayl0(l₃85) *************_*********_**********_****************************

Dayl0(1401) ***_****************************_***********_*********_*******_**

Dayl0(136₂) *****************************_*******************************

FW 3

Cos6(IgHV3-74) GCGGACTCCGTGAAGGGC CGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTAT 240

DayO ****************** * ** ********************_*****************

Dayl0(1385) ****************** ********************************_*********_*

Dayl0(1401) *****_************* ****************_**************************

Dayl0(136₂) ****************** ****************************************** FW__3

Cos6(IgHV3-74) CTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCAAGA 294

DayO _{******************************************************}

Dayl0(1385) _{******************************************************}

Dayl0(1401) _{******************************************************}

Dayl0(136₂) ************Q* ** ****************** **^***********^*>p

Stars indicate identities in nucleic acid sequence.

Table 5 above shows the nucleotide sequences of V_H genes identified in patients at day 0 and day 10 following exposure to porcine hepatocytes and their corresponding germline progenitors. The nomenclature of the germline genes is based on Tomlinson et al., The repertoire of human germline V_H sequences reveals about fifty groups of V_H segments with different hypervariable loops, J. Mol. Biol.227:116 (1992). The numbering and borders of the CDR regions are depicted according to the conventions set by Kabat et al., Sequences of proteins of immunological interest. US Dept. of Health and Human Services, US Government Printing Services (1987).

Table 6: Amino Acid Segments Expressed from V„ Genes FR1 CDRl FR2

I I I i

DP35 QVQLVESGGGLVKPGGSLR SCAASGFTFS DYYMS WIRQAPGKG EWVS

642 _**********_{******************** ***** **************}

103 _{***************************** ***** **************} 793 *_****_********_******_*****_****** *_**** ***********_***

802 *****_****_**p*****_**_******_***** _***** **************

Cos6 EVQLVESGGG VQPGGSLRLSCAASGFTFS SYWMH VRQAPGKGLVWVS

_DavO *_{****************}*_***_****_{***** ***** **************}

1385 _*******_******_******_****_***_{**** ***** **************} 1401 _{**************}***_*****_***_{***** ***** **************}

1₃62 _{********************}*_{********* *****} *_{*************}

CDR2 R3

I I

DP35 YISSSGSTIYYADSVKG RFTISRDNAKNS YLQMNSLRAEDTAVYYCAR ₆₄2 _****_*****Q**_***** *Q_**_***_*****_**_******_***_**********

1₀₃ ********_*Q***_**** _* Q* _{***************************** *}

793 _*****+_***g_******* _{* * ***************************** *}

802 *********<$******* * Q* * * + * + * + * * * * -* * *■ * * -k * * * * * * + * * * * * *

Cos6 RINSDGSSTSYADSVKG RFTISRDNAKNT YLQMNSLRAEDTAVYYCAR Dav_{O ***************** *********************************} 1₃85 _{***************** *********************************}

1401 _**********_{******* *********************************}

1362 _{***************** ********************} Q_{* ********** J}

Table 6 above shows the translated amino acid sequences of V_H genes isolated from patients at days 0 (Clone Nos.642) and day 10 (Clone Nos.103, 105,793,802, 1385, 1401, and 1362) following exposure to porcine hepatocytes. The translated sequences of the most closely- related germline genes are shown for comparison's sake in Table 6 and the nucleotide sequences in Table 5 above.

Tables 7 and 8 below show all presently known alleles of the IgHV3-l 1 and IgHV3-74 genes. FP6642745

Table 7: Alignment of the Human IGHV3-11 Alleles

1 2 3 4 5 6 7 8 9 10 11 1₂ 13 14 15 16 17 18 19 ₂0 Q V Q L V E S G G G L V K P G G S L R

M99652,IGHV3-11*01,V3-11 CAG GTG CAG CTG GTG GAG TCT GGG GGA .. GGC TTG GTC AAG CCT GGA GGG TCC CTG AGA Zl₂337, IGHV3-ll*01,DP-35 X9₂₂₂0, IGHV3 - 11*01, ₂₂-₂B

