Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5082—Supracellular entities, e.g. tissue, organisms
  • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/57536—Endothelin, vasoactive intestinal contractor [VIC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
  • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/575—Hormones
  • G01N2333/5754—Endothelin, vasoactive intestinal contractor [VIC]
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/24—Immunology or allergic disorders
  • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease

Definitions

• the field of the invention is inflammation and tissue and organ transplant rejection.
• Tissue, cardiac, and other solid-organ transplants are often compromised by acute or chronic rejection, typically characterized by the development of transplant- associated arteriosclerosis.
• This stenosis occurs only in the grafted tissue and not in host arteries, and indicates that the pathological inflammatory processes occur only in the donor tissue.
• Histologically, lesions related to graft rejection differ from common chronic arteriosclerosis, in that transplant arteriosclerosis involves the artery in a concentric rather than eccentric fashion; lipid accumulation is less common in the early development of the transplant- associated lesion, and the development of the disease is faster.
• Monocytes and macrophages accumulate in the early stages of graft rejection, followed by macrophage and smooth muscle cell accumulation, and in the later, more obliterative stage, smooth muscle cells predominate. It is likely that an immune response stimulates expression of a variety of growth factors and cytokines which are involved in the pathogenesis of graft rejection.
• ET-1 Endothelin-1 (ET-1) , a 21 a ino acid peptide, was first isolated from the supernatant of cultured endothelial cells. Besides having potent vasoconstrictive effects, ET-1 has been shown to exert a mitogenic effect on endothelial and smooth muscle cells in vitro and in the neointima after injury from balloon angioplasty in vivo.
• Plasma ET-1 levels are elevated at various time points after uncomplicated transplantation of hearts, other solid organs, and in patients with severe symptomatic atherosclerosis. In atherosclerotic lesions, ET-1 protein and mRNA are increased compared with normal vessels.
• ET-1 binds to two distinct G-protein coupled receptors, ET A and ET B . These receptors are expressed in endothelial cells and smooth muscle cells, but in the course of normal chronic atherosclerosis, receptor expression decreases.
• the invention features a method to reduce graft rejection, graft-induced arteriosclerosis, or other graft-induced chronic cell- mediated inflammatory processes in a mammal.
• the method(s) involve inhibiting the expression of ET-1 or ET-1 activity on the transplanted or inflamed tissue by administering a therapeutic compound to the graft either before or after implantation to the host mammal.
• a therapeutic compound is a drug, nucleic acid, chemical, or protein compounded in a suitable excipient which will have a beneficial effect, such as inhibiting graft rejection or arteriosclerosis.
• ⁇ grafted and “transplanted” are used interchangeably herein, and indicate a graft of any tissue transplanted between genetically nonidentical individuals or species. Allografts are between genetically nonidentical individuals of the same species, xenografts are between genetically nonidentical members of different species. The methods of the invention can be used for both types of grafts. These terms are distinguishable from an isograft, meaning a tissue or organ transplanted between genetically identical individuals or the same individual.
• ET-l activity means the biological functions of ET-1, vasoconstriction and mitogenic properties which are exerted on, e.g., smooth muscle cells and endothelial cells.
• an alloimmune-induced arteriosclerosis aspect of the graft rejection is treated by this method.
• alloimmune is meant an immunologic response induced by the engrafted tissue, regardless of its source (e.g., allografts, xenografts).
• the graft being spared from rejection is a solid organ, more preferably a heart.
• the source and host animal for the graft or inflammation is a mammal, preferably a rodent, and more preferably a human patient.
• transcription or translation of ET-1 is reduced or inhibited in mononuclear cells which have infiltrated the graft and/or endothelial cells.
• These cells are preferably host mononuclear cells but may also be donor mononuclear cells.
• mononuclear cells is meant macrophages, monocytes, lymphocytes, and any other cell types which are typically referred to as mononuclear in the art.
• the inhibition of the effects of ET-1 is brought about by inhibiting transcription or translation of ET-l mRNA.
• this regulation of expression occurs in mononuclear cells and/or endothelial cells, more preferably mononuclear cells which have infiltrated the graft or area of inflammation.
• the inhibition of ET-l effects is achieved by means of inhibiting translation of ET-l mRNA into ET-l polypeptide, preferably by introducing antisense DNA into ET-l expressing cells (e.g., mononuclear cells).
