METHODS FOR IDENTIFYING GRAFT REJECTION SUPPRESSING COMPOUNDS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S. Provisional Patent
Application Serial No. 60/629,538 (filed November 18, 2004), the disclosure of which is incorporated herein by reference in its entirety and for all puiposes.
FIELD OF THE INVENTION
[0001] The present invention generally relates to methods of identifying compounds for treating or preventing graft rejection. More particularly, the invention pertains to methods of screening test compounds for PKCβ inhibitors and to methods of using such inhibitors to suppress or prevent graft rejection in human or non-human subjects.
BACKGROUND OF THE INVENTION
[0002] The immune system is well equipped to rapidly identify foreign, diseased or inflamed tissue and rapidly destroys it. This has always been a major barrier to tissue, organ and cell transplantation as well as gene therapy. Graft rejection occurs when the T lymphocytes from the recipient recognize and respond to donor histocompatibility antigens which are present on the surface of donor-derived graft cells and tissues. Graft destruction which occurs within the first few weeks after transplantation is called "acute rejection". Usually, the use of immunosuppressive drugs temporarily prevents this result. Unfortunately, the grafts may eventually be destroyed weeks or months later. This failure of permanent graft acceptance is referred to as "chronic rejection."
[0003] Major problems are generally associated with chronic immunosuppression, encapsulation or immunoisolation. The unwanted side effects of chronic immunosuppression include increased susceptibility to opportunistic infection and tumor formation. The desire for long-term acceptance of grafted tissue in the absence of continuous immunosuppression is a long-standing goal in human medicine. Current immunosuppressive drugs are often ineffective in blocking chronic allograft rejection.
[0004] There is a need for new compounds and methods for preventing and treating graft rejection associated with tissue and organ transplant. The instant invention addresses this and other needs.
SUMMARY OF THE INVENTION
[0005] In one aspect, the present invention provides methods for identifying agents that prevent or suppress allogeneic graft rejection. The methods entail assaying test compounds for ability to modulate a biological activity or expression of PKCβ. Some of these methods further comprise testing the identified agents for ability to suppress allogeneic graft rejection in a test subject. In some methods, the test subject has received an allogeneic graft with minor histocompatibility antigen mismatches. In some of the screening methods, the identified agents inhibit the biological activity of PKCβ. The biological activity of PKCβ can be its kinase activity. In some of the methods, the identified agents inhibit expression of PKCβ.
[0006] In a related aspect, the invention provides methods for identifying agents that suppress or prevent allogeneic graft rejection. These methods involve (a) assaying a biological activity or expression of PKCβ, or a fragment thereof, in the presence of test compounds to identify one or more modulating agents that modulate the biological activity or expression of the PKCβ molecule; and (b) testing the identified modulating agents for ability to suppress graft rejection. In some of these methods, (b) comprises examining the identified modulating agent for ability to suppress graft rejection in a test subject. In some of the methods, the test subject has received an allogeneic graft with minor histocompatibility antigen mismatches. The PKCβ molecule employed can be a human PCKβ or a murine PKCβ. In some methods, the identified modulating agents inhibit the biological activity of PKCβ. The biological activity of PKCβ modulated in these methods can be its kinase activity. In some methods, the identified modulating agents inhibit expression of PKCβ. [0007] In another aspect, the invention provides methods for suppressing or preventing graft rejection in a subject. The methods involve administering to the subject a pharmaceutical composition comprising an effective amount of a PKCβ-antagonist. Some of these methods are directed to treating human subjects. In some of these methods, the PKCβ antagonist is identified by screening test compounds for ability to inhibit the kinase activity of human PKCβ. In some other methods, the PKCβ antagonist is a known PKCβ inhibitor, e.g., LY333531 or LY379196. Some of the methods are directed to treating subjects with a graft containing minor histocompatibility antigen mismatches. In some of these methods, the subjects have a skin graft.
[0008] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figures 1A-1C show rejection of skin allografts harboring minor histocompatibility antigen mismatches in PKCβ-deficient and control mice. '
[0010] (A) Graft survival. The percentage of unrejected skin grafts from male
C57BL/6 donor mice onto female PKCβ-deficient recipients (filled squares) or C57BL/6 controls (open squares) is shown at various times post grafting. All recipients were 12 weeks old when grafted. Group sizes were n=7 (KO) or n=9 (controls). MST, median survival time +/- standard deviation. The observed significance level p was determined via Student's t-test.
[0011] (B) Histological graft features on day 6 post grafting. Shown are H&E stained longitudinal sections of male allograft tissue for a C57BL/6 (left) or PKCβ-defϊcient (right) female recipient. Thickening of the graft epidermis (GE) and mononuclear infiltration of the area at the junction of graft (GD) and recipient (RD) dermal tissue are readily observable. Infiltration (large arrows) is comparable in grafts on C57BL/6 or PKCβ-deficient recipients. Shown is one representative out of three independently investigated recipients per genotype.
