COMPOSITIONS AND METHODS OF USING TERMINALLY LABELED PEPTIDES THAT BIND MHC CLASS I MOLECULES

Abstract: The present invention provides peptides that bind to MHC class I molecules. The peptide has a core of 7 to 14 amino acids linked at either its N- or C- terminus to at least two amino acids. The terminal-most additional amino acid contains a reporter group. Thepeptides may be used in a variety of methods, including for tissue typing, vaccine development, or to isolate MHC class I molecules.

Description: Description COMPOSITIONS AND METHODS OF USING TERMINALLY LABELED PEPTIDES THAT BIND MHC CLASS I MOLECULES Technical Field The present invention relates generally to peptides that bind MHC class I molecules and their use in a variety of methods, including for tissue typing, vaccine development and MHC class I purification. Background of the Invention MHC (major histocompatibility complex) class I molecules are highly polymorphic proteins, whose function it is to display peptides derived from intracellular proteins on the cell surface for recognition by T lymphocytes (T cells). The ability to bind and display a particular peptide is dependent upon the particular MHC class I molecule expressed in an individual. If a displayed peptide is derived from a foreign molecule, such as a viral protein, a cellular immune response may be evoked. In a transplant setting, disparity of MHC class I molecules leads to graft- versus-host disease or rejection of tissue transplants. In addition, certain disease susceptibilities correlate with MHC class I type. For these reasons, among others, it is important to determine the MHC class I type in an individual. Conventionally, tissue typing has been performed using antibodies that detect specific MHC class I molecules. This method suffers from the lack of antibody reagents that can finely discriminate between class I molecules having very similar amino acid sequences. More recently, polymerase chain reaction has been used in conjunction with DNA sequence determination or probe hybridization to identify and discriminate class I molecules. Whereas this latter method may be more accurate than antibody typing, it is laborious, time-consuming, typically requires the use of radioactive materials, and must be performed by highly skilled individuals. Because MHC class I molecules display peptides to T cells, a promising approach to developing vaccines to diseases and infections, such as those caused by viruses and bacteria, is to use peptides derived from highly immunogenic molecules expressed by the pathogen as immunogens. Once an immunogenic molecule has been identified, determining which peptide derived from that molecule is useful as a vaccine has previously been performed by functional assays. A series of overlapping peptides are synthesized and administered in vitro or in vivo and the resulting immune response is assayed. Problems with this method include that it is time-consuming and it does not readily lend itself to testing multitudes of peptides. The development of rapid, direct assays for both tissue typing and screening for peptide vaccines has been hampered by difficulties in labeling peptides while retaining the ability to bind class I. MHC class I molecules have a deep cleft into which peptides are anchored at both the N- and C-terminus. Thus, end labeling of a peptide would interfere with binding to MHC class I. Indeed, the addition of a tyrosine residue at the N-terminus of a peptide greatly reduced its binding to MHC class I (Christinck et al., Nature 352:67-70, 1991). Internal labeling of peptides requires a particular amino acid within the sequence (e.g., Tyr for iodination, Lys for biotinylation), limiting the generic usefulness of such methods. In view of the problems associated with determining the MHC class I type on cells from an individual, there is a compelling need for rapid, easily performed tissue typing and peptide screening methods which are more effective and are not associated with such disadvantages. The present invention fulfills these needs and further provides other related advantages. Summary of the Invention Briefly stated, the present invention exploits the use of peptides comprising a core which binds class I molecules and is terminally labeled at either the N- or C-terminus, and may be used to perform tissue typing and screening assays, while providing other related advantages. In one aspect, the present invention provides methods for typing a cell for expression of an MHC class I molecule. Within one embodiment, the method comprises the steps of: (a) contacting the cell with a peptide having a core of 7 to 14 amino acids that binds an MHC class I molecule, wherein the core is linked at its N-terminus to a plurality of amino acids and the N-terminal amino acid of the peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the cells express the MHC class I molecule. In another embodiment, the method comprises the steps of: (a) contacting the cell with a peptide having a core of 7 to 14 amino acids that binds an MHC class I molecule, wherein the core is linked at its C-terminus to a plurality of amino acids and the C-terminal amino acid of the peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the cells express the MHC class I molecule. In another embodiment, the method comprises the steps of: (a) contacting the cell with a reference peptide that binds MHC class I and with a peptide having a core of 7 to 14 amino acids that binds the MHC class I molecule, wherein the core is linked at its N-terminus to a plurality of amino acids and the N-terminal amino acid of the peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the cells express the MHC class I molecule. In yet another embodiment, the method comprises the steps of: (a) contacting the cell with a reference peptide that binds MHC class I and with a peptide having a core of 7 to 14 amino acids that binds the MHC class I molecule, wherein the core is linked at its C-terminus to a plurality of amino acids and the C-terminal amino acid of the peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the cells express the MHC class I molecule. In another aspect, the present invention generally provides methods for screening peptides that bind to MHC class I molecules. Within one embodiment, the method comprises the steps of: (a) contacting a cell expressing an MHC class I molecule with a candidate peptide having 7 to 14 amino acids that is linked at its N-terminus to a plurality of amino acids and the N-terminal amino acid of the candidate peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the candidate peptide binds the MHC class I molecule. In another embodiment, the method comprises the steps of: (a) contacting a cell expressing an MHC class I molecule with a candidate peptide having 7 to 14 amino acids that is linked at its C-terminus to a plurality of amino acids and the C-terminal amino acid of the candidate peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the candidate peptide binds the MHC class I molecule. In another embodiment, the method comprises the steps of: (a) contacting a cell expressing an MHC class I molecule with a candidate peptide having 7 to 14 amino acids and with a peptide having a core of 7 to 14 amino acids, wherein the core is linked at its N-terminus to a plurality of amino acids and the N-terminal amino acid of the peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the candidate peptide binds the MHC class I molecule. In yet another embodiment, the method comprises the steps of: (a) contacting a cell expressing an MHC class I molecule with a candidate peptide having 7 to 14 amino acids and with a peptide having a core of 7 to 14 amino acids, wherein the core is linked at its C-terminus to a plurality of amino acids and the C-terminal amino acid of the peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the candidate peptide binds the MHC class I molecule. The present invention also provides methods of purifying MHC class I molecules. Within one embodiment, the method comprises the steps of: (a) contacting a biological preparation containing an MHC class I molecule with a peptide having a core of 7 to 14 amino acids that binds the MHC class I molecule, wherein the core is linked at its N-terminus to a plurality of amino acids and the N-terminal amino