• antisense DNA is meant any DNA of 15 nucleotides or longer which is complementary to a region of the ET-l nucleotide sequence (SEQ ID NO: ), and can be identified and generated using methods well known in the art.
• the diagnostic assays and therapeutics of the invention encompass the use of isolated DNA containing part or all of the sequence of a mammalian ET-l.
• the DNA encodes human ET-l, however, any mammalian DNA encoding ET-l is included for use in the invention as long as it has preferably 50% sequence identity with the human ET-l sequence, more preferably 70% and most preferably over 90% sequence identity with the human sequence.
• vectors containing the isolated DNA cells, which can be prokaryotic or eukaryotic, containing the isolated DNA; and the use of methods of manufacturing recombinant ET-l known in the art, such as methods of culturing the cells containing isolated ET-l DNA under conditions permitting expression of the DNA.
• isolated DNA refers to a DNA sequence which may be single stranded or double stranded, sense or antisense and which has been removed from the sequences which flank it in a naturally occurring state, e.g., the sequences adjacent to the DNA sequence in a genome in which it naturally occurs.
• the term includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences.
• inventions include inhibiting ET-l activity by use of a blocking agent (e.g., physically), inhibiting agent (an agent that restrains or retards physiologic, chemical, or enzymatic action; depression or arrest of function) , or antagonizing agent (an agent which opposes or resists the action of another; agents that tend to neutralize or impede the action or effect of others; e.g., something which binds or interacts with the ET-l receptor and doesn't have the biological activity of ET-
• a blocking agent e.g., physically
• inhibiting agent an agent that restrains or retards physiologic, chemical, or enzymatic action; depression or arrest of function
• antagonizing agent an agent which opposes or resists the action of another; agents that tend to neutralize or impede the action or effect of others; e.g., something which binds or interacts with the ET-l receptor and doesn't have the biological activity of ET-
• ET A and ET B There are two types of G-protein linked ET-l receptors, ET A and ET B ; either or both of these receptor types are subject to blocking, inhibiting or antagonizing in preferred embodiments of the invention. In preferred embodiments, these receptor's function is inhibited by interfering with any portion of the G-protein cascade, including subsequent cellular effects (e.g., calcium influx into the receptor-bearing cell) . Agents working on the receptor may be peptide (at least 5 amino acids in length) or non-peptide. ET-l expression or activity preferably may be inhibited by agents which directly interact with a translated ET-l product, e.g., an endothelin converting enzyme inhibitor to prevent posttranslational modifications which may be essential for ET-l activity.
• whichever inhibitor of ET-l activity or expression is used results in a reduction of smooth muscle cell migration or proliferation in the intimal layer of arteries of the graft.
• the intima is defined as the region between the lumen and the internal elastic lamina of the artery; the media is defined as the region between the internal and external elastic laminae; and the adventitia is defined as the region outside the external elastic lamina of the artery.
• the graft may be treated prior to transplantation; preferably by suffusing, soaking, or perfusing it with a therapeutic composition of the invention.
• the invention also features a method of detecting graft rejection, graft-induced arteriosclerosis, or any other graft pathophysiology in a transplant recipient or patient.
• the method involves analyzing a sample from the patient for increased expression of ET-l in the graft, which, if present, indicates ongoing pathophysiology. This method can be performed and ET-l expression detected very soon following the transplant, within 1 day, preferably within 3 days, more preferably within 5 days. It can be performed throughout the life of the graft.
• the increased expression is detected in graft-infiltrating mononuclear cells.
• Another featured method to detect graft rejection, graft-induced arteriosclerosis, or any other graft pathophysiology in a transplant recipient is an in vivo method which involves detection of a labelled molecule which is targeted specifically for cells producing ET-l or ET-l itself.
• a detectable label is linked to an ET-l specific antibody or antibody fragment (Fab, Fab 2 ) , administered to a graft recipient, and the graft imaged.
• Fab, Fab 2 ET-l specific antibody or antibody fragment
• This label is preferably radioactive, more preferably has a short half-life, and most preferably is 1:ll In. Use of this label has been reported in Yasuda et al . , 1987, Circulation 76:306-311; Frist et al .
