WO1993016183A1

WO1993016183A1 - Cyclophilin associated membrane proteins dna sequences

Info

Publication number: WO1993016183A1
Application number: PCT/US1993/001123
Authority: WO
Inventors: Irving L. Weissman; Jeffrey S. Friedman
Original assignee: The Board Of Trustees Of The Leland Stanford Jr. University
Priority date: 1992-02-07
Filing date: 1993-02-08
Publication date: 1993-08-19

Abstract

A protein capable of binding with high affinity to cyclophilin C in the absence of cyclosporin A, cyclophilin associated membrane protein (CAMP-c), and DNA encoding such a protein is provided. Also provided are antibodies directed against CAMP-c and methods by which these reagents are used.

Description

CYCLOPHILIN ASSOCIATED MEMBRANE PROTEINS DNA SEQUENCES ^• 5

This invention was made in the course of work supported by the U.S. Government, which has certain rights in this invention.

10 BACKGROUND OF THE INVENTION

The immunosuppressive drug cyclosporin A (CsA) is an undecapeptide fungal product which has a highly specific inhibitory effect on T cell activation or differentiation in vivo and .in vitro. While CsA is currently widely used to

15 prevent allograft rejection, its utility as a therapeutic agent is also being explored in a variety of autoimmune and neoplastic conditions.

Currently, there is no widely accepted model which explains the immunosuppressive effects of CsA and the

20 functionally similar macrolide FK506. Each drug is a naturally occurring fungal product which binds specifically to a class of proteins termed immunophilins (Bierer et al. (1990) Science 250: 556; Schreiber et al. (1991) Science 251: 283). While the immunophilins thus far isolated are prolyl-isomerases in their

25 free form, the cognate drug:immunophilin complexes (CsA for cyclophilins, FK506 and rapamycin for the FKBP's) lack that activity.

The mechanisms by which these immunosuppressive ligands inhibit T cell signal transduction are not completely

30 understood, nor are the normal function(s) of the immunophilins in T cells and other tissues in which they are expressed. Evidence suggests that CsA and FK506 possess virtually identical bioactivities, blocking T cell activation by interfering with an intracellular signaling event distal to

35 both calcium flux and phosphatidyl inositol hydrolysis (Dumont et al. (1990) J. Immunol. 144: 1418; Tocci et al. (1989) J. Immunol. 143: 718) .

The most abundant intracellular receptor for CsA, cyclophilin, has been purified (Handschumacher et al. (1984) Science 226: 544) and its polynucleotide sequence cloned and sequenced (Haendler et al. (1987) EMBO J. §_ : 947) . The cyclophilin protein possesses an intrinsic enzymatic activity, peptidyl-prolyl isomerase PPIase (Fischer et al. (1989) Nature 337: 476; Takahashi et al. (1989) Nature 337: 473) , which is blocked by CsA binding. The FK binding protein, FKBP, a receptor for FK506, also possesses a PPIase activity (Harding et al. (1989) Nature 341: 758; Siekierka et al. (1989) Nature 341: 755) which is inhibited by FK506 binding. The role of immunophilin PPIase activity in immunosuppression has been investigated in a number of experiments. Studies with analogues of CsA P (Durette et al. (1988) Transplant Proc. 20: 51; Sigal et al. (1991) J■ Exp. Med. 173 : 619) demonstrated that some analog compounds inhibit the PPIase activity of cyclophilin A, but lack immunosuppressive activity, while others are relatively poor inhibitors of PPIase, but retain immunosuppressive action. Similar results have been obtained in the FKBP/FK506 system using a nonnatural immunophilin ligand, 506BD, which is an efficient inhibitor of the FKBP rotamase, has no effect on T cell activation, and is able to block the immunosuppressive effects of both FK506 and rapamycin (Bierer et al. (1990) op.cit. ) .

The cloning of a third mammalian cyclophilin, cyclophilin C (cyp C) exhibiting a high degree of ho ology with known cyclophilins has been described. Cyp C is most highly expressed in the kidney, and can be detected in activated T cells as well as in the bone marrow stromal line AC 6, and is a mediator for the immunosuppressive and nephrotoxic actions of CsA. Cyp C binds to various cytoplasmic proteins which are likely to be involved in its functions in the absence of CsA or in the presence of CsA.

A substantially pure complex comprising a cyp C polypeptide and a protein of about 77kD has been identified. This 77kD protein binds to a cyp C polypeptide in the absence of cyclosporin A, and such binding is calcium-independent. In the presence of CsA, the CsA:cyp C complex no longer binds the 77kD protein, but now binds a 55kD species, designated calcineurin, which is involved in events associated with signal transduction that are blocked by CsA and FK506.

The sequestration of the 55kD calcineurin protein into drug:receptor complexes and the potential alteration of its normal function are apparently important aspects of the mechanism of action of CsA. It is still possibile that the release of the 77kD protein from a normally bound conformation is the physiologically important event at the drug:receptor interface. However, the results of titration experiments suggest that at pharmacologically relevant concentrations of

CsA, significant amounts of the 55kD protein may be sequestered in drug:receptor complexes, while the pool of 77kD protein is relatively unaffected. The 77kD protein is apparently involved in the normal function of cyp C, and may be related to its isomerase activity. The 55kD protein is also recognized by the FK506:FKBP complex, and is itself a likely candidate for a critical step in a signal transduction pathway.

The 77kD protein is therefore likely to be a major substrate for the cyp C PPIase activity. It may aid cyp C in its normal function, perhaps serving as a chaperone molecule for cyp C substrates, or it may represent a natural agonist or antagonist of the activity of cyp C, among other possibilities. Alternatively, as the cyp C:CsA complex intersects and is likely to block a signal transduction pathway, the cyp C:77kD complex may also be part of a signaling pathway.

The immunosuppressive ligands CsA and FK506 reveal a potential role of the immunophilins in signal transduction. Endogenous molecules akin to CsA and FK506 likely are normally present in cells, and that these molecules regulate signal transduction pathways through interaction with the immunophilins. Studies of the 77kD molecule will help to clarify the normal role of the immunophilins in signal transduction and protein processing.

Other CsA receptors may also be present in the cell, perhaps with higher affinity or important subcellular localization. In Neurospora and Saccharomyces, CsA may exert its effect by forming a toxic 'complex' with its receptors and other as yet unidentified cellular components (Tropschug et al. (1989) J. Biol. Chem. 263 : 14433. By analogy, such a complex may be responsible for the inhibition of T cell activation, and the nephrotoxicity observed in mammals. These results support the notion that it is the association of the CsA:cyp C complex with the 55kD protein that results in the CsA effects observed. Additional mammalian CsA binding proteins have been identified. Cyclophilin A is expressed at relatively high levels in most cell types (Koletsky et al. (1986) J. Immunol. 137: 1054, although the effects of CsA seem restricted to T lymphocytes, renal, and neural tissues as evidenced by immunologic effects and clinical toxicity. The cyp C RNA is present at relatively high levels in the kidney, and the kidney is known to be susceptible to progressive damage during the course of CsA therapy. Cyp C RNA is also present in a variety of murine T and B cell lines, but is undetectable by Northern blot analysis in total thymic RNA. Thus, cyp C expression in lymphocytes may be regulated by their state of activation. Understanding the functional role of the 77kD and 55kD proteins and other associated proteins isolated from specific tissues will provide information relevant to the activity of CsA on T cell mediated immunity, nephrotoxicity, and other tissue specific effects of CsA such as mast cell degranulation (Hultsch et al. (1990) J. Immunol. 144: 2659.

The purification and cloning of polypeptides which bind to cyclophilin C or other cyclophilins would therefore be of great interest and utility for understanding the interaction of cyclosporin and cyclophilin C, and developing a screening method for pharmaceutical compounds which alter the binding interaction(s) . Such a protein or its fragments and modified forms thereof would be useful, for example, for immunomodulation or the treatment of neoplastic conditions, as well as a structural model for the rational design of a host of drugs likewise affecting, for example, the interaction of cyclosporin and cyp C, and other uses. One could also, for example, use polynucleotides encoding such proteins or antibodies directed against such proteins to obtain other genes encoding proteins associated with cyclophilins, and as probes to diagnose genetic diseases associated with the gene locus. Thus, there exists a need in the art for isolated polypeptides which bind to cyclophilins under physiological conditions, and polynucleotides encoding such polypeptides.

SUMMARY OF THE INVENTION

The present invention provides several novel methods and compositions for modulating the immune response and for screening for modulators of the immune response. These methods utilize polynucleotide sequences encoding a cyclophilin C- associated membrane protein (CAMP-c) recombinant polypeptide and complementary polynucleotides which are substantially identical to CAMP-c polynucleotide sequences.

The present invention provides isolated polypeptides which are substantially identical to a cyclophilin C-associated membrane protein (CAMP-c) . Also embodied within the present invention are compositions comprising substantially pure CAMP-c polypeptides, fragments and analogs thereof, especially cyclophilin C-binding poypeptides. The CAMP-c polypeptides of the invention comprise a polypeptide sequence which is substantially identical to a cloned CAMP-c sequence provided herein. In one aspect of the invention, CAMP-c polypeptides and compositions thereof are provided. CAMP-c polypeptides comprise polypeptide sequences which are substantially identical to a sequence shown in Fig. 2 or a cognate CAMP-c gene sequence.

The present invention also provides isolated polynucleotides which comprise a polynucleotide sequence that encodes a CAMP-c polypeptide and/or that is substantially identical to the cloned CAMP-c sequence provided herein. As desired, the polynucleotide may further comprise an operably linked promoter sequence to form an expression vector, which may be transformed into a prokaryotic or eukaryotic host. The characteristics of the cloned sequences are given, including the nucleotide and predicted amino acid sequence in Fig. 2. Polynucleotides comprising these sequences can serve as templates for the recombinant expression of quantities of CAMP- c polypeptides. Polynucleotides comprising these sequences can also serve as probes for nucleic acid hybridization to detect the transcription and mRNA abundance of CAMP-c mRNA in individual lymphocytes (or other cell types) by j-n situ hybridization, and in specific lymphocyte populations by Northern blot analysis and/or by jji situ hybridization (Alwine et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74: 5350) and/or PCR amplification. Such recombinant polypeptides and nucleic acid hybridization probes have utility for in vitro screening methods for immunomodulatory agents and for diagnosis and treatment of pathological conditions and genetic diseases. Methods of screening for an immunomodulating agent are also provided. These methods comprise contacting said agent with a CAMP-c polypeptide or cyclophilin C binding fragment thereof and assaying (1) for the presence of a complex between the agent and the CAMP-c polypeptide or cyclophilin C binding fragment thereof or (2) for the presence of a complex between the CAMP-c polypeptide and cyclophilin C. The CAMP-c polypeptides or cyclophilin C may be labeled. In one embodiment, candidate immunomodulatory agents are identified by their ability to block the binding of a CAMP-c polypeptide to cyclophilin (cyclophilin C) . The cyclophilin preferably includes one or more binding sites at which a CAMP-c polypeptide specifically binds. One means for detecting binding of a CAMP-c polypeptide to a cyclophilin is to immobilize the cyclophilin, such as by covalent or noncovalent chemical linkage (e.g., via a specific antibody, or avidin- biotin linkage) to a solid support, and to contact under binding conditions the immobilized cyclophilin with a CAMP-c polypeptide that has been labeled with a detectable marker (e.g., by incorporation of radiolabeled amino acid). Such contacting is typically performed in aqueous conditions which permit binding of a CAMP-c polypeptide to a target cyclophilin containing a CAMP-c binding site. Binding of the labeled CAMP- c to the immobilized cyclophilin is measured by determining the extent to which the labeled CAMP-c polypeptide is immobilized as a result of a specific binding interaction. Such specific binding may be reversible, or may be optionally irreversible if a cross-linking agent is added in appropriate experimental conditions. The invention also provides antisense polynucleotides complementary to CAMP-c sequences which are employed to inhibit transctipnion, processing, or translation of the cognate mRNA species and thereby effect a reduction in the amount of the respective CAMP-c protein in a cell (e.g., a lymphocyte of a patient) . Such antisense polynucleotides can function as immunomodulatory drugs by inhibiting the formation of CAMP-c protein required for signal transduction and activation.

In a variation of the invention, polynucleotides of the invention are employed for diagnosis of immunopathological conditions or genetic disease that involve alterations in the structure or abundance of CAMP-c.

The invention also provides antibodies which bind to CAMP-c with an affinity of about at least 1 x 10⁷ M^-1 and which lack specific high affinity binding for unrelated proteins (e.g., albumin). Such antibodies can be used as diagnostic reagents to identify pathological cells in a cellular sample from a patient (e.g., a lymphocyte sample) as being cells which contain an altered amount of CAMP-c protein and/or a variant CAMP-c protein (e.g., isoform or mutant) as compared to normal cells of the same cell type. These antibodies may be used, e.g., in identifying cells expressing CAMP-c. Frequently, anti-CAMP-c antibodies are included as diagnostic reagents for immunohistopathology staining of cellular samples in situ. Additionally, anti-CAMP-c antibodies may be used therapeutically by targeted delivery to cells (e.g., by cationization or by liposome/immunoliposome delivery) .

The invention also provides CAMP-c polynucleotide probes for diagnosis of disease by detection of CAMP-c mRNA, or rearrangements or amplification of the CAMP-c gene in cells explanted from a patient, or detection of a pathognomonic CAMP- c allele (e.g., by RFLP or allele-specific PCR analysis). Typically, the detection will be by in situ hybridization using a labeled (e.g., ³⁵S, ³²P, ³H, fluorescent, biotinylated, digoxigeninylated) CAMP-c polynucleotide, although Northern blotting, dot blotting, or solution hybridization on bulk RNA isolated from a cell sample may be used, as may PCR amplification using CAMP-c-specific primers. The detection of pathognomonic rearrangements or amplification of the CAMP-c locus or closely linked loci in a cell sample will identify the presence of a pathological condition or a predisposition to developing a pathological condition (e.g., a genetic disease). All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1. Duplicate samples showing specific adhesion of AC 6 proteins to cyp C-GST matrix. AC 6 cell lysate was incubated with GST alone, purified by adherence to glutathione agarose, and subjected to elution with cyclosporin A (30 μg/ l CsA) . Eluted proteins are shown in 'GST alone' lanes.

Proteins remaining bound to glutathione agarose after CsA elution were boiled in SDS sample buffer, shown in the next two lanes 'control SDS...'. Experimental lanes were prepared by incubating AC 6 cell lysate with cyp C-GST, followed by purification on glutathione agarose and subsequent elution with CsA as described above—lanes 'CsA elution of...'. Proteins of 77kD, ~60kD, -37kD, and ~25kD specifically eluted from cyp C- GST by CsA are visible. Proteins remaining bound after CsA elution were boiled in SDS sample buffer, shown in the 'SDS sample buffer...' lanes. The bulk of the proteins bound to cyp C are completely eluted by the addition of CsA.

