Isolated, MAGE-3 derived peptides which complex with HLA-A2 molecules and uses thereof

Tumor rejection antigens derived from tumor rejection precursor MAGE-3 have been identified. These "TRAS" bind to the MHC-class I molecule HLA-A2, and the resulting complexes stimulate the production of cytolytic T cell clones which lyse the presenting cells. The peptides and complexes may be used diagnostically, therapeutically, and as immunogens for the production of antibodies, or as targets for the generation of cytolytic T cell clones.

FIELD OF THE INVENTION 
This invention relates to immunogenetics and to peptide chemistry. More 
particularly, it relates to peptides, especially deca- and nonapeptides 
useful in various ways, including immunogens and as ligands for HLA-A2 
molecules. More particularly, it relates to a so-called "tumor rejection 
antigen", derived from the tumor rejection antigen precursor encoded by 
gene MAGE-3, and presented by the MHC-class I molecule HLA-A2. 
BACKGROUND AND PRIOR ART 
The study of the recognition or lack of recognition of cancer cells by a 
host organism has proceeded in many different directions. Understanding of 
the field presumes some understanding of both basic immunology and 
oncology. 
Early research on mouse tumors revealed that these displayed molecules 
which led to rejection of tumor cells when transplanted into syngeneic 
animals. These molecules are "recognized" by T-cells in the recipient 
animal, and provoke a cytolytic T-cell response with lysis of the 
transplanted cells. This evidence was first obtained with tumors induced 
in vitro by chemical carcinogens, such as methylcholanthrene. The antigens 
expressed by the tumors and which elicited the T-cell response were found 
to be different for each tumor. See Prehn, et al., J. Natl. Canc. Inst. 
18:769-778 (1957); Klein et al., Cancer Res. 20: 1561-1572 (1960); Gross, 
Cancer Res. 3: 326-333 (1943), Basombrio, Cancer Res. 30:2458-2462 (1970) 
for general teachings on inducing tumors with chemical carcinogens and 
differences in cell surface antigens. This class of antigens has come to 
be known as "tumor specific transplantation antigens" or "TSTAs". 
Following the observation of the presentation of such antigens when 
induced by chemical carcinogens, similar results were obtained when tumors 
were induced in vitro via ultraviolet radiation. See Kripke, J. Natl. 
Canc. Inst. 53:333-1336 (1974). 
While T-cell mediated immune responses were observed for the types of tumor 
described supra, spontaneous tumors were thought to be generally 
non-immunogenic. These were therefore believed not to present antigens 
which provoked a response to the tumor in the tumor carrying subject. See 
Hewitt, et al., Brit. J. Cancer 33: 241-259 (1976). 
The family of tum.sup.- antigen presenting cell lines are immunogenic 
variants obtained by mutagenesis of mouse tumor cells or cell lines, as 
described by Boon et al., J. Exp. Med. 152: 1184-1193 (1980), the 
disclosure of which is incorporated by reference. To elaborate, tum.sup.- 
antigens are obtained by mutating tumor cells which do not generate an 
immune response in syngeneic mice and will form tumors (i.e., "tum.sup.+ " 
cells). When these tum.sup.+ cells are mutagenized, they are rejected by 
syngeneic mice, and fail to form tumors (thus "tum.sup.- "). See Boon et 
al., Proc. Natl. Acad. Sci. USA 74:272 (1977), the disclosure of which is 
incorporated by reference. Many tumor types have been shown to exhibit 
this phenomenon. See, e.g., Frost et al., Cancer Res. 43:125 (1983). 
It appears that tum.sup.- variants fail to form progressive tumors because 
they initiate an immune rejection process. The evidence in favor of this 
hypothesis includes the ability of "tum.sup.- " variants of tumors, i.e., 
those which do not normally form tumors, to do so in mice with immune 
systems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl. 
Acad. Sci. USA 76: 5282-5285 (1979); and the observation that 
intraperitoneally injected tum.sup.- cells of mastocytoma P815 multiply 
exponentially for 12-15 days, and then are eliminated in only a few days 
in the midst of an influx of lymphocytes and macrophages (Uyttenhove et 
al., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes the 
observation that mice acquire an immune memory which permits them to 
resist subsequent challenge to the same tum.sup.- variant, even when 
immunosuppressive amounts of radiation are administered with the following 
challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA 74:272-275 
(1977); Van Pel et al., supra; Uyttenhove et al., supra). 
