TW419483B - Isolated tumor rejection antigen precursor mage-2 derived peptides, and uses thereof - Google Patents

Abstract

The invention describes peptides derived from tumor rejection antigen precursor mage-2. These peptides bind with HLA-A2 molecules, thus presenting complexes which provoke cytolytic T cell production. The resulting ''CTLs'' are specific for complexes of HLA-A2 and the peptide. The complexes can be used to generate monoclonal antibodies. The cytolytic T cells produced may be used in the context of immunotherapy, such as adoptive transfer.

Description

Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs I 419483 ΑΊ A7 B7 V. Description of the invention (1) Inventive jaw field The present invention relates to immunoencephalopathy and peptide chemistry. Specialties relate to peptides that can be used in a variety of ways including immunogens and as ligands for HLA-A2 molecules, in particular undecapeptides, decapeptides, and nonapeptides. More specifically, the present invention relates to a so-called π tumor rejection antigen derived from a tumor rejection antigen precursor (encoded by the gene MAGE-2) and expressed by the MHC class I molecule HLA-A2 ". The host organism is recognized or unrecognizable, and research is being conducted in a number of different directions. Understanding the field is believed to have a basic understanding of basic immunology and oncology .. Ill. A little understanding. Early research on mouse tumors showed that transplantation into syngeneic animals is possible Table shows the molecules that reject tumor cells. These molecules are "recognized" by the recipient animal's T cells and stimulate the cell-degrading T-cell response with the breakdown of the M transplant cells. This evidence was first attributed to chemical carcinogens such as methyl Raethylcholanthrene is obtained by inducing tumors in a test tube test. Antigens expressed by Xenopus and which can induce T-cell responses are found to be different from each other. See Prehn, et al., J. Natl. Cane. Inst. 18: 769 -778 ¢ 1957): Klein et at., Cancer Res. 20: 1561-1572 (1960); Gross, Cancer Res. 3: 326-333 (1943), B as ο π brio, Cancer R es, 3 0 : 2 4 5 8-2 4 6 2 (19 7 0) Teaching on the use of chemical carcinogens to induce differences in tumours and cell surface antigens. Such antigens are called "tumor-specific transplantation antigens" or "TSTAs". Observation by chemical carcinogens induced M antigens Later • Similar results were obtained when inducing tumors by UV radiation in a test tube test. See Kripke, J. Natl. Cane. -4-This paper size applies the Chinese National Standard (CNS) A4 (2 丨 〇X 297 mm) (Please read the cautions on the back before filling this page)-丨 Installation_ Order printed by the Central Consumers Bureau of the Ministry of Economic Affairs, printed by the Consumer Cooperatives 丨 419483 A7 B7 V. Description of the invention (2) I nst 5 3: 3 3 3-1 3 3 6 U 9 7 4) ° Although T cell-mediated immune responses have been observed for various types of tumors described above, it is believed that spontaneous tumors are usually non-immunogenic. Therefore, it is believed that they will not present tritium antigens to those with tumors. Individuals stimulate tumor response. See Hewitt, eta 1 ·, B t * i t. J. Cancer 3 3: 2 4 1-2 5 9 (1 9 7 6). The cell line showing turo_antigen is such as Boon et al ., J. Ekp. Med. 152: 1 184- 1 1 93 (1980) (also described here for reference) * The mutation resulting immunogenic mutant cells via tumor cell lines in mice or tin. It is further explained that the turn-antigen system is obtained by mutation of tumor cells. This kind of tumour cells does not produce an immune response in syngeneic mice and will generate tumors (ie, "turn cells"). When this tuf cell is mutated, the syngene is gene Mice reject and cannot produce tumors (hence becoming "tunT"). See Boon et al., Proc. Natl. Acad. Sci. USA 74: 272 (1 977) (also described here for reference). Many have been shown This type of swelling has this phenomenon. See, for example, Frost et al., Cancer Res. 43: 125 (1983) ° Gu Ran turn-the mutant strain cannot generate progressive sleep tumors because of triggering the immune rejection process. Evidence supporting this hypothesis includes sleep Tumor mutant strains (ie, cell lines that do not normally produce pupillary tumors) provide such evidence for mice that suppress the immune system by irradiation with M below a lethal dose, Van Pel et al., Proc. Natl. Acad. Sci. USA 76: 5282-5285 (1979); observe the intraperitoneal injection of breast cell tumor P815 tum-cells, M index multiply for 12 to 15 days * and then only need to inject human lymphocytes and macrophages for a few days to remove (U y 11 enh 〇veeta 1., J. EX p Med. 1 5 2: 1 1 7 5 -1183 (1980)). Further evidence includes observing that mice receive an exemption _ 5-This paper size applies Chinese National Standards (CNS > A4 specifications (2 丨 OX297 mm) ) --------- < ------ II ------ f — V.. (Please read the notice on the back before filling this page), Central Bureau of Standards, Ministry of Economic Affairs Printed by the Employee Consumption Cooperative 419483 A7 ____B7__ V. Description of the invention (3) Epidemic memory, even if the mice administer the immunosuppressive amount of radiation to challenge the cells' This immune memory still allows the mice to fight against subsequent tU BT Pick 9 of the mutant strain (Boon et al *, Proc * Hatl, Acad-Sci. USA 74: 272-275 (1977); Van Pel et al., Supra; Uyttenhove eta 1., Supra) ° Later studies found that when spontaneous When tumors are mutated, immunogenic mutant strains do respond. Indeed, these mutant strains can elicit an immunoprotective response to the original tumor. See Van Pel et al., J. Exp. Med. 157: 1992-2001 (1983) . In this way, it is shown that the so-called

"I tumor rejection antigen" is a target for gene rejection. Similar results can be obtained when foreign genes penetrate spontaneous tumors. See Fearson et al., Cancer Res. 48: 2975-1980 (1988). A class of antigens originate on the surface of tumor cells and are recognized by cytolytic T cells * leading to cell breakdown. Such antigens are hereinafter referred to as "tumor rejection antigens" or "TRAs". TRAs may or may not be mentioned The antibody response was elicited. These antigens have been studied in a test tube by characterizing the cell-decomposable T cells, that is, by using a special small group of cells that decompose T cells (hereinafter referred to as "CTL") to identify antigens. The small population of T cells proliferates when they recognize the tumor rejection antigen that is present, and the cells that present the antigen are broken down. Special activation studies identified CTL strains that can specifically decompose cells expressing these antigens. Examples of such research work are 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. Imnunol. 124: 1627-1634 (1980); Maryanski et al., Eur. J. -6-This paper size is applicable to the Chinese National Standard (CNS) A4 wash case (210 X 297 mm) ----- ---- ^ .------ IT ------ f — (Please read the back 4 / Attention is- before filling out this page)., A7 "419483 __ B7 V. & Description of Invention (&)

Immunol, 124: 1627-1834 (1980); Haryanski et al.,

Eur. J. Immunol. 12'406-412 (1982); Palladino et al., Cane. Res. 47: 5074-5079 (1987). This type of analysis requires multiple types of antigens recognized by CTLs, including minor compatible antigens, male-specific HY antigens, and a class of antigens called " turn- "antigens, which are discussed herein. The subject tumor paradigm is called P815. See Deplaeti 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) (also described herein for reference). P815 tumors are breast cell tumors. * Induced with methylcolanine in DBA / 2 mice and used in Tumor and cell line culture in vitro. P315 strains have been mutated to produce a variety of tusT variants *, including P91A (DePlaen, supra), 35B (Szikora, supra), and the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. P198 (Sibi 1) le, supra). In contrast to tumor rejection antigens (this is the difference), turn-antigens appear only after mutations in tumor cells. Tumor rejection antigens appear in specific tumor cells that are not mutated. Therefore, refer to the reference, cells The strain can be tum *, such as a cell line called " P1 " and Turn-variant strains are generated by stimulation. Since the tuni_ phenotype is different from the parent cell strain, it is expected that the DHA of the tunr cell line and its turn · mother cell line are different. This difference can be used to locate interest in the turn-cell It was found that tum-mutant strains such as P 9 U, 3 5 B and P 1 98 genes differ from their normal alleles by point mutations in the coding region of the gene. See Szikora and Sibille, s_u_gJl.a, and Lur gu in et a 1 _, Ce 1 1 58: -7- This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) A7 419483 B7 V. Description of the invention (5) 293-303 (1989) This is an example of the TRAs of the present invention. These reports also verify that peptides derived from the tuiT antigen are presented by Ld molecules for recognition by CTLs. P91A is presented by Ld and P35 is presented by DdSP198 by Kd. PCT application PCT / US92 / 04354 (application date: May 22, 1992, the same recipient as the case) teaches a class of human tumor rejection antigen precursor coding genes, called the MAGE family. Several of these genes are also described in van der ·

