JP2001509677A - Peptides for activating CTL and peptide-bearing antigen-presenting cells - Google Patents

Abstract

  (57) [Summary] Methods for generating antigen-specific cytotoxic T lymphocytes (CTLs) in vitro and using these CTLs for in vivo treatment are provided. Antigen presenting cells (APCs) for use in the present invention significantly include dendritic cells pretreated with GM-CSF and IL-4. Incubation of the CD8 preparation in the presence of antigen-presenting cells that bind the desired peptide resulted in antigen-specific CTL. IL-7 was generally added on the first day of incubation; IL-10 was added one day later during restimulation. The invention also includes a method of using peptide-pulsed dendritic cells to identify novel MHC class I-restricted tumor antigens and subdominant epitopes.

Description

DETAILED DESCRIPTION OF THE INVENTION Peptides for activating CTL and peptide-bearing antigen-presenting cells   This application is related to US patent application Ser. No. 60 / 036,691, filed Jan. 31, 1997. No. 6 claims priority and is a continuation-in-part thereof. Background of the Invention   The present invention provides for the ex vivo treatment of pathological conditions such as viral diseases and cancer. The present invention relates to compositions and methods to be prevented or treated by law. . In particular, the invention is optional Selected peptide bound to a major histocompatibility complex (MHC) molecule Inducing Cytotoxic T Lymphocytes (CTLs) Using Reducing Antigen Presenting Cells (APCs) How to guide.   CTL (or CD8, as CD8 is also known) is a cell containing foreign antigens. Specifically recognizes vesicles and kills them. They are disease states, such as viral infections And establish a major line of defense against conditions caused by tumor cells.   In contrast to antibodies, antigen must first be presented to T cells for activation to occur No. CTLs are not intact foreign antigens themselves, but rather MHC class I Of 8-12 residues bound to the molecule and displayed on the surface of antigen presenting cells (APC) Recognize peptides.   The presentation of antigen to T cells is mainly based on the HLA (human leukocyte antigen) complex, It is mediated by key histocompatibility complex (MHC) molecules. MHC gene products On the cell surface, and "non-self" He is largely responsible for the recognition of tissue grafts.   MHC is classified as a Class I, Class 11, or Class III molecule. Class I MHC molecules are expressed on almost all nucleated cells and Is recognized by Class III MHC molecules are involved in the initiation and persistence of the immune response Related cells, such as T lymphocytes, B lymphocytes, macrophages, and the like. It is mainly expressed on others. Class III MHC molecules become helper T lymphocytes Specific immunogens that are more recognized and displayed as helper T lymphocyte proliferation Induces an amplification of the immune response to the sex peptide.   In general, the CTL response is not directed against all possible determinants, Directed against only a few "immunodominant" epitopes. Sercarz   et al. , "Annu. Rev. Immunol." 11: 729 (19) According to the nomenclature developed by J. 93), the epitope is involved in the process of natural infection. When normally recognized, or with whole or naturally processed antigens When immunized, the epitope is immunodominant. 1) Synthetic peptide as immunogen Or 2) the dominant epitope is removed (or altered) from all antigens If so, the subdominant epitope mediates the immune response. Like a dominant epitope , The subdominant epitope is also processed by the APC and the MHC complex on the surface Presented as a body.   Treats diseases like cancer, AIDS, hepatitis and other infectious diseases, theoretically attractive A simple approach is to activate only CTL that recognize the diseased cells. 1 One possible approach is to immunize a healthy individual and isolate CTL from this individual. Injecting these cells into a person with the disease. However, donor MHC haplotype must be identical to that of the recipient. this is is important. Because the CTL of the recipient is Can it interact only with peptides bound to only one of the class I molecules? It is. Also, regardless of what peptide the Class I molecule contains, All of the class I molecules that are different from those expressed in the individual taking the D8 cells , CTL reacts violently. This reactivity is the root of immune rejection in the transplanted organ. The original cause.   It is difficult to find two unrelated persons with exactly the same class I molecule. As such, some therapeutic approaches involve converting existing CD8 cells in vitro. by incubation with IL-2, a T cell growth factor, at A non-specific approach to "promoting" CD8 cells is employed. However, this Protocol (known as LAK cell therapy or TIL [tumor infiltrating lymphocyte] therapy) Allows relatively non-specific expansion of previously activated CTLs Will only be. Most IL-2 stimulator cells are irrelevant for disease eradication . In addition, the side effects of LAK cell therapy are often severe. (Greenberg, P. , "Adv. In Immunol." 49: 281 (1991); Mel. ief, C, "Adv. Cancer Research" 58:14 (1992). ); Riddell et al. , "Science" 257: 238 (19 92).   The development of specific CTL-based immunotherapy has led to the development of molecules that trigger CTL responses. Requires identification. For example, tumor associated antigen (TAA). These TAAs are primary Both metastasis or recurrence of tumors after treatment and eradication of already established tumors An immune response of interest can be elicited (Boon, T. et al., " Annual Review of Immunology, "12: 237- 365 (1994); et al. , "Annual Rev view of Immunology ", 10: 617-644 (1994); Rosenberg, S.M. A. , "Immunology Today", 18 175-182 (1997)). Identification of CTL epitopes from various TAAs (Boon, T. et al., "Ann. Rev. Immun" ol. 12: 337-365 (1994); Urban, J. et al. L. et a l. , "Ann. Rev. Immunol." 10617-644 (1994). Rosenberg, S .; A. , "Immunology Today", 1 8: 175-182 (1997); et al. , "J. Exp . Med. 181: 2109-2117 (1995); Disis, M .; et   al. , "Cancer Research", 54: 1071-1076 ( 1994); Peoples, G .; E. FIG. et al. "Proc. Natl. Acad. Sci. USA ", 92 432-436 (1995); Tsang. , K .; Y. et al. , "J. Natl. Cancer Inst." 87: 982-990 (1995); Cox, A .; L. et al. , "Science e ", 264: 716-719 (1994); Skipper, J. et al. C. et al. , "J. Exp. Med." 193: 527-534 (1996)). Ho In most cases, using CTLs from cancer patients, tumor cell MHC molecules The library or peptide of the expressed gene eluted from it was screened. Most of these CTLs are from patients with melanoma, and to date Most of the harbored CTL epitopes are from melanoma. Malignant disease, example For example, to treat breast cancer, lung cancer, colon cancer and other more common types of other gastrointestinal cancers Only a few epitopes are available is there.   Recent Discovery of MHC Class I Allele Specific Motifs and Peptide-MHC Classes The development of the Las I binding assay has led to the identification of antigenic epitopes recognized by CTL Promoted. Rammensee, et al. "Immunogenet." 41: 178 (1995); Kubo, et al. "J. Immunol. 152: 3913 (1994); Ruppert, et al. , "Cell 74: 929 (1993); Kast, et al. , "J. Immunol . 152: 3904 (1994). Several studies have shown that The basis of immunodominant cells and molecules in the CTL response was probed. Ca o, et al. J. Immunol. 157: 505 (1996); ameson, et al. "Eur. J. Immunol." 22: 266. 3 (1992); Wipke, et al. "Eur. J. Immunol. 23: 2005 (1993); Chen, et al. , "J. Exp. Me d. 180: 1471 (1994); Oldstone, M .; A. , "J.V. irol. 65: 6381 (1991); Li, et al. "Eur. J . Immunol. 22: 943 (1992); Sigal, et al. , "Molec. Immunol." 32: 623 (1995); Ouka, e. t al. , "J. Immunol." 152: 4843 (1994). Contrast In humans, only dominant epitopes are generally identified in humans, and in humans The definition of a subdominant epitope was particularly problematic for the setting of the diseases involved. i Studies of n vitro immunization have generally produced impressive results in the past. Because, even in the case of known immunodominant epitopes, the derived CTL Has a low affinity (binding activity) and, after all, naturally Lack of provable recognition of the processed antigen. Wentworth Et al. , "Molec. Immunol." 32: 603 (1995). . In addition, plans to use the smallest epitope in only a few cases In vivo immunization was attempted. Vitiello, et al. , "J . Clin. Invest. 95: 341 (1995); Marchand, et al. , "Int. J. Cancer" 63: 883 (1995); Jo. eger, et al. , "Int. J. Cancer" 66: 162 (199 6); Mukherji, et al. , "Proc. Natl. Acad. S. ci. (USA) 92 8078 (1995).   However, in the past, it specifically activated CTLs associated with these diseases. To make it more reliable, no reliable approach was available. The present invention relates to this and other Of issues. Summary of the Invention   In general, the present invention provides for cytotoxic T cells (CD8 cells) in vitro or Relates to a method of activating in vivo. In one embodiment, this The method comprises presenting a desired immunogenic peptide from an antigen, pre-treated with a growth factor. Associates with class I MHC molecules on cells and causes antigen presenting cells to become cytotoxic Incubating the harmful T cells in the presence of one or more growth factors; This consists in producing activated cytotoxic T cells. Preferred A PC activates dendritic cells, especially dendritic cell precursors, in the presence of GM-CSF and IL-4. And cells produced by incubation. In a preferred embodiment One growth factor IL-7 and the other is IL-10. IL-7 is preferred Kuha Incub At the beginning of the incubation step, and IL-10 is added one day later. Was.   The present invention also provides a method for specifically killing target cells in a human patient. . This method involves taking a fluid sample containing cytotoxic T cells from a patient, Sex T cells with antigen presenting cells pretreated with one or more pretreatment growth factors Contacting, wherein said antigen presenting cells comprise a class I MHC molecule and In the presence of a progenic peptide and two or more incubation growth factors And incubating the pretreated antigen-presenting cells with cytotoxic T cells, This produces activated cytotoxic T cells and activates cytotoxic T cells. Contacting the cells with an acceptable carrier, thereby forming a pharmaceutical composition; Administering the drug composition to the patient.   Antigen presenting cells with empty MHC class I molecules on their surface Induce cytotoxic T cells that are effective in treating chronic infections and cancer Wear. Specifically, the present invention relates to an empty MHC class I molecule on an antigen presenting cell. And the immunogenic peptides selected for these empty MHC class I molecules Cytotoxic T cells that carry tides and are specific for killing specific antigen targets A method for activating is provided. The present invention relates to cancer, certain immune diseases and viruses. It has wide therapeutic use in the treatment of sexually transmitted diseases. As such, this method Activated C from antigen-presenting cells having tea MHC class I molecules on their surface TL is isolated and activated in a carrier or excipient acceptable as a pharmaceutical composition Suspending the CTL and administering the pharmaceutical composition to a patient having the disease May be further included.   In a preferred embodiment, IL-7, IL-10 and peptide- Primary cell injury in vitro using pulsed dendritic cells (DC) Induced T lymphocytes (CTL) were induced. DCs were isolated from normal volunteers CTL epitopes prepared from peripheral blood mononuclear cells and of known viruses and tumors Were pulsed with various HLA-A1 and -A2.1, corresponding to These anti Protopresenter cells are hepatitis B virus epitopes restricted by the HLA-A2 molecule. It was very efficient at inducing the response of CTL to CTL. Most of CTL Not only can kill peptide-coated target cells, but also CTLs have high specificity for immunogens and It has both initiative. Induction of CTL by DC is a known tumor Recombinant IL-7 and IL-10 by using peptides corresponding to the relevant antigen But was significantly enhanced by IL-12. Peptide induced CT L is very useful in recognizing and killing tumor cell lines expressing appropriate tumor antigens. In tissue culture without loss of activity and specificity. It could be magnified 0 times. Methods and methods for inducing CTL with peptide-coated DC Itokine combination identifies new CTL epitopes and is based on CTL There is value for both the development of immunotherapy.   The present invention also provides that certain epitopes are identified during the course of a natural disease or infection. Irrespective of whether it is recognized (ie, it is immunodominant or not) To determine the total immunogenic potential of an antigen of CTLs by the response of CTLs.   In one embodiment, the gp100 melanoma associated tumor antigen is selected as a model system We studied the diversity of responses of human antitumor cytotoxic T cells. Normal Volante Using lymphocytes from media and in vitro priming protocol And dominant gp100HLA -The peptide corresponding to the A2.1 restricted CTL epitope was tested. Said protocol Have utilized peptide-pulsed dendritic cells as APCs and have Utilizes IL-7 and IL-10 as itokines. Peptide-pulsed standard Cells and gp100⁺High CTL activity on both melanoma cells also Obtained using four of the five peptides tested. Therefore, the present invention Include peptides detected using the methods described herein.   Also, using an in vitro priming protocol, patients with melanoma HLA-A2 from gp100 does not appear to represent a CTL epitope in . One binding peptide was tested. Three of the six peptides tested were normal Induced CTLs in lymphocytes from volunteers. Of these peptides One is also immunogenic for lymphocytes from patients with relapsing melanoma. there were. These three CTL epitopes are responsible for natural immune response in melanoma patients. Using peptide to induce a T cell response When they did, they appeared to be immunogenic in nature, so they Can be thought of as a loop.   The present invention does not require the use of the patient's CTL, but instead uses the known TAA or Identification of MHC binding peptides and novel in vitro priming An alternative approach to identifying tumor epitopes, which relies on protocols. Specifically, the basis of MHC binding motifs and their binding to MHC molecules Peptides selected for potency were obtained using lymphocyte cultures from normal individuals. Test for their ability to elicit tumor-reactive CTLs in vitro I do. Follow this “reverse immunological” approach to melanoma Several CTL epitopes from TAA expressed in tumors have been identified Was. In the examples, CTLs derived from various TAAs expressed on solid adenocarcinoma were used. Pitopes, such as MAGE2, MAGE3, carcinoembryonic antigen (CEA), and Apply this method to identify HER2 / neu (Weynants, P. et al. , "International Journal of C ancer "56: 826-829 (1994); De Plaen, E .; et   al. , "Immunogenetics" 40 360-369 (1994). Vincent, R .; G. FIG. et al. , "Cardiovasc. Sur g. 66: 320 328 (1978); Shavery, J. et al. et al. , "CRC Critical Reviews in Oncology a nd Hematology "2: 355-399 (1985); Thomps. on, J.M. et al. , "Journal of Clinical and and   Laboratory Analytics "5: 344-366 (1991). Sikorska, H .; et al. , "Cancer Detectio n and Prevention, 12: 321-355 (1988); Mu. rano, R .; et al. , "Cancer Research" 45:57 69-5780 (1985); Steward, A .; M. et al. , "Ca ncer "33.1246-1252 (1974); Slamon, D .; J. e t al. , "Science" 244: 707-712 (1989); ota, J. et al. et al. , "Lancet" 1: 765-767 (1986). ). These results indicate a high incidence of tumors such as breast, lung, colon and stomach cancer Related to the development of immunotherapies based on the epitopes of E. coli. BRIEF DESCRIPTION OF THE FIGURES FIG. Antigen specificity in lymphocyte cultures after expansion in vitro And CTL response to MAGE-3 epitope.   As described in Materials and Methods, one positive CTL culture (Table III) From the experiments described in Section III), anti-CD3 monoclonal antibody (A) or MAGE-3 Expanded with 161 peptide (B). On day 13, cytotoxic activity against the following targets And tested: Δ, Steinlin + peptide; □, stain Phosphorus-peptide; ●, 938 mel (Al +, MAGE-3⁺); ○, peptide ,. 888mel (Al +, MAGE-3^-). On day 13, the anti-CD3 antibody (A ) Stimulated cells expanded 1300 times, and cells stimulated with peptide (B) Magnified 820 times. FIG. Antigen specificity and gp100 peptide C9₂₈₀CTL response to   CD8 from normal volunteers⁺Lymphocytes were converted to C9 in vitro._{Two 80} , -Pulsed autologous DC. A: every peptide-pulsed monocyte After establishing the CTL line by weekly restimulation, the following various target cell lines The lytic activity was measured using: ■,. 221 (A2.1) -no peptide; □ ,. 221 (A2.1) + G9₂₈₀○, 624 mel (A2⁺, Gp100⁺) ▲, A372mle (A2⁺, Gp100^-)); Δ, 697 mel (A2⁺, gp100⁺); Δ, 677 mel (A2⁺, Gp100⁺). B: Cold target inhibition Antigen specificity demonstrated by research. G9₂₈₀30: 1 by specific CTL line 624 mel (A2⁺, Gp100⁺) Is dissolved at the following low temperature ( (Unlabeled) blocked by target cells: □,. 221 (A2.1) + G9_{28 0} ■,. 221 (A2.1) + control HLA-A2.1 Compatible peptide (FLPSDFFPSV); 221 (A2.1) -peptide None. FIG. Antigen specificity and gp100 peptide G9₁₇₈(A) and G10₁₇₇( CTL response to B).   In vitro with gp100 peptide carried on autologous DC Stimulated CD8 + lymphocytes from normal volunteers. Produce CTL system And 20: 1 in the presence and absence of unlabeled cold target (inhibitor). Different "Cr-labeled target cell lines" were used at different inhibitor: target ratios. Tested. Experimental conditions for both panels: □,. 221 (A2.1) + g p100 peptide (G9 in A₁₇₈And in B, G10₁₇₇); 2 21 (A2.1) -no peptide; ▲, 662 mel (A2⁺, Gp100⁺); △, A375mel (A2⁺, Gp100); Δ, 624 mel + gp100 pairs Pulsed with peptide. 221 (A2.1) inhibitor (G9 in A₁₇₈So G10 in B₁₇₇); Δ, 624mel + control peptide (FLPSDF FPSV). 221 (A2.1) inhibitors. FIG. gp100 peptide G9₁₇₈And G10₁₇₇The CTL specific for Recognizes two distinct epitopes.   Peptide G9₁₇₈(A) or G10₁₇₇This is the CTL extracted in (B) Tested for their ability to kill their targets □,. 221 (A2.1) + " G10₁₇₇○,. 221 (A2.1) + G9₁₇₈△,. 221 (A2.1)- No peptide; ▲, 624 mel (A2⁺, Gp100⁺); △, A375mel (A2⁺Gp100). FIG. gp100 peptide G10₅₇₀CTL response to   Normal volunter was performed using peptide-pulsed autologous DC. CTLs were derived using CD8 + lymphocytes from A. G: A: G10₅₇₀Special Heterologous CTL lines were tested for their ability to kill the following targets: □,. 221 (A2.1) + G10₅₇₀○,. 221 (A2.1) -No peptide; Δ, 6 97 mel (A2 +, gp100 +): Δ, 624 mel (A2 +, gp100 +) +); ●, A375 mel (A2 + gp100); ▲, 677 mel (A2 +, gp100 +). B: Low temperature for lysis of 624mel (A2 +, gp100 +) cells Target inhibition.⁵¹Killing of Cr-labeled 624mel cells was performed using G10₅₇₀(▼) Low temperature (unlabeled) pulsed with control peptide (▽, FLPSDFFPSV). 221 (A2.1) cells. FIG. By lymphocytes of patients using peptides corresponding to subdominant epitopes Inhibition of melanoma reactive CTL.   Peptide G10₁₇₇In autologous DCin vitro pulsed by Stimulate blood lymphocytes from melanoma patients and after 3 cycles of antigen-restimulation, T cells Cell lines were assayed for their lytic activity against these targets: □,. 2 21 (A2.1) + G10₁₇₇■,. 221 (A2.1) -No peptide; ○ 624mel (A2 +, gp100 +); ▲, A375mel (A2 +, gp 100). 20: 1 excess G10₁₇₇Unlabeled pulsed with. 221 (A2.1 ) (◇); or unlabeled pulsed with HLA-A2.1 binding control peptide. 624mel by the addition of 221 (A2.1) (）, FLPSDFFPSV) Antigen specificity was further demonstrated by cold target inhibition of cell lysis. FIG. Recognition of various tumor types by MAGE2-specific CTL clones.   MAGE2 [10₁₅₇] The specific CTL clone is transformed into the following target cells: Were tested for their lytic activity against: O, MAGE2 [10₁₅₇] I did it. 221A2.1; ●, not containing peptide. 221A2.1; Δ, 62 4me1 (melanoma, A2⁺, MAGE2⁺); □, KATO-III (gastric Ca, A 2⁺, MAGE2⁺); ◇, SW403 (colon Ca, A2)⁺, MAGE2? ); ■ , WiDr (colon Ca, A2, MAGE2?); 、, 888 mel (melanoma, A 2, MAGE2^-). FIG. MAGE2 [10₁₅₇Demonstrates antigen specificity of specific CTL clone Cryotarget inhibition assay.   