Later research found that when spontaneous tumors were subjected to 
mutagenesis, immunogenic variants were produced which did generate a 
response. Indeed, these variants were able to elicit an immune protective 
response against the original tumor. See Van Pel et al., J. Exp. Med. 157: 
1992-2001 (1983). Thus, it has been shown that it is possible to elicit 
presentation of a so-called "tumor rejection antigen" in a tumor which is 
a target for a syngeneic rejection response. Similar results have been 
obtained when foreign genes have been transfected into spontaneous tumors. 
See Fearon et al., Cancer Res. 48: 2975-1980 (1988) in this regard. 
A class of antigens has been recognized which are presented on the surface 
of tumor cells and are recognized by cytolytic T cells, leading to lysis. 
This class of antigens will be referred to as "tumor rejection antigens" 
or "TRAs" hereafter. TRAs may or may not elicit antibody responses. The 
extent to which these antigens have been studied, has been via cytolytic T 
cell characterization studies, in vitro i.e., the study of the 
identification of the antigen by a particular cytolytic T cell ("CTL" 
hereafter) subset. The subset proliferates upon recognition of the 
presented tumor rejection antigen, and the cells presenting the antigen 
are lysed. Characterization studies have identified CTL clones which 
specifically lyse cells expressing the antigens. Examples of this work may 
be found in Levy et al., Adv. Cancer Res. 24: 1-59 (1977); Boon et al., J. 
Exp. Med. 152:1184-1193 (1980); Brunner et al., J. Immunol. 124: 1627-1634 
(1980); Maryanski et al., Eur. J. Immunol. 124: 1627-1634 (1980); 
Maryanski et al., Eur. J. Immunol. 126: 406-412 (1982); Palladino et al., 
Canc. Res. 47: 5074-5079 (1987). This type of analysis is required for 
other types of antigens recognized by CTLs, including minor 
histocompatibility antigens, the male specific H-Y antigens, and the class 
of antigens referred to as "tum-" antigens, and discussed herein. 
A tumor exemplary of the subject matter described supra is known as P815. 
See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988); 
Szikora et al., EMBO J 9: 1041-1050 (1990), and Sibille et al., J. Exp. 
Med. 172: 35-45 (1990), the disclosures of which are incorporated by 
reference. The P815 tumor is a mastocytoma, induced in a DBA/2 mouse with 
methylcholanthrene and cultured as both an in vitro tumor and a cell line. 
The P815 line has generated many tum.sup.- variants following mutagenesis, 
including variants referred to as P91A (DePlaen, supra), 35B (Szikora, 
supra), and P198 (Sibille, supra). In contrast to tumor rejection 
antigens--and this is a key distinction--the tum.sup.- antigens are only 
present after the tumor cells are mutagenized. Tumor rejection antigens 
are present on cells of a given tumor without mutagenesis. Hence, with 
reference to the literature, a cell line can be tum.sup.+, such as the 
line referred to as "P1", and can be provoked to produce tum.sup.- 
variants. Since the tum.sup.- phenotype differs from that of the parent 
cell line, one expects a difference in the DNA of tum.sup.- cell lines as 
compared to their tum.sup.+ parental lines, and this difference can be 
exploited to locate the gene of interest in tum.sup.- cells. As a result, 
it was found that genes of tum.sup.- variants such as P91A, 35B and P198 
differ from their normal alleles by point mutations in the coding regions 
of the gene. See Szikora and Sibille, supra, and Lurquin et al., Cell 58: 
293-303 (1989). This has proved not to be the case with the TRAs of this 
invention. These papers also demonstrated that peptides derived from the 
tum.sup.- antigen are presented by the L.sup.d molecule for recognition by 
CTLs. P91A is presented by L.sup.d, P35 by D.sup.d and P198 by K.sup.d. 
PCT application PCT/US92/04354, filed on May 22, 1992 assigned to the same 
assignee as the subject application, teaches a family of human tumor 
rejection antigen precursor coding genes, referred to as the MAGE family. 
Several of these genes are also discussed in van der Bruggen et al., 
Science 254: 1643 (1991). It is now clear that the various genes of the 
MAGE family are expressed in tumor cells, and can serve as markers for the 
diagnosis of such tumors, as well as for other purposes discussed therein. 
See also Traversari et al., Immunogenetics 35: 145 (1992); van der Bruggen 
et al., Science 254: 1643 (1991). The mechanism by which a protein is 
processed and presented on a cell surface has now been fairly well 
documented. A cursory review of the development of the field may be found 
in Barinaga, "Getting Some `Backbone`: How MHC Binds Peptides", Science 
257: 880 (1992); also, see Fremont et al., Science 257: 919 (1992); 
Matsumura et al., Science 257: 927 (1992); Latron et al., Science 257: 964 
(1992). These papers generally point to a requirement that the peptide 
which binds to an MHC/HLA molecule be nine amino acids long (a 
"nonapeptide"), and to the importance of the first and ninth residues of 
the nonapeptide. As described herein, while this "rule" is generally true, 
there is some leeway as to the length of peptides which MHC-class I 
molecules will bind. 