• the label can be a fluorescent label for in vitro use, or a spin label, or any other suitable label known to those skilled in the art. All methods are suitable for use in vitro, e.g., by in situ hybridization, and can be detected by any standard method, e.g., x-ray techniques, epifluorescence microscopy, NMR, etc. An elevated level of label in the graft is indicative of graft pathophysiology. Tissue can be obtained from the graft or from a blood sample which is then enriched using standard methods for mononuclear cells.
• Mononuclear cell activation or an upregulation of ET-l expression, can be assayed using standard techniques such as PCR, Northern, Southern, and dot blotting; in situ hybridization, antibody assays such as ELISAs, Western blots, etc., and such activation in peripheral blood samples will be indicative of graft rejection, graft-arteriosclerosis, or other graft pathophysiology.
• Another method featured in the invention is delivery of gene therapy to a grafted tissue, either prior to engraftment or after engraftment.
• the gene therapy may involve delivering a DNA encoding ET-l polypeptide, polypeptide analog, or a fragment thereof; or encoding an antisense DNA, which is complementary to at least 15 nucleotides of the sequence of ET-l, preferably SEQ ID NO:4. Delivery is effected by administering the nucleic acid in a suitable vector for expressing therapeutic amounts of gene transcript to the graft and/or infiltrating mononuclear cells.
• the method includes administering a test compound to a graft either prior to or following transplant into a mammal, allowing sufficient time for graft rejection to progress, if present, removing a sample of the graft and a sample of normal host tissue or tissue from a control animal, and comparing the expression of ET-l in the samples. Samples will be normalized to produce a value of ET- 1/cell.
• ET-l in the graft is less than 3-4 fold greater than the baseline expression in the control tissue ("differential expression") , it will indicate that the test compound has potential utility to inhibit expression or activity of ET-l and may further be useful to inhibit graft rejection, arteriosclerosis, and pathophysiology.
• the term “differentially expressed” refers to the ET-l gene transcript in a graft which is substantially greater or less than the amount of the same transcript found in the surrounding host tissue or in normal controls.
• gene transcript is meant a mRNA or cDNA.
• Peptides derived from the sequence of ET-l may be used to generate antibodies or directly for therapeutic purposes (e.g., competition with endogenous ET-l polypeptide for receptor sites) .
• Such peptides can be generated by methods known to those skilled in the art, including proteolytic cleavage of the protein, de novo synthesis of the fragment, or genetic engineering, e.g., cloning the gene or a portion of the gene encoding ET-l into an expression vector as described above.
• Also included in therapies of the invention are the use of analogs of the above peptides.
• Analogs can differ from the native peptide by conservative amino acid replacements which alter the sequence but do not adversely affect the functioning of the resulting polypeptide, or by modifications which do not affect the sequence, or by both.
• Modifications include in vivo or in vitro chemical derivitization of polypeptides, e.g., acetylation or carboxylation.
• modifications of glycosylation e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes.
• ET-l-related polypeptides which can be used in therapeutic compositions of the invention include polypeptide analogs in which one or more peptide bonds have been replaced with an alternative type of covalent bond (a "peptide mimetic") which is not susceptible to cleavage by peptidases.
• a peptide mimetic an alternative type of covalent bond
• proteolytic degradation of the peptides following injection into the subject is a problem
• replacement of a particularly sensitive peptide bond with a noncleavable peptide mimetic will make the resulting peptide more stable and thus more useful as a therapeutic.
• Such mimetics, and methods of incorporating them into polypeptides are well known in the art.
• amino-terminal blocking groups such as t- butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4,- dinitrophenyl.
• This invention offers improvements over the prior art in terms of more directed therapeutics, diagnostics, and screening for new therapeutics.
• the graft-rejecting screening tests can be performed very soon following transplantation, allowing a much sooner therapeutic intervention (e.g., immunosuppression, compositions of this invention) ; this will enhance graft survival and make possible grafts between individuals with larger histocompatibility differences than has been possible previously.
• G3PDH glyceraldehyde-3-phosphate dehydrogenase
• Fig. IB is a comparison of ET-l mRNA expression in allografts, isografts, and paired spleens in the acute (day 5 after transplantation) and the chronic allograft rejection (day 75 after engraftment) models.
• Fig. 2 is the amino acid sequence of human ET-l.
• Fig. 3 is the cDNA sequence of human mRNA for ET- 1.
• Acute and chronic rejection are major complications which develop following cardiac transplantation.
