Isolated peptide defined by SEQ ID NO: 17 and uses thereof

Tumor rejection antigens presented by HLA-B44 molecules are described. These peptides are useful in diagnostic and therapeutic methodologies. The tumor rejection antigens are not derived from tyrosinase, which has previously been identified as a tumor rejection antigen precursor processed to an antigen presented by HLA-B44.

FIELD OF THE INVENTION
 This invention relates to isolated peptides, derived from tumor rejection
 antigen precursors and presented by HLA molecules, HLA-B44 in particular,
 and uses thereof. In addition, it relates to the ability to identify those
 individuals diagnosed with conditions characterized by cellular
 abnormalities whose abnormal cells present complexes of these peptides and
 HLA molecules, the presented peptides, and the ramifications thereof.
 BACKGROUND AND PRIOR ART
 The process by which the mammalian immune system recognizes and reacts to
 foreign or alien materials is a complex one. An important facet of the
 system is the T cell response. This response requires that T cells
 recognize and interact with complexes of cell surface molecules, referred
 to as human leukocyte antigens ("HLA"), or major histocompatibility
 complexes ("MHCs"), and peptides. The peptides are derived from larger
 molecules which are processed by the cells which also present the HLA/MHC
 molecule. See in this regard Male et al., Advanced Immunology (J.P.
 Lipincott Company, 1987), especially chapters 6-10. The interaction of T
 cell and complexes of HLA/peptide is restricted, requiring a T cell
 specific for a particular combination of an HLA molecule and a peptide. If
 a specific T cell is not present, there is no T cell response even if its
 partner complex is present. Similarly, there is no response if the
 specific complex is absent, but the T cell is present. This mechanism is
 involved in the immune system's response to foreign materials, in
 autoimmune pathologies, and in responses to cellular abnormalities.
 Recently, much work has focused on the mechanisms by which proteins are
 processed into the HLA binding peptides. See, in this regard, Barinaga,
 Science 257: 880 (1992); Fremont et al., Science 257: 919 (1992);
 Matsumura et al., Science 257: 927 (1992); Latron et al., Science 257: 964
 (1992).
 The mechanism by which T cells recognize cellular abnormalities has also
 been implicated in cancer. For example, in PCT application PCT/US92/04354,
 filed May 22, 1992, published on Nov. 26, 1992, and incorporated by
 reference, a family of genes is disclosed, which are processed into
 peptides which, in turn, are expressed on cell surfaces, which can lead to
 lysis of the tumor cells by specific CTLs. The genes are said to code for
 "tumor rejection antigen precursors" or "TRAP" molecules, and the peptides
 derived therefrom are referred to as "tumor rejection antigens" or "TRAs".
 See Traversari et al., Immunogenetics 35: 145 (1992); van der Bruggen et
 al., Science 254: 1643 (1991), for further information on this family of
 genes. Also see U.S. Pat. No. 5,342,774, incorported by reference.
 In U.S. Pat. No. 5,405,940, the disclosure of which is incorporated by
 reference, nonapeptides are taught which bind to the HLA-A1 molecule. The
 reference teaches that given the known specificity of particular peptides
 for particular HLA molecules, one should expect a particular peptide to
 bind one HLA molecule, but not others. This is important, because
 different individuals possess different HLA phenotypes. As a result, while
 identification of a particular peptide as being a partner for a specific
 HLA molecule has diagnostic and therapeutic ramifications, these are only
 relevant for individuals with that particular HLA phenotype. There is a
 need for further work in the area, because cellular abnormalities are not
 restricted to one particular HLA phenotype, and targeted therapy requires
 some knowledge of the phenotype of the abnormal cells at issue.
 The enzyme tyrosinase catalyzes the reaction converting tyrosine to
 dehydroxyphenylalanine or "DOPA" and appears to be expressed selectively
 in melanocytes (Muller et al., EMBO J 7: 2715 (1988)). An early report of
 cDNA for the human enzyme is found in Kwon, U.S. Patent No. 4,898,814. A
 later report by Bouchard et al. , J. Exp. Med. 169: 2029 (1989) presents a
 slightly different sequence. A great deal of effort has gone into
 identifying inhibitors for this enzyme, as it has been implicated in
 pigmentation diseases. Some examples of this literature include Jinbow,
 W09116302; Mishima et al. , U.S. Pat. No. 5,077,059, and Nazzaropor, U.S.
 Pat. No. 4,818,768. The artisan will be familiar with other references
 which teach similar materials.
 U.S. patent application 08/081,673, filed Jun. 23, 1993 now U.S. Pat. No.
 5,487,974 and incorporated by reference, teaches that tyrosinase may be
 treated in a manner similar to a foreign antigen or a TRAP molecule--i.e.,
 it was found that in certain cellular abnormalities, such as melanoma,
 tyrosinase is processed and a peptide derived therefrom forms a complex
 with HLA molecules on certain abnormal cells. These complexes were found
 to be recognized by cytolytic T cells ("CTLs"), which then lyse the
 presenting cells. The ramifications of this surprising and unexpected
 phenomenon were discussed. Additional peptides have now been found which
 also act as tumor rejection antigens presented by HLA-A2 molecules. These
 are described in Serial No. 08/203,054, filed Feb. 28, 1994 now U.S. Pat.
 No. 5,530,096 and incorporated by reference.
 U.S. patent application Ser. No. 08/233,305 filed Apr. 26, 1994 now U.S.
 Pat. No. 5,519,117 and incorporated by reference, disclosed that
 tyrosinase is also processed to an antigen presented by HLA-B44 molecules.
 The finding was of importance, because not all individuals are
 HLA-A2.sup.+. The fact that tyrosinase is processed to an HLA-B44
 presented peptide, however, does not provide for a universal approach to
 diagnosis and treatment of all HLA-B44.sup.+ tumors, because tyrosinase
 expression is not universal. Further, the fact that tyrosinase is
 expressed by normal cells as well as tumor cells may suggest some caution
 in the therapeutic area.
 Khanna, et al., J. Exp. Med. 176: 169-179 (July 1992), disclose an HLA-B44
 binding peptide, which is discussed further infra. The Khanna peptide is
 not related to the peptides claimed herein.
 Kita, et al., Hepatology 18(5): 1039-1044 (1993), teach a 20 amino acid
 peptide alleged to bind to HLA-B44 and to provoke lysis.
 Thorpe, et al., Immunogenetics 40: 303-305 (1994), discuss alignment of two
 peptides found to bind to HLA-B44, and suggest a binding motif generally.
 The Thorpe disclosure speaks of a negatively charged amino acid at
 position 2, and one at position 9 which may be hydrophobic, or positively
 charged.
