Mutant MHC class I molecules

Methods of generating a conjugate of MHC class I molecule and a compound via a cysteine residue engineered into the .beta.2-M subunit. Also featured are uses of the conjugates.

BACKGROUND OF THE INVENTION
 The major histocompatibility complex ("MHC") plays a central role in the
 immune system. Antigen-specific T cells recognize antigenic peptides in
 association with MHC class I or II molecules on the cell surface. Class I
 molecules consist of two noncovalently associated subunits: a highly
 polymorphic .alpha. heavy chain and a conserved .beta.2-microglobulin
 (".beta.2-M") light chain. Two of the three extracellular domains of the
 heavy chain, i.e., domains .beta.1 and .alpha.2, are folded into a
 "groove" structure which anchors an antigenic peptide for presentation to
 T cells.
 Human class I molecules (or "complexes") have been refolded from E.
 coli-produced heavy chains and .beta.2-M subunits in the presence of
 synthetic peptides (Garboczi et al., Proc. Natl. Acad. Sci. USA,
 89:3429-3433, 1992). The three-dimensional structures of such recombinant
 complexes as determined by X-ray crystallography are virtually identical
 to the structure of the class I molecule as isolated from human cells
 (Madden et al., Cell, 75:693-708, 1993; Bjorkman et al., Nature,
 329:506-512, 1987). Further, subtype A0201* of HLA-A2 produced in E. coli
 and assembled with synthetic HIV-1 nonapeptides has been shown to elicit
 cytolytic CD8.sup.+ T cell responses (Walter et al., Int. Immunology,
 9:451-459, 1997).
 The classical class I gene family includes the highly polymorphic human
 class I molecules HLA-A, -B, and -C, and murine class I (i.e., H-2)
 molecules D, K, and L. A series of structural relatives (non-classical
 class I molecules) has been found in humans (e.g., HLA-E, -F, -G, -H, -I,
 and -J; and CD1) and mice (Q, T, M, and CD1) (Shawar et al., Annu. Rev.
 Immunol., 12:839-880, 1994). These molecules have the typical structure of
 an antigen-presenting molecule, where a polymorphic heavy chain is
 noncovalently associated with the conserved .beta.2-M subunit. The T cell
 repertoire reacting with these non-classical ligands has been
 characterized to only a limited extent.
 SUMMARY OF THE INVENTION
 The invention features a method of preparing a conjugate of an MHC class I
 molecule and a compound. In this method, one first obtains an MHC class I
 molecule, where a cysteine residue (i.e. a non-natural or new cysteine
 residue) has been engineered into its .beta.2-microglobulin subunit. The
 compound (e.g., a protein, a carbohydrate, a lipid molecule, or any other
 organic compound) is then conjugated to the mutant class I molecule
 specifically via a linkage formed between the sulfhydryl group of the new
 cysteine residue in the .beta.2-microglobulin subunit and a functional
 group of the compound. Alternatively, the compound can be first conjugated
 to the new .beta.2-M subunit, and then the subunit is mixed with an
 .alpha. heavy chain (from the same or different species as the .beta.2-M
 subunit) in the presence of an appropriate peptide to form a
 compound-class I conjugate. The cysteine residue is preferably introduced
 into a region of .beta.2-M that faces away from the interface between
 .beta.2-M and the .alpha. heavy chain. Exemplary regions are those
 corresponding to residues 15-23, 35-53, or 66-97 of SEQ ID NO: 1. SEQ ID
 NO: 1 shows the amino acid sequence of a human .beta.2-M. Minor sequence
 variations can exist among .beta.2-M molecules from different or the same
 species; and residues from two different .beta.2-M sequences are said to
 be corresponding to each other when they are equivalent in function or
 relative position to the conserved residues in the two .beta.2-M
 sequences, or both. The new residue, alone or together with one or more
 (e.g., two to five) amino acid residues, can be inserted into the
 .beta.2-M region without any deletion of the region, or replace one or
 more (e.g., two to five) residues of the region. For instance, the new
 cysteine residue can replace a residue that corresponds to serine 52,
 tyrosine 67, or lysine 91 of SEQ ID NO: 1.
 The compound can be, for example, a ligand for a multivalent binding
 molecule, an antibody (e.g., one that is specific for a tumor antigen), a
 molecule on the surface of a cell (e.g., an antigen-presenting cell or any
 other hematopoietic cell), or a ligand for a surface receptor of a cell.
 The new compound-class I conjugates have several uses. For instance, when a
 multivalent binding molecule is supplied, conjugates of the ligands for
 the binding molecule with the new, monomeric class I molecules (e.g., ones
 that consist of an .alpha. heavy chain, the new .beta.2-M, and a peptide
 associated with the heavy chain) can be multimerized. Multimeric class I
 molecules can be used to, e.g., label, isolate and quantitate specific T
 cells. Exemplary multivalent binding molecules are avidin (or a derivative
 thereof, e.g., streptavidin), whose ligands are biotin and biotin
 derivatives.
 A conjugate of the present invention can also be used to stimulate the
 immunity of an individual (e.g., a human or a mouse). To accomplish this,
 a conjugate of a new class I molecule and a cell (e.g., an
 antigen-presenting cell) is introduced into an individual, and the
 conjugate can stimulate the immune cells, particularly T cells specific
 for the peptide in the conjugate. In this method, the cell and the class I
 molecule in the conjugate are preferably syngeneic with this individual.
 Another conjugate of the present invention can be used to eradicate a tumor
 (or any other undesired cell) in an individual. In this method, a
 conjugate of a new class I molecule and an antibody specific for an
 antigen (or a ligand for a receptor) expressed exclusively or primarily on
 the cell is introduced into the individual. The .alpha. heavy chain in the
 conjugate can be allogeneic or xenogeneic) to the patient. If the heavy
 chain is syngeneic to the patient, the class I molecule would be
 associated with an antigenic peptide that can elicit a strong T cell
 response.