• Levels of endothelin-1 (ET-l) are elevated in plasma from patients with grafts and those with symptomatic vascular atherosclerosis but little is known about its role in these processes.
• ET-l endothelin-1
• the heterotopic cardiac transplant model allowed us to study the graft and the recipient's host heart (exposed to the same circulation but without development of vascular lesions) , which was not removed during transplantation.
• the neointimal lesions are composed of infiltrating inflammatory cells (rather than smooth muscle cells) , which are predominantly macrophages with fewer lymphocytes. Between 45 and 90 days, the infiltrating inflammatory cell population in the neointima decreases as intimal smooth muscle cells appear. In the last phase (beyond 90 days) , the neointima is maximally expanded, often obliterative, and composed predominantly of smooth muscle cells with fewer infiltrating mononuclear cells.
• Heterotopic abdominal cardiac transplantation was performed using Lewis donor hearts as described (Adams et al., 1992, supra) in an allogeneic combination involving F344 recipients for the chronic rejection model, and WF donor hearts into Lewis hosts for the acute rejection model.
• both the host (recipient) and the transplanted hearts were collected for histologic analysis and RNA extraction.
• the host heart served as a reference that had been exposed to the same circulation but was normal on histologic examination.
• ET-l oligonucleotides spanning exon/intron borders within the coding region were chosen using the MAC VECTOR* program (Int. Biotechnologies Inc, New Haven, CT) .
• the 5'primer (AT GGA TTA TTT TCC CGT GAT C; SEQ ID NO:l) and 3'primer (C TGT AGT CAA TGT GCT CGG; SEQ ID NO:2) generated a 614 bp fragment.
• PCR studies were carried out on a GeneAmp system 9600 (total vol, 25 ⁇ l) as described in (Russell et al., supra (1993); Russell et al., supra (1994)).
• Reaction conditions included 1.25 ⁇ l cDNA, l ⁇ M (each) 5'and 3'primer, lOmM TrisHCL/50mM KCl/1.5mM MgCl2/0.001% (wt/vol) gelatin/800/iM dNTPs/0.625 unit of A pliTaq DNA polymerase.
• the amplified product was cloned directly into the PCRII vector (Invitrogen, San Diego, CA) .
• Glyceraldehyde-3- phosphate dehydrogenase (G3PDH; amplification for 22 cycles at 94°C, 58°C and 72°C) , which represents an ubiquitously expressed mRNA, was used as an internal reference to reflect total cellular RNA, as previously described (Russell et al., 1993, Proc . Natl . Acad . Sci . USA. , supra , Russell et al., 1994, J. Clin . Invest , supra .
• Immunohi s tochemi s try Cardiac grafts, paired host hearts and spleens and isografts were embedded in OCT (Optimal Cutting Temperature compound) and stored at -70°C until sectioning on a cryostat.
• OCT Optimal Cutting Temperature compound
• ET-l staining was performed using a polyclonal rabbit anti-ET-1 antibody raised against human ET-l and kindly provided by Biomedica,
• EXAMPLE 1 ET-l mRNA expression ET-l mRNA expression increases in acute cardiac allograft rejection (WF into LEW) model :
• Figure 1A ET-l levels obtained from the allografts on day 7 were significantly lower than those on day 5 (p ⁇ 0.02). Transcripts from the paired host hearts were low and comparable at all time points (day 3,5,7).
• Figure 1C ET-l transcripts levels in allografted hearts at day 5 were significantly higher (p ⁇ 0.05), than in day 5 control isografts, suggesting that the rejection process rather than the surgical procedure is responsible for the ET-l increase.
• ET-l mRNA expression in matched recipient spleens during the rejection process was significantly less than in the graft (p ⁇ 0.02) and comparable to host heart and isograft transcript levels indicating that the microenvironment of the rejecting graft is crucial for the induction of ET-l synthesis in mononuclear cells.
• ET-l transcript levels were significantly higher in cardiac allografts compared with paired host hearts at the time points examined (day 7, 28, 75) . These points were chosen to examine three stages of arteriosclerosis (Day 7 - when the adhesion of scattered mononuclear cells to the lumen is seen; day 28 - when mild degrees of concentric intimal thickening are first apparent, and day 75 when neointimal thickening and smooth muscle cell proliferation is a typical feature (Johnson et al., J. Heart Transplant , 8:349-59 (1989)). The upregulation of ET-l was seen early (day 7) and was sustained over time (p ⁇ 0.003) (Figure 1C) .