Bruggen et al., Science 254: 1643 (1991). Learn about the expression of a variety of H AGE family genes in tumor cells today, and can be used as a tumor diagnostic marker 1 »1 M and for other uses discussed in this article. See also Traversari et al., I »πuηogenetics 35: 145 (1992); van der

Bruggen et al., Science 254: 1643 (1991). The processing of proteins and the mechanisms that emerge on the surface of cells have been well documented. A comprehensive review of developments in this field can be found in BarUasa "obtaining several" trunks ": how MHC binds to peptides", Science 257: 880 (1992); see also Fremont et al., Science 257: 919 (1992);

Matsuraura et al., Science 257; 927 (1992); Matron et al., Science 257: 964 (1992). These reports usually target the nine-amino acid ("Ninth Peptide") peptides that are bound to MHC / HLA molecules by the printed work requirements of the Ministry of Economic Affairs, China ’s Associated Bureau of Work and Consumer Cooperatives. The importance of the ninth residue. Studies on MAGE genes have shown that the special nonapeptide actually appears on the surface of several tumor cells, and the presence of the nonapeptide must have the molecule HLA-Al. HAGE-1 tumor rejection antigen complex (" TRA "or nonapeptide) causes TRA-presenting cells to be broken down by finely decomposed T cells (" CTLs "). -8- This paper is in accordance with the Chinese National Standard (CNS) A4 Specification (210 X 297 mm) It was stated to the germanium member whether the original substance of the case would be changed after the amendment of the case. Central Government Standards Bureau, Ministry of Economic Affairs, Star Elimination Ι 4 19483 No. 8410331 Patent Application No. 1-Chinese Manual Amendment Page (S7 yuan) Month ^ / 1, | V. Description of the invention (^) You one or two, for example, refer to the application No. 08/217 · 187 filed by Traversari et al. There are 88 other MAGE-derived peptides. For example, US Patent Application No. 07 / 938,334 (application date: August 31, 1992) and US Patent Application No. 073,103 (application date: June 7, 1993) The study described describes the degree of homology when comparing the homology regions of multiple M AGE genes with the homology regions encoding HAG E-1 genes that are similar to each other. Indeed, these observations have led to the disclosure of the present invention and its The appearance of claiming patents is superior to those of the first class of gold Identical N- and C-terminal amino acids. These nine forms are described as being used as immunogens for multiple purposes, alone or when coupled with a carrier. The size of the nine forms is large enough to constitute the site of the antigen's action, and the anti-Korean produced by this site The 撺 description can be used to identify 呔, regardless of whether the 肽 peptide is present alone or constitutively or part of a larger 呔. These references * Special Application No. 073.103 shows the gambling system between HLA-A1 and M AGE-3 However, only about 26% of white and U% black races revealed HLA-A1 molecules. Therefore, they have additional information provided by other MHC molecules, so that some races can be discussed in the previous paragraph. Benefits from research0 The discovery of the antigenic performance of MAGE-2 derived maggots listed below, the peptide complexed with the MHC class I molecule HLA-A2. The discovery includes therapeutic and diagnostic uses, and is used for the purpose of the present invention. (Details will be described later.) The simple and sharp part 1 of the circle i is the sharp and bright circle, which includes HLA-A2.1 up to 0.5. This paper uses the Chinese National Standard (CMS) Α4 Specification （210X297 公 嫠} ---------- Installation ------ Order ------ Taste > 1 {Please Read the notes on the back and then fill out this page) I 419483 A7 B7 V. Description of the invention (please read the back-ιδ winter * i matters _ then write the page of the book. Figure 2 shows borrowing 51 (^ release test analysis, measurement Comparative data on the response of pure HPV strains to a variety of substances. Example No. 1: Experimental conditions: Unless otherwise stated, experiments were performed at room temperature. All F mo c protected amino acids, synthetic polymers, peptides and TF A are stored at -20¾.状 ^^ — 成 _ The peptide was synthesized by solid-phase method in an automated peptide synthesizer (Abined AMS 422) (see Gausepohl and Frank Biotech, September, 1990; Gausephol et al., In E. G ira 11 and D. Andreu , (eds). Peptides 1990: 206-207 (1990)) ° Multi-round manufacturing of peptide M, in which 48 different peptides are synthesized simultaneously.

Tentagel S AC (Rapp et al ,, Reform and Perspective of Solid Phase Makeup Synthesis, 205-210 (1990); Sheppard and Williams, Int. J. Peptide Protein Res. 20: 451-454 (1982), Polyethylene Graft polymer with two fermented spacers on a polystyrene matrix is used as a resin (40-60 rag / leaf, 10u mol Fmoc amino acid loaded). Representative couplings printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs The method will be from 90w 1 0.67 H BOP (Gausephol et al ,, peptides 1988: 241-243 (1988) 5 Castro et al ,, Tet, Lett 14: 1219-1222 (1975)) to NMP, 20w l HMM to HMP 2/1 (v / v) and 100i / 1 0.60 M appropriate

Fmoc base acid (Fields and Noble, Int. J. Pep, Prot. -10 This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) A7 419483 _______ _B7_ V. Description of the invention (s)

Res. 35: 161-214 (1990Π A mixture of NHP (6-fold excess) solution was added to each reaction vessel. At 70% reaction time, about 50 μl of diazomethane was added to the reaction vessel.

Fmoc deprotection was performed by adding 3 times 0.8 ral hexahydropyridine / DMA 1/4 (v / v) to each reaction vessel. The coupling time and deprotection time started at 30 minutes and 3 X 3 minutes, respectively, and the progress of sleep synthesis was extended. 6x 1.2 nl DMA was used for coupling and washing after F mo c deprotection. After the required sequence is reached and the final »1» 0 (; is removed, the peptide-based resin is washed thoroughly with MDMA, diazomethane, dichloromethane / ether 1/1 (v / v), and ether. Dry. 刬 __Cleaving and tillering Cleavage by resin and removal of the side chain protection system 200 yl TFA / water '19/1 U / v) was added to each reaction vessel 6 times, with an interval of 5 minutes to obtain free carboxylic acid Acid peptide. As for the peptide containing 1 > 0? 6 / water / ethionase 18/1/1 (V / V / V) 0 2 hours after the first addition of TFA · Borrow 10 ml of ether / pentane 1/1 (v / v) and cool to-20t : Peptide was precipitated from the combined filtrate. Centrifuge (-20 scoops, 2500 g, 10 ιπίη) and divide into young ones. Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. Nine capsules of ether / pentane 1/1 ίν / ν) were processed and separated by the same centrifugation procedure, and the peptides were dried at 45¾ for 15 minutes. Each peptide was dissolved in 2 «1 water (or 2 ml 10 V»; acetic acid), and the solution was frozen in liquid nitrogen for 3 minutes and lyophilized while centrifuging (1300 γρβ, 8-16 -11- This paper is in accordance with Chinese national standards (CNS) A4 specification (210'〆297 mm) 419483 A7 B7 V. Description of the invention (9) The purity of the peptide was determined by reversed-phase HPLC; about 50 nmol of the whole was dissolved in 100 wl 30 vv acetic acid. 30 wl of the solution was applied to The RP-HPLC system uses a ternary solvent system; A: water · B: acetonitrile, C: 2 V% TFA in water. 30 minutes from 90% A, 5% B, 5% C to 20% Α · 75% B, 5% C was subjected to gradient dissolution (1.0 nl / inin). Detection was performed at 214 nm. Random samples were taken and analyzed by PDHS by mass spectrometry. All 31 棰 binding peptides were analyzed by PDMS by mass spectrometry and hydrolyzed by HP Amlnoquant. The analysis is based on the analysis of amino acids. The difference between the calculated value of all analyzed samples * and the measured values is within the experimental error (0.1%) specified by the instrument manufacturer. All amino acid compositions are as expected. 2

1 Of all 71 MAGE-2 peptides that had been lyophilized, 1 ag was weighed and dissolved in 10 w DMS0. All soluble peptides were used as 0.5 Bg / ml at 0.9%

Dilute NaCl, fHM5% acetic acid in distilled water (CH3C00H, Merck Darmstadt, Germany) or IN HaOH in distilled water (Merck Darmstadt, German) and neutralize to 7. Fine J & _ 174CEM.T2 cells were cultured in IscoveE modified Dulbecco's medium (Biochron KG Seromed BeYHn, Germany), supplemented with 100 Ιϋ / ml cyanogenin (Biocades Pharma, Leiderdorp, T he N ether 1 ands) * 1 0 0 ug / m 1 Kanamycin (Sigma St, Louis, USA), 2mM chain (ICN Biomedicals 12 This paper size applies to China National Standard {CNS) A4 specification (210X297 mm) Please read the back Note. Please fill in the nest again. This page is bound to be printed by the Consumer Cooperatives of the Central Government Bureau of the Ministry of Economic Affairs Γ 419483 Α7 Β7 V. Description of the Invention (10)