Fig. 2. Nucleotide sequence of CAMP-c cDNA, and deduced amoino acid sequence of CAMP-c [SEQ ID NOS:14 and 15]. Fig. 3. Sequence alignment at the amino acid level comparing the SRCR domain of CAMP-c with other SRCR containing proteins. The Genalign program (Intelligenetics, Mountain View, California) was used to align protein sequences of selected genes containing the Scavenger Receptor Cysteine Rich (SRCR) domain. The aligned sequences are: Human scavenger receptor [SEQ ID NO:16], CAMP-c [SEQ ID NO:17], Speract [SEQ ID NO:18] (a sea urchin protein). Human CD-6 [SEQ ID N0:19], Human CD-5 [SEQ ID N0:20]. The number 3 designates that the SRCR domain used in the alignment figure was the 3rd SRCR domain found in the protein. Speract, CD-5 and CD-6 have several repeats of the SRCR domain, while the scavenger receptor and CAMP-c have only one copy of this region. The six precisely spaced cysteine residues are highlighted. Fig. 4. Northern analysis of CAMP-c gene expression.

Northern analysis of CAMP-c expression in selected tissues shows that its expression profile matches that of cyp C. CAMP- c expression is induced by addition of IL-1 in AC 6 cells. It is detectable in lymphoid tissues and the kidney, but not in the liver.

Fig. 5a. Demonstration of the cyclosporin A sensitivity of binding of recombinant CAMP-c to cyp C. This figure shows that recombinant CAMP-c will bind to cyp C in the absence (lane 1) , but not in the presence of cyclosporin A (lane 2) . These results are in agreement with results obtained using native CAMP-c purified from AC 6 cells. The additional lanes on the left side of this figure demonstrate the sensitivity to cyclosporin of one of the monoclonal antibodies raised against cyp C. This antibody, D4 2A5, will precipitate cyp C in the absence (left lane) , but not the presence of cyclosporin (second lane from left) .

Fig. 5b. Demonstration that CAMP-c is a glycoprotein. This figure shows the results of treatment of recombinant CAMP-c produced in Cos 7 cells with glycosidases. In the first lane, protein is run without treatment and has an apparent molecular weight of ~77kD. When the protein is digested with endoglycosidase F or endoglycosidase H, its molecular weight changes to approximately 64kD, in agreement with the weight predicted from the amino acid sequence. Finally, treatment with O-glycanase has no effect on the protein. Thus, CAMP-c protein is a glycoprotein with N-linked sugars.

Fig. 6. Protein purification overview. This figure outlines the steps used in the large-scale purification of CAMP-c to generate material for protein sequence analysis.

Fig. 7. Elution conditions for CAMP-c proteins. Six identical samples of bound CAMP-c were subjected to various elution conditions (A-F) , through three rounds of elution. Addition of cyclosporin A was the most efficient method of removing CAMP-c from cyp C. A slight sensitivity to pH was also noted in lanes F, where some CAMP-c proteins are observed to elute in the absence of CsA. Fig. 8. In situ hybridization of cyclophilin C in murine kidney.

Fig. 9. Monoclonal antibodies against cyclophilin C (cyp C) . Antibody D4(l) co-immunoprecipitates the 77kD protein. Fig. 10. Immunofluorescence staining of kidney cross-sections using anti-cyp C antibody D4 2A5.

Fig. 11. Complete nucleotide sequence of CAMP-c cDNA.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly undertood by one of ordinary skill in the art to which this invention belongs. 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. For purposes of the present invention, the following terms are defined below. The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.

The term "recombinant" nucleic acid is one which is not naturally occurring, or is made by the artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA".

The terms "substantial homology" or "substantial identity" as used herein denotes a characteristic of a polypeptide sequence or nucleic acid sequence, wherein the polypeptide sequence has at least 60 percent sequence identity compared to a reference sequence, and the nucleic acid sequence has at least 70 percent sequence identity compared to a reference sequence. The percentage of sequence identity is calculated excluding small deletions or additions which total less than 25 percent of the reference sequence. The reference sequence may be a subset of a larger sequence, for example, as a segment of the full-length murine CAMP-c polypeptide sequence shown in Fig. 2; however, the reference sequence is at least 20 nucleotides long in the case of polynucleotides, and at least 6 amino residues long in the case of a polypeptide. Sequence homology, for polypeptides, is typically measured using sequence analysis software, see, e.g., Sequence Analysis Software Package of the Genetics Computer Group, 575 Science Dr., Madison, Wisconsin, 53711. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

Specific hybridization is defined herein as the formation of hybrids between a probe polynucleotide (e.g. , a polynucleotide of the invention which may include substitutions, deletion, and/or additions) and a specific target polynucleotide (e.g., a polynucleotide having the sequence in Fig. 2) , wherein the probe preferentially hybridizes to the specific target such that, for example, a single band corresponding to CAMP-c mRNA can be identified on a Northern blot of RNA prepared from T cells. Polynucleotides of the invention and recombinantly produced CAMP-c and fragments or analogs thereof may be prepared on the basis of the sequence data provided in Fig. 2 according to methods known in the art and described in Maniatis et al., Molecular Cloning: A

Laboratory Manual. 2nd Ed., (1989) , Cold Spring Harbor, N.Y. and Berger and Kimmel, Methods in Enzvmoloqy, Volume 152, Guide to Molecular Cloning Techniques (1987) , Academic Press, Inc. , San Diego, CA, which are incorporated herein by reference. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Generally, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.

The term "CAMP-c native protein" and "full-length CAMP-c protein" as used herein refers to a polypeptide of 575 amino acids corresponding to the deduced amino acid sequence shown in Fig. 2. or corresponding to the deduced amino acid sequence of a cognate full-length cDNA.

The term "fragment" as used herein refers to a polypeptide that has an amino-ter inal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the CAMP-c sequence deduced from a full-length cDNA sequence (e.g., the cDNA sequence shown in Fig. 2) . CAMP÷c fragments typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer.

The term "analog" as used herein refers to polypeptides which are comprised of a segment of at least 25 amino acids that has substantial similarity to a portion of the 575 residue long deduced amino acid sequence shown in Fig. 2, and which has at least one of the following properties: (1) binding to cyclophilin C under suitable binding conditions, (2) binding to cyclophilin C in the absence of CsA but having substantially reduced (by at least one log unit) binding affinity for cyclophilin C in the presence of 30 μg/ml CsA under suitable binding conditions, and (3) binding to an anti- CAMP-c antibody with an affinity of at least 1 x 10⁵ M^"1 or more. Typically, CAMP-c analog polypeptides comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally-occurring sequence. CAMP-c analogs typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer, most usually being as long as full-length naturally-occurring CAMP-c (e.g., 575 residues) .

The term "CAMP-c polypeptide" is used herein as generic terms to refer to native protein, fragments, or analogs of CAMP-c. Hence, native CAMP-c, fragments of CAMP-c, and analogs of CAMP-c are species of the CAMP-c polypeptide genus. The term "cognate" as used herein refers to a gene sequence that is evolutionarily and functionally related between species. For example but not limitation, in the human genome, the human CD4 gene is the cognate gene to the mouse CD4 gene, since the sequences and structures of these two genes indicate that they are highly homologous and both genes encode a protein which functions in signaling T cell activation through MHC class II-restricted antigen recognition. Thus, the cognate human gene to the murine CAMP-c gene is the human gene which encodes an expressed protein which has the greatest degree of sequence identity to the murine CAMP-c protein and which exhibits an expression pattern similar to that of the murine CAMP-c (e.g., expressed in lymphoid tissues and kidney, but not liver) . The term "altered ability to modulate" is used herein to refer to the capacity to either enhance transcription or inhibit transcription of a gene; such enhancement or inhibition may be contingent on the occurrence of a specific event, such as T cell stimulation. This alteration will be manifest as an inhibition of the transcriptional enhancement of the IL-2 gene that normally ensues following T cell stimulation. The altered ability to modulate transcriptional enhancement or inhibition may affect the inducible transcription of a gene, such as in the just-cited IL-2 example, or may effect the basal level ^' transcription of a gene, or both.

The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents are evaluated for potential activity as immunosuppressants by inclusion in screening assays described hereinbelow.

The terms "immunosuppressant" and "immunosuppressant agent" are used herein interchangeably to refer to agents that have the functional property of inhibiting an immune response in human, particularly an immune response that is mediated by activated T-cells.

The terms "candidate immunosuppressant" and "candidate immunosuppressant agent" are used herein interchangeably to refer to an agent which is identified by one or more screening method(s) of the invention as a putative inhibitor of T cell activation. Some candidate immunosuppressants may have therapeutic potential. As used herein, the terms "label" or "labeled" refers to incorporation of a detectable marker, e.g. , by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods) . Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes (e.g., ³H, ¹ C, ³⁵S, ³²P, ¹²⁵I, ¹³¹I) , fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase) , biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g. , leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags) . In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition) , and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. As used herein, the terms "isolated" is used interchangeably with the terms "substantially pure" and

"substantially homogenous" to describe a protein or polypeptide which has been separated from components which naturally accompany it. Typically, a monomeric protein is substantially pure when at least about 60 to 75% of a sample exhibits a single polypeptide backbone. Minor variants or chemical modifications typically share the same polypeptide sequence. A protein is substantially free of naturally associated components when it is separated from the native contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. The term is used to describe polypeptides and nucleic acids which have been synthesized in heterologous mammalian cells or plant cells, E. coli and other prokaryotes.

As used herein the terms "pathognomonic concentration", "pathognomonic amount", and "pathognomonic staining pattern" refer to a concentration, amount, or localization pattern, respectively, of a CAMP-c protein or mRNA in a sample, that indicates the presence of a pathologic condition or a predisposition to developing a immunologic disease, such as a lymphocytic leukemia, graft-versus-host reaction, allograft rejection, or autoimmune reaction. A pathognomonic amount is an amount of a CAMP-c protein or CAMP-c mRNA in a cell or cellular sample that falls outside the range of normal clinical values that is established by prospective and/or retrospective statistical clinical studies. Generally, an individual having a disease (e.g., lymphocytic leukemia, allograft rejection) will exhibit an amount of CAMP-c protein or mRNA in a cell or tissue sample that is outside the range of concentrations that characterize normal, undiseased individuals; typically the pathognomonic concentration is at least about one standard deviation outside of the mean normal value, more usually it is at least about two standard deviations or more outside the mean normal value. However, essentially all clinical diagnostic tests produce some percentage of false positives and false negatives. The sensitivity and selectivity of the diagnostic assay must be sufficient to satisfy the diagnostic objective and any relevant regulatory requirements. In general, the diagnostic methods of the invention are used to identify individuals as disease candidates, providing an additional parameter in a differential diagnosis of disease made by a competent health professional.

DETAILED DESCRIPTION

Generally, the nomenclature used hereafter and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation) . Generally enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see, generally,

Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference) which are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

Oligonucleotides can be synthesized on an Applied Bio Systems oligonucleotide synthesizer according to specifications provided by the manufacturer.

Methods for PCR amplification are described in the art (PCR Technology: Principles and Applications for DNA Amplification ed. HA Erlich, Stockton Press, New York, NY (1989) ; PCR Protocols: A Guide to Methods and Applications. eds. Innis, Gelfland, Snisky, and White, Academic Press, San Diego, CA (1990) ; Mattila et al. (1991) Nucleic Acids Res. 19: 4967; Eckert, K.A. and Kunkel, T.A. (1991) PCR Methods and Applications 1 : 17; U.S. Patent 4,683,202, which are incorporated herein by reference) and exemplified hereinbelow. A basis of the present invention is the isolation of novel polypeptides which bind to cyclophilin c in the absence of CsA, and which exhibit a reduced binding affinity for cyclophilin c in the presence of saturating concentrations of CsA. Complementary DNA (cDNA) clones which encode these polypeptides have been isolated and sequenced, and purified polypeptides expressed from recombinant polynucleotides containing such sequences are made.

The present invention provides purified polypeptide forms and cloned nucleic acids encoding the first set of proteins, CAMP-c, demonstrated to have a high affinity for a cyclophilin in the absence of the drug CsA. These proteins are fundamental to understanding the role(s) of cyclophilins, such as cyclophilin C, in organisms from yeast to higher eukaryotes. In addition, they will be of practical importance in, for example, immunomodulation or the treatment of neoplastic conditions, antisense polynucleotides for immunomodulation of T lymphocytes, diagnostic antibodies and polynucleotide probes, screening assays for developing novel immunosuppressants, as well as providing structural models for the rational design of a host of drugs likewise affecting, for example, the interaction of cyclosporin and cyp C. Polynucleotides encoding such proteins or antibodies directed against such proteins can be used to obtain other genes encoding proteins associated with cyclophilins.

Purification and cloning of cyp C binding proteins in the absence of cyclosporin c In considering the functions of mammalian cyclophilins, it is useful to distinguish between two states: the unbound conformation, i.e., absence of CsA or endogenous ligand, and the drug or endogenous ligand in a complex with the cyclophilin. Three mammalian cyclophilins, cyp A, cyp B, and cyp C may have similar properties in the presence of the drug

CsA. All three cyclophilins are capable of complexing with and inhibiting the activity of the calcium/calmodulin dependent phosphatase calcineurin (J. Liu et al. (1991) Cell 66: 807) . In the absence of CsA, the properties of the three mammalian cyclophilins diverge. Protein/protein interactions between cyp A or cyp B and other cellular proteins do not occur in the absence of CsA. Cyp C (Friedman and Weissman (1991) op.cit. ) , on the other hand, interacts with high affinity with at least three cellular proteins of molecular weights 77kD, 60kD and 37kD. This strong interaction can be disrupted by the addition of saturating amounts of CsA.

Molecules such as the 77kD, 60kD and 37kD proteins are the keys to an understanding of the roles cyclophilins are designed to play in organisms from yeast to higher eukaryotes. Protein/protein interactions between the 77kD, 60kD, and 37kD proteins and cyclophilins may serve to regulate the interaction of cyclophilins and calcineurin-like molecules.

A murine cDNA sequence which encodes the CAMP-c 77kD polypeptide is provided in Fig. 2. The full sequence of the CAMP-c cDNA, including untranslated sequences, is shown in Fig. 11. The cDNA sequence encodes a polypeptide of 64kD which is glycosylate to give a 77kD protein.

CAMP-c Polynucleotides

Genomic or cDNA clones encoding CAMP-c may be isolated from clone libraries (e.g. , available from Clontech, Palo Alto, CA) using hybridization probes designed on the basis of the nucleotide sequences shown in Fig. 2. Where a cDNA clone is desired, clone libraries containing cDNA derived from lymphocyte mRNA are preferred. Alternatively, synthetic polynucleotide sequences corresponding to all or part of the sequences shown in Fig. 2 may be constructed by chemical synthesis of oligonucleotides. Additionally, polymerase chain reaction (PCR) using primers based on the sequence data disclosed in Fig. 2 may be used to amplify DNA fragments from genomic DNA, mRNA pools, or from cDNA clone libraries. U.S. Patents 4,683,195 and 4,683,202 describe the PCR method. Additionally, PCR methods employing one primer that is based on the sequence data disclosed in Fig. 2 and a second primer that is not based on that sequence data may be used. For example, a second primer that is homologous to or complementary to a polyadenylation segment may be used.