M15496,IGHV3-ll*02,hv3.3 X92287, IGHV3-ll*03,VH3-8 -T--

CDR1-IMGT

21 22 23 24 25 26 27 28 29 30 3 311 3322 3 333 3 344 3 355 36 37 38 39 40

L S C A A S G F T F SS DD YY YY M S

M99652, IGHV3-11*01,V3-11 CTC TCC TGT GCA GCC TCT GGA TTC ACC TTC AGT GAC TAC TAC ATG AGC Z12337,IGHV3-ll*01,DP-35 X92220, IGHV3 -11*01, 22 -2B M1549S, IGHV3- 11*02, hv3.3 X92287,IGHV3-ll*03,VH3-8 CDR2 -

41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 W I R Q A P G K G L E W V S Y I S S S G

M99652,IGHV3-11*01,V3-11 TGG ATC CGC CAG GCT CCA GGG AAG GGG CTG GAG TGG GTT TCA TAC ATT AGT AGT AGT GGT Z12337,IGHV3-ll*01,DP-35 X92220, IGHV3 -11*01, 22 -2B M15496,IGHV3-ll*02,hv3.3 X92₂87, IGHV3-ll*03,VH3-8

IMGT

61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 S T I Y Y A D S V K G R F T I S R

M99S52, IGHV3- 11*01, V3 - 11 AGT ACC ATA TAC TAC GCA GAC TCT GTG AAG ... GGC CGA TTC ACC ATC TCC AGG Z12337,IGHV3-ll*01,DP-35 X92220, IGHV3 -11*01, 22 -2B M1549S,IGHV3-ll*02,hv3.3

FP6642745

X92287, IGHV3-ll*03,VH3-8 TA- -C-

81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100

D N A K N S L Y Q M N S R A E D T A

M99652,IGHV3-11*01,V3-11 GAC AAC GCC AAG AAC TCA CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC ACG GCC Z12337, IGHV3-ll*01,DP-35 X92₂₂0,IGHV3- 11*01, ₂2-₂B M15496, IGHV3-ll*0₂,hv3.3 X92287,IGHV3-ll*03,VH3-8

_CDR3 - IMGT 101 10₂ 103 104 105 106

V Y Y C A R

M99652, IGHV3- 11*01, V3 - 11 GTG TAT TAC TGT GCG AGA GA Z12337, IGHV3-ll*01,DP-35 X92220, IGHV3 -11*01, 22 -2B L T E M15496, IGHV3 - 11*02, hv3.3 T-A CT- ACT -C -A- X92287, IGHV3-ll*03,VH3-8

FP6642745

Table 8: Alignment of the human IGHV3-74 alleles

1 2 3 4 5 6 7 8 9 10 11 1₂ 13 14 15 16 17 18 19 ₂0 E V Q L V E S G G G V Q P G G S L R

Z12353,IGHV3-74*01,DP-53 GAG GTG CAG CTG GTG GAG TCC GGG GGA GGC TTA GTT CAG CCT GGG GGG TCC CTG AGA D16832,IGHV3-74*01,13G12 Z3008₂, IGHV3-74*01/0₂,DA-8 Z1739₂, IGHV3-74*0₂,COS-6 J00239,IGHV3-74*03,H11 CDRl - IMGT

21 _{22 2}3 24 ₂5 26 27 28 29 30 3 311 332₂ 3333 3344 35 36 37 38 39 40

L S C A A S G F T F SS SS YY M H

Zl₂353,IGHV3-74*01,DP-53 CTC TCC TGT GCA GCC TCT GGA TTC ACC TTC AGT AGC TAC TGG ATG CAC D16832, IGHV3-74*01, 13G12 Z3008₂,IGHV3-74*01/0₂,DA-8 Z17392, IGHV3-74*02,COS-6 J00239, IGHV3-74*03,H11 CDR2-

41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 W V R Q A P G K G L V V S R I N S D G