[0012] (C) Histological features on day 27 post grafting. Shown are H&E stained longitudinal sections of male allograft tissue (upper panels) or normal recipient skin (lower panels) adjacent to the graft for a C57BL/6 (left) or PKCβ-defϊcient (right) female recipient. Thickening of the epidermis (GE) and mononuclear infiltration of the dermis (GD) are readily observable in the grafts, but not in normal tissue (RE, RD). Infiltration is much more pronounced in grafts on C57BL/6 recipients. Shown is one representative out of three independently investigated recipients per genotype. GE, graft epidermis; GD, graft dermis; RE, recipient epidermis; RD, recipient dermis. Data are shown at 1Ox magnification.
[0013] Figures 2A-2B show serum immunoglobulin isotype levels in PKCβ-deficient and control mice.
[0014] (A) Baseline serum immunoglobulin isotype levels in ng/μl in 8 week old male C57BL/6 mice, a large cohort of randomly selected G3 males on a C57BL/6 background from a forward genetic ENU-mutagenesis screen, and in PKCβ-defϊcient males.
[0015] (B) Serum immunoglobulin isotype levels 74 days post grafting in PKCβ- deficient or control female recipients. P-values as determined via heteroscedastic t-test are shown for selected comparisons.
DESCRIPTION OF THE PREFEREED EMBODIMENTS
I. Overview
[0016] The invention is predicated in part on the discovery by the present inventors of a significant delay of graft rejection in mice lacking protein kinase C, beta isoform (PKCβ). Specifically, compared to control mice, PKCβ-deficient mice have delayed rejection of minor histocompatibility antigen disparate allografts. In addition, the present inventors also found that the delayed rejection is accompanied by a drastic reduction in IgM and THI -dependent immunoglobulin isotypes.
[0017] In accordance with these discoveries, the present invention provides methods for screening for novel compounds that prevent or suppress graft rejection in transplantation. Test compounds are first examined for their ability to modulate a biological activity of a PKCβ molecule, e.g., its kinase activity. The agents thus identified are then further tested for ability to suppress graft rejection in a test subject. Various PKCβ molecules can be employed in the screening assays. For example, PKCβ from human, rat or mouse can be used to screen the modulators. In preferred embodiments, a human PKCβ is used.
[0018] The methods of the present invention also find therapeutic applications.
Typically, the approach entails administering to a subject in need of treatment a known PKCβ antagonist or one that can be identified in accordance with the present invention. Pharmacological inhibition of PKCβ provides a novel approach for preventing or suppressing various types of graft rejections human and non-human subjects. In particular, subjects receiving allogeneic grafts that contain minor histocompatibility mismatches are suitable for treatment with PKCβ-mhibiting compounds. In addition, because the compounds likely suppress graft rejections by inhibiting T cell activation and T cell mediated immune responses, they are also useful for the treatment of other inflammatory disorders mediated by T cells. Examples include but are not limited to Rheumatoid Arthritis, Diabetes, SLE, Neurodegenerative Disorders, Psoriasis, Dermatitis, Allergy, Asthma, COPD and Anaphylaxis.
[0019] The following sections provide guidance for making and using the compositions of the invention, and for carrying out the methods of the invention.
I. Definitions
[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al. , DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991). In addition, the following definitions are provided to assist the reader in the practice of the invention.
[0021] The term "agent" or "test agent" includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms "agent", "substance", and "compound" can be used interchangeably.
[0022] The term "analog" is used herein to refer to a molecule that structurally resembles a reference molecule but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs, to identify variants of known compounds having improved traits (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.
[0023] As used herein, "contacting" has its normal meaning and refers to combining two or more agents (e.g., polypeptides or small molecule compounds) or combining agents and cells (e.g., a polypeptide and a cell). Contacting can occur in vitro, e.g., combining two or more agents or combining a test agent and a cell or a cell lysate in a test tube or other container. Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by coexpression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate.
[0024] The teπn "homologous" when referring to proteins and/or protein sequences indicates that they are derived, naturally or artificially, from a common ancestral protein or protein sequence. Similarly, nucleic acids and/or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of similarity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity is routinely used to establish homology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establish homology. Methods for determining sequence similarity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein or well known and readily available in the art.
[0025] A "host cell," as used herein, refers to a prokaryotic or eukaryotic cell that contains heterologous DNA that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and/or the like.
[0026] The terms "identical" or "sequence identity" in the context of two nucleic acid sequences or amino acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. A "comparison window", as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482; by the alignment algorithm of Needleman and Wunsch (1970) J. MoI. Biol. 48:443; by the search for similarity method of Pearson and Lipman (1988) Proc. Nat. Acad. Sci U.S.A. 85:2444; by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program by Intelligentics, Mountain View, CA; and GAP, BESTFIT, BLAST, FASTA, or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.). The CLUSTAL program is well described by Higgins and Sharp (1988) Gene 73:237-244; Higgins and Sharp (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-10890; Huang et al (1992) Computer Applications in the Biosciences 8:155-165; and Pearson et al. (1994) Methods in Molecular Biology 24:307- 331. Alignment is also often performed by inspection and manual alignment. In one class of embodiments, the polypeptides herein are at least 70%, generally at least 75%, optionally at least 80%, 85%, 90%, 95% or 99% or more identical to a reference polypeptide, as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to a reference nucleic acid, as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters.