Inc, Costa Mesa, CA, USA) and 10% fetal bovine serum (FCS, Hyc 1 ο ne L aboratories I nc. L og η, U tah, USA >. Cells at 37t: · 5% C02 in humidification Air M 2. 5x 10e / ml density culture 3 ° I knot i 17 4C EM. T2 cells were washed twice in FCS-free medium and placed in serum-free medium to a density of 2 × 10β cells / ml. 40wl suspension and 10wl of individual peptide dilutions (ranging from 500 iig / ϋΐ to 15.6tfg / ml) were placed in a V-bottom 96-well plate (G reine "G mb Η, F rickenhause η", together with 0.9% NaCl twice the serial dilutions. Gerbery >. Final concentration in the range of 200 yg / ntl to 3.1 ws / ml peptide containing 8xl04 174CEM.T2 cells. The solution was gently stirred for 3 minutes and then incubated at 37 ^ 0, 5% CO2 in humidified air for 16 hours. Then cells M 100 w 1 0.9% HaCl, 0.5% bovine serum albumin (Sigma St. Louis, USA) * 0.02% Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs ^^ 1- ^^^ 1 ^^^ 1 n H— · »ί— i-l-ID— nn In, laJ. · (Please read the notes on the back · Please fill in this page first)

NaN3 (Merck Darmstadt, Germany) was washed once. After one centrifugation at 1200 rpm, nine capsules were placed in 4 ΐ: once again floated in HLA-A2. 1 specific mouse monoclonal antibody BB7.2 at 50 «1 for 30 minutes. Cells were then washed twice and goat anti-mouse IgG F (ab) z segment (Tago Inc, Burlingane, CA, USA) that had been conjugated with luciferin isothiocyanate (Tago Inc, Burlingane, CA, USA) κ 1:40 dilution and total Volume 25 «1 for 30 minutes. After the last incubation, the cells were washed twice and the fluorescence was measured at 488 nm by a M FACS can flow cytometer (Becton Dickinson, Franklin Lakes, HJ, USA). The fluorescence index was used to plot the morphology, and the concentration of HLA-A2.1 on 174CEH.T2 cells when the highest rounding value was 0.5 was measured. -13- This paper is compatible with the Chinese National Standard (CNS) A4 specification (210X29 * 7 mm) 419483 A7 B7 5. Description of the invention (11). The results are shown in Table I.

Table I Binding affinities of human hormonal tumor-derived peptides and the protein MAGE-1 supporting the HLA-A2.1 mode (compilation of Falk et al., Nature 351: 290-296 (1992); Hunt et al., Science 255 : 1261-1263 (1993): Hijian et al., J. Immunotherapy 14: 121-126 (1993)) 0 (Please read the precautions on the back before filling this page)

、 -ST Consumption cooperation between employees of the Central Bureau of Standards of the Ministry of Economic Affairs Du Yin 褽 -14- This paper U applies the Chinese National Standard (CMS) A4 specification (2! OX 297 mm) 419483 Peptide number printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs Sequence residues induce 0.5 FI highest value GLEA ^ GEALGL GLEAXGZAL 15-25 > 100 ^ rg / ml 15- 23 60 pg / m! 4 ALGLVGAQA 22- 30 > 100 pg / ml GLVGAQAPA 24- 32 £ 5 uq / slL DLZSEFQAA αοο-108 > 100 μς / ml DLESEFQAAI AISRKHVELV 100-109 10Θ-117 > 100 μς / ιηΐ > 100 ^ g / ml AISRKliVEL 108-116 > 100 j / g / ml 2 KHVELVHFL 112- 120 40 KMVELVSnL KHVELVBFLLL 112-121 > 100 μς / ml 112-122 > 100 ^ g / ml LLLKYHAREPV 120-130 > 100 fjg / ml LLKYHAKEPV 121-130 > 100 jjg / nl VLRNCQDFFPV 139-149 > 100 μς / ml 3 VIFSKASEYL 149-158 35 μς / άΐ 4 YLQLVTGIEV 157-166 35 ^ g / ml 5 YLQLVFGIEW-QLVFGIEW 157-167 159-167 > 100 pg / ml 25 pg / ml 6 QLVTGIEWEV 159-169, 30 pg / ml GIEWEWPI 163-172 > 100 i; g / ml PISELYILV 171-X79 55 pg / ml ELYILVTCL 174-182 > 100 jjg / ml HLYI LVTCLGL 174-184 > 100 μς / ml YILVTCLGL 176-164 > 100 i; g / ml CLGLSYDGL 181-189 65 pg / ml CLGLSYDGLL 181-190 > 100 pg / ml VKPKTGLLI 195-203 > 200 i; g / ml VMPKTGLLII VKPKTGLLIIV 195-204 > 100 μς / ml 195-205 > 100 jjg / ml GLLIIVIAI 200-20B > 100 jjg / ml GLLIIV1AII 200-209 > 100 pg / ml GLLIIVIAI IA 200-210 > 100 pg / ml LLIIVXAII 201-209 > 100 pg / ml LLIIVLAIIA LLIIVIAIIAI LITVLAIIA 201-210 > 100 pg / ml 201-211 > 100 pg / jnl 202-210 > 100 yg / ml LIIVLAJIAI 202-211 > 100 pg / ml 7 IIVLMIAI 203-211 20 ^ g / ol IIAIEGDCA 208-216 > 100 jjg / ml KIKZELSHL KIKEELSMLEV 220-228 > 100 μς / ffil 8 220-230 25 ^ g / ml LHQDLVQENYL 246-256 > 100 pg / ml FLV7GPRALI 271-279 € 5 pg / ail 9 ALIETSYVKV ALIETSrVTVL 277-286 20 ^ g / ml 277-287 > 100 pg / al 10 LIETSYVKV LIETSYVKVL 278-2fi6 30 jjg / ml 278-287 55 yq / al TLKIGGEPHI 290-299 > 100 pg / ml EISrPPLHERA 298-308 > 100 pg / ml -15 Please read the 'Notes on the back' first_ item before f paper ruler Applicable to China National Standard (CNS) A4 (210 X 297 mm), printed by the Central Standards Bureau of the Ministry of Economic Affairs, Consumer Cooperatives, 4ΐ9483 Α7 Β7 ____ 5. Description of the invention (13) 1? 4CEM.T2 cell strains are expressed as & quot "Blank" 'When a peptide binds to a peptide that is found in these molecular grooves, the unstabilized HLA-A2.1 molecule can be stabilized. The stabilized HLA-A2.1 molecule is not to be degraded. This is based on the analysis of peptide binding results. This results in an increase in cell surface expression of HLA-A2.1 molecules. Fluorescence index is a measurement of HLA-A2.1 molecular up-regulation. The fluorescence index is calculated according to the following formula: MF = average fluorescence value ίΜΙΟ »! ® sister " * ίＭΡ) blank sister fluorescent index-(M F) blank sister The background fluorescent index is 0. To identify the MAG E-2 form of the HLA-A2.1 molecule that can bind to 174CEM.T2 cells, see the amino acid of MAGE-2 according to ν ander Bruggeneta 1 · Science 254: 1643-1647 (1991) sequence. View all peptides produced with nine, ten, or eleven amino acids in combination with the published HLA-A2.1 binding mode (Table I > Only peptides with sequence identification numbers in Table III: 1-11 can be raised at low peptide concentrations Adjusting the performance of the HLA-A2.1 molecule means that it binds to the HLA-A2.1 molecule as described in Example 2. None of the other 50 peptides can do this. The results of the fluorescence measurements are shown in Tables I and II. HLA-A2 The 0.5 maximum upward adjustment value of .1 molecule on 17 4 CEM.T2 cells * was determined by plotting the FI of individual peptides against the peptide intensity.