acid of the peptide contains a reporter group; and (b) isolating the MHC class I molecule bound to the peptide. In another embodiment, the method comprises the steps of: (a) contacting a biological preparation containing an MHC class I molecule with a peptide having a core of 7 to 14 amino acids that binds the MHC class I molecule, wherein the core is linked at its C-terminus to a plurality of amino acids and the C-terminal amino acid of the peptide contains a reporter group; and (b) isolating the MHC class I molecule bound to the peptide. In yet another aspect, the present invention also generally provides terminally-labeled peptides that bind a MHC class I molecule. Such a peptide has a core of 7 to 14 amino acids that binds the MHC class I molecule and is linked at either its N- or C-terminus to a plurality of amino acids, the terminal amino acid of which contains a reporter group. In one embodiment of this invention, the amino acids linked to the core comprise glycine residues. In preferred embodiments, three glycine residues are linked to either the N- or C-terminus of the core. In certain embodiments, the reporter group is biotin or fluorescein. Other aspects of the invention will become evident upon reference to the following detailed description and attached drawings. Brief Description of the Drawings Figure 1 contains graphs depicting that H-2 K d -restricted bcr-abl specific CTL can lyse syngeneic targets incubated with biotinylated bcr-abl peptides with biotin linked at either the C- or N-terminus. Chromium labeled LSTRA cells were incubated with varying concentrations of the biotinylated or nonbiotinylated bcr-abl peptides and used as CTL targets. K d -restricted bcr-abl peptide-specific CTL (BCA99) were used as effectors. Cytotoxicity assays were assessed at effector to target ratio of 5:1 in a standard 4 h chromium release assay. Figure 2 contains a graph showing that H-2 K b -restricted røs-L61 specific CTL can lyse syngeneic targets incubated with biotinylated røs-L61 peptides with biotin linked at either the C- or N-terminus. Chromium-labeled EL-4 cells were incubated with various concentrations of the biotinylated or nonbiotinylated ras- 6\ peptides and used as CTL targets. K b -restricted ras-L6\ peptide-specific CTL (BA572) were used as effectors. Cytotoxicity assays were assessed at effector to target ratio of 5:1 in a standard 4 h chromium release assay. Figure 3 contains graphs showing biotinylated peptides that bind to class I MHC molecules can be detected on target cells by flow cytometry using a fluorescence-labeled avidin-biotin detection method. LSTRA cells were incubated with various concentrations of the N- or C-terminus biotinylated bcr-abl peptides. The fluorescent staining was performed as described below and fluorescence intensity was measured by flow cytometry. Figure 4 contains flow cytometry tracings showing that the fluorescence intensity detected on target cells increases with time of incubation with biotinylated peptide. LSTRA cells were incubated with 100 μg/ml of N-terminus biotinylated bcr- abl peptide (B*-GGGGFKQSSKAL) for between 1 and 18 hours at 37°C. The fluorescent staining was performed as described below and fluorescence intensity was measured by flow cytometry. Figure 5 contains flow cytometry tracings demonstrating that biotinylated IFV and OVA peptides can be used to stain H-2 K d or K b positive cells, respectively. Biotinylated K d -specifιc bcr-abl peptide (GFKQSSKALGGGK-B*) or IFV peptide (IYSTVASSLGGGK-B*) was incubated with K d -positive LSTRA cells, and biotinylated K b -specifιc ras- 6l peptide (AGLEEYSAMGGG-B*), or OVA peptide (SI NFEKLGGGK-B*) was incubated with K b -positive EL-4 cells, respectively, at varying peptide concentrations. The fluorescent staining was performed as described below and fluorescence intensity was measured by flow cytometry. Figure 6 contains flow cytometry tracings showing that the biotinylated peptides can be used to discriminate murine cells expressing H-2 K d or K b MHC class I molecules. K d -positive LSTRA cells and K b -positive EL-4 cells were incubated with lOO μg/ml biotinylated bcr-abl peptide (GFKQSSKALGGGK-B*), IFV peptide (IYSTVASSLGGGK-B*), ras-L6l peptide (AGLEEYSAMGGGK-B*), or OVA peptide (SILNFEKLGGGK-B*), respectively. The fluorescent staining was performed as described below and fluorescence intensity was measured by flow cytometry. Figure 7 contains graphs showing that biotinylated peptides bind specifically to MHC class I molecules. Chromium-labeled LSTRA cells were incubated with bcr-abl (GFKQSSKAL), IFV (IYSTVASSL), or OVA (SILNFEKL) peptides at various concentrations (from 0.5 μg to 640 μg/ml), followed by incubation with 5 μg/ml of biotinylated bcr-abl peptide (B*-GGGGFKQSSKAL). K d -restricted bcr-abl peptide-specific CTL (BCA99) were used as effectors. Cytotoxicity assays were assessed at effector to target ratio of 5:1 in a standard 4 h chromium release assay (Fig. 6A). For peptide binding competition experiments (Fig. 6B), LSTRA cells were incubated with the bcr-abl, IFV, or OVA peptides at various concentrations (from 50 μg to 500 μg/ml), followed by incubation with 50 μg/ml of a biotinylated bcr-abl peptide (B*-GGGGFKQSSKAL). The fluorescent staining was performed as described below and fluorescence intensity was measured by flow cytometry. Detailed Description of the Invention Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms that will be used hereinafter. "Amino acids" refers to any one of the 20 L-amino acids commonly found in proteins, any one of the corresponding D-amino acids, rare amino acids, such as hydroxylysine, or a non-protein amino acid, such as homoserine. Also included within the scope of the present invention are amino acids which have been altered, e.g., by chemical means, such as methylation, amidation, or acylation. "MHC class I" refers to molecules expressed on the cell surface that bind peptides produced by intracellular degradation of proteins or introduced exogenously and display them on the cell surface. MHC class I molecules are heterodimers of a polymorphic heavy chain and β2-microglobulin. A "candidate peptide" refers to a 7 to 14 amino acid molecule to be tested for binding to MHC class I. This peptide may be used without further modification or, in the case of a direct screening assay for binding to MHC class I, it may be linked to a plurality of amino acids. A "core" refers to a 7 to 14 amino acid molecule that binds an MHC class I molecule. The sequence of the core and of the candidate peptide are derived from sequences of native proteins. The core or candidate peptide sequence may contain one or more amino acid substitutions as long as it retains binding to MHC class I. A "reference peptide" in the context of this invention refers to a molecule that binds MHC class I molecules. A "reporter group" refers to a compound conjugated to the terminal amino acid of a peptide and allows for detection of the peptide. Such compounds include radiolabels, fluorochromes, compounds which are chemiluminescent, enzymes which act upon a substrate to produce a detectable molecule, biotin and other such compounds which are detected upon the addition of a labeled ligand (e.g., avidin in the case of biotin, antibodies in the case of other molecules). As noted above, the present invention in one aspect generally provides peptides that bind to MHC class I molecules and contain a reporter group. The peptides are comprised of a core of 7 to 14 amino acids that bind an MHC class I molecule, wherein the core is linked at either its N-terminus or C-terminus to two or more amino acids. The terminal, additional amino acid also contains a reporter group. Peptides may be synthesized by standard chemistry techniques, including synthesis by an automated procedure (e.g., automated peptide synthesizers sold by Applied Biosystems, Inc., Foster City, CA). Briefly, in an automated method, peptides are prepared by solid-phase peptide synthesis methodology which involves coupling each protected amino acid residue to a resin support. If the reporter group is to be added at the N-terminus, the C-terminal peptide may be amidated or contain a free carboxylic acid. Peptides synthesized by an automated peptide synthesizer are synthesized using standard Fmoc chemistry (Fields and Noble, Int. J. Pept. Protein