It is apparent to one of skill in the art that nucleotide substitutions, deletions, and additions may be incorporated into the polynucleotides of the invention. However, such nucleotide substitutions, deletions, and additions should not substantially disrupt the ability of the polynucleotide to hybridize to one of the polynucleotide sequences shown in Fig. 2 under hybridization conditions that are sufficiently stringent to result in specific hybridization.

Specific hybridization is defined herein as the formation of hybrids between a probe polynucleotide (e.g., a polynucleotide of the invention which may include substitutions, deletion, and/or additions) and a specific target polynucleotide (e.g., a polynucleotide having the sequence in Fig. 2) , wherein the probe preferentially hybridizes to the specific target such that, for example, a single band corresponding to CAMP-c mRNA can be identified on a Northern blot of RNA prepared from a suitable cell source (e.g., a lymphocyte population). Polynucleotides of the invention and recombinantly produced CAMP-c, and fragments or analogs thereof, may be prepared on the basis of the sequence data provided in Fig. 2 according to methods known in the art and described in Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989), Cold Spring Harbor, N.Y. and Berger and Kimmel, Methods in Enzymology. Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, CA, which are incorporated herein by reference.

CAMp-c polynucleotides may be short oligonucleotides (e.g., 25-100 bases long), such as for use as hybridization probes and PCR (or LCR) primers. CAMP-c polynucleotide sequences may also comprise part of a larger polynucleotide

(e.g., a cloning vector comprising a CAMP-c clone) and may be fused, by polynucleotide linkage, in frame with another polynucleotide sequence encoding a different protein (e.g., glutathione S-transferase or -galactosidase) for encoding expression of a fusion protein. Typically, CAMP-c polynucleotides comprise at least 25 consecutive nucleotides which are substantially identical to a naturally-occurring CAMP-c sequence (e.g., Fig. 2), more usually CAMP-c polynucleotides comprise at least 50 to 100 consecutive nucleotides which are substantially identical to a naturally- occurring CAMP-c sequence. However, it will be recognized by those of skill that the minimum length of a CAMP-c polynucleotide required for specific hybridization to a CAMP-c target sequence will depend on several factors: G/C content, positioning of mismatched bases (if any) , degree of uniqueness of the sequence as compared to the population of target polynucleotides, and chemical nature of the polynucleotide (e.g., methylphosphonate backbone), among others.

The present invention provides nucleic acid sequences encoding CAMP-c polypeptide sequences. Nucleic acids according to the present invention will possess a sequence which is either derived from a natural source gene or one having substantial homology with a natural CAMP-c-encoding gene or a portion thereof, particularly fragments capable of binding cyp C.

The DNA compositions of this invention are typically derived from genomic DNA or cDNA, and may be a hybrid of the various combinations. They may also be chemically synthesized recombinant nucleic acids comprising sequences otherwise not naturally occurring are also provided by this invention. Although the wild type sequence may be employed, the wild type sequence will often be altered, e.g., by deletion, substitution, or insertion. cDNA or genomic libraries of various types may be screened as natural sources of the nucleic acids of the present invention, or such nucleic acids may be provided by amplification of sequences resident in genomic DNA or other natural sources, e.g., by the polymerase chain reaction (PCR). See, e.g., PCR Protocols: A Guide to Methods and Applications. Innis, M. , Gelfand, D. , Sninsky, J. and White, T. , eds.. Academic Press: San Diego (1990), incorporated herein by reference. The choice of cDNA libraries normally corresponds to a tissue source which is abundant in mRNA for the desired receptors. Phage libraries are normally preferred, but plasmid libraries may also be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured and probed for the presence of desired sequences. The DNA sequences used in this invention will usually comprise at least about 5 codons (15 nucleotides) , more usually at least about 7 codons, typically at least about 10 codons, preferably at least about 15 codons, more preferably at least about 25 codons and most preferably at least about 35 codons. One or more introns may also be present. This number of nucleotides is usually about the minimal length required for a successful probe that would hybridize specifically with a CAMP- c-encoding sequence.

Techniques for nucleic acid manipulation are described generally, for example, in Sambrook et al., 1989 or Ausubel et al., 1987, incorporated herein by reference. Reagents useful in applying such techniques, such as restriction enzymes and the like, are widely known in the art and commercially available from such vendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega Biotec, U. S. Biochemicals, New England Nuclear, and a number of other sources. The recombinant nucleic acid sequences used to produce fusion proteins of the present invention may be derived from natural or synthetic sequences. Many natural gene sequences are obtainable from various cDNA or from genomic libraries using appropriate probes. See, GenBank, National Institutes of Health. A nucleic acid or fragment thereof is substantially identical to another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand) , there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95 to 98% of the nucleotide bases.

Alternatively, substantial identity exists when a nucleic acid or fragment thereof will hybridize to another under nucleic acid (or a complementary strand thereof) under selective hybridization conditions, to a strand, or its complement. Typically, selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. See. Kanehisa (1984) Nuc. Acids Res. 12: 203, incorporated herein by reference. The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least about 17 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in excess of 30°C, typically in excess of 37°C, and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31: 349, incorporated herein by reference.

Nucleic acid probes may be prepared based on the sequence of the CAMP-c cDNA sequence provided by the present invention. The probes will include an isolated nucleic acid attached to a label or reporter molecule and may be used to isolate other nucleic acid sequences, having sequence similarity by standard methods. For techniques for preparing and labelling probes see, e.g., Sambrook et al. (1989) op.cit. or Ausubel et al. (1987) op.cit.. both incorporated herein by reference. Other similar nucleic acids may be selected by using homologous nucleic acids. Alternatively, nucleic acids encoding these same or similar polypeptides may be synthesized or selected by making use of the redundancy in the genetic code. Various codon substitutions may be introduced, e.g., silent changes thereby producing various restriction sites, or to optimize expression for a particular system. Mutations may be introduced to modify the properties of the polypeptide receptors, perhaps to change the ligand-binding affinities, the interchain affinities, or the polypeptide degradation or turnover rate. Probes comprising synthetic oligonucleotides or other nucleic acids of the present invention may be derived from naturally occurring or recombinant single or double stranded nucleic acids or be chemically synthesized. Probes may also be labelled by nick translation, Klenow fill-in reaction, or other methods known in the art.

Portions of the DNA sequence having at least about 15 nucleotides, usually at least about 15 nucleotides, and fewer than about 6 kb, usually fewer than about 1.0 kb, from a DNA sequence encoding CAMP-c are preferred as probes. The probes may also be used to determine whether mRNA encoding CAMP-c is present in a cell or tissue.

Production of CAMP-c Polypeptides

The nucleotide and amino acid sequences shown in Fig. 2 enable those of skill in the art to produce polypeptides corresponding to all or part of the full-length CAMP-c polypeptide sequence. Such polypeptides may be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding CAMP-c, or fragments and analogs thereof. Alternatively, such polypeptides may be synthesized by chemical methods or produced by in. vitro translation systems using a polynucleotide template to direct translation. Methods for expression of heterologous proteins in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are described further in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y. and Berger and Kimmel, Methods in Enzvmology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, CA.

Fragments or analogs of CAMP-c may be prepared by those of skill in the art. Preferred amino- and carboxy- termini of fragments or analogs of CAMP-c occur near boundaries of functional domains. For example, but not for limitation, such functional domains include: (1) domains conferring the property of binding to cyclophilin C, (2) domains comprising the amino acid sequence having structural similarity to the 55kD protein which binds cyclophilin C in the presence of CsA, and (3) domains to which contain peptide cleavage sites or phosphorylation sites.

One method by which structural and functional domains may be identified is by comparison of the nucleotide and/or amino acid sequence data shown in Fig. 2 to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. For example, the NAD-binding domains of dehydrogenases, particularly lactate dehydrogenase and malate dehydrogenase, are similar in conformation and have amino acid sequences that are detectably homologous (Proteins. Structures and Molecular Principles. (1984) Creighton (ed.), W.H. Freeman and Company, New York, which is incorporated herein by reference) . Further, a method to identify protein sequences that fold into a known three- dimensional structure are known (Bowie et al. (1991) Science 253: 164) . Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in the CAMP-c sequences of the invention.

Additionally, computerized comparison of sequences shown in Fig. 2 to existing sequence databases can identify sequence motifs and structural conformations found in other proteins or coding sequences that indicate similar domains of the CAMP-c protein. For example but not for limitation, the programs GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr. , Madison, WI) can be used to identify sequences in databases, such as GenBank/EMBL, that have regions of homology with a CAMP-c sequences. Such homologous regions are candidate structural or functional domains. Alternatively, other algorithms are provided for identifying such domains from sequence data. Further, neural network methods, whether implemented in hardware or software, may be used to: (1) identify related protein sequences and nucleotide sequences, and (2) define structural or functional domains in CAMP-c polypeptides (Brunak et al. (1991) J. Mol. Biol. 220: 49, which is incorporated herein by reference) .

Fragments or analogs comprising substantially one or more functional domain may be fused to heterologous polypeptide sequences, wherein the resultant fusion protein exhibits the functional property(ies) conferred by the CAMP-c fragment. Alternatively, CAMP-c polypeptides wherein one or more functional domain have been deleted will exhibit a loss of the property normally conferred by the missing fragment. By way of example and not limitation, the domain conferring the property of binding to cyclophilin C may be fused to -galactosidase to produce a fusion protein that can be used in an assay to measure binding to cyclophilin C and which can also enzymatically convert a chromogenic substrate to a chromophore.

Although one class of preferred embodiments are fragments having amino- and/or carboxy-termini corresponding to amino acid positions near functional domains borders, alternative CAMP-c fragments may be prepared. The choice of the amino- and carboxy-termini of such fragments rests with the discretion of the practitioner and will be made based on experimental considerations such as ease of construction, stability to proteolysis, thermal stability, immunological reactivity, amino- or carboxyl-ter inal residue modification, or other considerations.

In addition to fragments, analogs of CAMP-c can be made. Such analogs may include one or more deletions or additions of amino acid sequence, either at the amino- or carboxy-termini, or internally, or both; analogs may further include sequence transpositions. Analogs may also comprise amino acid substitutions, preferably conservative substitutions. Additionally, analogs may include heterologous sequences generally linked at the amino- or carboxy-terminus, wherein the heterologous sequence(s) confer a functional property to the resultant analog which is not indigenous to the native CAMP-c protein. However, CAMP-c analogs must comprise a segment of 25 amino acids that has substantial similarity to a portion of the amino acid sequence shown in Fig. 2, respectively, and which has at least one of the requisite functional properties enumerated in the Definitions (supra ) . Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter post-translational modification of the analog, possibly including phosphorylation, and (4) confer or modify other physicochemical or functional properties of such analogs. CAMP-c analogs include various muteins of a CAMP-c sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring CAMP-c sequence (preferably in the portion of the polypeptide outside the cyclophilin C binding domains) .

Conservative amino acid substitution is a substitution of an amino acid by a replacement amino acid which has similar characteristics (e.g., those with acidic properties: Asp and Glu) . A conservative (or synonymous) amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence) . Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins. Structures and Molecular Principles, (1984) Creighton (ed.), W.H. Freeman and Company, New York; Introduction to Protein Structure. (1991) , C. Branden and J. Tooze, Garland Publishing, New York, NY; and Thornton et al. (1991) Nature 354: 105; which are incorporated herein by reference) .

Native CAMP-c proteins, fragments thereof, or analogs thereof can be used as reagents in cyclophilin binding assays for identifying agents that interfere with CAMP-c binding and function, said agents are thereby identified as candidate immunosuppressant drugs which may be used, for example, to block lymphocyte activation. Typically, in vitro binding assays that measure binding of CAMP-c to cyclophilin employ cyclophilin C polypeptides that contain at least one CAMp-c binding site. The cyclophilin polypeptide (or the CAMP-c polypeptide) is typically linked (prior to, during, or subsequent to the interprotein binding reaction) to a solid substrate by any of various means known to those of skill in the art; such linkage may be noncovalent (e.g., binding to a highly charged surface such as PDVF) or may be by covalent bonding (e.g., typically by chemical crosslinkage or streptavidin-biotin linkage) . CAMP-c polypeptides are typically labeled by incorporation of a radiolabeled amino acid. The labeled CAMP-c polypeptide is contacted with a cyclophilin C polypeptide under aqueous conditions that permit specific binding in control binding reactions with a binding affinity of about 1 x 10⁵ M^-1 or greater (e.g., 20-200 M NaCl or KC1 and 5-100 mM Tris HCl pH 6-8) . Specificity of binding is typically established by adding unlabeled competitor at various concentrations selected at the discretion of the practitioner. Examples of unlabeled protein competitors include, but are not limited to, the following: unlabeled CAMP- c polypeptide, bovine serum albumin, and cytoplasmic protein extracts. Binding reactions wherein one or more agents are added are performed in parallel with a control binding reaction that does not include an agent. Agents which inhibit the specific binding of CAMP-c polypeptides to cyclophilin polypeptides, as compared to a control reaction, are identified as candidate immunosuppressant drugs. Also, agents which prevent downstream signal transduction (e.g., calcineurin activation) by cyclophilin C and CAMP-c in vitro are thereby identified as candidate immunosuppressant drugs. The present invention provides CAMP-c polypeptides.

Also included are homologous sequences, allelic variations, natural or induced mutants, alternatively expressed variants, and proteins encoded by DNA which hybridize under high or low stringency conditions, to CAMP-c-encoding nucleic acids retrieved from naturally occurring material. Closely related polypeptides or proteins retrieved by antisera to CAMP-c are also provided. Induced mutants may be derived from encoding nucleic acids using irradiation or exposure to chemical mutagens such as EMS, or may take the form of engineered changes by site-specific mutagenesis or other techniques of modern molecular biology. See, e.g., Sambrook, Fritsch and Maniatis (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. , CSH Press, incorporated herein by reference for all purposes. The present invention also provides for other polypeptides comprising fragments of CAMP-c polypeptides substantially homologous thereto, especially those capable of binding cyp C. The peptides of the present invention will generally exhibit at least about 80% sequence identity with naturally occurring sequences, typically at least about 85% sequence identity with a natural CAMP-c sequence, more typically at least about 90% sequence identity, usually at least about 95% sequence identity, and more usually at least about 97% sequence identity. The length of comparison sequences will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues.

Expression of CAMP-c Polypeptides The nucleic acid sequences of the present invention capable of ultimately expressing the desired CAMP-c polypeptides can be formed from a variety of different polynucleotides (genomic or cDNA, RNA, synthetic oligonucleotides, etc.) as well as by a variety of different techniques.

As stated previously, the DNA sequences will be expressed in hosts after the sequences have been operably linked to (i.e. , positioned to ensure the functioning of) an expression control sequence. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g.. tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired DNA sequences (see, e.g. , U.S. Patent 4,704,362, which is incorporated herein by reference) . E. coli is one prokaryotic host useful particularly for cloning the DNA sequences of the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia. and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g. , an origin of replication) . In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.