 Use of the new recombinant .beta.2-M, monomeric and multimeric class I
 molecules containing the new .beta.2-M, and the new compound-class I
 conjugates are also within the scope of the invention.
 The new .beta.2-M eliminates the need for genetically engineering MHC heavy
 chains to create a chemically reactive site in each of the differing MHC
 class I molecules. This is a significant advantage in view of the enormous
 polymorphism of MHC heavy chains.
 Other features and advantages of the invention will be apparent from the
 following detailed description, and from the claims.

DETAILED DESCRIPTION
 Methods are provided for preparing a conjugate of a MHC class I molecule
 and a compound. The MHC class I molecule contains a recombinant .beta.2-M
 subunit in which a cysteine residue has been introduced. This residue is
 preferably introduced into a region of .beta.2-M that does not interact
 with the .alpha. heavy chain. Standard mutagenesis techniques can be
 employed to generate DNA encoding such mutant .beta.2-M molecules. General
 guidance can be found in Sambrook et al., Molecular Cloning, A Laboratory
 Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
 1989; and Ausubel et al., Current Protocols in Molecular Biology, Greene
 Publishing and Wiley-Interscience, New York, N.Y., 1993. The cysteine
 residue introduced into the .beta.2-M subunit provides a convenient site
 for highly selective chemical modification. In addition, coupling between
 sulfhydryls and certain functional groups is reversible, allowing a
 compound to be released from the class I molecule to which it has been
 conjugated.
 The compound can be linked to the new cysteine residue in the class I
 molecule via a functional group of its own. Sulfhydryl-reactive functional
 groups include, but are not limited to, maleimides, pyridyl disulfide,
 .alpha.-haloacyl derivatives (e.g., iodoacetamides), alkyl halides, and
 aryl halides. Maleimides, alkyl and aryl halides, and .alpha.-haloacyls
 react with sulfhydryls to form thiol ether bonds. Pyridyl disulfides, on
 the other hand, react with sulfhydryls to produce mixed disulfides. The
 pyridyl disulfide product is cleavable. A sulfhydryl-reactive crosslinker
 can also be used, especially when the compound does not contain any
 sulfhydryl-reactive groups itself. Such a crosslinker possesses two or
 more different reactive groups that allow its sequential conjugations with
 two or more other molecules. For instance, crosslinkers that are
 amine-reactive at one part and sulfhydryl-reactive at another part (e.g.,
 some DOUBLE-AGENT.TM. crosslinkers available from PIERCE, Rockford, Ill.)
 can be used to link an amine-containing compound with the mutant class I
 molecule. By way of example, N-Succinimidyl 3-(2-pyridyldithio)-propionate
 ("SPDP") is a reversible NHS-ester (i.e., N-hydroxysuccinamide-ester),
 pyridyl disulfide cross-linker; the amine-reactive NHS-ester in SPDP can
 be reacted first with a compound of interest, and then with the free --SH
 group of the new class I molecule. Useful water-soluble SPDP analogs
 include sulfosuccinimidyl 6-(3-[2-pyridyldithio]propionamido) hexanoate
 (i.e., sulfonating long chain SPDP analog or Sulfo-LC-SPDP), LC-SPDP,
 4-Succinimidyloxycarbonyl-.alpha.-methyl-.alpha.-(2-pyridyldithio)-toluene
 ("SMPT"), and
 sulfosuccinimidyl-6-(.alpha.-methyl-.alpha.-[2-pyridyldithio]-toluamido)
 hexamoate ("Sulfo-LC-SMPT"). Additional sulfhydryl- and amine-reactive
 heterofunctional crosslinkers include
 succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate ("SMCC"),
 N-.gamma.-maleimidobutyryloxysulfosuccinimide ester,
 m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, sulfosuccinimidyl
 [4-iodoacetyl] aminobenzoate, and sulfosuccinimidyl 4-[p-maleimidophenyl]
 butyrate (Pierce, Ill.). Homobifunctional sulfhydryl-reactive crosslinkers
 such as Bis-maleimidohexane ("BMH"), 1,4-Di-(3'-[2'-pyridyldithio]
 propionamido-butane ("DPDPB"), and 1,5-difluoro-2,4-dinitrobenzene
 ("DFDNB") can also be used if the compound to be conjugated contains a
 sulfhydryl.
 The .alpha. and .beta.2-M subunits of the MHC class I protein can be from
 the same or different species (e.g., humans, rats, mice, hamsters, frogs,
 chickens, etc.). The transmembrane and optionally intracellular domains of
 the .alpha. subunit can be removed to promote proper in vitro folding.
 Methods for obtaining class I heavy chains and .beta.2-M subunits and for
 forming monomeric class I-peptide complexes are well known in the art
 (see, e.g., Zhang et al., Proc. Natl. Acad. Sci. USA, 89:8403-8407, 1992;
 and Garboczi et al., Proc. Natl. Acad. Sci. USA, 89:3429-3433, 1992). The
 .alpha. and .beta.2-M subunits obtained with a recombinant expression
 system (e.g., an E. coli or baculoviral system) can be refolded separately
 or together, and then associated in the presence of appropriate peptides
 (e.g., peptides of about 8-12 amino acid residues in length).
 Alternatively, the mutant .beta.2-M subunit can be associated with the
 .alpha. subunit of a pre-formed class I molecule via exchange with the
 .beta.2-M subunit in the pre-formed molecules; in such case, no peptide
 supply is needed in the association reaction if the pre-formed class I
 molecule is already occupied by a peptide.