Studies on the MAGE family of genes have now revealed that a particular 
nonapeptide is in fact presented on the surface of some tumor cells, and 
that the presentation of the requires that the presenting molecule be 
HLA-A1. Complexes of the MAGE-1 tumor rejection antigen (the "TRA" or 
nonapeptide") leads to lysis of the cell presenting it by cytolytic T 
cells ("CTLs"). 
Attention is drawn, e.g., to concurrently filed application Serial No. 
08/217,187 to Traversari et al., and Ser. No. 08/217,188 to Melief et al., 
both of which present work on other, MAGE-derived peptides. 
Research presented in, e.g., U.S. patent application Ser. No. 07/938,334 
filed Aug. 31, 1992, and in U.S. patent application Ser. No. 073,103, 
filed Jun. 7, 1993, showed that when comparing homologous regions of 
various MAGE genes to the region of the MAGE-1 gene coding for the 
relevant nonapeptide, there is a great deal of homology. Indeed, these 
observations lead to one of the aspects of the invention disclosed and 
claimed therein, which is a family of nonapeptides all of which have the 
same N-terminal and C-terminal amino acids. These nonapeptides were 
described as being useful for various purposes which includes their use as 
immunogens, either alone or coupled to carrier peptides. Nonapeptides are 
of sufficient size to constitute an antigenic epitope, and the antibodies 
generated thereto were described as being useful for identifying the 
nonapeptide, either as it exists alone, or as part of a larger 
polypeptide. 
These references, especially Ser. No. 073,103, showed a connection between 
HLA-A1 and MAGE-3; however, only about 26% of the caucasian population and 
17% of the negroid population presents HLA-A1 molecules on cell surfaces. 
Thus, it would be useful to have additional information on peptides 
presented by other types of MHC molecules, so that appropriate portions of 
the population may benefit from the research discussed supra. 
It has now been found that antigen presentation of MAGE-3 derived peptides 
is not limited to HLA-A1 molecules. The invention set forth, in the 
disclosure which follows, identifies peptides which complex with MHC class 
I molecule HLA-A2. The ramifications of this discovery, which include 
therapeutic and diagnostic uses, are among the subjects of the invention, 
set forth in the disclosure which follows.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
EXAMPLE 1 
The methodologies employed in this set of experiments are akin to those 
described by Elvin et al., J. Imm. Meth. 158: 161-171 (1993), Townsend et 
al., Nature 340:443-448 (Aug. 10, 1989), and Townsend et al., Cell 
62:285-290 (Jul. 27, 1990), all of which are incorporated by reference in 
their entirety. 
Cell line 0.174 as described was used. It is an HLA-A2 presenting cell line 
deficient in the pathway which supplies peptides to the endoplasmic 
reticulum, the site of assembly of MHC class-I heterodimers. The cell line 
can assemble MHC class-I molecules, but these are unstable and, on cell 
lysis, dissociate into free heavy and light chains during overnight 
incubation. The heterodimers can, however, be stabilized in vitro via 
addition of appropriate peptide ligands. (Townsend et al., Nature 340: 
443-448 (1989); Townsend et al., Cell 62: 285-295 (1990)). Thus, the 
stabilized molecules can be immunoprecipitated with antibodies specific 
for the MHC class-I molecule. 