Other microbes, such as yeast, may also be used for expression. Saccharo vces is a preferred host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.

In addition to microorganisms, mammalian tissue cell culture may also be used to express and produce the polypeptides of the present invention (see, Winnacker, "From Genes to Clones," VCH Publishers, N.Y., N.Y. (1987), which is incorporated herein by reference) . Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al. (1986) Immunol. Rev. 89: 49, which is incorporated herein by reference), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, and the like.

The vectors containing the DNA segments of interest (e.g. , polypeptides encoding a CAMP-c polypeptide) can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts. (See. generally, Maniatis, et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, (1982) , which is incorporated herein by reference.)

Eukaryotic cells which express recombinant CAMP-c protein and which express cyclophilin C may be used in screening assays to identify agents which inhibit binding of cyclophilin C to CAMP-c; such agents may be identified as candidate immunosuppressant agents.

Expression of recombinant CAMP-c protein in cells, particularly cells of the lymphopoietic lineage, may be used to identify and isolate genes that are transcriptionally modulated, either positively or negatively, by the presence of CAMP-c protein. Such genes are typically initially identified as cDNA clones isolated from subtractive cDNA libraries, wherein RNA isolated from cells expressing recombinant CAMP-c and RNA isolated from control cells (i.e., not expressing recombinant CAMP-c) are used to generate the subtractive libraries and screening probes. In such a manner, CAMP-c- dependent genes may be isolated. CAMP-c-dependent genes (or their regulatory sequences operably linked to a reporter gene) may be used as a component of an in vitro transcription assay employing a cell extract from cells expressing CAMP-c as a necessary component for efficient transcription; such transcription assays may be used to screen for agents which inhibit CAMP-c-dependent gene transcription and are thereby identified as candidate immunosuppressant agents.

Where desired, a signal or leader sequence can direct the polypeptide through the membrane of a cell. Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.

Preparation of recombinant or chemically synthesized nucleic acids; vectors, transformation, host cells Large amounts of the nucleic acids of the present invention may be produced by replication in a suitable host cell. The natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the DNA constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to, with and without and integration within the genome, cultured mammalian or plant or other eukaryotic cell lines. The purification of nucleic acids produced by the methods of the present invention are described, e.g., in Sambrook et al. (1989) op.cit. or Ausubel et al. (1987) op.cit.1.

The nucleic acids of the present invention may also be produced by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22: 1859 or the triester method according to Matteucci et al. (1981) J. Am. Chem. Soc. 103: 3185, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

DNA constructs prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system recognized by the host, including the intended DNA fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment. Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Secretion signals may also be included where appropriate, whether from a native CAMP-c protein or from other receptors or from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes, and thus attain its functional topology, or secreted from the cell. Such vectors may be prepared by means of standard recombinant techniques well known in the art and discussed, for example, in Sambrook et al. (1989) op.cit. or Ausubel et al. (1987) op.cit..

The selection of an appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host, and may, when appropriate, include those naturally associated with CAMP-c genes. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al. (1989) op.cit. or Ausubel et al. (1987) op.cit. ; see also, e.g., Metzger et al. (1988) Nature 334: 31. Many useful vectors are known in the art and may be obtained such vendors as Stratagene, New England Biolabs, Promega Biotech, and others. Promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts. Useful yeast promoters include the promoter regions for metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others. Appropriate nonnative mammalian promoters might include the early and late promoters from SV40 (Fiers et al. (1978) Nature 273: 113) or promoters derived from murine molony leukemia virus, mouse mammary tumor virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma. In addition, the construct may be joined to an amplifiable gene (e.g., DHFR) so that multiple copies of the gene may be made. For appropriate enhancer and other expression control sequences see also Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor Press, N.Y. (1983) , incorporated herein by reference.

While such expression vectors may replicate anonymously, they may less preferably replicate by being inserted into the genome of the host cell, by methods well known in the art.

Expression and cloning vectors will likely contain a selectable marker, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector. The presence of this gene ensures the growth of only those host cel_ls which express the inserts. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxic substances, e.g. a picillin, neomycin, methotrexate, etc.; (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g. the gene encoding D-alanine racemase for Bacilli. The choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art.

The vectors containing the nucleic acids of interest can be transcribed in vitro and the resulting RNA introduced into the host cell by well-known methods (e.g., by injection. See, Kubo et al. (1988) FEBS Lett. 241: 119, incorporated herein by reference) , or the vectors can be introduced directly into host cells by methods well known in the art, which vary depending on the type of cellular host, including electroporation; transfection employing calcium chloride, rubidium chloride calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome) ; and other methods. See generally, Sambrook et al. (1989) and Ausubel et al. (1987), both incorporated herein by reference. The cells into which have been introduced nucleic acids described above are meant to also include the progeny of such cells.

Large quantities of the nucleic acids and polypeptides of the present invention may be prepared by expressing the CAMP-c nucleic acids or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells. The most commonly used prokaryotic hosts are strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis or Pseudo onas may also be used. Mammalian or other eukaryotic host cells, such as those of yeast, filamentous fungi, plant, insect, amphibian or avian species, may also be useful for production of the proteins of the present invention. Propagation of mammalian cells in culture is per se well known. See, Tissue Culture. Kruse and Patterson, ed. , Academic Press (1973), incorporated herein by reference. Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although it will be appreciated by the skilled practitioner that other cell lines may be appropriate, e.g., to provide higher expression, desirable glycosylation patterns, or other features.

Clones are selected by using markers depending on the mode of the vector construction. The marker may be on the same or a different DNA molecule, preferably the same DNA molecule. With mammalian cells the CAMP-c gene itself may be the best marker. In prokaryotic hosts the transformant may be selected, e.g. , by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker.

Prokaryotic or eukaryotic cells transformed with the nucleic acids of the present invention will be useful not only for the production of the nucleic acids and polypeptides of the present invention, but also, for example, in studying the characteristics of CAMP-c polypeptides, including their ability to bind cyp C, or to evaluate the effectiveness of a substance as an antagonist or agonist. Protein modifications: fragments The present invention also provides for modified CAMP-c polypeptides, including chemical modifications, modifications of the CAMP-c primary polypeptide sequence, and polypeptides incorporating unusual amino acids. Chemical modifications or derivatizations may be accomplished n vivo or n vitro and include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labelling, e.g., with radionuclides, including enzymatic modifications, all by methods well known in the art. Also embraced by the present invention are versions of the same primary amino acid sequence which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine. Also included are genetic variants, both natural and induced. Induced mutants may be derived from various techniques including both random mutagenesis of the encoding nucleic acids using irradiation or exposure to EMS, or may take the form of engineered changes by site-specific mutagenesis or other techniques of modern molecular biology. See, Molecular Cloning: A Laboratory Manual, 2nd ed. , Vol. 1-3 , ed. Sambrook, et al. , Cold Spring Harbor Laboratory Press (1989) or Current Protocols in Molecular Biology, ed. F. Ausubel et al. , Greene Publishing and Wiley-Interscience: New York (1987 and updates) , both incorporated herein by reference.

Besides substantially full-length polypeptides, the present invention provides for biologically active fragments of the polypeptides. Significant biological activities include ligand-binding, immunological activity and other biological activities characteristic of CAMP-c polypeptides. Immunological activities include both immunogenic function in a target immune system, as well as sharing of immunological epitopes for binding, serving as either a competitor or substitute antigen for a CAMP-c epitope. As used herein, the term fragment or segment, as applied to a polypeptide, will ordinarily be at least about 5 contiguous amino acids, typically at least about 7 contiguous amino acids, more typically at least about 9 contiguous amino acids, usually at least about 11 contiguous amino acids, preferably at least about 13 contiguous amino acids, more preferably at least about 16 contiguous amino acids, and most preferably at least about 20 to 30 or more contiguous amino acids. Segments of a particular domain will be segments of the appropriate size within the corresponding domain.

For immunological purposes, immunogens may be produced which tandemly repeat polypeptide segments, thereby producing highly antigenic proteins. Alternatively, such polypeptides will serve as highly efficient competitors for specific binding. Production of antibodies specific for CAMP-c polypeptides or fragments thereof is described below.

The present invention also provides for fusion polypeptides comprising CAMP-c polypeptides and fragments. Homologous polypeptides may be fusions between two or more CAMP-c sequences or between the sequences of CAMP-c and a related cyclophilin binding protein. Likewise, heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins. For example, ligand-binding or other domains may be "swapped" between different new fusion polypeptides or fragments. Such homologous or heterologous fusion polypeptides may display, for example, altered strength or specificity of binding. Fusion partners include immunoglobulins, bacterial 3-galactosidase; trpE, Protein A, 0-lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor. See, e.g., Godowski et al. (1988) Science 241: 812, incorporated herein by reference. Fusion proteins will typically be made by either recombinant nucleic acid methods, as described below, but may be chemically synthesized. Techniques for synthesis of polypeptides are described, for example, in Merrifield (1963) J. Amer. Chem. Soc. 85: 2149, incorporated herein by reference.

Protein purification The present invention describes the purification of CAMP-c polypeptides from their natural source. Various methods for the isolation of the CAMP-c polypeptides from other biological material, such as from cells transformed with recombinant nucleic acids encoding CAMP-c, may be accomplished by various methods well known in the art. For example, such polypeptides may be purified by immunoaffinity chromatography employing, e.g., the antibodies provided by the present invention. Various methods of protein purification are well known in the art, and include those described, e.g., in Guide to Protein Purification, ed. M. Deutscher, vol. 182 of Methods in Enzymology (Academic Press, Inc.: San Diego, 1990) and R. Scopes, Protein Purification: Principles and Practice, (Springer-Verlag: New York, 1982) , both incorporated herein by reference.

Peptidomimetics In addition to polypeptides, the invention provides peptidomimetics of CAMP-c, some of which may be used therapeutically as competitive antagonists for CAMP-c binding to cyclophilin C. Peptidomimetics comprise polypeptide-like polymers that contain novel backbone structures or unnnatural amino acids (Ellman et al. (1992) Science 255: 197, which is incorporated herein by reference) , or other non-peptide chemical constituents, including peptoids (Simon et al. (1992) Proc. Natl. Acad. Sci. (U.S.A.) 89: 9367) . A consensus motif may form the basis for synthesis of peptidomimetics. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non- peptide compound are termed "peptide mimetics" or "peptidomimetics" (Fauchere, J. (1986) Adv. Drug Res. 15: 29; Veber and Freidinger (1985) TINS p.392; and Evans et al. (1987) J. Med. Chem 30: 1229, which are incorporated herein by reference) and are usually developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity) , but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: -CH₂NH-, -CH₂S-, -CH₂-CH₂~, -CH=CH- (cis and trans), -C0CH₂-, -CH(OH)CH₂-, and -CH₂S0~, by methods known in the art and further described in the following references: Spatola, A.F. in "Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins," B. Weinstein, eds.,

Marcel Dekker, New York, p. 267 (1983) ; Spatola, A.F., Vega Data (March 1983) , Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review); Morley, J.S., Trends Pharm Sci (1980) pp. 463-468 (general review) ; Hudson, D. et al. , Int J Peot Prot Res (1979) 14.: 177-185 (-CH₂NH-, CH₂CH₂~) ; Spatola, A.F. et al., Life Sci (1986) 3_8: 1243-1249 (-CH₂-S) ; Hann, M.M. , J Chem Soc Perkin Trans I (1982) 307-314 (-CH-CH-, cis and trans); Al quist, R.G. et al., J Med Chem (1980) 21:1392-1398 (-COCH₂-) ; Jennings-White, C. et al., Tetrahedron Lett (1982) 23_:2533 (-COCH₂-) ; Szelke, M. et al., European Appln. EP 45665 (1982) CA: 92:39405 (1982) (-CH(OH)CH₂-) ; Holladay, M.W. et al.. Tetrahedron Lett (1983) 14:4401-4404 (-C(OH)CH₂~) ; and Hruby, V.J., Life Sci (1982) 11:189-199 (-CH₂-S-) ; each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is -CH₂NH-. Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half- life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others. Labeling of peptidomimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling. Such non-interfering positions generally are positions that do not form direct contacts with the macromolecules(s) (e.g., immunoglobulin superfamily molecules) to which the peptidomimetic binds to produce the therapeutic effect. Derivitization (e.g., labelling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61: 387, incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

Peptides or peptidomimetics which are pharmaceutically active as immunosuppressants may be administered to a patient for prophylaxis and therapy, typically to prevent or reverse allograft (or xenograft) rejection.

For therapeutic or prophylactic uses, a sterile composition containing a pharmacologically effective dosage of one or more peptide or peptidomimetic is administered to a human patient or veterinary non-human patient for treatment of a immunopathological condition. Generally, the composition will comprise a peptide or peptidomimetic that is identical to or substantially similar to a CAMP-c polypeptide sequence that binds cyclophilin C. A pharmaceutically acceptable carrier or excipient is often employed in such sterile compositions. Routes of administration are typically intramuscular or intravenous injection or topical application, however some chemical forms of the invention may be effectively administered orally or by other routes. The compositions for parenteral administration will commonly comprise a solution of an peptide or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g. , water, buffered water, 0.9% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the peptide(s) or peptidomimetic(s) in these formulations can vary widely, i.e. , from less than about 0.01%, usually at least about 0.1% to as much as 5% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Thus, a typical pharmaceutical composition for intramuscular injection could be made up to contain 1 ml sterile buffered water, and about 10-1000 g of polypeptide. A typical composition for intravenous infusion can be made up to contain 250 ml of sterile Ringer's solution, and about 100-1000 mg of peptide. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th Ed. , Mack Publishing Company, Easton, Pennsylvania (1980) , which is incorporated herein by reference. Excipients should be chemically compatible with the peptide(s) or peptidomimetic(s) that are the active ingredient(s) of the preparation, and generally should not increase decomposition, denaturation, or aggregation of active ingredient(s) .

Antibodies The present invention also provides polyclonal and/or monoclonal antibodies capable of specifically binding to the CAMP-c polypeptides and fragments thereof. The term antibody is used both to refer to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities. Peptide fragments may be prepared synthetically in a peptide synthesizer and coupled to a carrier molecule (e.g., keyhole limpet hemocyanin) and injected into rabbits over several months. The rabbit sera is tested for immunoreactivity to the CAMP-c protein or fragment. Monoclonal antibodies may be made by injecting mice with the protein polypeptides, fusion proteins or fragments thereof. Monoclonal antibodies will be screened by ELISA and tested for specific immunoreactivity with the CAMP-c polypeptide or fragments thereof. See, E. Harlow and D. Lane, (1988) Antibodies: A Laboratory Manual. CSH Laboratories, incorporated herein by reference for all purposes. These antibodies will be useful in assays as well as pharmaceuticals.

Once a sufficient quantity of the desired polypeptide has been obtained, it may be used for various purposes. A typical use is the production of antibodies specific for binding. These antibodies may be either polyclonal or monoclonal and may be produced by in vitro or in vivo techniques well known in the art.