• ET-l transcript levels in the paired host hearts were low throughout the observation period and comparable to those seen in the acute rejection model (Figure IB) .
• the spleens of the allograft-rejecting animals expressed significantly less ET-l mRNA (p ⁇ 0.0007) than allografts at day 75, comparable to matched host heart and day 75 isografts ( Figure 1C) , again indicating that there is intragraft upregulation of ET-l by stimuli in the allograft.
• EXAMPLE 2 Immunohistochemical studies Expression of ET-l gene products as identified by a rabbit anti ET-l polyclonal antibody Host hearts
• Acute cardiac allograft rejection (WF into LEW) In contrast to normal hearts where only endothelial cells stained positive for ET-l, allografts undergoing acute rejection showed focal, positive staining for ET-l in mononuclear cells as well.
• ET-l immunopositive cells were seen as early as day 3, with a subsequent increase in the extent of labelling. At the later time point (day 7), the intensity of labelling per cell was reduced.
• Chronic cardiac allo ⁇ raft rejection (LEW into F344 )
• endothelial cells and many of the mononuclear cells showed a dense labelling with the anti-ET-1 antibody.
• the labelling of the endothelial cells was reduced in intensity and primarily restricted to endothelial cells adjacent to ET-l positive mononuclear cells and interstitial dendritic cells. Occasionally ET-l positive cells could be seen within the thickened intima.
• day 75 a marked increase in the intensity of discrete mononuclear cells labelling plus focal vascular intimal cell staining and medial smooth muscle cell staining was seen.
• ET-l mRNA The upregulation of ET-l mRNA is restricted to allografts and does not occur in the paired host spleens or hearts, nor in isografts, and indicates that the allogeneic stimulus plays a crucial role in the local upregulation of ET-l expression.
• IFN-gamma, TNF-alpha and IL-1 have been shown to induce ET-l production in cells maintained in culture (Lee et al., J. Biol . Chem . , 266:16188-192 (1991), Ohta et al., Biochem . Biophys . Res . Commun . , 169:578-84
• Macrophages and monocytes have been shown to express ET-l in vitro following stimulation (e.g., with phorbol esters) , but lymphocytes have not been shown to express ET-l (Ehrenreich et al . , 1990, supra) .
• Our findings suggest that lymphocytes may also play a role in the expression of ET-l in grafts. Taken together, these findings suggest that cytokines associated with the allogeneic milieu may contribute to increased ET-l production within the rejecting graft.
• Arteriosclerotic changes associated with chronic rejection develop in stages (Adams et al., 1992, supra) . Early in the course (day 7 to 14) mononuclear cells adhere to the vessel wall.
• ET-l immunostaining was observed in monocytes and macrophages in the neointima in addition to the strong staining of vascular endothelial cells.
• ET-l staining showed marked increases in both neointimal cell types and some staining in medial cells.
• Activated macrophages have been shown to play an important role in the development of graft arteriosclerosis (Russell et al., 1993, Proc . Natl . Acad. Sci. USA, supra , Russell et al., 1994, Transplantation, supra . It is likely that these macrophages might be supplying the necessary cytokines to stimulate endothelial cells to produce ET-l, or alternatively they could be contributing to the mononuclear cell production of ET-l.
• ET-l immunoreactivity within the arteriosclerotic lesions of transplant vessels suggests a common role for ET-l, as a vasoactive substance and as a potential mitogen, in the development of various forms of arteriosclerosis.
• EXAMPLE 3 Assays to detect rejection of a graft
• Methods to detect rejection or arteriosclerotic changes of a graft involve detection of expression of the ET-l gene either at the level of transcription, e.g, by PCR, Northern blot, differential mRNA display, or in situ hybridization, or at the level of translation/protein production, e.g., by FACS, Western blot, or in situ immunostaining.
• detection methods provide a means for early detection of events which lead to graft rejection, and thus facilitate early intervention to prevent or inhibit rejection of the transplanted organ (e.g., by immunosuppression) .
• ET-l transcript or protein levels in transplanted heart samples obtained by endomyocardial biopsy could serve as clinical markers of mononuclear cell infiltration.
• tissue which may be obtained using standard biopsy techniques known in the art.
• Specimens of the transplanted tissue can be obtained by biopsy using common medical practice and prepared for analysis using standard histological or nucleic acid methodology.