[0027] The terms "substantially identical" nucleic acid or amino acid sequences means that a nucleic acid or amino acid sequence comprises a sequence that has at least 90% sequence identity or more, preferably at least 95%, more preferably at least 98% and most preferably at least 99%, compared to a reference sequence using the programs described above (preferably BLAST) using standard parameters. For example, the BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions.
[0028] The term "modulate" with respect to biological activities of a PKCβ molecule refers to a change in the cellular level or other biological activities (e.g., its kinase activity) of PKCβ. Modulation of PKCβ activities can be up-regulation (i.e., activation or stimulation) or down-regulation (i.e. inhibition or suppression). For example, modulation may cause a change in cellular level of PKCβ, enzymatic modification (e.g., phosphorylation) of PKCβ, binding characteristics (e.g., binding to a substrate or ATP), or any other biological, functional, or immunological properties of PKCβ proteins. The change in activity can arise from, for example, an increase or decrease in expression of the PKCβ gene, the stability of mRNA that encodes the PKCβ protein, translation efficiency, or from a change in other bioactivities of the PKCβ enzymes (e.g., its kinase activity). The mode of action of a PKCβ modulator can be direct, e.g., through binding to the PKCβ protein or to a gene encoding the PKCβ protein. The change can also be indirect, e.g., through binding to and/or modifying (e.g., enzymatically) another molecule which otherwise modulates PKCβ.
[0029] The term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a PKCβ promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance. A polylinker provides a convenient location for inserting coding sequences so the genes are operably linked to the PKCβ promoter. Polylinkers are polynucleotide sequences that comprise a series of three or more closely spaced restriction endonuclease recognition sequences.
[0030] The term "subject" includes mammals, especially humans. It also encompasses other non-human animals such as cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys. A test subject is typically a non-human animal that expresses an endogenous PKCβ gene, e.g., a mouse.
[0031] A transcription regulatory element or sequence include, but is not limited to, a promoter sequence (e.g., the TATA box), an enhancer element, a signal sequence, or an array of transcription factor binding sites. It controls or regulates transcription of a gene operably linked to it.
[0032] A "variant" of a molecule such as a PKCβ is meant to refer to a molecule substantially similar in structure and biological activity to either the entire molecule, or to a fragment thereof. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the sequence of amino acid residues is not identical. III. Using PKCβ to Screen for Graft Rejection Suppressing Compounds: General Scheme [0033] PKCβ is an isoform of the PKC family of the serine/threonine protein kinases.
Members of the PKC kinase family are structurally and enzymatically distinct isoforms. They are divided into the classical α, β (βl and βll) and γ subfamilies, the novel δ, ε, η and θ subfamilies, and the atypical ζ and λ/i subfamilies. Most of the known PKCs are expressed in T lineage cells where they function as sensors of lipid second messengers and/or calcium ion concentration changes caused by engagement of the TCR and its coreceptors.
[0034] The present invention provides methods of using PKCβ to screen for compounds that suppress graft rejection in human or non-human subjects, especially rejection of grafts with minor histocompatibility antigen mismatches. As a consequence of the connection between PKCβ and graft rejection, inhibition of biological activities or cellular level of PKC βs can lead to suppression of graft rejection. Typically, test compounds are first screened for ability to modulate a biological activity of a PKCβ. Preferably, the biological activity of PKCβ to be monitored in the screening assays is its kinase activity. The biological activity of PKCB to be monitored can also be its expression or its cellular level, as well as a specific binding of PKCβ to a test compound. After test compounds that modulate a biological activity of PKCβ have been identified, they are typically further examined for ability to suppress or prevent graft rejection in a test subject (e.g., a mouse). This step serves to confirm that by modulating the biological activity of PKCβ, compounds identified in the first step are indeed useful to treat or prevent graft rejection. As a control, the compounds can also be examined for ability to modulate other members of the PKC kinase family (e.g., PKCα, PKCδ, or PKCγ). In some therapeutic applications of the present invention, compounds that show selective modulation (e.g., inhibition) of PKCβ over the other PKC isoforms are employed. In some methods, PKCβ- inhibiting compounds which can also inhibit one or more of the other PKC isoforms are used because such compounds could have higher therapeutic efficacy in treating certain diseases and conditions.
[0035] PKCβ from various species can be employed in screening the PKCB modulators of the present invention. These include PKCβ encoded by polynucleotides with accession numbers BC036472, NM_212535 and NM_002738 (human); NM_012713 (rat); and NM_008855 and BC038148 (mouse). Examples of PKCβ from other species are encoded by polynucleotides with accession numbers NM_200978, BC055154, NM_174587, XM_414868, and AY393849. Any of these PKCβ polynucleotide sequences and their corresponding polypeptides can be used to screen test agents for modulators in the present invention. Preferably, a human PKCβ molecule is used. Polynucleotide and polypeptide sequences encoding the other PKC isoforms from various species are also know in the art. Molecular structures and biochemical functions of these PKC molecules have all been well characterized in the art, e.g., Coussens et al., Science 233: 859-866, 1986; Kubo et al, FEBS Lett. 223: 138-142, 1987; Greenham et al., Hum. Genet. 103: 483-487, 1998; Feng et al., J. Biol. Chem. 275: 17024-17034, 2000; Ventura et al., Crit Rev Eukaryot Gene Expr. 11 : 243-67, 2001; and Spitaler et al., Nat Immunol. 5:785-90, 2004.