Res. 35:161-214, 1990). Peptides containing a C-terminal biotin group may be prepared using commercially available Fmoc-Lys-(ε-d-Biotin)-Wang resin (e.g., Anaspec, San Jose, CA). Similarly, peptides containing a biotin at the N-terminus may be synthesized using standard Fmoc Wang resins. Following synthesis, the N-terminal Fmoc group is removed, and the dry resin is transferred to a flask. A biotin reactive group, d-Biotin-4-nitrothenylester, is then added to the resin in the presence of NMP (N-methyl-pyrrolidone) and DIPEA (diisopropylethylamine) to obtain the corresponding N-terminal biotinylated peptides. After the last residue has been attached, and the N-terminal biotin group added, the protected peptide is cleaved from the resin. As an example, cleavage may be achieved with acid (0.5:0.95) mixture or 1, 2-ethanedithiol/thioanisole/water/trifluoroacetic acid (0.25:0.5:0.5:8.75) mixture if the peptides contain a methionine, cysteine, or arginine amino acid. Side chain functional groups are de-protected by treatment with CH2CI2. If desired, crude product may be then further purified by gel filtration, HPLC, partition chromatography, or ion exchange chromatography. As indicated above, the core of the peptide contains 7 to 14 amino acids and binds an MHC class I molecule. Preferred ranges of the core include 8 to 14 amino acids, 8 to 12, and 9 to 11 amino acids. The derivation of the amino acid sequence of the peptide core will depend upon the use of the peptide. For example, when the peptide is to be used as a vaccine, the core amino acid sequence may be derived from an antigenic molecule. When the vaccine is directed against a virus, the antigenic molecule may be any protein fragment that is presented by MHC class I molecules, including proteins not normally present at the cell surface except as peptides presented by MHC class I molecules. When the vaccine is directed against a tumor, the antigenic molecule may be any peptide or fragment of tumor specific or tumor associated proteins that is presented by MHC class I molecules, including proteins not normally present at the cell surface except when presented as peptides by MHC class I molecules. When the peptide is to be used for tissue typing or purification of MHC class I molecules, the core sequence may be derived from either an endogenous protein, foreign protein, or a native protein sequence, and may be altered to bind with greater affinity or specificity. Such alterations include amino acid substitutions, side chain modifications, insertions, deletions, and the like. Many amino acid sequences and motifs that bind particular MHC class I molecules have been identified (see Rammensee et al., Annu. Rev. Immunol. 11:213-44, 1993; Engelhard, Annu. Rev. Immunol. 72:181-207, 1994; Engelhard, Current Opinion in Immunol. 5:13-23, 1994). Additional amino acids are linked to either the N- or C-terminus of the core region. The additional amino acids are not derived from native amino acid sequence. At least two amino acids, and preferably not more than 25, are linked. Other ranges of amino acids that are suitable for linkage within the present invention are from 2 to 20, 2 to 15, and 2 to 10. Preferred numbers of additional amino acids are from 2 to 8. A particularly preferred number of amino acids is 3 or 4. These additional amino acids may be any of the amino acids discussed above. Preferably, the additional amino acids are small in structure (e.g., glycine, alanine, valine, serine, and threonine), with glycine being most preferred. Bulky amino acids, such as histidine, phenylalanine, tryptophan, and tyrosine are not preferred. The additional amino acids may be added at either the N-terminus or the C-terminus. While it is possible to add amino acids to both termini, it is preferable that they be added only to either the N- or C-terminus. It is also evident to one skilled in the art that the peptides may be multiply linked together containing either a single reporter group or multiple reporter groups. A reporter group or groups is added to the terminal, additional amino acid. The reporter group may be any molecule that is directly detectable or detectable upon the addition of one or more other compounds. For example, a radiolabel, such as 32p ; I25j 5 3fj ma y be used and is directly detectable. Radiolabels may be added by incorporating a labeled amino acid into the synthesis. Fluorescein or other fluorescent molecules, such as rhodamine, Texas red, phycoerythrin, or the like, may be added to the terminal residue. Fluorescein may be conveniently conjugated to a free amine group such as found at the N-terminus of the peptide or the side chain of a lysine residue. Briefly, by way of example, FITC (fluorescein isothiocyanate) is reacted with a suitable peptide in a basic buffer, such as boric acid, pH 9.2 at room temperature in the dark. Unbound FITC is subsequently removed by dialysis or by filtration on a sizing chromatography column. Using similar methods, the peptide may be labeled with Texas Red. Other protocols for labeling with various fluorescent molecules are within the purvey of one skilled in the art. A preferred reporter group for labeling peptides is biotin. As described above, biotin may be conveniently added during peptide synthesis to the C-terminus by using a commercially available biotinylated lysine residue coupled to resin. Alternatively, a biotin hydrazide compound can be used to label peptides at the C-terminus by a carbodiimide coupling of the hydrazide to carboxyl groups (Vector Laboratories, Burlingame, CA). For addition of biotin to the N-terminal amino acid, peptides may be synthesized using standard Fmoc resins. Following synthesis, the N-terminal Fmoc group is removed and a reactive biotin group is added to the resin. For example, d-Biotin-4-nitrothenylester may be reacted with the N-terminal amino group in the presence of NMP and DIPEA (diisopropylethylamine). N-hydroxysuccinimide derivatives of biotin are also suitable for attaching biotin to a primary amino group. Briefly, an N-hydroxysuccinimide ester is dissolved in either dimethylsulfoxide or dimethylformamide and reacted with the peptide in a suitable buffer, such as HEPES or a sodium bicarbonate buffer, pH 8.5. Following incubation at room temperature for 2 h, the reaction is terminated by the addition of glycine, ethanolamine, or other compound that contains free amino groups. Unreacted biotin may be removed by gel filtration or dialysis. Other biotin derivatives, such as biotin maleimide, may also be used. Biotin maleimide reacts under mild acid or neutral pH conditions in an aqueous solution with free sulfhydryl groups on a peptide, such as those found on a cysteine residue. If the peptide lacks cysteine, sulfhydryl groups may be introduced chemically. For example, primary amines may be modified to contain a free thiol group by using compounds such as 2-aminothiolane. The biotin maleimide is then reacted with the prepared peptide in a phosphate buffer, pH 7. Following incubation at room temperature for 3 h, the unreacted biotin maleimide is removed by gel filtration or by dialysis. Other suitable biotin derivatives may also be used. Reporter groups are detected by appropriately suitable means. In the case of fluorochrome reporter groups, detection may be by the well-known methods of flow cytometry, confocal microscopy, or fluorescence microscopy. Radiolabels are detectable by autoradiography, scintillation or gamma counting, or phosphor image analysis. When the peptide contains a biotin reporter group, detection is indirectly performed. A molecule, such as avidin or streptavidin, which contains a label is added to the reaction. The label is preferably a fluorescent molecule, such as fluorescein or phycoerythrin, but other types of labels, such as a radiolabel, are also suitable. With labeled avidin, the label is detected by appropriate means as described above. As noted above, this invention in another aspect provides methods for screening peptides for binding to MHC class I (e.g., to identify candidate vaccines) by either a direct assay or a competitive inhibition (indirect) assay. Within one embodiment, screening is performed by a direct binding assay using a candidate peptide having 7 to 14 amino acids that is linked at either its N- or C-terminus to at least