Z12353, IGHV3-74*01,DP-53 TGG GTC CGC CAA GCT CCA GGG AAG GGG CTG GTG TGG GTC TCA CGT ATT AAT AGT GAT GGG D16832,IGHV3-74*01,13G12 Z30082, IGHV3-74*01/02,DA-8 Z17392, IGHV3-74*02,COS-6 J00239,IGHV3-74*03,H11

IMGT

61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

S S T S Y A D S V K G R F T I S R

Z12353,IGHV3-74*01,DP-53 AGT AGC ACA AGC TAC GCG GAC TCC GTG AAG ... GGC CGA TTC ACC ATC TCC AGA D16832, IGHV3-74*01, 13G12 Z30082,IGHV3-74*01/02,DA-8 Z17392,IGHV3-74*02,COS-6

T J00239,IGHV3-74*03,H11 -CG

FP6642745

81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100

D N A K N T L Y Q M N S L R A E D T A

Z12353,IGHV3-74*01,DP-53 GAC AAC GCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGT CTG AGA GCC GAG GAC ACG GCT

D16832,IGHV3-74*01,13G12

Z30082,IGHV3-74*01/02,DA-8

Z17392,IGHV3-74*0₂,COS-6

J00239,IGHV3-74*03,H11

_CDR3 - IMGT 101 10₂ 103 104 105 106 V Y Y C A R Zl₂353,IGHV3-74*01,DP-53 GTG TAT TAC TGT GCA AGA

D1683₂,IGHV3-74*01,13G12

Z30082, IGHV3- 74*01/02, DA- 8

Z17392, IGHV3-74*02,COS-6

J00239,IGHV3-74*03,H11 GA

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Claims

1. A method of preventing, reducing or inhibiting expression of anti-xenogeneic antigen antibody, comprising selecting a gene(s), messenger ribonucleic acid(s) (mRNA), or protein(s) associated with the expression or regulation of antibody(ies) to xenogeneic tissue, organ or antigen (s) thereof; obtaining oligonucleotide(s) which are anti-sense to a target selected from the group consisting of gene(s), mRNA(s) and fragment(s) thereof which are associated with expression or regulation of expression of the antibodies or aromatic amino acid oligomers comprising an amino acid sequence selected from the group consisting of antibodies fragments targeted to minor microgrooves of the polynucleotide(s) encoding them having at least one aromatic amino acid; and administering the thus obtained oligonucleotide(s) to a subject in need of treatment in an amount and under conditions effective to bind to its target and prevent, reduce or inhibit expression of anti-xenogeneic antigen antibody.

2. The method of claim 1 , wherein the oligonucleotides are about 7 to 400 base pairs (bp) long.

3. The method of claim 2, wherein the oligonucleotides are about 10 to 200 bp long.

4. The method of claim 3, wherein the oligonucleotides are about 13 to 100 bp long.

5. The method of claim 4, wherein the oligonucleotides are up to about 40 bp long.

6. The method of claim 1, wherein the oligonucleotide targets are selected from the group consisting of transcription initiation, enhancer function, antigen contact sites that mediate antibody/xenoantigen interaction and intra-genic sequences.

7. The method of claim 6, wherein the oligonucleotide targets are selected from the group consisting of nucleotide segments located within the CDRl, CDR2, CDR3 and FR 3 regions of the immunoglobulin V_H gene (s) shown in Tables 4, 5 and 6.

8. The method of claim 7, wherein the oligonucleotide target is selected from the group consisting of nucleotide segments located within the CDRl, CDR2, FR 3 and CDR3 regions of the the immunoglobulin VH gene (s) shown Tables 4, 5 and 6, either alone or in combination.

9. The method of claim 7, wherein the transcription initiation target is selected from the group consisting of gene sequences in the promoter region between positions - 154 and +57 of nucleic acids DP35 (IgHV3-l 1) and V3-74 (IgHV3-74).

10. The method of claim 8, wherein the oligonucleotide target is selected from the group consisting of nucleotide segments located within the CDRl, CDR2, FR3 and CDR3 regions of the the immunoglobulin VH gene (s) encoded by nucleic acids DP35 (IgHV3-l 1) and V3-74 (IgHV3-74).