 Fleischhauer, et al., Tissue Antigens 44: 311-317 (1994) contains a survey
 of HLA-B44 binding peptides.
 It has now been found that the MAGE-3 also expresses a tumor rejection
 antigen precursor is processed to at least one tumor rejection antigen
 presented by HLA-B44 molecules. It is of interest that this peptide
 differs from a peptide also derived from MAGE-3 and known to bind to
 HLA-A1, by a single, added amino acid at the N-terminus. This, inter alia,
 is the subject of the invention disclosure which follows.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
 EXAMPLE 1
 Melanoma cell line LB33-MEL which has been available to researchers for
 many years, was used in the following experiments. A clone derived
 therefrom was also used. The clone is referred to hereafter as LB33-MELc1.
 Samples containing mononuclear blood cells were taken from patient LB33.
 The melanoma cell line was contacted to the mononuclear blood cell
 containing samples. The mixtures were observed for lysis of the melanoma
 cell lines, this lysis indicating that cytolytic T cells ("CTLs") specific
 for a complex of peptide and HLA molecule presented by the melanoma cells
 were present in the sample.
 The lysis assay employed was a chromium release assay following Herin et
 al., Int. J. Cancer 39:390-396 (1987), the disclosure of which is
 incorporated by reference. The assay, however, is described herein. The
 target melanoma cells were grown in vitro, and then resuspended at
 10.sup.7 cells/ml in DMEM, supplemented with 10 mM HEPES and 30% FCS
 (i.e., from fetal calf serum) and incubated for 45 minutes at 37.degree.
 C. with 200 .mu.Ci/ml of Na(.sup.51 Cr)O.sub.4. Labelled cells were washed
 three times with DMEM, supplemented with 10 mM Hepes. These were then
 resuspended in DMEM supplemented with 10 mM Hepes and 10% FCS, after which
 100 ul aliquots containing 10.sup.3 cells, were distributed into 96 well
 microplates. Samples of PBLs were added in 100 ul of the same medium, and
 assays were carried out in duplicate. Plates were centrifuged for 4
 minutes at 100 g, and incubated for four hours at 37.degree. C. in a 5.5%
 CO.sub.2 atmosphere.
 Plates were centrifuged again, and 100 ul aliquots of supernatant were
 collected and counted. Percentage of .sup.51 Cr release was calculated as
 follows:
 ##EQU1##
 where ER is observed, experimental .sup.51 Cr release, SR is spontaneous
 release measured by incubating 10.sup.3 labeled cells in 200 ul of medium
 alone, and MR is maximum release, obtained by adding 100 ul 0.3% Triton
 X-100 to target cells.
 Those mononuclear blood samples which showed high CTL activity were
 expanded and cloned via limiting dilution, and were screened again, using
 the same methodology.
 The same method was used to test target K562 cells. When EBV-B cells were
 used, the only change was the replacement of DMEM medium by Hank's medium,
 supplemented with 5% FCS.
 These experiments led to isolation of CTL clones LB33-CTL-159/5. FIG. 1
 shows that this clone lysed tumor cells, but not EBV-B cells, or K562
 cells.
 Following the same protocol, a second CTL clone, i.e., LB33-CTL-159/3 was
 isolated. These lines will be referred to as "159/5" and "159/3",
 respectively. This second CTL has specificity differing from 159/5. This
 was ascertained following isolation of two antigen loss variants which (i)
 are lysed by 159/5 but not 159/3 and (ii) are not lysed by 159/5 and are
 lysed by 159/3. These variants are referred to as A.sup.- and B.sup.-,
 respectively.
 The A.sup.- variant was then immunoselected with 159/5, and a third variant
 was obtained, which was not lysed by either 195/5 or 159/3. This variant
 is referred to as A.sup.- B.sup.-. FIG. 2 summarizes the results of the
 lysis assays, leading to isolation of the variants.
 EXAMPLE 2
 It was of interest to determine the pattern of HLA expression of variant
 A.sup.- B.sup.-. The patient from whom parent line LB33-MEL was derived
 was typed as HLA-A24, A28, B13, B44, Cw6, Cw7. When PCR expression
 analysis was carried out, it was found that both LB33-MELc1, and the
 B.sup.- variant express all six alleles; however, the A-B.sup.- variant
 does not express HLA-A28, B44, and Cw7. As a result, it was concluded that
 one of these HLA molecules presents the antigen leading to lysis by CTLs.
 The following example explores this further.
 EXAMPLE 3
 Samples of the A.sup.- B.sup.- variant were transfected by plasmid
 pcDNA-I/AmpI which had cloned therein, one of HLA-A28, HLA-B44, or
 HLA-Cw7. Following selection, the cells were tested in a TNF release
 assay, following Traversari, et al., Immunogenetics 35: 145-152 (1992),
 incorporated by reference herein. The results are summarized in FIG. 3,
 which shows that HLA-B44 is clearly implicated in the presentation of the
 antigen.
 EXAMPLE 4
 Once the presenting HLA molecule was identified, studies were carried out
 to identify the molecule, referred to hereafter as the "tumor rejection
 antigen precursor" or "TRAP" molecule which was the source of the
 presented peptide.
 To do this, total mRNA was isolated from cell line LB33-MELc1. The
 messenger RNA was isolated using an oligo-dT binding kit, following well
 recognized techniques. Once the messenger RNA was secured, it was
 transcribed into cDNA, again using standard methodologies. The cDNA was
 then ligated to EcoRI adaptors and cloned into the EcoRI site of plasmid
 pcDNA-I/Amp, in accordance with manufacturer's instructions. The
 recombinant plasmids were then electrophorated into DH5.alpha. E. coli
 (electroporation conditions: 1 pulse at 25 .mu.farads, 2500 V).
 The transfected bacteria were selected with ampicillin (50 .mu.g/ml), and
 then divided into pools of 100 bacteria each. Each pool represented about
 50 different cDNAs, as analysis showed that about 50% of plasmids
 contained an insert. Each pool was amplified to saturation, and plasmid
 DNA was isolated via alkaline lysis, potassium acetate precipitation and
 phenol extraction, following Maniatis et al., in Molecular Cloning: A
 Laboratory Manual (Cold Spring Harbor, N.Y., 1982). Cesium gradient
 centrifugation was not used.