two amino acids, the terminal amino acid of which contains a reporter group. In preferred embodiments, the candidate peptide is linked to three glycine residues at either its N- or C-terminus. The peptide is contacted with a cell expressing an MHC class I molecule. The presence of the reporter group is then detected on the cell, thereby determining whether the peptide binds the MHC class I molecule. In certain embodiments, the reporter group is biotin or fluorescein. In a preferred embodiment, the detection method uses flow cytometry. In related embodiments of the invention, methods of screening peptides for binding to MHC class I molecules is performed by an indirect assay. In this assay, the candidate peptide contains a core of 7 to 14 amino acids that binds MHC class I and contains at least two additional amino acids at the N- or C-terminus, the terminal of which contains a reporter group. In these methods, a cell expressing an MHC class I molecule is contacted with a candidate peptide having a core of 7 to 14 amino acids, in which the core is linked at its N- or C-terminus to at least two amino acids, the terminal amino acid of which contains a reporter group and with a reference peptide that is known to bind the MHC class I molecule. In preferred embodiments, the core is linked to three glycine residues at either its N- or C-terminus. The presence of the reporter group is detected on the cell, such as by flow cytometry, thereby determining whether the peptide with a reporter group binds the MHC class I molecule. In certain embodiments, the reporter group is biotin or fluorescein. Cells expressing MHC class I molecule may be obtained from a variety of sources. For example, for tissue typing uses, cells are preferably isolated from a subject. For vaccine development, cells may be isolated from a subject, typically with a predetermined MHC type, or from cultured cells. Nearly any tissue isolated from the individual may be used, as essentially all cells express MHC class I, the exceptions being sperm and red blood cells. Peripheral blood mononuclear cells (PBMC) are a preferred source. PBMC may be isolated from blood by hypotonic lysis of erythrocytes and subsequent washing of cells. Additionally, granulocytes may be removed by Ficoll-Hypaque density gradient. Cells suitable for the screening assay may also be obtained from a tissue culture cell line, such as EBV-immortalized B cells that express known MHC type or HLA-typed standard cell lines obtained from any blood bank. Such cells naturally express a particular MHC class I molecule. Cells that express a desired class I molecule following transfection with an expression vector containing the class I gene are also suitable for use within this invention. Sequences encoding class I genes may be obtained from publicly available databases, such as GENBANK or EMBL. Mutant cells deficient in class I expression or cells of a different species than the origin of the introduced class I genes are especially suitable for transfection. In order to express the class I molecule on the cell surface, the cell must also endogenously express a β2-microglobulin capable of complexing with the introduced class I molecule, or a β2-microglobulin gene must be co-transfected. Peptides within the present invention are assayed for binding by either a direct assay or a competition binding assay. Peptides which either bind directly to cells expressing an MHC class I molecule or inhibit binding of reference peptides known to bind the MHC class I haplotype are useful as vaccines. In addition, candidate peptides, which are shown by these assays to bind known MHC class I molecules, are useful in establishing a panel of peptides for MHC class I typing or isolation of MHC class I molecules. The core sequences for candidate vaccines will typically be derived from pathogenic organisms, such as viruses, bacteria, and parasites or will be derived from cancer antigens. Proteins of special interest are those with sequences that normally are processed in the MHC class I antigen processing pathway and presented by MHC class I molecules. The microbial or cancer protein of special interest to serve as vaccine targets for MHC class I restricted T cells are those with segments having a molecular configuration or "motif that will bind in the cleft of MHC class I molecules, those resulting in peptide/MHC molecule complexes present at the cell surface of the target cell in a concentration high enough to stimulate the specific T cell receptor, and those possessing a peptide/MHC molecule complex that has a molecular configuration that is recognized by a T cell receptor expressed by nominated individuals. Hepatitis viruses, HIV, rhinoviruses and the like are of special interest. Other pathogenic intracellular organisms, such as Plasmodium, which causes malaria, Leishmania, which causes leishmaniasis, and Tubercle bacillus, which causes tuberculosis, are also of interest. Cancer specific proteins, such as the mutated segment of oncogenic proteins, for example, ras and p53, and the joining region segment of chimeric proteins expressed by fusion genes and the like are of special interest. Cancer related proteins that are expressed by cancer cells in a higher concentration than expressed by normal cells are of interest as are other self proteins expressed selectively or aberrantly by cancer cells. As an aid to designing peptides for vaccines, certain sequence motifs have been identified as mediating binding to particular MHC class I molecules. (See, Rammensee et al., Annu. Rev. Immunol. 77:213-244, 1993.) Peptides derived from regions of a protein containing such a motif may be tested first in an initial round of screening. One means for screening candidate peptides is by a direct binding assay on whole cells. Briefly, cells expressing MHC class I molecules are contacted with the peptides and the presence of the reporter molecule detected. The binding of peptides to cells is preferably measured by relative fluorescence intensity. Briefly, by way of example, cells are contacted with peptides by mixing different concentrations of biotinylated peptides and incubating for 3 h at 37°C. Following incubation, cells are washed in a buffer, such as PBS containing 0.1% BSA and further incubated for 20 min at 4°C with fluorescein-conjugated avidin D (F idin D; Vector Laboratories, Burlingame, CA). Cells are washed two or three times before analysis by flow cytometry. Relative fluorescence intensity is calculated by subtracting the fluorescence value obtained from cells sustained with F idin D alone (control staining) from the fluorescence value obtained from biotin-labeled peptide plus Flavidin D (experimental staining). A peptide with a fluorescence value that is statistically significantly increased over background is further tested in a competitive binding assay as described below. The competition peptide used is preferably a reference peptide, although the core sequence may be used as well or alternatively. Competition by one of these peptides demonstrates that the candidate peptide is binding specifically to MHC class I molecules. As a preferred alternative to the direct binding assay described above, an indirect or competitive inhibition assay may be used. Briefly, for example, cells are incubated with varying concentrations of non-biotinylated candidate peptides in media for 1 h at 37°C and biotinylated reference peptides containing a core of 7 to 14 amino acids linked to at least two additional amino acids. In this assay, the competition peptide may be added before, concurrently, or after the biotinylated peptide. The biotinylated reference peptide is added at its optimum staining concentration, which will generally be between 1-100 μg/ml, and the candidate peptide is added relative to the biotinylated peptide in a range of ratios from 10 3 :1 to 1:10 3 in 10-fold increments. It is evident that one skilled in the art alternatively could fix the concentration of the candidate peptide and vary the concentration of biotinylated peptide. The cells are incubated for another three hours at 37°C and then washed three times in media. F idin D is added for an additional 20 min incubation. The cells are washed three times before analysis by flow cytometry. Fluorescence intensity is calculated by subtracting the fluorescence