 In general, the conjugation between the class I protein and the compound is
 performed after the .beta.2-M subunit has folded into a native or
 native-like conformation. In this conformation, the naturally occurring
 cysteine residues (4 in the heavy chain and 2 in .beta.2-M) are engaged in
 stable disulfide bonds and hence not accessible for chemical modification.
 Therefore, conjugation occurs specifically via the unpaired,
 surface-exposed cysteine.
 Described below are the generation and use of several conjugates of the
 invention. Unless otherwise defined, all technical and scientific terms
 used herein have the same meaning as commonly understood by one of
 ordinary skill in the art to which this invention belongs. Although
 methods and materials similar or equivalent to those described herein can
 be used in the practice or testing of the present invention, exemplary
 methods and materials are described below. These exemplary methods and
 materials are illustrative only and not intended to be limiting. All
 publications, patent applications, patents, and other references mentioned
 herein are incorporated by reference in their entirety. In case of
 conflict, the present specification, including definitions, will control.
 I. Cell-Class I Conjugates
 The new class I molecule can be conjugated to a compound (e.g., a protein,
 carbohydrate, or lipid molecule) on a cell surface via the new cysteine in
 the .beta.2-M subunit. To accomplish this, the surface of the cell can be
 reduced in mild conditions with a reducing reagent (e.g., dithiothreitol
 ("DDT"), 2-mercaptoethanol, or 2-mercaptoethylamine.HCl), resulting in
 reactive sites (e.g., sulfhydryl groups) on the cell surface. Such
 reactive sites will then be reacted directly, or linked through a
 bifunctional crosslinker, with the free sulfhydryl group in the class I
 molecule. By way of example, the sulfonyl groups attached to the
 succinimidyl rings in Sulfo-NHS esters (e.g., Sulfo-LC-SPDP or Sulfo-SMCC)
 make these crosslinkers membrane-impermeable and thus non-reactive with
 inner membrane proteins; thus, these crosslinkers are useful in
 crosslinking the new class I complexes to the cell surface. To determine
 optimal conjugation conditions, class I negative cells such as human
 HMy2.C1R cells (American Type Culture Collection CRL-1993) can be used.
 The density of the class I peptide complexes anchored on the cell surface
 can be determined by fluorescence-activating cell sorting ("FACS")
 analysis, using monoclonal antibodies ("MAb"s) against the class I
 molecule.
 Peptide-class I complexes conjugated to syngeneic cells can be used to
 stimulate the immunity in an individual. To do this, cells derived from
 this individual or an another with matching MHC haplotypes are conjugated
 in vitro to the new class I molecules that have been loaded with antigenic
 peptides of interest. Useful cells include, but are not limited to,
 peripheral blood lymphocytes taken from a Ficoll density gradient,
 purified antigen presenting cells such as macrophages/monocytes, dendritic
 cells, and B cells, or red blood cells. Antigenic peptides of interest
 are, for example, melanoma-associated immunodominant epitopes derived from
 melanoma-associated antigens such as MART-1/Melan A, gp 100/Pmel 17,
 tyrosinase, Mage 3, p15, TRP-1, and .beta.-catenin (Tsomides et al.,
 International Immunol., 9:327-338). The cell conjugates are then
 introduced into the individual. If the conjugates are used for
 vaccination, it may be preferred to use antigen-presenting cells as the
 conjugates' cellular components, since these cells can provide the
 requisite costimulatory signals for inducing an effective T cell response.
 The above immunization strategy allows the control of epitope density and
 circumvents a variety of problems associated with classical vaccination
 strategies. For instance, unlike traditional peptide vaccines, the
 peptides embedded in the present pre-formed, class I peptide complexes are
 protected from rapid enzymatic degradation or intracellular processing.
 Traditional peptide vaccines are generally not directly presented by class
 I molecules on the cell surface; instead, they are typically internalized
 and processed inside the cell for association with MHC class I molecules.
 Peptide presentation is thus dependent on a series of intracellular events
 including the rate of protein degradation, peptide transport, and
 competition with endogenous peptides.
 EXAMPLE 1
 Direct Conjugation of Class I Complexes with Cells
 10.sup.6 cells in PBS are reduced with 50 .mu.M DDT for 30 minutes.
 Consequently, reactive --SH groups on the cell surface are generated.
 After two washes with PBS, the reduced cells are incubated with the new
 class I complexes, resulting in formation of disulfide bonds between the
 --SH groups in the .beta.2-M subunits and the --SH groups on the surface
 of the reduced cells.
 EXAMPLE 2
 Indirect Conjugation of Class I Molecules with Cells
 10.sup.6 cells are suspended in 500 .mu.l PBS buffer (pH 7.2), and 1 mg of
 Sulfo-SMCC is added to the cell solution. The incubation proceeds for an
 hour at room temperature or 30 minutes at 37.degree. C., resulting in a
 covalent bond between the NHS-ester group in Sulfo-SMCC and a primary
 amine on the cell surface.
 The cells are then washed three times with PBS, and incubated with 0.5 mg
 of the new recombinant class I protein at 4.degree. C. for 1 hour. This
 step leads to formation of a covalent bond between the free --SH group in
 the new class I protein and the maleimide group in Sulfo-SMCC.
 II. Antibody-Class I Conjugates
 The new MHC class I molecule can be specifically targeted to a cell by
 conjugating to an antibody against an antigen expressed on the surface of
 the cell. For instance, non-self (e.g., allogeneic) class I molecules
 conjugated to antibodies specific for a tumor antigen (or any antigen
 exclusively or primarily expressed by any other undesired tissue) can be
 attached to tumor tissue (or the undesired tissue) in an individual. The
 tissue, which now bears foreign MHC molecules, becomes a target of
 allograft rejection, one of the strongest immune responses known, and can
 thereby be destroyed.