The experiment indicates that only a limited proportion of peptides that match the HLA-A2.1 mode can bind such HLA molecules with high affinity. Therefore, it may be a human CTL to be borrowed. -16- This paper uses Chinese National Standard (CNS) A4 specifications (210X297). (Mm) (Please read the precautions on the reverse side before filling out this page) Installation I 419483 A7 B7 V. Description of the invention (U) Candidates for the identified MAGE-2 protein, human CTL only when it binds to HLA molecule Recognizable peptide. Table II Binding affinities of other peptides derived from human melanoma to the binding of the protein MAGE-2 in coordination with the generalized HLA-A2.1 pattern (Ruppert et al Cell 74: 929 -937 (1 993)). Peptide concentration of 0.5 FI maximal base QTASSSSTL -QTASSSSTLV STLV3

jESVL VTCLGLSYDGL KTGLLIIVL KTGLLIIVLA KTGLLIIVLAI HIIKIGGEPHI 37-45 37-46 43-53 4B- 56 130-139 130-140 179-189 198-206 198-207 19Θ-208 289-299 > 100 vg / sil > 100 fig / ml j! 5 jjg / pl " > 100} iq / wl 70 vg / ol > 100 μς / αΐ > 100 μς / ^ 1 € 5 pg / nl 60 pg / ml > 100 > 100 μς / ιηΐ I ^ — ^ 1 ^ — ^ 1 ^ — ^ 1 ^ — ^ 1 I plil · »I .---- i ^ J. s. 、-"« (Please read the notes and items on the back first (Fill in this page again.) Du Yin printed by the staff of the Central Bureau of Standards of the Ministry of Economic Affairs -17- This paper applies the Chinese National Standard (CNS) A4 specification (2 [OX 297 mm) 419483 A7 B7 Staff consumption of the Central Bureau of Standards of the Ministry of Economic Affairs Printed by the cooperative V. Description of the invention (ο Table III Peptide derived from the melanoma protein MAGE-2 and HLA-A2.1 binding sequence identification number peptide number amino acid sequence 1 SILVEVTLGBV residues 43-53 1 meal LVEVTLGE7 residue Group 45-53 2 2 KHVSLVHIX residue 112-120 3 3 VIFSKASEYL residue 149-158 4 4-YLQLVFGIEV residue 157-166 5 QLVFGIEW residue 159-167 6 6 QLVFGIEWEV residue 159-169 7 7 .IXVLAIIAI KIWE2LSKLEV AL IETSYVKV residues 203-211 8 8 9 Box residues 220-230 277-286 9 10 10 LIETSYVKV residues 278-286 11 According to F a 1 lc eta 1., H ature 3 5 1: 2 9 0-2 9 6 (1 9 9 1);

Hunt et al ·, Science 255: 1261-1263 (1992); and Hijman et al., J. I * * uηotherapy 14: 121-126 (1993) found most HLA-A2.1 using the HLA-A2.1 model Binding peptide. Only another HLA-A2.1 binding peptide was discovered using the generalized HLA-A2.1 pattern of Ruppert et al., Cell 74: 929-937 (1993). Example 3 This example shows the induction of an immune response in a test tube test. As for an example of the possibility of fully inducing a single response (including the MAGE-2 peptide), it is shown that the treatment is 18- The paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210 × 7mm)---«mt ^ i— ^ ^ 1 I ^^ 1 I t * R ^^ 1 0¾, vs ** < Please read the notes on the back 1 before filling out this page) 419483 A7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs_B7___ 五Explanation of the invention (16) This response of the defective cell line 174CEM.T2 to HPV 呔. The performance of HLA-A2.1 on 174CEM.T2 cells (T2) was increased by culturing T2 cells in a medium containing related peptides. T2 cells can present HL / \ _ A2.1-bound related peptides in high amounts, and therefore are good antigen-presenting cells (APC). The reaction induction method recently described (Kast et al., J. Immunotherapy 14: 115-120 (1993)). Medium * T2 cell lines were used as APCs, and Ficoll post-cells were used as responder cells. 1) HU-A2. 1 pair of T2 peptide-loaded T2 cells Μ2χ 10β cells / m. Incubate at 37f in a T25 flask (Becton Dickinson, Falcon, Plymouth Engeland) and incubate for 13 hours in the following medium. The medium is serum-free IMDM (= Iscoves modified DuIbeccoK medium: Biochrom KG, Seromed Berlin, Germany) contains Charm (2mM, ICN Biochemicals Inc., Costa Meisa, ϋSA), antibiotics (100 IU / m 1 Penicillin (B 「Ocades P ha「 ma, 1 e ί de 「dorp, the N ether 1 ands）, 100 wg / m 1 (Convenin (Sigma, St. Louis, USA)) and 80 wg / ml From HPV's special peptide MLDLQPETT (sequence identification number: 12) 2) Mitomycin C treated T2 antigen-presenting cells (APCs) After incubation T2 cells were centrifuged * After M 20 x 1 〇β cells / m 1 density with Mitomycin C (50ug / ml) was resuspended in serum-free RPMI (Gibco Paisley, Scotland) medium and incubated at 37 ° C for 1 hour. T2 cells were then washed three times in RPMI. 3) Prepare to induce an immune response ^^ 1 — ^^ 1 H-—-I ^^^ 1 ^^^ 1 I:: ·· ϋ— 11_ ^ JS * νβ »(Please read the back first Please fill in this page again for attention) -19- This paper size applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) A7 419483 B7 V. Description of invention (〖7) ^^^ 1 ^^^ 1 ^^ ^ 1 ^^^ 1 ^^^ 1 tm · 1 * l ^ l —-- aJ * * (Please read the note on the back · Please note the item before filling out this page) 96-well U-shaped bottom plate (Costar, Cambridge, USA) Wells were filled with ΙΟΟ, ΟΟΟ Mitonycin C-treated T2 cells in 50 «1 serum-free complete RPMI medium (glutamine (2mM, ICN Biochemicals Inc., Costa Meisa, USA) antibiotics (100 IU / ml cyanogen I» (Brocades Pharma, Leiderdrop, The Netherlands), 100wg / ni 1 Conmycin (Sigma, St. Louis, USA)) and peptide MLDLQPETT (sequence identification number: 12) of 80. Wg / n 丨. 4) Responder cells Responder cells are HLA-A2.1 subtype donors (= CB,) of mononuclear peripheral blood lymphocytes (PBU. Ficoil procedure (Ficoll product: Lyraphoprep of Hycomedpharma, Oslo, Norway) by Yu Xi PBL was separated and washed twice in RPMI. After separation and washing, PBL was resuspended in complete RPMI medium containing 30% human pooled serum (HPS) (HPS was tested for inhibitory activity in mixed lymphocyte culture). 5) Once Incubation of 400,000 PBL-CB for immune response, add 50 «1 medium (the medium described in paragraph 4 above) to a 96-well U-bottom plate that has been filled with wells of M T2 cells • and 37T: at 5% CO2 R90% humidity incubator for 7 days. 6) Stimulation again (day 7) On the seventh day of the cultivation of PBL, peptide MLDLQPETT, and T2 cells from the Consumer Cooperative of the Central Beech Associate Bureau of the Ministry of Economic Affairs, PBL-C.B.JW peptide MLDLQPETT was again stimulated. For this purpose, 96-well cells and culture medium were recovered. Viable cells were isolated by the} Mcoll procedure and washed in RPMI. Each well of a new 96-well U-bottom plate was seeded with 50, 0 0 viable cells and 50 w 1 complete 1 ^ 141 medium containing 15% and evaluated 5. 20,000 non-homologous photos per hole -20- This paper is compliant with Chinese national standards (CNS > A4 size (210 × 297 mm) 1 419483 A7 _B7___ V. Description of the invention (18) Shooting (3000 Radiator) PBL And 50,000 non-homologous irradiated (10,000 Rads) EBV-transformed B lymphocytes (= EBV-C. B.) M and 50 W 1 containing 15% Η PS and 80US / ml peptide MLDLQPETT complete RPMI Culture medium. Cells were incubated at 37t for 7 days in an incubator at 5% CO2 and 90% humidity. 7) Restimulation (day 14) PBL, peptide MLDLQPETT and T2 cells were cultured on day 14, PBL-CBM-like MLDLQPETT was stimulated again. Repeat the previous re-stimulation procedure. 8) PBL, gHLDLQPETT and T2 cells were cloned by limiting dilution. On the 21st day, 96-well cells and culture medium were harvested. Viable cells were isolated by the Fi coll program and washed in complete RPMI containing 15% HPS. A large number of viable cells are selected by limiting dilution. One well of a chopped 96-well U-shaped bottom plate (Costar, Cambridge, USA, cat, nr. 3799) was added with 50wl of complete RPMI medium containing 15% HPS and 100 live cells (= HPV16 large amount of anti-MLDLQPETT) ). For other new 96-well U-bottom plates, the process is repeated exactly except for the number of cells in each well: each plate subsequently contains 10, 1 *, or 0.3 cells / well of cell dilution. In each well, 20,000 PBL and 10,000 hydroxyl pooled and irradiated (3,000 Rad) four different donors were added, and three different irradiated (10,000 Rads) were 111 ^-/ \ 2.1 donors (71) -5 / 518 / "¥) of EBV transformed B cells * and 50w complete RPMI medium containing 15% HPS, and the peptide MLDLQPETT at a concentration of 40 wg / ml * white lectin f L euc 〇agg 1 utini η} (P har * ai acia, ϋ ppsa 1 a, S wede η), human recombinant strain IL-2 (Eurocetus, Ansterdan, The Netherlands) at a concentration of 120 IU / ml ° -21- This paper size applies to Chinese national standards (CNS ) A4 specification (2IOX297 mm) A7 I 419483 ____B7_ V. Description of the invention (19) 9) Enlarged blunt plant 1 ^ 1 ^^^ 1 ^^^ 1 ^^^ 1 MI m · it ^ im V ^ ¾ HI 1 ^ 1!-1 HI t > * va.. (Please read the notes on the back before filling in this purchase) The final volume in each well is 100 «1 plus person = > -25,000 viable cells -20,000 irradiated PBL Pool (as described above) -10,000 irradiated EBV pools (as described above) -2 ju glfe MLDLQPETT -6 IU heavy sister strain IL-2 On day 49, 65 pure strains and a large sample of thief were used as performer cells And T2 (with or without gMLDLQPETT) as target cells for analysis of cytotoxicity. Background killing was defined as: T2 cells co-bred with GILGFVFTL were killed with non-coupling (but bound to HLA-A2.1) peptides. Such influenza matrix protein-derived peptides are HLA-A2.1-limited influenza-specific CTL antigen-acting sites and are known in the industry. HPV Langda (C.B.) anti-MLDLQPETT executor Niu appears to be specific for killing MLDLQPETT-sensitized cells. Restriction dilution assay using HPV large sleeping cells. After 23 days, five pure strains were used for cytotoxicity assay. The results of a representative pure strain are shown in FIG. 2. Printed by Shellfish Consumer Cooperative, Central Bureau of Standards, Ministry of Economic Affairs. The data indicates that the peptide sequence identification number: 1-11 is a single peptide of the recognition sequence. However, homologs of these peptides, heteromorphic or gene variants, can be found inside and outside the cellular environment. The present invention encompasses all homologs of the aforementioned peptides * heteromorphic or genetic variants, but must be bound to the HLA-A2.1 molecule. Polypeptides that are peptide homologs specifically contain at least about 40%, preferably at least about 60%, and more preferably at least about 75% of the amino acid sequences compared to the amino acid sequences listed in Table II. -22- This paper size applies to Chinese national standards (CNS > A4 size (210X297 mm) ί 419483 Α7 Β7 V. Description of invention (20) The industry will understand that other variants shown above are included in the scope of the present invention .Specifically includes variants which are different from the above and only rely on conservative amino acid substitution for synthetic amidine. In particular, it includes MA (alanine), S (serine), ct-aminobutyric acid and other alternatives C (cysteamine) Acids>, because cysteine-containing europium is known to be sensitive to (air) oxidation during synthesis and processing. A number of these conservative amino acid substitutions are described in Taylor J. Mol. B 丨 〇1. 188: 233- 258 (1986) ° The above shows that 呔 or its Η segment contains any changes in the amino acid sequence, regardless of κ conservative P acid substitution, loss, or other changes, but the polypeptide must be related to HLA-A2.1 Molecular binding. Peptide fragments can be small peptides with a sequence of at least five or more amino acids. When the polypeptide is bound to HLA-A2.1 molecules, the sequence is shown in Table II. Polypeptides larger than the indicated peptide, when available in HLA -A2.1 positive body induces MAGE-2 specific CTL responses and contains (part of) those listed in Table II The amino acid sequence and its conservative substitutions * are included within the scope of the present invention. These polypeptides may be from 9 to 12, preferably from 9 to 11, or even from 9 to 10 amino groups. Employees of the Central Bureau of Standards, Ministry of Economic Affairs The printing of the invention by a consumer cooperative includes the use of each means, regardless of genetic engineering> peptides produced by solid-phase technology or other peptide synthesis. The aforementioned peptides can have multiple chemical modifications at the ends and still fall within the scope of the invention. Other chemistry Modifications are also possible, particularly in actinides and binary configurations. The term "derivatives" encompasses all modified peptides. Many aspects of the invention can be used to prevent, diagnose, and / or treat or prevent MAGE-2 epidermal cells ( Contains melanoma cells and other cancer cells) related diseases 0 For all uses, it is not administered in the form of M immunogens. Because the peptides are quite short -23- This paper size is applicable to China National Standard (CNS) A4 specifications (210X 297) (Centi) r 419483 A7 B7 5. Description of the invention (), it needs to be mixed, compounded, coupled, conjugated or chemically combined with adjuvants with carrier materials that can provide immunogenicity such as lipids or other materials. Advance Or the range of therapeutic dose will of course vary with the diseased population (age, gender, weight, etc.), the nature and severity of the condition to be treated, the special features of the present invention and its route of administration. Any appropriate route of administration can be used to achieve an effective dose The essence of the invention. Mindopan, can also be used in any industry known-tincture form. In addition, peptides can also be administered by controlled release devices and / or drug delivery devices. Can be used with other active substances, such as * Special, T cell activation Agents such as interleukin 2 are co-administered. «Art * The peptide of the present invention can also be used for other purposes, such as diagnostics. For example, it can be checked whether the vaccination of the peptide of the present invention is successful. The test tube can be used to test whether the tadpole can activate the T cells of the immunized corpus callosum. Other aspects of the invention will be apparent to those skilled in the art. • There is no need to repeat them here. The terms and notations used for illustration are for description purposes only and are not intended to limit its scope. It is by no means intended to use these terms and notations to exclude any equivalents or parts of the features shown and described. Many modifications in the invention park are possible. Please read the memorandum, “Notes and Items” on this page. Η Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs

This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm). Economic. 郅 Staff of the Central Bureau of Standards, cooperation and printing I 419483 A7 B7 V. Description of Invention (K) U)-General Information (i) Application Human: Melief, Cornelis J'M. Visseren, Η. JW K ast; W. M. van der Brugen, Pierre Boon-Falleur, Thierry (il) Invention Name: Derived from isolated tumor rejection antigen precursor MAGE-2 State and its use (Isolated Tumor Rejection Antigen Precursor MAGE-2 Derived Peptides, and Uses Thereof) (iii) Serial number: 6 2 (iv) Mailing address: (A) Trustee: F e 1 fe & L yneh ( B) Street name: 8 0 5 T hird A venue (€) City name: New York City (D) Use name: New York (E) Country name: USA (F) Zip code: 10022 (v) Computer allows format : (A) Media type: Magnetic disk, 5. 2 5 inches * 3 6 0 li b Capacity (B) Computer: IBM PS / 2 (C) Operating system: PC-D0S (D) Software: W 〇rdperfect ( vi) Currently applying for resources: -25- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) -1 pack. Ϊ [-'(Please read the notes and items on the back before filling out this page) 419483 A7 B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of Invention (23) ίΑ) Application number: 08/217, 188 (Β) Application date: 24-MARCH-1994 (C) Category: 435 (vOi) Agent information: (Α) Name: Hanson, Norman D. (Β) Registration number: 30,946 (C) File No .: LUD 5340 (ix) Telex information⑴ Phone: (212)688-9200 m Fax: (212)838-3884 (2) Serial identification number: 1 Information: (i) Sequence characteristics (A) Length: 11 Amino acid residue (B) Category: Amino acid (D) Form: Linear (ii) Molecular category: Protein U 丨) Sequence description: Sequence identification number: 1: Ser Th r Leu Va 1 Glu Val Thr Leu Gly Glu Val 1 5 10 < 2) Sequence identification number: 2 Information: (\) Sequence characteristics ⑴ Length: 9 Amino acid residue (B) Category: Amino acid (D) Form: Linear -26- Please Read the items on the back first-The paper size is applicable to the Chinese National Standard (CNS) A4 (210 X 297) (B) Binding 419483 A7 Printed by B7 of the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (24) (π) Molecular type: Protein U i) Sequence description: Sequence identification number: 2 Glu Val 1 5 (2) Information of sequence identification number: 3: (1) Sequence characteristics: (A) Length: 9 amino acid residue (B) Category: amino acid < D) Form: linear (ii) Molecular class ·· Protein U i) Sequence description: Sequence identification number: 3: Lys Met Val Glu Leu Val His Phe Leu 1 5 (2) Sequence identification number: 4 Information: (i) Sequence characteristics: (A) Length: 10 amino acid residues (B) category: amino acid (D) morphology: linear (ii) molecular category: protein (Xi) sequence description: sequence identification number: 4: Val lie Phe Ser Lys Ala Ser Glu Tyr Leu 1 5 10 (2) Sequence identification number: 5 Information: (i) Sequence characteristics: ^^^ 1 ^^^^ 1 1 · ii I ^^^^ 1 I mk ^ i— "'J $ Ttherapy (Please read the “Notes on the back” before filling out this page) -27- This paper size applies to Chinese National Standard (CNS) Α4ϋ 格 (210X 2ί »7 mm) 419483 A7 B7 V. Description of the invention (25) Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (A) (B) (D) (ii) Molecular Class (X i) Sequence Tyr Leu Gin 1 (2) Sequence Identification (i) f Column (A) (B) (D) i ii) molecular class (xi) sequence theory Gin Leu V a 1 1 i 2) sequence recognition (ϊ) sequence (A) (B) (D) (ii) molecular class (xi) sequence theory Length: 10 amino acid residue category: amino acid form: linear gender: protein identification: sequence identification number: 5: Leu Val Phe Gly lie Glu Vai 5 10 number: 6 information: special feature: length: 9 amino group Acid residue type: amino acid form: linear gender: protein: sequence identification number: 6: Phe Gly lie Glu Val Val 5 number: 7 information: characteristics: length: 11 amino acid residue type: amino acid Form: Line Gender: Protein Identification: Sequence Identification Number: 7: 28- This paper size applies to China National Standard (CNS) Α4 size (210X29? Mm) Please read the back of τ--item _ 419483 A7 B7 Ministry of Economic Affairs Printed by the Consumers' Cooperative of China Standards Bureau V. Invention Description (26)