11. The method of claim 10, wherein the target region is selected from the group consisting of nucleotide sequences shown in Table 5, and amino acid sequences shown in Table 6.

12. The method of claim 11, wherein the target region is selected from the group consisting of amino acids 24-35 and 50-68 shown in Table 6.

13. The method of claim 12, wherein the tissue or organ is a swine tissue or organ, and the subject is a human subject.

14. A method of preventing, reducing or inhibiting xenograft rejection, comprising the method of claim 1 , and further comprising transplanting into the subject a tissue or organ comprising the xenogeneic antigen.

15. The method of claim 14, wherein the xenograft is selected from the group consisting of liver, heart and kidney organs, and islets of Langerhans tissues.

16. A method of treating autoimmune diseases or conditions, comprising the method of claim 1, wherein the target antigen comprises an endogenous antigen which is not recognized as a self antigen by the subject.

17. The method of claim 16, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, anti-╬▓ amyloid antibodies (Alzheimer's disease) and anti-insulin auto-antibodies (diabetes).

18. The method of claim 15, wherein the thus obtained oligonucleotide (s) is exposed to the tissue or organ comprising a xenogeneic antigen prior to transplantation.

19. The method of claim 15, wherein the thus obtained oligonucleotide (s) are administered to the subject prior and subsequent to transplantation.

20. The method of claim 1 , further comprising operatively linking the oligonucleotide to a vector or a ribozyme.

21. The method of claim 20, wherein the vector is selected from the group of viruses and plasmids.

22. The method of claim 21, wherein the vector is a murine leukemia virus or an adeno-associated viral vector.

23. The method of claim 1 , wherein the target sequences are selected to contain at least one unmethylated CpG.

24. The method of claim 1, wherein at least one CpG of the oligonucleotide is demethylated.

25. The method of claim 1, wherein the aromatic amino acid oligomer (s) comprise (s) about 5 to 50 amino acids.

26. The method of claim 25, wherein the aromatic amino acid oligomer (s) comprise (s) up to about 40 amino acids.

27. The method of claim 26, wherein the aromatic amino acid oligomer (s) comprise (s) up to about 15 amino acids.

28. The method of claim 1, wherein the aromatic amino acid oligomer comprises about 2 to 10 aromatic amino acids.

29. The method of claim 28, wherein the aromatic amino acid oligomer comprises about 4 to 8 aromatic amino acids.

30. The method of claim 1, wherein the aromatic amino acid oligomer comprises an aromatic amino acid selected from the group consisting of natural aromatic amino acids and natural amino acids substituted by halogen, alkyl, alkenyl, alkynyl, azido, amino, primary and secondary amines, alkoxy, thiol, thioalkyl, azo and alkylazo.

31. A protein, comprising a polypeptide selected from the group consisting of polypeptides encoded by an oligonucleotide(s) which are anti-sense to a target selected from the group consisting of gene(s), mRNA(s) and fragment(s) thereof which are associated with expression or regulation of expression of the antibodies or aromatic amino acid oligomers comprising an amino acid sequence selected from the group consisting of antibodies fragments targeted to minor microgrooves of the polynucleotide(s) encoding them having at least one aromatic amino acid.

32. The protein of claim 31, wherein the oligonucleotides are about 7 to 400 base pairs (bp) long.

33. The protein of claim 32, wherein the oligonucleotides are about 10 to 200 bp long.

34. The protein of claim 33, wherein the oligonucleotides are about 13 to 100 bp long.

35. The protein of claim 34, wherein the oligonucleotides are up to about 40 bp long.

36. The protein of claim 31 , wherein the oligonucleotide are anti-sense to a target selected from the group consisting of transcription initiation, enhancer function, antigen contact sites that mediate antibody/xenoantigen interaction and intra-genic sequences.