Tumor cell line 624mel (A), SW403 (B) or KATO-III (C)⁵¹Cr-labeled and the following low-temperature targets at various inhibitor / target ratios: 的, MAGE2 [10₁₅₇]. 221A2.1; Irrelevant A2.1 binding peptide (HBc_18-27); ●, not containing peptide . 221A2.1. The effector / target ratio used was 8: 1 (A), 4: 1 ( B) or 5: 1 (C). FIG. 9. Recognition of various tumor types by MAGE3-specific CTL clones.   MAGE3 [9₁₂₁Specific CTL clones were cytotoxic using the following targets: Tested for the properties: o, MAGE3 [9₁₁₂]. 221A2.1 ; ●, not containing peptides. 221A2.1; Δ, 624mel (melanoma, A2⁺ , MAGE3⁺); □, KATO-III (gastric Ca, A2⁺, MAGE3⁺); ■ , SW403 (colonic Ca, A2⁺, MAGE3⁺); ■, WiDr (colon Ca, A 2^-, MAGE3⁺); ▲, 888me1 (melanoma, A2^-, MAGE3⁺). FIG. MAGE3 [9₁₁₂] Antigen specificity of specific CTL clone Low-temperature target inhibition assay to prove its ability.   Tumor cell line 624mel (A), SW403 (B) or KATO-III (C)⁵¹Cr-labeled and the following low-temperature targets at various inhibitor / target ratios: 的, MAGE3 [9₁₁₂]. 221A2.1; Pulsed with an irrelevant A2.1 binding peptide (HBc18-27). 221A2. 1; ●, not containing peptide. 221A2.1. Of the effector / target used The ratio was 8: 1 (A), 8: 1 (B) or 5: 1 (C). FIG. Recognition of various tumor types by CEA-specific CTL clone   CEA [9₆₉₁] The specific CTL clone was cloned using its target Tested for harm: ○, CEA [9₆₉₁]. 221A2.1; ● Does not contain peptides. 221A2.1; △, KATO-III (gastric Ca, A 2⁺, CEA⁺); ◇, SW403 (colon Ca, A2)⁺, CEA⁺); ▲, HT-2 9 (colonic Ca, A2^-, CEA⁺). FIG. Cold Target Inhibition Protocol Demonstrating Antigen Specificity of CEA-Specific CTL Clones Essay.   CEA [9₆₉₁Specific CTL at an effector / target ratio of 15: 1 Tested in both experiments. Tumor cell line KATO-III (A) or SW 403 (B)⁵¹Cr labeled and at different inhibitor / target ratios: Mixed with cold target: ○, CEA [9,₆₉₁ ⁺]. 221A2.1; ▲, irrelevant A2.1 binding peptide (HBc_18-27). 221A 2.1. FIG. HER2 [9₄₃₅] Specific CTLs can kill tumor cells.   (A): HER2 [9₄₃₅Using specific CTL as an effector, The following target cell lines were tested for lysis: 〇, HER2 [9₄₃₅Pulse with Was. 221A2.1; ●, not containing peptide. 221A2.1; Δ, SW40 3 (colon Ca, A2⁺HEL-2 / neu⁺); ▲, HT-29 (colon Ca, A 2^-HEL-2 / neu⁺). (B) Antibody demonstrated by cold target inhibition assay Protospecificity. HER2 [9₄₃₅] 10: 1 effector / target with specific CTL In the ratio of targets⁵¹The dissolution of Cr-labeled SW403 varies depending on the low-temperature target described below. Blocked at inhibitor / target ratio: (D) HER2 [9₄₃₅]so Pulsed. 221A2.1; ▲, unrelated A2.1 binding peptide (HBc_{18 -27} ). 221A2.1; ●, not containing peptide. 221A2. One. FIG. HEL2 [9_Five] Demonstration of tumor reactivity of specific CTL.   (A): HEL2 [9_FiveUsing specific CTL as an effector, Were tested for lysis of target cell lines: o, HEL2 [9_Five]. 221A2.1; ●, not containing peptide. 221A2.1; $, SW403 ( Colon Ca, A2⁺HEL-2 / neu⁺); 、, HT-29 (colon Ca, A2^- HEL-2 / neu⁺). (B): Antibody proved by cold target inhibition assay Protospecificity. HEL2 [9_Five] 10: 1 effector / specific CTL strain / In the target ratio⁵¹The dissolution of Cr-labeled SW403 varies depending on the following low-temperature targets: Were blocked at an inhibitor / target ratio of: 〇, HEL2 [9_Five] Russ. 221A2.1; ▲, unrelated A2.1-binding pepsid (HBc_{18-2 7} ). 221A2.1; ●, not containing peptide. 221A2.1 . FIG. Peptide analogs can induce tumor-reactive CTL.   (A): CEA [9_{twenty four}] M2V9-specific CTLs against the following target cell lines: Tested for its cytolytic activity: o, CEA [9_{twenty four}] Pulse on M2V9 did. 221A2.1; ●, not containing peptide. 221A2.1; Δ, SW4 03 (colonic Ca, A2⁺, CEA⁺); ■, KATO-III (gastric Ca, A2⁺, CEA⁺); 、, HT-29 (colon Ca, A2^-, CEA⁺).   (B) Antigen specificity demonstrated by cold target inhibition assay. CEA [9_{twenty four}] M At a 2: 1 effector / target ratio with the 2V9-specific CTL line⁵¹Cr Lysis of labeled SW403 is dependent on the low temperature target of various inhibitors / targets Blocked in ratio: ○, CEA [9_{twenty four}] Pulsed with M2V9. 221 A2.1; ▲, irrelevant A2.1 binding peptide (HBc_18-27) Pulsed . 221A2.1; ●, not containing peptide. 221A2.1. Description of the preferred embodiment   The term “peptide” typically refers to the amino acid alpha-amino group of adjacent amino acids. A series of peptides, one connected to the other, by a peptide bond to the rubonyl group In order to denote the residues, typically L-amino acids, "oligope" is used herein. Used interchangeably with "peptide".   An “immunogenic peptide” is a peptide in which the peptide binds to MHC alleles and reacts with CTL. A peptide that contains an allele-specific motif so that You. Thus, the immunogenic peptide binds to the appropriate class I MHC molecule and Inducing the response of cytotoxic T cells to the antigen from which the immunogenic peptide is derived Can be.   The term “residue” is used in an oligopeptide by an amide bond or a mimetic amide bond. Means amino acids incorporated or mimetic amino acids in .   The term “pretreatment growth factor” refers to the use of CTLs, peptides and CTLs Before incubating with growth factors used to expand, antigen presenting cells Mean added growth factor. The term “incubation growth factor” refers to AP CTLs are added together as C and are incubated together to expand the specific CTL. Means growth factors that are   In addition, the present invention provides a general strategy for determining the full immunological potential of a given antigen. Including. The results, and in combination with data derived from clinical samples, Conditions, such as the dominance and CTL epitopes associated with chronic viral infection and cancer. And sub-dominance can be determined this time. Similar to that detailed herein Experimental strategies have multiple mechanisms involved in the pathogenesis of infection, cancer and autoimmune diseases. We look forward to at least being able to facilitate clarification .   The present invention provides a novel method for identifying human CTL epitopes derived from tumor-associated antigens. An in vitro primary immunization assay and its application are provided. In one aspect In the present invention, the present invention relates to human PBMC in the presence of IL-7 and IL-10. With stimulation with peptide-pulsed dendritic cells. Using this protocol We report that gp100 melanoma-associated tumor antigen (G9₁₇₈, G10₁₇₇And G10₅₇₀ ) Were defined as three novel subdominant HLA-A2.1 restricted CTL epitopes I did. Following our dendritic cell priming in vitro immunization protocol, 1 Only six of the two potential subdominant epitopes have been tested to date So additional gp100 The derived epitope may also be identifiable. Experiments described herein The CTL epitope contained in a given tumor antigen was identified using the protocol of (Regardless of whether the CTL epitope is immunodominant or not) To take full advantage of the immunological potential of the tumor antigen itself.   The present invention also describes the role of immunodominance in specific areas of the anti-tumor CTL response. Provide a way to check. Some of the CTL epitopes recognized by cancer patients Are relatively weaker than recognized epitopes, for example, in acute viral infections Appear to bind to their MHC class I restrictors (Kawakami, et al., J. Immunol. . 154; 3961 (1995); Celis, et al. , Sem. Ca ncer Biol. 6: 329 (1995)). With these results, Some of the CTL reactivity directed against MHC class I binding epitopes Inactivated by education or peripheral resistance of the thymus And dominant tumor epitopes may in fact be relatively low binders It was suggested. In fact, in a recent overview we have found that pepti recognized by cancer patients Have an average affinity of 170 ± 52 nM (n = 2) for their corresponding MHC molecules. Found that it binds with Slightly lower (49 ± 15 nM (n = 15)) Affinity). Binding of 11-500 nM epitopes is dominant and subdominant Found in both groups (Tables II and IV) and the two highest Affinity binding peptide G9₁₅₄And G10₄₇₆(Table I) shows melanoma patients Corresponds to the immunodominant GTL epitope recognized by TIL. Furthermore, dominance The gp100 epitope is Elicit anti-melanoma CTL response in 10-36% of all lymphocyte cultures However, subdominant epitopes were less frequent (2-3% of culture) Induced CTLs that can kill.   In vivo and using both dominant and subdominant epitopes And induction of in vitro CTL results in a more intense, diverse immune response . A multivalent vaccine containing a mixture of epitopes from several TAAs, The need for complex mixtures of proteins or genes can be overcome. Subdominance Immunization using epitopes has also been used to direct the response of CTLs to specific conserved sequences. And thus prevent the emergence of tumor variants during the course of the disease Will be able to.   The present invention also provides for specificity for both dominant and subdominant epitopes. To induce tumor-specific CTLs from the blood of cancer patients. this These CTLs were approximately 1 × 10⁹To cells (data not shown) ) And still retain antigen specificity, and thus can be used for adoptive therapy. Can be. Convenient immunotherapy using peptide-primed CTL from blood collection (Since it does not require tumor biopsy) and the already promising current T Reliable (due to a high degree of specificity) alternative to IL-based immunotherapy (Rosenberg, et al., "J. Natl. Cancer." Inst. 86: 1159 (1994)).   The technique of the present invention is capable of eliminating, in part, target infected cells. Depending on the determination of the epitope recognized by Identify these epitopes One approach is to characterize subtypes of human class I MHC alleles. Identify all specific peptide motifs associated with a given disease . MHC class S antigens are encoded by the HLA-A, B and C loci. HLA- A and B antigens are expressed at approximately equal density on the cell surface, but HLA-C Is significantly lower (perhaps less than 10-fold). Each of these loci Has a number of alleles. Has defined MHC molecules, especially MHC class I molecules A large number of cells are known and are readily available. These cells are Use to identify specific allele-specific motifs associated with the target disease Can be.   Using the allele-specific motif, the amino acid sequence of the potential antigen target is then Known and desired antigens, particularly antigens associated with human viral diseases or cancers The T cell epitope from is determined. This general approach involves simultaneous continuation and Commonly assigned U.S. patent application Ser. No. 07 / 926,666 and U.S. Pat. / 027,146, which are incorporated herein by reference. In more detail.   Identification of potential epitopes on multiple target proteins in this way can be it can. Examples of suitable antigens include prostate specific antigen (PSA), hepatitis B core, surface And polymerase antigens (HBVc, HBVs, HBVp), hepatitis C antigens, Epstein-Barr virus antigen, melanoma antigen (eg, MAGE-1) Antigen of human immunodeficiency virus (HIV), anti-human papilloma virus (HPV) , Cytomegalovirus (CMV), herpes simplex virus (HSV), and And other oncogene products (c-Erb B, CEA, p53-breast / ovary) I do.   These approaches typically involve isolating peptides from specific MHC molecules. And sequencing the peptide to determine the relevant motif . See Busus et al. , "Scie nce "242: 1065 (1988) describes the conversion of MHC to acid from bound peptides. The elution method was described first. Subsequently, Rammensee and his co-workers (Falk et al., Nature, 351: 290 (19) 91) characterizes peptides bound to naturally processed class I molecules. Developed a new approach. Other investigators have reported that the eluted paper from type B class I molecules. Routine automated sequencing of peptides allows for more abundance in various HPLC fractions. Peptides (Jardetsky, et al., "Nature" 353: 32). 6 (1991) and by mass spectrometry, type A2.1 (Hunt, et al., " Science "225 1261 (1992). Succeeded. An overview of the characterization of naturally processed peptides can be found in Rtzs Immunol. Today "12: 447 (1991).   The definition of motifs specific for different class I alleles is based on the knowledge of the amino acid sequence. Allows identification of potential epitopes from antigenic peptides that have been established. Typically The identification of potential peptide epitopes was first determined using a computer. It is performed by scanning the amino acid sequence of the desired antigen for the presence of the motif. Then The sequence of the epitope is synthesized. The ability to bind to MHC class molecules may vary. In some methods, for example, purified class I molecules and radioiodinated peptides For example, using cells that express tide and / or empty class I molecules, For example, immunofluorescence staining and flow microfluorimetry, peptide-dependent class 1 Measured by assembly assays and inhibition of CTL recognition by peptide competition Is done. Another alternative described in the literature is the inhibition of antigen presentation (Sette, et al.   al. , "J. Immunol." 141: 3893 (1991), in v. In vitro assembly assays (Townsend, et al., Cell, 62: 285 (1990)), and mutated cells, such as RMA. S The FACS-based assay used (Melief, et al., "Eu t. J. Immunol. 21: 2963 [1991]).   Next, peptides that tested positive in the MHC class I binding assay were identified as i Ability of peptides to induce primary or secondary CTL responses in vitro Assay. For example, antigen presenting cells incubated with peptides Is assayed for the ability to induce a CTL response in a responder cell population. Can be. For secondary responses, antigen presenting cells are normal cells, for example, peripheral blood cells. It can be a nuclear cell or a dendritic cell (Inab, et al., "JE xp. Med. 166: 182 (1987); Boog, "Eur. J. Im. munol. 18: 219 [1998]).   It also carries a broad class I molecule with internally processed peptides Mutant mammalian cell lines lacking the ability, for example, cure, 319: 675 (1986); Ljunggren, et al. , "Eur. J. Immunol." 21: 2963-2970 (1991)), And human somatic T cell hybridoma, T-2 (Cerundolo, et al.) . Nature 345: 449-452 (1990)) and suitable humans. Cell lines transfected with the class I gene can When tested for the ability to induce an in vitro primary CTL response. Used conveniently for Those empty MH C cells have a higher density of MHC-peptide complexes on the surface of antigen presenting cells Therefore, it is preferable to induce a primary response. Other eukaryotic cell lines that can be used The lineage is based on various insect cell lines, such as mosquito larvae (ATCC cell line CCL125, 126, 1660, 1591, 6585, 6586), silkworm (ATTC CR) L8851), armyworm (ATTC CRL1711), moth (ATTC CC) L80) and a suitable human class I MHC allele record gene and human B_Two Drosophila transfected with the microglobulin gene (Dr. osophila) cell lines, including, for example, the Schneider cell line .   Once the appropriate epitope is determined, the motif required for MHC binding And an immunogenic peptide containing an epitope recognized by CTL. Immunogenic peptides may be produced synthetically or by recombinant DNA technology, or Alternatively, it can be isolated from natural sources, such as whole virus or tumor. Business As one will appreciate, immunogenic peptides are of various lengths and are neutral (uncharged). Can be in either form or salt form, and can be modified, e.g., by glycosylation. Contains no silylation, side chain oxidation, or phosphorylation, or any modifications thereof. Polypeptides as described herein. Must not destroy the biological activity of   Desirably, the peptide is as small as possible, but still Will retain substantially all of the activity. If possible, the peptide of the invention is Or optimized for 10 amino acid residues in length to reduce MHC class I molecules on the cell surface Binding, endogenously processed viral peptides or tumor cell peptides It may be desirable for the size to be balanced with the size.   The peptide having the desired activity may be optionally modified to provide certain desired attributes, For example, it provides improved pharmacological properties while binding to the desired MHC molecule. And substantially all of the biological activity of the unmodified peptide that activates the appropriate T cells It can be increased or at least retained. For example, the peptide Various changes, for example, substitutions, which can be conservative or non-conservative, Where such changes may have certain advantages in their use, such as , MHC binding can be provided. Conservative substitutions replace amino acid residues in organisms Replacing one with another chemically and / or chemically similar, eg one hydrophobic Replaces a residue with another or one polar residue with another You. Substitutions can be combined, eg, Gly, Ala; Val, Ile, Leu, Me. t; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; And Phe and Tyr. The effect of a single amino acid substitution is to replace D-amino acids. Can also be used to probe. Such modifications are well known in peptide synthesis techniques. Can be done using   The peptides of the present invention can be produced in a wide variety of methods. Those Due to the relatively short size of the peptide, the peptide is in solution or Can be synthesized on a solid support. Various automatic synthesizers are commercially available. It is available and can be used according to known protocols. For example See, Stewart and Young, "Solid. Phase Peptide Synthesis "2nd edition, Pierce C chemical Co. (1984) supra.   Thus, recombinant DNA technology can be used, where the immunogenicity of interest is Expression vector containing nucleotide sequence encoding peptide And transformed or transfected into a suitable host cell. And culture under conditions suitable for expression. These techniques are generally It is known in the field and is generally described in the following literature: Sambrook k et al. "Molecular Cloning, A Labora tory Manual "Cold Spring Harbor Press , Cold Spring Harbor, New York (1982) Which are incorporated herein by reference). One or more books Using the fusion protein comprising the peptide sequence of the invention, the appropriate epithelium of T cells Can also be presented.   The CTL is then activated ex vivo using an immunogenic peptide. The ex vivo therapy and the pharmaceutical composition thereof of the present invention can be used for viral infections, immune disorders. And useful for treating mammals, especially humans, for treating and / or preventing cancer It is. Examples of diseases that can be treated using the ex vivo therapy of the invention Means prostate cancer, hepatitis B, hepatitis C, AIDS, kidney cancer, cervical cancer, lymphoma, CM V, condyloma acuminatum, including breast and ovarian cancer, colon cancer, lung cancer and HSV You.   Detection or removal of first signs of viral infection or tumor for therapeutic use Start therapy at or within a short time after diagnosis in the case of acute infection Should. Then at least until the symptoms substantially disappear and then During a period of time, the level of CTL is increased. In chronic infection, carry the dose, It may then be necessary to increase the dose.   Treatment of an infected individual with the methods of the invention results in infection in the acutely infected individual. Can be dissipated faster. Against progressive chronic infection For sensitive and susceptible (or susceptible) individuals, these methods It is effective for preventing the evolution from infection to chronic infection. Susceptibility of infection If identified before or during, target the compositions to them and The need for administration to a population can be minimized.   The method of the present invention is used to treat chronic infections and to stimulate the immune system to carry Can be used to eliminate virus-infected cells in the cell.   