• tissues can be set into OCT or paraffin, or frozen prior to cutting into thin sections for histological evaluation, or the RNA and protein extracted and analyzed (e.g., by PCR, in situ hybridization, Western, Northern, or Southern blotting, etc.) .
• One method involves linking a detectable label to a molecule which will target ET-l producing cell or ET-l itself.
• a detectable label e.g., antibodies or fragmented antibodies (Fab, Fab 2 ) specific for ET-l can be linked to a label (e.g., a radiolabel, a fluorescent label, a spin label, etc.) and administered to the human or animal which has received a tissue graft (or to a sample retrieved from the patient) .
• a label e.g., a radiolabel, a fluorescent label, a spin label, etc.
• appropriate means are used to image or detect the label (e.g., epifluorescence, x-ray detection, NMR) .
• the grafted tissue will be imaged, and the presence of detectable label in the graft as compared to control tissues (e.g., normal host tissue) will be indicative of upregulation of ET-l in the graft, and initiation of rejection or graft-induced arteriosclerosis. Jn vitro, standard in situ hybridization techniques can be used, or ELISA or Western blotting assays to detect an increase in expression of ET-l in the grafted tissue.
• Fab fragments can have two morphologies: a single binding site (univalent Fab) or two linked binding sites (bivalent Fab 2 ) . These can be produced and labelled using methods well known in the art (e.g. , papain digestion) , and the term Fab is used to include either form.
• ET-l gene transcript in a graft indicates graft rejection.
• graft rejection in patients may be decreased or inhibited using gene therapy in which the antisense strand of the upregulated ET-l gene is introduced into the cells in which the gene is transcribed.
• the antisense strand (either RNA or DNA) may be directly introduced into the cells in a form that is capable of binding to the transcripts, or a vector containing sequence which, once within the target cells, is transcribed into the appropriate antisense mRNA, may be the therapeutic administered to the patient's cells.
• Antisense nucleic acid which hybridizes to the complementary coding strand of DNA can decrease or inhibit production of the polypeptide product encoded by the upregulated ET-l gene, by associating with the normally single-stranded mRNA transcript, and thereby interfere with translation.
• Other gene therapies may involve the use of recombinant mutants (e.g., to compete with endogenous peptide for binding on ET-l receptors, or interfere with ET-l effects at the level of the gene promoter or enhancers) .
• the isolated DNA may be introduced into target cells of the patient by standard vectors and/or gene delivery systems. Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, and adenoviruses, among others.
• a therapeutic composition which includes a pharmaceutically acceptable carrier and a therapeutically effective amount of a nucleic acid which is capable of inhibiting expression of ET-l gene in grafts and other inflammatory conditions, either directly or by encoding a transcript which inhibits expression of the gene.
• the therapeutic composition may also include a gene delivery system as described above.
• compositions are provided which are capable of inhibiting or reducing the effects of ET-l (e.g., the ET-l receptor antagonist SB 209670; Douglas et al . , 1994, Circ . Res . 75:190) on allografts, xenografts, or in other inflammatory conditions, such as autoimmune disorders, myocarditis, endocarditis, Lyme disease, and other chronic cell-mediated inflammatory conditions.
• ET-l e.g., the ET-l receptor antagonist SB 209670; Douglas et al . , 1994, Circ . Res . 75:190
• the compounds may be peptide or non-peptide, and have actions on the expression of ET-l (e.g., transcription or translation of the gene) ; act on the ET-l receptors ET A and ET B at any level (e.g., binding or antagonism of the binding site, interference with the G-protein/kinase cascade which is activated following receptor binding) ; or act as posttranslational inhibitors on ET-l (e.g., Endothelin converting enzyme inhibitors) .
• ET-l e.g., transcription or translation of the gene
• ET A and ET B at any level (e.g., binding or antagonism of the binding site, interference with the G-protein/kinase cascade which is activated following receptor binding)
• posttranslational inhibitors on ET-l e.g., Endothelin converting enzyme inhibitors
• ET-l receptors may also be upregulated in inflamed or allografted tissues (e.g., on migrating or proliferating smooth muscle cells and endothelial cells) , so drugs and therapies directed to ET-l receptors are likely to be particularly useful in reducing the effects of ET-l in these conditions.
• Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal: e.g., physiological saline.