 The amplified plasmids were then transfected into eukaryotic cells. Samples
 of COS-7 cells were seeded, at 15,000 cells/well into tissue culture flat
 bottom microwells, in Dulbecco's modified Eagles Medium ("DMEM")
 supplemented with 10% fetal calf serum. The cells were incubated overnight
 at 37.degree. C., medium was removed and then replaced by 30 .mu.l/well of
 DMEM medium containing 10% Nu serum, 400 .mu.g/ml DEAE-dextran, 100 .mu.M
 chloroquine, and 100 ng of a plasmid containing cDNA for HLA-B44 from
 LB33. Following four hours of incubation at 37.degree. C., the medium was
 removed, and replaced by 50 .mu.l of PBS containing 10% DMSO. This medium
 was removed after two minutes and replaced by 200 .mu.l of DMEM
 supplemented with 10% of FCS.
 Following this change in medium, COS cells were incubated for 48 hours at
 37.degree. C. Medium was then discarded, and 2000 cells of 159/5 were
 added, in 100 .mu.l of Iscove's medium containing 10% pooled human serum
 and 25 U/ml IL-2. Supernatant was removed after 24 hours, and TNF content
 was determined in an assay on WEHI cells, as described by Traversari et
 al., Immunogenetics 35: 145-152 (1992), the disclosure of which is
 incorporated by reference. One pool stimulated TNF release above
 background, and these bacteria were cloned, and used in the following
 experiment.
 EXAMPLE 5
 Plasmid DNA was extracted from the bacteria cloned in Example 4,
 transfected into a new sample of COS cells in the same manner as described
 supra, and the cells were again tested for stimulation of 159/5. A
 positive clone was found in clone 350/2, as demonstrated by data
 summarized in FIG. 4.
 In order to confirm the results obtained to this point, the human
 choriocarcinoma cell line JAR, which is readily available from the
 American Type Culture Collection, was used. This cell line does not
 express HLA molecules, nor is it recognized by CTL 159/5. When JAR was
 transfected with HLA-B44 cDNA, it was still not recognized by CTL 159/5.
 Co-transfection with HLA-B44 and 350/2 cDNAs, however, led to lysis, as is
 seen in FIG. 4.
 The plasmid from the positive clone was removed, and sequenced following
 art known techniques. Information shows that the plasmid insert was 1896
 base pairs long, and showed no homology with any sequences in data banks.
 The nucleotide sequence is set forth herein as SEQ ID NO: 1.
 EXAMPLE 6
 In order to ascertain the peptide which was the tumor rejection antigen,
 fragments of SEQ ID NO: 1, averaging about 300 base pairs, were amplified
 via PCR, cloned into PcDNAI 1/Amp, and then cotransfected into COS cells
 with plasmid encoding HLA-B44, following the protocols of the preceding
 examples. These experiments led to identifying the region corresponding to
 amino acid residues 683-955 of SEQ ID NO: 1 as encoding the antigenic
 peptide. This region was compared to the peptide described by Khanna, et
 al., J. Exp. Med. 176: 169-176 (7/92), and the peptide described in Ser.
 No. 08/233,305, filed Apr. 26, 1994, now U.S. Pat. No. 5,519,117 i.e.:
 Glu Glu Lys Leu Ile Val Val Leu Phe
 corresponds to these residues. As such, a peptide corresponding to this
 sequence was synthesized, and used to sensitize HLA-B44.sup.+ cell lines.
 The results are shown in FIGS. 6A and 6B, which depict the results of a
 .sup.51 Cr release assay using EBV transformed B cells (FIG. 6A), and the
 B.sup.- variant described supra (FIG. 6B). The cells were incubated with
 varying concentrations of the peptide for 30 minutes at 37.degree. C.,
 before adding CTL 159/5 (effector/target ratio: 10:1). Half maximal lysis
 was obtained with 100-200 ng/ml of peptide.
 EXAMPLE 7
 Examples 1-6, set forth supra, describe work using the cell line
 LB33-Melc1. Additional cell lines were also derived from a cutaneous
 metastasis from patient LB33. One such line is LB33-MEL.A-1, which is used
 in the example which follows.
 First, the cell line was used, in the same manner that the cell line of
 examples 1-6 was used (Herin et al., supra). Blood mononuclear cells
 (10.sup.6 /well), were stimulated with irradiated tumor cells (3/10.sup.5
 cells/well), in 2 ml of Iscove's medium, supplemented with 10% pooled
 human serum, asparagine-glutamine-arginine (36 mg/ml, 216 mg/ml, 116
 mg/ml, respectively), 2-mercaptoethanol (0.05 mM), and 5 U/ml of human
 IL-4. IL-2 (10 U/ml) was added on the third day of cultivation.
 Sensitivity of the tumor cells to autologous CTLs was determined as in
 example 1, supra. The experiment yielded 82 stable cytolytic T
 lymphocytes, derived from seven independent cultures. All of these CTLs
 were CD8.sup.+. They were specific for tumor cells in that they lysed
 LB33-MEL.A-1 cells, but not K562, or autologous, EBV transformed cells.
 EXAMPLE 8
 The fact that LB33-MEL.A-1 cells were lysed by autologous CTLs suggested
 the next experiment, which was to identify the antigens recognized by
 establishing antigen loss variants.
 To do this, samples of the cell line were selected, four times, with the
 autologous CTL clone LB33-CTL 159/3, described supra. Each round of
 selection involved incubating, for 2-6 hours, 2-3.times.10.sup.7 adherent
 tumor cells with a similar number of CTLs, in the same manner described
 supra. In each round, CTLs were washed away following the incubation, and
 the surviving adherent tumor cells were amplified prior to the next round
 of selection.
 This procedure resulted in a clone resistant to CTL 159/3; however, when
 tested with additional autologous CTLs, it was found that CTL 159/5,
 described supra, did lyse the loss variant, as did additional CTL clones,
 including 204/26, and 202/1. Please see FIG. 6, the column labelled
 "MEL.A-1.1". Similarly, additional cell lines were established which were
 not lysed by one of these four CTL clones, but was lysed by the others.
 Note FIG. 6. Thus, at least four different antigens were found to be
 presented on the surface of LB33-MEL.A-1, because four distinct
 antigen-loss variants were identified. As set forth in FIG. 6, then,
 LB33-MEL.A-1 is considered "A.sup.+ B.sup.+ C.sup.+ D.sup.+ " for antigen
 expression (lysed by all of CTL 159/3, 159/5, 204/26, and 202/1);
 MEL.A-1.1 is A.sup.- B.sup.+ C.sup.+ D.sup.+ (not lysed by 159/3, lysed by
 others); MEL.A-1.2 is A.sup.+ B.sup.- C.sup.+ D.sup.+ (not lysed by 159/5;
 lysed by others), MEL.A-1.3 is A.sup.+ B.sup.+ C.sup.- D.sup.+ (not lysed
 by 204/26; lysed by others), and MEL.A-1.4 is A.sup.- B.sup.+ C.sup.+
 D.sup.- (not lysed by 202/1 or 159/3). Further, cell line MEL.A-1.1.1 was
 isolated, which was A.sup.- B.sup.- C.sup.+ D.sup.- (lysed only by
 204/26).