 To elicit a strong immune response against undesired tissue in an
 individual, an antibody specific for the tissue can also be conjugated to
 a syngeneic MHC class I molecule that is associated with a potent T cell
 epitope (e.g., HLA-A2 with the influenza matrix protein 59-68 (ILGFVFTLTV,
 SEQ ID NO: 2), or the influenza B NP 85-94 (KLGEFYNQMM; SEQ ID NO: 3)).
 Such a conjugate can elicit a strong zcytotoxic T cell response that
 eradicates the undesired tissue.
 A variety of monoclonal antibodies can be used to target tumor tissue.
 Exemplary tumor-associated antigens include, but are not limited to, the
 Lewisy-related carbohydrate (found on epithelial carcinomas), the IL-2
 receptor p55 subunit (expressed on leukemia and lymphoma cells), the
 erbB2/pl85 carcinoma-related proto-oncogene (overexpressed in breast
 cancer), gangliosides (e.g., GM2, GD2, and GD3), epithelial tumor mucin
 (i.e., MUC-1), carcinoembryonic antigen, ovarian carcinoma antigen MOv-18,
 squamous carcinoma antigen 17-1A, and malignant melanoma antigen MAGE. To
 extend the serum half life of the MHC-antibody conjugate, the antibody may
 be humanized if the tumor to be treated is in a human.
 The antibody and the MHC components in the present conjugate are conjugated
 via a covalent bond between the new cysteine residue in the mutant
 .beta.2-M and a functional group in the antibody. Intermediary
 crosslinkers can be used to form the covalent bond. For example, mild
 oxidation of the sugar moieties in an antibody, using, e.g., sodium
 metaperiodate, will convert vicinal hydroxyls to aldehydes or ketones. The
 reaction will be restricted to sialic acid residues when 1 mM sodium
 metaperiodate is used at 0.degree. C. Subsequent reaction of the aldehyde
 or ketone group with a sulfhydryl-reactive, hydrazide-containing
 crosslinker (e.g., 3-(2-Pyridyldithiol)propionyl hydrazide ("PDPH"),
 4-(N-maleimidomethyl)cyclohexane-1-carboxylhydrazide hydrochloride, or
 4-(4-N-maleimidophenyl)butyric acid hydrazide hydrochloride) results in
 the formation of a hydrazone bond. The antibody can then be bonded to the
 new MHC class I molecule via a disulfide bond formed between the new
 cysteine residue in the .beta.2-M and the sulfhydryl-reactive group in the
 crosslinker.
 Alternatively, mild reduction of an immunoglobulin can generate free
 sulfhydryl groups from the disulfide bonds in the hinge region. The
 sulfhydryl groups can then be reacted with the free sulfhydryl group of
 the class I complex, either directly or via a homobifunctional
 crosslinker.
 Antibody fragments (e.g., Fab, or F(ab)'.sub.2 both of which lack the
 glycosylated Fc-portion) can also be conjugated to the class I complex.
 F(ab)'.sub.2 fragments can be generated from antibody molecules by pepsin
 cleavage; these fragments can be linked through heterofunctional
 crosslinkers such as those that are amine- and sulfhydryl-reactive.
 Reduction of F(ab) .sub.2 fragments generates Fab' fragments, which
 contain free sulfhydryl groups. These free sulfhydryl groups can be
 utilized in conjugation with the new class I proteins.
 EXAMPLE
 Generation of an Antibody-Class I Conjugate
 The following is an exemplary protocol for conjugating an antibody to the
 new MHC class I molecule.
 An immunoglobulin G ("IgG") stock solution is first prepared. It contains
 2mg/ml IgG (i.e., 13 .mu.M) in 0.1 M sodium acetate buffer (pH 5.5). The
 solution is stored at 0.degree. C. To oxidize the antibody, 1 ml of the
 IgG stock solution is mixed with 0.1 ml of cold sodium metaperiodate
 solution (stock: 1 mM sodium periodate in 0.1 M sodium acetate buffer, pH
 5.5) for 20 min at 0.degree. C. in the dark. To stop the oxidation
 reaction, glycerol is added to reach a final concentration of 15 mM, and
 the incubation proceeds for 5 additional minutes at 0.degree. C.
 The IgG sample is then dialyzed over night against 0.1 M sodium acetate
 buffer (pH 5.5) and concentrated in a CENTRICON 30. The crosslinker PDPH
 is then added to the sample to a final concentration of 5 mM, and the
 sample is incubated for approximately 2 hours at room temperature.
 The IgG protein attached to PDPH is purified by HPLC (i.e., high
 performance liquid chromatography) gel filtration. The HPLC running buffer
 is 0.1 M Tris-Cl (pH 8). The IgG peak fraction is concentrated with
 CENTRICON 30 to a final volume of 100 .mu.l.
 To prepare for the class I molecule in the MHC-antibody conjugate, 2 mg of
 HPLC-purified class I molecule is mildly reduced with a solution
 containing 0.1 mM DTT and 0.1 M Tris-Cl (pH 8) for 1 hour at room
 temperature. This will maintain the cysteine in a reduced state. The class
 I molecule preparation is then purified by HPLC, which removes DTT, and
 concentrated with CENTRICON 30.
 2 mg of the resulting class I molecule is dissolved in 0.5 ml Tris-Cl (pH
 8), and added to 0.5 ml of PDPH-modified IgG for overnight incubation at
 4.degree. C. The sample is then concentrated with CENTRICON 100 and the
 MHC-antibody conjugate so obtained, which is approximately 200 kD in size,
 is purified by HPLC gel filtration or fast protein liquid chromatography.
 The conjugate is concentrated with CENTRICON 100. Excess peptide (e.g.,
 5-15 fold molar excess) is added to the concentrated conjugate preparation
 to stabilize the class I molecule in the conjugate.