value obtained from cells stained with fluorescinated avidin alone from the fluorescence value obtained from biotin labeled peptide plus fluorescinated avidin. Staining without competitive inhibitor establishes maximal fluorescence. A peptide with a reduced fluorescence value compared to no inhibitor that is statistically significantly different is considered to bind MHC class I. Alternatively, purified MHC class I molecules may be used as targets in these assays, although not preferable. Briefly, for example, purified MHC class I molecules may be obtained from cells expressing MHC class I by affinity chromatography, immunoprecipitation with antibodies, or other suitable purification schemes. The purified class I molecules are used to coat ELISA plates. Unbound sites are blocked by the addition of buffer containing BSA or non-fat milk. Biotinylated candidate peptides are added to the wells and unbound peptides are washed out. Avidin that is conjugated to an enzyme, such as horseradish peroxidase, alkaline phosphatase, or β-galactosidase, is added and further incubated for an hour. A suitable chromogenic substrate is added to the wells. Absorbance is subsequently measured. An absorbance value that is statistically significant compared to the absorbance value obtained for a known MHC class I binder establishes that the peptide binds MHC class I. Alternatively, a competition binding ELISA may be performed. In such an assay, non¬ biotinylated candidate peptides are added first. Inhibition of binding of the biotinylated reference peptides, which is statistically significantly different compared to no competition added, establishes that the candidate peptides bind the MHC class I molecule. As noted above, the present invention in another aspect provides methods for typing a cell for expression of an MHC class I molecule. Tissue typing is an essential procedure performed in a pretransplant setting to establish donor and host compatibility. Tissue typing is also used in a post-transplant setting to establish chimerism or the source of a graft, especially in the context of bone marrow transplantation. Additionally, tissue typing may be used to establish paternity of a child, susceptibility to diseases and syndromes, disease associations, and suppression or enhancement of class I molecules indicative of a disease or syndrome, and the like. Within one embodiment, typing is performed by a direct binding assay. Within a related embodiment, typing is performed by an indirect binding assay. In certain embodiments, the methods use a peptide having a core of 7 to 14 amino acids that binds an MHC class I molecule, and is further linked at either its N- or C-terminus to at least two amino acids, the terminal amino acid of which contains a reporter group. In the direct assay, which is a preferred assay, the peptide is contacted with a cell expressing an MHC class I molecule. In the indirect assay methods, the cell is contacted with an unlabeled reference peptide and with the labeled peptide having a core of 7 to 14 amino acids that binds an MHC class I molecule, and is further linked at either its N- or C-terminus to at least two amino acids, the terminal amino acid of which contains a reporter group. In preferred embodiments, the core is fused to three glycine residues at either its N- or C-terminus. After contacting, the presence of the reporter group is detected on the cell, thereby determining whether the cell expresses the MHC class I molecule. In certain embodiments, the reporter group is biotin or fluorescein. The preferred detection method is flow cytometry. The peptides used in typing have a core of 7 to 14 amino acids that bind an MHC class I molecule. The core is linked at its N- or C-terminus to at least two amino acids, the terminal amino acid of which contains a reporter group. A panel of peptides that bind to all known MHC class I molecules is used in tissue typing. The panel may be generated by determining peptides that bind particular MHC class I molecules as described above. Alternatively, or in addition, peptides that have been previously established to bind particular MHC class I molecules may be used (see Engelhard, Curr. Opinion in Immunol. 6:13-23,1994). In general, typing will be performed on cells isolated from a subject. Preferably, cells are isolated from blood, bone marrow, lymphoid organs, or skin, although any diploid tissue may be used. Most preferably, cells are isolated from peripheral blood. For ease of analysis, either mononuclear cells obtained following hypotonic lysis of erythrocytes and subsequent washing or obtained from a Ficoll- Hypaque gradient may be used. The assays for tissue typing are performed as described above. A direct binding assay is preferable. A competition binding assay may also be used once binding is established in the direct assay in order to verify the specificity of binding. For a direct assay, a fluorescent assay in which biotinylated peptides are bound to cells and detected by fluorescein-conjugated avidin or streptavidin is preferred. Criteria for establishing binding are identical to those described above. In the competition binding assay, the competing peptide is preferably a reference peptide, but a peptide composed of core sequences may also be used. In tissue typing, either the entire panel of peptides may be used on cells from each individual, or a subset of peptides may be employed. A subset may be chosen based on the race and ethnicity of the subject. If the MHC type is not determined by the subset, the remaining peptides in the panel are used. As previously noted, the present invention in yet another aspect provides methods for isolating and purifying MHC class I molecules. In these methods, a biological preparation isolated from tissue, peripheral blood cells, or other cells which express an MHC class I molecule are contacted with a peptide having a core of 7 to 14 amino acids that binds an MHC class I molecule and is linked at either its N- or C-terminus to at least two amino acids, the terminal amino acid of which contains a reporter group. The MHC class I molecule bound to the peptide is subsequently isolated. In preferred embodiments, the reporter group is biotin and the substrate used for isolation contains avidin. In these methods, class I molecules are isolated from biological preparations, from in vivo or in vitro sources. Biological preparations may be obtained by solubilizing cells expressing MHC class I either endogenously or by transfection. For transfected cells, the MHC class I molecules may be found on the cell surface or in the cell culture supernatant depending on the nature of the construct. Prior to purification, an enrichment of the molecules may be preliminarily performed. When MHC class I is isolated from membranes, the membranes are first solubilized in buffer containing 1% NP-40™. MHC class I may be enriched by removal of undesired molecules by size exclusion chromatography, centrifugation, precipitation, and the like. Purification of class I molecules using a peptide of the present invention may be performed by affinity chromatography after coupling the peptide to a solid substrate. Briefly, for example, a biotinylated peptide may be coupled to avidin- Sepharose™ or streptavidin-Sepharose™. The Sepharose™ beads are then placed in a suitable column. The biological preparation is passed over the column and unwanted proteins and peptides flow through. Elution of the bound MHC class I may be performed using salt or trifluoracetic acid at pH 2.0. The isolated material may be characterized by SDS-PAGE analysis or further purified by HPLC or similar methods. Alternatively, the MHC class I molecule may be isolated by first contacting the biotinylated peptide with the biological preparation and subsequently isolating the peptide. An incubation step of 3 h may increase yields of MHC class I. Avidin coupled to a solid substrate, such as Sepharose™ beads, is then added to the mixture and the beads removed by centrifugation. Alternatively, avidin coupled to beads containing ferromagnetic particles may be contacted with the biological preparations, which are then separated out by applying a magnetic field. After washing the beads extensively, the MHC class I molecules may be eluted from the peptide with either salt or pH changes as described