In the first part of these experiments, peptides were tested to determine 
if they facilitated assembly of HLA-A2 in the cell line. The peptides 
tested included the following: 
SEQ ID NO: 1 Gly Leu Glu Ala Arg Gly Glu Ala Leu 
SEQ ID NO: 2 Ala Leu Ser Arg Lys Val Ala Glu Leu 
SEQ ID NO: 3 Cys Leu Gly Leu Ser Tyr Asp Gly Leu 
SEQ ID NO: 4 Ile Leu Gly Asp Pro Lys Lys Leu Leu 
SEQ ID NO: 5 His Leu Try Ile Phe Ala Thr Cys Leu 
SEQ ID NO: 6 Phe Leu Trp Gly Pro Arg Ala Leu Val 
SEQ ID NO: 7 Thr Leu Val Glu Val Thr Leu Gly Glu Val 
SEQ ID NO: 8 Ala Leu Ser Arg Lys Val Ala Glu Leu Val 
SEQ ID NO: 9 Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val 
SEQ ID NO: 10 Ala Leu Val Glu Thr Ser Yr Val Lys Val 
Cells were labelled by exposure to [.sup.35 S] methionine (aliquots of 
1--2.times.10.sup.7 cells, labeled with 100-200 .mu.Ci, 60 minutes of 
contact). The cells were then washed, once, with phosphate buffered 
saline, and then resuspended in 10 ml of lysis buffer (0.5% NP-40; 0.5% 
Mega 9, 150 mM NaCl, 5 mM EDTA, 50 mM Tris [pH 7.5], 2 mM 
phenylmethylsulfonylflouride, 5 mM iodoacetamide). The lysates were then 
incubated with peptide (10 .mu.M and 20 .mu.M), for 15-18 hours. Nuclei 
were then pelleted in a microfuge, and the lysates were precleared, 
overnight, at 4.degree. C. with 0.2 ml of washed, 10% (w/v) Staphylococcus 
A organisms. Lysates were divided into two portions, and monoclonal 
antibody BB7.2 was added to a final concentration of 5 ug/ml. This mAb is 
a conformation specific, HLA-A2 recognizing mAb described by Parham et 
al., Hum. Immunol. 3: 277-299 (1981). The mixtures were incubated for 90 
minutes on ice, followed by addition of bovine serum albumin to 1% (w/v), 
and 100 ul of 5% (w/v) protein-A Sepharose beads. Tubes were rotated for 
45 minutes, after which beads were washed, four times, with 1 ml wash 
buffer (0.5% NP-40, 150 mM NaCl, 5 mM EDTA, 50 mM Tris [PH 7.5]). Samples 
were eluted, and analyzed on 12% polyacrylamide gels, in accordance with 
Townsend et al., Nature 340: 443-448 (1989) . 
FIG. 1 shows results from these experiments for the peptides which gave 
positive results. These were SEQ ID NOS: 2, 6, 7, 8 and 10, as is 
evidenced by the dark band, indicated by HC (heavy chain) common to all of 
the gels, and represents immunoprecipitated MHC molecule (HLA-A2) that had 
complexed with the peptide prior to electrophoresis. 
The figure shows work with SEQ ID NOS: 2, 6, 7, 8 and 10, running from left 
to right. The vertical bar separates SEQ ID NO: 5 from gels marked 
"0.174", "A2 line", and "0.174 matrix". 0.174 is a "negative" control for 
the heavy chain of the MHC class I molecule. As noted supra, this cell 
line does not present stable MHC-class I molecules without exogenous 
peptide, and as mAb BB7.2 is conformation specific, it should not 
precipitate uncomplexed MHC-class I molecules. "A2" refers to a known cell 
line presenting HLA-A2 (the line is LBL 721, described by DeMars et al., 
Hum. Immunol. 11: 77 (1984)), but any cell presenting stable HLA-A2 
molecules would function in the same way. "0.174 matrix" shows results 
when 0.174 cell line was incubated together with the control peptide 
GILGFVFTL (SEQ ID NO: 11), which is derived from influenza virus and is 
known to be presented by HLA-A2. 
The results show the stabilization of the MHC-class I molecule, by the fact 
that the bands for "HC" (heavy chain) are comparable to those obtained for 
A2 and 0.174 matrix. In fact, the MHC molecule is disrupted by the 
reducing gel; however, the heavy chain molecules will be bound by the 
comformation specific mAb if stabilized prior to reduction. This is in 
fact what the gels show --i.e.--that the recited peptides bound to the 
HLA-A2 molecules, and stabilized them. 
EXAMPLE 2 
Once binding peptides were identified, a series of titration experiments 
were carried out. In these, varying concentrations of peptides, in 
accordance with Townsend et al., Cell 62: 285-295 (Jul. 27, 1990) at 293, 
incorporated by reference herein, were added to lysates of the cell line 
referred to supra, and immunoprecipitated to determine the concentration 
which was the best concentration for the binding of the peptide. 
FIG. 2 shows the results obtained for two of the peptides i.e., SEQ ID NO: 
2 and 6. The peptides were titrated against a known HLA-A2 binding peptide 
SEQ ID NO: 11, with 10 fold dilutions starting at 20 .mu.M, and decreasing 
to 2, 0.2 and 0.002 .mu.M. 
Experiments were carried out with these peptides (i.e., SEQ ID NOS: 2 and 
6). In the case of SEQ ID NO: 2, in experiments not reported here the 
peptide titrated at 5-10 nM. This was comparable to the control (SEQ ID 
NO: 11). 