 III. Multimeric MHC Class I Complexes
 The new MHC class I molecule can also be used to form a multimeric MHC
 class I complex. To do so, one can first obtain a conjugate of a monomeric
 class I molecule and a ligand for a multivalent binding molecule. The
 conjugate is formed specifically via a linkage between the sulfhydryl
 group of the new cysteine in the .beta.2-M subunit and a functional group
 of the ligand. Useful ligands include, but are not limited to,
 iodoacetyl-LC-biotin, N-(6-[Biotinamido]
 hexyl)-3'-(2'pyridyldithio)-propionamide ("biotin-HPDP"), and
 1-Biotinamido-4-(4'-[maleimidomethyl] cyclohexane-carboxamido)butane
 ("Biotin-BMCC"). All these biotin derivatives bind to avidin (or a
 derivative thereof such as streptavidin), a tetravalent molecule.
 Quantitative blocking of the biotin-binding sites on avidin will render
 the avidin molecule mono-, bi-, or tri-valent. To generate the conjugate,
 the .alpha. and .beta.2-M subunits may be allowed to associate in the
 presence of a peptide of interest to form a stable heteroduplex complex,
 and then be linked to the ligand; alternatively, the .beta.2-M subunit may
 be-first linked to the ligand, and then allowed to associate with the
 .alpha. chain in the presence of the peptide. Multimers of these
 conjugates can be formed by supplying to these conjugates the multivalent
 binding molecule to which the ligand binds.
 The multimeric MHC class I complexes can be used for labeling,
 quantitation, isolation, and stimulation of T cells. By way of example,
 the class I multimers can be immobilized on solid-phase matrices,
 generating an affinity support for the enrichment and isolation of T cells
 harboring the corresponding antigen receptors. The matrices may be, for
 instance, agarose gel, beaded polymers, polystyrene plates, glass slides,
 nitrocellulose membrane, or columns. Immobilization can be effected by
 non-covalent coupling (e.g., between biotin-avidin/streptavidin
 interaction, or through magnetic field), or covalent coupling. Certain
 reversible coupling can allow the captured cells to be eluded from the
 affinity support. For instance, if the complex is biotinylated with Biotin
 HPDP, the biotin moiety can be cleaved from the reacted sulfhydryl with a
 reducing reagent (e.g., DDT, or .beta.-mercaptoethanol).
 The new class I multimers can also be used to characterize T cells that
 recognize non-classical class I proteins. This is because the new
 .beta.2-M can assemble with a wide variety of class I heavy chains, and
 thus a wide variety of class I multimer probes can be generated using the
 new .beta.2-M.
 The HLA class I gene family (see, e.g., "Phylogeny of the Major
 Histocompatibility Complex," in Immunological Reviews, 113:1-241, 1990)
 includes the highly polymorphic class I genes HLA-A, HLA-B and HLA-C, all
 of which show widespread tissue expression. At least three additional
 class I genes HLA-E, HLA-F and HLA-G (known as non-classical class I b
 genes) have been identified; these genes are highly homologous to the
 classical HLA class I genes, and their polypeptide products are all
 associated with .beta.2-M. The family of non-classical class I genes also
 includes members that do not reside in the MHC genomic complex, such as
 the subfamily of CD1 genes in both mouse and human, and the mouse thymus
 leukemia (TL) antigen. Both the TL antigen and CD1 proteins have the
 typical structure of an antigen-presenting class I molecule. They form
 heterodimers at the cell surface in which .alpha. heavy chain of
 approximately 38-50 kDa interacts with .beta.2-M. Another notable
 non-classical class I gene in mice is the Qa-2 gene. The Qa-2 products,
 like other class I heavy chains, are associated with .beta.2-M. The
 function of these non-classical molecules and the responding T cells are
 largely unknown.
 The ability of .beta.2-M to associate with diverse heavy chains, and the
 use of these complexes as signal (e.g., fluorescence)-producing probes,
 can facilitate the characterization of the T cells that react with these
 class I molecules. Information about the corresponding T cells may provide
 important clues about their specialized function in the immune system.
 Monomeric class I complexes conjugated to a solid support are in effect
 multimerized due to their physical proximity. Thus, the new, monomeric
 class I complexes conjugated to a solid surface via their free --SH groups
 can also be used for the same purposes as those described above for
 multimeric class I complexes.
 The following examples describe the generation and some uses of several new
 class I MHC-peptide tetramers. The examples serve to illustrate, but not
 limit, the new methods and reagents.
 In these examples, CTL clones from asymptomatic HIV-1 seropositive patients
 were established and maintained as described (Johnson et al., J. Immunol,
 147:1512-1521, 1991). HLA-typing was performed by the Massachusetts
 General Hospital Tissue Typing Laboratory using standard serological
 techniques. H-2K.sup.b -SV9 specific CTL clones (syngeneic reactive) were
 generated in H-2.sup.b mice (K.sup.b D.sup.b) as described (Zhou et al.,
 J. Immunol Methods, 153:193-200, 1992). The alloreactive CTL were derived
 from H-2.sup.dm2 mice (K.sup.d D.sup.d). All CTLs were maintained in
 culture by periodic stimulation with RMA-S cells (H-2.sup.b ; see, e.g.,
 Townsend et al., Cold Spring Harb. Symp. Quant. Biol., 54 Pt
 1:299-308,1989) loaded with SV9 peptide. CTL clone 4G3, which arose in an
 H-2K.sup.b mouse, reacts specifically with the ovalbumin octapeptide pOVA
 (SIINFEKL; SEQ ID NO: 12) in association with H-2K.sup.b (Walden et al.,
 Proc. Natl. Acad. Sci. USA, 87:9015-9019, 1990). Cell surface MHC class I
 molecules on RMA-S cells are largely devoid of peptide unless loaded with
 peptides from the external medium (Heemels et al., Ann. Rev. Biochem.,
 64:463-491 1995).