above. The following examples are offered by way of example and not by way of limitation. EXAMPLES EXAMPLE 1 SYNTHESIS OF PEPTIDES All peptides are synthesized (Biopolymer Facility, Dept. of Immunology, University of Washington) using FMOC Chemistry in an automated peptide synthesizer (Model 433A; Applied Biosystems, Inc., Foster City, CA), which can monitor the progress of synthesis and confirm the peptide integrity. Peptides synthesized in this way usually have purity greater than 95%, as revealed by the HPLC chromatography. Peptides containing a C-terminal biotin are constructed using commercially available Fmoc-Lys-(ε-d-Biotin)-Wang resin (Anaspec, San Jose, CA). Peptides containing an N-terminal biotin are constructed using standard Fmoc Wang resins. The N-terminal Fmoc group is removed at the end of synthesis, and the dry resin (50 mg, 0.025 mmole) transferred to a dry flask. A biotin group is added by reacting 0.25 mmole of d-Biotin-4-Nitrothenyl ester (Anaspec, San Jose, CA) with the resin in the presence of 5 ml NMP (N-methyl-pyrrolidone) and 0.25 ml DIPEA (diisopropylethylamine). Peptides are then released from the peptide resin by cleavage with either 3 ml of H2θ/trifluoroacetic acid (0.5:0.95) mixture or 3 ml of 1,2- ethanedithiol/thioanisole/Η2θ/trifluoroacetic acid (0.25:0.5:0.5:8.75) mixture if the peptides contain a methionine, cysteine, or arginine amino acid. The mixture is then evacuated. The dry residue is transferred to a filter funnel and washed repeatedly with CH2CI2 to remove deprotection groups and scavengers. The peptides are further extracted with 5% acetic acid and lyophilized. The correct masses of each peptide was verified using a Sciex API III Atmospheric Pressure Ionization Mass Spectrometer. The measured mass was typically within 0.1 % of the predicted mass. EXAMPLE 2 SPECIFICITY OF DIFFERENT LENGTH PEPTIDES The amino acid sequences of synthetic røs-L61 or bcr-abl peptides used are shown in Tables 1, 2, and 3. H-2 K b -specific ovalbumin (OVA) p257-264 (SIINFEKL) peptide, H-2 K d -specific influenza virus (IFV) A/PR 8 HA p518-526 (IYSTVASSL) peptide and H-2 D b -specific influenza NP 1934 p366-374 (ASNENMETM) peptide are constructed as control peptides. In addition, the ras-L6\ amino acid sequence was contained in 7-mers to 14-mers centering on the known binding sequence; the bcr-abl binding sequence was contained in peptides varying from 8 to 14 amino acids. The peptides were tested in a cytotoxicity assay. Cytotoxicity was measured using the target cells LSTRA (H-2K d ) or EL-4 (H-2K b ). Target cells (LSTRA) or EL-4) were harvested and resuspended at 10 8 cells/ml in 20% FCS RPMI 1640 media. 250 μCi of 51 Cr (New England Nuclear, Boston, MA) were added to each tube and incubated for 45 min. at 37°C. Labeled targets were washed 3 times with HBSS, and resuspended in 20% RPMI at lxl 0 5 cells/ml. Antigen-specific CTL were used as effectors on day 5 of stimulation. For peptide specific lysis, 51 Cr-labeled target cells were incubated with designated peptide (50 μg/ml) for 1 hr at 37°C. Effector cells were plated at 5x10 4 , 10 4 and 0.2x10 4 cells (100 μl) per well of a 96 well plate. Target cells, 10 4 cells per 100 μl, were added. Plates were incubated at 37°C for 4 h. The amount of 51 Cr released into 100 μl of the supernatant from each well was determined. The percent specific lysis was calculated by the following formula: n , . _ . . test counts - spontaneous release counts , .„ % specific lysis = x 100 maximal releasable counts - spontaneous release counts All determinations of cytotoxicity were carried out in triplicate and at a minimum of three effector to target ratios. Lytic units (LU) were calculated using the exponential fit equation (Pross et al., J. Clin. Immunol. 7:51-63, 1981). One LU is defined as the number of effector cells required to cause 33% lysis of target cells. LU are expressed as the number of LU/10 6 lymphocytes. For the competition assay, lxlOVml chromium labeled target cells (LSTRA or EL-4) were incubated with varying concentrations of reference peptides (from 5 μg to 640 μg/ml) in 15 ml tubes for 30 min. at 37°C, followed by 3 h incubation with 50 μg/ml of biotinylated peptides at 37°C. 100 μl of labeled target (10 4 cells) were plated into each well of 96 well plates and then dilutions of effectors were added. Plates were incubated for 4 hours at 37°C. 100 μl of supernatant was harvested from each well and counted in a gamma counter. The nonamer core regions were tested for recognition by syngeneic or allogeneic target cells in the cytotoxicity assay described above. As shown in Table 1, the bcr-abl core region was K d restricted, whereas, the ras- 6\ core region was K b restricted. Table 1. Specific Cytotoxicity of H-2K d Restricted Bcr-abl-Specific CTL Clone and H-2K b Restricted Rαs-L61-Specific CTL Clone Targeting Name of Target Cell MHC Class I Bcr-abl Ras-161 Peptide Amino and Peptide Restriction CTL Clone CTL Clone Acid Sequence EL-4 --- --- <1 <1 EL-4 + 0VA(p257-264) SIINFEKL K b <1 <1 EL-4 + IFV (p518-526) IYSTVASSL κ d <1 <1 EL-4 + IFV(p366-374) ASNENMETM D <1 <1 EL-4 + føs-L61(p59-67) AGLEEYSAM K <1 64.88 EL-4 + Bcr3-abl2(p922-930) GFKQSSKAL K <1 <1 LSTRA — — <1 <1 LSTRA + 0VA(p257-264) SIINFEKL κ b <1 <1 LSTRA + IFV (p518-526) IYSTVASSL κ d <1 <1 LSTRA + IFV(p366-374) ASNENMETM D <1 <1 LSTRA + føs-L61(p59-67) AGLEEYSAM K b <1 <1 LSTRA + Bcr3-abl2(p922-930) GFKQSSKAL K a 42.95 <1 Cytotoxicity assays were assessed at effector to target ratios of 25:1, 5: 1, 1 :1 and 0.2:1 in a standard 4 h chromium release assay. LU were calculated as described in Material and Methods. Targets included LSTRA tumor of Balb/c mouse origin, and EL-4 tumor of B6 mouse origin. Eight to 14 amino acid long bcr-abl and rαs-L6\ sequences containing the nonamer sequence were synthesized with the additional amino acids derived from the natural sequence of p210 bcr-abl or rαs, respectively. The results presented in Table 2 demonstrate that the nonamer bcr-abl sequence, GFKQSSKAL, is required for recognition, but that cells incubated with longer sequences, up to 14 amino acids, are equivalently lysed. Table 2. H-2 K d Restricted Bcr-abl-Specific CTL Clone Can Lyse LSTRA (H-2D) Cells Loaded with Varying Lengths of Bcr-abl Peptides Targeting Length of Specific Lvsis (%) Amino Acid Sequence Bcr-abl Peptide Peptide 5:1 1:1 0.2:1 None --- ... 6 2 1 P917-930 VHSATGFKQSSKAL 14 er 72 51 23 P918-930 HSATGFKQSSKAL 13 mer 75 53 21 P919-930 SATGFKQSSKAL 12 mer 75 50 18 P920-930 ATGFKQSSKAL 11 mer 67 42 15 P921-930 TGFKQSSKAL 10 mer 83 56 14 P922-930 GFKQSSKAL 9 mer 86 54 21 P923-930 FKQSSKAL 8 mer 13 8 3 P922-931 GFKQSSKALQ 10 mer 83 56 14 P922-932 GFKQSSKALQR 11 mer 67 42 15 P922-933 GFKQSSKALQRP 12 mer 75 50 18 P922-934 GFKQSSKALQRPV 13 mer 75 53 21 P922-935 GFKQSSKALQRPVA 14 mer 72 51 23 Chromium-labeled LSTRA tumor cells were pre-incubated with designated bcr-abl peptide (50 μg/ml) for 1 h at 37°C before used as targets. Cytotoxicity assays were assessed at effector to target ratios of 5:1, 1:1 and 0.2:1 in a standard 4 h 5l Cr -release assay. Similarly, cells incubated with 8 to 14 amino acids of the ras-L61 sequence are equivalently lysed, as shown in Table 3. Table 3. H-2 K b Restricted Rαs-L61 -Specific CTL Clone Can Lyse EL-4 (H-2B) Cells Loaded with Varying Lengths of Rαs-L61 Peptides Targeti ng . . Seαuence Length of Speci fic Lysi s (X) Ras Pepti de Am n0 ACi α ^ uence Peptide 25: 1 5: 1 1 : 1 None - -- --- 4 2 2 P54-67 DILDTAGLEEYSAM 14 mer 57 18 7 P55-67 ILDTAGLEEYSAM 13 mer 60 33 16 p56-67 LDTAGLEEYSAM 12 mer 67 52 23 p57-67 DTAGLEEYSAM 11 mer 60 31 13 p58-67 TAGLEEYSAM 10 mer 60 43 22 p59-67 AGLEEYSAM 9 mer 63 52 27 p60-67 GLEEYSAM 8 mer 57 46 26 p59-68 AGLEEYSAMR 10 mer 38 12 1 P59-69 AGLEEYSAMRD 11 mer 28 20 9 P59-70 AGLEEYSAMRDQ 12 mer 57 46 27 P59-71 AGLEEYSAMRDQY 13 mer 50 33 17 P59-72 AGLEEYSAMRDQYM 14 mer 20 13 2 Chromium-labeled EL-4 tumor cells were pre-incubated with designated Rαs-L6l peptide (50 μg/ml) for 1 h at 37°C before used as targets. Cytotoxicity assays were assessed at effector to target ratios of 25:1, 5:1 and 1:1 in a standard 4 h 51 Cr -release assay. Labeling the core region with a biotin moiety at the N-terminus dramatically reduced specific lysis for both the bcr-abl and rαs-L6l sequence (Figures IA, IB). However, in contrast, the