37. The method of claim 36, wherein the oligonucleotide target is selected from the group consisting of nucleotide segments located within the CDRl, CDR2, CDR3 and FR 3 regions of the immunoglobulin V_H gene (s) shown in Tables 4, 5 and 6.

38. The protein of claim 37, wherein the oligonucleotide target is selected from the group consisting of nucleotide segments located within the CDRl, CDR2, FR 3 and CDR3 regions of the the immunoglobulin VH gene (s) shown Tables 4, 5 and 6, either alone or in combination.

39. The protein of claim 37, wherein the transcription initiation target is selected from the group consisting of gene sequences in the promoter region between positions - 154 and +57 of nucleic acids DP35 (IgHV3-l 1) and V3-74 (IgHV3-74).

40. The protein of claim 38, wherein the oligonucleotide target is selected from the group consisting of nucleotide segments located within the CDRl, CDR2, FR3 and CDR3 regions of the the immunoglobulin VH gene (s) encoded by nucleic acids DP35 (IgHV3-l 1) and V3-74 (IgHV3-74).

41. The protein of claim 40, wherein the target region is selected from the group consisting of nucleotide sequences shown in Table 5, and amino acid sequences shown in Table 6.

42. The protein of claim 41, wherein the target region is selected from the group consisting of amino acids 24-35 and 50-68 shown in Table 6.

43. The protein of claim 31, wherein the polypeptide is encoded by a polynucleotide selected from the group consisting of nudeotides 1 to 294 of an IgVH-311 allele sequence plus a nucleotide sequence encoding a CR3, nudeotides 1 to 294 of an IgVH-74 allele sequence pies a nucleotide sequence encoding a CDR3, and fragments thereof about 9 to 56 nudeotides long.

44. The protein of claim 31, wherein the polypeptide is encoded by nudeotides 1 to 294 of an IgVH-311 allele sequence plus a nucleotide sequence encoding a CR3 or fragments thereof about 9 to 56 nudeotides long.

45. The protein of claim 31, wherein the polypeptide is encoded by nudeotides nudeotides 1 to 294 of an IgVH-74 allele sequence pies a nucleotide sequence encoding a CDR3 orfragments thereof about 9 to 56 nudeotides long.

46. A nucleic acid comprising a polynucleotide encoding the polypeptide of claim 31.

47. The nucleic acid of claim 46, wherein the polynucleotide is nudeotides 1 to 294 of an IgVH- 311 allele sequence plus a nucleotide sequence encoding a CR3 or fragments thereof about 9 to 56 nudeotides long.

48. The nucleic acid of claim 46, wherein the polynucleotide is nudeotides nudeotides 1 to 294 of an IgVH-74 allele sequence pies a nucleotide sequence encoding a CDR3 or fragments thereof about 9 to 56 nudeotides long.

49. The nucleic acid of claim 46, being a DNA.

50. A nucleic acid, which is anti-sense to the nucleic acid of claim 46.

51. The nucleic acid of claim 50, which is a DNA.

52. The nucleic acid of claim 50, which is an RNA.

53. A hybrid vector, comprising a vactor and the nucleic acid of claim 46 operatively linked thereto.

54. The hybrid vector of claim 53, which is a hybrid eukaryotic or prokaryotic vector.

55. A transfected cell, comprising a host cell, carrying the nucleic acid of claim 46 alone or operatively linked to a vector.

56. A method of producing the protein associated with an anti-xenogeneic antigen antibody, comprising obtaining an oligonucleotide(s) which is(are) anti-sense to a target selected from the group consisting of gene(s), mRNA(s) and fragment(s) thereof which are associated with expression or regulation of expression of the antibodies or aromatic amino acid oligomers comprising an amino acid sequence selected from the group consisting of antibodies fragments targeted to minor microgrooves of the polynucleotide(s) encoding them having at least one aromatic amino acid; obtaining a nucleic acid comprising the polynucleotide; operatively linking the nucleic acid to a vector to obtain a hybrid vector; transfecting a host cell with the thus obtained hybrid vector; culturing the transfected cell in an expression medium under conditions effective to express the polynucleotide as a protein; and allowing the protein to accumulate.