Antigen-presenting cells (APC) carrying an appropriate immunogenic peptide in tissue culture Incubating the patient's CTL precursor cells (CTLp) with the source Thus, ex vivo CT against a specific pathogen (infectious agent or tumor antigen) An L response is induced. CTLp is activated, matures and spreads to effector CTL After a large, suitable incubation time (typically 3-12 weeks), cells are infected. Injected back into the patient, where the cells are specific target cells (infected cells or tumor cells) Will destroy. Infusion of cells into a patient can be a T cell growth factor, such as Interleukin-2 (IL-2). Specific cytotoxic T cells Cultivation of stimulator cells to optimize in vitro conditions for the development of Is maintained in a suitable serum-free medium, wherein the serum-free medium comprises one or more growth factors. For example, IL-2, IL-4, IL-7 and IL-12. .   Peripheral blood lymphocytes following venipuncture or leukapheresis of a normal donor or patient Is conveniently isolated and used as a source of CTLp response factors. Especially secondary C In one embodiment for the TL response, the appropriate APC is Incubate with 100 μM peptide under appropriate culture conditions for 4 hours. Then Peptide loading APC with response factor cell populations and in vitro optimized culture conditions Incubate for 7-10 days. For the primary CTL incubation, APC expressing empty MHC is used to stimulate native CTLp. this In some cases, CTLs are stimulated more frequently (1-2 times).   Radiolabeled target cells, both specific peptide-pulsed targets and pepti Endogenous processing of the relevant virus or tumor antigen from which the derived sequence was derived The culture can be assayed for the presence of CTL that kills the target cells expressing the morphology. Thus, positive CTL activation can be measured.   The CTL specificity and MHC restriction of a patient is known in many areas in the art. Can be measured by the method described in For example, the HLA phenotype of the donor CTL Different peptide-bearing target cells expressing shared human MHC class I alleles By testing against, the CTL limit can be measured. MHC binding Peptides that test positive in the assay and produce a specific CTL response are immune Identified as an epidemiological peptide.   As mentioned above, in vitro induction of CTLs is based on allele specific on APCs. Requires specific recognition of peptides that bind to specific MHC class I molecules. APC The number of specific MHC / peptide complexes may vary, especially during the primary immune response. Determine the level of CTL stimulation. Low amount of peptide / MHC complex per cell Is sufficient to make the cells susceptible to lysis by CTL, or Sufficient to stimulate a secondary CTL response, but the success of CTLp during the primary response Significant activation requires a significantly higher number of MHC / peptide complexes You.   Mutant cell lines capable of expressing empty MHC As it is not present for all human MHC alleles, the endogenous MHC-related peptide Is removed from the surface of the APC and the resulting empty MHC molecules are then It is advantageous to use techniques to carry the native peptide. Non-transformed (non Tumorigenic), uninfected cells, and, preferably, autologous cells of the patient to AP Use as C was directed to the development of ex vivo CTL therapy Desirable for designing CTL induction protocols. The present invention relates to endogenous MHC Strip the peptide from the surface of the APC or incubate at low temperature. (37 ° C. → 26 ° C.), and then carry the desired peptide, Empty class I MHC (which then carries the appropriate immunogenic peptide A new method for generating the same.   A stable MHC class I molecule is a complex of trimers formed from the following factors: 1) a peptide of usually 8 to 10 residues, 2) its α1 and α2 domains A transmembrane polymorphic protein heavy chain having a peptide binding site, and 3) non-covalent binding Associated non-polymorphic light chain, β_TwoMicroglobulin. Bound peptide And / or β from the complex_TwoWhen microglobulin is dissociated, MH C-Class I molecules become non-functional and unstable and undergo rapid degradation at 37 ° C. Cheating. Almost all MHC class I molecules isolated from PBMCs Has an endogenous peptide attached to it. Therefore, A for primary CTL induction The first step in making PCs is to degrade before exogenous peptides can be added. Or bound to MHC class I molecules on APC without causing cell death To remove all endogenous peptides.   Two possible ways to generate free MHC class I molecules are to increase the culture temperature to 37 ° C. From overnight to 26 ° C., peptide-free M Express HC class I and convert endogenous peptides to cells using mild acid treatment Is to strip from. Mild acid treatment converts previously bound peptides into cells Release into the outside environment to release new exogenous peptides into empty MHC class I Attach to the molecule. A low temperature overnight incubation at 26 ° C. Metabolic rate can be slowed, generating stable empty class I molecules The class I molecules can then efficiently bind to the exogenous peptide. Also, Cells that do not actively synthesize MHC molecules (eg, resting PBMCs) can Will not produce large quantities of empty surface MHC molecules.   By severe acid stripping using trifluoroacetic acid, pH 2, or Alternatively, acid denaturation of immunoaffinity purified class I-peptide complexes Thus, peptide extraction is achieved. These methods are not feasible for CTL guidance Noh. This is because at the same time as removing endogenous peptides, APC's ability to live It is important to preserve optimal metabolic state, which is critical for antigen presentation Because. Identify endogenous peptides and identify tumor-associated T cell epitopes A mild acid solution at pH 3 such as glycine or citrate-phosphate Buffers have been used. This treatment is particularly effective, as MHC class I molecules Only is destabilized (and the associated peptide is released), but other Surface antigens remain intact and include MHC class II molecules (Suguwara , S.P. Et al. , "J. Immunol. Meth." 100: 83 (1 987)). Most importantly, treatment of cells using a mild acid solution Does not affect the ability to live or metabolic status. Stripping of endogenous peptides Occurs in 2 minutes at 4 ° C., so the treatment of mild acid is rapid and Carry peptide After holding, the APC is functional. Utilizing this technology in the present invention, Generate peptide-specific APCs for generation of pro-specific CTL. The resulting APC is It is efficient in inducing peptide-specific CTL.   Typically, prior to incubation of the APC with CTLp to be activated, In the subsequent response, it is carried on a human class I molecule to be expressed on the surface of the APC. Antigenic peptide in sufficient amounts to become APCs or stimulator cells Added to culture. In the present invention, a sufficient amount of the peptide is sufficient for each stimulating factor. Approximately 200 or more class I molecules carrying peptides to be expressed on the surface of cells It is the amount to be done. Preferably, the cells of the stimulating factor are 5-100 μg / ml pepti Incubate with   The resting or precursor CTLs are then cultured in culture with the appropriate APC and CTLs. Incubate for a time sufficient to activate. CTL is antigen-specific Activated by law. The ratio of precursor CTL / APC may vary from individual to individual, In addition, variables such as the compliance of an individual's lymphocytes with culture conditions and disease May depend on symptoms or other conditions used in physiotherapy for the described treatment . However, preferably, the CTL: APC (ie, the response / stimulator) ) The ratio ranges from about 10: 1 to 100: 1. Therapeutically usable or effective number Maintain the CTL / APC culture for the long period of time necessary to stimulate You can have.   Activated CTLs can be effectively removed from APCs using one of a variety of known methods. Can be separated. For example, APC, pepti carried on stimulator cells Or a monoclonal antibody specific for CTL (or a segment thereof) The body can be used to bind to their appropriate complementary ligand. Next In, antibody labeling The transformed cells are removed from the mixture by any suitable means, such as well-known immunoprecipitation or immunoassay. It can be extracted by a method.   An effective, cytotoxic amount of activated CTL can be used in vitro and in vivo. o between use, as well as the amount and quantity of cells that are ultimate targets for these killer cells And can change with type. This amount also varies depending on the patient's condition. And should be determined by the practitioner considering all appropriate factors. . However, preferably, about 1 × 10⁶~ About 1 × 10¹²And more preferably about 1 × 10⁸~ About 1 × 10¹¹And even more preferably about 1 × 10⁹~ About 1 × 10^TenActivity Sexed CTL was used on adult humans, compared to about 5 × 10⁶~ 5 × 10⁷ Cells are used in mice.   As described above, after the activated CTLs have been collected from the cell culture, the treated individual Administer cells. However, unlike the physiotherapy of other current treatments, the present invention Uses a cell culture system that does not contain transformed cells or tumor cells. It is important to be careful. Thus, antigen presenting cells and activated CTL complete If good separation is not achieved, it may be related to the administration of small numbers of stimulator cells. No known inherent dangers exist, but administration of mammalian tumor-promoting cells is extremely high Can be dangerous.   One aspect of the present invention is an AP generated by the in vitro technology of this application. C is used for therapy against CTL in vivo. In this aspect , APCs remove native antigenic peptides and toxins (eg, lysine). A-chain or Pseudomonas toxin) Cells of a patient (eg, peripheral blood cells). Then re-direct the APC to the patient Where the endogenous CTL specific for the antigenic peptide binds to the APC Will. Coupling toxin is harmful Activated CTL, an activity that stimulates graft rejection after binding to APC Will kill sex CTL. Such a directed CT L killing is a CTL-mediated rejection of tissue transplants and autoimmune diseases. It is widely effective for treating cases. The regimen of treatment depends on the specific disease to be treated and And will depend on the judgment of the treating physician.   Methods for reintroducing cellular components are known in the art and are described in U.S. Pat. No. 4,844,893 (Honsik, et al.) And US Pat. No. 690,915 (Rosenberg), which are hereby incorporated by reference. (Incorporated as part of the handbook). For example, activated CT Administration by intravenous infusion of L is appropriate.   Against CTL precursors (CTLp) selected from viruses and neoplastic diseases Identification of peptide sequences that provide a potential immunogenic potential is Constant immunotherapy, for example, peptide-based vaccines and antigen-specific adoptive Will promote the development of vesicle therapy.   Pan DR-binding peptide of the present invention and pharmaceutical composition and vaccine set thereof The composition can be administered to a mammal, especially a human, for prophylactic and / or therapeutic purposes. it can. Using Pan DR Peptide Against Other Immunogens Administered Peptide Immune response can be enhanced. For example, to treat and treat viral infections and cancer To prevent and / or prevent a CTL / Bread DR mixture can be used. Also, immunogens that induce an antibody response can be used. Bread DR peptide Examples of diseases that can be treated using immunogenic mixtures of and other immunogens are , Prostate cancer, hepatitis B, hepatitis C, AIDS, kidney cancer, cervical cancer, lymphoma, CMV And the condyloma Include.   Pan DR-binding peptides also include species that include unwanted T cell reactivity. Can be used to treat various conditions. Using pan DR binding peptide Examples of diseases that can be treated for use include autoimmune diseases (eg, rheumatoid arthritis). Gusset, multiple sclerosis, and myasthenia gravis), allograft rejection, Rugie (eg, pollen allergy), Lyme disease, hepatitis, LCMV, hemolytic streptococci Includes bacterial endocarditis, or glomerulonephritis, and food sensitivity.   In therapeutic applications, immunogenic compositions or pan DR binding peptides of the invention If you already have cancer, an autoimmune disease, or are infected with the virus in question To any individual. Pan-DR-binding disease during incubation or acute phase Treat with peptide or immune complex, separately or together with other treatments as appropriate .   Effective therapeutic CTL response to viral or tumor antigens in therapeutic applications Withdraws and cures or at least partially inhibits symptoms and / or complications A composition comprising an immunogenic composition in an amount sufficient to administer to a patient . Similarly, cure or at least partially suppress the symptoms of the disease and its complications Administering a composition comprising a pan DR binding peptide in an amount sufficient to I do. The appropriate amount to achieve this is defined as "therapeutically effective dose" Is done. Effective amounts for this use include, for example, peptide compositions, methods of administration, therapeutics. Disease stage and severity, patient weight and general health, and It depends on the judgment of the teacher.   A therapeutically effective amount of an immunogenic composition of the invention will generally be sufficient for initial immunization. About 1.0 μg per 70 kg patient (for therapeutic or prophylactic administration) To about 10,000 μg, usually about 100 to about 8000 μg, preferably about 200 to About 6000 μg of peptide Range. Following these administrations, specific CTL activity in the patient's blood Measurements can take weeks to months, depending on patient response and symptoms About 1.0 μg to about 1000 μg of peptide enhancement according to the enhancement regimen. Administration is performed.   The compositions of the present invention generally have a significant disease state, i.e., life-threatening or potential. It must be kept in mind that it will be used in potentially life-threatening situations. This In such cases, the minimization of external substances and the relative non-toxic nature of the complex In view of the fact, it is possible to administer a substantial excess of these compositions, and The treating physician may find it desirable.   For prophylactic use, administration should be to the at-risk group. For example, malaria, For protection against hepatitis or AIDS, the composition of the present invention is administered prophylactically, This can be achieved by increasing the immune capacity. First signs of the disease Or at the time of tumor detection or surgical removal, or in case of acute infection Shortly after disconnection, therapeutic administration can begin. Then at least the symptoms The dosage is increased until substantially disappeared and for a period thereafter. Chronic infection In some cases, it may be necessary to carry a dose and subsequently increase the dose.   When an infected individual is treated with a composition of the present invention, It can speed up the resolution of the infection. Susceptibility (or To prevent the evolution of acute to chronic infections for individuals. Thus, the composition is particularly effective. Susceptible individuals are identified before or during infection In some cases, the compositions may be targeted to them, for example, as described herein. And the need for administration to a larger population can be minimized.   Also, the peptide mixture or conjugate may be used to treat chronic infections. To stimulate the immune system to eliminate virus-infected cells in the carrier Can be used for To effectively stimulate the response of cytotoxic T cells It is important to provide a sufficient amount of the immunopotentiating peptide to the formulation and mode of administration. It is important. Thus, for the treatment of chronic infections, a typical dose is 70 kg About 1.0 μg to about 5000 μg per dose per patient, preferably Ranges from about 5 μg to about 1000 μg. Effectively immunize potential individuals Immunization administration and subsequent established intervals for an extended period of time, such as For example, administration may need to be increased in the first four weeks. Smell in case of chronic infection Whether at least clinical symptoms or laboratory tests have ruled out the virus infection, Alternatively, dosing until indicated to be substantially extinct, and for a period thereafter Should continue.   Pharmaceutical compositions for therapeutic or prophylactic treatment may be parenteral, topical orally or Intended for local administration. Typically, the pharmaceutical composition is administered parenterally, for example, intravenously. Administered intra-, subcutaneously, intradermally, or intramuscularly. For ease of administration, the invention The vaccine composition is particularly suitable for oral administration. Therefore, the present invention is not Of the peptide or conjugate dissolved or dispersed in a carrier, preferably an aqueous carrier. A composition for parenteral administration comprising a solution is provided. Various aqueous carriers, e.g. For example, water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic Acids and others can be used. These compositions are conventional, well-known It can be sterilized by bacterial techniques or can be sterile filtered. Raw The solution can be packaged for immediate use or lyophilized. The resulting, lyophilized preparation is combined with a sterile solution before administration. The composition is a pharmaceutical Ancillary substances that are acceptable, for example, approximate physiological conditions Substances required to perform, such as pH and buffering agents, tonicity adjusting agents, Lubricants and others, for example, sodium acetate, sodium lactate, sodium chloride , Potassium chloride, calcium chloride, sorbitan monolaurate, triethylamido Oleate, and others.   The concentration of the CTL stimulating peptide of the present invention in a pharmaceutical formulation can vary widely. Yes, ie less than about 0.1% by weight, usually about 2% by weight or at least double % To as high as 20% by weight or 50% by weight or higher Depending on the particular mode of administration chosen, primarily fluid volume, viscosity Degree, and others.   In addition, the peptides and complexes of the present invention can be administered via liposomes. Wear. Liposomes target the complex to specific tissues, such as lymphoid tissues. Or selectively target infected cells, and It serves to increase the half-life of the composition. Liposomes include emulsions, foams, micelles , Insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and others. this In these preparations, the peptide to be delivered is used alone as part of the liposome. Contaminates or binds to dominant receptors, eg, in lymphoid cells Combined with a molecule that binds to the CD45 antigen, e.g. Or in combination with other therapeutic or immunogenic compositions . Therefore, a liposome enriched with the desired peptide or complex of the present invention is prepared. Can be directed to lymphoid cells, where liposomes are then selected Deliver the therapeutic / immunogenic peptide composition. Liposomes used in the present invention Are standard vesicle-forming lipids, which are generally neutral and negatively charged phospholipids and And sterols, for example Including resterol). The choice of lipids will generally be Depends on somal size, liposome acid instability and stability in the bloodstream You. Various methods for producing liposomes are available and are described, for example, in the following documents: Szoka, et al. , "Ann. Rev. Biohys. B ioeng. 9, 467 (1980); U.S. Pat. No. 4,235,871, U.S. Pat. No. 4,501,728, U.S. Pat. No. 4,837,028 and U.S. Pat. No. 5,019,369 (incorporated herein by reference).   Ligands to be incorporated into liposomes to target immune cells For example, antibodies or antibodies specific for cell surface determinants of the desired immune system cells. Can be included. Lipo containing peptide or complex Sosome suspensions can be administered intravenously, locally, topically, and otherwise. The dosage may vary, among others, depending on the method of administration, the complex delivered, and It changes according to the stage of the disease.   