[0036] In addition to an intact PKCβ molecule or a polynucleotide encoding the intact PKCβ molecule, a PKCβ fragment, analog, or a functional derivative can also be used. The PKCβ fragments that can be employed in these assays usually retain one or more of the biological activities of the PKCβ molecule (typically, its kinase activity). PKCβs from the various species have already been sequenced and well characterized. Therefore, their fragments, analogs, derivatives, or fusion proteins can be easily obtained using methods well known in the art. For example, a functional derivative of a PKCβ can be prepared from a naturally occurring or recombinantly expressed protein by proteolytic cleavage followed by conventional purification procedures known to those skilled in the art. Alternatively, the functional derivative can be produced by recombinant DNA technology by expressing only fragments of a PKCβ that retain its kinase activity.
IV. Test Compounds
[0037] Test agents that can be screened with methods of the present invention include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines, oligocarbamates, polypeptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Some test agents are synthetic molecules, and others natural molecules.
[0038] Test agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. Combinatorial libraries can be produced for many types of compound that can be synthesized in a step-by-step fashion. Large combinatorial libraries of compounds can be constructed by the encoded synthetic libraries (ESL) method described in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642. Peptide libraries can also be generated by phage display methods (see, e.g., Devlin, WO 91/18980). Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field. Known pharmacological agents can be subject to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
[0039] Combinatorial libraries of peptides or other compounds can be fully randomized, with no sequence preferences or constants at any position. Alternatively, the library can be biased, i.e., some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, in some cases, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, or to purines.
[0040] The test agents can be naturally occurring proteins or their fragments. Such test agents can be obtained from a natural source, e.g., a cell or tissue lysate. Libraries of polypeptide agents can also be prepared, e.g., from a cDNA library commercially available or generated with routine methods. The test agents can also be peptides, e.g., peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides can be digests of naturally occurring proteins, random peptides, or "biased" random peptides. In some methods, the test agents are polypeptides or proteins.
[0041] The test agents can also be nucleic acids. Nucleic acid test agents can be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be similarly used as described above for proteins.
[0042] In some preferred methods, the test agents are small molecules (e.g., molecules with a molecular weight of not more than about 500 or 1,000). Preferably, high throughput assays are adapted and used to screen for such small molecules. In some methods, combinatorial libraries of small molecule test agents as described above can be readily employed to screen for small molecule modulators of PKCβs. A number of assays are available for such screening, e.g., as described in Schultz (1998) Bioorg Med Chem Lett 8:2409-2414; Weller (1997) MoI Divers. 3:61-70; Fernandes (1998) Curr Opin Chem Biol 2:597-603; and Sittampalam (1997) Curr Opin Chem Biol 1 :384-91.
[0043] Libraries of test agents to be screened with the claimed methods can also be generated based on structural studies of a PKCβ polypeptide, their fragments or analogs. Such structural studies allow the identification of test agents that are more likely to bind to a PKCβ polypeptide. The three-dimensional structure of a PKCβ polypeptide can be studied in a number of ways, e.g., crystal structure and molecular modeling. Methods of studying protein structures using x-ray crystallography are well known in the literature. See Physical Bio-chemistry, Van Holde, K. E. (Prentice-Hall, New Jersey 1971), pp. 221-239, and Physical Chemistry with Applications to the Life Sciences, D. Eisenberg & D. C. Crothers (Benjamin Cummings, Menlo Park 1979). Computer modeling of a PKCβ polypeptide structure provides another means for designing test agents for screening PKCβ modulators. Methods of molecular modeling have been described in the literature, e.g., U.S. Patent No. 5,612,894 entitled "System and method for molecular modeling utilizing a sensitivity factor", and U.S. Patent No. 5,583,973 entitled "Molecular modeling method and system". In addition, protein structures can also be determined by neutron diffraction and nuclear magnetic resonance (NMR). See, e.g., Physical Chemistry, 4th Ed. Moore, W. J. (Prentice- Hall, New Jersey 1972), and NMR of Proteins and Nucleic Acids, K. Wuthrich (Wiley- Interscience, New York 1986).
[0044] Modulators of the present invention also include antibodies that specifically bind to a PKCβ polypeptide. Such antibodies can be monoclonal or polyclonal. Such antibodies can be generated using methods well known in the art. For example, the production of non-human monoclonal antibodies, e.g., murine or rat, can be accomplished by, for example, immunizing the animal with a PKCβ polypeptide or its fragment (See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York). Such an immunogen can be obtained from a natural source, by peptides synthesis or by recombinant expression. [0045] Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989) and WO 90/07861. Human antibodies can be obtained using phage-display methods. See, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047. In these methods, libraries of phage are produced in which members display different antibodies on their outer surfaces. Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies with a desired specificity are selected by affinity enrichment to a PKCβ polypeptide of the present invention.