• a therapeutically effective amount is an amount of the DNA of the invention which is capable of producing a medically desirable result in a treated animal, e.g., downregulation of the differentially expressed allograft gene.
• compositions of the invention can be formulated for pharmaceutical, veterinary, and organ culture use (e.g., treatment of tissue or organ prior to transplantation) , optionally together with an acceptable diluent, carrier or excipient and/or in unit dosage form.
• organ culture use e.g., treatment of tissue or organ prior to transplantation
• an acceptable diluent, carrier or excipient and/or in unit dosage form e.g., in unit dosage form.
• conventional pharmaceutical, veterinary, or culture practice may be employed to provide suitable formulations or compositions.
• the formulations of this invention can be administered by parenteral administration, for example, intravenous, subcutaneous, intramuscular, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, topical, intranasal, aerosol, scarification, and also oral, buccal, rectal, vaginal, or topical administration.
• parenteral administration for example, intravenous, subcutaneous, intramuscular, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, topical, intranasal, aerosol, scarification, and also oral, buccal, rectal, vaginal, or topical administration.
• formulations of this invention may also be administered by the use of surgical implants which release the compounds of the invention. These devices could be readily implanted into the graft prior to transplantation, and could be mechanical or passive. Mechanical devices, such as pumps, are well known in the art, as are passive devices (e.g., consisting of a polymer matrix which contains therapeutic formulations; these polymers may slowly dissolve or degrade to release the compound, or may be porous and allow release via pores) .
• Parenteral formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
• Formulations for parenteral administration may, for example, contain as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors.
• polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes
• biocompatible, biodegradable lactide polymer or polyoxyethylene-polyoxypropylene copolymers
• Other potentially useful parenteral delivery systems for the factors include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
• Formulations for inhalation may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
• Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration.
• the concentration of the compound in the formulations of the invention will vary depending upon a number of factors, including the dosage to be administered, and the route of administration.
• the non-nucleotide therapeutics of the invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration.
• General dose ranges are from about 0.01 mg/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day.
• the preferred dosage to be administered is likely to depend upon the extent of progression of rejection, the overall health of the patient, and the route of administration.
• Peptides may be administered to the patient intravenously in a pharmaceutically acceptable carrier such as physiological saline.
• a pharmaceutically acceptable carrier such as physiological saline.
• parenteral administration such as intravenous, subcutaneous, intramuscular, intraperitoneal, and inhalation.
• nucleic acid therapies of the invention will vary, but a preferred dosage for intravenous administration of nucleic acid therapies is from approximately 10 6 to 10 22 copies of the nucleic acid molecule.
• EXAMPLE 5 Screening Assay for ET-l Inhibitors/Antagonists Screening for ET-l inhibitors and ET-l receptor antagonists can also be accomplished in vivo using the rat heterotopic cardiac transplant model described herein and in Sayegh et al., 1991, supra , and Adams et al., 1992, supra .
• the organ to be grafted can be perfused or soaked in a solution containing a candidate compound prior to transplantation. The organ can then transplanted and monitored for indications of rejection.
• Transplant rejection can be monitored using conventional methods, e.g., sacrifice of the animal followed by gross examination of the tissue and histological studies, as well as the diagnostic assays of the invention, e.g., evaluating a tissue biopsy for the differential expression of ET-l in host and graft tissue.
• a decrease in gene expression over non-treated graft controls or a reduction in the physical characteristics of transplant rejection would indicate that the candidate compound inhibits graft rejection and could be useful in inhibiting symptoms of other chronic inflammatory conditions (e.g., autoimmune disorders, carditis, etc.).
• Test compounds may be any sort of drug, chemical, nucleic acid, polypeptide.
• a potential source of non- nucleic acid test compounds are those related to known immunosuppressive compounds (e.g., cyclosporin) and those related to known ET-l inhibitors, antagonist, and blockers (e.g., SB 209670), although any compound may be chosen for use in this screening assay.
• immunosuppressive compounds e.g., cyclosporin
• ET-l inhibitors, antagonist, and blockers e.g., SB 209670
• Nucleic acid test compounds are likely to be composed of nucleotides which are complementary (antisense DNA) to some portion of the ET-l expression machinery (e.g., the coding region of the ET-l gene, signal sequences, promoters, etc.) or encode a polypeptide which may mimic ET-l polypeptide but is biologically inactive (e.g., to compete for receptor binding with endogenous ET-l) ; the encoded polypeptide may have a tertiary structure which physically blocks the receptor or binds the ET-l polypeptide or gene.