 When the 82 CTLs identified via example 7 were tested on these lines, 29
 anti-A, 29 anti-B, 10 anti-C, and 14 anti-D clones were identified,
 suggesting that there were no other antigens being presented.
 Selection with anti-D CTL clone 202/1 led to identification of a line which
 was also resistant to the anti-A CTL clone (159/3), as did selection with
 anti-B CTL (i.e., the resulting A.sup.- B.sup.- C.sup.+ D.sup.- line).
 This result suggests that A.sup.- D.sup.- and A.sup.- B.sup.- D.sup.-
 antigen loss variants were actually HLA loss variants, with antigens A, B
 and D sharing the same HLA presenting molecule, or that different class I
 molecules had been lost together with the antigen loss variants. The
 following experiments pursued this issue.
 EXAMPLE 9
 The patient from whom the LB33 cell lines had been developed had been
 serologically typed, previously, as HLA-A24, A28, B13, B44, Cw6, Cw7.
 Studies were then carried out to determine the expression of HLA class I
 genes by the cell lines.
 Semi-quantitative conditions for DNA amplification by PCR were established
 in order to assess the expression of each of the six class I alleles by
 the different LB33-MEL tumor cell clones. The Amplification Refractory
 Mutation System (ARMS) PCR methodology proposed by Browning et al, that
 relies on the perfect nucleotide matched needed at the 3' end of primers
 to ensure specificity of DNA amplifications was used. See Browning et al,
 Proc. Natl. Acad. Sci. USA 90: 2842 (1993) incorporated by reference
 herein. On the basis of sequences obtained in typing LB33, allele-specific
 primers that enabled discrimination of each one of the six alleles from
 the five others (5' primer followed by 3' primer) were synthesized.
 for A24: 5'-GCCGGAGTATTGGGACGA and 5'-GGCCGCCTCCCACTTGC (SEQ ID NOS: 5 and
 6)
 for A28: 5'-GGAGTATTGGGACCGGAAG and 5'-GGCCGCCTCCCACTTGT (SEQ ID NOS: 7 and
 8)
 for B13: 5'-CGCCACGAGTCCGAGGAT and 5'-CCTTGCCGTCGTAGGCTA (SEQ ID NOS. 9 and
 10)
 for B44: 5'-CGCCACGAGTCCGAGGAA and 5'-CCTTGCCGTCGTAGGCGT (SEQ ID NOS. 11
 and 12)
 for Cw6: 5'-CCGAGTGAACCTGCGGAAA and 5'-GGTCGCAGCCATACATCCA (SEQ ID NOS. 13
 and 14)
 for Cw7: 5'-TACAAGCGCCAGGCACAGG and 5'-CTCCAGGTAGGCTCTGTC (SEQ ID NOS. 15
 and 16)
 To carry out semi-quantitative measurements of expression, 27 cycles of PCR
 amplification of reverse transcribed RNA were carried out with each set of
 primers and DNA amplification was found to be in the linear range
 observed. The quantity of the amplified DNA was visually assessed with
 agarose gels stained with ethidium bromide. These quantities were compared
 to those obtained with a standard curve containing the products of RT-PCR
 amplification of serial dilutions of RNA from LB33-MEL.A-1 cells. The
 expression of samples was normalized for RNA integrity by taking into
 account the expression level of the .beta.-actin gene. The results were
 expressed relative to the level of expression by LB33-MEL.A-1 cells. The
 results of this work are set forth in Table 1, which follows. A "+++"
 indicates expression corresponding to more than half that of the
 LB33-MEL.A-1 cells, "++" means that expression was between 1/8 and 1/2 of
 that of LB33-MEL.A-1, a "+" means that expression was less than 1/8 of
 that of LB33-MEL.A-1 expressed and "-" means there was no expression.
 TABLE 1
 Expression of HLA class I by the antigen-loss
 variants derived from LB33-MEL.A-1 cells.
 LB33-MEL.A tumor cells
 LB33-MEL.A-1 Antigen-loss variants
 Expression
 of A.sup.- B.sup.- C.sup.- A.sup.- D.sup.- A.sup.-
 B.sup.- D.sup.-
 A. Gene Expression
 A24 +++ +++ +++ - ++ +++
 A28 +++ +++ +++ +++ + -
 B13 +++ +++ +++ + +++ +++
 B44 +++ +++ +++ +++ ++ -
 Cw6 +++ +++ +++ + +++ +++
 Cw7 +++ ++ +++ +++ + -
 As seen, both MEL.A-1 cells, and B.sup.- variant expressed similar levels
 of all six HLA alleles. The A variant showed an approximately 4-fold
 decrease in expression of Cw7. The remaining antigen loss variants showed
 decreases in expression of sets of three alleles. For C.sup.- cells,
 reduced levels of expression for HLA-A24, B13, and Cw6 were found, while
 A.sup.- D.sup.-, and A.sup.- B.sup.- D.sup.- variants showed reduction in
 A28, B44, and Cw7 expression. This suggests that A24-B13-Cw6, and
 A28-B44-Cw7 constitute two HLA class I haplotypes of patient LB33, and
 that reduced expression of these haplotype probably accounted for loss of
 antigen expression by the immunoselected tumor cells.
 EXAMPLE 10
 The next experiments were designed to confirm a correlation between HLA
 gene expression, and lysis by CTLs. To do this, the expression of a given
 HLA gene, as determined supra, was compared with the results obtained
 using a standard antibody assay. Only A24, A28 and B13 were tested, using
 murine antibodies specific thereto (C7709A1 for A24; 2.28M1 for A28, and
 TU48 for B13). Binding of antibody was determined by incubation with
 antibody, washing and then contacting with goat anti-mouse Ig antibodies,
 coupled to fluorescein. The cells were then analyzed by flow cytometry, a
 standard technique.
 Table 2 summarizes the results, which are also shown in FIG. 7. In table 2
 that follows, the indicated level of HLA expression corresponds to the
 mean intensity of fluorescence shown in FIG. 6. Values are expressed
 relative to levels found in LB33-MEL.A-1 cells.
 It appears from these results that when levels of HLA expression estimated
 to range below 1/8 of that of LB33-MEL.A-1 cells, undetectable or barely
 detectable levels of HLA surface molecules are found, thus suggesting that
 antigen presentation to CTL was unlikely for the given HLA molecule.