Also, one or more PADRE peptides and one or more C Encoding a TL epitope or a polypeptide containing an antibody-derived epitope DNA or RNA into a patient to transform the nucleic acid-encoded polypeptide An immune response can be induced. Wolff, et al. , "Sci 247 ": 1465-1468 (1990) describes the genetics encoded by nucleic acids. It describes the use of nucleic acids to produce offspring expression.   For solid compositions, conventional non-toxic solid carriers can be used. Solid charge The body can be, for example, pharmaceutical grades of mannitol, lactose, starch, stearic acid Magnesium, sodium saccharin, talc, cellulose, glucose, Loin, magnesium carbonate , And others. Non-toxic pharmaceutically acceptable compositions for oral administration Is a commonly used excipient, such as the aforementioned carriers, and generally 10-95%, More preferably 25-75% of the active ingredient, ie one or more of the invention's It is formed by mixing the complex.   For aerosol administration, the peptide is preferably in finely divided form in a surfactant or And with the propellant. Typical percentages of the complex are 0.01-20% by weight %, Preferably 1 to 10% by weight. Surfactants, of course, are non-toxic, Preferably it must be stable in the propellant. Representative examples of such surfactants Are fatty acids containing 6 to 22 carbon atoms, such as caproic acid, octanoic acid , Lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, ores Of the thearic and oleic acids with aliphatic polyhydric alcohols or their cyclic anhydrides Ester or partial ester. Mixed esters, for example mixed or natural Glycerides can be used. Surfactant is 0.1 to 20% by weight of the composition %, Preferably from 0.25 to 5% by weight. The balance of the composition is usually a propellant. You. In addition, the carrier may be optionally used for intranasal delivery, for example, with lecithin. Can be included.   In another aspect, the present invention provides an immunogenic amount of an immunogenic amount as an active ingredient. Pan DR peptide or CTL pan DR peptide conjugate described herein Related vaccines. One or more complexes, including human In the host to be used, either bound to its own carrier, or It can be introduced as a polymer or a heteropolymer. Such poly Mers have the advantage of increasing the immunological response, and If different peptides are used for Introduce antibodies and / or CTLs that react with different antigenic determinants of tumor cells Have the additional ability to Useful carriers are well known in the art, and For example, thyroglobin, albumin, for example, bovine serum albumin, injured Wind toxoid, polyamino acids such as poly (lysine: glutamic acid), type B liver Influenza virus core protein, hepatitis B virus recombinant vaccine and others Include. Vaccines may also contain physiologically acceptable (acceptable) diluents, such as Contains water, phosphate buffered saline, or saline, and is more typically Specifically, an adjuvant can be included. Adjuvants, for example, incomplete froi Adjuvant, aluminum phosphate, aluminum hydroxide, or alum, It is a substance well known in this field. And, as mentioned above, The light peptide is converted to a lipid, eg, P_ThreeCTL response by binding to CSS Can be primed. Injection, aero, as described herein Immunization with the peptide composition via sol, oral, transdermal, or other route The primary immune system produces large amounts of CTL specific for the desired antigen Respond to the vaccine, and the host is at least partially immune to subsequent infection. Be epidemiological or resistant to the development of chronic infections.   Are or are susceptible to a disease, for example, a viral infection or cancer Otherwise, a patient at risk of disease is given a vaccine containing the pan DR peptide of the present invention. Administration of an anti-tin composition to elicit an immune response to the antigen and thus the patient's own Enhances the ability of the immune response. Such an amount is an “immunologically effective dose”. Is defined as In this use, the precise amounts again depend on the patient's health and weight. , Depending on the mode of administration, the nature of the formulation, and the like, but is generally g to about 5000 μg / 70 kg, more usually It ranges from about 10 μg to about 500 μg / 70 kg body weight.   In some cases, a vaccine of the peptide of the invention is administered with the virus in question, Vaccines and combinations induce neutralization of antibody responses to the envelope antigen of irs May be desirable. For example, a PADRE peptide is Can increase the potency or broaden the population You. A suitable hepatitis vaccine that can be used in this method is Reco mit-Kline).   For therapeutic or immunization purposes, the peptides of the invention may also be used to attenuate viral hosts. For example, it can be expressed by vaccinia or fowlpox. This approach H is a vector that expresses a nucleotide sequence encoding the peptide of the present invention. And using vaccinia virus. Acute or chronic infection When introduced into an infected host or into an uninfected host, recombinant vaccinia virus Express the immunogenic peptide, thereby eliciting a CTL response in the host. Immunity Vaccinia vectors and methods useful in the Patent No. 4,722,848 (incorporated herein by reference) It is described in. Another vector is BCG (Bacille Calmette).   Guerin). The BCG vector is described by Stover et al. , "N atature 351: 456-460 (1991) (this is by reference. Which are incorporated herein by reference. Therapeutic administration of the peptides of the invention Alternatively, a wide variety of other vectors useful for immunization, such as Salmonella Typhi (Salmonella typhi) vectors and others It will be apparent to those skilled in the art from the description in the detailed description.   Moreover, CTLs can be derived using the antigenic complex ex vivo. Wear. The resulting CTLs do not respond or respond to other favorable forms of therapy Is chronic in patients who do not respond to the therapeutic peptide vaccine approach Can be used to treat infections (viral or bacterial) or tumors You. In tissue culture, patient CTL precursor cells (CTLp) were converted to antigen-presenting cells (AP C) by incubating with the source and the appropriate immunogenic peptide Ex vivo CTL response to specific pathogens (infectious agents or tumor antigens) The answer is guided. CTLp is activated, matures and becomes effector CTL After a suitable incubation time (typically 1 to 4 weeks) for expansion, the cells are infected. Injected back into the patient, where the cells have their specific target cells (infected cells or Tumor cells).   Peripheral blood monocytes (PBMC) from normal volunteers in vitro Class I MHC binding for the ability to elicit a CTL response using We have previously described techniques for screening peptides (Celis, et al.   al. , "Proc. Natl. Acad. Sci." (USA) 91:21. 05 (1994)). This method utilizes CD8 purified lymphocytes as a source of CTLp. And Staphylococcus aureus Prepared by activating PBMC with Cowan-I (SAC-1), Peptide-pulsed antigen presenting cells (APC) were utilized. According to this protocol And we and other groups have identified several new tumor-specific CTL epitopes. Could be identified. HLA-A2.1 binding from hepatitis B virus (HBV) In an experimental model system using an ionic peptide, this approach would be peptide-pulsed. Very important in inducing a primary CTL response to target cells But only a small amount (about 10%) of the resulting CTL is produced naturally. We have observed that targets that process antigen can be killed. A place In some cases, peptide-reactive CTLs recognize endogenously processed antigens. That the interaction of the CTL target cells could be attributed to low affinity (Wentworth, et al., "Molec. Immunol.") 32: 603 (1995)).   In order to optimize the in vitro CTL printing technique, Using dendritic cells (DCs) and participating in CTL responses We also assessed the addition of IL-7, IL-10 and IL-12 ( Celis, et al. , "Proc. Natl. Acad. Sci. (US A) "912105 (1994); Alderson, et al. , "J.E. xp. Med. 173: 923; Kos, et al. "Eur. J. Im munol. 22: 3183 (1992); Chen, et al. "J. Immunol. 147: 528 (1991); Yang, et al. , " J. Immunol. 155: 3897 (1995); Ferrari, et.   al. , "Clin. Exp. Immunol." 101: 239 (1995). ); Mehrotra, et al. "J. Immunol." 151: 24. 44 (1993), Mehrotra, et al. "J. Immunol. 154: 5093 (1995); Gatey, et al. , "Cell I mmunol. 143: 127 (1992); Chouaib, et al. , "Proc. Natl. Acad. Sci. (USA)" 91: 12659 ( 1994); Fallarino, et al. , IJ. Immunol. "1 56: 1095 (1996). Primary lymphocyte culture Antigen-specific CTLs that can recognize naturally processed antigens from In effect, DC is actually more effective than SAC-1 generated APC. Our results show that Furthermore, instead of IL-12, IL-7 and IL-7 Using -10 greatly increases the DC's ability to trigger these CTL responses. Will be strengthened. Example   The following examples are illustrative, but do not limit the invention. "Materials and methods" Synthetic peptide   Peptides were purchased from Applied Biosystems. ems) equipment (Foster City, CA, USA). simply In particular, after removal of the α-amino-t-butylcarbonyl protecting group, phenylamine was removed. Preformed symmetrical anhydride in 4 fold excess of cetamidomethyl resin restricted peptide (Arginine, histidine, asparagine, and glutamine 1 hour in dimethylporumamide with cibenzyltriazole ester) I did. Arginine, histidine, asparagine, glutamine and histidine For coupling residues, the coupling step was repeated to obtain high coupling efficiency. Suitable Cleavage of the peptide by treatment with hydrogen fluoride in the presence of the appropriate scavenger Let go. Synthetic peptides by reversed-phase high-pressure liquid chromatography (RV-HPLC) The tide was purified. The purity and identity of the peptide were It was routinely> 95% as determined by precipitation and mass spectrometry. MHC binding assay   Peptide binding assays for purified HLA-A2.1 molecules were previously described. Measured as done. Rupert et al. , “Cell” 74:92 9 (1993); Sette et al. , "Molec. Immunol. 31: 813 (1994). Briefly, this assay uses detergent solubilized M Based on inhibiting the binding of the radiolabeled standard peptide to the HC molecule. For example, the standard peptide used for HLA-A2.1 binding studies was FLPSDY FPSV (HBC_18-27), From hepatitis B virus core (nucleoprotein) antigen to C TL epitope, positions 18-27 (Bertolletti, et al., "J. . Virol. 67: 2376 (1996). Peptide MAGE3₁₆₁(EV DPIGLY) is a CTL episode recognized in the context of the HLA-A1 allele. Corresponding to the toup (Celis, et al., Proc. Natl. Aca d. Sci. (USA) "91: 2105 (1994); Gaugulet, et.   al. , "J. Exp. Med." 179: 921 (1994). Peptide G 9₂₈₀(YLEGPPVTA) is recognized by HLA-A2.1 melanoma patients Corresponding to a CTL epitope from the melanosome gp100 molecule (Cox e t al. , "Science" 264: 716 (1994); Kawakawa. i, et al. , "J. Immunol." 154: 3961 (1995)). . The standard peptide for the A68.2 binding assay was FTQAGYPAL. Was.   Standard peptide is prepared by chloramine T method¹²⁵I (ICN, CA, CA) -Iodinated). Almost 15% of the bound peptide (almost HLA-A2.1 concentration (in the range of 10 nM) was determined in the inhibition assay. Used. Various doses of the test peptide (1.0 μM to 1 nM) were added to Protea -Zeinhibita -And 1 nM cocktail in the presence of β2-microglobulin at 5 nM At room temperature together with a radiolabeled standard peptide and HLA-A2.1 molecule For two days. At the end of the incubation period, M The% HC bound radioactivity was determined by gel filtration and 50% of the binding of the labeled peptide Concentration of the test peptide that inhibits₅₀) Was calculated. Cell lineage   HLA-A2.1 gene is replaced with HLA-A, -B, -C null mutant human B lymphocyte Blast cell line. 221 by transfection. 221 (A 2.1) A cell line was generated (Celis, E. et al., "Proceed" edings of Natural Academy of Science s United State of America "91: 2105-21 09 (1994)). Flow cytometry of binding of anti-class I MHC antibodies Allows. 221 (A2.1) cells are cells that normal lymphoblasts Expressing a single copy of the same HLA allele at a chromosomal location It was shown to express as much as 0% of cell surface HLA-A molecules. . 221 ( A2.1) Cells are prepared by flow cytometry using anti-class I MHC-specific antibodies. About 50-100% of normal lymphoblast cell expression as measured by tree Expresses frequently cell surface HLA-A molecules. As described (Went worth, et al. , "Molec. Immunol." 32: 603 ( 1995)) a gene encoding hepatitis B virus core (nucleoprotein) antigen . 221 (A2.1) cell line was transfected.   Steimlin EBV-LCL (HLA-A1 / 1, B8 / 8) Test CTL recognition of peptides in the context of HLA-A1 molecule using did.   The HLA-type melanoma cell line used in these studies (624mel (A2 . 1, A3, B7, B14, gp100⁺); A375mel (A1, A2.1) , B17, gp100^-); 938mel (A1, A24, B7, B8, MAG E-3⁺); 888 mel (A1, A24, B52, B55, MAGE-3)^-) Is Previously described. Kawakami, et al. "Proc. Natl. Acad. Sci. 91: 6458 (1994); Kawakami, et. al. , "J. Immunol." 154: 3691 (1995). Colon adenocarcinoma Cell lines SW403 and HT-29 were transferred to American Type Culture Collection. (American Type Culture Collection) ) (Rockville, Maryland) and as recommended by the manufacturer Maintained in culture. The gastric cancer cell line KATO-III was purchased from Japan Cancer Research Bank ( Japan Cancer Research Resources Ba nk). All tumor lines were treated with antibiotics and 10% (v / v) F 400 μg / ml G41 for BS (and .221 (A2.1) cell line 8) maintained in culture using RPMI-1640 medium supplemented. Black Tumor colon and gastric cancer cells were treated with 100 U / ml γ-IFN (Genzyme) for 3 hours. After 48 treatments at 7 ° C, their susceptibility to lysis by CTL was tested. Tested. In all cases, cells treated with γ-IFN produced MHC class I development. Increased current level 2-3 times (unpublished). γ-IFN treatment is CTL-mediated cytotoxicity Although the level of harm was increased, untreated melanoma cells were still recognized by CTL ( Data not shown).   Expression of MAGE2 and MAGE3 was determined by RT-PCR as described. (De Smet, C. et al., "Immunogeneti"). cs "39: 121-129 (1994); Gaugler, B .; et al. , "J. Exp. Med." 179: 921-930 (1994); Zakut. , R.A. et al. , "Cancer Research" 53: 5-8 (19 93)). Furthermore, using specific monoclonal antibodies by immunohistochemistry, MAGE3 protein expression was measured (Kocher, T. et al., " Cancer Research 55: 2236-2239 (1995)). Monoclonal antibodies CEMO10 (Takara Shuzo, Japan) and c-ne, respectively u Ab-5 (Oncogene Science, Unionde, NY) CEA and HEL2 / neu by cytofluorimetric analysis using Was measured. Purification of peripheral blood mononuclear cells   Peripheral blood mononuclear cells (PBMC) for venipuncture or leukocyte export from HLA-type patients More (depending on the initial amount of CTLp required) and Ficoll-Pa Purified by gradient centrifugation using que (Pharmacia). Typically , 1 × 10⁶Of PBMC can be obtained from 1 ml of each peripheral blood, or A typical leukocyte export method is 1 to 10 × 10^TenLive up to PBMC be able to.   For example, HLA-A2.1⁺Peripheral from normal volunteers and melanoma patients Blood mononuclear cells (PBMCs) were prepared by Ficoll-Paque (Pharmacia, D.). (Piscataway, D.J.) from the product of leukocyte export. Also Diagnosed with cutaneous melanoma PBMC were obtained from a 51-year-old male patient who had been rejected. 18 out of 43 nodes Was found to be positive for tumor cells.   The isolated and purified PBMC is combined with an appropriate amount of synthetic peptide (HLA-binding mochi). (Containing the sequence of the antigen and the antigen of interest) Co-culture with an appropriate number of APCs that express empty MHC molecules. You. PBMC is usually 1-3 × 10⁶Medium in cells / ml, eg, RPMI -1640 (with autologous serum or plasma) or serum-free medium AIM-V (G ibco). In vitro generation of dendritic cells   APC can be 1 × 10, depending on the type of cells used.^Four~ 1 × 10⁶Cells / ml Use in the concentration range. Possible APC sources include: Autoro Gas PBMC, SAC-I activated PBMC, PHA blasts: P as described Autologous dendritic cells (DC) isolated from BMC and purified (Inaba, et al. , "J. Exp. Med.", 166: 182 (1987)); And mutant and genetically engineered mammalian cells, such as patient Allele H Suitable MH expressing an "empty" HLA molecule that is syngeneic to the LA type Mouse RMA-S cell line transfected with C gene or human T2 cells Cell line. APCs containing empty HLA molecules are likely to contain An empty MHC molecule is more likely than an MHC molecule occupied by a peptide. A potent inducer of the CTL response because it can be so easily associated Are known to be children (DeBruijin, et al., "Eur. J. Immunol. 21: 2963-2970 (1991)).   Dendritic cells (DC) from tissue culture were used to trigger a CTL response . Dendritic cells (DCs) are generated from the monocyte-rich cell fraction as described ( Tsai, et al. , "Cell Immunol." 144: 203 (1 992)) or adhere to plastic (at 37 ° C in serum-containing medium) 2 hours). Briefly, PBMC is converted to multi-stage percoll ( Percoll) gradients (30, 40, 50.5 and 70%) and Monocyte-rich populations were collected at 40-50.5% interface in the light density fraction . Washed monocytes containing DC precursors were prepared as described in Romani, et al. , "E xp. Med. 18083 (1994); Sallusto, et al. , As described in “J. Exp. Med.” 179: 1109 (1994). In addition, 50 ng / ml GM-CSF and 1,000 U / ml IL-4 (both Was obtained from R & D Systems, Minneapolis, MN) 5 × 10^FiveCells / ml in complete medium (see below) Nourished. The adherent monocyte population was treated in the same way. After 7 days, DC typical Cells displaying cell surface markers (CD3^-, CD14^-, CD88^-, HLA -A, -B, -C^HI, HLA-DQ⁺; Data not shown).   "Fresh" DCs were isolated directly from the pheresis product as described (Ts ai, et al. , "J. Clin. Invest." 82: 1731 (19 88)). Briefly, monocytes and DCs were first converted to Percoll gradient as described above. And most of the monocytes were applied to plastic for 2 hours at 37 ° C. Removed by deposition. Single-step Metrizamide gradient By eliminating contaminating monocytes and lymphocytes using The DC fraction released from the substrate was further purified. 50-70% of the resulting cells Has a typical DC marker and is distinguished from other cell types by phase contrast microscopy. Can be different. CTL induction using synthetic peptides and dendritic cells   Riddell, et al. , "Nature Medicine" 2: 2 16 (1996), similar to the method described for CTL clones. CTL lines were expanded in tissue culture. 25 × 10⁶Autrogas Illumination Gamma-irradiation (4200 rad) PBMC (APC to prevent their growth, CTLp expansion) and 5 × 10⁶Allogeneic (non-HLA-A2) and 25 ml containing EB virus-transformed lymphoblast B cells 30 ng / ml anti-CD3 monoclonal antibody (OKT3) in complete medium 5 × 10 in the presence of^FourThe CTL was resuspended. Also, peptide-pal Using Sudo PBMC (instead of anti-CD3 antibody) to expand the CTL lineage, Antigen specificity was maintained.   The mixed PBMC, APC and peptide are placed in a suitable culture vessel, for example, plastic. 3 in a T-flask, gas-impermeable plastic bag, or roller bottle Humidified air / CO at 7 ° C_TwoIt was kept in the incubator. After the start of culture On day 1, 200 IU / ml of recombinant IL-2 was added to the culture. 5 in culture Fresh media containing 0 IU / ml IL-2 was supplied on days 5, 8 and 11. , T cell concentration is number> 1.5 × 10⁶When / ml was reached, it was split.   After an active period of culture, usually occurring during the first 3-5 days, the recombinant growth factor For example, interleukin-2 (IL-2), interleukin-4 (IL- 4), interleukin-7 (IL-7 ) Or interleukin-10 (IL-10) to the culture. Thus, the resulting effector CTL can be further expanded. Required for specific patients Expansion cultures may be further cultured for 5-12 days, depending on the number of effector CTLs required. Can be held. In addition, hollow fiber artificial capillary systems (Cellco ) Can be used to perform expansion cultures, where a larger number (1 × 10¹¹ Cells) can be maintained.   Irradiated, autologous, peptide to obtain the required number of cells for treatment The need to restimulate the culture 2-4 times with pulsed adherent PBMC There is. By day 14-16, on average almost 5 × 10⁷CTL was obtained . 