57. The method of claim 56, wherein the vector is a prokaryotic or eukaryotic vector.

58. The method of claim 56, wherein the polynucleotide is selected from the group consisting of nudeotides 1 to 294 of an IgVH-311 allele sequence plus a nucleotide sequence encoding a CR3, nudeotides 1 to 294 of an IgVH-74 allele sequence pies a nucleotide sequence encoding a CDR3, and fragments thereof about 9 to 56 nudeotides long.

59. The method of claim 46, wherein the polynucleotide is nudeotides 1 to 294 of an IgVH-311 allele sequence plus a nucleotide sequence encoding a CR3 or fragments thereof about 9 to 56 nudeotides long.

60. The method of claim 46, wherein the polynucleotide is nudeotides nudeotides 1 to 294 of an IgVH-74 allele sequence pies a nucleotide sequence encoding a CDR3 or fragments thereof about 9 to 56 nudeotides long.

61. The method of claim 56, wherein the nucleic acid is a DNA.

62. The method of claim 56, wherein the nucleic acid is an RNA.

63. The method of claim 56, further comprising isolating the protein.

64. The method of claim 56, further comprising forming a salt of the protein by addition of an acid.

65. The method of claim 64, wherein the acid is selected from inorganic and organic acids.

66. The method of claim 63, further comprising forming a salt of the protein by addition of an acid.

67. The method of claim 64, wherein the acid is selected from inorganic and organic acids.

68. An antibody selectively binding to the polypeptide of claim 31.

69. The antibody of claim 68, which is a monoclonal antibody.

70. The antibody of claim 68, which selectively binds to an oligopeptide about 3 to about 20 amino acids long.

71. The antibody of claim 68, selectively binding to an oligopeptide of the polypeptide encoded by nudeotides 1 to 294 of an IgVH-31 1 allele sequence plus a nucleotide sequence encoding a CR3, nudeotides 1 to 294 of an IgVH-74 allele sequence pies a nucleotide sequence encoding a CDR3, or fragments thereof about 9 to 56 nudeotides long.

72. The antibodyof claim 71, selectively binding to an oligopeptide of the polypeptide encoded by nudeotides 1 to 294 of an IgVH-31 1 allele sequence plus a nucleotide sequence encoding a CR3 or fragments thereof about 9 to 56 nudeotides long.

73. The antibodyof claim 71 , selectively binding to an oligopeptide of the polypeptide encoded by nudeotides nudeotides 1 to 294 of an IgVH-74 allele sequence pies a nucleotide sequence encoding a CDR3 or fragments thereof about 9 to 56 nudeotides long.

74. A method of diagnosing an anti-xenogeneic antigen antibody, comprising contacting a sample with the antibody of claim 68; allowing for antibody-anti-xenogeneic antigen antibody complex(es) to form; and detecting the presence of any antibody-anti-xenogeneic antigen antibody complex(es) formed and comparing it(them) to a control conducted in the absence of anti-xenogeneic antigen antibody.

75. A method of detecting a nucleic acid encoding an anti-xenogeneic antigen antibody or associated with its regulation in a subject or fragment thereof, comprising contacting a sample suspected of comprising the nucleic acid with an oligonucleotide(s) which is(are) anti-sense to a target selected from the group consisting of gene(s), mRNA(s) and fragment(s) thereof which are associated with expression or regulation of expression of the antibodies or aromatic amino acid oligomers comprising an amino acid sequence selected from the group consisting of antibodies fragments targeted to minor microgrooves of the polynucleotide(s) encoding them having at least one aromatic amino acid under hybridizing conditions; allowing for hybridization to occur; and detecting the presence of any nucleic acid-oligonucleotide hybrid(s) formed and comparing it(them) to a control conducted in the absence of the nucleic acid.

76. A method of inhibiting xenogeneic transplant rejection in a subject, comprising conducting the method of claim 1.

77. The method of claim 76, wherein the oligonucleotide(s) is(are) administered to the subject prior to, during and/or subsequent to transplantation.