[0046] Human antibodies against a PKCβ polypeptide can also be produced from non-human transgenic mammals having transgenes encoding at least a segment of the human immunoglobulin locus and an inactivated endogenous immunoglobulin locus. See, e.g., Lonberg et al., WO93/12227 (1993); Kucherlapati, WO 91/10741 (1991). Human antibodies can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. Such antibodies are particularly likely to share the useful functional properties of the mouse antibodies. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using a PKCβ polypeptide or its fragment.
V. Screen Test Compounds for PKCβ Modulators
[0047] To identify modulators of T cell activation and function, test compounds are first screened for ability to modulate a biological activity of PKCβ as described herein. In some preferred embodiments, test compounds are examined for ability to modulate (e.g., inhibit) the kinase activity of PKCβ. Many assays are known in the art which can be used to monitor the kinase activity of PKCβ in the presence of test compounds. These include both cell based assays and non-cell based assays. For example, PKC activity can be measured with commercially available assays kits, e.g., the PKC Enzyme Assay Kits from PanVera/Invitrogen (Carlsbad, California) or Amersham (Bucks, UK). An example of cell based assay is described in Koya et al. (J. Clin. Invest. 100: 115-126, 1997) which examined the inhibitory effect of a compound (LY333531) on PKCβ in mesangial cells using a PKC- specific peptide substrate. Other assays for monitoring PKCβ activities are also described in the ait, e.g., Khayat et al., Am J Physiol Cell Physiol 275: C1487-C1497, 1998; Nitti et al. Biochem Biophys Res Commun.294: 547-52, 2002; and Guo et al., Eur J Immunol. 33:928- 38, 2003.
[0048] In some embodiments, test compounds can be first screened for their ability to bind to a PKCβ polypeptide. Compounds thus identified can be further subject to assay for ability to modulate (e.g., to inhibit) PKCβ kinase activity as described above. Binding of test agents to a PKCβ polypeptide can be assayed by a number of methods including, e.g., labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.), and the like. See, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168; and also Bevan et al., Trends in Biotechnology 13: 115-122, 1995; Ecker et al., Bio/Technology 13:351-360, 1995; and Hodgson, Bio/Technology 10:973-980, 1992. The test agent can be identified by detecting a direct binding to the PKCβ polypeptide, e.g., co- immunoprecipitation with the PKCβ polypeptide by an antibody directed to the PKCβ polypeptide. The test agent can also be identified by detecting a signal that indicates that the agent binds to the PKCβ polypeptide, e.g., fluorescence quenching.
[0049] In some other methods, test agents are assayed for activity to modulate cellular level of PKCβ, e.g., transcription or translation. The test agent can also be assayed for activities in modulating expression level or stability of the PKCβ polypeptide, e.g., post- translational modification or proteolysis. Various biochemical and molecular biology techniques well known in the art can be employed to practice the present invention. Such techniques are described in, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N. Y., Second (1989) and Third (2000) Editions; and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1987-1999). In some embodiments, endogenous levels of a PKCβ can be directly monitored in cells normally expressing this enzyme (e.g., T cells). In some embodiments, expression or cellular level of a PKCβ can be examined in an expression system using cloned cDNA or genomic sequence encoding the PKCβ.
[0050] Alternatively, modulation of expression of a PKCβ gene can be examined in a cell-based system by transient or stable transfection of an expression vector into cultured cell lines. Assay vectors bearing transcription regulatory sequence (e.g., promoter) of a PKCβ gene operably linked to reporter genes can be transfected into any mammalian host cell line for assays of promoter activity. Constructs containing a PKCβ gene (or a transcription regulatory element of a PKCβ gene) operably linked to a reporter gene can be prepared using only routinely practiced techniques and methods of molecular biology (see, e.g., Sambrook et al. and Ausubel et al., supra). General methods of cell culture, transfection, and reporter gene assay have been described in the art, e.g., Ausubel, supra; and Transfection Guide, Promega Corporation, Madison, WI (1998). Any readily transferable mammalian cell line may be used to assay PKCβ promoter function, e.g., CHO, HCTl 16, HEK 293, MCF-7, and HepG2 are all suitable cell lines.
[0051] When inserted into the appropriate host cell, the transcription regulatory elements in the expression vector induces transcription of the reporter gene by host RNA polymerases. Reporter genes typically encode polypeptides with an easily assayed enzymatic activity that is naturally absent from the host cell. Typical reporter polypeptides for eukaryotic promoters include, e.g., chloramphenicol acetyltransferase (CAT), firefly or Renilla luciferase, beta-galactosidase, beta-glucuronidase, alkaline phosphatase, and green fluorescent protein (GFP).