For production of polyclonal antibodies, an appropriate target immune system is selected, typically a mouse or rabbit. The substantially purified antigen is presented to the immune system in a fashion determined by methods appropriate for the animal and other parameters well known to immunologists. Typical sites for injection are in the footpads, intramuscularly, intraperitoneally, or intradermally. Of course, another species may be substituted for a mouse or rabbit.

An immunological response is usually assayed with an immunoassay. Normally such immunoassays involve some purification of a source of antigen, for example, produced by the same cells and in the same fashion as the antigen was produced. A variety of immunoassay methods are well known in the art. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual. CSH Laboratory; or Goding (1986) Monoclonal Antibodies: Principles and Practice, 2d ed, Academic Press, New York, both incorporated herein by reference for all purposes.

Monoclonal antibodies with affinities of 10⁸ M^"1 preferably 10⁹ to 10¹⁰, or stronger will typically be made by standard procedures as described, e.g., in Harlow and Lane (1988) or Goding (1986) , which are hereby incorporated herein by reference. Briefly, appropriate animals will be selected and the desired immunization protocol followed. After the appropriate period of time, the spleens of such animals are excised and individual spleen cells fused, typically, to immortalized myeloma cells under appropriate selection conditions. Thereafter the cells are clonally separated and the supernatants of each clone are tested for their production of an appropriate antibody specific for the desired region of the antigen. Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors. See Huse et al. (1989) Science 2_4_6:1275-1281, incorporated herein by reference. The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or noncovalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminent agents, magnetic particles and the like.

Patents, teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see Cabilly, U.S. Patent No. 4,816,567, incorporated herein by reference.

Native CAMP-c proteins, fragments thereof, or analogs thereof, may be used to immunize an animal for the production of specific antibodies. These antibodies may comprise a polyclonal antiserum or may comprise a monoclonal antibody produced by hybridoma cells. For general methods to prepare antibodies, see Antibodies: A Laboratory Manual. (1988) E. Harlow and D. Lane, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, which is incorporated herein by reference.

For example but not for limitation, a recombinantly produced fragment of murine CAMP-c can be injected into a rat along with an adjuvant so as to generate an immune response. Rat immunoglobulins which bind the recombinant fragment with a binding affinity of at least 1 x 10⁷ M"¹ can be harvested from the immunized rat as an antiserum, and may be further purified by immunoaffinity chromatography or other means. Additionally, spleen cells are harvested from the rat and fused to myeloma cells to produce a bank of antibody-secreting hybridoma cells. The bank of hybridomas can be screened for clones that secrete immunoglobulins which bind the recombinantly produced fragment with an affinity of at least 1 x 10⁵ M"¹.

For some applications of these antibodies, such as identifying immunocrossreactive proteins, the desired antiserum or monoclonal antibody(ies) are not monospecific. In these instances, it may be preferable to use a synthetic or recombinant fragment of CAMP-c as an antigen rather than using the entire native protein. More specifically, where the object is to identify immunocrossreactive polypeptides that comprise a particular structural moiety, such as a cyclophilin-c-binding domain, it is preferable to use as an antigen a fragment corresponding to part or all of a commensurate structural domain in the CAMP-c protein. Production of recombinant or synthetic fragments having such defined amino- and carboxy- termini is provided by the CAMP-c sequences shown in Fig. 2. One use of such antibodies is to screen cDNA expression libraries, preferably containing cDNA derived from human or murine mRNA from various tissues, for identifying clones containing cDNA inserts which encode structurally- related, immunocrossreactive proteins, that are candidate novel cyclophilin-binding proteins. Such screening of cDNA expression libraries is well known in the art, and is further described in Young et al., Proc. Natl. Acad. Sci. U.S.A. 8_0_.:1194-1198 (1983), which is incorporated herein by reference] as well as other published sources. Another use of such antibodies is to identify and/or purify immunocrossreactive proteins that are structurally or evolutionarily related to the native CAMP-c protein or to the corresponding CAMP-c fragment (e.g., functional domain; cyclophilin-c-binding domain) used to generate the antibody.

Various other uses of such antibodies are to diagnose and/or stage leukemias or other neoplasms, and for therapeutic application (e.g., as cationized antibodies or by targeted liposomal delivery) to treat immunological diseases and neoplasia.

Screening Assays: Drug Identification This invention is particularly useful for screening compounds by using the CAMP-c polypeptide or binding fragment thereof in any of a variety of drug screening techniques.

The CAMP-c polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, or borne on a cell surface. One method of drug screening utilizes eucaryotic or procaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, for the formation of complexes between an CAMP-c polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between a CAMP-c polypeptide or fragment and cyp C is interfered with by the agent being tested.

Thus, the present invention provides methods of screening for drugs, e.g., immunomodulating agents, comprising contacting such an agent with a CAMP-c polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the CAMP-c polypeptide or fragment, or (ii) for the presence of a complex between the CAMP-c polypeptide or fragment and cyp C, by methods well known in the art. In such competitive binding assays CAMP-c polypeptide or fragment is typically labeled. Free CAMP-c polypeptide or fragment is separated from that present in a protein:protein complex, and the amount of free (i.e., uncomplexed) label is a measure of the binding of the agent being tested to CAMP-c or its interference with CAMP-c:cyp C binding, respectively.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the CAMP-c polypeptides and is described in detail in Geysen, European Patent Application 84/03564, published on September 13, 1984, incorporated herein by reference. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with CAMP-c polypeptide and washed. Bound CAMP-c polypeptide is then detected by methods well known in the art.

Purified CAMP-c can be coated directly onto plates for use in the aforementioned drug screening techniques.

However, non-neutralizing antibodies to the polypeptide can be used to capture antibodies to immobilize the CAMP-c polypeptide on the solid phase.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding CAMP-c compete with a test compound for binding to CAMP-c polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants of CAMP-c.

The screening assays of the present invention may utilize isolated or purified forms of these assay components. This refers to nucleic acid segments, polypeptides and the like of the present invention which have been separated from their native environment (e.g., a cytoplasmic or nuclear fraction of a cell) , to at least about 10-50% purity. A substantially pure composition includes such agents that are approaching homogeneity, i.e., about 80-90% pure, preferably 95-99% pure.

Methods for Assaying Heterodimerization

Methods of screening for agents that reduce the binding of a CAMP-c polypeptide to a cyclophilin protein, and more particularly that prevent the specific heterodimerization of native CAMP-c to native cyclophilin C, also can identify novel candidate immunosuppressants. Heterodimerization assays involve in vitro binding assays comprising CAMP-c and cyclophilin polypeptides (native, fragments, or analogs) , wherein test agents can be added to the binding reaction(s) and tested for their ability to inhibit heterodimer formation or reduce the affinity of binding. Agents which interfere with the intermolecular binding between the CAMP-c protein (or fragment or analog thereof) and the cyclophilin C protein (or fragment or analog thereof) are thereby identified as candidate immunosuppressants.

These methods of screening may involve labelling CAMP-c and/or cyclophilin C polypeptides, or corresponding fragments or analogs with any of a myriad of suitable markers, including radiolabels (e.g., ¹²⁵I, ¹⁴C, or ³²P) , or various fluorescent labels and enzymes (e.g., glutathione-S- transferase, luciferase, and /3-galactosidase) . If desired for basic binding assays, one of the components may be immobilized by standard techniques. For example but not for limitation, such immobilization may be effected by linkage to a solid support, such as a chromatographic matrix, or by binding to a charged surface, such as a plastic 96-well microtiter dish. In one class of embodiments, parallel heterodimerization reactions are conducted, wherein one set of reactions serves as control and at least one other set of reactions include various quantities of agents, mixtures of agents, or biological extracts, that are being tested for the capacity to inhibit pairwise heterodimerization between a CAMP- c polypeptide (native, fragment, or analog) and a cyclophilin C polypeptide (native, fragment, or analog) . Agents that inhibit heterodimerization relative to the control reaction(s) are thereby identified as candidate immunosuppressants.

In each case, the labeled polypeptide is contacted with the immobilized polypeptide under aqueous conditions that permit specific binding of the CAMP-c to cyclophilin C. Particular aqueous conditions may be selected by the practitioner according to conventional methods. However, preferable embodiments utilize the following buffered aqueous conditions: 10-250 mM NaCl, 5-100 mM Tris HC1, pH 5-8. It is appreciated by those in the art that additions, deletions, modifications (such as pH) and substitutions (such as KC1 substituting for NaCl or buffer substitution) or additions

(e.g., addition of a mild detergent such as Tween) may be made to these conditions. Modifications can be made to the basic binding reaction conditions so long as specific binding of CAMP-c protein to cyclophilin C occurs in the test reaction. Conditions that do not permit specific binding in control reactions (no agent included) are not suitable for use in binding assays.

Preferred embodiments include heterodimerization assays which use CAMP-c and cyclophiln C polypeptides which are produced by recombinant methods or chemically synthesized.

Additional preferred embodiments comprise CAMP-c and cyclophilin C analogs that have superior stabilities as experimental reagents. For example, preferred analogs may be resistant to degradation by proteolytic activities present in the binding reaction(s) , and/or may be resistant to oxidative inactivation. Such analogs may include amino acid substitutions which remove proteolytic cleavage sites and/or replace residues responsible for oxidative inactivation (e.g., methionine, cysteine) . However, the analogs must be functional in at least the control heterodimerization assay(s); therefore, analogs comprising amino acid substitutions which destroy or significantly degrade the functional utility of the analog in the heterodimerization assay are not employed for such assays. Preferable embodiments employ a reaction temperature of at least 4 degrees Centigrade, more preferably 25 to 42 degrees Centigrade, and a time of incubation of at least 15 seconds, although longer incubation periods are preferable so that, in some embodiments, a binding equilibrium is attained.

Binding kinetics and the ther odynamic stability of bound CAMP- c:cyclophilin C complexes determine the latitude available for varying the time, temperature, salt, pH, and other reaction conditions. However, for any particular embodiment, desired binding reaction conditions can be calibrated readily by the practitioner using conventional methods in the art, which may include binding analysis using Scatchard analysis, Hill analysis, and other methods (Proteins, Structures and Molecular Principles, (1984) Creighton (ed.), W.H. Freeman and Company, New York) .

Specific binding of labeled CAMP-c protein to cyclophilin is determined by including unlabeled competitor protein(s) (e.g., albumin) and/or unlabeled competitor CAMP-c or competitor cyclophilin. After a binding reaction is completed, labeled CAMP-c protein that is specifically bound to cyclophilin C is detected. For example and not for limitation, after a suitable incubation period for binding, the aqueous phase containing a non-immobilized CAMP-c protein is removed and the substrate containing an immobilized cyclophilin C and any labeled protein bound to the cyclophilin C is washed with a suitable buffer, optionally containing unlabeled blocking agent(s) , and the wash buffer(s) removed. After washing, the amount of detectable label remaining specifically bound to the immobilized cyclophilin C is determined (e.g., by optical, enzymatic, autoradiographic, or other radiochemical methods) . In some embodiments, addition of unlabeled blocking agents that inhibit non-specific binding are included. Examples of such blocking agents include, but are not limited to, the following: CsA, bovine serum albumin, nonionic detergents (NP-40, Tween, Triton X-100, etc.), nonfat dry milk proteins, Denhardt's reagent, polyvinylpyrrolidone, Ficoll, and other blocking agents. Practioners may, in their discretion, select blocking agents at suitable concentrations to be included in binding assays; however, reaction conditions are selected so as to permit specific binding between a CAMP-c protein and cyclophilin C protein in a control binding reaction. Blocking agents are included to inhibit nonspecific binding of labeled CAMP-c protein to immobilized cyclophilin C (or other protein) and/or to inhibit nonspecific binding of labeled cyclophilin to immobilized CAMP-c protein (in such alternative embodiments.

In embodiments where protein is immobilized, covalent or noncovalent linkage to a substrate may be used. Covalent linkage chemistries include, but are not limited to, well- characterized methods known in the art (Kadonaga and Tijan, Proc. Natl. Acad. Sci. (U.S.A.) 83: 5889-5893 (1986) , which is incorporated herein by reference) . One example, not for limitation, is covalent linkage to a substrate derivatized with cyanogen bromide (such as CNBr-derivatized Sepharose 4B) . It may be desirable to use a spacer to reduce potential steric hindrance from the substrate. Noncovalent bonding of proteins to a substrate include, but are not limited to, bonding of the protein to a charged surface and binding with specific antibodies. Polypeptides are typically labeled by incorporation of a radiolabeled nucleotide (H³, C¹⁴, S³⁵, P³²) or a biotinyl moiety that can be detected by labeled avidin (e.g., avidin containing a fluorescent marker or enzymatic activity) .

Methods Relating to Genetic Disease In one preferred embodiment of the invention, hybridization probes that specifically identify the CAMP-c gene may be used in methods for diagnosing genetic disease. For example, but not for limitation, the genetic disease thus diagnosed may involve a lesion in the relevant CAMP-c structural or regulatory sequences, or may involve a lesion in a genetic locus closely linked to the CAMP-c locus and which can be identified by restriction fragment length polymorphism or DNA sequence polymorphism at the linked CAMP-c locus. In a further preferred embodiment, CAMP-c gene probes are used to diagnose or identify a genetic disease, wherein the amount or functionality of endogenous CAMP-c is sufficient for the individual to exhibit an increased probability of developing a genetic condition, particularly a immunological condition.

Antisense Polynucleotides

Additional embodiments directed to modulation of immune system function (e.g. , lymphocyte activation) include methods that employ specific antisense polynucleotides complementary to all or part of the sequences shown in Figs. 2 or 11. Such complementary antisense polynucleotides may include nucleotide substitutions, additions, deletions, or transpositions, so long as specific hybridization to the relevant target sequence corresponding to Figs. 2 or 11 is retained as a functional property of the polynucleotide. Complementary antisense polynucleotides include soluble antisense RNA or DNA oligonucleotides which can hybridize specifically to CAMP-c mRNA species and prevent transcription of the mRNA species and/or translation of the encoded polypeptide (Ching et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 16: 10006; Broder et al. (1990) Ann. Int. Med. 113: 604; Loreau et al. (1990) FEBS Letters 274: 53; Holcenberg et al., W091/11535; U.S.S.N. 07/530,165 ("New human CRIPTO gene"); WO91/09865; WO91/04753; WO90/13641; and EP 386563, each of which is incorporated herein by reference) . The antisense polynucleotides therefore inhibit production of CAMP-c polypeptides. Antisense polynucleotides that prevent transcription and/or translation of mRNA corresponding to CAMP- c polypeptides may inhibit lymphocyte activation and/or reverse the activated phenotype of T cells. Compositions containing a therapeutically effective dosage of CAMP-c antisense polynucleotides may be administered for treatment of immune conditions, particularly allotype graft rejections and lymphocytic leukemias. Antisense polynucleotides of various lengths may be produced, although such antisense polynucleotides typically comprise a sequence of about at least 25 consecutive nucleotides which are substantially identical to a naturally-occurring CAMP-c polynucleotide sequence, and typically which are identical to a sequence shown in Figs. 2 or 11.