 In view of this, and assuming that C.sup.-, A.sup.- D.sup.- and A.sup.-
 B.sup.- D.sup.- selected cells had lost expression of antigen because of
 lack of HLA molecules, it appeared to be the case that the class I
 presenting molecules for antigen A were A28 or Cw7, B44 for antigen B, A24
 or B13 or Cw6 for antigen C, and A28 or Cw7 for antigen D.
 TABLE 2
 LB33-MEL.A-1 Antigen-loss variants
 Expression of A.sup.- B.sup.- C.sup.- A.sup.- D.sup.- A.sup.-
 B.sup.- D.sup.-
 Expression of surface antigen
 A24 100 33 13 4 41 95
 A28 100 29 14 3 1 1
 B13 100 27 22 1 40 230
 EXAMPLE 11
 The experiments detailed above were followed by additional work to
 determine, definitively, the presenting molecules for the antigens
 expressed by the LB33-MEL.A cells. To do this, tumor cells which had lost
 expression of particular HLA class I molecules were transfected, using the
 classic calcium phosphate precipitation method, with expression vector
 pcDNA3, into which the particular class I cDNA was cloned. This vector
 contains the neo.sup.R marker. Transfectants were selected with 1.5 mg/ml
 of G418, and were then used to stimulate CTL clones, using the TNF assay
 set forth in the previous examples.
 FIG. 8 depicts these results. Expression of antigen B was restored in
 A.sup.- B.sup.- D.sup.- cells by transfection with a plasmid carrying
 HLA-B44, but not with plasmids containing HLA-A28 or HLA-Cw7. The
 expression of antigen C was restored in C.sup.- cells by transfection with
 HLA B13. Four other anti-C CTL clones also recognized C.sup.- cells, but
 five other anti-C CTL clones, including depicted CTL 179C/50, did not;
 rather, these CTLs recognized C.sup.- cells transfected with HLA-Cw6.
 Thus, it may be concluded that there are two groups of anti-C CTL clones.
 One recognizes an antigen presented by HLA-B13, and the other an antigen
 presented by HLA-Cw6. As for antigen D, A.sup.- D.sup.- cells were
 restored to A.sup.- D.sup.+ via transfection with HLA-A28. None of the
 cDNA restored expression of antigen A (i.e., tested HLA A28, B44, Cw7),
 although it clearly is presented by HLA-class I molecules, because lysis
 by anti-A CTLs is completely inhibited by anti-class I monoclonal antibody
 W6/32. It is possible that this antigen may be presented by a non-A, B, C
 class I molecule, of which two alleles were present in patient LB33, one
 of these being lost, together with the A28-B44-Cw7 haplotype in A.sup.-
 D.sup.-, A.sup.- B.sup.- D.sup.- cells.
 The results for antigen C have led to a change in nomenclature. There are
 two antigens referred to as Antigen, Ca1 and antigen Cb, hereafter.
 EXAMPLE 12
 In further experiments, the question of whether or not cells of the line
 LB33-MEL.B could be recognized by autologous cell lines, was addressed.
 Irradiated LB33-MEL.B.1 cells were used in the same manner as was used,
 supra (Herin, et al), to stimulate autologous lymphocytes. The lymphocytes
 had been taken from patient LB33 in 1990 or 1994.
 As is shown in FIG. 9, only the lymphocytes from 1994 lysed LB33-MEL.B-1
 cells; however, they did not lyse LB33-MEL.A cells. Thus, the LB33-MEL.B-1
 line presents an antigen not found on LB33-MEL.A.
 The experiments described herein parallel those described supra and, as in
 the prior experiments, another panel of CD8.sup.+ CTL clones were
 established. The panel of reactivity of CTL 269/1 is shown in FIG. 10A.
 Note reaction with "MEL.B-1", but not "MEL.A-1". The new antigen defined
 thereby is referred to as LB33-E.
 In antibody inhibitory experiments, mAbs to HLA-A24 inhibited lysis. This
 is shown in FIG. 10B. Hence, the "E" antigen is presented by HLA-A24.
 EXAMPLE 13
 Fleischhauer et al., Tissue Antigens 44: 311-317 (1994), incorporated by
 reference, teach a consensus motif for HLA-B44 binding. This motif is
 described as a nine or ten amino acid polypeptide, where Glu predominates
 at second position, Tyr or Phe is present at the last position (position 9
 or 10), and hydrophobic residues, such as Met, are at the third position.
 The MAGE-3 TRAP amino acid sequence contains a stretch of amino acids at
 position 167-176, which corresponds to this motif. The amino acid sequence
 is:
 Met Glu Val Asp Pro Ile Gly His Leu Tyr (SEQ ID NO: 17).
 The HLA-B44 motif is known to contain at least two major subtypes, referred
 to as HLA-B* 4402 and HLA-B* 4403. The MHC molecule appears on 23% of all
 Caucasians. When this figure is combined with standard analyses of
 melanoma, it is concluded that 15% of Caucasian melanoma patients should
 present HLA-B44 on the surface of their melanoma cells. Thus, it is of
 great interest to determine if the peptide of SEQ ID NO: 17 or related
 molecules can in fact be used to identify HLA-B44 cells, and to provoke
 their lysis following binding to the MHC molecule. As noted in prior
 examples, the peptide of SEQ ID NO: 2 was shown to bind to HLA-B44
 positive cells. A peptide was designed with was similar to SEQ ID NO: 2,
 except for having Ala at position 8, rather than Leu. This new peptide,
 i.e.:
 Glu Glu Lys Leu Ile Val Val Ala Phe (SEQ ID NO: 18),
 was tested in a competition assay with SEQ ID NO: 17. This peptide was used
 in view of result obtained in experiments not reported here. Briefly,
 derivatives of SEQ ID NO:17 were prepared, wherein each derivative
 contained an Ala at a position not occupied by Ala in SEQ ID NO:17. CTL
 clone 159/5 was slightly better at recognizing complexes containing SEQ ID
 NO:18 than SEQ ID NO:17, making it an excellent reagent for competitive
 assays. Competition was carried out using C1R cells, described by Storkus
 et al., J. Immunol 138:1657-1659 (1987). These C1R cells are MHC class I
 negative, lymphoblastoid cells. The C1R cells were transfected with cDNA
 for HLA-B*4402, or genomic DNA HLA-B*4403, using the same methodology
 given supra. The cDNA for HLA-B*4402 is set forth by Fleischhauer, et al,
 Tissue Antigens 44: 311-317 (1994), while that for HLA-B*4403 is given by
 Fleischhauer, et al. (1990) New Eng. J. Med 323:1818-1822 (1990). Both
 papers are incorporated by reference.