Vl. Screen for Compounds that Suppress Graft Rejection
[0052] To identify novel compounds that suppress graft rejection, a PKCβ modulator described above can be further examined to confirm its ability to suppress or prevent graft rejection. This typically involves testing the compounds for ability to suppress or delay graft rejection in a test subject that has received an allogeneic graft. Using non-human animals to study transplantation and graft rejection is a technique that has long been practiced in the art. General guidance on various techniques of employing small animals (e.g., rats, mice, and rabbits) in transplantation study is provided in Experimental Transplantation Models in Small Animals, Green et al. (Eds.), published by T&F STM (March 1995); Pathology and Immunology of Transplantation and Rejection, Thiru et al. (Eds.), published by Blackwell Publishers (February 2001); and Handbook of Animal Models in Transplantation Research, Cramer et al. (Eds.), published by CRC Press (December 1993). The present disclosure also provides specific procedures of using mice to study graft rejection, as detailed in the Examples below.
[0053] Ability to suppress graft rejection by the PKCβ-modulating compounds identified as described above can be examined using any of the methods known in the art or described herein. In some preferred embodiments, mice transplanted with skin grafts with minor histocompatibility antigen mismatches are used. As exemplified in the Examples below, a PKCβ-inhibiting compound can be administered to C57BL/6 recipient mice receiving a skin graft from C57BL/6 donor mice. In some methods, the donor mice can have a minor histocompatibility antigen mismatch, e.g., by having the male-specific H-Y alloantigen.
[0054] Administration of the compound to the test subject can take place prior to, simultaneously with, or subsequent to the transplantation. Preferably, the recipient mice are administered with the compound from the pregrafting stage to the end of the screening process. Detailed guidance of the specific formulations, dosages, and mode of administration for administering PKCβ-inhibiting compounds are provided in the sections below. In the screening, graft rejection in the treated mice is typically compared to control mice that have not been receiving the PKCβ-inhibiting compound. If administration of a PKCβ-inhibiting compound results in a significant delay or inhibition of graft rejection, it is identified as a novel agent that suppresses or prevents graft rejection.
[0055] Other than the animal model described in the Examples herein, various other animal systems for studying graft rejections are also known in the art. For example, Ehst et al. (Am J Transplant. 3:1355-62, 2003) described an experimental animal system to study cooperation between CD4 and CD8 T cells in rejection of skin grafts with a single minor histocompatibility antigen mismatch. Other animal models for studying various other allogeneic or xenogeneic graft rejections have also been described in the art, e.g., Nicholls et al., Invest Ophthalmol Vis Sci. 32:2729-34, 1991 (rat model for corneal graft); Pierson et al., J Exp Med. 170:991-6, 1989 (mouse model for xenogeneic skin graft); and Shenoy et al., Clin Exp Immunol. 112:188-95, 1998 (mouse model for bone marrow transplantation). Any of these animal systems can also be used to examine potential graft rejection- suppressing activities of PKCβ-inhibiting compounds. VH. Therapeutic Applications
[0056] The present invention finds therapeutic applications in treating and preventing graft rejection in subjects that received tissue or organ transplants. By specific and selective inhibition of PKCβ kinase, compounds of the present invention can be used to suppress immune responses elicited by allogeneic transplantation. The compounds can be identified using the screening methods described above. Other than the PKCβ antagonizing compounds that can be identified in accordance with the present invention, various other PKCβ-inhibiting agents that are known in the art can also be used in the therapeutic embodiments of the invention (See, e.g., Gordge et al., Cell Signal 6: 871-882, 1994). An example of such agents the selective PKCβ inhibitor (£)~13-[(dimethylamino)methyl]- 10,11,14,15-tetrahydro-4,9 : 16,21 -dimetheno- IH, 13H-dibenzo[e,/c]pyrrolo [3 ,4- /z][l,4,13]oxadiazacyclohexadecene-l,3(2H)-dione (LY333531). This compound is a potent protein kinase C (PKC) inhibitor developed by Eli Lilly for the treatment of diabetic complications (Engel et al., Int'l J. Pharma. 198: 239-247, 2000; and Jirousek et al., J. Med. Chem. 39: 2664-2671, 1996). A related compound, LY379196, also selectively inhibits PKCβ over other PKC isoforms (Slosberg et al., MoI Carcinog. 27:166-76, 2000; and Dang et al., Biochem Pharmacol. 67:855-64, 2004). Other PKCβ inhibitors known in the art include, e.g., staurosporine, CGP41251, K252a, UCN-01, Go6976 (Ηofmann, FASEB J. 11 : 649-669, 1997; and Gordge et al., Cell Signal 6: 871-882, 1994). In addition, a cyclo- oxygenase-2 inhibitor, SC-236, also inhibits PKCβ expression and activity (Jiang et al., Oncogene 21: 6113-22, 2002).
[0057] The PKCβ modulators of the present invention can be directly administered under sterile conditions to the subject to be treated. The modulators can be administered alone or as the active ingredient of a pharmaceutical composition. To suppress or prevent graft rejection in a subject, the PKCβ-inhibitory compounds can also be used alone or in combination with other immune-modulating agents. For example, a PKCβ-modulating compound of the present invention can also be used in conjunction with known immunosuppressive drugs such as cyclosporin. In some applications, a first PKCβ- inhibiting compound is used in combination with a second PKCβ inhibitor in order to suppress or prevent graft rejection to a more extensive degree than cannot be achieved when one PKCβ modulator is used individually.