Antisense polynucleotides may be produced from a heterologous expression cassette in a transfectant cell or transgenic cell, such as a transgenic pluripotent hematopoietic stem cell used to reconstitute all or part of the hematopoietic stem cell population of an individual. Alternatively, the antisense polynucleotides may comprise soluble oligonucleotides that are administered to the external milieu, either in the culture medium in vitro or in the circulatory system or interstitial fluid in vivo. Soluble antisense polynucleotides present in the external milieu have been shown to gain access to the cytoplasm and inhibit translation of specific mRNA species. In some embodiments the antisense polynucleotides comprise methylphosphonate moieties. For general methods relating to antisense polynucleotides, see Antisense RNA and DNA, (1988), D.A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) .

Isolation of the Cognate Human CAMP-c Gene The human homolog of the murine CAMP-c gene is identified and isolated by screening a human genomic clone library, such as a human genomic library in yeast artificial chromosomes, cosmids, or bacteriophage λ (e.g., λ Charon 35), with a polynucleotide probe comprising a sequence of about at least 20 contiguous nucleotides (or their complement) of the cDNA sequence shown in Figs. 2 and 11. Typically, hybridization and washing conditions are performed at high stringency according to conventional hybridization procedures. Positive clones are isolated and sequenced. For illustration and not for limitation, a full-length polynucleotide corresponding to the sequence of Fig. 2 may be labeled and used as a hybridization probe to isolate genomic clones from a human or murine genomic clone libary in λEMBL4 or λGEMll (Promega Corporation, Madison, Wisconsin) ; typical hybridization conditions for screening plaque lifts (Benton and Davis (1978) Science 196: 180) can be: 50% formamide, 5 x SSC or SSPE, 1-5 x Denhardt's solution, 0.1-1% SDS, 100-200 μg sheared heterologous DNA or tRNA , 0-10% dextran sulfate, 1 xlO⁵ to 1 x 10⁷ cpm/ml of denatured probe with a specific activity of about 1 x 10⁸ cpm/μg, and incubation at 42°C for about 6-36 hours. Prehybridization conditions are essentially identical except that probe is not included and incubation time is typically reduced. Washing conditions are typically 1-3 x SSC, 0.1-1% SDS, 50-70°C with change of wash solution at about 5-30 minutes.

Nonhuman CAMP-c cDNAs and genomic clones (i.e., cognate nonhuman CAMP-c genes) can be analogously isolated from various nonhuman cDNA and genomic clone libraries available in the art (e.g., Clontech, Palo Alto, CA) by using probes based on the sequences shown in Figs. 2 and 11, with hybridization and washing conditions typically being less stringent than for isolation of human CAMP-c clones. Polynucleotides corresponding to or complementary to the nucleotide sequences shown in Figs. 2 or 11 can serve as PCR primers and/or hybridization probes for identifying and isolating germline genes corresponding to CAMP-c. These germline genes may be human or may be from a related mammalian species, preferably rodents or primates. Such germline genes may be isolated by various methods conventional in the art, including, but not limited to, by hybridization screening of genomic libraries in bacteriophage λ or cosmid libraries, or by PCR amplification of genomic sequences using primers derived from the sequences shown in Figs. 2 or 11. Human genomic libraries are publicly available or may be constructed de novo from human DNA.

Genomic clones of CAMP-c, particularly of the murine CAMP-c gene, may be used to construct homologous targeting constructs for generating cells and transgenic nonhuman animals having at least one functionally disrupted CAMP-c allele. Guidance for construction of homologous targeting constructs may be found in the art, including: Rahemtulla et al. (1991) Nature 353: 180; Jasin et al. (1990) Genes Devel. 4_.: 157; Koh et al. (1992) Science 256: 1210; Molina et al. (1992) Nature 357: 161; Grusby et al. (1991) Science 253: 1417; Bradley et al. (1992) Bio/Technology 10: 534, incorporated herein by reference) . Homologous targeting can be used to generate so- called "knockout" mice, which are heterozygous or homozygous for an inactivated CAMP-c allele. Such mice may be sold commercially as research animals for investigation of immune system development, neoplasia, immunodeficiency, transplantation hosts, and other uses.

Chimeric targeted mice are derived according to Hogan, et al., Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E.J. Robertson, ed. , IRL Press, Washington, D.C., (1987) which are incorporated herein by reference. Embryonic stem cells are manipulated according to published procedures (Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E.J. Robertson, ed. , IRL Press, Washington, D.C. (1987); Zjilstra et al. (1989) Nature 342:435; and

Schwartzberg et al. (1989) Science 246: 799, each of which is incorporated herein by reference) .

Additionally, a CAMP-c cDNA or genomic gene copy may be used to construct transgenes for expressing CAMP-c polypeptides at high levels and/or under the transcriptional control of transcription control sequences which do not naturally occur adjacent to the CAMp-c gene. For example but not limitation, a constitutive promoter (e.g., a ESV-tk or pgk promoter) or a cell-lineage specific transcriptional regulatory sequence (e.g., a CD4 or CD8 gene promoter/enhancer) may be operably linked to a CAMP-c-encoding polynucleotide sequence to form a transgene (typically in combination with a selectable marker such as a neo gene expression cassette) . Such transgenes can be introduced into cells (e.g., ES cells, hematopoietic stem cells) and transgenic cells and transgenic nonhuman animals may be obtained according to conventional methods. Transgenic cells and/or transgenic nonhuman animals may be used to screen for immunological enhancing agents and/or to screen for potential immunosuppressants, as overexpression of CAMP-c or inappropriate expression of CAMP-c may result in a altered lymphocyte activation responses.

Identification and Isolation of Proteins That Bind CAMP-c Proteins that bind to CAMP-c and/or a CAMP-c: cyclophilin complex are potentially important signal transduction and regulatory proteins. Such proteins may be targets for novel immunosuppressant agents. These proteins are referred to herein as accessory proteins. Accessory proteins may be isolated by various methods known in the art.

One preferred method of isolating accessory proteins is by contacting a CAMP-c polypeptide to an antibody that binds the CAMP-c polypeptide, and isolating resultant immune complexes. These immune complexes may contain accessory proteins bound to the CAMP-c polypeptide. The accessory proteins may be identified and isolated by denaturing the immune complexes with a denaturing agent and, preferably, a reducing agent. The denatured, and preferably reduced, proteins can be electrophoresed on a polyacrylamide gel. Putative accessory proteins can be identified on the polyacrylamide gel by one or more of various well known methods (e.g. , Coomassie staining, Western blotting, silver staining, etc. ) , and isolated by resection of a portion of the polyacrylamide gel containing the relevant identified polypeptide and elution of the polypeptide from the gel portion.

A putative accessory protein may be identified as an accessory protein by demonstration that the protein binds to CAMP-c and/or a CAMP-c:cyclophilin complex. Such binding may be shown in vitro by various means, including, but not limited to, binding assays employing a putative accessory protein that has been renatured subsequent to isolation by a polyacrylamide gel electrophoresis method. Alternatively, binding assays employing recombinant or chemically synthesized putative accessory protein may be used. For example, a putative accessory protein may be isolated and all or part of its amino acid sequence determined by chemical sequencing, such as Edman degradation. The amino acid sequence information may be used to chemically synthesize the putative accessory protein. The amino acid sequence may also be used to produce a recombinant putative accessory protein by: (1) isolating a cDNA clone encoding the putative accessory protein by screening a cDNA library with degenerate oligonucleotide probes according to the amino acid sequence data, (2) expressing the cDNA in a host cell, and (3) isolating the putative accessory protein. Alternatively, a polynucleotide encoding a CAMP-c accessory polypeptide may be constructed by oligonucleotide synthesis, placed in an expression vector, and expressed in a host cell.

Putative accessory proteins that bind CAMP-c and/or a CAMP-c:cyclophilin complex in vitro are identified as accessory proteins. Accessory proteins may also be identified by crosslinking in vivo with bifunctional crosslinking reagents (e.g., dimethylsuberimidate, glutaraldehyde, etc.) and subsequent isolation of crosslinked products that include a CAMP-c polypeptide. For a general discussion of cross-linking, see Kunkel et al. (1981) Mol. Cell. Biochem. 34: 3, which is incorporated herein by reference. Preferably, the bifunctional crosslinking reagent will produce crosslinks which may be reversed under specific conditions after isolation of the crosslinked complex so as to facilitate isolation of the accessory protein from the CAMP-c polypeptide. Isolation of crosslinked complexes that include a CAMP-c polypeptide is preferably accomplished by binding an antibody that binds a CAMP-c polypeptide with an affinity of at least 1 x 10⁷ M^"1 to a population of crosslinked complexes and recovering only those complexes that bind to the antibody with an affinity of at least 1 x 10⁷ M^"1. Polypeptides that are crosslinked to a CAMP-c polypeptide are identified as accessory proteins.

Screening assays can be developed for identifying candidate immunosuppressant agents as being agents which inhibit binding of CAMP-c to an accessory protein under suitable binding conditions.

Rational Drug Design The goal of rational drug design is to produce structural analogues of biologically active polypeptides of interest or of small molecules with which they interact, e.g., agonists, antagonists, inhibitors, in order to fashion drugs which are, e.g., more active or stabile forms of the polypeptide, or which, e.g. , enhance or interfere with the function of a polypeptide in vivo. See, e.g. , Hodgson (1991) Bio/Technology 9_: 19, incorporated herein by reference. In one approach, one first determines the three-dimensional structure of a protein of interest or, for example, of a protein- inhibitor complex, by x-ray crystallography, by computer modelling or, most typically, by a combination of the two approaches. Less often, useful information regarding the structure of a polypeptide may be gained by modelling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (J. Erickson et al. (1990) Science 249: 527, incorporated herein by reference) .

It is also possible to isolate a target-specific antibody, selected by a functional assay, e.g., inhibition of elastase, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating antiidiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analogue of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. The selected peptides would then act as the pharmacore.

Thus, one may design drugs which have, e.g., improved CAMP-c activity or stability or which act as inhibitors, agonists, antagonists, etc. By virtue of the present invention, sufficient amount of polypeptide may be made available to perform such analytical studies as X-ray crystallography. In addition, the knowledge of the CAMP-c protein sequence provided herein will guide those employing computer modelling techniques in place of or in addition to x- ray crystallography.

CAMP-c polypeptides can be used for rational drug design of candidate immunosuppressants. The CAMP-c cloned sequence provided herein permits production of substantially pure CAMP-c polypeptides which can be used for protein X-ray crystallography or other structure analysis methods. Alternatively, or in conjunction with physical methods, the sequence information provided herein can be analyzed by computer methods used to predict structures of CAMP-c polypeptides and protein complexes. Potential therapeutic drugs may be designed rationally on the basis of structural information thus provided. In one embodiment, such drugs are designed to prevent formation of a CAMP-c protein:cyclophilin C complex.

Thus, the present invention may be used to design drugs, including drugs with a capacity to inhibit binding of CAMP-c to cyclophilins, particularly to cyclohilin C having at least one CAMP-c binding site. The design of compounds that interact preferentially with a CAMP-c polypeptide can be developed using computer analysis of three dimensional structures. A set of molecular coordinates can be determined using: (1) crystallographic data, (2) data obtained by other physical methods, (3) data generated by computerized structure prediction programs operating on the deduced amino acid sequence data provided herein, or, preferably, a combination of these data. Examples of physical methods that may be used to define structure are, for example, two-dimensional ho onuclear correlated spectroscopy (COSY) . For those skilled in the art with one-dimensional NMR spectroscopy, COSY provides the kind of information available from a single-frequency decoupling experiment (e.g., which spins are scalar coupled to one another) . In a COSY plot, the ID spectrum lies along the diagonal, and the off-diagonal elements are present at the intersection of chemical shifts of groups that are J coupled. The "fingerprint" region contains i¹^ , ¹H^α) cross-peaks from the peptide backbone. The degree of resolution of the "fingerprint" region of the COSY map obtained in H₂0 is a good predictor of the success of sequence-specific assignments to be obtained without recourse to isotopic labeling.

Transferred nuclear Overhauser effect (TRNOE) spectra (¹H NMR) relies on different 2D NOE spectra, and, in essence, looks at the conformation of the ligand just after it has dissociated from the protein. The use of TRNOE presumes, however, that the bound and free ligands are in fast exchange on the chemical shift time scale, which translates to a ligand K_D greater than or equal to about 1 x 10^"4 M. TRNOE methods are useful to crosscheck and augment the distance information obtained by other approaches.

It is not intended that the present invention be limited by the particular method used to obtain structural information. Furthermore, it is not intended that the present invention be limited to a search for any one type of drug; one or more of the molecules may be naturally-occurring or may be synthetic, or may be a chemically-modified form of a naturally- occurring molecule. In some embodiments, it is desirable to compare the structure of CAMP-c protein(s) to the structure(s) of other proteins, particularly cyclophilin-associated factors. This will aid in the identification of and selection of drugs that either selectively affect CAMP-c, or have a broad-spectrum effect on more than one species of cyclophilin-binding factor. In some embodiments, it is desired that drugs that interfere with the binding of CAMP-c protein(s) to cyclophilin be developed, particularly involving binding to cyclophilin C that contains at least one CAMP-c binding sequence. In such a case, structural information concerning CAMP-c:cyclophilin complexes may be obtained in several ways known in the art.

In addition to physical methods, the deduced amino acid sequence data of CAMP-c may be used for computer structure modeling (molecular modeling) using three-dimensional techniques. The invention will better be understood by reference to the following illustrative examples. The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL EXAMPLES

Purification of 77, 60 and 37 kD proteins and cloning of CAMP-c cDNA The 77kD, 60kD and 37kD proteins are encoded by a single gene, designated the "cyclophilin C associated membrane protein" (or "CAMP-c") . Transfection studies using COS7 cells reveal that the CAMP-c gene produces a glycoprotein of 77kD, in agreement with initial results using AC6 cells. Treatment with glycosidases Endo F or Endo H reveals a core polypeptide of

64kD, in agreement with the predicted weight based on sequence. The CAMP-c gene belongs to a family of genes encoding proteins containing a cysteine rich domain which is found on a variety of cell surface molecules (Freeman et al. (1990) Proc. Natl. Acad. Sci. USA 87: 8810) . The CAMP-c protein may also be present on the cell surface. Surprisingly, this result places cyp C directly in line as part of a potential signalling pathway which spans the cell membrane. Previous studies have demonstrated that cyp C (as well as cyp A) has the potential to intersect and inhibit an intracellular signalling pathway in the presence of the drug CsA.