 EXAMPLE 1
 Production of a Mutant .beta.2-M
 The ribosome-binding site and coding region from a .beta.2-M expression
 plasmid (Garboczi et al., Proc. Natl. Acad. Sci. USA, 89:3429-2433, 1992)
 was cloned into the E. coli expression vector pLM1 (Garboczi et al., J.
 Immunol., 157:5403-5410, 1996) to achieve a higher protein yield. The
 .beta.2-M polypeptide is of human origin and has 99 amino acid residues
 (SEQ ID No: 1) plus one additional methionine residue at the N-terminus
 that results from the expression in E. coli.
 A cysteine residue was substituted for the tyrosine 67 residue in the
 polypeptide by using overlap extension PCRs (i.e., polymerase chain
 reactions). Four PCR primers were used. The first two were used at the 5'
 and 3' ends of the .beta.2-M coding region in the pHN1 plasmid (Garboczi
 et al., Proc. Natl. Acad. Sci. USA, 89:3429-3433, 1992). These two primers
 were the 5' primer CTAGAGGATCCTCACACAGGAAACAGAATTTCGAG (SEQ ID NO: 4), and
 the 3' primer CCACCGCGCTACTGCCGCCAGGC (SEQ ID NO: 5). The 5' primer
 introduced a new BamHI site at the 5' end of the .beta.2-M coding region.
 The other two primers were used for mutagenesis. They were the top strand
 primer CTC TTG TAC TGC ACT GAA TTC ACC CCC (SEQ ID NO: 6), and the bottom
 strand primer GAA TTC AGT GCA GTA CAA GAG ATA GAA (SEQ ID NO: 7). The new
 cysteine codon and its complement are underlined.
 To generate the cysteine mutation, the following two pairs of primers were
 first used on the pHN1 template to yield two PCR products: (i) the 5'
 primer and the bottom strand primer, and (ii) the 3' primer and the top
 strand primer. The two PCR products were electrophoresed and purified from
 agarose gel. They were then subjected together to a PCR reaction using the
 5' and 3' primers. The final PCR product, which contains the .beta.2-M
 coding region with the cysteine mutation, was digested with BamHI and
 HindIII and cloned into the same sites in pLM1. The mutated .beta.2-M
 (i.e., ".beta.2-M(Cys67)") sequence was confirmed by DNA sequencing.
 Plasmid pLM1 bearing the .beta.2-M(Cys67)-coding sequence was transformed
 into E. coli host BL21(DE3)plysS (Studier et al., Methods Enzymol.,
 185:60-89,1990), and a large amount of .beta.2-M(Cys67) was obtained in
 the form of inclusion body protein.
 EXAMPLE 2
 Generation of Peptide-MHC Tetramer
 To generate a class I MHC-peptide tetramer, the tyrosine residue at
 position 67 (SEQ ID NO: 1) of human .beta.2-M was first replaced with a
 cysteine residue, using standard mutagenesis techniques (see above). There
 are two naturally occurring cysteine residues in .beta.2-M, which maintain
 the immunoglobulin structure of .beta.2-M by forming a di-sulfide bond.
 The new cysteine residue did not bond with any one of the natural cysteine
 residues, allowing the proper folding of the mutant .beta.2-M
 (".beta.2-M(Cys67)"). The free sulfhydryl group in the new cysteine
 residue was to be used for subsequent chemical modifications of the
 .beta.2-M subunit.
 A monomeric mutant HLA-A2 containing .beta.2-M(Cys67) was then generated.
 The HLA-A2 heavy chain and the mutant .beta.2-M subunit was obtained in
 large amounts from E. coli host cells transformed with the respective
 expression vectors. The formation of HLA-A2 (subtype A0201) was initiated
 in a dilute solution containing a denatured HLA-A2 heavy chain (1 .mu.M),
 the denatured .beta..sub.2 -M(Cys67) polypeptide (2 .mu.M), and a
 synthetic peptide (10 .mu.M) (see, e.g., Garboczi et al., Proc. Natl.
 Acad. Sci. USA, 89:3429-3433, 1992). The folded MHC-peptide complexes were
 purified by HPLC (i.e., high performance liquid chromatography) gel
 filtration. The protein concentration of the purified complex was
 determined by measuring its optical density. For HLA-A2, 1 A.sub.280 unit
 represents 0.67 mg ml.sup.-1 of the protein. The solvent-accessible
 cysteine 67 in the mutant .beta.2-M subunit was maintained in a reduced
 state by 0.1 mM DDT.
 The monomeric mutant HLA-A2 was then biotinylated with iodoacetyl-LC-biotin
 ("ILB"; Pierce), a sulfhydryl-reactive reagent, at the cysteine 67 residue
 of .beta.2-M(Cys67). ILB, which was dissolved in N,N-dimethyl-formamide,
 was applied at a 5-fold molar excess over the total amount of cysteine in
 solution. The biotinylation reaction was carried out in Tris-HCl (pH 8.0)
 and 0.1 mM DDT, and the reaction lasted for 1 hour in the dark at room
 temperature. Subsequent to the reaction, the volume of the reaction mix
 was reduced with CENTRICON 30 from 0.5-1 ml to 50 .mu.l, diluted again in
 1 ml Tris-HCl (pH 8.0), concentrated to about 100 .mu.l, and purified by
 HPLC gel filtration. In the biotinylated .beta.2-M(Cys67), the biotin
 moiety of ILB was separated from the sulfhydryl group of the cysteine 67
 residue by the long iodoacetyl-LC arm.