 The cells were labelled with .sup.51 Cr for one hour at 37.degree. C., in
 the presence of anti-HLA class I monoclonal antibody W6/32 (30% (v/v) of
 culture medium of the hybridoma cells). This increases the ability of the
 cells to present antigenic peptides to T cells.
 Labelled cells were washed, and incubated for 30 minutes at 20.degree. C.,
 in serum free medium, together with various concentrations of competitor
 peptides. These peptides included:
 Ser Glu Ile Trp Arg Asp Ile Asp Phe (SEQ ID NO: 3)
 which binds to HLA-B44 molecules, as discussed, supra
EQU Phe Leu Arg Gly Arg Ala Tyr Gly Leu (SEQ ID NO: 19),
 which is encoded by EBV gene EBNA-3A and binds to HLA-B8 (Burrows, J. Exp
 Med 171:345-349 (1990)), and SEQ ID NO: 17.
 The peptide of SEQ ID NO: 18 was then added in the serum free culture
 medium at a final concentration of 45 ng/ml, (C1R-B4402.sup.+ cells), or
 160 ng/ml (C1R-B4403.sup.+ cells). The cells were incubated for 30 minutes
 at 20 C, and washed twice in Iscove's medium plus 2% fetal calf serum. The
 CTL clone LB33-CTL 159/5 was added in Iscove's medium and 10% human serum,
 at an E:T ratio of 20. The release of .sup.51 Cr was measured after three
 hours, and is shown in FIGS. 11A and 11B, for C1R-B*4402 and C1R-B*4403
 cells. The data presented in FIG. 11, show clear evidence of competition.
 EXAMPLE 14
 Additional experiments were then carried out following those described in
 Example 13.
 Cytolytic T cell clones (CTLs) were derived from two subjects, referred to
 as LB 816 and LB 822, respectively. These subjects showed no evidence of
 cancer.
 Blood mononuclear cells (BMCs) were isolated from the subjects, using
 density gradient centrifugation. T lymphocytes in the BMCs were purified
 by resetting, using sheep red blood cells which had been treated with
 aminoethylisothiouronium bromide, and then labelled with an anti-CD8
 monoclonal antibody coupled to magnetic microbeads. The CD8.sup.+ cells
 were sorted by passage through a magnetized area, and then stored at
 -80.degree. C. in Iscove's culture medium, supplemented with 10% human
 serum, 116 mg/ml L-arginine, 36 mg/ml L-asparagine, and 216 mg/ml of
 L-glutamine, 0.05M 2-mercaptoethanol, and 10% DMSO.
 Any non-rosetting BMCS were left to adhere for two hours at 37.degree. C.
 on tissue culture plates. Non-adherent cells were discarded, and adherent
 cells cultured for seven days in the presence of IL-4 (50 U/ml), and
 GM-CSF (100 ng/ml). The resulting population was enriched for antigen
 presenting cells ("APCs"; in this case, dendritic cells or macrophages).
 Then, from 5.times.10.sup.5 to 10.sup.6 of these cells were incubated in 2
 ml wells for four hours, at 37.degree. C., in 400 ul Iscove's medium
 supplemented with 2.5 ug/ml of human B2 microglobulin, and 50 ug/ml of the
 peptide of SEQ ID NO: 17. Adherent, peptide pulsed cells were then
 irradiated at 5000 rads, and washed. Next, 2.times.10.sup.6 autologous
 CD8.sup.+ T cells were added, in culture medium, supplemented with 1000
 U/ml of IL-6, and 5 ng/ml of IL-12.
 Seven days later, lymphocytes were restimulated with adherent, autologous
 BMCs, pulsed with peptide as above. 5.times.10.sup.6 BMCs were left to
 adhere for two hours at 37.degree. C., in 400 ul Iscove's medium
 containing B2-microglobulin and SEQ ID NO: 17, as discussed above. Any
 peptide pulsed, adherent cells, were irradiated and washed. Responder
 cells were then added, in culture medium supplemented with 10 U/ml of
 IL-2, and 5 ng/ml of IL-7.
 On day 14, the lymphocytes were restimulated with autologous BMCs pulsed
 with SEQ ID NO: 17. The BMCs were incubated, at 2.times.10.sup.7 cells/ml,
 in the augmented Iscove's medium discussed supra but without 10% DMSO.
 After two hours of incubation (20.degree. C.), peptide pulsed BMCs were
 irradiated, washed, and resuspended at 2.times.10.sup.6 cells /ml in
 culture medium augmented with IL-2 and IL-7, as above. Samples of these
 stimulator cells (2.times.10.sup.6), were added to each well which
 contained responder cells.
 The responder lymphocytes were cloned on day 21. Anywhere from 10 to 0.3
 cells/well were seeded in microwells, in culture medium which had been
 supplemented with 50 U/ml of IL-2, and 5 U/ml of IL-4. These were then
 stimulated by adding allogenic EBV transformed B cells (LG2-EBV) and
 irradiated at 10,000 rads, at 20,000 cells per well, one of (i) allogeneic
 EBV-transformed B cells, (ii) peptide pulsed HLA-B4402.sup.+ cells, or
 (iii) peptide pulsed HLA-B4403.sup.+ cells. For (ii) or (iii), irradiation
 was at 15,000 rads, at 8000 cells per well.
 Microcultures were restimulated every week in the same way they were on the
 21st day. The one change was that at days 28 and 35, 40,000 and 60,000
 EBV-B cells respectively were added per well, as compared to 20,000 at day
 21.
 Between days 41 and 52, aliquots of the proliferating microcultures were
 transferred into V-bottom microwells, in order to test for lytic activity
 against HLA-B4402.sup.+ or HLA-B4403.sup.+ target cells, pulsed and not
 pulsed with SEQ ID NO: 17.
 Any microcultures which showed anti-peptide lytic activity were
 restimulated with 5.times.10.sup.4 irradiated, peptide pulsed B4402.sup.+
 or B4403.sup.+ cells, plus 5.times.10.sup.5 irradiated LG2-EBV-B cells, in
 800 ul of culture medium augmented with 50 U/ml of IL-2, and 5 U/ml of
 IL-4.
 After seven days, the CTL clones were restimulated every week with
 2.times.10.sup.5 irradiated peptide pulsed B4402.sup.+ or B4403.sup.+
 cells, together with 10.sup.6 irradiated LG2-EBV-B cells, as described
 supra. In this way, CTLs LB 816-CTL-340 A/1, and LB822-CTL-346A/1 were
 obtained. These CTLs are specific for complexes of SEQ ID NO: 17 and
 either HLA-B*4402, or HLA-B4403, respectively.