Northern analysis of CAMP-c mRNA (Fig. 4) reveals that it is expressed in a pattern virtually identical to that of cyp C mRNA. CAMP-c is found in AC 6 cells and is induced by IL-1. It is also expressed at relatively high levels in the kidney and spleen, but not in the liver. This evidence of co- expression at the message level suggests that in addition to interacting at the protein level, the genes for CAMP-c and cyp C may be coordinately regulated. Protein sequence determination To determine the actual amino acid sequence or to obtain polypeptide fragments of CAMP-c, the protein may be digested with enzymes such as trypsin, clostripain, or Staphylococcus protease or with chemical agents such as cyanogen bromide, O-iodosobenzoate, hydroxylamine or 2-nitro-5-thiocyanobenzoate. Peptide fragments may be separated by reversed-phase high performance liquid chromatography (HPLC) and analyzed by gas-phase sequencing. Other sequencing methods known in the art may also be used. The CAMP-c polypeptides or specific fragments thereof may be used to affinity purify respective members.

Examples

1. Large Scale culture of AC 6 AC 6 cells were grown in standard bone marrow medium consisting of RPMI 1640, 5% fetal bovine serum (Sigma), lmM sodium pyruvate (Irvine Scientific) , 2mM L-glutamine (Irvine Scientific) and 10^"5M β-mercaptoethanol. Cells were cultured in humidified incubators at 37 ° C with 7% C0₂. Cells were passed using trypsin:EDTA and expanded by splitting at a 1:10 dilution every 7 days. Large-scale preparations consisted of 100-200 150 mm dia tissue culture dishes. Cells were grown to confluence and refed with bone marrow medium containing 10% FCS to increase cell density. Some cultures were grown in the presence of IL-1 prior to harvesting in an attempt to increase the yield of CAMP-c. Addition of IL-1 did not appear to affect the overall yield of CAMP-c.

2. Protein harvesting and purification Confluent AC 6 monolayers were washed twice with cold

PBS and placed on ice. Cells were harvested by addition of 5 ml of lysis buffer (0.5% triton X-100, 150mM NaCl, 25 mM TrisHCl pH 6.5 or 25 mM Hepes pH 6.6, lmM MgCl₂, 2mM CaCl₂, lmM PMSF, lmM DTT) per plate on ice, followed by cell scraping and collection into centrifugation bottles. Crude lysate was spun at 6,000 rpm in a GSA rotor for 30 minutes at 4'C to pellet nuclei and large debris.

Lysates were then divided up into 200 ml aliquots and precleared by addition of 300 μg of glutathione-s-transferase plus 5 ml of a 50% solution of glutathione agarose (Sigma) . Incubation was performed at 4'C with gentle agitation for a minimum of 3 hours, a maximum of 10 hours. Reactions were then spun for 15 minutes at 2000 rpm in a Beckman centrifuge, and the supernatants were transferred to fresh tubes. Specific absorption of CAMP-c was performed by adding 200 μg of cyp C- GST fusion and 5 ml of a 50% solution of glutathione agarose. Affinity reactions were performed at 4'C with gentle agitation for 1-3 hours. Proteins were recovered by spinning tubes at 2000 rpm in a Beckman centrifuge and retaining the glutathione agarose pellets. Pellets were washed three times with 50 ml of fresh lysis buffer followed by recentrifugation and discarding of the supernatant. Pellets were then resuspended in 10 ml of lysis buffer and transferred to 15 ml tubes for centrifugation. Pellets were washed IX with 3 volumes of 50 mM TrisHCl pH 6.5. Pellets were then resuspended in 2 volumes of 50 mM TrisHCl plus 30 μg/ml CsA and allowed to incubate on ice for 15-30 minutes. Supernatant was collected, and the pellets were washed again with 2 volumes of 50 mM Tris-HCl and supernatants were combined. An equal volume of cold 20% TCA was added to the supernatants, and this mixture was allowed to remain on ice for 20 minutes before centrifugation at 10,000 rpm in a Sorval SS34 rotor. Supernatants were discarded, and pellets were washed 2 times with 5 ml of cold acetone before being vacuum dried. Samples were resuspended in SDS-PAGE sample buffer containing 50 mM DTT and held at 65°C for 15 minutes before loading on either 9.5% or 11% SDS-PAGE gels.

Following electrophoresis, proteins were electroblotted onto PVDF membranes (Millipore) using a Biorad apparatus and a transfer buffer consisting of 129 mM glycine and 25 mM Tris base. Proteins were transferred at 4'C with a current of .85 amps for 1-2 hours. Membranes were stained/fixed/ estained in standard coomassie blue, methanol, acetic acid solutions to visualize transferred proteins. Bands of interest were excised for further processing.

3. Preparation of proteins for sequence determination Chips of PVDF membrane containing the proteins of interest were chopped into small squares and placed into a methanol solution containing .5% PVP-40 for 1 hour at room temperature in order to block the membrane prior to in-situ protease digestion. Chips were washed extensively with water to remove excess PVP 40. Chips were then placed in a minimum volume (approximately 100 μl) of trypsin digestion buffer (lOOmM Tris-HCl pH 8.5, 5% acetonitrile) and incubated with freshly prepared trypsin (Boehringer/Mannheim) at an approximate substrate to enzyme ratio of 20:1 (weight/weight) at 37"C overnight. Digest supernatant was transferred to a separate tube, and membranes were washed IX with 200 μl of 80% formic acid (vol/vol) , followed by a single wash with distilled water. Washes were combined with the original supernatant and samples were prepared for microbore HPLC by reducing volume to 200 μl or less and removing formic acid by lyophilization. Subsequent sample preparation and sequence analysis were performed in the PAN core facility.

Peptides were loaded onto a microbore reversed-phase HPLC column eluted with a linearly increasing gradient of acetonitrile in 0.1% triflouroacetic acid. Peaks were followed by in-line UV detection and were collected by hand into microfuge tubes.

4. Isolation of partial cDNA clone by PCR

Oligonucleotides corresponding to both sense and antisense strands were synthesized for several stretches of protein sequence. These oligonucleotides were used in PCR reactions in all possible pairwise combinations (using oligonucleotides of opposite polarity) . Template for PCR reactions was plasmid prep DNA from AC6 cell cDNA libraries 3. Approximately 100 ng of plasmid library was used per PCR reaction in a total volume of 100 μl. Standard PCR buffer with 1.5mM MgCl₂ was used for all reactions. The successful primer pair and the successful reaction conditions were arrived at empirically. The PCR conditions were an initial 30 cycles with a denaturation step at 94°C for 1 minute followed by cooling down to 37°C and remaining at 37°C for 2 seconds, followed by a 72°C step of 1 minute. No PCR products were detectable (by ethidium bromide staining of agarose gels) between any pair of primers after 30 cycles of amplification. A second round of amplification was performed by transferring .5ul of reaction mix into a fresh 100 μl reaction containing buffer, enzyme. nucleotide and the same primer pair. The second PCR reactions were performed with a denaturation step at 94°C for 1 minute, an annealing step of 1 minute at 55°C, and a polymerization step of 1 minute at 72°C for 35 additional cycles. Under such conditions, it was obvious that there were a number of incorrect PCR products. PCR products were cloned either by blunt end ligation or by first cutting with restriction enzymes at sites provided by the primers, followed by ligation into appropriately prepared pBS SK- plasmid vectors (Stratagene) . Numerous cloned PCR products (approximately 30) were partially sequenced before the isolation of the correct clone. We were unable to find any conditions which amplified a 'correct' ethidium staining band without using two rounds of amplification. We suspect that this difficulty is due either to the specific sequence of our primers, or more likely to their carrying inosines and/or degenerate nucleotides at certain positions.

The successful primer pair is listed in Table 2. DNA was ethanol precipitated from the PCR reaction between these two primers, and cut with the restriction enzymes BamHl and EcoRl. Digests were run on a 1.2% low melt agarose gel (FMC Bioproducts) and excised and purified by organic extraction and precipitation before being ligated into BamHl/EcoRl cut pBS SK. Plasmids containing inserts were grown up and sequenced using the dideoxy chain termination method and Sequenase reagents (United States Biochemical Corp.).

5. Screening AC.6 plasmid library for longer cDNA clones An AC.6 cDNA library of complexity 4xl0⁶ in an XL-1 blue host was plated at a density of approximately 50,000 colonies per plate on 150 mm LB/amp plates. Colonies were lifted off the plates onto nitrocellulose filters (Scleicher and Schule) and processed sequentially by placing filters onto Whatman paper saturated with 1% SDS, then denaturing solution followed by neutralization solution. Filters were then dried and baked in a vacuum oven for 2 hours. Filters were prewashed in .1% SSC, .5% SDS at 65°C for at least one hour prior to prehybridization. Prehybridization and hybridization were carried out in a solution of 5XSSC, 1%SDS, 0,5% nonfat dry milk, 100 μg/ml prepared salmon sperm DNA at 65°C for 16 hours. Filters were washed at 65°C in 0.1XSSC, 0.1%SDS for 30 minutes as a final wash before autoradiography. P³² labeled probes were prepared by excising the 664 bp BamHl/EcoRl PCR fragment from pBS SK-. Gel purified fragment was labeled by random priming in the presence of 50μCi of [ -³²P]dCTP (Amersham) followed by chromatography over a sephadex G-50 column. Labeled DNA was boiled before addition to hybridization reaction.

Regions of bacterial plates containing positive colonies were cored with a pasteur pipette and diluted in 1ml of fresh L broth. Dilutions of this resuspension were plated and the entire process was repeated until isolated positive colonies were obtained. Screening time and procedure could be reduced in secondary and tertiary platings by testing individual bacterial colonies by PCR. This was accomplished by innoculating bacteria into a PCR reaction mixture using a toothpick, followed by making a record of the colony with the same toothpick (or pipette tip) on a fresh LB/amp plate.

Primers used for this screening were internal to the primers used in isolating the original 664 bp partial cDNA clone, and had the following sequence: CAMP-c #2: 5' GGCCAGTTGCAGATCCCTG 3* [SEQ ID NO:l] and CAMP-c #4 5' CACTCTCATGATGACGCTG 3' [SEQ ID NO:2]. These primers amplify a fragment of approximately 250 bp. PCR conditions for these reactions were: 94°C, 1 minute; 55°C, 1 minute; and 72°C, 1 minute for 30 cycles. Using PCR as part of the screening procedure was a significant time saver both from the perspective of shortening the time required to isolate a single positive clone, and for elimination of false positives.

6. Northern analysis of CAMP-c expression

Northern analysis was performed as described in J. Friedman and I. Weissman (1991) Cell 6>:799-806, incorporated herein for all purposes. Labeled probe was prepared as described above using the 650 bp partial cDNA isolated by PCR. 7. Transfection experiments using pSR-alpha CAMP-c Constructs containing the CAMP-c coding region in pSR alpha were produced by digesting the CAMP-c cDNA clone with Sal 1, followed by blunt ending of the DNA ends using Klenow and all four dNTPs. Blunt ended DNA was then digested with Not 1, and the resulting fragments were resolved by agarose gel electrophoresis. The band of interest was excised from the low-melt agarose gel, melted, organically extracted and ethanol precipitated. This Not 1/ blunt fragment was ligated into pSRalpha cut with Not 1 and Stu 1. Bacteria were transformed with the ligation products, and individual colonies were assayed for the presence of the desired expression construct. Transfection of Cos 7 cells was performed by the DEAE-Dextran method, and labeled proteins were prepared from transfected cells essentially as described (Friedman and Weissman, 1991, incorporated herein by reference) .

8. DNA Sequence analysis and Homology Comparisons Homology searches were performed using the FASTDB program of Intelligenetics software. Sequence alignments and protein structure analysis were performed using the GENALIGN and PEP programs from Intelligenetics, and the PROSITE program. All programs were accessed via the Beckman Center for Molecular and Genetic Medicine computer system at Stanford.

9. Identification and purification of CAMP-c Initial experiments utilizing metabolically labeled extracts from AC 6 revealed that in the absence of drug a protein of approximately 77kD bound to cyp C (Friedman and Weissman, 1991, incorporated herein by reference) . In addition, when CsA was present during the period of incubation, this 77kD protein was no longer bound, but a new protein of 55kD was bound. A variety of experiments (including protein sequencing) showed that these proteins were unrelated to each other. When affinity purification reactions were scaled up and performed on unlabeled cell extracts, additional protein bands were observed adherent to cyp C in the absence of CsA (Figure 1) . These additional proteins had apparent molecular weights of 60kD, 37kD and approximately 25kD, and ran as diffuse bands on SDS gels. Of particular note was the observation that these proteins could be eluted from cyp C by the addition of saturating amounts of CsA. The ability to elute these partner proteins by the addition of CsA proved to be an essential step in the purification process.

Figure 1 demonstrates the spectrum of AC 6 cell lysate proteins bound to either glutathione-s-transferase (GST) or cyp C-GST. Adherent proteins are subjected to elution first by saturating amounts of CsA, and then by boiling in SDS sample buffer plus DTT. Four proteins are observed to be eluted specifically from cyp C-GST by CsA. The approximate molecular weights of the eluted proteins are 77kD, 60kD, 37kD and 25kD. The 77kD protein corresponds to the protein previously observed binding to cyp C in the absence of drug in ³⁵S labeling experiments (Friedman and Weissman, 1991, incorporated herein by reference) . Protein purification reactions were modified and scaled up as described herein in order to purify sufficient material for protein sequence determination. In brief, protein was prepared in batches by combining detergent lysate from 100- 150 cm² tissue culture dishes of superconfluent AC 6 cells. Affinity absorption reactions were run on lysate volumes of approximately one liter using 200-500 μg of cyp C-GST as affinity absorbent. Cyp C-GST was repurified by addition of glutathione agarose and subsequent centrifugation. The agarose was extensively washed with lysis buffer and then subjected to elution with CsA. Eluate was concentrated by TCA precipitation and loaded on 9.5% SDS-PAGE gels. Proteins were electroblotted onto PVDF membranes and fixed/stained/destained for visualization. Bands of interest were excised, digested in situ with trypsin and eluted from PVDF for microbore HPLC separation followed by protein sequence determination using an ABI protein sequencer.

10. Protein Sequences determined from 77kD, 60kD and 37kD proteins

We were particularly interested in the possible sequence relationship between the various proteins observed adhering to cyp C in the absence of CsA. The 60kD and 37kD proteins were the most abundant in our purification, and these proteins generated the most useable sequence for cloning purposes. The 77kD protein was of interest because it had originally been identified in labeled protein preparations, while the 60kD and 37kD proteins were either absent or difficult to detect in labeled lysates. Protein sequence from the "25kD protein has not been obtained as this protein comigrates with significant contaminating bands on SDS gels. Table 1 is a partial listing of the sequences obtained from the 77kD, 60kD and 37kD proteins.

Table 1

77kD #1 MQALEF [SEQ ID NO:3] 60kD #1 TMQALEFHTVPVEVLAK [SEQ ID NO:4]

37kD #1 TMQALEFHTVPVEVLAK

60kD #2 AV7QMSTEEIA [SEQ ID NO:5]

37kD #2 AV7QQSTEEIA [SEQ ID NO:6]

60kD #3 VEIFYR [SEQ ID NO:7] 60kD #4 ALGYE7ATQALGR [SEQ ID NO:8]

60kd #5 LFAT7QDPTFIT [SEQ ID NO:9]

From this table it is clear that all these proteins share certain sequences in common. Sequencing results suggest a relationship among the three proteins, and the possibility that they arise from a common precursor.