Gin Leu Val Phe Gly lie Glu Val Val Glu Val 1 5 10 (2) Sequence identification number: 8 information: (i) Sequence characteristics: (/ 0 length: 9 amino acid residue (B) class 刖: amine Form of basic acid (D): linear (ii) Molecular type: protein (X i) sequence ^ Description: sequence identification number: 8: lie lie Val Leu Ala lie lie Ala lie 1 5 f 2) information of sequence identification number: 9 : (I) Sequence excitement: (A) Length: 11 Amino acid residue (B) Category: Amino acid (D) Form: Linear (ii) Molecular category: Protein (X i) Sequence description: Sequence recognition 煸No. 9:

Lys lie Trp Glu Glu Leu Ser Met Leu Glu Val 1 5 10 (2) Sequence identification number: 1 0 Information: (i) Sequence characteristics: (A) Length: 10 Amino acid residue (B) Category: Amine Acid-29- This paper size is in accordance with China National Standard (CNS) A4. (210X297 mm) m »ttn Is --- 1 0¾- " (Please read the back of the" Notes on the back of the ® "before filling out this page ) 419483 A7 B7 V. Description of the invention (27) (D) Morphology: linear (ii) Molecular type: protein (X Π sequence description: sequence identification number: 1 0:

Ala Leu lie Glu Thr Ser Tyr Val Lys Val 1 5 10 (2) Sequence identification number: 11 Information: (ΐ) Sequence characteristics: (A) Degree of fear: 9 amino acid residues 2) Category: amino acids (D) Morphology: linear (Η) Molecular type: protein (X i) Sequence description: sequence identification number: 11:

Leu lie Glu Thr Ser Tyr Val Lys Val 1 5 (2) Serial identification number: 1 2 Information: (i) Serial characteristics: Central Standards Bureau of the Ministry of Economic Affairs—Industrial and Consumer Cooperatives ’Seal ^ n I In n ^ i (n ^ i 1 --11 u HJH 11 ^ 1 ^^^ 1 1 -¾ T ”4 (Please read the notes on the back first, and then fill in this page) (length: 11 amino acid residue (B) category : Amino acid (D) form: linear (ii) molecular type: protein (X i) sequence description: sequence identification number: 1 2:

Gly Leu Glu Ala Arg Gly Glu Ala Leu Gly Leu 1 5 10 (2) Serial identification number: 1 3 Information: -30- This paper size applies to China National Standard (CNS) A4 specifications (210 X 297 mm) A7 B7 419483 V. Description of the invention (28 mfl ^ nn 11 m «Εί * ^ — -i kcl ^ t ^ 1 0¾,-*.. (Please read the back of the ©. Note Ϋ. Before filling this page) Ministry of Economic Affairs Printed on (i) sequence characteristics (A) length (B) category (D) morphology (Π) molecular category: (Xi) sequence description: Gly Leu Glu Ala 1 (2) sequence g Number (i) Sequence feature U) Length (B) Category (D) Morphology (ii) Molecular category: (X i) Sequence description: Ala Leu Gly Leu 1 (2) Sequence identification number (i) Sequence feature (A) length (B) Class (D) Form (ii) Molecular class: (X i) Sequence description: 9 amino acid residue amino acid linear protein sequence identification number: 13: Arg Gly Glu Ala Le 5 14 Information: 9 amine Amino acid residue amino acid linear protein sequence identification number: 14: ValGly Ala Gin All 5 15 Information: 9 amino acid residue amino acid linear protein sequence identification number: 15 : -31- This paper size applies to Chinese National Standard (CNS) A4 (210X297mm) A7 419463 B7 V. Description of Invention (29)

Gly Leu Val Gly Ala Gin Ala Pro Ala 1 5 (2) Sequence identification number: 1 6 Information: (i) Sequence excitement: (A) Length: 9 Amino acid residue (B) Category: Amino acid (D) Morphology: Linear (ii) Molecular type: Protein (X i) Sequence description · Sequence identification number: 1 6: ._n

Asp Leu Glu Ser Glu Phe Gin Ala Ala 1 5 (2) Sequence identification number: 1 7 Information: (i) Sequence characteristics: U) Length: 10 amino acid residue (B) Category: amino acid (D) Form: Linear (ii) Molecule Category: Protein (Xi) Sequence Description: Sequence Identification Number: 1 7:

Asp Leu Glu Ser Glu Phe Gin Ala Ala lie 1 5 10 (2) Sequence identification number: 1 8 Information: (i) Sequence characteristics: (A) Length: 10 amino acid residues (B) Class 刖: amine Base acid-32- This paper size is in accordance with Chinese National Standard (CNS) A4 (210X297 mm) 41 948 A7 B7 V. Description of invention (30) Printed (D) by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (ii) ) Molecular category: (X ί) Sequence description: Ala lie Ser Arg 1 ί 2) Sequence identification number (i) Sequence characteristics (A) Length 2) Category (D) Morphology (ii) Molecular category: (X i) sequence Description: Ala lie Ser Arg 1 (2) sequence identification number (i) sequence feature (A) length (B) category (D) morphology Ui) molecular category: ί X i) sequence description: Lys Met Val Glu 1 (2) Sequence identification number: Linear protein sequence identification number: 18: Lys Met Val Glu Leu Val 5 10 1 9 Information: 9 Amino acid residues Amino acid linear protein sequence identification number: 19: Lys Met Val Glu Leu 5 20 Information: 10 amino acid residues amino acid linear protein sequence identification number: 20: Leu Val His Phe Leu Leu 5 10: 2 1 of News: 33- This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) Please read back fir. Note-item before filling out this page Printed by the Central Consumers ’Cooperative Bureau of the Ministry of Economic Affairs 41 94t5J A7 B7 V. Description of the invention (31) (i) Sequence characteristics: U) Length: 11 Amino acid residue (B) Category: Amino acid (D) Form: Linear (Η) Molecular category: Protein U i) Sequence description: sequence identification number: 2 1: Lys Met Val Glu Leu Val His Phe Leu Leu Leu 1 5 10 (2) sequence identification number: 22 information: ii) JT column characteristics: (A) length: 11 amino acid Residue U) Category: Amino acid (D) Form: Linear (ii) Molecule category: Protein (Xi) Sequence description: Sequence identification number: 2 2: Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val 1 5 10 (2) Sequence identification number: 2 3 Information: (i) Sequence characteristics: U) Length: 10 amino acid residues (B) Species: Amino acid (D) Form: Linear (ii) Molecular category: Protein (X i) sequence description: Sequence identification number: 2 3: nn In nn ^^^^ 1 ^ ^^^^ 1 He—-s,-° '-(Please read the back first. Note-please fill out this page again) -34- This paper size is applicable to Chinese National Standard (CNS) A4 (2 丨 0 X 297 mm) 4ί9483 Α7 Β7 V. Description of Invention (32 Employees' Cooperatives of Central Standards Bureau, Ministry of Economic Affairs Print

Leu Leu Lys Tyr 1 (2) sequence identification number (i) sequence feature (A) length (B) category (D) morphology (ii) molecular category: (xi) sequence $ Description: Val Leu Arg Asn 1 f 2) Sequence identification number (i) Sequence characteristics (A) Length (B) Category (D) Morphology (ii) Molecular category: (χ Π Sequence description: Tyr Leu G 1 n Leu 1 (2) Sequence identification number (i) Sequence characteristics (A) Length (B) Category