[0058] Pharmaceutical compositions of the present invention typically comprise at least one active ingredient together with one or more acceptable carriers thereof. Pharmaceutically carriers enhance or stabilize the composition, or to facilitate preparation of the composition. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered {e.g., nucleic acid, protein, modulatory compounds or transduced cells), as well as by the particular method used to administer the composition. They should also be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. This carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral, sublingual, rectal, nasal, or parenteral. For example, the PKCβ modulator can be complexed with carrier proteins such as ovalbumin or serum albumin prior to their administration in order to enhance stability or pharmacological properties.
[0059] There are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000). Without limitation, they include syrup, water, isotonic saline solution, 5% dextrose in water or buffered sodium or ammonium acetate solution, oils, glycerin, alcohols, flavoring agents, preservatives, coloring agents starches, sugars, diluents, granulating agents, lubricants, and binders, among others. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
[0060] The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100% by weight. Therapeutic formulations are prepared by any methods well known in the art of pharmacy. See, e.g., Gilman et al., eds., Goodman and Gilman's: The Pharmacological Bases of Therapeutics , 8th ed., Pergamon Press, 1990; Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; Avis et al., eds., Pharmaceutical Dosage Forms: Parenteral Medications, published by Marcel Dekker, Inc., N.Y., 1993; Lieberman et al., eds., Pharmaceutical Dosage Forms: Tablets, published by Marcel Dekker, Inc., N. Y., 1990; and Lieberman et al., eds., Pharmaceutical Dosage Forms: Disperse Systems, published by Marcel Dekker, Inc., N. Y., 1990.
[0061] The therapeutic formulations can be delivered by any effective means which could be used for treatment. Depending on the specific PKCβ modulators to be administered, the suitable means include oral, rectal, vaginal, nasal, pulmonary administration, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) infusion into the bloodstream. They can also be administered in eye drops or topical skin application.
[0062] For parenteral administration, PKCβ modulators of the present invention may be formulated in a variety of ways. Aqueous solutions of the modulators may be encapsulated in polymeric beads, liposomes, nanoparticles or other injectable depot formulations known to those of skill in the art. The nucleic acids may also be encapsulated in a viral coat.
[0063] Additionally, the compounds of the present invention may also be administered encapsulated in liposomes. The compositions, depending upon its solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally teπned a liposomic suspension. The hydrophobic layer, generally but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such a diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
[0064] The compositions may be supplemented by active pharmaceutical ingredients, where desired. Optional antibacterial, antiseptic, and antioxidant agents may also be present in the compositions where they will perform their ordinary functions.
[0065] The therapeutic formulations can conveniently be presented in unit dosage form and administered in a suitable therapeutic dose. A suitable therapeutic dose can be determined by any of the well known methods such as clinical studies on mammalian species to determine maximum tolerable dose and on normal human subjects to determine safe dosage. Except under certain circumstances when higher dosages may be required, the preferred dosage of a PKCβ modulator usually lies within the range of from about 0.001 to about 1000 mg, more usually from about 0.01 to about 500 mg per day. [0066] The preferred dosage and mode of administration of a PKCβ modulator can vary for different subjects, depending upon factors that can be individually reviewed by the treating physician, such as the condition or conditions to be treated, the choice of composition to be administered, including the particular PKCβ modulator, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the chosen route of administration. As a general rule, the quantity of a PKCβ modulator administered is the smallest dosage which effectively and reliably prevents or minimizes the conditions of the subjects. Therefore, the above dosage ranges are intended to provide general guidance and support for the teachings herein, but are not intended to limit the scope of the invention.
EXAMPLES
[0067] The following examples are offered to illustrate, but not to limit the present invention.
Example 1 . Material and Methods for Skin Grafting in Mice [0068] All mice used in transplant rejection experiments were maintained under specific pathogen free conditions in the GNF barrier facility. C57BL/6J and BALB/cJ mice were obtained from the Jackson labs and intercrossed to generate C57BL/6xBALB/cJ Fl donor animals. The PKCβ-deficient mice (Leitges et al., Science 273: 788-791, 1996) used had been backcrossed onto a C57BL/6 background for more than 10 generations.
[0069] Skin grafting was performed as follows. Following shaving and disinfection in 70% Ethanol, 1 cm2 (acute rejection) or 2 cm2 (minor mismatch rejection) dorsal skin patches were removed from 10-12 weeks old female C57BL/6 or PKCβ- knockout recipients under anesthesia and replaced by equally sized, shaved and disinfected abdominal donor skin patches which were obtained from freshly euthanized female (C57BL/6xBALB/c) Fl (acute rejection) or male C57BL/6 (minor mismatch rejection) donors. Grafts were fixed with 7-0 Prolene sutures and monitored daily for the first 2 weeks post grafting and thereafter weekly for another 5-8 weeks. Graft condition was classified as follows: (1) ++, no signs of rejection and good hair growth; (2) +, no signs of rejection but no hair growth, some mild swelling and erythema may occur; (3) +/-, foci of necrosis and/or a reduction in graft size, significant swelling and erythema; and (4) -, full rejection. Graft size reduced to less than 20%, massive scab formation, no viable graft tissue left. For graft survival plots, the percentage of un-rejected grafts per group was plotted versus time post grafting, ^-values for statistical significance were calculated in Student's t-tests. Median survival times (MST) were calculated from the observed full rejection times for all animals of each group and are shown +/- standard deviation.