EXAMPLE 3 
A series of experiments were carried out to show the ability of the peptide 
SEQ ID NO: 6 to provoke lysis by cytolytic T lymphocytes ("CTLs") specific 
to complexes of the peptide and HLA-A2. The first steps in these 
experiments are described herein. 
Peripheral blood lymphocytes ("PBLs") were taken from a normal donor, i.e., 
one without any cancer tumors. The donor, referred to as "LB705" was typed 
as HLA-A1, A2, B8, B27. At the start ("day 0"), PBLs from the donor were 
suspended, at 10.sup.6 cells/ml, in Iscove's medium and 10% fetal calf 
serum and "AAG" (asparagine+arginine+glutamine), and 20 ug/ml of rabbit 
antihuman IgM antibody, and 20 ng/ml recombinant human IL-4 ("r-hu-IL4"), 
and 0.005% Pansorbin cells. The mixture was distributed into 24-well 
tissue culture plates (2 ml per well). 
At day 3, the cells were centrifuged and resuspended in Iscove's medium and 
10% human serum and AAG and 20 ng/ml r-hu-IL4. 
Two days later, on day 5, the cells were again centrifuged, and resuspended 
in fresh Iscove's medium and 10% human serum and AAG and 20 ng/ml 
r-hu-IL-4, and 20 U/ml recombinant human .gamma.-interferon. 
On day 6, the cells were again centrifuged, and resuspended at 
5.times.10.sup.6 cells/ml in Iscove's medium without serum, and 50 ug/ml 
of the peptide of SEQ ID NO: 6, plus 2.5 ug/ml of human .beta.2 
microglobulin. The cells were incubated in this mixture for four hours at 
37.degree. C., and were then irradiated at 50 Gy. The cells were then 
centrifuged again, and resuspended in Iscove's medium and 10% human 
serum+AAG. The cells were then placed in individual wells of 24 well 
tissue culture plates, at 1 million cells per well. 
Responder cells were then added. These were CD8.sup.+ T cells also obtained 
from donor LB705. Fractions of CD8.sup.+ cells had been secured from the 
donors' PBLs using well known techniques for separating T cell fractions. 
The responder cells were added to the wells, at 5.times.10.sup.6 
cells/well. Final volume was 2 ml. Following the addition of the cells, 
1000 U/ml of recombinant human IL-6 ("r-hu-IL-6"), and 10 ng/ml of 
recombinant human IL-12 ("r-hu-IL-12") were added. 
Seven days later, i.e., on day 13, the responder cells, i.e., the CD8.sup.+ 
cells, were restimulated. This was accomplished by transferring the mixed 
culture discussed above to autologous adherent cells, together with 10 
U/ml r-hu-IL-2, and 5 ng/ml of r-hu-IL-7. The autologous adherent cells 
had been prepared previously, by incubating 5.times.10.sup.6 irradiated 
(50 Gy) PBLs from LB705, in 1 ml Iscove's medium+10% human serum+AAG, at 
37.degree. C. for two hours. Any non-adherent cells were removed and 50 
ug/ml of the peptide of SEQ ID NO: 6 and 2.5 ug/ml of human .beta.2 
microglobulin were added, in 0.5 ml of serum free medium. This mixture was 
incubated for two hours at 37.degree. C., and then washed. The responder 
CD8.sup.30 cells were then added to them. 
At day 21, another stimulation of the responder cells took place, by adding 
2.times.10.sup.6 PBLs, irradiated at 50 Gy, which had been incubated in 
serum free medium+50 ug/ml human .beta.2 microglobulin plus 50 ug/ml of 
SEQ ID NO: 6 for two hours, followed by washing. 
This protocol resulted in the generation of CTLs specific for complexes of 
SEQ ID NO: 6 and HLA-A2, which is shown in the following example. 
EXAMPLE 4 
Experiments were carried out on day 28 to determine if peptides in 
accordance with the invention, when complexed to HLA-A2 molecules, would 
provoke lysis by CTLs. 
Cells of line T2 were used. This cell line presents HLA-A2 molecules on its 
surface, but has an antigen processing defect which results in increased 
capacity for presenting exogenous peptides. See Cerundolo, et al, Nature 
345: 449 (1990), which describes this cell line. Other equivalent cell 
lines are also available. 
Samples of T2 cells were labelled with radioactive chromium (.sup.51 Cr), 
and incubated together with 1 .mu.m of the peptide of SEQ ID NO: 6. The 
preincubation took place for one hour prior to introduction of CTLs. 