Arg Ala Arg Glu Pro Val 5 10 24 Information: 11 amino acid residues amino acid linear protein sequence identification number: 24: Cys Gin Asp Phe Phe Pro Val 5 10 25 Information: 11 amino acid residues amino groups Acid linear protein sequence identification number: 2 5: Val Phe Gly lie Glu Val Val 5 10 26 Information: 10 Amino Acid Residues Amino Acid 35 This paper size applies to Chinese National Standard (CNS) A4 specification (210X 297 mm) ) Please read it first. Attention-item and then fill out this page 丨 419483 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (33) (D) Form: Linear i ii) Molecular category: protein & lt X i) Sequence description: Sequence identification number: 26: G 1 y lie Q l'u Val Val Glu Val Val Pro lie 1 1 10 (2) Sequence identification number: 27 Information: (i) Sequence characteristics: (i) A) Length: 9 amino acid residues 2) Category: amino acid (D) Form: linear (ii) Molecular category: protein (X i) Sequence description: sequence identification number: 2 7: Pro lie Ser His Leu Tyr lie Leu Val 1 5 (2) Sequence identification number: 2 8 Information: (i) Sequence characteristics : (Ί \) length: 9 amino acid residues (B) category: amino acid (D) morphology: linear (ii) molecular category: protein (X Π sequence description: sequence identification number: 2 δ: His Leu Tyr lie Leu Val Thr Cys Leu 1 5 (2) Serial identification number: 2 9 Information: (Please read the note on the back Ϋ · item and then fill out this page} This paper size applies Chinese National Standard (CNS) Α4 specifications (2 丨0 × 297 mm) 41 9483 A7 B7 V. Description of the invention (34 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (i) Sequence characteristics (A) Length (B) Category (D) Morphology (ii) Molecular category: (X Π sequence description: His Leu Tyr lie 1 (2) sequence g type number (i) sequence characteristics (A) length (B) category (D) morphology (ii) molecular category: (Xi) sequence description: Tyr lie Leu Val 1 (2) sequence identification number (i) sequence emblem (A) length (B) category (D) morphology (ii) molecular category: (Xi) sequence description: 11 amino acid residue: amino acid: Linear protein sequence identification number: 2 9: Leu Val Thr Cys Leu Gly L e 5 10 30 Information: 9 amino acid residues amino acid linear protein sequence recognition edition : 30: Thr Cys Leu Gly Leu 5 3 1 Information: 9 Amino acid residues Amino acid linear protein sequence identification number: 31: -37 This paper size applies to China National Standard (CNS) Α4 size (210X 297 mm) } Please read the back first. Ιέ Winter · Italian% Then write 'Si. This page η 419483 A7 Printed by B7 Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (35) Cys Leu Gly Leu Ser T yr Asp Gly Leu 1 5 (2) Sequence identification number: 3 2 Information ... (i) Sequence characteristics: (A) Length: 10 Amino acid residue (B) Category: Amino acid (D) Form: Linear (ii) ) Molecular category: Protein (X i) Sequence description: Sequence identification number: 3 2: Cys Leu Gly Leu Ser T yr Asp G1y Leu Leu 1 5 10 Γ2) Sequence identification number: 3 3 Information: (i) Sequence characteristics: (A) Length: 9 amino acid residues (B) category: amino acid (D) morphology: linear (Π) molecular type · protein (X i) sequence description: sequence identification S number: 3 3: Val Met Pro Lys Thr Gly Leu Leu lie 1 5 (2) Sequence identification number: 34 Information: (i) Sequence characteristics: (A) Length: 10 amino acid residues (B) Category: Amino acid (Please read the note on the back &-; then fill out this page) -38- This paper size applies to China National Standard (CNS) A4 specification (2t × 297mm) 4194 83 A7 Printed by B7 Consumer Cooperatives of the Bureau of Standards V. Invention Description (36) (D) Form: Linear i ii) Molecular Type: Protein (X Π Sequence Description: Sequence Identification Number: 3 4: Val Met Pro Lys Thr Gly Leu Leu lie lie 1 5 10 (2) Sequence identification number: 3 5 Information: (i) Sequence characteristics: (A) Length: 11 Amino acid residue (B) Category: Amino acid (D) Form: Linear (ii) ) Molecular category: Protein (X i) Sequence description: Sequence identification number: 35: Val Met Pro Lys Thr Gly Leu Leu lie lie Val 1 5 10 (2) Sequence identification number: 3 6 Information: (i) Sequence characteristics : (A) Length: 9 Amino acid residues (B) Category: Amino acid (D) Form: Linear (ii) Molecular category: Protein U i) Sequence description: Sequence identification number: 3 6: Gly Leu Leu lie lie Val Leu Ala lie 1 5 (2) Serial identification number: 37 information: (Please read the note on the back of the item before filling in this page) -39 -This paper size applies to China National Standard (CNS) A4 specifications (2 [0 × 297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs-(9 4 8 3 A7 B7 V. Description of the invention (37) (i) Sequence characteristics : (A) Length: 10 amino acid residues (B) Category: Amino acid (D) Form: Linear (ii) Molecular category: Protein (X Π Sequence description: Sequence identification number: 37: Gly Leu Leu lie lie Val Leu Ala lie lie 1 5 10 (2) Sequence identification number: 3 8 Information: (i) Sequence characteristics: (A) Length: 11 Amino acid residue (B) Category: Amino acid (D) Form: Linear (ii) molecular type: protein (X i) sequence description: sequence identification number: 3 8: Gly Leu Leu lie lie Val Leu Aia lie lie Ala 1 5 10 (2) sequence identification number: 39 information: (i ) Sequence excitement: (A) length: 9 amino acid residues ί B) category: amino acid (D) morphology: linear (ii) molecular category: protein (X i) sequence description: sequence identification number: 39 : ^ 11 »it ί ^^^ 1 1-^^ 1 ^^^ 1) * (please read the ^ -notes carefully before filling out this page) -40- This paper size applies to Chinese national standard ( CNS > Μ Grid (210 'qe 297mm> 4 1 9 4 8 3 a7 B7 V. invention is described in (38)

Leu Leu lie lie Val Leu Ala lie lie 1 5 (2) Sequence identification number: 40 information: (i) Sequence characteristics: (A) Length: 10 amino acid residue (B) Category: amino acid (D) Morphology: Linear (ii) Molecule category: Protein (xi) Sequence description: Sequence identification #: 40:

Leu Leu lie lie Val Leu Ala lie lie Ala 1 5 10 f 2) Sequence identification number: 4 1 Information: (i) Sequence characteristics: (A) Length: 11 Amino acid residue (B) Category: Amine Acid (D) form: Linear (ii) Molecular category: Protein (Xi) Sequence description: Sequence identification number: 41: Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (Please read the precautions on the back before filling out this page )

Leu Leu lie lie Val Leu Ala lie lie Ala lie 1 5 10 (2) Sequence identification number: 42 information: (i) Sequence characteristics: (A) Length: 9 amino acid residue (B) category: amino acid -4 1-This paper size applies Chinese National Standard (CNS) A4 (210 X 297 mm) 419483 A7 B7 V. Description of the invention (39) (D) Form: Linear (ii) Molecular category: Protein (X Π sequence Description: Sequence identification number: 42:

Leu lie lie Val Leu Ala lie lie Ala 1 5 (2) Sequence identification number: 43 information: (i) Sequence characteristics: (A) Length: 1 0 Amino acid residue (B) Category: Amino acid (D ) Morphology: Linear (ii) Molecule category: Protein (X Π Sequence description: Sequence identification #: 4 3:

Leu lie lie Val Leu Ala lie lie Ala lie 1 5 10 (2) Serial identification number: 4 4 Information: ii) Sequence characteristics: Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs ^ Λ (Please read the precautions on the back first Refill this page) U) Length: 9 Amino acid residues (B) Category: Amino acid (D) Form: Linear (U) Molecule category: Protein (Xi) Sequence description: Sequence identification number: 44: lie lie Ala lie Glu Gly Asp Cys Ala 1 5 (2) Serial identification number: 45 Information: -42- This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) 419483 A7 B7 Zhongli Bureau of Standards, Ministry of Economic Affairs Printed by the Employee Consumption Cooperative 5. Description of the invention (called) (i) Sequence characteristics: U) Length: 9 amino acid residues (B) Category: Amino acid (D) Form: Linear (ii) Molecular category: Protein ( xi) Sequence description: Sequence identification number: 45: Lyslie Trp Glu Glu Leu Ser Met Leu 1 5 (2) Sequence identification number: 46 Information: (i) Sequence characteristics: U) Length: 11 amino acid residues ( B) Category: Amino acid (D) Form: Linear (线性) Molecular category: Protein (X Π Sequence description Sequence identification number: 46: Leu Met Gin Asp Leu Val Gin Glu Asn Tyr Leu 1 5 10 (2) Sequence identification number: 47 Information: (i) Sequence characteristics: (A) Length: 9 amino acid residues (B ) Category: Amino acid (D) Form: Linear (ϋ) Molecular category: Protein (χΠ Sequence description: Sequence identification number: 47: (Please read the notes on the back ^ before filling this page) This paper is for China National Standard (CNS) Α4 Specification (2 丨 〇 X 297 mm) 4194 83 A7 B7 V. Invention Description Ui)

Phe Leu Trp Gly Pro Arg Ala Leu lie 1 5 i 2) Serial identification number: 4 8 Information: (i) Sequence characteristics: (A) Length: 9 amino acid residue (B) Category: amino acid (D ) Morphology: linear (ii) molecular category: protein (xi) sequence description: sequence identification number: 48:

Leu lie Glu Thr Ser Tyr Val Lys Val 1 5 (2) Sequence identification number: 49 Information: (i) Sequence characteristics: (A) Length: 11 Amino acid residue (B) Category: Amino acid (D) Morphology: linear (ϋ) Molecular type: protein (X i) Sequence description: sequence identification 煸 number: 49:

Ala Leu lie Glu Thr Ser Tyr Val Lys Val Leu 1 5 10 (2) Sequence identification number: 5 0 Information: (i) Sequence characteristics: (A) Length: 10 Amino acid residue (B) Category: Amine Base acid-44- This paper size is in accordance with Chinese National Standard (CNS) A4 (210X29? Mm) I 'Order < Please read the precautions on the back before filling this page) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 419483 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (42) 1 1 1 (D) Morphology: Linear 1 1 | (h) Molecular category: Protein 1 I Please 1 (X ί) Sequence description: Sequence identification number: 50: Read first 1 I Thr Leu Lys I 1 e Gly Gly Glu Pro H is lie Read back 1 side-I 1 5 10 of 1 Note 1 (2) Serial identification number: 51 Information: meaning, 1 (i) sequence specific item ii 1 (A) length: 11 amino acid residues in writing (B) category: amino acid page 1 | (D) morphology: linear 1 (ii) molecular category: protein 1 1 (xi) Sequence description: Sequence identification number: 51: ί Order H is lie Ser Tyr Pro Pro Leu His Glu A rg Ala 1 1 5 10 1 I (2) Sequence ID: 52 Information: 1 1 (i) Sequence specific 1 I (A) Length: 9 amino acid residue 1 ί B) Category: Amino acid 1 I (D) Form: Linear 1 1 (ii) Molecular category: Protein 1 丨 (X i) Sequence description: Sequence identification number: 52: i Gin Thr Ala Ser Ser Ser Ser Thr Leu i 1 5 1 m Serial identification number: 53 Information: 1 1 -45- 1 1 1 1 This paper size applies to the Chinese national standard i CNS) A4 specification (210X297 mm > I 4194 8 3 A7 B7 V. Invention Explanation (C) (i) Sequence characteristics: U) Length: [0] Amino acid residue (B) Category: Amino acid (D) Form: Linear (线性) Molecular category: Protein f X i) Sequence description: Sequence Identification Number: 5 3:

Gin Thr Ala Ser Ser Ser Ser Thr Leu Val 1 5 10 (2) Sequence identification number: 5 4 Information: (i) Sequence characteristics: (A) Length: 9 amino acid residue (B) Category: amino acid (D) Morphology: Linear (U) Molecular type: Protein (X i) Sequence description: Sequence identification number: 5 4:

Val Thr Leu Gly Glu Val Pro Ala Ala 1 5 (2) Serial identification number: 5 5 information ... (i) 'Sequence special excitement: Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. 1 ^ 1 ^ 1 ^^ ^^ 1 ^^^^ 1 ^^^^ 1 ^^ — 1— \ J (Please read the note τι on the back before filling this page) U) Length: 10 amino acid residues (B) Category: Amino acid (D) form: linear < ii) molecular type: protein (X i) sequence description: sequence identification number: 5 5: -46- This paper size applies Chinese National Standard (CNS) Α4 specification (210 X 297 mm) A7 B7 Five 'invention description (

Val Thr Lys Ala Glu Met Leu Glu Ser Val 1 5 10 (2) Sequence identification number: 5 6 Information: ii) Sequence characteristics: (A) Length: 11 Amino acid residue (B) Category: Amino acid ( D) Morphology: Linear (ii) Molecule category: Protein (X i) Sequence j Description: Sequence identification number: 5 6:

Val Thr Lys Ala Glu Met Leu Glu Ser Val Leu 1 5 10 (2) Sequence identification number: 5 7 Information: (i) Sequence special emblem: (A) Length: 11 limb acid residue (B) Category: Amine Form of basic acid (D): Linear (ii) Molecular type: Protein (Xi) Sequence description: Sequence identification number: 57: Printed by the Machining Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the note t on the back before filling in) (This page)

Val Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu 1 5 10 (2) Sequence identification number: 5 3 Information: (i) Sequence characteristics: (A) Length: 9 Amino acid residue (B) Category: Amine Acid-47- This paper size applies to China National Standards (CNS) A4 specifications (210X297 mm). 9483 A7 B7 Printed by the Consumer Cooperatives of the China Standards Bureau of the Ministry of Economic Affairs. 5. Description of the invention (45) (D) Form: Linear (ϋ) Molecule category: Protein (X ί) Sequence description: Sequence identification number: 5δ:

Lys Thr Gly Leu Leu lie lie Val Leu 1 5 (2) Sequence identification ffi number: 5 9 Information: (i) Sequence characteristics: (A) Length: 10 amino acid residues (B 1 category: amino acids ( D) 'morphology: linear (ii) molecular type: protein (X i) sequence description: sequence identification number: 5 9:

Lys Thr Gly Leu Leu lie lie Val Leu Ala 1 5 10 (2) Sequence identification number: 60 information: (i) Sequence characteristics: (A) Length: 11 Amino acid residue (B) Category: Amino acid ( D) Morphology: Linear (ϋ) Molecular category: Protein (X i) Sequence description: Sequence identification number: 6 0:

Lys Thr Gly Leu Leu Ile * Ile Val Leu Ala lie 1 5 10 (2) Serial identification number: 6 1 Information: -4 8-This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) — -i I — ^ 1 ^ — ^ 1 * k Shiyue ··! 1 »-mil ^ — ^ 1 ^^^ 1 Ti. (Please read the note on the back first: item before filling out this page) 19 4 8 3 A7 B7 V. Description of the invention (46)

(i) Sequence characteristics: (A) Length: 11 Amino acid residues (B) Category: Amino acid (D) Morphology: Linear (ii) Molecular category: Protein (X i) Sequence description: Sequence identification number: 6 1: His Thr Leu Lys lie Gly Gly GIu Pro His II 1 5 10 62 Information: 9 Amino acid residues Amino acid (2) Sequence identification number (i) Lecitex U) Length (B) Category (D ) Morphology: Linear (U) Molecular category: Protein (X i) Sequence description: Sequence identification number: 62: Met Leu Asp Leu Gin Pro Glu Thr Th 1 5 --------- 求 ----- -Order ------ Guang— * · (Please read 41 * Note on the back side first and then fill out this page) '' Printed by the Consumers' Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 49- This paper size applies to Chinese National Standards (CNS) A4 size (210 X 297 mm)

Claims (1)

^ 4 1 9 4 8 3 A8 BS C8 D8 Hang No. 84103311 Li Application Chinese Patent Application 锪 Original (December 88) Patent Application Announcement This Year iAM 1. A hydroxyl-isolated hydrazone, which is selected From sequence_other number: 1, sequence identification number: 2, sequence identification number: 4 * sequence identification number: 8, sequence identification number: 9, and sequence identification number: 11: sequence miscellaneous number: 1 Ser Thr Leu Val Glu Val Thr Leu Gly Glu Val 15 10 sequence identification number: Leu Val Glu Val. Thr Leu Gly Glu Val 1 5 sequence identification number: 4 Val He Phe Ser Lys Ala Ser Glu Tyr Leu 1 5 10 sequence identification number: 8 lie lie Val Leu Ala lie lie Ala lie 1 5 Sequence identification number: 9 Lys lie Trp Glu Glu Leu Ser Met Leu Glu Val 15 10 Sequence identification number: 11 (Please read the notes on the back before filling this page) 1 · Leu He Glu Thr Ser Tyr Val Lys Val " 〇1 5 The isolated peptide according to item 1 of the scope of patent application is marked with a sequence identification number: 1. The isolated peptide according to item 1 of the scope of patent application is designated as sequence identification number: 9. -An isolated non-covalent complex of HLA-A2 with an isolated peptide as described in claim 1 of the scope of patent application. According to the separated non-covalent complex in item 4 of the scope of patent application, this paper uses Zhonggu II Home Standard (CNS) A4 format (210X297 mm) I 419483 ABCD. The peptide is labeled with a sequence identification number: 1. 6. The isolated non-covalent complex according to item 4 of the application for patent, wherein the peptide is labeled with a sequence identification number: 2. 7. The isolated non-covalent complex according to item 4 of the scope of the patent application, wherein the peptide is identified by a sequence identification number: 4. 8. The isolated non-covalent complex according to item 4 of the scope of patent application | wherein the peptide is marked with a sequence identification number: 8. 9. The isolated non-covalent complex according to item 4 of the scope of the patent application, wherein the peptide is identified by a sequence identification number: 9. 10. The isolated non-covalent complex according to item 4 of the scope of the patent application, wherein the peptide is identified by a sequence identification number: 11. (Please read the notes on the back before filling out this page.) Pack-'11 Printing policy of the Consumers' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs This paper scale is applicable to the Chinese National Standard (CNS) Α4 ^ grid (210 × 297 mm)