 EXAMPLE 15
 In a further set of experiments, HLA-B4402.sup.+ or HLA-B4403.sup.+ EBV
 transformed B cells which do not express MAGE-3 were labelled with .sup.51
 Cr in the presence of monoclonal antibody W6/32, for 1 hour, at 37.degree.
 C., following Brodsky, et al, J. Immunol 128:135 (1982). The cells were
 washed, and incubated for 30 minutes at 20.degree. C. in serum free
 medium, using varied concentrations of SEQ ID NO: 17. Each CTL described
 in Example 14 was tested in a .sup.53 Cr release assay, also as described,
 with chromium release being measured after four hours.
 The results, set forth in FIG. 12, shows that the peptide did, in fact,
 provoke lysis.
 EXAMPLE 16
 In the following experiments, additional tumor cell lines which are HLA-B44
 positive were examined.
 All cell lines tested were labelled with .sup.51 Cr for one hour, at
 37.degree. C. They were then added in Iscove's medium plus 2% fetal calf
 serum, to various numbers of the two CTL clones discussed in example 14.
 The amount of .sup.51 Cr released was measured after four hours. Controls
 were also used, as indicated in FIG. 13. Note that the cell line LB33-MEL
 was incubated with IFN-.gamma. (50 U/ml), for 48 hours before the assay.
 The results of these experiments are shown in FIG. 13. CTL clones
 LB816-CTL-340 A/1 and LB822-CTL-346 A/1 lysed tumor cells expressing
 MAGE-3, but did not lyse LB33-EBv B cells which did not express the MAGE
 gene. The CTL clone LB822-346 A/1 lysed the HLA-B*4403.sup.+ tumor cell
 line MZ2-MEL, which expresses MAGE-3, but did not lyse the antigen loss
 variant MZ2-MEL.61.2D.sup.-.
 EXAMPLE 17
 As a final test to determine if the peptide of SEQ ID NO: 17, in complexes
 with HLA-B44, stimulated CTLs, experiments were carried out to determine
 if tumor necrosis factor release was stimulated.
 First, COS-7 cells were transfected by cDNA encoding MAGE-3 following
 Gaugler, et al, J. Exp. Med 179:921-930 (1994), in the expression vector
 PcDNA-I/AMP, and one of HLA-B*4402 cDNA cloned into vector pcDNA3, or
 HLA-B 4403 cDNA cloned into pcDNA1/AMP. The DEAE dextran chloroquine
 method of Aruffo, Proc. Natl. Acad. Sci. USA 84:3365-3369 (1987) was used.
 Transfectants were incubated for 24 hours, at 37.degree. C., then 3000
 CTLs/well were added. Materials were incubated for 18 hours, at 37.degree.
 C. Supernatants were then collected, and TNF content was determined by
 tested the cytolytic effect on TNF sensitive WEHI-16 clone 13 cells,
 following Espevik et al, J. Immunol. Meth 95:99-105 (1986).
 Table 3, which follows, shows the results, wherein TNF release is expressed
 in pg/ml. LB33-MEL and LB494 MEL were incubated with IFN.gamma. at 100
 U/ml for 24 hours prior to the assay. Tumor Cell lines LB33-MEL,
 LB494-MEL, and MZ2-MEL were also tested. These cell lines all express
 MAGE-3 cDNA, and are either HLA-B*4402.sup.+ (LB33-MEL, LB494-MEL), or
 HLA-B*4403.sup.+ (MZ2-MEL). Hence, no transfection was necessary for these
 cells. The results show that TNF was released. Hence, one concludes that
 SEQ ID NO: 17 is being presented by HLA-B44 MHC molecules, and these
 complexes provoke CTL activity.
 TABLE 3
 TNF production of anti-MAGE-3.B44 CTL clones
 TNF
 CTL Clones Stimulator cells (pg/ml)
 A LB816-CTL-340A/1 COS 0.7
 (B4402) COS+MAGE-3 0.6
 COS+HLA-B4402 0.5
 COS+HLA+B4402+MAGE-3 33.7
 LB33-MEL (B4402, MAGE-3.sup.+++) 74.9
 LB494-MEL (B4402, MAGE-3.sup.+++) 32.3
 B LB822-CTL-346A/1 COS 1.2
 (B4403) COS+MAGE-3 1
 COS+HLA-B4403 1.2
 COS+HLA-B4403+MAGE-3 26.6
 MZ2-MEL (B4403, MAGE-3.sup.+++) 67.3
 The foregoing experiments describe isolated nucleic acid molecules coding
 for a tumor rejection antigen precursor, a "TRAP" molecule. The protein
 molecule for which these code is processed intracellularly in a manner
 which leads to production of at least one tumor rejection antigen, or
 "TRA", which is presented by HLA-B44 molecules. While it has been observed
 previously that HLA-B44 molecules present peptides derived from
 tyrosinase, the nucleic acid molecules of the invention do not code for
 tyrosinase, and the TRAs are not tyrosinase derived.
 The tumor rejection antigens of the invention are isolated nonapeptides
 which have a Glu residue at the 2nd position, and a Phe or Tyr residue at
 the 9th or 10th position. Especially preferred are the nonamer of SEQ ID
 NO: 2, i.e.:
 Glu Glu Lys Leu Ile Val Val Leu Phe as well as the nonamer
 Glu Glu Lys Leu Ile Val Val Ala Phe (SEQ ID NO: 18)
 and the decamer:
 Met Glu Val Asp Pro Ile Gly His Leu Tyr (SEQ ID NO: 17).
 Also useful are nonapeptides which, in addition to the required residues at
 positions 2 and 9 or 10, have one or more These are set forth in SEQ ID
 NOS: 20, 21, 22, and 23 of the following defined residues:
 position 1: Glu or Met
 position 3: Lys or Val
 position 4: Leu or Asp
 position 5: Ile or Pro
 position 6: Val or Ile
 position 7: Val or Gly
 position 8: Leu, Ala or His
 position 9: Leu (when the peptide is a decamer)
 Of particular interest are peptides which satisfy certain consensus motifs.
 These motifs include:
 Xaa Glu (Xaa).sub.3 Val (Xaa).sub.2 Phe (SEQ ID NO:24)
 and
 Xaa Glu (Xaa), Val Xaa Phe (SEQ ID NO:25)
 wherein Xaa is any amino acid, as well as the consensus motif
 Xaa Glu (Xaa).sub.2,3 Ile (Xaa).sub.3 Xaa (SEQ ID NO:26 and 27)
 wherein the first, second, and third occurrences of Xaa refer to any amino
 acids, and the fourth one, i.e., the carboxy terminal amino acid,
 represents tyrosine or phenylalanine. Especially preferred are the
 peptides of SEQ ID NOS: 17 and 18, and the variations discussed, supra.