Control samples of T2 cells were not incubated with peptide. 
CTLs were prepared by stimulating CD8.sup.+ cells with autologous APCs, 
preincubated with the peptide of SEQ ID NO: 6 for a period of 21 days, in 
accordance with example 3, supra. 
FIG. 3 shows the results. The X-axis shows the ratio of effector/target 
cells, while the Y axis depicts the percent of specific lysis, determined 
by measuring chromium release, in accordance with Boon, et al., J. Exp. 
Med. 152: 1184 (1980), incorporated by reference in its entirety. In each 
test well, non-specific lysis was eliminated by adding 50,000 K562 cells 
to the 1,000 .sup.51 Cr labelled T2 target cells employed. The K562 cells 
act to eliminate non-specific lysis, as Natural Killer, or "NK" cells 
preferentially lyse this line. 
It is clear that the peptide, when presented by HLA-A2, provoked lysis of 
the T2 cells. 
EXAMPLE 5 
The work described in example 4 led to the generation of a mixed culture of 
CD8.sup.+ T cells specific for complexes of SEQ ID NO: 6 and its 
presenting HLA-A2 molecules, and non-specific CTLs. To isolate CTL clones 
of the desired specificity, a limiting dilution assay was carried out in 
accordance with Herin, et al., Int. J. Cancer 39: 390-396 (1987), 
incorporated by reference, but summarized herein. 
On day 29, following Herin, et al., supra, irradiated SK23-MEL cells, known 
to express MAGE-3 and HLA-A2, were combined with the CTL mixed culture, 
together with LG2-EBV cells, which acted as feeder cells. Also, 50 U/ml of 
IL-2, and 5 U/ml IL-4 were added to the mixture. This resulted in the 
generation of CTL clones 297/19, CTL 297/22, CTL 297/27, and CTL 297/36. 
EXAMPLE 6 
In example 4, the induction of lysis of cells presenting the peptide of SEQ 
ID NO: 6 was shown, using a fixed amount of peptide with varying 
effector/target ratios. In these experiments, the effector/target ratio 
was kept constant, and the amount of peptide varied. Again, the .sup.51 Cr 
release assay of example 4 was used, as were the T2 cells and CTLs of 
example 4. The four different CTLs of example 5 were used. 
FIGS. 4A, 4B, 4C and 4D show these results, and indicate some dose 
dependency of lysis. When T2 cells were not incubated with peptide, lysis 
was always below 2%. 
EXAMPLE 7 
In another set of experiments, the lytic effect of the peptide of SEQ ID 
NO: 6 was tested in a model where the host cells did not inherently 
express HLA-A2. 
Samples of cell line COS-7 were used. The cells were transfected with one 
of (i) genomic DNA for HLA-A2.01 and cDNA for MAGE-3, (ii) genomic DNA for 
HLA-A2.01 only, or (iii) cDNA for MAGE-3 only. In each case, the 
transfecting vector was pcDNA/AmpI, where the DNA was ligated to EcoRI 
adaptors, and cloned into the EcoRI site of the plasmid in accordance with 
manufacturer's instructions. The recipient 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. 
Cells were incubated overnight, and medium was removed and replaced by 30 
ul/well of DMEM containing 10% Nu serum, 400 ug/ml DEAE dextran, 100 .mu.M 
chloroquine, and 100 ng of the plasmids made as described herein. As a 
further control, COS-7 cells transfected with HLA-A2 alone (via HLA-A2.01 
in pcDNA/AmpI), were preincubated for one hour with 1 .mu.M of the peptide 
of SEQ ID NO: 6, following example 4, supra. 
In addition, melanoma cell lines which were known to express MAGE-3 and 
which were HLA-A2.sup.+ were used. These cell lines are identified in the 
figures as LB 373, LB43, LB24 clone 409, and SK23. Cell line MZ2-MEL.43 is 
HLA-A2.sup.-. In additional tests the line was also transfected with the 
HLA-A2 gene in pcDNA/AmpI. 
A TNF release assay was used, following Traversari, et al, Immunogenetics 
35: 145-152 (1992), incorporated by reference herein, but outlined below, 
together with the modifications thereto. Specifically, 1500 CTLs were 
combined with 30,000 target cells (CTLs were one of the clones discussed 
supra). The cells were cultured together, in the presence of 25 u/ml of 
IL-2. Twenty-four hours later, supernatants from the cultures were tested 
against WEHI 164 clone 13 cells, which are sensitive to TNF. Sensitivity 
was increased by adding LiCl to the WEHI 164 clone 13 cells (20 mM), in 
accordance with Beyart, at al, PNAS 86: 9494-9498 (1989). 