1-2 × 10⁶/ Ml aliquots of DC at 20 ° C. with 3 μg / ml β_Two In the presence of microglobulin, 40 μg / ml peptide was separately packaged for 4 hours. Loose and irradiated (4,200 rad) before use. Purify with immunomagnetic beads CD8⁺T cells (Dynabooks M-450 and Detachabbe nd; Dynal) was used as the response factor. Typically, a 48-well pre- 200-250 x 10 to obtain enough cells for cell culture⁶PBMC 24 × 10⁶CD8⁺T cells were obtained. 10 ng / m in each well 0.25 ml of cytokine in the presence of 1 IL-7 (Genzyme) Generated DC (1 × 10^Five0.25 ml of CD8 (in cells / ml)⁺T thin CTL primers in 48-well tissue culture plates by mixing with vesicles. Cultures were constructed. 24 hours after the start of culture, recombination at 10 ng / ml Add IL-10 (Endogen, Cambridge, MA) to each well Was added. Irradiation (3,300 rad) oh pulsed with peptide as described The CTL cultures (each individual well) Was restimulated every 7 days (up to 6 cycles). After each restimulation cycle, recombinant I L-10 (10 ng / ml) and recombinant IL-2 (10 IU / ml) They were added on days 1 and 2, respectively. For one experiment (Table II), the second antibody Immediately before the primary restimulation cycle and 24 hours after antigen restimulation, the culture supernatant was collected (1 00 μl) to measure the production of GM-CSF. This measurement was performed using an ELISA kit (R & D System, Minneapolis, MN). Complete medium Shows 5% of AB male human serum, L-glucmin, RP supplemented with sodium ruvinate non-essential amino acids (all from Gibco) MI-1640 (Gibco, Grand Island, NY) Was. CTL cytotoxicity assay   Activate expanded cells before using them (eg, injecting them into the patient) , Viability and sterility.   Standard⁵¹Cr release assay (Biddison, WE (1991), "C current protocol in Immunology "p7, 17. 1-17.5, J.I. Coligan et al. Hen, Wiley and S ons, New York) using target cells expressing the appropriate HLA molecule. CTL cytotoxicity that occurs in the presence and absence of immunogenic peptides The harmful activity can be measured. For example, about 5 × 10^Five10 cells / ml cells Incubate overnight at 37 ° C. with μg / ml synthetic peptide to obtain peptide -A pulsed target was prepared. All cells are 100-300 μCi⁵¹Cr (I CN) / 3 × 10⁶Cells were labeled for 1 hour at 37 ° C. Adherent target cells ( For example, melanoma) was separated from tissue culture flasks with trypsin-EDTA. Mark Cells are washed by centrifugation and placed in a round bottom microtiter plate. Final volume 0.2 m containing 10% fetal calf serum (FBS, GIBCO) Mixed with effector in 1 RPMI-1640. Centrifuge the plate (5 minutes, 400 × g) to increase cell-to-cell contact,_Twoincubator I put it inside. After 4-6 hours of incubation, 0.1 ml of supernatant was added to each well. Radioactivity was measured with a gamma counter. 48 well mini culture After first screening for cytotoxic activity in each well of the -Cells were not routinely counted. However, in counting these cell samples, Thus, the effector / target ratio in these assays is between 10: 1 and 1: 1. Range. Low temperature target inhibition assay blocks lysis of melanoma cells Peptide-pulsed unlabeled. 221 (A2.1) Measure the capacity of cells To determine antigen specificity.   Specific by the following formula⁵¹By calculating the% release of Cr, specific cytotoxicity The% harm was determined: [(cpm of test sample-spontaneous⁵¹Cr release cpm) / (most Big⁵¹Cr release cpm-spontaneous⁵¹Cr release cpm)] × 100. Target alone By incubating in the absence of effectors,⁵¹Cr solution Release is measured and the target is 0.1% Triton-X100 (Sigma Che m. Co. , St. Louis, Mo.) Big⁵¹A cpm of Cr release was obtained. The measurements were performed in duplicate. Average mark Sub-errors were typically less than 10% of the mean blood.   Viability was measured by exclusion of trypan blue dye by live cells. Custom The cells were tested for the presence of endotoxin by the appropriate technique. Finally, a proper Bacterial or fungal contamination by microbiological methods (chocolate agar and others) The presence of dye was determined. One When a spontaneous cell passes all quality control and safety tests, And wash with appropriate injection solution (Ringer / glucose lactate / human serum album) Min), which can include T cell growth factors, eg, IL-2. And injected intravenously into the patient.                                 Example 1            By acid stripping and subsequent peptide loading                Preparation of Effective HLA Allele Specific Antigen Presenting Cells   In this example, low temperature incubation and acid stripping were performed. Used to generate empty MHC class I molecules to implement the peptide loading method. For use in diagnostic or ex vivo therapy applications. Demonstrates preparing effective HLA allele specific antigen presenting cells (APC). same Such a method is disclosed in US patent application Ser. No. 08 / 468,454, filed Jun. 6, 1995. (This is a continuation of US patent application Ser. No. 08 / 103,401, filed Aug. 6, 1993. (Both of which are incorporated herein by reference) )It is described in. Using APC in this example, antigen-specific cytotoxicity Cytotoxic T lymphocytes were sensitized for the development of harmful cells. This is HLA-A2 . Staphylococcus aureus (Stap) as APC in the presence of HLA-1 and HLA-A1 (Hylococcus aureus) cowan I SAC I activated PB MC, phytohemaglinin (PHA) T cell blasts or peripheral blood mononuclear cells ( PBMC). The result is to other APC and other MHC allele Applicable. Culture medium   2 mM L-glutamine (Irvine Scientific) ), 50 μg / ml gentamicin (Gibco) and 5% heat inactivation Pooled human AB serum (Gemini Bioproducts) [RP [MI / 5% HS] supplemented with RPMI 1640 + Hepes + Glutamine (G In ibco), incubation of PHA blasts and CTL was performed. L-glutamine and gentamicin as described above and 10% heat inactivated pool Fetal bovine serum (Irvine Scientific) cts) [RPMI / 10% FCS] supplemented with RPMI 1640 + Hepes + Glutamine (B ioWhittaker), EBV-transformed lymphoblastoid cell line (LCL) Was maintained. The chromium release assay was performed in RPMI / 10% FCS. Cultured cell line   JY, HLAA2.1 expressing human EBV transformed B cell line Grow in RPMI / 10% FCS. K562, ie NK cell sensitivity The erythroblastoma line was grown in RPMI / 10% FCS. Chrome release K562 Used in assays, NK and LAK cells provide background Reduced ring. peptide   As described above, using the HLA allele motif for specific antigens, U.S.A. transferred simultaneously S. S. N. No. 07 / 926,666 and U.S. Pat. S . S. N. As described in 08 / 027,146, these studies were The immunogenic peptide used was synthesized. The sequences are shown in Table I. Pepti Is routinely dissolved in 100% DMSO at 20 mg / ml and And stored at -20 ° C. Isolation of peripheral blood mononuclear cells (PBMC)   Collect whole blood in a syringe containing heparin (10 U / ml) and make a 50 cc circle. 1600 rpm (Beckman GS-6K) in a conical centrifuge tube (Falcon) R) for 15 minutes. The plasma layer is then removed and 1 A 10 ml buffy coat was collected with a 0 ml pipette. Buffy coat Mix well and dilute with an equal volume of serum-free RPMI 1640. Then buff Eeecoat over 20 ml Ficoll-Paque (Pharmacia) Stratified into 50 cc conical tube and break-off Without centrifugation at 400 xg for 20 minutes at room temperature. Transfer pipette ( 2 interfaces / 50 cc tube) to collect the interface containing PBMCs and use 50 ml of 3x 10 minutes in serum free RPMI (1770, 1500 and 1300 rpm) Washed. Freezing and thawing of PBMC   30 × 10 PBMC⁶Cells / ml 90% FCS + 10% DMSO (Sig ma) using a cryovial (Nalge) in 1 ml aliquots Frozen. Cryo 1 ° C freezing volume containing isopropanol (Fisher) Place the cryovials in a vessel (Nalge) for 4 hours at -70 ° C (min. ) ~ Overnight (maximum) arranged. The isopropanol was changed after every fifth use. Long term The cryovials were transferred to liquid nitrogen for interim storage. Crystals almost melt The PBMC were thawed by continuous shaking in a 37 ° C. water bath until D Nase 30 μg / ml (for dead cells Serum-free RPM containing (Calbiochem) to avoid aggregation due to The cells were immediately diluted in I medium and washed twice. Preparation of CD4 + wasting response factor cell population   CD4 + lymphocyte depletion was performed using antibody-coated flasks: According to the instructions for use, 25 ml of PBS CMF (without calcium magnesium) I) CD4 by swirling the flask with +1 mM EDTA (Sigma) + Mi for selection of cells (Applied Immunity Science) Wash the croCELLector T-150 flask for 30 seconds, then flatten Incubated for 1 hour at room temperature on a clean surface. Aspirate buffer and flask By shaking for 30 minutes to maintain the coating on the binding surface, Washed twice. To each washed flask, add 25 ml of medium and place on a flat surface For 20 minutes at room temperature. Medium prepared to receive cells Medium was left in the flask until it was ready. Contains 30 μg / ml DNase The PBMC in the medium having the medium was thawed and washed twice. For one flask, the maximum Large 12 × 10⁷Cells were resuspended in 25 ml of medium. Medium from flask Aspirate, then add cell suspension gently to MicrCELLector . Incubate flask containing cells for 1 hour at room temperature on flat surface did. At the end of the incubation, gently flank the flask For 10 minutes to resuspend non-adherent cells. Non-adherent CD4 + T cells Collect the attrition cells and then wash the flask twice with PBS CMF to remove non-adherent cells. Was collected. The collected CD4 + T cell depleted cells are spun down by centrifugation, And resuspended in it. Development of PHA blasts   PBMCs were isolated using the standard Ficoll-Paque protocol. Was. Frozen cells were washed twice before use. 1 μg / ml PHA (Wellco me) and RPMI / 5% HS containing 10 U / ml rIL-2. 2 × 10 cells⁶/ Ml. Contains 10 U / ml rIL-2 PHA blasts were maintained in the medium, feeding and splitting as needed. P HA blasts were used as APC on day 6 of culture. Use PBMC as APC When used, the generation of empty class I molecules and the loading of peptides Performed only by the tap method. Acid stripping / peptide loading of PBMC and PHA blasts   PBMC were isolated using the Ficoll-Paque protocol. Frozen When using colony cells, PBMC were washed twice before use. PH as described above A blasts were prepared and washed before use. Once the cells are prepared, cool the cells Washed once in 0.9% NaCl (JT Baker) + 1% BSA. 5 In a 0 cc conical centrifuge tube, transfer cells to 10⁷Sterile quench cold in / ml Phosphate-phosphate buffer [0.13 M citric acid (JT Baker), 0.0 6M sodium phosphate monobasic (Sigma) pH 3, 1% BSA, 3 μg / ml of β_TwoMicroglobulin (Scripps Labs)] And incubated on ice for 2 minutes. Immediately, 5 volumes of cold sterile neutralizing buffer # 1 [0.15 M sodium phosphate monobasic pH 7.5, 1% BSA, 3 μg / Ml β_TwoMicroglobulin, 10 μg / ml peptide ” Cells were sedimented at 1500 rpm for 5 minutes at 4 ° C. 1 cell cold Sterile neutralization buffer # 2 [PBS CMF 1% BSA, 3 μg / ml DNase, 3 μg / ml β_TwoMicroglob Phosphorus, 40 μg / ml peptide] at 20 ° C. for 4 hours. Incubated. Cells are grown in medium at approximately 5 × 10⁶/ Ml and diluted at 6000 rad Irradiated. The cells are then centrifuged at 1500 rpm for 5 minutes at room temperature, Was resuspended. In CTL-derived cultures (below), acid stripped / pepti The cells carrying the cells were used immediately. Binding assays using intact cells and radiolabeled peptides   JY cells are acid stripped (ie, citrate-phosphate as described above). (Treat with salt buffer and neutralization buffer # 1) or Cubbed. JY control cells were left untreated in tissue culture medium. After processing, One of the cell populations was washed twice with serum-free RPMI,¹²⁵I radioactive labeling 941.01 The (HBc18-27) peptide (standard chloramine T iodination) was loaded. To measure binding specificity,¹²⁵I-941.01 (10^Fivecpms) +/- 1 200 μl of neutralization buffer # 2 containing 00 μg of unlabeled 941.01 (see above) 2 × 10 inside⁶The cells were resuspended. Incubate cells at 20 ° C for 4 hours. Bait and washing twice with serum-free RPMI to remove free peptide. 20 cells Resuspended in 0 μl serum-free RPMI. In a microfuge tube, The cell suspension is layered on 800 μl FCS and sedimented by centrifugation for 5 seconds I let it. The supernatant was aspirated and the radioactivity remaining in the pellet was measured (Micro medic automatic gamma counter, 1 minute / tube). Binding of radiolabeled peptides to empty MHC molecules   Low temperature incubation or acid stripping peptide loading protocol In order to determine the efficiency of peptide loading using Coal, JY cells (HLA- A2.1 EBV transformed B cell line) at 26 ° C. overnight Or acid stripping to remove endogenous MHC-associated peptides, and hand¹²⁵I. Exogenous Peptides Using Radiolabeled HLA-A2.1 Binding Peptides The loading of the metal was measured. Inhibition of labeled peptides using cold peptides of identical sequence The specificity of this reaction was determined by measuring. The results shown in Table 2 prove Thus, acid treatment of cells significantly (approximately 1) the amount of labeled cells that bind to JY cells. (By a factor of 0). Furthermore, the binding of the labeled peptide can be achieved by adding a low-temperature peptide. Completely blocked, demonstrating specific binding (data not shown). FACS analysis   For each antibody to be tested, approximately 10⁶Cells were used. Transfer cells to PBS C Washed twice with MF + 0.1% BSA. Each sample contains 100 μl PBS, CMF + 0.1% BSA + primary antibody at 2 μg / ml (BB7.2, ATCC) Or (9.12.1, INSERM-CNRS, Marseille) or ( LB3.1, Children's Hospital, Pittsburgh) did. Negative controls were always included. Incubate cells on ice for 20 minutes, PBS   Washed twice with CMF + 0.1% BSA. 100 μl of anti-mouse IgG cells   Resuspend in FITC complex (Sigma), PBS CMF + 0.1% Diluted 1:50 in BSA and incubated on ice for 20 minutes. PB cells Washed twice with S CMFH-0.1% BSA, FACScan (Becton) (Dickinson) analysis. analysis Cells needed to be extended to subsequent days when PBS was added to PBS / 1% Parapol Fixed with aldehyde (Fisher) and analyzed within one week. Analysis by FACS analysis   The PHA-induced T cell blasts were subjected to acid strip / peptide loading according to the method described above. did. The resulting cells were subjected to anti-HLA-A2 (BB7.2) and FACS analysis. And anti-HLA alpha chain specific (9.12.1) monoclonal antibody . The control for this experiment was not treated at pH 3 (but PBS buffer pH 7. 2) and treated with citrate-phosphate buffer (MH). C to strip C)_TwoAbsence of microglobulins and peptides And neutralized cells. The results indicate that these cells were citrate- When treated with a phosphate (pH 3) buffer, both anti-HLA class I antibodies alone (anti- HEL-2 and alpha chain specific) significantly (10-fold) Reduced, but monoclonal antibodies specific for class II MHC molecules ( (HLA-DR) did not decrease. The most important thing And β_TwoAcid stripping in the presence of microglobulins and peptides Neutralization of surviving cells results in the conservation of significant amounts of class I MHC antibody reactive sites, The decrease in light intensity was only 2.5 times. Acid treated cells are trypan blue It remained viable as measured by exclusion and forward / side scatter analysis. EBV type Transformed B cell lines, fresh (or frozen) PBMC and other peptides Binds to HLA-A2.1 or HLA-A1) with similar results Obtained (data not shown). Stimulates autologous PBMC or PHA blasts carrying acid stripped / peptide Induction of primary CTL for use as a factor   Acid stripping / peptide loading of PBMC and PHA blasts is described above. I have. While incubating the stimulator cells with the peptide for 4 hours, the responder cells Prepared: Response factor was PBMC depleted of CD4 + T cells (described above). 3 × 10 response factor cells in medium⁶Resuspend in 1 ml / ml and 1 ml Of the response factor suspension in a 24-well tissue culture plate (Falcon, Becto) n Dickinson) in each well. Incubate plate 37 ° C, 5% CO_TwoIn, until a population of stimulators is prepared, I put it. Once irradiated, in a medium containing 20 ng / ml rIL-2 Stimulating factor APC to PBMC for 10⁶/ Ml or PHA bud 3 × 10 for cells^FiveResuspended in 1 / ml and containing the response factor 1 ml / well of stimulator cell suspension was added to the cells. After 7 days of induction, 200 100 μl of medium containing ng / ml rIL-7 was added to each well (maximum). Final 10 ng / ml rIL-7). After 10 days of induction, 200 U / ml rIL 100 μl of medium containing -2 was added to each well (final 10 U / ml r). IL-2).   Additional critical parameters for the induction of primary CTL are: Enrichment of CD8 + T cells in the response factor cell population (due to CD4 + T cell depletion) 2) Addition of rIL-7 to CTL-induced cultures from day 0, and 3) Antigens on days 12-14 using autologous adherent cells pulsed with peptide Culture restimulation. Restimulation of CTL antigen   12-14 days after induction, primary CTLs were generated using autologous adherent APCs. Restimulated with peptide. Melt and wash autologous PBMC as described above. Was. Cells were irradiated at 6000 rads. Cells are spheronized and placed in medium × 10⁶/ Ml and resuspend 1 ml of cell suspension in 24 wells Add to each well of the tissue culture plate, 37 ° C, 5% CO_Two2 hours in Cubbed. Unwashed by washing each well three times with serum-free RPMI The adherent cells were removed. After this step, 3 μg / ml β_TwoMicroglobulin and And 0.5 μl of medium containing 20 μg / ml total peptide to each well. Added. APC at 37 ° C. with 5% CO_TwoPeptide and β below_TwoMicro gro Incubated with Brin for 2 hours. Aspirate wells and in 1 ml of medium 1.5 × 10⁶/ Ml of response factor cells was added to each well. Two days later, 20U / 1 ml of medium containing ml of rIL-2 was added to each well. Every three days thereafter The culture was supplemented with 10 U / ml rIL-2 (final). Cytotoxic chromium release assay   Seven days after restimulation of the primary induction, the cultures were evaluated for cytotoxic activity.   a. Preparation of effector cells: centrifuge the response factor, RPMI / 10% FCS 10 in⁷/ Ml. Perform a 3-fold serial dilution of the effector, Produce effector / target ratios of 100: 1, 33: 1, 11: 1 and 3: 1. Was. In duplicate, an aliquot of effector cells was 100 μl / well. On a 96-well U-shaped bottom cluster plate (Costar). Was.   b. Preparation of target cells: approximately 16-20 hours prior to the assay, 3 μg / ml β_TwoMicroglobulin and 10 μg / ml whole peptide In the presence or absence, target cells were 3 × 10⁶/ Ml at RPMI / 1 Resuspended in 0% FCS. After pre-incubation, centrifuge the target cells, The pellet was quenched with 200 μl (300 μCi) sodium (⁵¹Cr) chromate (NE N). Incubate cells with antigen at 37 ° C. for 1 hour Was. The labeled target cells were washed three times with RPMI / 10% FCS.   c. Assay configuration: Target cell concentration was 10% in RPMI / 10% FCS.^Five / Ml and add 100 μl aliquots to each well containing the response factor Was added. K562 cells (for cold target, blocking NK and LAK activity) ) Is washed and 10% in RPMI / 10% FCS.⁷Resuspended in Was. 20 μl aliquots are added per well and 20: 1 cold target / labeling A target ratio was generated. Voluntary⁵¹To determine the release of Cr, 100 μl / well Of RPMI / 10% FCS with 100 μl / well of labeled target cells and 20 Added to μl / well of K562. Biggest⁵¹100 μl for release of Cr 1% Triton-X100 (Sigma) in PBS CMF at 100 μl / well Wells were added to labeled target cells and 20 μl / well of K562. plate Was centrifuged at 1200 rpm for 2 minutes to accelerate the formation of cellular complexes. A 37 ° C, 5% CO_TwoFor 5 hours. Plate 1 Centrifuge at 200 rpm for 2 minutes and collect 100 μl / well supernatant The assay was harvested. Specific lysis using standard gamma counting techniques % Was measured (Micromedic automatic gamma counter, 0.5 min / tube). The% specific lysis was determined by the following formula: release of cpm experiment-spontaneous release of cpm / Cpm max release-cpm voluntary release x 100.                                 