 The biotinylated MHC-peptide complexes were purified by HPLC gel
 filtration. The purified complexes were then multimerized in the presence
 of deglycosylated avidin-phycoerythrin ("avidin-PE"; Molecular Probes).
 Deglycosylated avidin-PE binds more than 12 .mu.g of biotin per mg
 protein. The resulting tetrameric MHC-peptide complexes were subjected to
 HPLC gel filtration (TSK G 3000SW, TOSO HAAS, Gel Filtration
 Standard/BIO-RAD) and then concentrated (CENTRICON 100, Amicon).
 EXAMPLE 3
 Labeling of T cells with a Peptide-Class I Tetramer
 A peptide-MHC class I tetramer was prepared with a HIV-1 gag peptide (i.e.,
 SL9, whose sequence is SLYNTVATL (SEQ ID NO: 8)) (Johnson et al., J.
 Immunol, 147:1512-1521, 1991), and a control peptide from HIV reverse
 transcriptase (i.e., IV9, whose sequence is ILKEPVHGV (SEQ ID NO: 9))
 (Tsomides et al., Proc. Natl. Acad. Sci. USA, 88:11276-11280 1991). The
 tetramer was formed by use of avidin-PE, as described in the preceding
 Example. The binding of the tetramer to cytotoxic T lymphocyte ("CTL")
 clones with known antigen-binding specificity was monitored by flow
 cytometry.
 Specifically, 5.times.10.sup.5 cells from each of three human CTL clones
 specific for HIV-1 derived peptides were prestained with an anti-CD8
 monoclonal antibody ("mAb") Cy-Chrome (Pharmingen). These three clones,
 i.e., 68A62 (Tsomides et al., Proc. Natl. Acad. Sci. USA, 88:11276-11280,
 1991), 18030 (Johnson et al., J. Immunol., 147:1512-1521, 1991), 63D35 are
 specific for HLA-A2-IV9 (i.e., IV9 presented by HLA-A2), HLA-A2-SL9, and
 HLA-B11-AK9 (AK9: a HIV-1 reverse transcriptase peptide with the sequence
 of AIFQSSMTK (SEQ ID NO: 10)), respectively.
 The pre-stained cells were then incubated with the soluble, PE-labeled
 tetrameric HLA-A2-SL9 complex at a concentration of 50 .mu.g/100 .mu.l
 RPMI at 4.degree. C. for an hour. The cells were washed and subjected to
 FACS (i.e., fluorescence-activating cell sorting) analysis using a FACSCAN
 flow cytometer (Becton Dickinson).
 In the FACS analysis, contour plots were based on 10,000 events gated on
 forward- versus side-scatter. An arbitrarily set boundary on the
 PE-fluorescence intensity of a negative control was the position of the
 X-axis quadrant marker, implying that about 80% of the cells can be
 considered as positive for PE-fluorescence.
 The data showed that there was a 10-fold increase in the PE fluorescence
 intensity when the PE-labeled tetramer stained CTLs of clone 18030 (FIG.
 1B), as compared to CTLs of clones 68A82 and 63D35 (FIGS. 1A and 1C).
 Thus, the staining of CTLs by the HLA-A2-SL9 tetramer was specific.
 A similar shift of PE fluorescence intensity was observed with CTLs of
 clone 68A62 when those CTLs were stained with a HLA-A2-IV9 tetramer.
 Based on quadrant markers, about 80% of the CTLs specific for HLA-A2-SL9
 were stained positively with the phycoerythrin-labeled tetramer.
 EXAMPLE 4
 Labeling of T Cells with a Hybrid Peptide-Class I Complex
 The mutant human .beta.2-M in which Tyr 67 has been replaced with a
 cysteine residue was also shown to bind stably to the heavy chains of the
 mouse MHC class I molecule K.sup.b.
 Tetrameric MHC class I molecules were generated with use of a purified
 murine H-2K.sup.b ("K.sup.b ") heavy chain (Zhang et al., Proc. Natl.
 Acad. Sci. USA, 89:8403-8407, 1992), the mutant human .beta.2-M, and SV9,
 a Sendai virus nucleoprotein-derived peptide (FAPGNY, SEQ ID NO: 11)
 (Kast et al., Proc. Natl. Acad. Sci. USA, 88:2283-2287, 1991; and
 Schumacher et al., Nature, 350:703-706, 1991). SV9 promoted folding of the
 hybrid K.sup.b molecule with an efficiency similar to that of SL9 for
 promoting folding of HLA-A2. The purified K.sup.b tetramer migrated as a
 single peak on gel filtration HPLC. The presence of the heavy chain and
 the .beta.2-M subunit in the peak fractions was confirmed by SDS
 polyacrylamide gel electrophoresis. 5.times.10.sup.5 murine CTLs of three
 different clones were pre-stained with anti-CD8 TRI-COLOR (CALTAG), and
 then incubated with 50 .mu.g/100 .mu.l RPMI PE-labeled chimeric H-2K.sup.b
 -SV9 tetramer for an hour at 4.degree. C. The three CTL clones 2F3, 3C2,
 and 4G3 (Walden et al., Proc. Natl. Acad. Sci. USA, 87:9015-9019,1990) are
 specific for SV9 bound to H-2K.sup.b, SV9 bound to H-2Db, and OVA
 (SIINFEKL, SEQ ID NO: 12); derived from ovalbumin; Walden et al., Proc.
 Natl. Acad. Sci. USA, 87:9015-9019) bound to H-2K.sup.b, respectively.
 (The specificity of the clones is determined on the basis of their lysis
 of target cells loaded with the corresponding peptide.) After the
 incubation, the cells were washed and subjected to FACS analysis.