The results of these experiments are depicted in FIGS. 5A-5D. Each figure 
presents TNF release (pg/ml), for a different CTL clone, for transfectants 
(top panel, each figure), and tumor cell lines (bottom panel). The figures 
show that MAGE-3 transfection alone is insufficient to provoke lysis, nor 
is HLA-A2 transfection. Transfection with both MAGE-3 and HLA-A2 was 
sufficient, which is not surprising. What is unexpected, however, is the 
increase in lysis secured when peptide SEQ ID NO: 6 is added to cells 
transfected with HLA-A2 alone. Note this pattern in the second panel as 
well, where the data for "MZ2-MEL.43/HLA-A2+1 .mu.M SEQ ID NO: 6" 
demonstrated superior lysis to all others. These patterns, as indicated, 
are repeated over all CTL clones tested. 
EXAMPLE 8 
An additional set of experiments were carried out to test the lytic affect 
of the peptide in a chromium release assay on tumor cells. The TNF assay 
of example 6, supra, is more sensitive than a chromium release assay, so 
the latter would confirm results of the TNF assay. 
The nature of the .sup.51 Cr release assay was described supra, with 
reference to Boon, et al J. Exp. Med 152: 1184-1193 (1980). Using the same 
assay, CTL clones 297/19 and 297/22 were used, with a series of target 
cells known to be HLA-A2 positive. The cell lines LB43 and SK23 are also 
known to express MAGE-3. Cell line T2 is HLA-A2.sup.+, and MAGE-3.sup.-. 
Even at low E/T ratios, there was significant lysis, showing the ability to 
induce lysis. 
The foregoing describes the identification of peptides derived from the 
MAGE-3 tumor rejection antigen precursor which interact with MHC class I 
molecule HLA-A2. Of particular interest, and a part of the subject matter 
of the present invention, are the peptides represented by SEQ ID NOS: 
1-10. These peptides are easily synthesized by Merrifield Synthesis or 
other peptide synthesis methodologies. 
Of special interest are peptides which satisfy the following formulas: 
##STR1## 
where n is 4 or 5, and Xaa is any amino acid. Especially preferred are 
peptides such as SEQ ID NO: 6, which is exemplary of this class. 
The peptides, as indicated, complex with HLA-A2 molecules, and these 
complexes have been immunoprecipitated, thus leading to another feature of 
the invention, which is isolated complexes of the HLA-A2 molecule and 
either one of these peptides. 
Both the peptides and the complexes are useful in various ways. As was 
shown, the peptides bind to the HLA-A2 molecule, and thus they are useful 
in assays to determine whether or not HLA-A2 presenting cells are present 
in a sample. The peptide is contacted to the sample of interest in some 
determinable form, such as a labelled peptide (radiolabel, chromophoric 
label, and so forth), or bound to a solid phase, such as a column or 
agarose or SEPHAROSE bead, and the binding of cells thereto determined, 
using standard analytical methods. 
Both the peptides and the isolated complexes may be used in the generation 
of monoclonal antibodies or cytolytic T cell clones specific for the 
aforementioned complexes. Those skilled in the art are very familiar with 
the methodologies necessary to accomplish this, and the generation of a 
cytolytic T cell clone is exemplified supra. As cancer cells present 
complexes of MAGE-3 derived peptides such as SEQ ID NOS: 2, 6, 7, 8 and 10 
and HLA-A2, these monoclonal antibodies and cytolytic T cells clones serve 
as reagents which are useful in diagnosing cancer. The chromium release 
assay discussed supra is exemplary of assays which use CTLs to determine 
targets of interest, and the art is quite familiar with immunoassays and 
how to carry these out. 
Cytolytic T cell clones thus derived are useful in therapeutic milieux such 
as adoptive transfer. See Greenberg et al., J. Immunol. 136(5): 1917 
(1986); Reddel et al., Science 257: 238 (1992); Lynch et al., Eur. J. 