Example 2     Screening for peptides identifying CTL epitopes   To identify CTL epitopes, SAC-I activated PBMC as APC Stimulated CTL. Cold temperatures enhance the expression of empty MHC, SAC-I-activated PBMC APC was generated to allow the loading of the functional peptide . The method comprises generating an APC for use in stimulating CTL. Provide another protocol. This embodiment also embeds CTL by APC. Provide another intense protocol. Complete medium   The tissue culture media used in this study was Hepes and L-glutamine. (Gibco) and 2 mM L-glutamine (Irvine Scientific) ific), 0.5 mM sodium pyruvate (Gibco), 100 U / μ g / ml penicillin / streptomycin (Irvine) and 5% heat Inactivated human serum type AB (RPMI / 5% HS; Gemini Bioprod ucts) supplemented with RPMI 1640. EBV transformed line Is 10% heat-inactivated fetal bovine serum (RPMI / 10% F) instead of human serum. CS, Irvine). Cytokine   Recombinant human interleukin-2 (rIL-2) and interleukin-4 (RIL-4) was obtained from Sandoz, each at 10 U / ml And a final concentration of 10 ng / ml. Human interferon-γ (IF N-γ) and recombinant human interleukin-7 (rIL-7) Genzym e) and used at final concentrations of 20 U / ml and 10 ng / ml, respectively. Used. peptide   The peptides were synthesized as described above and are listed in Table I. 10 daily peptides Dissolve at 0 mg / ml in 0% DMSO, aliquot and use Until storage at -20 ° C. Cell lineage   JY, Steinlein, EHM, BVR, and KT3 are each H LA, A_2.1, A₁, A_Three, A₁₁, And A_{twenty four}Homozygous human EBV form expressing Transformed B cell line. They were grown in RPMI / 10% FCS and CT Used as a target in the L assay. K562, ie, RPMI / 10 NK cell sensitive, erythroblastoma lines grown in% FCS were assayed for CTL assays. Used to reduce background killing. MAGE antigen, m melanoma HLA A1 + cell lines expressing e397 and mel 938 Or MAGE antigen, which does not express mel 888, and RPMI / 10 Grow in% FCS. Isolation of peripheral blood mononuclear cells (PBMC)   Whole blood was collected in a syringe containing heparin and 1600 in a 50 cc tube. Centrifuged at RPM (Beckman GS-6KR) for 15 minutes. Then blood Remove the serosa and pipette 10 ml of buffy coat using circular motion. I did. Buffy coat was mixed well and diluted with an equal volume of serum-free RPMI . Next, Fee coat (30 ml) was added to 20 ml of Ficoll-Paque (Pharm acia) at 1850 RPM at room temperature without break-off (400 xg) for 20 minutes at 25 ° C. Transfer pipette ( Between Ficol and plasma containing PBMC using 2 interfaces / 50 cc tube) Was collected and washed three times with 50 ml of RPMI (1770, 1500, And 1300 rpm for 10 minutes). Cells are resuspended in 10-20 ml of medium. And counted and adjusted to the appropriate concentration. Freezing of PBMC   Nalgene Cryo 1 ° C. containing isopropanol (Fisher) 30 × 10 in a freezing container⁶Cells / tube (90% FCS / 10% DMSO; S igma) and placed at -70 ° C for 4 hours (minimum) to overnight (maximum). The isopropanol was changed after every fifth use. Tube for liquid storage for long term storage Moved to elementary. In order to melt, the PBMC is used until the last crystal is almost melted. Shake continuously in a 37 ° C. water bath (tubes either in a water bath or at room temperature). Did not place during that time). Bloodless containing 30 μg / ml DNase Diluting the cells in Qing RPMI to avoid aggregation of dead cells by DNA, Washed twice. Induction of primary CTL using SAC-I activated PBMC as APC   a. Preparation of SAC-I activated PBMC as APC:   Purify PBMC using standard Ficoll-Paque protocol, Then, 0.005% of Pansobin cells (SAC- I cell; Calbiochem), 20 μg / ml immunobeads (rabbit anti-human IgM; Biorad), and 20 resuspended in RPMI / 5% FCS containing ng / ml human rIL-4 Was. 2 ml / well cells were plated in a 24-well plate (Falcon, Becto) n Dickinson) and cultured at 37 ° C. 3 days later, medium Removed, cells were washed three times, and then RPMI / 10% HS was added. RPM After a further 2 days of culture in I / 10% HS, the cells were used.   b. Expression of empty class I molecules on APC and peptide loading of APC Holding         1. Low temperature incubation:             a. Expression of empty MHC in APC:   10 ng / ml rIL-4, 20 U / ml human IFN-γ, and 3 μg / Ml β_Two-Microglobulin (β_Twom; Contains Scripts Labs) APC in 2 x 10⁶/ Ml. Then the cells 5% CO_TwoOvernight at 26 ° C in the presence of These details The vesicle only expresses a class I molecule fraction in the empty state (about 10%) It should be noted that             b. Loading of peptides on APC stimulator cells:   Empty class I expressing APC was converted to serum-free RPMI (1 + L-glutami And Hepes) 1-2 times, and 50 μg / ml total peptide Pool (ie, 16.7 μg / ml of each peptide in pool of 3; 2 pools of 25 μg / ml of each peptide in the pool; 50 μg / ml individual peptide), 30 μg / ml DNase and 3 μg / ml β_Twom containing serum-free R 1 × 10 in PMI⁷Was resuspended. Incubate at 20 ° C for 4 hours After irradiating the cells with 6100 rads (5 × 10⁶/ M l; 25 × 10⁶Cells / tubes), wash and adjust to appropriate concentration for addition to induction culture Adjusted (see below).         2. Acid stripping:   It is used as another method to generate empty MHC on the surface of APC did. Low temperature 0.9% sodium chloride containing 1% BSA (JT Baker ), The SAC-I activated PBMC was washed once. 1% BSA and 3 μg / ml of β_Twom citrate-phosphate buffer (0.13 M citrate) Acid [J. T. Baker], 0.06 M sodium phosphate monobasic [Sigma a], 10 cells in pH 3)⁷/ Ml and incubate on ice did. After 2 minutes, 1% BSA, 3 μg / ml β_Twom, and 10 μg / ml 5 volumes of cold 0.15M sodium phosphate containing peptide [neutralization buffer # 1] Buffer, pH 7.5, and cells are incubated at 1500 RPM at 4 ° C. Centrifuge for 5 minutes. 1% BSA, 30 μg / ml DNase, 3 μg / ml β_Two m, and 1 ml of 50 μg / ml peptide [neutralization buffer # 1]. The cells were resuspended in PBS and incubated at 20 ° C. for 4 hours. After 4 hours incubation at 20 ° C., the cells were Irradiate with rad (5 × 10⁶/ Ml; 25 × 10⁶Cells / tubes), wash, and induce culture Was adjusted to the appropriate concentration for the addition of (see below). Preparation of CD4 + depleted PBMC response factor cell population (phosphorus using AIS flask Consumption of the sub-ball group)   Add 25 ml of PBS / 1 mM EDTA and make sure all surfaces are wet For 30 seconds, then incubate for 1 hour at room temperature with the binding surface down. AIS Micr oCell T-150 flask (particularly against CD4 + T cell depletion) Unusual; Menlo Park, California). This incubation After the incubation, shake the flask vigorously for 30 seconds, wash once with PBS / EDTA, Were washed twice with PBS and then incubated with 25 ml of medium for 15 minutes . Serum-free RPMI containing 30 μg / ml DNase (+ L-glutamine + Thaw PBMC in Hepes, wash once, incubate in medium for 15 min. I did it. After aspirating the medium from the flask, 180 × 10^FourPBMC up to 3 Added in 25 ml of medium containing 0 μg / ml of DNase. At room temperature After 1 hour, gently rock the flask for 10 seconds to resuspend non-adherent cells. I let you. The suspension of non-adherent cells containing CD8 + T cells is collected and the flask is Washed twice with BS. CD4 + T cell depleted PBMC is centrifuged and loaded on induction culture Was counted. The CD4 + and CD8 + phenotypes of the CD4 + Determined by ACS analysis (see below). In general, this technology uses CD8 + T cells were enriched 2-fold, and after exhaustion of CD4 + T cells, on average approximately 40-50% CD There were 8+ T cells and 15-20% residual CD4 + T cells. CD4 + T Cell depletion can also be caused by antibody and complementary methods or antibody-coated magnetic beads (Dyn). abeads). CD4 + T cell depletion is CT Lp was concentrated and cells that competed for cytonutrients were removed. Induction of primary CTL   During the 4-hour peptide loading of the stimulator APC, CD4 + T cells (see above) )) Utilizing AIS flasks to select CD8 + T cells through wasting; CD4 + to be used as a response factor population Consumed PBMC was prepared. 3 × 10 response factor cells⁶1 ml of body per ml Peptide loaded stimulator at 37 ° C. Placed until APC was prepared. Irradiated, peptide-loaded APC was serum-free R Wash once in PMI (+ L-glutamine and Hepes) and Adjust to the appropriate concentration and place in a 24-well plate at 1 ml / plate : 1 × 10 for PBMC and SAC-I activated PBMC as APC⁶ Stimulator cells (1 ml volume) were placed in wells containing responder cells For PHA blasts as APC, 1 ml of 3 × 10⁶Stimulator cells Well. Add rIL-7 (total volume of 2 ml) at a final concentration of 10 ng / ml. Added. On day 7, seven additional 10 μg / ml rIL-7 were added to the culture. Then, every 3 days, 10 U / ml rIL-2 was added. On day 12, culture Were restimulated with peptide-pulsed adherent cells and tested for cytolytic activity after 7 days. (See below). Protocol for restimulation of primary CTL using autologous adherent APC   Serum-free RPMI (+ L-glutamine and 30 μg / ml DNase) And Hepes), melt the autologous PBMC, wash twice, 5 × 10 5 in medium containing DNA⁶/ Ml. PBMC (in 5 ml 25 × 10⁶Cells / tube) were irradiated at 6100R. After washing once, the PBMCs were Resuspend in 4x10⁶/ Ml, and add 1 ml of irradiated PBMC Added to each well of a 24-well plate. PBMC at 37 ° C for 2 hours Incubate and wash three times to remove non-adherent cells and add in a 0.5 ml volume. 20 μg / ml total peptide added And 3 μg / ml β_TwoCulture in a medium containing microglobulin and re- Incubated at 37 ° C for 2 hours. Aspirate peptide and resuspend in medium 1.5 × 10 cloudy⁶Of response factor cells were added in a volume of 1 ml. 2 days later, 2 1 ml of medium containing 0 U / ml rIL-2 was added ... FACS analysis   1 × 10⁶Centrifuge cells / tubes, 100 μl / tube PBS / 0.1% BSA / 0 . 02% sodium azide (Sigma) +10 μl / tube directly bound anti Resuspend in body (Becton Dickinson) and place on ice for 15-20 Incubated for minutes. The cells were then washed with PBS / 0.1% BSA / 0.02% Wash twice with sodium silicide, resuspend in PBS, FACScan (B (Ecton Dickinson). Samples can be analyzed within 1-2 minutes If not, fix cells with PBS containing 1% paraformaldehyde, Analyzed within a minute. Cytotoxicity assay   a. Preparation of target cells   Approximately 16-20 hours prior to the CTL assay, target cells (Class I matched EBV type Was washed once and 3 × 10 5 in a volume of 10 ml.^Five/ Ml RPMI / 5 In the presence or absence of 10 μg / ml total peptide in% FCS. Turned cloudy.   b. Labeling of target cells   Centrifuge target cells, 200 μl / tube⁵¹In Cr chromate (NEN) And incubated for 1 hour on a shaking incubator at 37 ° C. . Target 3 times (10 ml / wash) RP After washing with MI / 10% FCS, the cells were resuspended in 10 ml (the efficiency of labeling was measured). 50 μl / target with Micromedic automatic gamma counter to determine Counted).   c. CTL assay   2 x 10 target cells^Five/ Ml and add 50 μl of cell culture to the U-bottom of 9 1 × 10 6 per well of a 6-well plate (Costar Corp.)^Four/ U It was added to the final concentration of the well. Wash K562 once, 4 × 10⁶/ Ml And add 50 μl / well to 2 × 10^Five/ Well to the final concentration (low The warm K562 / target ratio was 20: 1). Wash the response factor cells once, 9x 10⁶/ Ml and three-fold serial dilutions 90: 1, 30: 1, 10: Runs were performed at effector / target ratios of 1 and 3: 1. Double response factor cells In a duplicate well, a volume of 100 μl was added. 5 for voluntary release 0 μl / well labeled target cells, 50 μl / well K562, and 100 μl / well of medium was added. To maximize release 50 μl / well Labeled target cells, 50 μl / well K562, and 100 μl / well 0 . 1% Triton-X100 (Sigma) was added. 1200RP plate Centrifuged at M for 5 minutes. After incubating at 37 ° C for 5 hours, The plate was centrifuged again at 1200 RPM for 5 minutes and 100 μl / well The supernatant was collected. Standard gamma counting technology (Micromedic automatic gamma cow) % Specific lysis was determined according to the following formula: % Specific lysis = release of cpm experiment-spontaneous release of cpm / maximum release of cpm-cpm Spontaneous release x 100. Characteristic at the two highest effector / target (E: T) ratios Lysis of the target sensitized with the heterologous peptide by CTL is the control target (ie, pepti Does not contain If it is greater than 15% than the target cell, the cytotoxicity assay (CTL Say) was considered positive. Two highest effector / target (E: T) ratios Lysis of the target sensitized with the specific peptide by CTL in the control target (ie, 6% greater than (target cells without peptide) The assay (CTL assay) was considered borderline. result   Of the peptides that bind to the indicated alleles, 60 MAGEs tested to date 12, 25 of the HIV peptides, 13, 25 of the HIV peptides Three of the tides and seven of the 28 HBV peptides are in vitro. Primary CTLs were induced in this manner.                                 Example 3   Peptide-pulsed dendritic cells together with IL-7 and IL-10     Of CTL response stimulating CTL epitope using   SAC-I stimulated PBMCs show high affinity MHC binding from HBV. It has been shown that working effectively to generate a CTL response to They have proven. Nevertheless, peptide reactive C derived from this approach The majority (about 90%) of the TLs are transfected and express HBV antigen Target cells could not be recognized (Wentworth, et al. , "Molec. Immunol." 32: 603 (1995)). This technique To improve, we first studied normal (HBV negative) donors and previously A using PBMC from the same HBV peptide to generate a primary CTL response DC's ability to work as a PC Was studied. Because the number of DC circulating in peripheral blood is very low (<1%), we Is in a 7 day culture of monocytes purified in the presence of GM-CSF and I1-4 DC was generated (Romant, et al., “Exp. Med.” 18). 0:83 (1994); Sallusto, et al. , "J. Exp. Me d. 179: 1109 (1994)).   Twelve HLA-A2.1 normal volunteers (all HBV serological markers Known HBV CTL epitome using PBMC from A DC and CTL response to the loop was generated. kDC generated in vitro The HBV peptide HBC₁₈(This leads to HLA-A2.1 with high affinity Followed by autologous CD8 + purified lymphocytes from C Used to induce TL. Lymphocyte cultures were treated with antigen (peptide pers On day 7 and day 14, and cell lysis activity Sex was measured on day 21. CT in each experiment (and for each blood donor) The whole procedure was performed in separate microcultures (48-well plates) in L-derived cultures. Maintained throughout. Pulsed or not with peptide. 221 (A2.1 ) And the HBV core gene. Use 221 (A2.1) And 4-6 hours⁵¹Cytolytic activity was measured in a Cr release assay. table As the results shown in I show, all the runs performed using 12 individuals In experiments, at least one culture recognizes peptide-pulsed target cells CTL. TABLE I Peptides against known HBV epitopes in normal individuals       -Obtained in the induction of CTL using pulsed DC and IL-7       Summary of the results obtained ^aAll blood donors were HLA-A2 + and were directed against HBV serum markers It was negative.^*Donor No. The results obtained using No. 6 are given in Table II.^b % Specific lysis in the presence of the peptide is the value obtained in the absence of the peptide The culture was positive for CTL when> 20.^c When the% specific lysis was> 20 over untransfected cultures, Nourishment was positive for killing of HBc transfected target cell line .^d Numbers in parentheses represent percentages.   More importantly, most of the resulting peptide-reactive CTL (65-100% ) Is also HBC₁₈Express, process and naturally present CTL epitopes It is. 221 (A2.1). HBC target cells could be recognized. Table II As shown, one example of these experiments was constructed for that particular experiment. Shows cytolytic activity of 14 of 48 microcultures. These results show Thus, seven of these microcultures ("Table II"^**Is marked with ) Are peptide-pulsed and HBV core transfected target cells. Vesicle. 221 (A2.1). High specific lytic activity was demonstrated for both HBCs. Table II HBc₁₈Peptide-pulsed DC in the induction of CTL against       Examples of results obtained using ^aFourteen results out of a total of 48 cultures were selected as examples. "^*The cultures marked with "" are GM-CSF (> 3 fold) as a result of Ag restimulation. But did not produce cytolytic activity. "^**Cultures marked with GM-CSF was generated and peptide-pulsed and transfected It had significant specific lytic activity on both target cells.^b Prior to and 24 hours after Ag restimulation, human GM-CS Using a specific ELISA for F, the GM-CSF concentration in the culture supernatant was determined. It was measured. Results are expressed as pg / ml.^cGM-CSF ratio = raw after Ag restimulation GM-CSF / Ag generated GM-CSF generated before restimulation.^d With and without peptides. 221 (A2.1) cells and HBV core Gene [. 221 (A2.1). HBc]. 221 (A2 . 1) Using cells for 5 hours⁵¹Cytolytic activity in Cr release assay It was measured. The effector / target ratio was 10: 1 to 1: 1.   To predict which microcultures can have antigen-specific CTL Immediately before and two days after the second antigen restimulation, the cell cultures from each well were Washes were removed and they were analyzed for the presence of GM-CSF. This cytokine Can be generated by class I restricted CTL as a result of antigen challenge. Wear. Also, as shown by the results presented in Table II, G M-CSF production was induced by peptide restimulation and measured one week later. Vesolytic activity correlated to a large extent. All seven cultures with cytolytic activity Increased at least 5-fold the production of GM-CSF as a result of antigen restimulation . However, some cultures ("^*) During the restimulation procedure Produced GM-CSF but had no specific cytolytic activity. 48 cultures Of these, only four representing this pattern of reactivity were present (three of them). One is described in Table II). Therefore, the result of the culture reacting with the antigen Produce GM-CSF as their antigen-specific CTLs Can be predicted with some accuracy. Effect of addition of lymphocytes on induction of CTL by peptide-pulsed DC   IL-7 enhances the in vitro generation of CTLs and promotes their proliferation (Celis, et al., "Proc. Natl. Acad. Sci. (U. SA) "91: 2105-2109 (1994)); Wentworth, et. al. , "Molec. Immunol." 32: 603 (1995); Ald. erson, et al. , "J. Exp. Med." 173: 923)). Ma In addition, other lymphokines, such as IL-12 and IL-10, produce a CTL response. Have been reported to enhance growth and function as growth factors for CTLs (Chen, et al. , "J. Immunol." 147: 528 (1991); Yan. g, et al. , "J. Immunol." 155: 3897 (1995)). . IL-7, IL-10 and IL-12 are CT by peptide-pulsed DC To evaluate whether the induction of L can be further induced, our experiments In, we select an experimental model system that generally gives a suboptimal CTL response. I chose.   CT against melanoma-related peptides using SAC-I generated APC or DC L-lead is generally HBC₁₈To achieve better than CTL response to peptides Is more difficult (V. Tsai, unpublished). This is for melanoma-associated antigens Because of the lower frequency of precursors for Tissue-specific "can induce a certain level of immunological resistance in the case of Stands for "self" antigen. First, the HLA-A2. The effect of IL-10 was studied using DC pulsed with one binding peptide. As the results presented in Table III clearly demonstrate, the peptide-pulsed standard Cells and melanoma cells IL-10 significantly increased the number of microcultures with antigen-specific CTL for both Increased. In the absence of IL-10, only one of the 48 microcultures had C Cultures that had TL, but the addition of IL-10 resulted in MAGE-3-specific CTL Was increased 10-fold. Table III When using peptide-pulsed DC as antigen, IL-10       Enhances the induction of tumor-specific CTL ^aDCs were prepared from PBMC as described in Materials and Methods, and MAGE -3₁₆₁Pulsed with peptide. IL-10 (10 ng / ml) was cultured in CTL induction medium. Added during the first day of feeding and during the antigen restimulation procedure,^b "^*Cultures marked with "" are directed against both peptide sensitized targets and melanoma cells. Specific CTL activity.^c Stenilin (HLA-A1 +) cells without and without peptide Using MAGE-3₁₆₁Specific lysis for the peptide was measured. Dissolve the result Expressed as the difference (%) in% solution + peptide-% lysis without peptide.