 Histogram plots (FIGS. 2A-2C) for the FACS analysis were based on 10,000
 events gated on CD8 fluorescence. As shown in the plots, the K.sup.b -SV9
 tetramer stained only 2F3 CTLs (FIG. 2A), but not 3C2 (FIG. 2B) or 4G3
 (FIG. 2C) CTLs.
 In the FACS analysis, markers (M1, M2) were placed to delineate a region of
 positive intensity relative to a control (FIGS. 2B and 2C). Based on these
 markers, 78% of 2F3 CTLs (FIG. 2A) were found to stain positive with the
 K.sup.b -SV9 tetramer.
 EXAMPLE 5
 Binding of T Cells by Allogeneic Peptide-Class I Complexes
 Allograft rejection is probably the most powerful T cell reaction known.
 During such a rejection, an individual's T cells respond strongly to
 target cells (e.g., a skin graft from a genetically disparate individual
 of the same species) that bear allogeneic MHC molecules. It has been
 suggested that the affinities of T cell receptors for MHC-peptide
 complexes be higher when the MHC component is allogeneic (i.e., non-self)
 than syngeneic (i.e., self) Sykulev et al., Proc. Natl. Acad. Sci. USA,
 91:11487-11491, 1994 and Eisen et al., Advances in Protein Chemistry,
 49:1-56, 1996).
 FIGS. 3A-3D compare the reactivity of the K.sup.b -SV9 tetramer with murine
 CD8.sup.+ CTLs from two different mouse lines. Briefly, 5.times.10.sup.5
 syngeneic MHC- and allogeneic MHC-CTLs were stained with anti-CD8
 TRI-COLOR and anti-H-2K.sup.b -FITC mAbs (CALTAG), washed and FACS
 analysed. The CTLs were then incubated with the H-2K.sup.b SV9 tetramer-PE
 reagent (50 .mu.g/100 .mu.l RPMI, 4.degree. C., 1 h), washed, and again
 subjected to FACS analysis. All three fluorochromes were excited with an
 argon laser in a single laser instrument (total of 10,000 events
 collected).
 The CTLs in FIG. 3A were derived from a K.sup.b -positive mouse and the
 reaction shown is syngeneic; whereas in FIG. 3C, the CTLs were derived
 from a K.sup.b -negative mouse and the reaction shown is allogeneic. The
 tetramer bound specifically to CTLs from both mouse lines (FIGS. 3B and
 3D), but it distinguished between two subsets in the alloreactive CTLs
 (FIG. 3D), the better binding subset amounting to about 12-15% of the
 total CTL population. The observed difference in staining intensities of
 CTLs (FIG. 3D) might be influenced by differences in cell surface density
 of TCR and perhaps CD8 molecules. Assuming a 1:1 ratio of the TCR
 heterodimer with the CD3 complex, CD3 and CD8 expression levels were
 analyzed. The analysis showed that the cellular subsets of the allogeneic
 CTL line that stained with multimeric K.sup.b -SV9 of different
 intensities had the same amount of CD8 and CD3 per cell.
 OTHER EMBODIMENTS
 It is to be understood that while the invention has been described in
 conjunction with the detailed description thereof, the foregoing
 description is intended to illustrate and not limit the scope of the
 invention, which is defined by the scope of the appended claims. For
 example, to attach a foreign class I molecule (e.g., one with an
 allogeneic or even xenogeneic heavy chain) to a tumor cell, a ligand for a
 receptor specifically expressed on the tumor cell, instead of a
 tumor-specific antibody, is conjugated to the foreign MHC class I molecule
 to direct an alloresponse to the tumor tissue.
 Other aspects, advantages, and modifications are within the scope of the
 following claims.
 SEQUENCE LISTING
 &lt;100&gt; GENERAL INFORMATION:
 &lt;160&gt; NUMBER OF SEQ ID NOS: 12
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 1
 &lt;211&gt; LENGTH: 100
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 1
 Met Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala
 1 5 10 15
 Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His
 20 25 30
 Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu
 35 40 45
 Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr
 50 55 60
 Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala
 65 70 75 80
 Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp
 85 90 95
 Asp Arg Asp Met
 100
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 2
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Influenza virus
 &lt;400&gt; SEQUENCE: 2
 Ile Leu Gly Phe Val Phe Thr Leu Thr Val
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 3
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Influenza virus
 &lt;400&gt; SEQUENCE: 3
 Lys Leu Gly Glu Phe Tyr Asn Gln Met Met
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 4
 &lt;211&gt; LENGTH: 35
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 4
 ctagaggatc ctcacacagg aaacagaatt tcgag 35
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 5
 &lt;211&gt; LENGTH: 23
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 5
 ccaccgcgct actgccgcca ggc 23
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 6
 &lt;211&gt; LENGTH: 27
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 6
 ctcttgtact gcactgaatt caccccc 27
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 7
 &lt;211&gt; LENGTH: 27
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 7
 gaattcagtg cagtacaaga gatagaa 27
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 8
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Human immunodeficiency virus type 1
 &lt;400&gt; SEQUENCE: 8
 Ser Leu Tyr Asn Thr Val Ala Thr Leu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 9
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Human immunodeficiency virus type 1
 &lt;400&gt; SEQUENCE: 9
 Ile Leu Lys Glu Pro Val His Gly Val
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 10
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Human immunodeficiency virus type 1
 &lt;400&gt; SEQUENCE: 10
 Ala Ile Phe Gln Ser Ser Met Thr Lys
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 11
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Sendai virus
 &lt;400&gt; SEQUENCE: 11
 Phe Ala Pro Gly Asn Tyr Pro Ala Leu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 12
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Mus musculus
 &lt;400&gt; SEQUENCE: 12
 Ser Ile Ile Asn Phe Glu Lys Leu
 1 5