addition of three glycine residues plus a biotin to the 9-mer bcr-abl recognition sequence allowed substantially better lysis than the equivalent length native sequence containing a biotin. Linking the additional residues GGGK and biotin to the C-terminus gave nearly equivalent lysis of target cells as the native, unlabeled peptide (Figure IB). Similar results were obtained when the rαs-L61 core region had either GGG plus biotin added to the N-terminus or GGGK plus biotin added to the C-terminus (Figure 2). EXAMPLE 3 BIOTINYLATED-PEPTIDE/MHC CLASS I DIRECT BINDING ASSAY Detection of biotinylated peptide bound to MHC class I molecules on living cells is performed by incubating target cells (lxl0 6 /tube) in 15 ml plastic tubes with biotinylated peptides (from 0.1 μg/ml to 100 μg/ml) for 3 h at 37°C. After incubation, cells are washed two times in the 15 ml plastic tubes with 15 ml ice-cooled PBS containing 0.1% BSA, 0.08% sodium azide, pH 7.2. The cells are then incubated for 20 min at 4°C with 0.5 μg of fluorescein-conjugated avidin D (Vector Laboratories, Burlingame, CA) in 100 μl of PBS. After incubation, cells are washed two times, resuspended and fixed in 500 μl PBS containing 1% paraformaldehyde. Fluorescence intensity of living cells is measured by flow cytometry (Coulter Instruments). This assay may also be performed in 96-well plates. For staining in 96- well plates, cells (lOVwell) are incubated with biotinylated peptides for 3 h at 37°C in a volume of 100 μl. After incubation, cells are washed three times with 200 μl cold PBS (0.1% BSA, 0.08% sodium azide, pH 7.2). The cells are then incubated for 20 min at 4°C with 0.5 μg of FITC-avidin-D in 100 μl of PBS on ice. After incubation, cells are washed three times, resuspended and fixed in 500 μl PBS with 1% paraformaldehyde. Fluorescence intensity is measured on a flow cytometer (Coulter). LSTRA cells were incubated with varying concentrations (from 25 μg/ml to 200 μg/ml) of N- or C-terminus biotinylated peptides or core regions as described above. A shown in Figures 3A and 3B, N-terminus labeled 9-mer bcr-abl peptide (B*-GFKQSSKAL) and the C-terminus biotin-labeled 10-mer bcr-abl peptide (GFKQSSKALK-B*) failed to stain K d -positive cells. The natural sequence N-terminus biotin-labeled 12-mer (B*-SATGFKQSSKAL), which was weakly recognized by peptide-specific CTL in functional recognition assays, also failed to stain K d -positive cells in the fluorescence assay (Fig. 3C). By contrast, the 12-mers linked with biotinylated polyglycine at either the N-terminus (B*-GGGGFKQSSKAL) or C-terminus (GFKQSSKALGGGK-B*), which were strongly recognized by peptide specific CTL, bind and stain K d -positive LSTRA cells in a dose-dependent manner (Fig. 3D, E). Further studies showed that substantial fluorescence could be detected with an incubation time as short as one hour and increased with up to 18 hours (Fig. 4). Similar studies using 12-mer ras-L6\ peptides linked with biotinylated polyglycine at either the N-terminus (B* -GGG AGLEEYSAM) or C-terminus (AGLEEYSAMGGGK-B*) which were strongly recognized by the ras-L6\ peptide specific CTL were shown to bind and stain K d -positive EL-4 cells substantially by the fluorescence assay in a peptide dose-dependent manner (data not shown). The ability of biotinylated peptides to bind to viable cells was predicted to be directly proportional to the affinity of core sequence peptide to the designated class I MHC molecule. K d -restricted influenza core sequence (p518-526) and K b -restricted OVA core sequence (p257-264) are known to bind with high affinity. These cores were linked at their C-terminus with GGG plus biotin and tested at varying peptide concentrations in the direct fluorescence assay. Both biotinylated K d -specific bcr-abl peptide and K b -specific ras-L6\ peptides could be detected on K d -positive LSTRA or K b -positive EL-4 cells (Fig. 5 A, B), respectively, at a peptide concentration of 100 μg/ml, but only minimum fluorescence intensity was detectable at 10 μg/ml. By contrast, the biotinylated K d -specific influenza peptide (IYSTVASSLGGGK-B*) and K b -specific OVA peptide (SIINFEKLGGGK-B*) each could be readily detected at peptide concentrations as low as 0.1 μg/ml. Thus, biotinylated peptides containing higher affinity core sequence for the class I MHC molecules can be used to stain cells expressing the appropriate restricting class I MHC molecules at much lower peptide concentration. Moreover, biotinylated K d -specific and K b -specific peptides could distinguish between cells expressing K d and K b class I MHC molecules in the fluorescence staining assay. Biotinylated K d -specific bcr-abl peptide (GFKQSSKALGGGK-B*) and IFV peptide (IYSTVASSLGGGK-B*) specifically stained LSTRA (H-2K d ) cells, but failed to stain EL-4 (H-2K b ) cells. Conversely, biotinylated K b -specific ras-L6l peptide (AGLEEYSAMGGGK-B*) and OVA peptide (SILNFEKLGGGK-B*) specifically stained EL-4 cells (H-2K b ), but failed to stain LSTRA (H-2K d ) (Fig. 6A, B). EXAMPLE 4 BIOTINYLATED-PEPTIDE/MHC CLASS I INDIRECT BINDING ASSAY For competition experiments, cells (lxl0 6 /tube) were incubated first with varying concentrations of non-biotinylated competition peptides in 100 μl of RPMI 1640 complete medium in 15 ml plastic tubes for 1 h at 37°C. Cells were then incubated for 3 h with 50 μg/ml of biotinylated peptides at 37°C, following which, cells were washed 3 times in PBS and incubated for 20 min with 0.5 μg of fluorescein- conjugated avidin D (Vector Laboratories, Burlingame, CA) in 100 μl PBS. Cells were then washed 3 times and resuspended in 500 μl PBS with 1% paraformaldehyde. Fluorescence intensity was measured on a flow cytometer. Peptide competition experiments examined both CTL lysis (Fig. 7A) and fluorescence staining (Fig. 7B). In functional CTL assays the high affinity K d -specific influenza peptide (IYSTVASSL) substantially blocked lysis of target cells (LSTRA) incubated with the K d -specific biotinylated bcr-abl peptide (B*-GGGGFKQSSKAL) (Fig. 7A). By contrast, the K b -specific OVA peptide (SIINFEKL) and the nonamer bcr-abl peptide (GFKQSSKAL) had no effect. The results of fluorescence assays (Fig. 7B) showed that binding of the biotinylated peptides (B*-GGGGFKQSSKAL) was blocked by both the K d -specific influenza peptide (IYSTVASSL) and by the non¬ biotinylated nonamer bcr-abl peptide (GFKQSSKAL). The influenza peptide (IYSTVASSL), known to bind with high affinity to K d , was substantially more effective in blocking than was bcr-abl peptide (GFKQSSKAL). Thus, the results demonstrate that biotinylated peptides bind in a specific manner to class I MHC molecules. EXAMPLE 5 TISSUE TYPING USING BIOTINYLATED PEPTIDES Peripheral blood is drawn from the subject for a determination of the MHC class I type. Mononucleocells are isolated from the interface following Ficoll- Hypaque gradient centrifugation. PBMC (peripheral blood mononuclear cells) are washed in PBS containing 1% BSA. MHC class I type is assayed by incubation of live cells with biotinylated peptide panel and subsequent detection of binding using fluorescinated avidin. PBMC are aliquoted at 50,000 cells/tubes into 5 ml tubes or wells of a 96-well plate. Biotinylated peptides (from 0.1 μg/ml to 100 μg/ml, depending upon previously determined optimum staining) are added to the cells and incubated for 3 h at 37°C. As a negative control, one aliquot of cells receives buffer only. Following incubation, cells are washed three times with 1 ml of ice cold PBS containing 0.1% BSA and 0.08% sodium azide. Cells are then resuspended in 100 μl of PBS containing 0.5 μg of FITC-avidin-D (Vector Laboratories, Burlingame, CA), incubated for 20 min at 4°C and washed three times. Finally, cells are resuspended and fixed in 1% paraformaldehyde in PBS. Stained cells are examined by flow cytometry. Using forward and side scatter parameters, live cells are examined for relative fluorescence intensity. The negative control, cells incubated with fluorescinated avidin alone, serves to establish background staining. Positive staining, indicating the presence of a particular MHC class I molecule on the cell surface, has a statistically significant shift of staining from the negative control peak. Further verification of MHC class I type may be pursued by comparing staining patterns with