Immunol. 21: 1403 (1991); Kast et al., Cell 59: 603 (1989), all of which 
are incorporated by reference herein. In this methodology, the peptides 
set forth supra are combined with antigen presenting cells ("APCs") to 
form stable complexes. Many such methodologies are known, for example, 
those disclosed in Leuscher et al., Nature 351: 72-74 (1991); Romero et 
al., J. Exp. Med. 174: 603-612 (1991); Leuscher et al., J. Immunol. 148: 
1003-1011 (1992); Romero et al., J. Immunol. 150: 3825-3831 (1993); Romero 
et al., J. Exp. Med. 177: 1247-1256 (1993), and incorporated by reference 
herein. Following this, the presenting cells are contacted to a source of 
cytolytic T cells to generate cytolytic T cell clones specific for the 
complex of interest. Preferably, this is done via the use of an autologous 
T cell clone found in, for example, a blood sample, taken from the patient 
to be treated with the CTLs. Once the CTLs are generated, these are 
reperfused into the subject to be treated in an amount sufficient to 
ameliorate the cancerous condition, such as inhibiting their 
proliferation, etcetera by lysing cancer cells. 
Another aspect of the invention, shown in the examples, is the use of the 
combination of IL-6 and IL-12 to activate T cells, cytolytic T cells in 
particular "Activation" as used herein, refers to the ability to cause the 
T cells to carry out their intended function. In the case of CTLs, of 
course, this is the recognition and lysis of cells presenting on their 
surfaces appropriate combinations of peptide and MHC molecule. The 
activated T cells can then be used diagnostically, e.g., to determine 
whether a particular peptide/MHC combination is present on a cell 
subpopulation in a test sample. Also the use of the combined cytokines can 
facilitate the identification of particular CTLs. It is known that in a 
CTL sample, only a very small subpopulation is available which is specific 
to an MHC/peptide combination. By using the cytokines in combination with 
a sample presenting the desired combination, one can determine activation, 
via lysis, and compare it to a control value obtained where everything is 
kept constant except the sample is not mixed with the additional 
materials. This provides the requisite control value. 
Another feature of the invention is a kit useful in the activation of T 
cells, the kit comprising in separate portions, interleukin-6 and 
interleukin-12, the two separate portions being contained within a 
container means. The kits of interest may also include, e.g., a separate 
portion of peptide to be presented, and/or a vector or coding region for 
an MHC molecule, or even a vector or coding sequence which codes for both 
the MHC molecule and the peptide. For example, the peptide may be SEQ ID 
NO: 6. The vectors may contain an HLA-A2 coding region or a combination of 
HLA-A2 and SEQ ID NO: 6. Another feature of the invention is a composition 
consisting essentially of IL-6 and IL-12, in amounts sufficient to 
activate T cells, such as CTLs. 
"IL-6" and "IL-12" as used herein refer to all forms of these molecules, be 
they naturally occurring or produced recombinantly, human, murine, or any 
other species, as well as all variations of the molecule which have the 
same activating properties of IL-6 and IL-12. 
The amount of IL-6 and IL-12 used may vary; however, it is preferred to use 
from about 500 to about 1000 u/ml of IL-6, and from about 1 to about 10 
ng/ml of IL-12, although these ranges may vary, in accordance with the 
artisan's findings. 
Other aspects of the invention will be clear to the skilled artisan and 
need not be reiterated 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. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 13 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
GlyLeuGluAlaArgGlyGluAlaLeu 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
AlaLeuSerArgLysValAlaGluLeu 
5 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
CysLeuGlyLeuSerTyrAspGlyLeu 
5 
(2) INFORMATION FOR SEQ ID NO: 4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 
IleLeuGlyAspProLysLysLeuLeu 
5 
(2) INFORMATION FOR SEQ ID NO: 5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 
HisLeuTyrIlePheAlaThrCysLeu 
5 
(2) INFORMATION FOR SEQ ID NO: 6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 
PheLeuTrpGlyProArgAlaLeuVal 
5 
(2) INFORMATION FOR SEQ ID NO: 7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 
ThrLeuValGluValThrLeuGlyGluVal 
510 
(2) INFORMATION FOR SEQ ID NO: 8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 
AlaLeuSerArgLysValAlaGluLeuVal 
510 
(2) INFORMATION FOR SEQ ID NO: 9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 
LeuLeuLysTyrArgAlaArgGluProVal 
510 
(2) INFORMATION FOR SEQ ID NO: 10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 
AlaLeuValGluThrSerTyrValLysVal 
510 
(2) INFORMATION FOR SEQ ID NO: 11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 
GlyIleLeuGlyPheValPheThrLeu 
5 
(2) INFORMATION FOR SEQ ID NO: 12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(D) OTHER INFORMATION: The third Xaa is any 4 or 5 
amino acids 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 
XaaLeuXaaGlyXaaLeu 
5 
(2) INFORMATION FOR SEQ ID NO: 13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(D) OTHER INFORMATION: The third Xaa is any 4 or 5 
amino acids 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 
XaaLeuXaaGlyXaaVal 
5 
__________________________________________________________________________