• Potential protein test compounds may have these features as well, although any nucleic acid or protein compound or molecule may be used in the screening assay.
• the methods of the invention should enhance long term graft survival due to early diagnosis and effective counter-measures to acute and chronic rejection by inhibition of ET-l activity and expression.
• Our findings show that rodent cardiac allograft rejection models may be used to elucidate the precise in-vivo role of ET-l as a vasoactive and/or mitogenic factor after cardiac transplantation.
• the availability of specific ET- receptor antagonists will provide potential therapeutic strategies to prevent or ameliorate graft arteriosclerosis.
• ET-l gene transcript or polypeptide product levels may serve as clinical or diagnostic indicators of mononuclear cell infiltration, chronic inflammation, transplant rejection, and arteriosclerosis.
• ET-l may be used to determine that mononuclear cells are activated, given that the results described herein show that ET-l transcripts are expressed in mononuclear cells in the cardiac allograft, but not in host tissue.
• All or part of the human or any mammalian ET-l DNA sequence which has at least 50% sequence identity, preferably 70%, more preferably 90% sequence identity with the human ET-l sequence can be used as a hybridization probe to identify ET-l upregulation for the purpose of diagnosing transplant rejection.
• Portions of the DNA can also be used as PCR primers to amplify ET-l sequences to identify expression of these genes in grafts for the purpose of diagnosing rejection, using e.g., differential display technology (Russell et al . , 1993, supra) .
• ET-l DNA can be cloned into an expression vector and used to produce polypeptides of ET- 1 for the purpose of immunizing animals to generate polyclonal or monoclonal antibodies. Such antibodies can then be used for therapeutic applications as described above or for diagnostic applications such as identification of ET-l polypeptides in grafts indicating ongoing transplant rejection. Fusion proteins of ET-l containing components known to block specific inflammatory factors may also serve as a way of modulating the inflammatory response. DNA containing a sequence that encodes part or all of the amino acid sequence of ET-l can be subcloned into an expression vector, using a variety of methods known in the art.
• a recombinant polypeptide can be expressed as a fusion protein with maltose binding protein produced in E. coli .
• maltose binding protein fusion and purification system New England Biolabs
• the cloned human cDNA sequence can be inserted downstream and in frame of the gene encoding maltose binding protein (malE) , and the alE fusion protein can then be overexpressed.
• PCR can be used to introduce restriction sites compatible with the vector at the 5' and 3' end of the cDNA fragment to facilitate insertion of the cDNA fragment into the vector.
• affinity chromatography the fusion protein can be purified by virtue of the ability of the maltose binding protein portion of the fusion protein to bind to a ylose immobilized on a column.
• the pMalE plasmid contains a factor Xa cleavage site upstream of the site into which the cDNA is inserted into the vector.
• the fusion protein purified as described above can then be cleaved with factor Xa to separate the maltose binding protein from recombinant human cDNA gene product.
• the cleavage products can be subjected to further chromatography to purify recombinant polypeptide from the maltose binding protein.
• the purified recombinant gene product can then be used to raise polyclonal or monoclonal antibodies against the ET-l using well-known methods (see Coligan et al., eds., Current Protocols in Immunology, 1992, Greene Publishing Associates and Wiley-Interscience) .
• a mouse can be immunized with the recombinant protein, and antibody-secreting B cells isolated and immortalized with a non-secretory myeloma cell fusion partner. Hybridomas are then screened for production of ET-1-specific antibody and cloned to obtain a homogenous cell population which produces a monoclonal antibody.
• ET-l expression in grafts will enhance the longevity of transplant survival and survival of the transplant recipient. Regulation of ET-l expression in inflammatory conditions such as autoimmune disorders, cardiac inflammation, Lyme disease, or other chronic cell-mediated inflammatory process will lead to better management of these conditions, resulting in less tissue scarring and degradation, and improvement in quality and length of life.
• inflammatory conditions such as autoimmune disorders, cardiac inflammation, Lyme disease, or other chronic cell-mediated inflammatory process
• TTACTTCCCA CAAAGGCAAC AGACCGTGAG AATAGATGCC AATGTGCTAG CCAAAAAGAC 600

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