that obtained using cells with known MHC types of the same putative MHC type as the cells from the subject. EXAMPLE 6 PURIFICATION OF MHC CLASS I Peripheral blood cells were isolated from blood by Ficoll-hypaque gradient centrifugation and washed in PBS/5% FBS. Cells are incubated with 50 μg/ml of the biotinylated peptides for 3 h at 37°C. Following incubation, cells were washed twice in PBS. Pelleted cells are lysed by resuspension in 15 ml of 0.1% (vol/vol) TFA (trifluoroacetic acid), by use of ultrasonic cell disrupter (Model W-380), or by incubation with ice-cold PBS/1% Nonidet P-40™ for 1 h at 4°C. The lysate is clarified by centrifugation. A column is constructed using avidin-Sepharose™ beads. The centrifuged cell free supernatant which contains the biotinylated peptide/MHC class I molecule complex is passed over the column. The column is washed with 20 bed volumes of PBS. After the unwanted proteins and peptides flow through, the specifically bound MHC class I molecules are eluted from the column by the addition of 1.0% TFA buffer, pH 2.0. The isolated fractions containing the MHC class I molecules may be characterized by SDS-PAGE analysis and further purified by HPLC or similar methods. All publications and patent applications mentioned in this specification are herein 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. From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

Claims: Claims 1. A method of typing a cell for expression of an MHC class I molecule, comprising the steps of: (a) contacting the cell with a peptide having a core of 7 to 14 amino acids that binds an MHC class I molecule, wherein the core is linked at its N-terminus to a plurality of amino acids and the N-terminal amino acid of the peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the cells express the MHC class I molecule. 2. A method of typing a cell for expression of an MHC class I molecule, comprising the steps of: (a) contacting the cell with a peptide having a core of 7 to 14 amino acids that binds an MHC class I molecule, wherein the core is linked at its C-terminus to a plurality of amino acids and the C-terminal amino acid of the peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the cells express the MHC class I molecule. 3. A method of typing a cell for expression of an MHC class I molecule, comprising the steps of: (a) contacting the cell with a reference peptide that binds MHC class I and with a peptide having a core of 7 to 14 amino acids that binds the MHC class I molecule, wherein the core is linked at its N-terminus to a plurality of amino acids and the N-terminal amino acid of the peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the cells express the MHC class I molecule. 4. A method of typing a cell for expression of an MHC class I molecule, comprising the steps of: (a) contacting the cell with a reference peptide that binds MHC class I and with a peptide having a core of 7 to 14 amino acids that binds the MHC class I molecule, wherein the core is linked at its C-terminus to a plurality of amino acids and the C-terminal amino acid of the peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the cells express the MHC class I molecule. 5. A method of screening peptides for binding to MHC class I molecules, comprising the steps of: (a) contacting a cell expressing an MHC class I molecule with a candidate peptide having 7 to 14 amino acids that is linked at its N-terminus to a plurality of amino acids and the N-terminal amino acid of the candidate peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the candidate peptide binds the MHC class I molecule. 6. A method of screening peptides for binding to MHC class I molecules, comprising the steps of: (a) contacting a cell expressing an MHC class I molecule with a candidate peptide having 7 to 14 amino acids that is linked at its C-terminus to a plurality of amino acids and the C-terminal amino acid of the candidate peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the candidate peptide binds the MHC class I molecule. 7. A method of screening peptides for binding to MHC class I molecules, comprising the steps of: (a) contacting a cell expressing an MHC class I molecule with a candidate peptide having 7 to 14 amino acids and with a peptide having a core of 7 to 14 amino acids, wherein the core is linked at its N-terminus to a plurality of amino acids and the N-terminal amino acid of the peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the candidate peptide binds the MHC class I molecule, 8. A method of screening peptides for binding to MHC class I molecules, comprising the steps of: (a) contacting a cell expressing an MHC class I molecule with a candidate peptide having 7 to 14 amino acids and with a peptide having a core of 7 to 14 amino acids, wherein the core is linked at its C-terminus to a plurality of amino acids and the C-terminal amino acid of the peptide contains a reporter group; and (b) detecting the presence of the reporter group on the cell, therefrom determining whether the candidate peptide binds the MHC class I molecule. 9. The method according to any one of claims 1-8 wherein the reporter group is fluorescein. 10. The method according to any one of claims 1, 3, 5, or 7 wherein the core is linked at its N-terminus to Gly-Gly-Gly. 11. The method according to any one of claims 2, 4, 6, or 8 wherein the core is linked at its C-terminus to Gly-Gly-Gly. 12. The method according to any one of claims 1-8 wherein the reporter group is detected by flow cytometry. 13. A method of purifying MHC class I molecules, comprising the steps of: (a) contacting a biological preparation containing an MHC class I molecule with a peptide having a core of 7 to 14 amino acids that binds the MHC class I molecule, wherein the core is linked at its N-terminus to a plurality of amino acids and the N- terminal amino acid of the peptide contains a reporter group; and (b) isolating the MHC class I molecule bound to the peptide. 14. A method of purifying MHC class I molecules, comprising the steps of: (a) contacting a biological preparation containing an MHC class I molecule with a peptide having a core of 7 to 14 amino acids that binds the MHC class I molecule, wherein the core is linked at its C-terminus to a plurality of amino acids and the C- terminal amino acid of the peptide contains a reporter group; and (b) isolating the MHC class I molecule bound to the peptide. 15. The method according to either of claims 13 or 14 wherein the MHC class I molecule of step (b) is isolated by a substrate comprising avidin. 16. The method according to any of claims 1-8, 13, or 14 wherein the reporter group is biotin. 17. A method according to any of claims 1-8, 13, or 14 wherein the core is linked to between 2 to 8 amino acids. 18. A peptide having a core of 7 to 14 amino acids that binds an MHC class I molecule, wherein the core is linked at its N-terminus to a plurality of amino acids and the N-terminal amino acid of the peptide contains a reporter group. 19. A peptide having a core of 7 to 14 amino acids that binds an MHC class I molecule, wherein the core is linked at its C-terminus to a plurality of amino acids and the C-terminal amino acid of the peptide contains a reporter group. 20. The peptide according to either of claims 18 or 19 wherein the plurality of amino acids comprises glycine. 21. A peptide according to either of claims 18 or 19 wherein the core is linked to between 2 to 8 amino acids. 22. A peptide having a core of 7 to 14 amino acids that binds an MHC class I molecule, wherein the core is linked at its N-terminus to three glycine residues and the N-terminal amino acid of the peptide contains a reporter group. 23. A peptide having a core of 7 to 14 amino acids that binds an MHC class I molecule, wherein the core is linked at its C-terminus to three glycine residues and the C-terminal amino acid of the peptide contains a reporter group. 24. The peptide according to any of claims 18, 19, 22, or 23 wherein the reporter group is biotin. 25. The peptide according to any of claims 18, 19, 22, or 23 wherein the reporter group is fluorescein.

Inventors: CHEEVER MARTIN A, CHEN WEI, CHEEVER, MARTIN, A., CHEN, WEI
Applicants: UNIV WASHINGTON, UNIVERSITY OF WASHINGTON
Application year: 1996
Main classification: G01N33
IPC codes: C07K1, C07K4, G01N33