11. Cloning Strategy Several regions of protein sequence used for the generation of oligonucleotides for both PCR based cloning and direct oligonucleotide screening. Oligonucleotides were ordered corresponding to both the sense and antisense polarities of the protein sequence. Oligonucleotides were used in all possible pairwise combinations in PCR reactions (see experimental procedures) , and products were analyzed by agarose gel electrophoresis. The template for the PCR reactions was provided by an AC 6 cDNA library 3. PCR amplification proved to be successful in isolating a partial cDNA. However, PCR conditions and false positives were a significant problem (see experimental procedures) . In brief, no PCR products were detectable by ethidium staining between any pair of primers after 30 cycles of amplification. A second round of amplification was necessary to generate ethidium bromide-staining material. Under such conditions, it was obvious that there were a number of 'incorrect' PCR products. PCR products were cloned either by blunt end ligation or by first cutting with restriction enzymes at sites provided by the primers, followed by ligation into appropriately prepared pBS SK- plasmid vectors. Numerous cloned products were sequenced before the isolation of the correct clone. Table 2 lists the successful primer combination and the clone sequence corresponding to the primer regions. These two primers amplify a band of 664 bp. This band was purified and labeled for use as a probe to isolate longer cDNA clones from the AC 6 library. Several clones were isolated, and the sequence of the longest clone is presented in Fig. 2. An additional partial clone isolated from a cDNA library prepared from the EL-4 murine T cell line was also sequenced and found to be identical in the region of overlap with sequence in Fig. 2.

Table 2

Sense primer: (with BamHl site)

5^»CCGGATCCGTIGA(G/A)ATITT(C/T)TA 3' [SEQ ID NO: 10] Clone sequence:

5'GTGGA G ATCTT C TA 3 * [SEQ ID NO: 11] Antisense primer: (with EcoRI Site)

5'CAGAATTCAAAIGTIGG(G/A)TC(C/T)TGIGG 3' [SEQ ID NO: 12] Clone Sequence:

5'AAAGTGGG G TC T TGGGG 3' [SEQ ID NO: 13]

The CAMP-c cDNA is 2,171 nucleotides long and encodes a protein of 575 amino acids with a predicted molecular weight of 64kD and a predicted pi of 4.7. Homology searches at the amino acid level reveal that the CAMP-c contains a cysteine rich region found on the ectodomain of several cell surface proteins (Aruffo et al. (1991) J. Exp. Med. 174: 949; Freeman et al. 1990 op.cit. ; Kodama et al. (1990) Nature 343: 531; Matsumoto et al. (1990) Proc. Natl. Acad. Sci. USA 87: 9133; and Rohrer et al. (1990) Nature 343: 570; incorporated herein by reference) . An alignment of this region is shown in Figure 3. Thus far, no function has been ascribed to this cysteine rich domain, and it is known to be dispensable for the function of at least one of the proteins in which it occurs, the macrophage scavenger receptor (Rohrer et al. (1990) op.cit. ) . Other members of this family of proteins may have as many as four copies of this cysteine rich domain, while CAMP-c possesses only one copy. Other features of the CAMP-c sequence which are evident from sequence analysis are: several potential n-linked glycosylation sites, several potential phosphorylation sites for PKC and CK 2 kinases, and potential myristylation sites. Analysis of the CAMP-c protein sequence for predicted secondary structure reveals that CAMP-c possesses a single potential transmembrane spanning region near the carboxyl terminus (residues 468-491) In addition, the amino terminus of CAMP-c consists of 18 uncharged and largely hydrophobic residues which are likely to serve as a signal sequence.

Figure 4 shows a northern analysis of CAMP-c mRNA expression. Labeled full-length CAMP-c cDNA hybridizes to a single message of approximately 2,400 nucleotides. Interestingly, CAMP-c appears to be expressed in much the same tissue distribution as cyp C. CAMP-c is expressed by AC 6, appears to be inducible by IL-1 in AC 6 cells, is present in kidney and spleen, but absent or weakly expressed in the liver. These results suggest that expression of CAMP-c mRNA may be coupled in some way to expression of cyp C mRNA.

12. Transfection of CAMP-c cDNA into COS cells directs the production of a 77kD glycoprotein which binds to

CYP ^~

CAMP-c cDNA was cloned into a modified pSR-alpha expression vector (Takebe et al. (1988) Mol. Cell Bio. 8_: 466,

_SU_BSTITUTESHEET incorporated herein by reference) and transfected into C0S7 cells using the DEAE-Dextran method. ³⁵S labeled proteins were prepared by labeling the transfected COS cells 72 hours after transfection with methionine and cysteine for 3 hours. Figs. 5a and 5b show the results of standard affinity reactions using cyp C-GST as the affinity ligand. Panel 5a demonstrates that the observed 77kD protein present in CAMP-c transfected cells will bind to cyp C in the absence (lane 1) but not the presence (lane 2) of CsA. This is in agreement with the observed behavior of the native CAMP-c protein from AC6 cells. Fig. 5b shows the effect of glycosidase treatment on the recombinant CAMP-c protein. Lane 1 shows untreated protein, lane 2 shows protein after treatment with endoglycosidase F , lane 3 shows protein after treatment with endoglycosidase H, and lane 4 shows protein after treatment with o-glycanase. Both endo H and endo F treatments reveal a core polypeptide of 64kD, in agreement with the predicted molecular weight.

13• CAMP-c as an integral membrane protein On the basis of the results of homology comparisons, secondary structure analyses and the demonstration that CAMP-c is a glycoprotein, CAMP-c is an integral membrane protein. In support of this possibility, early purification attempts revealed the necessity for detergent in the lysis buffer in order to solubilize CAMP-c.

14. Production of monoclonal antibodies against Cyp C The antigen, recombinant cyp C-GST protein, was prepared as described (Friedman and Weissman, 1991, incorporated herein by reference) .

Fischer rats were immunized with 100-200 μg of purified cyp C-GST at approximately two week intervals. Animals received antigen in either saline or incomplete adjuvant carrier. After two immunizations, a serum sample from each animal was tested for reactivity against cyp C-GST in an ELISA assay. The animal with the highest reactivity was boosted with purified cyp C (purified away from GST after cleaving cyp C-GST with thrombin) , and was sacrificed three days later for isolation of splenic cells for use in a fusion reaction to produce hybridomas.

In order to produce hybridomas, immunized Fischer rat splenic cells were fused with the partner cell line FoxNY

(Taggart and Samloff (1982) Science 2_19:1228-1230, incorporated herein by reference) for the production of hybridomas. The technique used is basically that described by Foung et al. (1982) Proc. Natl . Acad. Sci . USA 79:7484-7488, incorporated herein by reference.

Individual clones growing in 96 well plates were screened by taking a sample of hybridoma conditioned medium and assaying for the presence of anti-cyp C antibodies using an ELISA reaction. In brief, cyp C-GST was immobilized on plastic wells of a 96 well plate. Hybridoma supernatants were incubated in these wells, giving any antibodies reactive with cyp C a chance to bind to the immobilized protein. The wells were thoroughly washed, and then incubated with an enzyme linked antibody which binds to rat immunoglobulin (alkaline phosphatase-conjugated goat anti-rat antibody from Fisher Co.). Again, the wells were thoroughly washed to remove unbound antibodies. Finally, the enzymatic reaction was commenced with the addition of a substrate for the alkaline phosphatase (p- nitrophenyl phosphate, Sigma Corp.), and the reactions were scored. Hybridomas scoring positive in this assay were further screened by ELISA against GST alone, in order to confirm that the antigen recognized was in fact cyp C. Hybridomas surviving this test were expanded and subcloned to ensure that a stable cell line was obtained. General techniques for the expansion and growing of hybridoma cells are described in E. Harlow and D. Lane, eds. (1988) Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory Press, chapter 7, incorporated herein by reference.

Using this technique, we have isolated 6 independent monoclonal antibodies which react with cyp C. These monoclonals have been further characterized (1) by immunofluorescence, in order to determine whether they are capable of staining tissues expressing cyp C; (2) by immunoprecipitation, to determine whether they are capable of precipitating cyp C from cell lysates; (3) and by western blotting, to determine whether they can recognize cyp C. Table 3 below summarizes the results of these characterizations, and provides names for the antibodies we have isolated.

Table 3

The antibody D4(l) has the property of being able to co-immunoprecipitate the CAMP-c protein along with cyp C. Thus, this particular antibody recognizes a different portion of the cyp C molecule than is recognized by the other antibodies which can immunoprecipitate cyp C, as is shown in the accompanying figures. Antibodies E6, Gil and D4 2A5 will not precipitate cyp C in the presence of cyclosporin A, while D4(l) will precipitate cyp C in the presence or absence of cyclosporin A. This suggests that the antibodies which are sensitive to cyclosporin may bind to the same region of cyp C that is bound by the CAMP-c protein and that this same region is bound by cyclosporin itself. Thus, the idiotypes of the antibodies which are cyclosporin sensitive may themselves resemble the structure of cyclosporin and the structure of that region of the CAMP-c protein which binds to cyp C.

15. Immunofluorescence staining using anti-cyp C antibodies

Freshly isolated murine tissues were frozen in OCT embedding medium on a block of dry ice. Subsequently, frozen tissue blocks were sectioned with a Hacker cryostat to yield 6 micron thick sections. Sections were affixed to glass slides and allowed to dry prior to fixation. Tissue sections were fixed for 10 minutes at room temperature in acetone, and subsequently allowed to dry.

A drop of antibody containing supernatant produced by the anti-cyp C hybridomas was placed on the tissue section and allowed to incubate at room temperature for 20 minutes.

Sections were then washed in PBS to remove unbound antibody.

A second stage goat anti-rat antibody conjugated to fluorescein (Caltag Co.) was incubated with the tissue sections for an additional 20 minutes in the dark. Excess antibody was again washed off with PBS.

Tissue sections were analyzed using a Nikon fluorescence microscope equipped with a 35 mm camera.

The immunofluorescence staining experiments verified the results of the in-situ hybridization work described above. There is detectable cyp C expression in kidney, testis and ovary. Further, these experiments allowed a much more detailed mapping of the expression of cyp C within the kidney than was possible using in-situ hybridization.

We have demonstrated that cyp C is expressed in a very well defined subset of kidney cells. It will be possible for us to express any gene of interest in this subset of kidney cells by using the promoter elements of the cyp C gene to drive gene expression. This may prove useful for the correction of defects in kidney function. Figure 10 includes photos of immunofluorescent staining using the anti-cyp C antibody D4 2A5 on sections of murine kidney.

16. Immunoprecipitation experiments

Immunoprecipitation takes advantage of the specificity of an antibody in order to purify a protein of interest from a complex mixture of proteins. The immunoprecipitation technique employed is essentially as described in Antibodies, A Laboratory Manual (1988) chapter 11, ed. E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, incorporated herein by reference.

In brief, a cell lysate containing labeled protein is prepared as described in Friedman and Weissman, 1991, incorporated herein by reference. Lysates were precleared by incubation with protein-A sepharose (Sigma Co.), followed by centrifugation. Specific antibody was then added to the supernatant, along with fresh protein-A sepharose coupled to a rabbit-anti-rat immunoglobulin (Pel-Freeze Co.). Proteins of interest were recovered along with the protein-A sepharose, which was washed extensively to remove unbound protein. Samples were then analyzed by electrophoresis on SDS-PAGE gels followed by autoradiography to detect labeled protein bands. Figure 9 shows the various antibodies in an immunoprecipitation reaction.

17• Antibodies reactive with Murine CAMP-c

Monoclonal antibodies which bind specifically to the CAMP-c polypeptides were produced by immunizing a rat with CAMP-c polypeptides and making hybridomas from splenocytes from immunized rats after a suitable period. A rat monoclonal antibody that specifically binds the 77kD, 60kD, and 37kD CAMP- c polypeptides was generated.

Potential function of cyp C revealed by interaction with CAMP-c Isolated cyp c is active as a prolyl-isomerase in the absence of CsA. CAMP-c may act as a molecular bridge between cyp C and another signalling molecule such as calcineurin. In this role, CAMP-c would be acting as an endogenous CsA-like molecule. In support of this notion are initial results showing that CsA and CAMP-c may compete for the same binding site on cyp C. Based upon results of crystal structure studies of cyp A, cyclophilins are not thought to undergo a conformational change upon the binding of CsA 7. Therefore, the possibility exists that the binding site for these proteins overlaps with the binding site for CsA, and their elution represents competitive binding of CsA.

It is possible that the ultimate effector of cyclophilin function is a molecule such as calcineurin. If this is the case, the protein/protein interactions we have observed may serve to regulate the interaction of cyclophilins and calcineurin-like molecules.

Should CAMP-c prove to mediate an interaction between cyp C and a molecule such as calcineurin, inappropriate regulation of this interaction, as might happen in the presence of CsA, may help to explain why CsA has such a specific effect on T cells and renal cells, while most other tissues which express both calcineurin and cyclophilins are unaffected. Site directed mutagenesis of both cyp C and CAMP-c will more clearly define the regions of contact. Crystallography of cyp C complexed to CAMP-c and complexed to CsA with or without calcineurin will also help to determine whether these apparently diverse ligands share common three-dimensional determinants.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

CLAIMS :

1. An isolated polynucleotide encoding a CAMP-c polypeptide or cyclophilin C binding fragment thereof.

2. A polynucleotide of Claim 1 which is substantially identical to the DNA sequence of Figure 2.

3. The polynucleotide of Claim 2 further comprising an operably linked promoter sequence.

4. An isolated recombinant vector which comprises a polynucleotide of Claim 1.

5. A prokaryotic or eukaryotic host transformed with the vector of Claim 4.

6. A substantially pure CAMP-c polypeptide or cyclophilin C binding fragment thereof.

7. A polypeptide of Claim 6 substantially identical to the amino acid sequence of Figure 2.

8. An antibody capable of specifically binding to a polypeptide or fragment of Claim 6.

9. A method of screening for an immunomodulating agent comprising contacting said agent with a CAMP-c polypeptide or cyclophilin C binding fragment thereof and assaying (i) for the presence of a complex between the agent and the CAMP-c polypeptide or cyclophilin C binding fragment thereof or (ii) for the presence of a complex between the CAMP- c polypeptide or cyclophilin C binding fragment thereof and cyclophilin C.

10. The method of Claim 9 wherein the polypeptide is labelled.

11. A composition comprising an antibody capable of binding to a CAMP-c polypeptide or a cyclophilin C binding fragment thereof.

12. A method of identifying cells expressing CAMP-c comprising contacting said cells with an antibody of Claim 11.

13. A method of screening for candidate immunosuppressants, comprising: performing a heterodimerization assay which includes a CAMP-c polypeptide with a cyclophilin C polypeptide and an agent; and determining whether the agent inhibits the heterodimerization.

14. A method of claim 13, wherein the heterodimerization assay comprises an in vitro binding reaction.

15. A method of claim 14, wherein one of the polypeptides is immobilized.