[0070] For histological analyses, 2x5 mm2 biopsies of the recipient skin-graft border area were obtained from selected mice, fixed in paraformaldehyde or formalin prior to paraffin embedding. Longitudinal sections were stained with Hematoxylin and Eosin (H&E) or Giemsa and analyzed via bright-field microscopy.
[0071] For serum immunoglobulin isotyping, 200 μl blood per mouse were obtained via retro-orbital bleeding at the indicated time points. Serum Immunoglobulin concentrations were determined via cytometric bead assay (Spherotech) using anti-isotype antibodies from Phamiingen. Heteroscedastic t-tests of statistical differences among groups were performed using Excel.
Example 2. PKC β deficiency results in delayed graft rejection
[0072] We investigated skin allograft rejection in age-matched female PKCβ- deficient or C57BL/6 control mice. If the donors were C57BL/6xBALB/c Fl mice1 harboring major histocompatibility mismatches, acute rejection was indistinguishable between the female PKCβ-deficient recipients and C57BL/6 control recipients. The PKCβ- deficient recipients and C57BL/6 control recipients showed comparable courses of graft deterioration and median survival times (MST) of 9 +/- 0.5 days respectively 8.5 +/- 0.5 days. However, if grafts were from male C57BL/6 donors which display only a minor histocompatibility antigen mismatch due to the presence of the male-specific H-Y alloantigen, rejection was significantly delayed in PKCβ-deficient female recipients (n=7) compared to C57BL/6 controls (n=9). The median survival times for the PKCβ-deficient recipients and the C57BL/6 controls were 48 +/- 8 days and 27 +/- 11 days, respectively (p<0.005, Fig. IA).
[0073] Histological analyses of skin biopsies taken 6 days post grafting revealed a thickened epidermis and massive mononuclear infiltration at the junction of recipient connective tissue and graft as described previously for rejection of MHC-I disparate grafts by C57BL/6 females (Kobayashi, Kawai et al. 1990) to comparable degrees in control and PKCβ-deficient recipients (Fig. IB). At later time points, histological features reflected progressive rejection with overall similar characteristics to those described in (Kobayashi, Kawai et al. 1990), in particular epidermal thickening and a mononuclear infiltration of the dermis which peaked a few days before full rejection of a given graft was observed. Histological features were generally consistent with the visual appearance of individual grafts. Mononuclear infiltration of the graft dermis, however, was generally more prevalent on control recipients than on PKCβ-knockout recipients. For example on day 27, the observed MST for C57BL/6J recipients, most grafts on C57BL/6J recipients showed significant to strong features of rejection and massive mononuclear infiltration of the dermis (Fig. 1C). In contrast, the grafts on PKCβ- knockout recipients showed milder features of rejection and less mononuclear infiltration of the dermis.
[0074] Even on day 41 , closer to the observed MST for PKCβ- knockout recipients, the mononuclear graft infiltration on PKCβ- knockout recipients was still smaller than that observed on C57BL/6 recipients at their MST or on day 41 for grafts that had not been entirely lost yet (data not shown). Normal recipient skin adjacent to the grafts showed comparable frequencies of mononuclear cells in both genotypes at all time points. B cells play a non-essential, supportive role in allograft rejection via the production of alloantibodies directed against determinants presented by the graft (Le Moine et al., Transplantation 73: 1373-81, 2002). PKCβ-deficient mice on a 129/Sv background showed strongly reduced serum levels of IgM and IgG, but relatively normal levels of other immunoglobulin isotypes compared to 129/Sv mice (Leitges et al., Science 273: 788-791, 1996). We determined serum Ig isotype levels in untreated mice and in allograft recipients 74 days post grafting and found that on a C57BL/6 background, PKCβ-deficient mice have strongly reduced basal levels of IgM and also the THI- dependent isotypes IgG3 and IgG2a compared to controls (Fig. 2A). These differences became larger and much more significant in mice which had undergone allograft rejection (Fig. 2B). In conclusion, PKCβ-deficient mice on a C57BL/6 background show a significant delay in the rejection of minor histocompatibility antigen disparate allografts which is accompanied by a drastic reduction in IgM and THI -dependent immunoblobulin isotypes compared to control mice. The fact that THI -dependent Ig isotypes were reduced is one observation which suggests that T cell function is impaired in PKCβ-deficient mice and that this T cell defect could contribute to the observed delay in minor histocompatibility antigen mismatched graft rejection.
***
[0075] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.
[0076] All publications, GenBank sequences, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes as if each is individually so denoted