 The peptides of the invention are similar to the peptide disclosed in Ser.
 No. 08/233,305 now U.S. Pat. No. 5,519,117, co-assigned to the assignee of
 the subject application, i.e.:
 Ser Glu Ile Trp Arg Asp Ile Asp Phe (SEQ ID NO: 3)
 Khanna, et al., supra, teaches a decamer, i.e.:
 Glu Glu Asn Leu Leu Asp Phe Val Arg Phe (SEQ ID NO: 4)
 but does not discuss how modification of the decamer could lead to an
 effective nonamer.
 The invention thus involves tumor rejection antigens which bind to HLA-B44
 molecules, and then provoke lysis by CTLs.
 As indicated, the complexes of TRA and HLA molecule provoke a cytolytic T
 cell response, and as such isolated complexes of the tumor rejection
 antigen and an HLA-B44 molecule are also encompassed by the invention, as
 are isolated tumor rejection antigen precursors coded for by the
 previously described nucleic acid molecules. Given the binding
 specificity, the peptides may also be used, simply to identify HLA-B44
 positive cells.
 The invention as described herein has a number of uses, some of which are
 described herein. First, the identification of a tumor rejection antigen
 which is specifically presented by an HLA-B44 molecule, as well as a
 nucleic acid molecule coding for its parallel tumor rejection antigen
 precursor permits the artisan to diagnose a disorder characterized by
 expression of the TRAP. These methods involve determining expression of
 the TRAP gene, and/or TRAs derived therefrom, such as TRA presented by HLA
 molecules. Other TRAs may also be derived from the TRAPs of the invention
 and presented by different HLA molecules. In the former situation, such
 determinations can be carried out via any standard nucleic acid
 determination assay, including the polymerase chain reaction, or assaying
 with labelled hybridization probes. In the latter situation, assaying with
 binding partners for complexes of TRA and HLA, such as antibodies, is
 especially preferred.
 The isolation of the TRAP gene also makes it possible to isolate the TRAP
 molecule itself, especially TRAP molecules containing the amino acid
 sequence of SEQ ID NO: 1. Fragments of peptides of these isolated
 molecules when presented as the TRA, or as complexes of TRA and HLA, B44,
 may be combined with materials such as adjuvants to produce vaccines
 useful in treating disorders characterized by expression of the TRAP
 molecule. In addition, vaccines can be prepared from cells which present
 the TRA/HLA complexes on their surface, such as non-proliferative cancer
 cells, non-proliferative transfectants, etcetera. In all cases where cells
 are used as a vaccine, these can be cells transfected with coding
 sequences for one or both of the components necessary to prove a CTL
 response, or be cells which express both molecules without transfection.
 Further, the TRAP molecule, its associated TRAs, as well as complexes of
 TRA and HLA, may be used to produce antibodies, using standard techniques
 well known to the art.
 When "disorder" is used herein, it refers to any pathological condition
 where the tumor rejection antigen precursor is expressed. An example of
 such a disorder is cancer, melanoma in particular.
 Therapeutic approaches based upon the disclosure are premised on a response
 by a subject's immune system, leading to lysis of TRA presenting cells,
 such as cells presenting the relevant HLA molecule. One such approach is
 the administration of CTLs specific to the complex to a subject with
 abnormal cells of the phenotype at issue. it is within the skill of the
 artisan to develop such CTLs in vitro. Specifically, a sample of cells,
 such as blood cells, are contacted to a cell presenting the complex and
 capable of provoking a specific CTL to proliferate. The target cell can be
 a transfectant, such as a COS cell of the type described supra. These
 transfectants present the desired complex on their surface and, when
 combined with a CTL of interest, stimulate its proliferation. COS cells,
 such as those used herein are widely available, as are other suitable host
 cells.
 To detail the therapeutic methodology, referred to as adoptive transfer
 (Greenberg, J. Immunol. 136(5): 1917 (1986); Reddel et al., Science 257:
 238 (7-10-92); Lynch et al., Eur. J. Immunol. 21: 1403-1410 (1991); Kast
 et al., Cell 59: 603-614 (11-17-89)), cells presenting the desired complex
 are combined with CTLs leading to proliferation of the CTLs specific
 thereto. The proliferated CTLs are then administered to a subject with a
 cellular abnormality which is characterized by certain of the abnormal
 cells presenting the particular complex. The CTLs then lyse the abnormal
 cells, thereby achieving the desired therapeutic goal.
 The foregoing therapy assumes that at least some of the subject's abnormal
 cells present the HLA/TRA complex. This can be determined very easily, as
 the art is very familiar with methods for identifying cells which present
 a particular HLA molecule, as well as how to identify cells expressing DNA
 containing the indicated sequences. Once isolated, such cells can be used
 with a sample of a subject's abnormal cells to determine lysis in vitro.
 If lysis is observed, then the use of specific CTLs in such a therapy may
 alleviate the condition associated with the abnormal cells. A less
 involved methodology examines the abnormal cells for HLA phenotyping,
 using standard assays, and determines expression via amplification using,
 e.g., PCR.
 Adoptive transfer is not the only form of therapy that is available in
 accordance with the invention. CTLs can also be provoked in vivo, using a
 number of approaches. One approach, i.e., the use of non-proliferative
 cells expressing the complex, has been elaborated upon supra. The cells
 used in this approach may be those that normally express the complex, such
 as irradiated melanoma cells or cells transfected with one or both of the
 genes necessary for presentation of the complex. Chen et al., Proc. Natl.
 Acad. Sci. USA 88: 110-114 (January, 1991) exemplifies this approach,
 showing the use of transfected cells expressing HPVE7 peptides in a
 therapeutic regime. Various cell types may be used. Similarly, vectors
 carrying one or both of the genes of interest may be used. Viral or
 bacterial vectors are especially preferred. In these systems, the gene of
 interest is carried by, e.g., a Vaccinia virus or the bacteria BCG, and
 the materials de facto "infect" host cells. The cells which result present
 the complex of interest, and are recognized by autologous CTLs, which then
 proliferate. A similar effect can be achieved by combining the tumor
 rejection antigen or the precursor itself with an adjuvant to facilitate
 incorporation into cells which present the HLA molecule of interest. The
 TRAP is processed to yield the peptide partner of the HLA molecule while
 the TRA is presented without the need for further processing.
 Other aspects of the invention will be clear to the skilled artisan and
 need not be repeated here.
 The terms and expressions which have been employed are used as terms of
 description and not of limitation, and there is no intention in the use of
 such terms and expressions of excluding any equivalents of the features
 shown and described or portions thereof, it being recognized that various
 modifications are possible within the scope of the invention.