^d 938mel (MAGE-3⁺) And 888mel (MAGE-3)^-) Cell line Specific lysis was measured using a standard. The result was 938mel dissolution-888me. Expressed as the difference in dissolution of 1 (ｌ).   Next, HLA-A from melanoma-associated antigen gp100 in two blood donors 2.1 Various doses of IL-10 and IL-7 using binding peptides Requirements were evaluated. The results shown in Table IV are for C using DC as APC. The following important implications for the role of these cytokines in the TL induction protocol A) In the absence of both IL-7 and IL-10, CT No L response was observed; b) IL-10 alone vs. gp100 Was not effective in inducing CTLs; c) when using IL-7 alone Some CTL response observed in one of the donors in absence of IL-10 D) when both IL-7 and IL-10 were used in combination, The frequency of microcultures displaying antigen-specific CTL in human blood donors And e) the optimal dose of IL-10 in this system is approximately 10 ng / ml. is there. Table IV Peptide-loaded DCs using melanoma epitopes from gp100       Of IL-7 and IL-10 in the induction of CTLs       Requirements ^aRepresents% of wells from a total of 48 configured for each condition.^b Ratio obtained using peptide-loaded target⁵¹Cr release%-including peptide If the value obtained using no target is 20% or more, then culture the peptide Positive for reactive CTL.^c gp100⁺Ratio obtained using melanoma cells⁶¹Cr release% -melanoma gp100 If the value obtained using cells is 20% or more, the culture is melanoma-reactive. Positive for CTL.   Finally, IL-12, another reported to enhance T cell activation and proliferation. Cytokines (Mehrotra, et al., " J. Immunol. 151: 2444 (1993); Mehrotra, e. t al. Gat., "J. Immunol." 154: 5093 (1995); ely, et al. , "Cell Immunol." 143: 127 (19 92); Chouaib, et al. , "Proc. Natl. Acad. S. ci. (USA) "91: 12659 (1994); Fallarino, et.   al. , "J. Immunol." 156: 1095 (1996)) Peptide-pulsed DC, IL-7 in eliciting primary CTL response in vitro We sought to determine whether to further increase the effects of and IL-10. In three different experiments, the frequency of cultures displaying IL-12 specific CTL activity Not only does it significantly increase, and in some cases, this Inhibited the CTL response. One of these experiments using "freshly isolated" DCs One embodiment is shown in Table V. Table V Example: IL-12 by peptide-pulsed "fresh" DC       Inhibits the development of antigen-specific CTL a) gp100 peptide G9 as described in Materials and Methods₂₈₀Pulsed at "Fresh" DC was prepared from PBMC. IL-12 (10 ng / ml) was CT Added on day 0 of the L-induced culture and not used during the antigen restimulation procedure. b) "^*Cultures marked with "" can be used for both peptide sensitized targets and melanoma cells. It is thought to have specific CTL activity against it. c) With and without peptide. Using 221 (A2.1) cells, C9₂₈₀ Specific lysis for the peptide was measured. Results are expressed as% dissolution + peptide-pepti The difference is expressed as the difference (%) in% dissolution without the use of a solution. d) 624 mel (gp100⁺) And A375mel (gp100^-) Cell line A specific lysis against melanoma cells was measured using a cell line. Result 624mel Dissolution-expressed as the difference (Δ) in the dissolution of A375mel. Expansion of peptide-induced CTL in tissue culture   One application of the methods described herein uses antigen-specific CTL. In the area of adoptive therapy. Peptide-induced CTLs are associated with tumor-infiltrating lymphocytes (T IL) and expanded in tissue culture in a manner similar to the T cell clone, And those against antigen If the specificity of these cells can be maintained, Can be used for medical treatment.   For these studies, T-cell mitogens (anti-CD3 antibodies) or antigens (particularly A different peptide) to activate CTL induced with peptide-pulsed DC and We appreciated using their ability to expand. As shown in Figure 1 As the results show, using any one of the T cell activators It was possible to efficiently activate and expand the MAGE-3-specific CTL line. Anti-CD3 antibodies are better at promoting cell expansion than peptide-pulsed PBMCs. It was very efficient (1,300 times / 820 times on the 13th) The cell culture used is higher than CTL grown in the presence of mitogen. The lytic activity was indicated. These results were completely unexpected. Why The T cell lines resulting from these cultures are not monoclonal, and T cells are activated and expanded by mitogens for their specificity This is because the ability exists. Consistent with this, in many cases, Show that repeated stimulation of the T cell line by phenotype results in complete loss of cytolytic activity. Was observed (data not shown). The problem is that T cells are cleared by limiting dilution. Can be overcome by cloning (data not shown) .   In addition, the ability of anti-CD3 antibodies to expand CTLs is dependent on some gp100-specific C Proven using the TL line. As the results presented in Table VI prove, 600- 1500 times expansion can be achieved over 15 days and high Specific lytic activity was retained. Table VI gp100 using anti-CD3 monoclonal antibody and IL-2       Expansion efficiency of specific CTL line  Anti-CD3 antibody, IL-2 and feeder cells described in Materials and Methods Using cells, four randomly selected Cs from the experiments described in Table VI The TL line was expanded.                                 Example 4   Methods for identifying CTL-stimulated subdominant and immunodominant epitopes Role of MHC binding affinity in immunogenicity and immunodominance   In previous studies, HLA-A2 restricted CTL lines from melanoma patients were g It has been shown to recognize various peptides derived from p100 monocyte-derived proteins. Was. Specifically, gp100 derived from tumor infiltrating lymphocytes (TIL) in four patients The reactive CTL line was changed to the size of the peptide containing the HLA-A2.1 binding motif. Panels were screened for reactivity (Kawakami, et al. , "J. Immunol." 154: 3961 (1995)). 1 Only 7 out of 69 peptides were recognized by at least one CTL line . Interestingly, G9₂₈₀Is recognized by CTL from 5 melanoma patients Have been reported by other researchers (Cox, et al., "Science". e "264 716 (1994)).   In this example, the HLA- contained within the sequence of a known TAAgp100 9- and 10-mer peptides corresponding to the A2.1 binding motif were synthesized and And tested for binding to purified HLA-A2.1 molecules. Tested 1 A total of 17 (12.5%) of the 36 peptides were in vitro Significant binding to HLA-A2.1 (IC₅₀<500 nM) (Table VI) I). Table VII HLA-A2.1 binding peptides from melanoma-associated antigen gp100       list ^aThe underlined sequences represent the CTL epitopes found in melanoma patients. (Kawakami, et al., “J. Immunol.” 154: 3). 961 (1995); Bakker, et al. "Int. J. Cance r "62:97 (1995) Cox, et al.). , "Science" 264: 716 (1994).^b Given the concentration (nM) required to inhibit 50% of the binding of the standard peptide Was measured. See Materials and Methods for more information.   Five known A2 restricted gp100-derived peptides are in fact contained within this set, The corresponding sequences in Table VII are underlined. The remaining two conventional The identified epitope (G10₁₆₄And G10₂₀₈), They are bugs Low MHC binding affinity was indicated (1010 nM and 2 080 nM, not included in Table VII). However, these two 10mer is two 9mer G9₁₇₈And G9₁₅₄Containing the corresponding 1 With a much higher binding affinity than the 0 mer and in fact the smallest epitope Could be represented.   From the results presented in Table VII, most importantly, the five known A2 Besides the dominant epitope there are at least 12 other peptides, these are It is observed that it binds to the HLA-A2.1 molecule with good affinity. this The following series of experiments were designed to determine the immunological potential of these peptides. Normal individuals to gp100 using peptides corresponding to immunodominant epitopes In vitro CTL induction of the body   MHC binding pepti corresponding to lymphocytes and melanoma TAA from normal individuals We report that can be used to induce melanoma-reactive CTL (Celis, et al., Proc. Natl. Acad. Sci. USA) 91: 2105 (1994)). Similar observations have been made to other groups. More reported (Houbiers, et al., "Eur. J. Immunol." 2 3: 2072 (1993); Van, et al. "Eur. J. Immun ol. 24: 3038 (1994); Gagliardi, et al. , " Int. Immunol. 7: 1741 (1995); Bakker, et. al. , "Cancer Res." 55: 5330 (1995); Pleba. nski, et al. , "Eur. J. Immunol." 25 1783 ( 1994)).   However, secondary culture protocols to identify CTL epitopes in humans The utility of the tool was limited by technical difficulties. For more information, these Cole results in the induction of low activity CTL, and eventually these epitopes Lack of provable recognition of processing cells was observed (Wentwo rth, et al. , "Molec. Immunol." 32: 603 (19) 95)).   To solve these difficulties, an improved in vitro CTL sensitization method has been developed. Designed. This key feature is characterized by dendritic cells (Bakker, et al., "C. cancel Res. 55: 5330 (1995); Young, et al. . , "J. Exp. Med." 171: 1315 (1990): Mehta-D. amani, et al. , "J. Immunol." 153: 996 (199). 4); Nair, et al. , "J. Virol." 67: 4062 (199). 3)) in combination with IL-7 as a source of peptide-pulsed APC (Kos, et al. "Eur. J. Immunol." 22: 3183 (1992). Used in the priming step and IL-10 (Chen, et al. , "J. Immunol." 147.528 (1991); Yang, et a. l. , "J. Immunol." 155: 3897 (1995)). Is to add during the cycle. This protocol covers known epitopes A higher binding activity to More effective (E. Celis, et al., In preparation).   Using this method, five previously reported reports are underlined in Table VII. The reported dominant gp100 peptide was replaced by at least two normal HLA-A2.1 They were tested for their ability to induce CTL in blood donors. Table VI The results in II are for a single well after three weekly cycles of challenge. 2 illustrates the type of CTL response obtained in the culture of Example 1. Table VIII Known epitoes from gp100 observed in normal individuals         Example of CTL response to ^a5 hours for 4 targets⁵¹Individual culture in Cr release The cytolytic activity (from the 48-well plate) was measured: 221 (A2.1 ) -1-Peptide,. 221 (A2.1) -no peptide, 624 mel (A2 +, Gp100 +) and A375mel (A2 +, gp100-). Effek The target / target ratio was 1: 1 to 10: 1. Numbers are in "Materials and Methods" % Specific cytotoxicity as defined by The result in each column is Figure 3 represents an example of an individual well, where the same experiment was performed using tide.^b This CTL line was stimulated with antigen and expanded for further characterization (second Figure). Also, a summary of the overall results from these experiments is presented in Table IX. Table IX. In vitro CTL from normal individuals against known epitopes       Response summary ^aThese CTLs are peptide-pulsed targets and melanoma (gp100⁺) Target Killed both sides.^b These CTLs killed only the peptide-pulsed target.^c Percentages represent the number of positive wells / total x 100.^d Each culture represents an individual well of a 48 well culture plate.   Taken together, the results show that 4 of the 5 tested peptides were of all cultures. Induce melanoma-reactive CTL in almost 10% It is shown that it was completed. gp100⁺Induces CTL response to melanoma cells The four peptides obtained were G9₁₅₄, G10₄₇₆, G9₂₀₉, And G9₂₈₀Met . Lowest affinity HLA-A2.1 binding peptide G10₄₅₇Only This type of CTL could not be induced. Also, these results show Thus, all five peptides recognized peptide-sensitized target cells, but did not At least some CTL specificity was elicited that did not lyse the cells. G9₂₈₀Excellent antigen specificity of CTL response   Peptide G9₂₈₀One of the antigen specificities of the CTL line derived in (Marked in footnotes) recognize various HLA-A2.1 melanoma cell lines Test their ability to⁺Pep inhibits lysis of melanoma cell lines By measuring the ability of a tide-pulsed, non-radiolabeled (cold) target, Further study. As shown in the results shown in FIG. 2A, G9₂₈₀Reactivity C TL kills several HLA-A2.1 melanoma cell lines expressing gp100 Could kill melanoma cell lines that are negative for gp100 could not. In addition, this reactivity to melanoma cells indicates that G9₂₈₀Peptide Almost completely blocked by the supported cold target, but irrelevant HLA-A 2.1 Not blocked by cold target pulsed with binding peptide, Specific (FIG. 2B). Identification of gp100-specific subdominant CTL epitope   As shown in Table VII, some gp100-derived peptides were purified. HLA-A2.1 molecule that binds And has not been reported as a CTL epitope recognized by melanoma patients. Specifically, all of the peptides in Table VII were replaced with melanoma TILs from some patients. Was tested for reactivity, but only five A2.1 binding peptides were It was proved to function as an epitope. Next, the six remaining gp100 Incoming peptides were tested according to an in vitro priming protocol. Table X As evidenced by the results in Table 3, three of the six peptides tested were melanoma Trigger reactive CTL and 5 of these peptides express gp100 Peptide-specific CTL that did not react with melanoma cells could be induced. TABLE X New epitopes from gp100 observed in normal individuals       Example of CTL response to ^a5 hours for 4 targets⁵¹Individual culture in Cr release The cytolytic activity (from the 48-well plate) was measured: 221 (A2.1 ) + Peptide,. 221 (A2.1) -No peptide, 624mel (A2 +, gp100 +) and A375mel (A2 +, gp100-). effector The / target ratio was 1: 1 to 10: 1. Figures are defined in Materials and Methods Represents the defined% specific cytotoxicity. The results in each column are for a specific peptide Represents an example of an individual well where the same experiment was performed using.^b Peptide G10₂₂₄All results obtained using are negative and are shown in this table. Absent.^c The CTL lines were restimulated with antigen and expanded for further characterization (FIGS. 3 to 5).   As shown in Table XI, CTL responses to these peptides were Often more than a response to the immunodominant epitope recognized by III) TCs that are somewhat more difficult and perhaps directed against the specificity of these peptides Indicates that the R repertoire is not abundant. Table XI From a normal individual to a new gp100 epitope         Summary of in vitro CTL response ^aThese CTLs are peptide-pulsed and melanoma (gp100 +) targets Killed both sides.^b These CTLs killed only the peptide-pulsed target.^c Percentages represent the number of positive wells / total x 100.^d Each culture represents an individual well of a 48 well culture plate. Antigen specificity of CTL derived with HLA-A2.1 binding peptide   As shown in Table X, in many cases, the ratio of peptide-induced CTL lines The level of cytotoxicity is not high, and moreover, in many cases, significant Background lytic activity (killing of antigen-negative targets) was observed. these The CTL response elicited using the newly defined gp100 epitope is To determine that it is actually specific, culture of lymphocyte cultures (legs in Table X) The antigen (peptide) and APC (two additional cycles) Re-stimulation, followed by various effector / target ratios. And tested. In addition, the ability to kill melanoma cells expressing gp100 is a function of synthetic peptides. Blocked with cold lymphoblast targets pulsed with tide.   As shown in the results shown in FIG.₇₈And G10₁₇₇CT induced by L is gp100⁺It is highly specific for melanoma cells and this activity is Inhibitable by unlabeled (cold) target cells pulsed with the relevant gp100 peptide But derived from the hepatitis B core protein sequence, positions 18-27. Inhibited by cold target cells pulsed with control HEL-2-high affinity peptide Was not.   Peptide G9₁₇₈And G10₁₇₇Is G10₁₇₇The amino terminus of Only depending on the presence of alanine residues contained in III). Whether these peptides represent a single or two separate epitopes G9 to determine₁₇₈And G10₁₇₇CTL lines induced in Tested against peptide. As shown in the results shown in FIG. 4, peptide G10_{17 7} CTLs induced by peptide G9₁₇₈Could not be recognized. To the other Then, as expected, G9₁₇₈CTLs induced by 10-mer pepti Recognizes both peptides since it contains all the residues present on the 9-mer I was able to. As these results show, those two overlaps Peptides can elicit two unique CTL specificities, thus Represents separate epitopes.   In addition, peptide G10₅₇₀Is effective in eliciting anti-melanoma-specific CTL Was. As shown by the data shown in FIG. 5, G10₆₇₀CFL strains release gp100 The emerging melanoma cell line can be killed and this lytic activity is G10₅₇₀Carry Low temperature target that cannot be inhibited by the low temperature target but is sensitized by an irrelevant peptide. Inhibited by target. G10₁₇₇In melanoma patients against gp100 using epitope In vitro CTL induction   Using lymphocytes obtained from HLA-A2.1 melanoma patients, G10₁₇₇D The pitope was tested for its ability to induce melanoma-reactive CTL. 48 pieces After establishing three CTL cultures and three rounds of antigen challenge, one culture was Responsive CTLs were given. As shown in the results shown in FIG. 6, peptide G10₁₇₇Invite CTLs in induced melanoma patients have peptide sensitizing targets and gp100⁺Melanoma cells Effectively and specifically recognizes both, and its lytic activity on melanoma cells is Blocked by cold target cells pulsed with the relevant peptide.   The present invention relates to at least three new tumor-reactive CTLs. Gp100 epitope. 6 of the 12 potential subdominant epitopes Only to date is our dendritic cell priming in vitro immunization protocol Gp100-derived epitopes could be identified Exists. The protocol of the experiments described herein is for a given tumor antigen. CTL epitopes contained in E. coli. Irrespective of gender) and complete the immunological potential of the tumor antigen itself Use for   In vivo and in vitro using dominant and subdominant epitopes RoCTL induction will produce a more intense and diverse immune response. Go A multivalent vaccine containing a mixture of epitopes from several TAAs will Would overcome the need for complex mixtures of proteins or genes. Sub-dominant epitome Immunization using a probe focuses the CTL response on a specific conserved sequence, This prevents the appearance of tumor variants during the course of the disease.   Furthermore, the presented results also indicate that the dominant and subdominant epitopes To be able to induce tumor-reactive CTL from the blood of cancer patients specific to both Prove it. These CTLs were cultured in culture at approximately 1 × 10⁹Can spread to cells Yes (data not shown) and still retain antigen specificity. Could be used for adoptive therapy. Peptide primed from blood Immunotherapy using CTL is convenient (does not require tumor biopsy) ) And reliable (because of its high specificity) Is an alternative to the current TIL-based immunotherapy (Rosenbe) rg, et al. , "J. Natl. Cancer Inst." 86:11. 59 (1994)).                                 Example 5                    Expansion of CTL recognizing MAGE antigen   The MAGE2 and MAGE3 antigens are only frequently expressed in melanomas Absent and also expressed in almost 40-60% of epithelial tumors (colon, breast, lung) (Weynants, P., et al., "International."   Journal of Cancer "56: 826-829 (1994); De Plaen, E.A. Et al. , "Immunogenetics" 4 0: 360-369 (1994); Russo, V .; Et al. , "Int international Journal of Cancer "64: 216- 221 (1995)). HLA-A1 (Celis, E. et al., "Pr receiveds of the Natal Academy of Sciences the United State of America 91: 2105-2109 (1994); Gaugler, B .; , Et al . , "Eur. J. Immunol." 179: 921-930 (1994)). And HLA-A2.1 (van der Brugen, P., et al. . , "Eur. J. Immunol." 24: 3038-3043 (1994). 3.) A CTL epitope from restricted MAGE3 was reported. Those studies are pepti EVDPIGLY (in the case of HLA-A1) and FLWGPRAL V (in the case of HLA-A2.1) induced in vitro CTLs are transformed into peptide-sensitized EBV Recognize cell lineage and⁺Proved how to kill melanoma cell lines ( Celis, E .; et al. , "Proceedings of the N atinal Academy of Sciences the Unite d State of America "91: 2105-2109 (1994) Gaugler, B .; Et al. "Eur. J. Immunol." 179: 921-930 (1994)) and HLA-A2.1 (van de r Bruggen, P .; Et al. "Eur. J. Immunol." 24: 3038-3043 (1994)).   Additional HLA-A2.1 restricted CTL from MAGE2 and MAGE3 To identify epitopes, the combined from MAGE2 and MAGE3, respectively, A total of 81 and 89 synthetic peptides, 9-11 residues long, all HLA-A2. 1 binding motif (Rupert, J., et al., Cell 74: 929-937 (1993); Rammensee, H .; -G. , Et   al. , "Immunogenetics" 41: 178-228 (1995). )) Binds to the purified HLA-A2.1 molecule in vitro We tested for their ability. 8 of the 81 peptides from MAGE2 and 89 pep from MAGE3 (Table XII), which is an affinity generally associated with CTL immunogenicity. Approximate limit values (Sette, A., et al., “Journal of   Immunology "153: 5586-5592 (1994)). Table XII HLA-A2.1 binding peptides from MAGE-2 and MAGE-3       List of ^aNumbers in parentheses indicate peptide size and position on protein sequence (subscript) Represent.^b The concentration (nM) of the test peptide required to inhibit 50% of the standard binding Measured. See Materials and Methods for more information. See also.^c Features directed to antigen-containing targets (+ peptide or MAGE + tumor, 624 mel) When the differential cytotoxicity is> 15% higher than the cytotoxicity using the negative control target, The culture was considered to contain CTL. Values are positive cultures out of 48 tested Represents the number of^d These cultures are> 10% specific for tumors after 2 rounds of restimulation It was cytotoxic. However, after cloning of T cells, The intensity of the response increased (FIGS. 1 and 2).^e Not tested.^f This peptide has been described as an HLA-A2 restricted epitope (41). Pep After 4 rounds of restimulation using tide, cytotoxicity on tumor cells was observed .^g In three of four different lymphocyte donors, CT L was obtained.   Primary in vitro immunization protocol for peptide-pulsed dendritic cells (DC) HLA-A2.1 binding pairs from MAGE2 and MAGE3 using Some of the peptides were tested (Tsai, V., et al., Journa. l of Immunology "158: 1796-1802 (1997)) . Most of the peptides (5 out of 5 from MAGE2 and 6 out of MAGE3) 5) induces CTL that kills peptide sensitized (EBV transformation) target cells (Table XII).   Selected cultures are expanded by repeated antigen restimulation (peptide and APC) Sufficient effector cells were obtained for a more extensive cytotoxicity assay. Details Alternatively, the ability to recognize tumor cells expressing the corresponding MAGE antigen Peptide-reactive CTL line Studied. In most cases, the reactivity towards the peptide-sensitized target cells is determined. It remained easily. Furthermore, MAGE2 [10₁₅₇] The reactive CTL line is natural Demonstrated ability to kill melanoma cells that produce and process MAGE2 protein Was. Similarly, of the six HLA-A2.1 binding peptides from MAGE3 Two, namely MAGE3 [9₂₇₁] And MAGE3 [9₁₁₂] Is anti-melanoma It was found to elicit refractory CTL (Table XII).   MAGE2 [10₁₅₇] And MAGE3 [9₁₁₂] Reactive CTL Antigen Specificity Cultures with anti-melanoma reactivity to study tumor and tumor responsiveness in more detail A T cell clone was isolated from the product. On the other hand, MAGE3 [9₂₇₁] Is black It has already been reported as an epitope of melanoma-reactive CTL (van der B Ruggen, P.R. Et al. "Eur. J. Immunol." 24: 3038-3043 (1994)), so that the corresponding C The TL line was further characterized.   As the results shown in FIG. 7 prove, MAGE2 [10₁₅₇] By peptide Induced CTLs are not only melanoma cells, but also colon and stomach tumor cells Strains were killed effectively, provided they were both HLA-A2 and MAGE2 Is expressed. In addition, melanoma, colon cancer cells and, to some extent, gastric cancer cells Knowledge is MAGE2 [10₁₅₇By unlabeled (cold) target cells pulsed at Could be locked, thus confirming the antigen specificity of this CTL clone ( (Fig. 6).   In addition, anti-MAGE3 [9₁₁₂] Antigen specificity and antitumor response of CTL clone The properties were measured. As the results shown in FIGS. 9 and 10 prove, this CTL Clones are also highly specific, HLA-expressing the MAGE3 protein. A2-positive melanoma, colon and The stomach tumor cells could be recognized.   In summary, these results indicate that antigen-specific CTLs can be 3 illustrates the identification of two novel HLA-A2.1 restricted MAGE-derived epitopes. This These CTLs are not only peptide sensitized target cells, but also various histological types Of tumor cell lines were recognized and lysed.                                 Example 6                     Expansion of CTL that recognizes CEA antigen   CEA (Carcinoembryonic Antigen) is used in epithelial tumors such as lung, stomach and breast cancer. Is a frequently expressed TAA (Vincent, RG, et al., "Cardiovasc. Surg." 66: 320-328 (1978); S havely, J .; Et al. "CRC Critical Review s in Oncology and Hematology "2: 355-3 99 (1985); Thompson, J.M. Et al. , "Journal   of Clinical and Laboratory Analysis 5: 344-366 (1991); Sikorska, H .; Et al. , "Cancer Detection and Prevention" 12: 321-355 (1988); Murano, R .; Et al. , "Canc er Research "45: 5770-5780 (1985); Stewa rd, A.I. M. Et al. , "Cancer" 33: 1246-1252 ( 1985)). MAGE2 and MAGE3 were outlined in the previous section. Following the approach and creating a secondary anchor related to affinity MHC binding CEA was scanned for the HLA-A2.1 binding motif (Ru pert, J. et al. Et al. , "Cell" 74: 929-937 (199). 3)). Contains a total of 18 motifs tested for HLA-A2.1 binding Of the 9 peptides (9-mer and 10-mer), 9 were 50-fold in HLA-A2.1. 0 nM or less IC₅₀Joined. Table XIII List of HLA-A2.1 binding peptides from CEA ^aNumbers in parentheses indicate peptide size and position on protein sequence (subscript) Represents^b The concentration of test peptide required to inhibit 50% of the binding of the standard peptide ( nM). See Materials and Methods for more information.^c Specific towards antigen-containing target (+ peptide or CEA + tumor, SW403) Culture when cytotoxicity> 15% higher than cytotoxicity using negative control target The product was considered to contain CTL. value is Represents the number of positive cultures out of 48 tested.^d This peptide has been described as an HLA-A2 restricted epitope (13). cell Ten damaging tumor cells were observed, but specificity was evidenced by inhibition of the cold target Could not be performed (not shown).^e Obtained from two (out of three) different lymphocyte donors. 4 rounds Cytotoxicity to tumor cells observed after restimulation with peptide .^f Not tested.   Interestingly, in cancer patients after vaccination with the recombinant CEA construct, Peptide CEA [9₆₀₅] Was recently reported as a CTL epitope (Tsan g, K. Et al. , "Journal of the National   Cancer Institute, 87: 982-990 (1995)).   Several other HLA-A2.1 binding peptides derived from CEA Using DC as the presenting cells, the ability to elicit CTL was studied. test Three of the five peptides were CTL-responsive to recognize peptide-sensitized target cells. Stimulated the answer. Moreover, after restimulation with antigen and APC, tumor cells expressing CEA The killing of the vesicle is CEA [9₆₀₅] And CEA [9₆₉₁Evidence for specific CTL (Table XIII). Peptide CEA [9₆₉₁CTL line reactive with Roaning and further study by specificity and recognition of various CEA expressing tumor cells did. CEA [9₆₉₁The cloned CTL specific for HLA- High specificity for tumor target cells (colon and stomach) expressing A2 and CEA (See FIG. 11). Furthermore, it is suitable for tumor cells of the stomach and colon. The killing activity of the peptide CEA [9₆₉₁Pulse with Was blocked with a “cold target” but was blocked with a control HLA-A2.1 binding peptide. It was specific because it was not blocked by the loose target (FIG. 12).                                 Example 7                Expansion of CTL recognizing HEL2 / neu antigen   Another TAA frequently found on epithelial tumors, also known as cerB-2 This TAA, which is the product of the HEL2 / neu gene, Cell surface overexpressed by cancer and also expressed by colon and lung cancer (Slamon, DJ, et al., Science e "244707-712 (1989); Yokota, J. et al. Et al. , "Lancet" 1: 765-767 (1986)). HLA-A2.1 binding module Of the 165 peptides (9-mer and 10-mer) containing the chief, 23 were purified IC to HLA-A2.1 molecule₅₀Found to bind at ≦ 500 nM (Table XIV). Table XIV List of HLA-A2.1 binding peptides from HEL2 / neu ^aNumbers in parentheses indicate peptide size and position on protein sequence (subscript) Represent.^b The concentration of test peptide required to inhibit 50% of the binding of the standard peptide ( nM). See Materials and Methods for more information.^c Specific towards antigen-containing target (+ peptide or HER + tumor, SW403) Culture when cytotoxicity> 15% higher than cytotoxicity using negative control target The product was considered to contain CTL. Values are the number of positive cultures out of 48 tested Represents^d These peptides were described as HLA-A2 restricted epitopes (10, 1 1, 47).   Among the highest affinity HLA-A2.1 binding peptides, it was HER2 [9, reported as CTL epitopes in₃₆₉] The peptide (Fisk, B., et al., "J. Exp. Med." 181). : 2109-2117 (1995)).   To identify additional HEL2 / neu-derived CTL epitopes, HLA-A 2.1 Four of the binding peptides (HER2 [9₄₃₅], HER2 [9₇₈₉], HER2 [9₄₈] And HER2 [9_Five]) For in vitro immunization Tested in the protocol. HER2 [9₃₆₉] In this analysis , This epitope can induce tumor-reactive CTL in our system I confirmed that. All five HLA-A2.1 binding peptides were tested The CTL that killed the tide-sensitized target cells were derived. Three of them (HER2 [ 9₃₆₉], HER2 [9₄₃₅] And HER2 [9_Five]) Are HLA-A2.1 and And CTLs that recognize and specifically kill tumor cells that express HEL2 / neu antigen drawer (Table XIV). HER2 [9₂₆₉] Is an epitome of tumor-reactive CTL in the past (Fisk, B., et al., “J. Exp. Med. . 181: 2109-2117 (1995)), so we use these CTLs. No further characterization was done. In contrast, HER2 [9₄₃₅] And HER2 [9_FiveThe CTL line specific for was expanded for further analysis.   As shown in FIG. 13, HER2 [9₄₃₅CTL lines induced in Indicates target cells for peptide sensitization and HLA-A2.1, HEL2 / neu-positive tumor cells. Both vesicles were killed (FIG. 13A). Furthermore, the recognition of the tumor cell target is again ER2 [9₄₃₅Was significantly inhibited by the unlabeled (cold) target pulsed at Not pulsed or pulsed with control HLA-A2.1 binding peptide Was not inhibited by the unlabeled target (FIG. 13B). These results prove As such, the recognition of tumor cells by these CTLs is indeed antigen specific. same Was tested for tumor cell reactivity and antigen specificity as described above._Five] Similar results were obtained using specific CTL lines (FIG. 14).                                 Example 8           Engineered CEA-specific CTL epitope   As previous studies have shown, primary or secondary anchor modifications can increase antigenicity. (Parkhurst, MR, et al., "Jo urnal of Immunology "157: 2539-2548 (19 96); Topalian, S.M. L. Et al. , "Journal of   Experimental Medicine "183: 1965-1971. (1996)). As shown in Table XIII, the "normative" anchor CEA "9" lacking one of the amino acids (V at the carboxyl terminus)_{twenty four}] Is a pool distribution Determined by column determination analysis (Rammensee, H.-G., et al. , "Immunogenetics" 41: 178-228 (1995)). Non To determine whether to replace the defined C-terminal anchor (T) with a defined residue , CEA [9_{twenty four}Were prepared and purified HLA-A 2. Tested for binding to molecule. These two analogs are located at position 2. Residues, especially the naturally occurring “L” (CEA [9_{twenty four}] -L2V9 distribution Row: LLTFWNPPV) or “M” residue (CEA [9_{twenty four}] -M2V9, sequence LMFWNPPV), which is most likely to have an “M” at position 2. Based on the finding that it is often associated with a suitable HLA-A2.1 binding capacity (Ru pert, J. et al. Et al. , "Cell" 74: 929-937 (199). 3); del Guercio,. M. F. Et al. , "Journal   of Immunology "154 686-693 (1995)). Natural Sequence CEA [9_{twenty four}] (IC₅₀176.6 nM) compared to the analogous peptide Added CEA [9 to purified HLA-A2.1 molecules._{twenty four}] -L2V9 (IC₅₀16 nM) with almost 10-fold increased affinity and CEA [9_{twenty four} ] -M2V9 (IC₅₀(4.5 nM) with a 4-fold increase.   Peptide CEA [9_{twenty four}], At least according to the protocol of our experiment, i Inability to trigger a CTL response in vitro (Table XIII) It was noteworthy that both peptide analogs were immunogenic by the CTL response. CTL lines induced by both analogs have CEA and HLA-A2 antigens. Expressed SW403 colon carcinoma cells were specifically recognized and killed (data shown). Absent). CEA [9_{twenty four}]-Cloning of M2V9 specific CTL and further characterization Were determined. As evidenced by the results shown in FIG. 15, these CTLs were in fact antigen specific. HLA-A2 is anomalous and expresses CEA⁺Stomach and colon tumor cell lines identified I knew.                                 Example 9            Tumor CTL epitope to HLA supertype molecule                              Cross-reactive binding   The above results illustrate the identification of multiple epitopes from various TAAs. This The combined use of such epitopes has led to the development of major breast, ovarian, lung and colon carcinomas. It is expected that effective coverage can be provided.   To examine the coverage of their relative potential population, we Check if the tope can bind to other ordinary alleles of the A2 supertype (Del Guercio, MF, et al., “Journal o f Immunology "154: 685-693 (1995); Sidne. y. Grey, et al. , "Immunology Today" 1 7: 261-266: 1996)). HLA-A2.2 and -A68.2 are Selected because it has a high frequency in the black population, and -A2.3 and And -A2.6 are Oriental ethnicities (del Guerci) because it has a high frequency in o, M.S. F. Et al. , "Journal of Immunology 154: 685-693 (1995); Sidney, J. et al. , Gray, et   al. , "Immunology Today" 17: 261-266: 19. 96)).   Peptides displaying CTL epitopes in addition to binding to HLA-A2.1 Pide also binds to at least other members of the HLA-A2 supertype. (Table XV). Table XV A2 supertype binding data from various CTL epitopes         Data a) Data is again from Tables 1, 2 and 3 (for comparison). b) IC out of 5 tested₅₀≦ 500 nM represents the number of binding alleles.   Specifically, an epitope from MAGE3, MAGE3 [9₂₇₁] And MAGE3 [9₁₁₂] Are both well (IC₅₀≦ 500 nM) binding, A2 All five members of the supertype were studied. Similarly, the peptide MAGE2 [10₁₅₇] Bound to three of the five alleles of the A2 supertype (Table XV). For the CTL epitope from CEA, the peptide CEA [9₆₀₅] And CEA [9₆₉₁] Are 4 and 5 of A2 super type respectively. It was found to bind (Table XV). Most importantly, CEA [9_{twenty four}] Although it was possible to bind only two of the five alleles, CEA [9_{twenty four}] M2 V9 and CEA [9_{twenty four}L2V9 is immunogenic in the studies of the present invention. And of the five A2 supertypes tested, 3 and And 4 (Table XV). Finally, various CTL episodes from HEL2 / neu Peptides displaying toupes bind to two or more of the A2 supertype alleles. (Table XV).   In summary, these results show that the present invention and earlier Most of the HLA-A2.1 restricted CTL epitopes Have the ability to bind to some of the members of the group. The operation approach is also Peptides such as HER2 [9_FiveCross-reactivity of (sequence ALCRWGLLLL) Is further increased by replacement of the primary and / or secondary binding anchors Research is ongoing to determine if it can be done.   The invention is described in some detail by way of illustration and example for purposes of clarity and understanding. However, certain changes and modifications can be made within the scope of the present invention. Will be apparent to those skilled in the art. All references cited herein And patents are incorporated herein by reference.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/18 A61P 33/06 33/06 A61K 39/00 Z // A61K 38/00 C12N 5/00 E 39/00 A61K 37/02 (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, C , CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Sydney, John United States of America, California 92037, La Jolla, Villa La Jolla Drive 8541 Dee. (72) Inventor Set, Alesandro United States of America, California 92037, La Jolla, Linda Rosa Avenue 5551 (72) Inventor Celis, Esteban United States, California 92130, San Diego, Lindfair Road 13644

Claims (1)

[Claims]   1. Pretreatment of immunogenic peptides from antigens that bind to class I MHC molecules Contacting antigen presenting cells pretreated with the long factor, and   Incubating the antigen presenting cells with purified CD8 and two or more Incubation in the presence of growth factors, which results in antigen-specific cytotoxicity. Produce T cells, A method for producing antigen-specific cytotoxic T cells in vitro.   2. 2. The method according to claim 1, wherein the antigen presenting cells are dendritic cells.   3. The dendritic cell precursor is selected from the group consisting of GM-CSF and IL-4 The dendritic cells by incubating in the presence of a pretreated growth factor. The method of claim 2, wherein is prepared.   4. The antigen-presenting cells are prepared by incubating about 10 to 50 μg / ml of an immunogenic peptide with ink. By contacting the desired immunogenic peptide with the MHC molecule The method of claim 1.   5. Peptides from pathogens or tumor-associated antigens with the desired MHC binding motif 2. The method according to claim 1, wherein the immunogenic peptide is identified by selecting a peptide. the method of.   6. The method of claim 1, wherein one of the incubation growth factors is IL-7. Method.   7. The method of claim 1, wherein one of the incubation growth factors is IL-10. the method of.   8. Incubation of growth factor IL-7 at the start of the incubation step And the incubation growth factor IL-10 is added one day later. The method of claim 1, wherein   9. Contacting said antigen-specific cytotoxic T cells with a pharmaceutically acceptable carrier; Thereby forming a pharmaceutical composition and administering said pharmaceutical composition to a patient. The method of claim 1, further comprising:   10. The cytotoxic T cells are cancer, AIDS, hepatitis, bacterial infection, fungal infection, mala 2. The method of claim 1, wherein the method is effective in the treatment of ria or tuberculosis.   11. Process   Taking a sample containing cytotoxic T cells from a first patient,   Contacting said cytotoxic T cells with antigen presenting cells pre-treated with a pre-treated growth factor Wherein the antigen presenting cell comprises a class I MHC molecule,   Desired immunogenic peptide and two or more incubation growth factors In the presence of the offspring, the pretreated antigen-presenting cells are combined with the cytotoxic T cells. Incubating, thereby producing activated cytotoxic T cells,   Contacting said activated cytotoxic T cells with an acceptable carrier, whereby Form a pharmaceutical composition, and   Administering the pharmaceutical composition to a patient, A method of specifically killing target cells in a human patient.   12. The method according to claim 11, wherein the antigen-presenting cell is a dendritic cell.   13. The dendritic cell precursor is selected from the group consisting of GM-CSF and IL-4 By incubating in the presence of pretreated growth factors. 13. The method of claim 12, wherein the vesicle is prepared.   14． The antigen-presenting cells are treated with about 10 to 50 μg / ml of the immunogenic peptide. By cuvating, the desired immunogenic pepti 12. The method of claim 11, wherein the contact is with an MHC molecule.   15. Selecting peptides from pathogens having the desired HLA binding motif The method of claim 11, wherein the immunogenic peptide is identified.   16. The method of claim 11, wherein one incubation growth factor is IL-7. The method described.   17. The method of claim 11, wherein one of the incubation growth factors is IL-10. The described method.   18. Initiation of incubation step with incubation growth factor IL-7 And the incubation growth factor IL-10 is added one day later The method of claim 11, wherein   19. The cytotoxic T cells are cancer, AIDS, hepatitis, bacterial infection, fungal infection, mala 12. The method of claim 11, wherein the method is effective in treating rear or tuberculosis.